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Comparison of activities of purified Cphy3925 AdhE from wild-type and ET strains. (A) SDS-PAGE gel of purified Cphy3925 from the wild-type (WT) and ET (G609D) strains showing single bands of the expected 95-kDa molecular mass. (B to E) Reactions for the two-step, bidirectional interconversion of acetyl-CoA acetaldehyde and ethanol: reduction of 300 M acetyl-CoA to acetaldehyde (red) (B), reduction of 18 mM acetaldehyde to ethanol (green) (C), oxidation of 2 M ethanol to acetaldehyde (purple) (D), and oxidation of 18 mM acetaldehyde to acetyl-CoA (blue) (E). Enzyme activities are shown in millimoles of NAD(P)H per micromole of enzyme per second measured using NADH(P)H or NAD(P) ϩ cofactors. Bar heights represent averages of duplicate activity measurements, and error bars represent 1 standard deviation.
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Novel processing strategies for hydrolysis and fermentation of lignocellulosic biomass in a single reactor offer large potential cost savings for production of biocommodities and biofuels. One critical challenge is retaining high enzyme production in the presence of elevated product titers. Toward this goal, the cellulolytic, ethanol-producing bact...
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Context 1
... ET strain also has mutations in two transporters putatively involved in cation homeostasis. Cphy0543 is homologous to MgtA, a P-type ATPase upregulated at low ambient Mg 2 ϩ concentrations (35) to mediate Mg 2 ϩ uptake (36) or Ca 2 ϩ /Mg 2 ϩ antiport (37). Cphy3778, the membrane component of an ABC transporter (PFAM accession no. PF06182), appears to be cotrans- cribed with Cphy3780, an ABC-type Na ϩ efflux protein (NCBI accession no. cd03267). In Bacillus subtilis , this Na ϩ efflux system is induced by ethanol and is proposed to compensate for an influx of extracellular Na ϩ resulting from a weakened membrane barrier (38). The variants in these cation transporters may increase their activities to alleviate cation leakage due to ethanol stress. AdhE activities. The ET strain has a G609D variant in Cphy3925 AdhE, a putative acetaldehyde-CoA dehydrogenase and alcohol dehydrogenase (ADH). The G609D mutation is in a conserved position in the active site of the C-terminal ADH do- main (NCBI accession no. cd08178). A previous study reported an ethanol-tolerant C. thermocellum strain with AdhE mutations (P704L and H735R) that shifted the cofactor specificity from NADH to NADPH, which was proposed to confer ethanol resistance by altering the internal redox balance (9). To deter- mine the effect of the G609D mutation on Cphy3925 enzyme activity, we purified WT and ET versions of the enzyme (Fig. 3A) and tested their in vitro catalysis of the two-step, bidirectional reactions converting acetyl-CoA to ethanol using either NADH or NADPH cofactors. The mutated Cphy3925 lost NAD(H)-dependent activities, but, unlike the mutated AdhE in C. thermocellum , the G609D mutation did not result in NADPH-dependent ADH activity (Fig. 3B to E). Instead, our results support the notion that the ET strain arrested AdhE-mediated interconversion of acetyl-CoA, acetaldehyde, and ethanol, which helps explain why the C. phytofermentans ET strain had lower ethanol yield. AdhE loss of function could mitigate ethanol stress by reducing intracellular levels of ethanol and its highly toxic precursor, acetaldehyde. C. phytofermentans encodes four Fe-dependent ADHs in addition to Cphy3925 as well as a Zn-dependent ADH. All 6 ADHs are expressed, and Cphy3925 and Cphy1029 are among the most highly expressed proteins on all tested carbon sources (19, 39). C. phytofermentans thus likely produces ethanol by the concerted action of multiple ADHs, and these other ADHs, especially Cphy1029, are responsi- ble for ethanol produced by the ET strain. Ethanol pathway engineering. To augment ethanol production by the ET strain, an alternative ethanol production