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Overproduction of geraniol by enhanced precursor supply in Saccharomyces cerevisiae
... Genetic truncated 3-hydroxyl-3-methylglutaryl-CoA reductase gene (tHMG1) [6] or a mutated HMG2 K6G [7] has been overexpressed to release the restriction on mevalonate node. The interconversion between IPP and DMAPP by isopentenyl-diphosphate isomerase (IDI1) was further enhanced to improve the formation of GPP [8]. ...
... Then, ERG20 ww -(GS) 4 and (GS) 4 -ERG20 ww fragments were cloned into the sites of BamH I and Not I of p424-tPt1 ( Fig. 2a) to construct p424-ERG20 ww -(GS) 4 -tPt1 and p424-tPt1-(GS) 4 -ERG20 ww , respectively. Further ERG20 ww -(G) 8 , ERG20 ww -(GGGS) 2 , ERG20 ww -(GSG) 3 , ERG20 ww -(PT) 4 , ERG20 ww -(EAAAK) 2 fragments were into the BamH I site of the p424-tPt1 to construct p424-ERG20 ww -(G) 8 ...
... Then, ERG20 ww -(GS) 4 and (GS) 4 -ERG20 ww fragments were cloned into the sites of BamH I and Not I of p424-tPt1 ( Fig. 2a) to construct p424-ERG20 ww -(GS) 4 -tPt1 and p424-tPt1-(GS) 4 -ERG20 ww , respectively. Further ERG20 ww -(G) 8 , ERG20 ww -(GGGS) 2 , ERG20 ww -(GSG) 3 , ERG20 ww -(PT) 4 , ERG20 ww -(EAAAK) 2 fragments were into the BamH I site of the p424-tPt1 to construct p424-ERG20 ww -(G) 8 ...
Pinene is an active natural monoterpene from plants and has important applications in flavorings, fragrances,and pesticides. Especially, pinene dimers are regarded as renewable fuels with high density. However, the microbial pinene production was limited by the low activity pinene synthase. In this study, the pinene synthase activity was improved by fusion linker optimization and chaperon coexpression. To construct the pinene pathway in Saccharomyces cerevisiae, YPL062W gene was deleted to increase the MVA pathway precursor acetyl-CoA. Truncated 3-hydroxyl-3-methylglutaryl-CoA reductase (tHMG1), isopentenyl-diphosphate isomerase (IDI1), and farnesyl diphosphate synthase mutant (ERG20F96W−N127W) were then integrated to improve the GPP pool. Pinene synthase tPt1 was expressed in the constructed engineered yeast, and the titer of pinene reached 0.166 mg/L. GPP is the direct precursor of pinene, ERG20ww and tPt1 were fused by different linkers and orders to improve the accessibility of GPP. Pinene titer reached 9.94 mg/L by fusion these proteins in the order of ERG20ww and tPt1 and with a flexible linker (G)8. After that, several chaperons were coexpressed and the chaperon Sil1p improved the pinene titer to 10.2 mg/L with a yield of 1.63 mg/L·OD600. The results presented here provide novel information on the applications of protein fusion and protein chaperons in microbial pinene production.
... Previous work has shown that engineered E. coli could produce 400 mg/L of limonene and approximately 100 mg/L of perillyl alcohol [13], and engineered yeasts were able to produce 95 µg/L of linalool [15], as well as 36.04 mg/L-2.0 g/L of geraniol [16,17]. ...
... On the other hand, HMG-CoA reductase was identified as a key rate-limiting enzyme in the MVA pathway of S. cerevisiae [23,24], and a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase gene (tHMGR) was overexpressed to increase the supply of mevalonate. DMAPP is the common substrate for the biosynthesis of both GPP and tRNA, and MAF1 represses the transcriptional activity of RNA polymerase III, serving as a negative regulator in the biosynthesis of tRNA [16]. The isoprenoid diphosphate isomerase IDI1 catalyzes the interconversion between DMAPP and IPP [25], but the formation of one molecule of GPP requires two molecules of IPP and one molecule of DMAPP. ...
... The isoprenoid diphosphate isomerase IDI1 catalyzes the interconversion between DMAPP and IPP [25], but the formation of one molecule of GPP requires two molecules of IPP and one molecule of DMAPP. Since the ratio of IPP to DMAPP is not optimal for GPP biosynthesis, Jingwen Zhou's research indicated that the isomerase IDI1 was the rate-limiting enzyme in geraniol production [16]. Thus, overexpression of truncated HMG-CoA (tHMG1), IDI1 and MAF1 could improve the production of monoterpenes. ...
Background
Geranyl acetate is widely used in the fragrance and cosmetic industries, and thus has great economic value. However, plants naturally produce a mixture of hundreds of esters, and geranyl acetate is usually only present in trace amounts, which makes its economical extraction from plant sources practically impossible. As an ideal host for heterologous production of fragrance compound, the Saccharomyces cerevisiae has never been engineered to produce the esters, such as geranyl acetate. ResultsIn this study, a heterologous geranyl acetate synthesis pathway was constructed in S. cerevisiae for the first time, and a titer of 0.63 mg/L geranyl acetate was achieved. By expressing an Erg20 mutant to divert carbon flux from FPP to GPP, the geranyl acetate production increased to 2.64 mg/L. However, the expression of heterologous GPP had limited effect. The highest production of 13.27 mg/L geranyl acetate was achieved by additional integration and expression of tHMG1, IDI1 and MAF1. Furthermore, through optimizing fermentation conditions, the geranyl acetate titer increased to 22.49 mg/L. Conclusions
We constructed a monoterpene ester producing cell factory in S. cerevisiae for the first time, and demonstrated the great potential of this system for the heterologous production of a large group of economically important fragrance compounds.
... Geraniol production has been demonstrated in S. cerevisiae. Early work focused on enhancing the mevalonate pathway flux, (Liu et al., 2013), followed by enhancing the supply of geranyl diphosphate selection of geraniol synthases and fusion to a geranyl pyrophosphate favoring mutant of the farnesyl-diphosphate synthase, resulting in modest titers (Zhao et al., 2016).Focusing on alternative sources of geraniol synthase, activating truncations of geraniol synthase, combined with ERG20 mutants favoring geranyl pyrophosphate, the highest shake flask titers in a eukaryote were reported (523.96 mg/L) (Jiang et al., 2017). ...
... MVA pathway (Liu et al., 2013), dynamic control of ERG20 expression and OYE2 deletion (Zhao et al., 2017), increasing GPP pool, minimizing dehydrogenation of geraniol (Zhou et al., 2014) construction of fusion proteins to facilitate substrate channeling (Zhao et al., 2016), compartmentalization of the geraniol pathway in mitochondria (Yee et al., 2019), peroxisomal localization and improvement of geraniol tolerance (Gerke et al., 2020), optimization of fermentation conditions, and interconversion of geranyl acetate and geraniol (Liu et al., 2016) with titers less than 525 mg/L in shake-flask fermentation in most papers. Our shake-flask results of 1 g/L geraniol exceeds shake-flask fermentation titers produced by these engineered S. cerevisiae, suggesting that Y. lipolytica is a preferred host for the production of monoterpenoids. ...
Geraniol is a monoterpene with wide applications in the food, cosmetics, and pharmaceutical industries. Microbial production has largely used model organisms lacking favorable properties for monoterpene production. In this work, we produced geraniol in metabolically engineered Yarrowia lipolytica. First, two plant-derived geraniol synthases (GES) from Catharanthus roseus (Cr) and Valeriana officinalis (Vo) were tested based on previous reports of activity. Both wild type and truncated mutants of GES (without signal peptide targeting chloroplast) were examined by co-expressing with MVA pathway enzymes tHMG1 and IDI1. Truncated CrGES (tCrGES) produced the most geraniol and thus was used for further experimentation. The initial strain was obtained by overexpression of the truncated HMG1, IDI and tCrGES. The acetyl-CoA precursor pool was enhanced by overexpressing mevalonate pathway genes such as ERG10, HMGS or MVK, PMK. The final strain overexpressing 3 copies of tCrGES and single copies of ERG10, HMGS, tHMG1, IDI produced approximately 1 g/L in shake-flask fermentation. This is the first demonstration of geraniol production in Yarrowia lipolytica and the highest de novo titer reported to date in yeast.
... Geraniol production has been demonstrated in S. cerevisiae. Early work focused on enhancing the mevalonate pathway flux, (Liu et al., 2013), followed by enhancing the supply of geranyl diphosphate selection of geraniol synthases and fusion to a geranyl pyrophosphate favoring mutant of the farnesyldiphosphate synthase, resulting in modest titers (Zhao et al., 2016).Focusing on alternative sources of geraniol synthase, activating truncations of geraniol synthase, combined with ERG20 mutants favoring geranyl pyrophosphate, the highest shake flask titers in a eukaryote were reported (523.96 mg/L) (Jiang et al., 2017). ...
... Many studies of geraniol production in E. coli and S. cerevisiae have been published based on overexpression of rate-limiting enzymes in the MVA pathway (Liu et al., 2013), dynamic control of ERG20 expression and OYE2 deletion (Zhao et al., 2017), increasing GPP pool, minimizing dehydrogenation of geraniol (Zhou et al., 2014) construction of fusion proteins to facilitate substrate . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. ...
Geraniol is a monoterpene with wide applications in the food, cosmetics, and pharmaceutical industries. Microbial production has largely used model organisms lacking favorable properties for monoterpene production. In this work, we produced geraniol in metabolically engineered Yarrowia lipolytica . First, two plant-derived geraniol synthases (GES) from Catharanthus roseus (Cr) and Valeriana officinalis (Vo) were tested based on previous reports of activity. Both wild type and truncated mutants of GES (without signal peptide targeting chloroplast) were examined by co-expressing with MVA pathway enzymes tHMG1 and IDI1. Truncated CrGES (tCrGES) produced the most geraniol and thus was used for further experimentation. The initial strain was obtained by overexpression of the truncated HMG1, IDI and tCrGES. The acetyl-CoA precursor pool was enhanced by overexpressing mevalonate pathway genes such as ERG10, HMGS or MVK, PMK. The final strain overexpressing 3 copies of tCrGES and single copies of ERG10, HMGS, tHMG1, IDI produced approximately 1 g/L in shake-flask fermentation. This is the first demonstration of geraniol production in Yarrowia lipolytica and the highest de novo titer reported to date in yeast.
... However, some aspects remain to be improved: the effective use of carbon/sugar sources, the redirection of the host metabolism towards an effective production of the compound of interest (i.e., without compromising the vitality of the host itself), modification of compounds mediated by plant enzymes absent in the host, etc. Overexpression of E. coli FPPS (IspA); introduction of plant (Clausena lansium) STS; optimization of RBSs; heterologous expression of MVA pathway; removal of competitive indole synthesis by tnaA deletion 0.60 g/L [44] S. cerevisiae concern enhancing the flow of the MVA pathway by overexpressing the key genes; downregulating the competitive pathways by replacing the native promoters with inducible ones; knocking-out or inhibiting some negative regulators; and strengthening terpene synthases or other enzymes associated with them, through overexpression or protein engineering. In this regard, Liu et al. (2013) [48] and Zhou et al. (2020) [51] tried to overcome the problem by manipulating three genes that have been shown to be related to an increase in the flow of the MVA pathway in order to produce geraniol and linalool, two monoterpenes which are widely used as heart notes in the perfume industry due to their pleasant rose and floral/spicy smell, respectively. The three genes in question are tHMG1, IDI1 and ERG20. ...