pathway comprised of pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (AdhB) from Zymomonas mobilis (Fig. 4A) was transferred into C. phytofermentans on the replicating pQexpE plasmid (Fig. 4B). Together, these enzymes couple decarboxylation of pyruvate to ethanol with the oxidation of NADH and thus represent an alternative to the AdhE ethanol formation pathway. We chose to express foreign enzymes rather than a WT copy of Cphy3925 because AdhE multimerizes (40) such that the mutant AdhE could have a dominant-negative effect in a merodiploid. Expression of pQexpE increased cellulolysis by ϳ 30% in both the WT and ET strains (Fig. 5A) and boosted ethanol production by 70% relative to the ET strain ( P Ͻ 0.01), thereby restoring ethanol yields to WT levels (Fig. 5B). CO 2 production increased disproportionately relative to H 2 production in WT and ET strains expressing pQexpE (Table 3). Elevated CO 2 synthesis is likely due to increased pyruvate decarboxylation by the Pdc enzyme. Previous results showed that Pdc/AdhB expression enhanced cellulolysis and ethanol production in WT C. cellulolyticum , which was proposed to result from consumption of excess pyruvate that otherwise leads to metabolic arrest (14). Increased metabolism (cellulolysis and production of CO and ethanol) by C. phytofermentans expressing Pdc/AdhB might be due to allevia- tion of inhibition by excess pyruvate. Alternatively, expression or activity of glycolytic enzymes might be regulated by NADH levels such that NADH reoxidation by Pdc/AdhB stimulates glycolysis, which results in increased substrate utilization. Conclusions. In this study, we investigated the genetic basis and phenotypic consequences of microbial ethanol tolerance by isolating, characterizing, and engineering an ethanol-resistant (ET) strain of Clostridium phytofermentans . The ET strain grows at higher ethanol concentrations than the wild-type strain (Fig. 1) and continues to produce ethanol at a 7% ambient ethanol concentration (Fig. 2C) but has impaired growth (Fig. 1) and ethanol yield (Fig. 2A) relative to the wild type. The genome sequence of the ET strain revealed 12 mutations in genes involved in diverse aspects of metabolism (Table 2), including a G609D variant in the bifunctional acetaldehyde CoA/alcohol dehydrogenase AdhE that abolishes its activity (Fig. 3). We complemented the AdhE mutation in the ET strain by expressing pyruvate decarboxylase (Pdc) and alcohol dehydrogenase B (AdhB) from Zymomonas mobilis on the pQexpE plasmid (Fig. 4), which boosted substrate conversion (Fig. 5A) and restored ethanol production (Fig. 5B). Additional work is needed to enhance C. phytofermentans ’ plant biomass degradation and ethanol formation rates and product titers. Recently, improvement of C. phytofermentans ’ growth on cellobiose, cellulose, and xylan by experimental evolution yielded strains that also produced ethanol more quickly (41). The genome sequence of the ET strain presented here suggests other novel approaches to potentially improve ethanol resistance and production. For example, our results suggest that further studies on ethanol resistance should focus on PlsD-mediated fatty acid incorporation into phospholipids, LysR-regulated gene expression patterns, overexpression of the Rnf complex to stimulate AdhE-mediated ethanol production, and prevention of cation ...
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Citations
... C. phytofermentans is remarkable among the Clostridium genus due to its ability to catabolize a broad range of substrates. Its genome encodes over 169 carbohydrate-active enzymes, the largest number among sequenced clostridia, and its efficient ethanol production makes it a model system for cellulosic biofuel production 21,23,[34][35][36][37] . E. coli is a well studied, facultative anaerobe capable of fermenting a broad range of substrates including glucose and glycerol which is a widely available waste product from biodiesel production 21,23,38 . ...