... However, some aspects remain to be improved: the effective use of carbon/sugar sources, the redirection of the host metabolism towards an effective production of the compound of interest (i.e., without compromising the vitality of the host itself), modification of compounds mediated by plant enzymes absent in the host, etc. Overexpression of E. coli FPPS (IspA); introduction of plant (Clausena lansium) STS; optimization of RBSs; heterologous expression of MVA pathway; removal of competitive indole synthesis by tnaA deletion 0.60 g/L [44] S. cerevisiae concern enhancing the flow of the MVA pathway by overexpressing the key genes; downregulating the competitive pathways by replacing the native promoters with inducible ones; knocking-out or inhibiting some negative regulators; and strengthening terpene synthases or other enzymes associated with them, through overexpression or protein engineering. In this regard, Liu et al. (2013) [48] and Zhou et al. (2020) [51] tried to overcome the problem by manipulating three genes that have been shown to be related to an increase in the flow of the MVA pathway in order to produce geraniol and linalool, two monoterpenes which are widely used as heart notes in the perfume industry due to their pleasant rose and floral/spicy smell, respectively. The three genes in question are tHMG1, IDI1 and ERG20. ...
The fragrance industry is increasingly turning to biotechnology to produce sustainable and high-quality fragrance ingredients. Microbial-based approaches have been found to be particularly promising, as they offer a more practical, economical and sustainable alternative to plant-based biotechnological methods for producing terpene derivatives of perfumery interest. Among the evaluated works, the heterologous expression of both terpene synthase and mevalonate pathway into Escherichia coli has shown the highest yields. Biotechnology solutions have the potential to help address the growing demand for sustainable and high-quality fragrance ingredients in an economically viable and responsible manner. These approaches can help compensate for supply issues of rare or impermanent raw materials, while also meeting the increasing demand for sustainable ingredients and processes. Although scaling up biotransformation processes can present challenges, they also offer advantages in terms of safety and energy savings. Exploring microbial cell factories for the production of natural fragrance compounds is a promising solution to both supply difficulties and the demand for sustainable ingredients and processes in the fragrance industry.
... Recently, the production of squalene with Saccharomyces cerevisiae has received considerable attention, because S. cerevisiae has a natural metabolic pathway for squalene synthesis, i.e., the mevalonate (MVA) pathway. In addition, S. cerevisiae is a well-developed eukaryotic producer microorganism and has been applied to produce many valuable plant natural products, including linalool, artemisinic acid and ginsenoside (Liu et al., 2013;Paramasivan and Mutturi, 2017a;Lian et al., 2018). ...
... When the glucose in the culture medium is exhausted, expression of ERG1 is inhibited, thereby increasing the accumulation of squalene in the yeast cells (Zhao et al., 2017). Many reports have highlighted the importance of tHMG1 and IDI1 in increasing mevalonate (MVA) pathway flux and promoting squalene production (Liu et al., 2013). In this study, making various combinations of different copy numbers of tHMG1 and IDI1 showed that the production of squalene was proportional to their copy numbers. ...
Squalene is an important polyunsaturated triterpene with wide applications in the food, cosmetics, and pharmaceutical industries. Currently, the main method for squalene production is extraction from oil-producing plants, but the scale is limited. The microbial fermentation with Saccharomyces cerevisiae still needs improvement to be economically viable. This study aimed to improve squalene production by metabolic engineering and random mutagenesis. First, the mevalonate (MVA) pathway was enhanced, by integrating tHMG1 and IDI1 into multi-copy site Ty2. Subsequently, the ACL gene from Yarrowia lipolytica, encoding citrate lyase was introduced and the β-oxidation pathway was enhanced with multiple copies of key genes. In addition, a high throughput screening strategy based on Nile red staining was established for high squalene-producer screening. After treatment with ARTP mutagenesis, a higher-producing mutant was obtained, with squalene production enhanced by 18.4%. A two-stage fermentation of this mutant in a 5 L bioreactor produced 8.2 g/L of squalene. These findings may facilitate the development of industrial squalene production by fermentation and potentially, other terpenoids.
... In order to improve the yield of monoterpene in yeast, the upstream MVA pathway (before ERG20) genes, tHMGI, IDI1, and even ERG20, are frequently overexpressed [11]. MAF1, a negative regulator of tRNA biosynthesis that shares the same precursors as GPP, was reported to have positive effects on geraniol production when deleted in S. cerevisiae [18]. The downstream MVA pathway (after ERG20) is often weakened by ERG9 down-regulation or upc2.1 (transcription factor depressing ergosterol biosynthesis) overexpression [14]. ...
... The low GPP content in W303-1a was supposed to result in low α-Terpineol production which was below detection limit. In order to increase the GPP pool, the MVA pathway rate-limiting genes such as tHMG1, IDI1 and ERG20 were often overexpressed [18]. Hence, the strain LCB01 was used as the original strain for α-Terpineol production. ...
Background:
Alpha-Terpineol (α-Terpineol), a C10 monoterpenoid alcohol, is widely used in the cosmetic and pharmaceutical industries. Construction Saccharomyces cerevisiae cell factories for producing monoterpenes offers a promising means to substitute chemical synthesis or phytoextraction.
Results:
α-Terpineol was produced by expressing the truncated α-Terpineol synthase (tVvTS) from Vitis vinifera in S. cerevisiae. The α-Terpineol titer was increased to 0.83 mg/L with overexpression of the rate-limiting genes tHMG1, IDI1 and ERG20F96W-N127W. A GSGSGSGSGS linker was applied to fuse ERG20F96W-N127W with tVvTS, and expressing the fusion protein increased the α-Terpineol production by 2.87-fold to 2.39 mg/L when compared with the parental strain. In addition, we found that farnesyl diphosphate (FPP) accumulation by down-regulation of ERG9 expression and deletion of LPP1 and DPP1 did not improve α-Terpineol production. Therefore, ERG9 was overexpressed and the α-Terpineol titer was further increased to 3.32 mg/L. The best α-Terpineol producing strain LCB08 was then used for batch and fed-batch fermentation in a 5 L bioreactor, and the production of α-Terpineol was ultimately improved to 21.88 mg/L.
Conclusions:
An efficient α-Terpineol production cell factory was constructed by engineering the S. cerevisiae mevalonate pathway, and the metabolic engineering strategies could also be applied to produce other valuable monoterpene compounds in yeast.
... In S. cerevisiae, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR) is a ratelimiting enzyme in the MVA pathway ( Supplementary Fig. 1), and overexpression of a truncated HMG-CoA variant (tHMG1), which is released from negative feedback regulation, increases the production of the triterpenoid protopanaxadiol 9 . In addition, it was reported that the production of a sesquiterpene amorphadiene, a precursor for artemisinin, was increased by overexpression of multiple genes involved in the MVA pathway 10 , which has been also applied for various terpene and terpenoid production [11][12][13][14][15][16] . Moreover, activation of the pyruvate dehydrogenase (PDH) bypass, which is a cytosolic pathway to synthesise acetyl-CoA from pyruvate via acetaldehyde and acetate ( Supplementary Fig. 1), overexpression of an alcohol dehydrogenase gene ADH2 involved in reverse reaction of ethanol fermentation, and inhibition of glyoxylate cycle showed as additive effect on production of a sesquiterpene α-santalene with modification targeting the MVA pathway 17 . ...
Natural secondary metabolites are medically, agriculturally, and industrially beneficial to humans. For mass production, a heterologous production system is required, and various metabolic engineering trials have been reported in Escherichia coli and Saccharomyces cerevisiae to increase their production levels. Recently, filamentous fungi, especially Aspergillus oryzae, have been expected to be excellent hosts for the heterologous production of natural products; however, large-scale metabolic engineering has hardly been reported. Here, we elucidated candidate metabolic pathways to be modified for increased model terpene production by RNA-seq and metabolome analyses in A. oryzae and selected pathways such as ethanol fermentation, cytosolic acetyl-CoA production from citrate, and the mevalonate pathway. We performed metabolic modifications targeting these pathways using CRISPR/Cas9 genome editing and demonstrated their effectiveness in heterologous terpene production. Finally, a strain containing 13 metabolic modifications was generated, which showed enhanced heterologous production of pleuromutilin (8.5-fold), aphidicolin (65.6-fold), and ophiobolin C (28.5-fold) compared to the unmodified A. oryzae strain. Therefore, the strain generated by engineering multiple metabolic pathways can be employed as a versatile highly-producing host for a wide variety of terpenes.
... The comparable titers observed in strains with cytosolic and peroxisomal t43CrGE-S-ERG20 ww indicated the ability of GPP and geraniol to traverse the peroxisomal membrane. Although isopentenyl diphosphate isomerase (IDI1) was identified as a rate-limiting enzyme (Liu et al., 2013) in MVA pathway, overexpression of ePTS1-tagged IDI1 resulted in an 11.1% decrease in geraniol titer, while the expression of cytosolic IDI1 increased the titer by 41.9% ( Fig. 2A). Moreover, to further enhance geraniol production, we integrated two additional copies of t43CrGE-S-ERG20 ww encoding gene and one copy of truncated 3-hydroxy-3-methylglutaryl-coenzyme reductase (tHMGR) (Donald et al., 1997;Ro et al., 2006) encoding gene into GERC2 and GERP2, leading to a 2.0-fold and 3.0-fold increase in geraniol titer in GERC5 and GERP5, respectively. ...
... Accumulation of sufficient geraniol is also critical for improving the yield of 8-hydroxygerainol, which often requires metabolic engineering of the biosynthesis pathway. Several strategies have been shown to increase the supply of geraniol, including truncation of GES enzyme or utilization of fusion tag 12 to improve its heterologous expression 13,14 , engineering of mevalonate (MVA) pathway to enhance GPP concentration 15,16 , and directed evolution of GES to improve its catalytic activity 17,18 . Despites these successes, little efforts focused on systematic optimization of the metabolic network to minimize the incompatibility between the heterologous biosynthesis pathway and the chassis. ...
Microbial production of monoterpenoid indole alkaloids (MIAs) provides a sustainable and eco‐friendly means to obtain compounds with high pharmaceutical values. However, efficient biosynthesis of MIAs in heterologous microorganisms is hindered due to low supply of key precursors such as geraniol and its derivative 8‐hydroxygeraniol catalyzed by geraniol 8‐hydroxylase (G8H). In this study, we developed a facile evolution platform to screen strains with improved yield of geraniol by using the SCRaMbLE system embedded in the Sc2.0 synthetic yeast and confirmed the causal role of relevant genomic targets. Through genome mining, we identified several G8H enzymes that perform much better than the commonly used CrG8H for 8‐hydroxygeraniol production in vivo. We further showed that the N‐terminus of these G8H enzymes plays an important role in cellular activity by swapping experiments. Finally, the combination of the engineered chassis, optimized biosynthesis pathway, and utilization of G8H led to the final strain with more than 30‐fold improvement in producing 8‐hydroxygeraniol compared with the starting strain. Overall, this study will provide insights into the construction and optimization of yeast cells for efficient biosynthesis of 8‐hydroxygeraniol and its derivatives.
... 5,6 First, Liu et al. reported that co-overexpression of isopentenyl diphosphate isomerase (IDI1) as well as the truncated HMG-CoA reductase (tHMG1) is an effective approach to increase the FPP pool in yeast. 7 Furthermore, the effective conversion of FPP into lycopene would also limit the final yield of lycopene. To solve this problem, many strategies including screening crucial genes (CrtE, CrtB, and CrtI) from diverse species and regulating their expression levels have been employed. ...
Lycopene is a red carotenoid with remarkable antioxidant activity, which has been widely used in food, cosmetics, medicine, and other industries. Production of lycopene in Saccharomyces cerevisiae provides an economic and sustainable means. Many efforts have been done in recent years, but the titer of lycopene seems to reach a ceiling. Enhancing the supply and utilization of farnesyl diphosphate (FPP) is generally regarded as an efficient strategy for terpenoid production. Herein, an integrated strategy by means of atmospheric and room-temperature plasma (ARTP) mutagenesis combined with H2O2-induced adaptive laboratory evolution (ALE) was proposed to improve the supply of upstream metabolic flux toward FPP. Enhancing the expression of CrtE and introducing an engineered CrtI mutant (Y160F&N576S) increased the utilization of FPP toward lycopene. Consequently, the titer of lycopene in the strain harboring the Ura3 marker was increased by 60% to 703 mg/L (89.3 mg/g DCW) at the shake-flask level. Eventually, the highest reported titer of 8.15 g/L of lycopene in S. cerevisiae was achieved in a 7 L bioreactor. The study highlights an effective strategy that the synergistic complementarity of metabolic engineering and adaptive evolution facilitates natural product synthesis.
... For de novo biosynthesis of lipids, the glycolytic fluxes to fatty acid biosynthesis, specifically to enhance the supply of precursors in S. cerevisiae has been considered the main strategy for metabolic engineering [10,11], and high yields of fatty acids with different chain lengths, or its derivatives such as fatty alcohols, fatty acid esters or others have also achieved [4,[12][13][14]. In addition, the mevalonate (MVA) pathway presented in yeast as typically existed in most eukaryotic cells, providing the de novo biosynthetic route of high value terpenoids from acetyl-CoA [2], including sterols (stigmasterol, sitosterol, cholesterol), lycopene [15], β-carotene [16], arteannuic acid [17], taxene [18], α-santalene [19], ginsenoside [20], geraniol [21], etc., that can be obtained in S. cerevisiae cell factory fermented from simple carbon source. ...
With staggering progress on genetic manipulation strategies, Saccharomyces cerevisiae is becoming an ideal cell factory for the de novo biosynthesis of lipid compounds. However, due to their hydrophobicity, lipids tend to be accumulated within intracellular spaces and cause a high burden on cell activity and induce product inhibition effect, which ultimately restricted the lipids biomanufacturing for industrial application. Herein, an oleic acid stress (OAS) model was applied for the long-time domestication of BY4741 cells, and a subclone of A-22 was obtained through a series of acclimation (0.1% glucose and 0.2% oleic acid), showing increased accumulation of both biomass and intracellular lipid droplets compared to WT. Comparative transcriptome analysis indicated that compared to fatty acid metabolism, most transcripts enriched in the pathways of glucose catabolism (glycolysis and citrate cycle) and lipid synthesis (phospholipid and sterol) were down-regulated under OAS. While interestingly, most the above transcripts tended to be ‘restored’ in adapted strain A-22. In addition, for physical adaptation, significant increase of phosphatidylcholines was identified by lipidomic analysis, which probably caused the subsequent subcellular expansion of peroxisomes and lipid droplets as observed in the adapted strain, since phosphatidylcholines are the major constituent of their membranes. The present study systematically investigated both the phenotype change and molecular mechanism on adaptation of S. cerevisiae towards oily environment. Detailed information on functional transcripts may provide novel rational modification targets to reinforce the hydrophobic lipids biosynthesis within S. cerevisiae engineered cell factory.
... 30 IDI1 was suggested to be another rate-limiting enzyme, which could catalyze the conversion of IPP into its allylic isomer DMAPP. 31 Thus, a copy of GgbAs1 and the two key genes in the MVA pathway (tHmg1 and Idi1) were integrated into the starting strain S. cerevisiae CEN.PK2-1C to construct a β-amyrin-producing strain B01. The strain produced 13.7 ± 0.2 mg/g DCW (dry cell weight) (70.9 ± 0.7 mg/L) β-amyrin and 142.7 ± 2.0 mg/g DCW (736.7 ± 1.4 mg/L) squalene ( Figure 2C). ...
... One of the more prominent examples that have been documented would be the overexpression of IDI1 (encoding isopentenyl diphosphate isomerase) that has achieved an approximate fivefold increase in cineole production (Ignea et al., 2011). When the same strategy was employed in geraniol production, a 1.45-fold titer increase was observed (Liu et al., 2013a). Another such example would involve the most frequently treated and highly regulated rate-limiting enzyme, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), which converts HMG-CoA to MVA. ...
Monoterpenoids are an important class of natural products that are derived from the condensation of two five‑carbon isoprene subunits. They are widely used for flavouring, fragrances, colourants, cosmetics, fuels, chemicals, and pharmaceuticals in various industries. They can also serve as precursors for the production of many industrially important products. Currently, monoterpenoids are produced predominantly through extraction from plant sources. However, the small quantity of monoterpenoids in nature renders this method of isolation non-economically viable. Similarly impractical is the chemical synthesis of these compounds as they suffer from high energy consumption and pollutant discharge. Microbial biosynthesis, however, exists as a potential solution to these hindrances, but the transformation of cells into efficient factories remains a major impediment. Here, we critically review the recent advances in engineering microbes for monoterpenoid production, with an emphasis on categorized strategies, and discuss the challenges and perspectives to offer guidance for future engineering.
... By introducing geraniol synthase and optimizing the key regulatory points in the mevalonate pathway, Liu et al. obtained 36.04 mg/L geraniol in S. cerevisiae (Liu, Zhang, Du, Chen, & Zhou, 2013). Jiang et al. expressed key enzymes, including geraniol synthase (Rossi et al.), farnesyl diphosphate synthase (ERG20), truncated 3-hydroxy-3-methylglutaryl-coenzyme reductase (tHMGR), and isopentenyl diphosphate isomerase (IDI1), with the Gal promoter to improve flux towards geraniol from GPP (geranyl diphosphate) and alleviate product toxicity. ...
Isoprenoids, as the largest group of chemicals in the domains of life, constitute more than 50,000 members. These compounds consist of different numbers of isoprene units (C5H8), by which they are typically classified into hemiterpenoids (C5), monoterpenoids (C10), sesquiterpenoids (C15), diterpenoids (C20), triterpenoids (C30), and tetraterpenoids (C40). In recent years, isoprenoids have been employed as food additives, in the pharmaceutical industry, as advanced biofuels, and so on. To realize the sufficient and efficient production of valuable isoprenoids on an industrial scale, fermentation using engineered microorganisms is a promising strategy compared to traditional plant extraction and chemical synthesis. Due to the advantages of mature genetic manipulation, robustness and applicability to industrial bioprocesses, Saccharomyces cerevisiae has become an attractive microbial host for biochemical production, including that of various isoprenoids. In this review, we summarized the advances in the biosynthesis of isoprenoids in engineered S. cerevisiae over several decades, including synthetic pathway engineering, microbial host engineering, and central carbon pathway engineering. Furthermore, the challenges and corresponding strategies towards improving isoprenoid production in engineered S. cerevisiae were also summarized. Finally, suggestions and directions for isoprenoid production in engineered S. cerevisiae in the future are discussed.
... In our previous study, NPP could be used as an optimal precursor to produce d-limonene in S. cerevisiae [22]. NPP is synthesized through the MVA pathway in yeast, which involves a multi-step reaction and multiple rate-limiting enzymes [23][24][25][26]. As a critical intermediate, the HMG-CoA will cause the toxicity to the bottom MVA pathway because of the over-accumulation of HMG-CoA. ...
d-Limonene, a cyclized monoterpene, possesses citrus-like olfactory property and multi-physiological functions, which can be used as a bioactive compound and flavor to improve the overall quality of alcoholic beverages. In our previous study, we established an orthogonal pathway of d-limonene synthesis by introducing neryl diphosphate synthase 1 (tNDPS1) and d-limonene synthase (tLS) in Saccharomyces cerevisiae. To further increase d-limonene formation, the metabolic flux of the mevalonate (MVA) pathway was enhanced by overexpressing the key genes tHMGR1, ERG12, IDI1, and IDI1WWW, respectively, or co-overexpressing. The results showed that strengthening the MVA pathway significantly improved d-limonene production, while the best strain yielded 62.31 mg/L d-limonene by co-expressing tHMGR1, ERG12, and IDI1WWW genes in alcoholic beverages. Furthermore, we also studied the effect of enhancing the MVA pathway on the growth and fermentation of engineered yeasts during alcoholic beverage fermentation. Besides, to further resolve the problem of yeast growth inhibition, we separately investigated transporter proteins of the high-yielding d-limonene yeasts and the parental strain under the stress of different d-limonene concentration, suggesting that the transporters of Aus1p, Pdr18p, Pdr5p, Pdr3p, Pdr11p, Pdr15p, Tpo1p, and Ste6p might play a more critical role in alleviating cytotoxicity and improving the tolerance to d-limonene. Finally, we verified the functions of three transporter proteins, finding that the transporter of Aus1p failed to transport d-limonene, and the others (Pdr5p and Pdr15p) could improve the tolerance of yeast to d-limonene. This study provided a valuable platform for other monoterpenes’ biosynthesis in yeast during alcoholic beverage fermentation.
... Geraniol is acyclic monoterpene alcohol obtained from the essential oils of aromatic plants (6,7). Rose, lavender and lemon are some of these plants (8). ...
Endometrial cancer is the most common type of cancer in the female reproductive system. Geraniol is acyclic monoterpene alcohol derived from essential oils of aromatic plants. This study aimed to investigate the apoptosis pathway of geraniol on Ishikawa cells. The cytotoxic effects of Geraniol on Ishikawa cells were determined by an MTT test. Ishikawa cells were seeded on cover slips, the IC50 dose was applied, and the cells were incubated with antibodies against Bax, Bcl-2, and TUNEL Assay. mRNA expression analysis of apoptosis-related genes was determined by RT-qPCR with an IC50 dose of Geraniol. The IC50 dose of Geraniol decreased Bcl-2 staining significantly, but it significantly increased Bax staining and TUNEL positive cells. A significant increase in the Bax, caspase3, caspase-8, cytochrome C and Fas genes and a significant decrease in the Bcl-2 gene was observed when the IC50 dose group was compared to the cells in the control group based on their mRNA expression levels.Analysis of expression of genes whose products are involved in apoptosis suggests the involvement of the mitochondrial pathway.
... The nucleotide insertions induce a frameshift, leading to the insertion of an early stop-codon in addition to 5 mutated amino acids at the end of the truncated protein. Rico et al., 2010); the isopentenyl diphosphate (IPP) isomerase Idi1 (Ignea et al., 2011); the negative regulator of RNA polymerase III Maf1, which represses tRNA biosynthesis from IPP (Liu et al., 2013); and the FPP-synthase from Gallus gallus carrying a point mutation for the N144W amino acid substitution (mFPS N144W ), which produces high amounts of GPP (Stanley Fernandez et al., 2000). The resulting strains were named Sen ("Sensitive, " in pex30 /pex31 /atg36 with low geraniol tolerance) and Tol ("Tolerant, " in pex30 /pex32 with high geraniol tolerance). ...
Monoterpenoids, such as the plant metabolite geraniol, are of high industrial relevance since they are important fragrance materials for perfumes, cosmetics, and household products. Chemical synthesis or extraction from plant material for industry purposes are complex, environmentally harmful or expensive and depend on seasonal variations. Heterologous microbial production offers a cost-efficient and sustainable alternative but suffers from low metabolic flux of the precursors and toxicity of the monoterpenoid to the cells. In this study, we evaluated two approaches to counteract both issues by compartmentalizing the biosynthetic enzymes for geraniol to the peroxisomes of Saccharomyces cerevisiae as production sites and by improving the geraniol tolerance of the yeast cells. The combination of both approaches led to an 80% increase in the geraniol titers. In the future, the inclusion of product tolerance and peroxisomal compartmentalization into the general chassis engineering toolbox for monoterpenoids or other host-damaging, industrially relevant metabolites may lead to an efficient, low-cost, and eco-friendly microbial production for industrial purposes.
... However, when plant-derived proteins are expressed in microorganisms, their expression level and enzyme activity are often limited due to improper folding and presence of transit peptides, leading to metabolic bottlenecks in the pathway 25,26 . Moreover, the compatibility of the heterologous pathway with the native metabolism should also be taken into consideration, such as concerns of cofactor imbalance, precursor competition, redox imbalance, and intermediate toxicity, all of which would result in low biosynthetic efficiency [27][28][29] . Therefore, to construct an unimpeded tocotrienols synthetic pathway in S. cerevisiae, efforts are needed to debug the target pathway and balance it with the related native pathways. ...