Planktonic cultures, of a rationally designed consortium, demonstrated emergent properties that exceeded the sums of monoculture properties, including a >200% increase in cellobiose catabolism, a >100% increase in glycerol catabolism, a >800% increase in ethanol production, and a >120% increase in biomass productivity. The consortium was designed to have a primary and secondary-resource specialist that used crossfeeding with a positive feedback mechanism, division of labor, and nutrient and energy transfer via necromass catabolism. The primary resource specialist was Clostridium phytofermentans ( a.k.a. Lachnoclostridium phytofermentans ), a cellulolytic, obligate anaerobe. The secondary-resource specialist was Escherichia coli , a versatile, facultative anaerobe, which can ferment glycerol and byproducts of cellobiose catabolism. The consortium also demonstrated emergent properties of enhanced biomass accumulation when grown as biofilms, which created high cell density communities with gradients of species along the vertical axis. Consortium biofilms were robust to oxic perturbations with E. coli consuming O 2 , creating an anoxic environment for C. phytofermentans . Anoxic/oxic cycling further enhanced biomass productivity of the biofilm consortium, increasing biomass accumulation ~250% over the sum of the monoculture biofilms. Consortium emergent properties were credited to several synergistic mechanisms. E. coli consumed inhibitory byproducts from cellobiose catabolism, driving higher C. phytofermentans growth and higher cellulolytic enzyme production, which in turn provided more substrate for E. coli . E. coli necromass enhanced C. phytofermentans growth while C. phytofermentans necromass aided E. coli growth via the release of peptides and amino acids, respectively. In aggregate, temporal cycling of necromass constituents increased flux of cellulose-derived resources through the consortium. The study establishes a consortia-based, bioprocessing strategy built on naturally occurring interactions for improved conversion of cellulose-derived sugars into bioproducts.
... In 2012 Li et al. inactivated lactate and malate dehydrogenase in C. cellulolyticum resulting in a titer of 2.7 g/L ethanol from cellulose [41]. Tolonen et al. increased the ethanol tolerance of Clostridium phytofermentans by serial transfer and followed an approach similar to Guedon et al., leading to an ethanol titer of around 0.9 g/L [42]. Chung et al. engineered the hyperthermophile Caldicellulosiruptor bescii to produce 0.6 g/L ethanol from switchgrass by heterologously expressing adhE from C. thermocellum [43]. ...
Background:
Engineering efforts targeted at increasing ethanol by modifying the central fermentative metabolism of Clostridium thermocellum have been variably successful. Here, we aim to understand this variation by a multifaceted approach including genomic and transcriptomic analysis combined with chemostat cultivation and high solids cellulose fermentation. Three strain lineages comprising 16 strains total were examined. Two strain lineages in which genes involved in pathways leading to organic acids and/or sporulation had been knocked out resulted in four end-strains after adaptive laboratory evolution (ALE). A third strain lineage recapitulated mutations involving adhE that occurred spontaneously in some of the engineered strains.
Results:
Contrary to lactate dehydrogenase, deleting phosphotransacetylase (pta, acetate) negatively affected steady-state biomass concentration and caused increased extracellular levels of free amino acids and pyruvate, while no increase in ethanol was detected. Adaptive laboratory evolution (ALE) improved growth and shifted elevated levels of amino acids and pyruvate towards ethanol, but not for all strain lineages. Three out of four end-strains produced ethanol at higher yield, and one did not. The occurrence of a mutation in the adhE gene, expanding its nicotinamide-cofactor compatibility, enabled two end-strains to produce more ethanol. A disruption in the hfsB hydrogenase is likely the reason why a third end-strain was able to make more ethanol. RNAseq analysis showed that the distribution of fermentation products was generally not regulated at the transcript level. At 120 g/L cellulose loadings, deletions of spo0A, ldh and pta and adaptive evolution did not negatively influence cellulose solubilization and utilization capabilities. Strains with a disruption in hfsB or a mutation in adhE produced more ethanol, isobutanol and 2,3-butanediol under these conditions and the highest isobutanol and ethanol titers reached were 5.1 and 29.9 g/L, respectively.
Conclusions:
Modifications in the organic acid fermentative pathways in Clostridium thermocellum caused an increase in extracellular pyruvate and free amino acids. Adaptive laboratory evolution led to improved growth, and an increase in ethanol yield and production due a mutation in adhE or a disruption in hfsB. Strains with deletions in ldh and pta pathways and subjected to ALE demonstrated undiminished cellulolytic capabilities when cultured on high cellulose loadings.