The diverse physiological functions of tocotrienols have listed them as valuable supplementations to α-tocopherol-dominated Vitamin E products. To make tocotrienols more readily available, tocotrienols-producing S. cerevisiae has been constructed by combining the heterologous genes from photosynthetic organisms with the endogenous shikimate pathway and mevalonate pathway. After identification and elimination of metabolic bottlenecks and enhancement of precursors supply, the engineered yeast can produce tocotrienols at yield of up to 7.6 mg/g dry cell weight (DCW). In particular, proper truncation of the N-terminal transit peptide from the plant-sourced enzymes is crucial. To further solve the conflict between cell growth and tocotrienols accumulation so as to enable high-density fermentation, a cold-shock-triggered temperature control system is designed for efficient control of two-stage fermentation, leading to production of 320 mg/L tocotrienols. The success in high-density fermentation of tocotrienols by engineered yeast sheds light on the potential of fermentative production of vitamin E tocochromanols. Tocotrienols are valuable supplementations to α-tocopherol-dominated Vitamin E products. Here, the authors engineer baker’s yeast by combining the heterologous genes from photosynthetic organisms with the endogenous pathway for the production of tocotrienols under cold-shock-triggered temperature control.
... (IPP) isomerase (IDI1), which catalyzes the conversion of IPP to DMAPP, contributed to significant enhancement of monoterpene titers [85]. Alternatively, DMAPP supply can be increased by overexpression of MAF1 [86], which is a negative regulator of another enzyme utilizing DMAPP, namely tRNA isopentenyltransferase (MOD5) [87]. Furthermore, DMAPP supply may be improved by deletion of ROX1, a transcription factor that represses many enzymes in the mevalonate and ergosterol biosynthesis pathway [88]. ...
In plants, prenylation of aromatic compounds, such as (iso)flavonoids and stilbenoids, by membrane-bound prenyltransferases (PTs), is an essential step in the biosynthesis of many bioactive compounds. Prenylated aromatic compounds have various health-beneficial properties that are interesting for industrial applications, but their exploitation is limited due to their low abundance in nature. Harnessing plant aromatic PTs for prenylation in microbial cell factories may be a sustainable and economically viable alternative. Limitations in prenylated aromatic compound production have been identified, including availability of prenyl donor substrate. In this review, we summarize the current knowledge about plant aromatic PTs and discuss promising strategies towards the optimized production of prenylated aromatic compounds by microbial cell factories.
... Previous studies have shown that HMG-CoA reductase is an important rate-limiting enzyme of the MVA pathway. 7,10 Hence, in the present study, tHMG1 was overexpressed in S. cerevisiae BY47419-2 under the control of an inducible GAL promoter (P GAL1 ) ( Table 1) (Supporting information, Figure S1), which could be in response to the glucose concentration in the medium in the GAL80 disruption strain. 20 The resulting strain, SE1, produced 298.43 mg/L squalene, which was almost 33-fold greater than that of the host strain BY47419-2 ( Figure 2B). ...
... However, compared to strain BJM-15 which possessed the best valencene production in 31 mutant strains (Fig. 4), the yield only increased by 1.7-fold, which was not in accordance with expectation. In a previous study, geraniol production was increased by 1.4-fold through overexpression of only tHMG1 [38]. In other works, augmentation of the native yeast genes in MVA pathway increased nerolidol production by 3.5-fold [39]. ...
Background:
The biological synthesis of high value compounds in industry through metabolically engineered microorganism factories has received increasing attention in recent years. Valencene is a high value ingredient in the flavor and fragrance industry, but the low concentration in nature and high cost of extraction limits its application. Saccharomyces cerevisiae, generally recognized as safe, is one of the most commonly used gene expression hosts. Construction of S. cerevisiae cell factory to achieve high production of valencene will be attractive.
Results:
Valencene was successfully biosynthesized after introducing valencene synthase into S. cerevisiae BJ5464. A significant increase in valencene yield was observed after down-regulation or knock-out of squalene synthesis and other inhibiting factors (such as erg9, rox1) in mevalonate (MVA) pathway using a recyclable CRISPR/Cas9 system constructed in this study through the introduction of Cre/loxP. To increase the supplement of the precursor farnesyl pyrophosphate (FPP), all the genes of FPP upstream in MVA pathway were overexpressed in yeast genome. Furthermore, valencene expression cassettes containing different promoters and terminators were compared, and PHXT7-VS-TTPI1 was found to have excellent performance in valencene production. Finally, after fed-batch fermentation in 3 L bioreactor, valencene production titer reached 539.3 mg/L with about 160-fold improvement compared to the initial titer, which is the highest reported valencene yield.
Conclusions:
This study achieved high production of valencene in S. cerevisiae through metabolic engineering and optimization of expression cassette, providing good example of microbial overproduction of valuable chemical products. The construction of recyclable plasmid was useful for multiple gene editing as well.
... [111]. Esteya vermicola, an endoparasitic fungus of pine wood nematode, has also been reported to produce monoterpenes as a mechanism to predate the nematode [112]. Production and export of monoterpenes by the fungus Ceratocystis moniliformis has also been reported [113]. ...
Yeast had participated with humans in food fermentation since the production of wine and bread, more than 10,000 years of shared history. It is well understood that fungi diversity is still underestimated and that we are far from understanding its importance and potential impact in biotechnology. Flavor compounds as “secondary metabolism” are very sensitive to fermentation conditions and mixed cultures, and although we had experience an exponential development of molecular biology in the last 30 years, metabolomics is still in its infancy. It was demonstrated in recent years that increase strain and species yeast diversity in a fermentation system increases sensory complexity and chemical aroma compound diversity in the final fermented product. Flavor compounds had many key functions for yeast, such as for survival and dispersion strategies, pheromone and defense mechanisms, and “quorum sensing” mechanisms for cell communication. Humans had taken advantage of many of these functions to increase taste and food sensory pleasure for a more exigent consumer, a phenomenon called “yeast domestication.” We focus this chapter mainly in the recent discussed yeast synthetic pathways for the formation of phenylpropanoid and terpenoid aroma compounds. In addition, we will emphasize the current knowledge that grape and wine microbiology research has contributed to understand how complex natural and inoculated yeast flora can affect flavor quality. The flavor phenotype concept and how to screen natural flora and develop consortia starters to innovate in food biotechnology are discussed.
... 49 Alternatively, ergosterol biosynthesis can also be downregulated chemically, as was demonstrated for the production of lupulones in yeast. 50 Several other strategies that could improve prenyl donor availability have been described, including overexpression of IDI1 (IPP isomerase) 51 and disruption of the polyprenyl transferase COQ2 gene, which disables the use of prenyl diphosphates for ubiquinone synthesis. 34 Subcellular Location of Enzymes May Affect 8-Prenylnaringenin Production. ...
Prenylated flavonoids possess a wide variety of biological activities, including estrogenic, antioxidant, antimicrobial and anticancer activity. Hence, they have potential applications in food products, medicines or supplements with health-promoting activities. However, the low abundance of prenylated flavonoids in nature is limiting their exploitation. Therefore, we investigated the prospect of producing prenylated flavonoids in the yeast Saccharomyces cerevisiae. As a proof of concept, we focused on the production of the potent phytoestrogen 8-prenylnaringenin. Introduction of the flavonoid prenyltransferase SfFPT from Sophora flavescens in naringenin-producing yeast strains resulted in de novo production of 8-prenylnaringenin. We generated several strains with increased production of the intermediate precursor naringenin, which finally resulted in a production of 0.12 mg L-1 (0.35 µM) of 8-prenylnaringenin under shake flask conditions. A number of bottlenecks in prenylated flavonoid production were identified and are discussed.
... This expanded catalogue of natural variation, combined with additional metabolic engineering, has been used to further improve heterologous geraniol production in S. cerevisiae. By introducing a fusion protein composed of geraniol synthase from Valeriana officinalis (tVoGES) that lacks plastid targeting signal and a mutant farnesyl diphosphate synthase ERG20p (F96W-N127W) , in a strain with a metabolically-engineered high-flux mevalonate pathway, 239 mg/L of geraniol was achieved in fed-batch cultures [41][42][43]. However, this concentration was still deemed inadequate for industrialization of the system. ...
Over the last two decades, rapid progress in the field of synthetic biology has opened several avenues for the heterologous de novo production of complex biological compounds, such as biofuels, pharmaceuticals, and food additives in microbial hosts. This minireview addresses the usage of the yeast Saccharomyces cerevisiae as a microbial cell factory for the production of flavour and aroma compounds, thereby providing a path towards a sustainable and efficient means of producing what are normally rare, and often expensive plant-derived chemicals.
... The oil of ginger, lemon, lavender, orange, rose, palmarosa and other plants, is acyclic monoterpene alcohol [9]. Geraniol is a colorless, odor pleasant compound (aroma of roses) [2]. ...
The aim of this work was to assess the performance of a vacuum fractionating column for the fractionation of Java Citronella Oil (Cymbopogon winterianus) and citronellal purification during batch mode operation at vacuum -76 cmHg and reflux ratios 5:1. Based on GC-MS analysis of Java Citronella Oil is known that citronellal, citronellol, and geraniol has yielded 21,59%; 7,43%; and 34,27%, respectively. Fractional distillation under reduced pressure and continued redistilled are needed to isolate the component of Java Citronella Oil. Redistilled can improve the purity, then distillate collected while the temperature changed. In the first distillate yielded citronellal with a purity of 75.67%. The first distillate obtained residue rhodinol product will then be carried back to separation into citronellol and geraniol. The purity of citronellol reached 80,65% purity, whereas geraniol reached 76.63% purity. Citronellal Purification resulting citronellal to 95.10% purity and p-menthane-3,8-diol reached 75.95% purity.
Geraniol, an acyclic monoterpene alcohol, has significant potential applications in various fields, including: food, cosmetics, biofuels, and pharmaceuticals. However, the current sources of geraniol mainly include plant tissue extraction or chemical synthesis, which are unsustainable and suffer severely from high energy consumption and severe environmental problems. The process of microbial production of geraniol has recently undergone vigorous development. Particularly, the sustainable construction of recombinant Escherichia coli (13.2 g/L) and Saccharomyces cerevisiae (5.5 g/L) laid a solid foundation for the microbial production of geraniol. In this review, recent advances in the development of geraniol-producing strains, including: metabolic pathway construction, key enzyme improvement, genetic modification strategies, and cytotoxicity alleviation, are critically summarized. Furthermore, the key challenges in scaling up geraniol production and future perspectives for the development of robust geraniol-producing strains are suggested. This review provides theoretical guidance for the industrial production of geraniol using microbial cell factories.
Lycopene has been widely used in the food industry and medical field due to its antioxidant, anti-cancer, and anti-inflammatory properties. However, achieving efficient manufacture of lycopene using chassis cells on an industrial scale remains a major challenge. Herein, we attempted to integrate multiple metabolic engineering strategies to establish an efficient and balanced lycopene biosynthetic system in Saccharomyces cerevisiae. First, the lycopene synthesis pathway was modularized to sequentially enhance the metabolic flux of the mevalonate pathway, the acetyl-CoA supply module, and lycopene exogenous enzymatic module. The modular operation enabled the efficient conversion of acetyl-CoA to downstream pathway of lycopene synthesis, resulting in a 3.1-fold increase of lycopene yield. Second, we introduced acetate as an exogenous carbon source and utilized an acetate-repressible promoter to replace the natural ERG9 promoter. This approach not only enhanced the supply of acetyl-CoA but also concurrently diminished the flux toward the competitive ergosterol pathway. As a result, a further 42.3% increase in lycopene production was observed. Third, we optimized NADPH supply and mitigated cytotoxicity by overexpressing ABC transporters to promote lycopene efflux. The obtained strain YLY-PDR11 showed a 12.7-fold increase in extracellular lycopene level compared to the control strain. Finally, the total lycopene yield reached 343.7 mg/L, which was 4.3 times higher than that of the initial strain YLY-04. Our results demonstrate that combining multi-modular metabolic engineering with efflux engineering is an effective approach to improve the production of lycopene. This strategy can also be applied to the overproduction of other desirable isoprenoid compounds with similar synthesis and storage patterns in S. cerevisiae.
One-Sentence Summary
In this research, lycopene production in yeast was markedly enhanced by integrating a multi-modular approach, acetate signaling-based down-regulation of competitive pathways, and an efflux optimization strategy.
Reconstruction and optimization of biosynthetic pathways can help to overproduce target chemicals in microbial cell factories based on genetic engineering. However, the perturbation of biosynthetic pathways on cellular metabolism is not well investigated and profiling the engineered microbes remains challenging. The rapid development of omics tools has the potential to characterize the engineered microbial cell factory. Here, we performed label‐free quantitative proteomic analysis and metabolomic analysis of engineered sabinene overproducing Saccharomyces cerevisiae strains. Combined metabolic analysis andproteomic analysis of targeted mevalonate (MVA) pathway showed that co‐ordination of cytosolic and mitochondrial pathways had balanced metabolism, and genome integration of biosynthetic genes had higher sabinene production with less MVA enzymes. Furthermore, comparative proteomic analysis showed that compartmentalized mitochondria pathway had perturbation on central cellular metabolism. This study provided an omics analysis example for characterizing engineered cell factory, which can guide future regulation of the cellular metabolism and maintaining optimal protein expression levels for the synthesis of target products.
Microbial cell factories, renowned for their economic and environmental benefits, have emerged as a key trend in academic and industrial areas, particularly in the fermentation of natural compounds. Among these, plant-derived terpenes stand out as a significant class of bioactive natural products. The large-scale production of such terpenes, exemplified by artemisinic acid—a crucial precursor to artemisinin—is now feasible through microbial cell factories. In the fermentation of terpenes, two-phase fermentation technology has been widely applied due to its unique advantages. It facilitates in situ product extraction or adsorption, effectively mitigating the detrimental impact of product accumulation on microbial cells, thereby significantly bolstering the efficiency of microbial production of plant-derived terpenes. This paper reviews the latest developments in two-phase fermentation system applications, focusing on microbial fermentation of plant-derived terpenes. It also discusses the mechanisms influencing microbial biosynthesis of terpenes. Moreover, we introduce some new two-phase fermentation techniques, currently unexplored in terpene fermentation, with the aim of providing more thoughts and explorations on the future applications of two-phase fermentation technology. Lastly, we discuss several challenges in the industrial application of two-phase fermentation systems, especially in downstream processing.
Secondary natural products (NPs) are a rich source for drug discovery. However, the low abundance of NPs makes their extraction from nature inefficient, while chemical synthesis is challenging and unsustainable. Saccharomyces cerevisiae and Pichia pastoris are excellent manufacturing systems for the production of NPs. This Perspective discusses a comprehensive platform for sustainable production of NPs in the two yeasts through system-associated optimization at four levels: genetics, temporal controllers, productivity screening, and scalability. Additionally, it is pointed out critical metabolic building blocks in NP bioengineering can be identified through connecting multilevel data of the optimized system using deep learning.
Geraniol is an acyclic isoprenoid monoterpene that is biosynthesised by a large number of aromatic plants. It is widely used in cosmetics, household products and pharmaceuticals, because of its low toxicity and environmentally-friendly profile. An increasing demand for the compound has encouraged research into alternative synthetic routes, and great efforts are still aimed to increase the compound yield through heterologous production in micro-organisms, using a variety of different strategies. Geraniol is proven to exhibit various biological activities, including antitumour, anti-inflammatory, anti-oxidant, antimicrobial, hepatoprotective, cardioprotective and neuroprotective effects. The lipophilic compound is a relatively good penetration enhancer and can be used to increase transdermal drug delivery. Encapsulation of the compound using different methods has circumvented problems associated with the dispersal of the non-polar entity within aqueous matrices, such as foods, to prevent spoilage. The volatile nature of the compound lends itself to applications such as postharvest fumigation of grain crops to protect against invading insects and mycotoxin-producing fungi, and as a mosquito repellent. In addition, administration via inhalation can combat respiratory pathogens susceptible to geraniol, while combating inflammation. Using the multidisciplinary Scopus database, it became evident through a bibliometric analysis of the available literature spanning the past 12 years that, despite a host of research reports documenting the anticancer activity of the compound, research is limited to in vitro, in silico and in vivo experiments, while clinical trials are completely lacking. This review is an update to our 2010 publication on the same topic and aims to reflect and compare the progress, and to highlight the potential and opportunities for further evidence based research.
Geraniol is a rose-scented monoterpene with significant commercial and industrial value in medicine, condiments, cosmetics, and bioenergy. Here, we first targeted geraniol as a reporter metabolite and explored the suitability and potential of Candida glycerinogenes as a heterologous host for monoterpenoid production. Subsequently, dual-pathway engineering was employed to improve the production of geraniol with a geraniol titer of 858.4 mg/L. We then applied a synthetic hybrid promoter approach to develop a decane-responsive hybrid promoter based on the native promoter PGAP derived from C. glycerinogenes itself. The hybrid promoter was able to be induced by n-decane with 3.6 times higher transcriptional intensity than the natural promoter PGAP. In particular, the hybrid promoter effectively reduces the conflict between cell growth and product formation in the production of geraniol. Ultimately, 1194.6 mg/L geraniol was obtained at the shake flask level. The strong and tunable decane-responsive hybrid promoter developed in this study provides an important tool for fine regulation of toxic terpenoid production in cells.
Geraniol has versatile applications in the fragrance, pharmacy, and biofuel industries. This monoterpene is produced via the mevalonate pathway in the yeast Saccharomyces cerevisiae. However, carbon flux leakage restricts the efficient biosynthesis of target compounds via this pathway. In this work, the mevalonate pathway of the recombinant S. cerevisiae was reprogrammed by introducing an isopentenyl phosphate kinase (IPK) and to establish an isopentenyl phosphate utilization (IPU) bypass that can utilise the isopentenyl phosphate (IP) produced from phosphomevalonate decarboxylase (PMD). Geraniol production was increased by 225% in the engineered strain. The yield of geraniol was further enhanced to 156.2 ± 6.6 mg/L through optimisation of key rate-limiting enzymes of the mevalonate pathway. Using pretreated molasses as a carbon source yielded 180.9 ± 2.9 mg/L of geraniol under optimal fermentation conditions, the highest reported titre to date using molasses medium. Modification of the mevalonate pathway can provide a commercially feasible strategy for monoterpene synthesis in engineered S. cerevisiae.
Monoterpenoids that belong to the terpenoids family are usually volatile and have strong aroma. Some monoterpenoids also have antioxidant, antibacterial and anti-inflammatory activities, which make them important raw materials for medicine, food and cosmetics industry. In recent years, the heterologous synthesis of monoterpenoids by microorganisms has attracted extensive attention. However, its large-scale application is greatly hampered by the low yield and high production cost. Nowadays, the rapid development of synthetic biology provides new approaches for enhancing the production of monoterpenoids by microorganisms. Different kinds of recombinant strains can be obtained via engineering of microbial cells to produce a variety of monoterpenoids with different properties. This paper summarized the latest strategies and progress in the application of synthetic biology to produce monoterpenoids by microorganisms, including the design and modification of biosynthetic pathway, as well as the design and optimization of high-yield monoterpenoids producing chassis cells.
While nepetalactone, the active ingredient in catnip, is a potent insect repellent, its low in planta accumulation limits its commercial viability as an alternative repellent. Here we describe for the first time de novo nepetalactone synthesis in Saccharomyces cerevisiae, enabling sustainable and scalable production. Nepetalactone production required introducing eight exogenous genes including the cytochrome P450 geraniol-8-hydroxylase, the bottleneck of the heterologous pathway. Combinatorial assessment of geraniol-8-hydroxylase and cytochrome P450 reductase variants, and copy-number variations were used to overcome this bottleneck. We found that several reductases improved hydroxylation activity and increasing geraniol-8-hydroxylase gene copy number improved 8-hydroxygeraniol titers. The accumulation of an unwanted metabolite implied inefficient channeling of carbon through the pathway. With the native yeast old yellow enzymes previously shown to use monoterpene intermediates as substrates, both homologues were deleted. These deletions increased 8-hydroxygeraniol yield, resulting in 3.10 mg/L/OD600 of nepetalactone from simple sugar in microtiter plates. This optimized pathway will benefit the development of high yielding strains for the scale up production of nepetalactone.
While nepetalactone, the active ingredient in catnip, is a potent insect repellent, its low in planta accumulation limits its commercial viability as an alternative repellent. Here we describe a platform for de novo nepetalactone production in Saccharomyces cerevisiae , enabling sustainable and scalable production. Nepetalactone production required introduction of eight exogenous genes including the cytochrome P450 geraniol-8-hydroxylase, which represented the bottleneck of the heterologous pathway. Combinatorial assessment of geraniol-8-hydroxylase and cytochrome P450 reductase variants, as well as copy-number variations were used to overcome this bottleneck. We found that several reductases improved hydroxylation activity, with a higher geraniol-8-hydroxylase ratio further increasing 8-hydroxygeraniol titers. Another roadblock was the accumulation of an unwanted metabolite that implied inefficient channelling of carbon through the pathway. With the native yeast old yellow enzymes previously shown to use monoterpene intermediates as substrates, both homologs were deleted. These deletions increased 8-hydroxygeraniol yield, resulting in a final de novo accumulation of 3.10 mg/L/OD600 of nepetalactone from simple sugar in microtiter plates. Our pathway optimization will aid in the development of high yielding monoterpene S. cerevisiae strains.
Terpenes are a major class of natural aromatic compounds in grapes and wines to offer the characteristic flavor and aroma, serving as important quality traits of wine products. Saccharomyces cerevisiae represents an excellent cell factory platform for large-scale bio-based terpene production. This review describes the biosynthetic pathways of terpenes in different organisms. The metabolic engineering of S. cerevisiae for promoting terpene biosynthesis and the alternative microbial engineering platforms including filamentous fungi and Escherichia coli are also elaborated. Additionally, the potential applications of the terpene products from engineered microorganisms in food and beverage industries are also discussed. This review provides comprehensive information for an innovative supply way of terpene via microbial cell factory, which could facilitate the development and application of this technique at the industrial scale.
Microbial production of monoterpenes has attracted increasing attention in recent years. Up to date, there are only few reports on the biosynthesis of the monoterpene alcohol citronellol that is widely used as fragrant and pharmaceutical intermediates. Here, we engineered Saccharomyces cerevisiae by employing a "push-pull-restrain" strategy to improve citronellol production based on the reduction of geraniol. Starting from a engineered geraniol-producing strain, different reductases were investigated and the best performing iridoid synthase from Catharanthus roseus (CrIS) resulted in 285.89 mg/L enantiomerically pure S-citronellol in shake flasks. Geranyl diphosphate (GPP), the most important precursor for monoterpenes, was enhanced by replacing the wild farnesyl diphosphate synthase (Erg20) with the mutant Erg20F96W, increasing the citronellol titer to 406.01 mg/L without negative influence on cell growth. Moreover, we employed synthetic protein scaffolds and protein fusion to colocalize four sequential enzymes to achieve better substrate channeling along with the deletion of an intermediate degradation pathway gene ATF1, which elevated the citronellol titer to 972.02 mg/L with the proportion of 97.8% of total monoterpenes in YPD medium. Finally, the engineered strain with complemented auxotrophic markers produced 8.30 g/L of citronellol by fed-batch fermentation, which was the highest citronellol titer reported to date. The multi-level engineering strategies developed here demonstrate the potential of monoterpenes overproduction in yeast, which can serve as a generally applicable platform for overproduction of other monoterpenes.