... The targeted intron was PCR amplified and inserted between the NdeI and BsrGI sites of pQint (15). Targeted pQint plasmids were transferred into C. phytofermentans by conjugation with Escherichia coli under anaerobic conditions (15,36,37). Colonies on GS2 plates containing the 40 g ml Ϫ1 erythromycin were picked, and the genomic intron insertion was confirmed by PCR and sequencing using primers in Table S3. ...
Plant-fermenting Clostridia are anaerobic bacteria that recycle plant matter in soil and promote human health by fermenting dietary fiber in the intestine. Clostridia degrade plant biomass using extracellular enzymes and then uptake the liberated sugars for fermentation. The main sugars in plant biomass are hexoses, and here, we identify how hexoses are taken in to the cell by the model organism Clostridium phytofermentans . We show that this bacterium uptakes hexoses using a set of highly specific, nonredundant ABC transporters. Once in the cell, the hexoses are phosphorylated by intracellular hexokinases. This study provides insight into the functioning of abundant members of soil and intestinal microbiomes and identifies gene targets to engineer strains for industrial lignocellulosic fermentation.
... Evolution in continuous culture setups was applied to obtain biotechnological platform strains like Saccharomyces cerevisiae, Escherichia coli and Clostridium phytofermentans with enhanced ethanol tolerance (Ma and Liu, 2010;Tolonen et al., 2015;Yomano et al., 1998). ...
The objective of this project was the development of enhanced methylotrophic chassis strains capable of converting methanol as carbon and energy source into biomass and ultimately into commodity chemicals under industrial conditions. Methanol is an alternative to carbohydrates as feedstock in industrial biotechnology as its use does not interfere with food supply and its production can start from CO2.A prerequisite for an efficient and large scale industrial fermentation is stable growth of the methylotrophic producer strain on high methanol concentrations. For this purpose, two closely related methylotrophic strains, Methylobacterium extorquens AM1 and TK 0001, which both have a growth optimum at about 1% methanol, were adapted in continuous culture to proliferate stably in the presence of methanol of up to 10% (v/v). The adaptations were conducted using GM3 devices enabling automated long term cultivation of microorganisms.Growth curves recorded for isolates obtained from evolved populations showed enhanced proliferation in the presence of methanol at 5% as compared with wild type cells. The isolates showed comparable albeit not identical growth pointing to heterogeneity among the adapted cells in the population.Genomic sequencing of isolated clones at different steps of the adaptation revealed differences in their mutation profiles. The gene metY coding for O-acetyl-L-homoserine sulfhydrylase was found to be mutated in all isolates. This enzyme undergoes a side reaction with methanol leading to the production of the methionine analogue methoxinine known to be toxic through incorporation into proteins.Enzymatic tests conducted with these mutants showed an almost complete loss of activity even with their natural substrates, validating the involvement of MetY in methanol toxicity.Transcriptomic analysis was performed to study the gene expression response of an evolved derivative of M. extorquens TK 0001 to short and long term exposure to high methanol and compared with the response of the ancestor strain. Genes implicated in cell division, ribosomal and flagellar structures, protein stability and iron uptake showed differences in expression patterns between the strains.The M. extorquens TK 0001 cells adapted to high methanol produced more biomass from methanol than the wildtype cells. This suggests that a compound synthesized through a pathway branching from the central metabolism would be produced in higher yield from methanol by the adapted cells compared to the wildtype cells. The production of D-lactate was tested for wildtype and evolved cells both overexpressing native lactate dehydrogenase. The evolved cells produced more lactate than the control cells, confirming the interest of this methanol adaptation.
... Although ethanol tolerance has often been studied as a proxy for ethanol production, many studies have found that increases in ethanol tolerance have no effect on ethanol production [26][27][28], including studies of C. thermocellum [29]. Furthermore, in cases where ethanol tolerance has been improved by selection, many of the improvements appear to be due to idiosyncratic mutations whose effects are not generalizable to other strain backgrounds or growth conditions [17,26]. ...
Background
Clostridium thermocellum is a promising microorganism for conversion of cellulosic biomass to biofuel, without added enzymes; however, the low ethanol titer produced by strains developed thus far is an obstacle to industrial application.