Geraniol is a monoterpenoid alcohol with various applications in food, cosmetics, and healthcare. Corynebacterium glutamicum is a potential platform for terpenoids production because it harbors the methylerythritol phosphate pathway. To engineer C. glutamicum to produce geraniol, two different truncated geraniol synthases (GESs) were respectively expressed, and strain LX02 expressing the truncated GESs from Valeriana officinalis (tVoGES) produced 0.3 mg/L of geraniol. Then, three geranyl diphosphate synthases (GPPSs) were combinatorially co-expressed with tVoGES to improve geraniol production. The amounts of produced geraniol were all higher than that produced by strain LX02. Strain LX03 co-expressing ERG20F96W–N127W (ERG20WW) and tVoGES produced the highest amount, 5.4 mg/L. Subsequently, the co-overexpression of 1-deoxy-d-xylulose-5-phosphate synthase (dxs) and isopentenyl diphosphate isomerase (idi) further increased the production to 12.2 mg/L in strain LX03. Lastly, the production of geraniol was increased to 15.2 mg/L via fermentation optimization. To our knowledge, this is the first report on the engineering of C. glutamicum to produce geraniol and thus can serve as a reference for other monoterpenoid production studies.
Dextranase from Chaetomium gracile is generally considered safe for use in the sugarcane industry. Herein, a truncated and codon-optimised α-dextranase gene from C. gracile was successfully cloned and expressed in Saccharomyces cerevisiae for the first time. The optimum conditions of fermentation was achieved when the maximum dextranase activity reached to 58.45 U/mL after 48 h in shake flasks. The optimal pH and temperature were 5.5 and 60 °C, respectively. The recombinant dextranase remained stable between pH 4 and 6 and temperature between 55 and 60 °C. The findings in the present study could facilitate large-scale production of food-grade recombinant dextranase for use in the sugar industry.
Monoterpene indole alkaloids (MIAs) from plants encompass a broad class of structurally complex and medicinally valuable natural products. MIAs are biologically derived from the universal precursor strictosidine. Although the strictosidine biosynthetic pathway has been identified and reconstituted, extensive work is required to optimize production of strictosidine and its precursors in yeast. In this study, we engineered a fully integrated and plasmid-free yeast strain with enhanced production of the monoterpene precursor geraniol. The geraniol biosynthetic pathway was targeted to the mitochondria to protect the GPP pool from consumption by the cytosolic ergosterol pathway. The mitochondrial geraniol producer showed a 6-fold increase in geraniol production compared to cytosolic producing strains. We further engineered the monoterpene-producing strain to synthesize the next intermediates in the strictosidine pathway: 8-hydroxygeraniol and nepetalactol. Integration of geraniol hydroxylase (G8H) from Catharanthus roseus led to essentially quantitative conversion of geraniol to 8-hydroxygeraniol at a titer of 227 mg/L in a fed-batch fermentation. Further introduction of geraniol oxidoreductase (GOR) and iridoid synthase (ISY) from C. roseus and tuning of the relative expression levels resulted in the first de novo nepetalactol production. The strategies developed in this work can facilitate future strain engineering for yeast production of later intermediates in the strictosidine biosynthetic pathway.
Terpenoids and polyphenols are high-valued plant secondary metabolites. Their high antimicrobial activities demonstrate their huge potential as natural preservatives in the food industry. With the rapid development of metabolic engineering, it has become possible to realize large-scale production of non-native terpenoids and polyphenols by using the generally recognized as safe (GRAS) strain, Saccharomyces cerevisiae, as a cell factory. This review will summarize the major terpenoid and polyphenol compounds with high antimicrobial properties, describe their native metabolic pathways as well as antimicrobial mechanisms, and highlight current progress on their heterologous biosynthesis in S. cerevisiae. Current challenges and perspectives for the sustainable production of terpenoid and polyphenol as natural food preservatives via S. cerevisiae will also be discussed herein.
Food spoilage is a global issue, and millions of people suffer from food-borne infections. So it is urgent to search for novel, effective and safe antimicrobial agents. A novel essential oil was obtained from Eremothecium ashbyii H4565, which was identified and conserved in the China Center for Type Culture Collection. The chemical components of the essential oil were analyzed by gas chromatography-mass spectrometry. The results showed that 22 components were identified, and the major compounds were geraniol, citronellol, phenylethyl alcohol, longifolene, linalool and nerolidol. By disc diffusion assay and double-broth dilution method, the essential oil exhibited antibacterial activities against food spoilage microbes, such as Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Listeria monocytogenes, Escherichia coli, Salmonella typhimurium, Zygosaccharomyces bailii, Saccharomyces cerevisiae, and Candida utilis. The minimum inhibition concentrations of the essential oil against these microorganisms were 15.6, 15.6, 15.6, 15.6, 7.8, 7.8, 31.2, 15.6, and 31.2 μg/mL, respectively. Based on the results of electron microscopy, protein and DNA leakage, and cytoplasmic membrane permeability, the possible antibacterial mechanism was indicated that cell wall and cytoplasmic membrane were destroyed as the targets by the essential oil. Therefore, it was suggested that essential oil might be used as a potential antimicrobial agent in food.
Covering: 2006 to 2018
Phytochemicals are important sources for the discovery and development of agricultural and pharmaceutical compounds, such as pesticides and medicines. However, these compounds are typically present in low abundance in nature, and the biosynthetic pathways for most phytochemicals are not fully elucidated. Heterologous production of phytochemicals in plant, bacterial, and yeast hosts has been pursued as a potential approach to address sourcing issues associated with many valuable phytochemicals, and more recently has been utilized as a tool to aid in the elucidation of plant biosynthetic pathways. Due to the structural complexity of certain phytochemicals and the associated biosynthetic pathways, reconstitution of plant pathways in heterologous hosts can encounter numerous challenges. Synthetic biology approaches have been developed to address these challenges in areas such as precise control over heterologous gene expression, improving functional expression of heterologous enzymes, and modifying central metabolism to increase the supply of precursor compounds into the pathway. These strategies have been applied to advance plant pathway reconstitution and phytochemical production in a wide variety of heterologous hosts. Here, we review synthetic biology strategies that have been recently applied to advance complex phytochemical production in heterologous hosts.
Terpenoids constitute a large family of natural products, attracting commercial interest for a variety of uses as flavours, fragrances, drugs and alternative fuels. Saccharomyces cerevisiae offers a versatile cell factory, as the precursors of terpenoid biosynthesis are naturally synthesized by the sterol biosynthetic pathway.
S. cerevisiae wild type yeast cells, selected for their capacity to produce high sterol levels were targeted for improvement aiming to increase production. Recyclable integration cassettes were developed which enable the unlimited sequential integration of desirable genetic elements (promoters, genes, termination sequence) at any desired locus in the yeast genome. The approach was applied on the yeast sterol biosynthetic pathway genes HMG2, ERG20 and IDI1 resulting in several-fold increase in plant monoterpene and sesquiterpene production. The improved strains were robust and could sustain high terpenoid production levels for an extended period. Simultaneous plasmid-driven co-expression of IDI1 and the HMG2 (K6R) variant, in the improved strain background, maximized monoterpene production levels. Expression of two terpene synthase enzymes from the sage species Salvia fruticosa and S. pomifera (SfCinS1, SpP330) in the modified yeast cells identified a range of terpenoids which are also present in the plant essential oils. Co-expression of the putative interacting protein HSP90 with cineole synthase 1 (SfCinS1) also improved production levels, pointing to an additional means to improve production.
Using the developed molecular tools, new yeast strains were generated with increased capacity to produce plant terpenoids. The approach taken and the durability of the strains allow successive rounds of improvement to maximize yields.
Linalool production was evaluated in different Saccharomyces cerevisiae strains expressing the Clarkia breweri linalool synthase gene (LIS). The wine strain T73 was shown to produce higher levels of linalool than conventional laboratory strains (i.e., almost three times the amount).
The performance of this strain was further enhanced by manipulating the endogenous mevalonate (MVA) pathway: deregulated overexpression
of the rate-limiting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) doubled linalool production. In a
haploid laboratory strain, engineering of this key step also improved linalool yield.
We have isolated two genes from yeast encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase [hydroxymethylglutaryl-coenzyme A reductase (NADPH); HMG-CoA reductase; EC 1.1.1.34], the rate-limiting enzyme of sterol biosynthesis. These genes, HMG1 and HMG2, were identified by hybridization to a cDNA clone encoding hamster HMG-CoA reductase. DNA sequence analysis reveals homology between the amino acid sequence of the proteins encoded by the two yeast genes and the carboxyl-terminal half of the hamster protein. Cells containing mutant alleles of both HMG1 and HMG2 are unable to undergo spore germination and vegetative growth. However, cells containing a mutant allele of either HMG1 or HMG2 are viable but are more sensitive to compactin, a competitive inhibitor of HMG-CoA reductase, than are wild-type cells. Assays of HMG-CoA reductase activity in extracts from hmg1- and hmg2- mutants indicate that HMG1 contributes at least 83% of the activity found in wild-type cells.
Gene expression profiling is rapidly becoming a mainstay of functional genomic studies. However, there have been relatively few studies of how the data from expression profiles integrate with more classic approaches to examine gene expression. This study used gene expression profiling of a portion of the genome of Saccharomyces cerevisiae to explore the impact of blocks in the isoprenoid biosynthetic pathway on the expression of genes and the regulation of this pathway. Approximately 50% of the genes whose expression was altered by blocks in isoprenoid biosynthesis were genes previously known to participate in the pathway. In contrast to this simple correspondence, the regulatory patterns revealed by different blocks, and in particular by antifungal azoles, was complex in a manner not anticipated by earlier studies.
Isopentenyl diphosphate:dimethylallyl diphosphate (IPP:DMAPP) isomerase catalyses a crucial activation step in the isoprenoid biosynthesis pathway. This enzyme is responsible for the isomerization of the carbon-carbon double bond of IPP to create the potent electrophile DMAPP. DMAPP then alkylates other molecules, including IPP, to initiate the extraordinary variety of isoprenoid compounds found in nature. The crystal structures of free and metal-bound Escherichia coli IPP isomerase reveal critical active site features underlying its catalytic mechanism. The enzyme requires one Mn(2+) or Mg(2+) ion to fold in its active conformation, forming a distorted octahedral metal coordination site composed of three histidines and two glutamates and located in the active site. Two critical residues, C67 and E116, face each other within the active site, close to the metal-binding site. The structures are compatible with a mechanism in which the cysteine initiates the reaction by protonating the carbon-carbon double bond, with the antarafacial rearrangement ultimately achieved by one of the glutamates involved in the metal coordination sphere. W161 may stabilize the highly reactive carbocation generated during the reaction through quadrupole- charge interaction.
Isopentenyl diphosphate and dimethylallyl diphosphate serve as the universal precursors for the biosynthesis of terpenes. Although their biosynthesis by means of mevalonate has been studied in detail, a second biosynthetic pathway for their formation by means of 1-deoxy-D-xylulose 5-phosphate has been discovered only recently in plants and certain eubacteria. Earlier in vivo experiments with recombinant Escherichia coli strains showed that exogenous 1-deoxy-D-xylulose can be converted into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate by the consecutive action of enzymes specified by the xylB and ispCDEFG genes. This article describes the transformation of exogenous [U-(13)C(5)]1-deoxy-D-xylulose into a 5:1 mixture of [U-(13)C(5)]isopentenyl diphosphate and [U-(13)C(5)]dimethylallyl diphosphate by an E. coli strain engineered for the expression of the ispH (lytB) gene in addition to recombinant xylB and ispCDEFG genes.
In Saccharomyces cerevisiae, Maf1 is essential for mediating the repression of transcription by RNA polymerase (pol) III in response to diverse cellular conditions. These conditions activate distinct signaling pathways that converge at or above Maf1. Thus, Maf1-dependent repression is thought to involve a common set of downstream inhibitory effects on the pol III machinery. Here we provide support for this view and define two steps in Maf1-dependent transcriptional repression. We show that chlorpromazine (CPZ)-induced repression of pol III transcription is achieved by inhibiting de novo assembly of transcription factor (TF) IIIB onto DNA as well as the recruitment of pol III to preassembled TFIIIB.DNA complexes. Additionally Brf1 was identified as a target of repression in extracts of CPZ-treated cells. Maf1-Brf1 and Maf1-pol III interactions were implicated in the inhibition of TFIIIB.DNA complex assembly and polymerase recruitment by recombinant Maf1. Co-immunoprecipitation experiments confirmed these interactions in yeast extracts and demonstrated that Maf1 does not differentially sequester Brf1 or pol III under repressing conditions. The results suggest that Maf1 functions by a non-stoichiometric mechanism to repress pol III transcription.
This paper reports the production of monoterpenes, which elicit a floral aroma in wine, by strains of the yeast Saccharomyces cerevisiae. Terpenes, which are typical components of the essential oils of flowers and fruits, are also present as free and glycosylated conjugates amongst the secondary metabolites of certain wine grape varieties of Vitis vinifera. Hence, when these compounds are present in wine they are considered to originate from grape and not fermentation. However, the biosynthesis of monoterpenes by S. cerevisiae in the absence of grape derived precursors is shown here to be of de novo origin in wine yeast strains. Higher concentration of assimilable nitrogen increased accumulation of linalool and citronellol. Microaerobic compared with anaerobic conditions favored terpene accumulation in the ferment. The amount of linalool produced by some strains of S. cerevisiae could be of sensory importance in wine production. These unexpected results are discussed in relation to the known sterol biosynthetic pathway and to an alternative pathway for terpene biosynthesis not previously described in yeast.
Malaria is a global health problem that threatens 300-500 million people and kills more than one million people annually. Disease control is hampered by the occurrence of multi-drug-resistant strains of the malaria parasite Plasmodium falciparum. Synthetic antimalarial drugs and malarial vaccines are currently being developed, but their efficacy against malaria awaits rigorous clinical testing. Artemisinin, a sesquiterpene lactone endoperoxide extracted from Artemisia annua L (family Asteraceae; commonly known as sweet wormwood), is highly effective against multi-drug-resistant Plasmodium spp., but is in short supply and unaffordable to most malaria sufferers. Although total synthesis of artemisinin is difficult and costly, the semi-synthesis of artemisinin or any derivative from microbially sourced artemisinic acid, its immediate precursor, could be a cost-effective, environmentally friendly, high-quality and reliable source of artemisinin. Here we report the engineering of Saccharomyces cerevisiae to produce high titres (up to 100 mg l(-1)) of artemisinic acid using an engineered mevalonate pathway, amorphadiene synthase, and a novel cytochrome P450 monooxygenase (CYP71AV1) from A. annua that performs a three-step oxidation of amorpha-4,11-diene to artemisinic acid. The synthesized artemisinic acid is transported out and retained on the outside of the engineered yeast, meaning that a simple and inexpensive purification process can be used to obtain the desired product. Although the engineered yeast is already capable of producing artemisinic acid at a significantly higher specific productivity than A. annua, yield optimization and industrial scale-up will be required to raise artemisinic acid production to a level high enough to reduce artemisinin combination therapies to significantly below their current prices.
Isoprenoid secondary metabolites are a rich source of commercial products that have not been fully explored. At present, there are isoprenoid products used in cancer therapy, the treatment of infectious diseases, and crop protection. All isoprenoids share universal prenyl diphosphate precursors synthesized via two distinct pathways. From these universal precursors, the biosynthetic pathways to specific isoprenoids diverge resulting in a staggering array of products. Taking advantage of this diversity has been the focus of much effort in metabolic engineering heterologous hosts. In addition, the engineering of the mevalonate pathway has increased levels of the universal precursors available for heterologous production. Finally, we will describe the efforts to produce to commercial terpenoids, paclitaxel and artemisinin.
Using a degenerate primer designed from triterpene synthase sequences, we have isolated a new gene from the medicinal plant Artemisia annua. The predicted protein is highly similar to beta-amyrin synthases (EC 5.4.99.-), sharing amino acid sequence identities of up to 86%. Expression of the gene, designated AaBAS, in Saccharomyces cerevisiae, followed by GC/MS analysis, confirmed the encoded enzyme as a beta-amyrin synthase. Through engineering the sterol pathway in S. cerevisiae, we explore strategies for increasing triterpene production, using AaBAS as a test case. By manipulation of two key enzymes in the pathway, 3-hydroxy-3-methylglutaryl-CoA reductase and lanosterol synthase, we have improved beta-amyrin production by 50%, achieving levels of 6 mg.L(-1) culture. As we have observed a 12-fold increase in squalene levels, it appears that this strain has the capacity for even higher beta-amyrin production. Options for further engineering efforts are explored.
Geraniol is a commercially important terpene alcohol occurring in the essential oils of several aromatic plants. It is one of the most important molecules in the flavour and fragrance industries and is a common ingredient in consumer products produced by these industries. In addition to its pleasant odour, geraniol is known to exhibit insecticidal and repellent properties and used as a natural pest control agent exhibiting low toxicity. Geraniol has been suggested to represent a new class of chemoprevention agents for cancer. Other biological activities such as antimicrobial, anti-oxidant, anti-inflammatory and some vascular effects have also been investigated. The effect of geraniol as a penetration enhancer for transdermal drug delivery has also attracted the attention of researchers and formulation scientists. This review aims to coherently discuss some of the most important applications of geraniol and unites the results obtained from several studies reporting the biological properties of this molecule.
Enhancement of the tolerance of Saccharomyces cerevisiae to monoterpenes has the potential to improve the de novo biosynthesis of these chemicals as well as the efficient utilization of monoterpene-containing citrus waste. The aims of the current work are to demonstrate the mechanisms by which ergosterol, an important component of cell membranes, protects S. cerevisiae from d-limonene stress and to provide some useful information for further metabolic engineering of the yeast.
Saccharomyces cerevisiae cells were treated with a sublethal dose of d-limonene for 2 h, and then ergosterol was added to investigate the physiological responses of S. cerevisiae. In d-limonene-treated cells, the membrane fluidity, permeability and saturated fatty acid ratio increased, whereas the intracellular ergosterol concentration decreased sharply. Addition of ergosterol restored membrane and intracellular ergosterol to normal levels. Exogenous ergosterol triggered nearly all of the genes that encode the biosynthesis of ergosterol.
In S. cerevisiae, the cell membrane is the target of d-limonene. Intracellular ergosterol availability is correlated with the d-limonene tolerance of the cells.
The results indicate that modification of the ergosterol biosynthesis pathway could be a promising strategy for constructing a robust yeast strain with enhanced tolerance.
Metabolic engineering of microorganisms is an alternative and attractive route for production of valuable terpenoids that are usually extracted from plant sources. Tanshinones are the bioactive components of Salvia miltiorrhizha Bunge, which is a well-known traditional Chinese medicine widely used for treatment of many cardiovascular diseases. As a step toward microbial production of tanshinones, copalyl diphosphate (CPP) synthase, and normal CPP kaurene synthase-like genes, which convert the universal diterpenoid precursor geranylgeranyl diphosphate (GGPP) to miltiradiene (an important intermediate of the tanshinones synthetic pathway), was introduced into Saccharomyces cerevisiae, resulting in production of 4.2 mg/L miltiradiene. Improving supplies of isoprenoid precursors was then investigated for increasing miltiradiene production. Although over-expression of a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase (tHMGR) and a mutated global regulatory factor (upc2.1) gene did improve supply of farnesyl diphosphate (FPP), production of miltiradiene was not increased while large amounts of squalene (78 mg/L) were accumulated. In contrast, miltiradiene production increased to 8.8 mg/L by improving supply of GGPP through over-expression of a fusion gene of FPP synthase (ERG20) and endogenous GGPP synthase (BTS1) together with a heterologous GGPP synthase from Sulfolobus acidocaldarius (SaGGPS). Auxotrophic markers in the episomal plasmids were then replaced by antibiotic markers, so that engineered yeast strains could use rich medium to obtain better cell growth while keeping plasmid stabilities. Over-expressing ERG20-BTS1 and SaGGPS genes increased miltiradiene production from 5.4 to 28.2 mg/L. Combinatorial over-expression of tHMGR-upc2.1 and ERG20-BTS1-SaGGPS genes had a synergetic effects on miltiradiene production, increasing titer to 61.8 mg/L. Finally, fed-batch fermentation was performed, and 488 mg/L miltiradiene was produced. The yeast strains engineered in this work provide a basis for creating an alternative way for production of tanshinones in place of extraction from plant sources. Biotechnol. Bioeng. 2012; 109: 2845-2853. © 2012 Wiley Periodicals, Inc.
Microbial cells engineered for efficient production of plant sesquiterpenes may allow for sustainable and scalable production of these compounds that can be used as e.g. perfumes and pharmaceuticals. Here, for the first time a Saccharomyces cerevisiae strain capable of producing high levels of α-santalene, the precursor of a commercially interesting compound, was constructed through a rationally designed metabolic engineering approach. Optimal sesquiterpene production was obtained by modulating the expression of one of the key metabolic steps of the mevalonate (MVA) pathway, squalene synthase (Erg9). To couple ERG9 expression to glucose concentration its promoter was replaced by the HXT1 promoter. In a second approach, the HXT2 promoter was used to express an ERG9 antisense construct. Using the HXT1 promoter to control ERG9 expression, it was possible to divert the carbon flux from sterol synthesis towards α-santalene improving the productivity by 3.4 fold. Combining this approach together with the overexpression of a truncated form of 3-hydroxyl-3-methyl-glutaryl-CoA reductase (HMGR) and deletion of lipid phosphate phosphatase encoded by LPP1 led to a strain with a productivity of 0.18mg/gDCWh. The titer was further increased by deleting DPP1 encoding a second FPP consuming pyrophosphate phosphatase yielding a final productivity and titer, respectively, of 0.21mg/gDCWh and 92mg/l of α-santalene.
Terpenoids are one of the largest and most diverse families of natural compounds. They are heavily used in industry, and the trend is toward engineering modified microorganisms that produce high levels of specific terpenoids. Most studies have focused on creating specific heterologous pathways for sesquiterpenes in Escherichia coli or yeast. We subjected the Saccharomyces cerevisiae ERG20 gene (encoding farnesyl diphosphate synthase) to a set of amino acid mutations in the catalytic site at position K197. Mutated strains have been shown to exhibit various growth rate, sterol amount, and monoterpenol-producing capacities. These results are discussed in the context of the potential use of these mutated strains for heterologous expression of monoterpenoid synthases, which was investigated using Ocimum basilicum geraniol synthase. The results obtained with up to 5 mg/L geraniol suggest a major improvement compared with previous available expression systems like Escherichia coli or yeast strains with an unmodified ERG20 gene that respectively delivered amounts in the 10 and 500 µg/L range or even a previously characterized K197E mutation that delivered amounts in the 1 mg/L range.
The progression of prostate cancer is associated with escape from cell cycle arrest and apoptosis under androgen-depleted conditions. Here, we found that geraniol, a naturally occurring monoterpene, induces cell cycle arrest and apoptosis in cultured cells and tumor grafted mice using PC-3 prostate cancer cells. Geraniol modulated the expression of various cell cycle regulators and Bcl-2 family proteins in PC-3 cells in vitro and in vivo. Furthermore, we showed that the combination of sub-optimal doses of geraniol and docetaxel noticeably suppresses prostate cancer growth in cultured cells and tumor xenograft mice. Therefore, our findings provide insight into unraveling the mechanisms underlying escape from cell cycle arrest and apoptosis and developing therapeutic strategies against prostate cancer.