Results
Here, we analyzed changes in the relative concentration of intracellular metabolites in response to gradual addition of ethanol to growing cultures. For C. thermocellum, we observed that ethanol tolerance, in experiments with gradual ethanol addition, was twofold higher than previously observed in response to a stepwise increase in the ethanol concentration, and appears to be due to a mechanism other than mutation. As ethanol concentrations increased, we found accumulation of metabolites upstream of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) reaction and depletion of metabolites downstream of that reaction. This pattern was not observed in the more ethanol-tolerant organism Thermoanaerobacterium saccharolyticum. We hypothesize that the Gapdh enzyme may have different properties in the two organisms. Our hypothesis is supported by enzyme assays showing greater sensitivity of the C. thermocellum enzyme to high levels of NADH, and by the increase in ethanol tolerance and production when the T. saccharolyticum gapdh was expressed in C. thermocellum.
Conclusions
We have demonstrated that a metabolic bottleneck occurs at the GAPDH reaction when the growth of C. thermocellum is inhibited by high levels of ethanol. We then showed that this bottleneck could be relieved by expression of the gapdh gene from T. saccharolyticum. This enzyme is a promising target for future metabolic engineering work.
Electronic supplementary material
The online version of this article (10.1186/s13068-017-0961-3) contains supplementary material, which is available to authorized users.
... In this approach, hydrogenase active site assembly is eliminated for hydrogen production that redirected carbon flux towards ethanol production [94]. A genetic change in C. phytofermentans demonstrated to improve its ethanol tolerance [95]. E. coli harboring engineered C. acetobutylicum butanol biosynthetic pathway efficiently converted n-butyric acid to n-butanol [96]. ...
... Extensive studies on protein catabolism-directed solventogenesis and acidogenesis are needed for guiding metabolic engineering of C. sticklandii to improve the yield of biofuel. Metabolic engineering targets for biofuel production have been evaluated from several Clostridial strains using conventional molecular approaches [1,95,100,123]. Systems-level characterization of C. sticklandii comprehensively examines metabolic networks to be engineered and provides a scaffold for more effective bioprocess development. Molecular complexity and the functional behavior of engineered C. sticklandii that plays a role in the response to alternative substrates have been efficiently explored with advances in an integrated knowledge base of its metabolic interaction networks. ...
Model-driven systems engineering has been more fascinating process for the microbial production of biofuel and bio-refineries in chemical and pharmaceutical industries. Genome-scale modeling and simulations have been guided for metabolic engineering of Clostridium species for the production of organic solvents and organic acids. Among them, Clostridium sticklandii is one of the potential organisms to be exploited as a microbial cell factory for biofuel production. It is a hyper-ammonia producing bacterium and is able to catabolize amino acids as important carbon and energy sources via Stickland reactions and the development of the specific pathways. Current genomic and metabolic aspects of this bacterium are comprehensively reviewed herein, which provided information for learning about protein catabolism-directed biofuel production. It has a metabolic potential to drive energy and direct solventogenesis as well as acidogenesis from protein catabolism. It produces by-products such as ethanol, acetate, n-butanol, n-butyrate and hydrogen from amino acid catabolism. Model-driven systems engineering of this organism would improve the performance of the industrial sectors and enhance the industrial economy by using protein-based waste in environment-friendly ways.
... using Bowtie 2 (53). Sequence variants (single nucleotide polymorphisms [SNPs], indels) in the CFY strains relative to the reference genome were identified using GATK (56) as described previously (57). Structural variations were detected using the breseq split-read analysis tool (58). ...
Increasing the resistance of plant-fermenting bacteria to lignocellulosic inhibitors is useful to understand microbial adaptation and to develop candidate strains for consolidated bioprocessing. Here, we study and improve inhibitor resistance in Clostridium phytofermentans (also called Lachnoclostridium phytofermentans), a model anaerobe that ferments lignocellulosic biomass. We survey the resistance of this bacterium to a panel of biomass inhibitors and then evolve strains that grow in increasing concentrations of the lignin phenolic, ferulic acid, by automated, longterm growth selection in an anaerobic GM3 automat. Ultimately, strains resist multiple inhibitors and grow robustly at the solubility limit of ferulate while retaining the ability to ferment cellulose. We analyze genome-wide transcription patterns during ferulate stress and genomic variants that arose along the ferulate growth selection, revealing how cells adapt to inhibitors through changes in gene dosage and regulation, membrane fatty acid structure, and the surface layer. Collectively, this study demonstrates an automated framework for in vivo directed evolution of anaerobes and gives insight into the genetic mechanisms by which bacteria survive exposure to chemical inhibitors.