A series of yeast shuttle vectors and host strains has been created to allow more efficient manipulation of DNA in Saccharomyces cerevisiae. Transplacement vectors were constructed and used to derive yeast strains containing nonreverting his3, trp1, leu2 and ura3 mutations. A set of YCp and YIp vectors (pRS series) was then made based on the backbone of the multipurpose plasmid pBLUESCRIPT. These pRS vectors are all uniform in structure and differ only in the yeast selectable marker gene used (HIS3, TRP1, LEU2 and URA3). They possess all of the attributes of pBLUESCRIPT and several yeast-specific features as well. Using a pRS vector, one can perform most standard DNA manipulations in the same plasmid that is introduced into yeast.
Hydroxymethylglutaryl-CoA reductase (HMG-R) is a key enzyme in the mevalonate pathway, from which thousands of molecules are derived including cholesterol and prenyl moieties. The regulation of HMG-R is complex and includes feedback control, cross-regulation by independent bio-chemical processes and contra-regulation of separate isozymes. From studies in yeast, these separate modes of regulation can be considered in an integrated fashion.
tRNA isopentenylation is a branch of an isoprenoid pathway in yeast. There is a competition for a substrate between isoprenoid biosynthetic enzyme Erg20p and tRNA isopentenyltransferase. Here we studied the direct effect of elevated tRNA biosynthesis on ERG20 expression. The maf1-1 mutant of Saccharomyces cerevisiae that has enhanced cellular tRNA levels was used. We show that both ERG20 transcript and Erg20 protein levels are increased in maf1-1. Additionally, maf1-1 leads to decreased ergosterol content in the cells. These effects of maf1-1 are dependent on functional tRNA isopentenyltransferase. Our results indicate that a complex regulation of the isoprenoid pathway involves also an effect of changes in tRNA biosynthesis.
The yeast farnesyl diphosphate synthase (FPPS) gene was engineered so as to construct allelic forms giving various activities of the enzyme. One of the substitutions was F96W in the chain length determination region. The other, K197, conserved within a consensus sequence found in the majority of FPP and GGPP synthases, was substituted by R, E and V. An intricate correlation has been found between the FPPS activity, the amount of ergosterol synthesized and cell growth of a mutant strain defective in FPPS. About 40% of wt FPPS activity was sufficient to support normal growth of the mutant. With further decline of FPPS activity (20 down to 3%) the amount of ergosterol remained unchanged at approximately 0.16% (vs dry weight), whereas growth yield decreased and lag times increased. We postulate that, in addition to ergosterol initiating and maintaining growth of yeast cells, FPP and/or its derivatives participate in these processes.
Saving energy, cost efficiency, producing less waste, improving the biodegradability of products, potential for producing novel and complex molecules with improved properties, and reducing the dependency on fossil fuels as raw materials are the main advantages of using biotechnological processes to produce chemicals. Such processes are often referred to as green chemistry or white biotechnology. Metabolic engineering, which permits the rational design of cell factories using directed genetic modifications, is an indispensable strategy for expanding green chemistry. In this chapter, the benefits of using metabolic engineering approaches for the development of green chemistry are illustrated by the recent advances in microbial production of isoprenoids, a diverse and important group of natural compounds with numerous existing and potential commercial applications. Accumulated knowledge on the metabolic pathways leading to the synthesis of the principal precursors of isoprenoids is reviewed, and recent investigations into isoprenoid production using engineered cell factories are described.
Plant monoterpenoids belong to a large family of plant secondary metabolites with valuable applications in cosmetics and medicine. Their usual low levels and difficult purification justify the need for alternative fermentative processes for large-scale production. Geranyl diphosphate is the universal precursor of monoterpenoids. In yeast it occurs exclusively as an intermediate of farnesyl diphosphate synthesis. In the present study we investigated the potential use of Saccharomyces cerevisiae as an alternative engineering tool. The expression of geraniol synthase of Ocimum basilicum in yeast allowed a strong and specific excretion of geraniol to the growth medium, in contrast to mutants defective in farnesyl diphosphate synthase which excreted geraniol and linalool in similar amounts. A further increase of geraniol synthesis was obtained using yeast mutants defective in farnesyl diphosphate synthase. We also showed that geraniol synthase expression affects the general ergosterol pathway, but in a manner dependent on the genetic background of the strain.
Amorphadiene, a sesquiterpene precursor to the anti-malarial drug artemisinin, is synthesized by the cyclization of farnesyl pyrophosphate (FPP). Saccharomyces cerevisiae produces FPP through the mevalonate pathway using acetyl-CoA as a starting compound. In order to enhance the supply of acetyl-CoA to the mevalonate pathway and achieve high-level production of amorphadiene, we engineered the pyruvate dehydrogenase bypass in S. cerevisiae. Overproduction of acetaldehyde dehydrogenase and introduction of a Salmonella enterica acetyl-CoA synthetase variant increased the carbon flux into the mevalonate pathway resulting in increased amorphadiene production. This work will be generally applicable to the production of a broad range of isoprenoids in yeast.
The yeast Saccharomyces cerevisiae was chosen as a microbial host for heterologous biosynthesis of three different plant sesquiterpenes, namely valencene, cubebol, and patchoulol. The volatility and low solubility of the sesquiterpenes were major practical problems for quantification of the excreted sesquiterpenes. In situ separation of sesquiterpenes in a two-phase fermentation using dodecane as the secondary phase was therefore performed in order to enable quantitative evaluation of different strains. In order to enhance the availability of the precursor for synthesis of sesquiterpenes, farnesyl diphosphate (FPP), the ERG9 gene which is responsible for conversion of FPP to squalene was downregulated by replacing the native ERG9 promoter with the regulatable MET3 promoter combined with addition of 2 mM methionine to the medium. This strategy led to a reduced ergosterol content of the cells and accumulation of FPP derived compounds like target sesquiterpenes and farnesol. Adjustment of the methionine level during fermentations prevented relieving MET3 promoter repression and resulted in further improved sesquiterpene production. Thus, the final titer of patchoulol and farnesol in the ERG9 downregulated strain reached 16.9 and 20.2 mg/L, respectively. The results obtained in this study revealed the great potential of yeast as a cell factory for production of sesquiterpenes.
Since the middle ages, essential oils have been widely used for bactericidal, virucidal, fungicidal, antiparasitical, insecticidal, medicinal and cosmetic applications, especially nowadays in pharmaceutical, sanitary, cosmetic, agricultural and food industries. Because of the mode of extraction, mostly by distillation from aromatic plants, they contain a variety of volatile molecules such as terpenes and terpenoids, phenol-derived aromatic components and aliphatic components. In vitro physicochemical assays characterise most of them as antioxidants. However, recent work shows that in eukaryotic cells, essential oils can act as prooxidants affecting inner cell membranes and organelles such as mitochondria. Depending on type and concentration, they exhibit cytotoxic effects on living cells but are usually non-genotoxic. In some cases, changes in intracellular redox potential and mitochondrial dysfunction induced by essential oils can be associated with their capacity to exert antigenotoxic effects. These findings suggest that, at least in part, the encountered beneficial effects of essential oils are due to prooxidant effects on the cellular level.
The GAL1 and GAL10 genes of Saccharomyces cerevisiae are transcribed divergently and transcription of both genes can be induced by galactose and repressed by glucose. This study describes the construction and characterization of 8 bidirectional expression vectors. These vectors carry both a modified inducible GAL promoter in one direction and a constitutive GPD promoter in the reverse direction. When the gene-encoded alpha-galactosidase was cloned into the modified GAL1 and GAL10 vectors, promoter activity was 85% of wild-type for the GAL1 promoter and 90% of wild-type for the GAL10 promoter, respectively. The modified GAL promoters and GPD promoter did not interfere with one another in the bidirectional vectors. Furthermore, yeast overexpressing human Bax under the control of either modified GAL1 or modified GAL10 in a bidirectional vector conferred a lethal phenotype that was rescued by coexpression of human Bcl-2 under the control of the GPD promoter in the same vector. These eight vectors can be used to express lethal genes and screen for genes that rescue the yeast from the lethal gene product.
Grape musts contain a variety of terpenols that significantly affect wine aroma. The amounts of these metabolites depend on the grape variety, and many cultivars are non-aromatic. Yeasts like Saccharomyces cerevisiae cannot produce and excrete monoterpenes efficiently, mainly due to their lack of monoterpene synthases. By metabolic engineering we have modified the isoprenoid biosynthesis pathway in a wine yeast strain of S. cerevisiae expressing the Clarkia breweri S-linalool synthase gene. Under microvinification conditions, without compromising other desirable and useful fermentative traits, the recombinant yeast efficiently excreted linalool to levels exceeding the threshold of human perception. Bearing in mind the possibility of (co-)expressing other genes that encode enzymes leading to the production of various aroma compounds and the feasibility of controlling the levels of their expression, the potential of this achievement for future genetic manipulation of wine varietal aroma or for use in other alcoholic drinks seems very promising.
Saccharomyces cerevisiae utilizes several regulatory mechanisms to maintain tight control over the intracellular level of farnesyl diphosphate (FPP), the central precursor to nearly all yeast isoprenoid products. High-level production of non-native isoprenoid products requires that FPP flux be diverted from production of sterols to the heterologous metabolic reactions. To do so, expression of the gene encoding squalene synthase (ERG9), the first committed step in sterol biosynthesis, was down-regulated by replacing its native promoter with the methionine-repressible MET3 promoter. The intracellular levels of FPP were then assayed by expressing the gene encoding amorphadiene synthase (ADS) and converting the FPP to amorphadiene. Under certain culture conditions amorphadiene production increased fivefold upon ERG9 repression. With increasing flux to amorphadiene, squalene and ergosterol production each decreased. The levels of these three metabolites were dependent not only upon the level of ERG9 repression, but also the timing of its repression relative to the induction of ADS and genes responsible for enhancing flux to FPP.
Metabolic engineering in microbes could be used to produce large amounts of valuable metabolites that are difficult to extract from their natural sources and too expensive or complex to produce by chemical synthesis. As a step towards the production of Taxol in the yeast Saccharomyces cerevisiae, we introduced heterologous genes encoding biosynthetic enzymes from the early part of the taxoid biosynthetic pathway, isoprenoid pathway, as well as a regulatory factor to inhibit competitive pathways, and studied their impact on taxadiene synthesis. Expression of Taxus chinensis taxadiene synthase alone did not increase taxadiene levels because of insufficient levels of the universal diterpenoid precursor geranylgeranyl diphosphate. Coexpression of T. chinensis taxadiene synthase and geranylgeranyl diphosphate synthase failed to increase levels, probably due to steroid-based negative feedback, so we also expressed a truncated version of 3-hydroxyl-3-methylglutaryl-CoA reductase (HMG-CoA reductase) isoenzyme 1 that is not subject to feedback inhibition and a mutant regulatory protein, UPC2-1, to allow steroid uptake under aerobic conditions, resulting in a 50% increase in taxadiene. Finally, we replaced the T. chinensis geranylgeranyl diphosphate synthase with its counterpart from Sulfolobus acidocaldarius, which does not compete with steroid synthesis, and codon optimized the T. chinensis taxadiene synthase gene to ensure high-level expression, resulting in a 40-fold increase in taxadiene to 8.7+/-0.85mg/l as well as significant amounts of geranylgeraniol (33.1+/-5.6mg/l), suggesting taxadiene levels could be increased even further. This is the first demonstration of such enhanced taxadiene levels in yeast and offers the prospect for Taxol production in recombinant microbes.
Biological effects of essential oils-a review
- F Bakkali
- S Averbeck
- D Averbeck
- M Waomar
Bakkali, F., Averbeck, S., Averbeck, D., Waomar, M., 2008. Biological effects of essential oils-a review. Food Chem. Toxicol. 46, 446-475.