... and C. thermocellum [27,46]. Supporting its role as a key enzyme in ethanol production, several interesting mutations have been found in adhE in strains that have been engineered for high ethanol production or tolerance [7,34,39,41,49]. Based on its amino acid sequence, AdhE is thought to be a bifunctional enzyme, responsible for both the ALDH and ADH reactions [12,16,23]. ...
Thermoanaerobacter ethanolicus is a promising candidate for biofuel production due to the broad range of substrates it can utilize and its high ethanol yield compared to other thermophilic bacteria, such as Clostridium thermocellum. Three alcohol dehydrogenases, AdhA, AdhB and AdhE, play key roles in ethanol formation. To study their physiological roles during ethanol formation, we deleted them separately and in combination. Previously, it has been thought that both AdhB and AdhE were bifunctional alcohol dehydrogenases. Here we show that AdhE has primarily acetyl-CoA reduction activity (ALDH) and almost no acetaldehyde reduction (ADH) activity, whereas AdhB has no ALDH activity and but high ADH activity. We found that AdhA and AdhB have similar patterns of activity. Interestingly, although deletion of both adhA and adhB reduced ethanol production, a single deletion of either one actually increased ethanol yields by 60-70%.
... Duplicate cultures were sampled in mid-log phase or after 2 days (RAC) or 3 days (stover). Fermentation products were quantified by HPLC 43 . ...
Bacteria respond to their environment by regulating mRNA synthesis, often by altering the genomic sites at which RNA polymerase initiates transcription. Here, we investigate genome-wide changes in transcription start site (TSS) usage by Clostridium phytofermentans, a model bacterium for fermentation of lignocellulosic biomass. We quantify expression of nearly 10,000 TSS at single base resolution by Capp-Switch sequencing, which combines capture of synthetically capped 5′ mRNA fragments with template-switching reverse transcription. We find the locations and expression levels of TSS for hundreds of genes change during metabolism of different plant substrates. We show that TSS reveals riboswitches, non-coding RNA and novel transcription units. We identify sequence motifs associated with carbon source-specific TSS and use them for regulon discovery, implicating a LacI/GalR protein in control of pectin metabolism. We discuss how the high resolution and specificity of Capp-Switch enables study of condition-specific changes in transcription initiation in bacteria.
... The saccharification is followed by simultaneous fermentation of xylose and glucose to ethanol using a genetically modified strain of brewer's yeast (Saccharomyces cerevisiae). Other organisms such as Clostridium and Pseudomonas can be used to produce alternative biochemicals (Ragauskas et al., 2014;Tolonen et al., 2015;Ramos et al., 2016;Sanford et al., 2016). ...
The production of liquid biofuels to blend with gasoline is of worldwide importance to secure the energy supply while reducing the use of fossil fuels, supporting the development of rural technology with knowledge-based jobs and mitigating greenhouse gas emissions. Today, engineering for plant construction is accessible and new processes using agricultural residues and municipal solid wastes have reached a good degree of maturity and high conversion yields (almost 90% of polysaccharides are converted into monosaccharides ready for fermentation). For the complete success of the 2G technology, it is still necessary to overcome a number of limitations that prevent a first-of-a-kind plant from operating at nominal capacity. We also claim that the triumph of 2G technology requires the development of favourable logistics to guarantee biomass supply and make all actors (farmers, investors, industrial entrepreneurs, government, others) aware that success relies on agreement advances. The growth of ethanol production for 2020 seems to be secured with a number of 2G plants, but public/private investments are still necessary to enable 2G technology to move on ahead from its very early stages to a more mature consolidated technology.
