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Bioupcycling methane into triacylglycerol for the production of sustainable aviation fuel by methanotrophic bacteria
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Many economies set climate mitigation targets for 2020 at the 2009 15th Conference of the Parties conference of the United Nations Framework Convention on Climate Change in Copenhagen. Yet no retrospective review of the implementation and actual mitigation associated with these targets has materialized. Here we track the national CO2 emissions from both territory and consumption (trade adjusted) perspectives to assess socioeconomic factors affecting changes in emissions. Among the 34 countries analysed, 12 failed to meet their targets (among them Portugal, Spain and Japan) and 7 achieved the target for territorial emissions, albeit with carbon leakage through international trade to meet domestic demand while increasing emissions in other countries. Key factors in meeting targets were intensity reduction of energy and the improvement of the energy mix. However, many countries efforts fell short of their latest nationally determined contributions. Timely tracking and review of mitigation efforts are critical for meeting the Paris Agreement targets.
The essential forces stabilizing membrane proteins and governing their folding and unfolding are difficult to decipher. Single-molecule atomic force spectroscopy mechanically unfolds individual membrane proteins and quantifies their dynamics and energetics. However, it remains challenging to structurally assign unfolding intermediates precisely and to deduce dominant interactions between specific residues that facilitate either the localized stabilization of these intermediates or the global assembly of membrane proteins. Here, we performed force spectroscopy experiments and multiscale molecular dynamics simulations to study the unfolding pathway of diacylglycerol kinase (DGK), a small trimeric multispan transmembrane enzyme. The remarkable agreement between experiments and simulations allowed precise structural assignment and interaction analysis of unfolding intermediates, bypassing existing limitations on structural mapping, and thus provided mechanistic explanations for the formation of these states. DGK unfolding was found to proceed with structural segments varying in size that do not correlate with its secondary structure. We identified intermolecular side-chain packing interactions as one of the major contributions to the stability of unfolding intermediates. Mutagenesis creating packing defects induced a dramatic decrease in the mechano-stability of corresponding intermediates and also in the thermo-stability of DGK trimer, in good agreement with predictions from simulations. Hence, the molecular determinants of the mechano- and thermo-stability of a membrane protein can be identified at residue resolution. The accurate structural assignment established and microscopic mechanism revealed in this work may substantially expand the scope of single-molecule studies of membrane proteins.
Sesquiterpenes have been identified as promising ingredients for aviation fuels due to their high energy density and combustion heat properties. Despite the characterization of numerous sesquiterpene structures, studies testing their performance properties and feasibility as fuels are scarce. In this study, 122 sesquiterpenoid skeleton compounds, obtained from existing literature reports, are tested using group contribution and gaussian quantum chemistry methods to assess their potential as high‐energy aviation fuels. Seventeen sesquiterpene compounds exhibit good predictive performance and nine compounds are further selected for overproduction in yeast. Through fed‐batch fermentation, all compounds achieve the highest reported titers to date. Subsequently, three representative products, pentalenene, presilphiperfol‐1‐ene, and α‐farnesene, are selected, produced, purified in large quantities, and tested for use as potential fuels. The performance of pentalenene, presilphiperfol‐1‐ene, and their derivatives reveals favorable prospects as high‐energy aviation fuels.
The recent unprecedented increase in energy demand has led to a growing interest in emerging alternatives such as the production of microbial lipids with high energy density and environmentally-friendly characteristics. Oleaginous yeasts represent a versatile and attractive tool for the accumulation of such lipids, also known as single cell oils (SCOs), used to manufacture biofuels (e.g., biodiesel, aviation fuel) and bioproducts. This review provides an overview of the most common oleaginous species, analysing the viability of typical feedstocks and their effect on lipid accumulation. The best results in terms of lipid content using glucose, glycerol, lignocellulose, or acetic acid as substrates are 81.4, 70, 68.2 and 73.4% (w/w), respectively. Besides, an analysis of the parameters that can affect lipid production is also presented. For instance, the optimum conditions for lipid accumulation are usually a C/N ratio between 100 and 200, pH between 5 and 6 (being more alkaline if acids are used as substrates) and temperature around 30 °C. Besides, genetic modifications generally allow to increase the lipid yield, even by up to 400%. Finally, some cost analysis is provided for scaling-up, with feedstock costs estimated at 50–80%, followed by fermenter costs, and downstream costs estimated at around 13%.
The aviation industry is responsible for approximately 2% of total anthropogenic greenhouse gas emissions. Having an expected four to six-fold growth by 2050, increased attention has been placed to reduce...
Due to continuously increasing population, industrialization, and environmental pollution, lead to generating high energy demand which suitable for our environment. Biodiesel is an alternative renewable fuel source. According to the feedstock of production, biodiesel has been categorized into four generations. The main disadvantage of the first and second generation is the raw material processing cost that the challenge for its industrial-level production. Oleaginous bacteria that contain more than 20% lipid of their cellular biomass can be a good alternative and sustainable feedstock. Oleaginous bacteria used as feedstock have numerous advantages, such as their high growth rate, being easy to cultivate, utilizing various substrates for growth, genetic or metabolic modifications possible. In addition, some species of bacteria are capable of carbon dioxide sequestration. Therefore, oleaginous bacteria can be a significant resource for the upcoming generation’s biodiesel production. This review discusses the biochemistry of lipid accumulation, screening techniques, and lipid accumulation factors of oleaginous bacteria, in addition to the overall general biodiesel production process. This review also highlights the biotechnological approach for oleaginous bacteria strain improvement that can be future used for biodiesel production and the advantages of using general biodiesel in place of conventional fuel, along with the discussion about global policies and the prospect that promotes biodiesel production from oleaginous bacteria.
Graphical Abstract
Methanotrophs capable of converting C1-based substrates play an important role in the global carbon cycle. As one of the essential macronutrient components in the medium, the uptake of nitrogen sources severely regulates the cell’s metabolism. Although the feasibility of utilizing nitrogen gas (N2) by methanotrophs has been predicted, the mechanism remains unclear. Herein, the regulation of nitrogen fixation by an essential nitrogen-fixing regulator (NifA) was explored based on transcriptomic analyses of Methylomicrobium buryatense 5GB1. A deletion mutant of the nitrogen global regulator NifA was constructed, and the growth of M. buryatense 5GB1ΔnifA exhibited significant growth inhibition compared with wild-type strain after the depletion of nitrate source in the medium. Our transcriptome analyses elucidated that 22.0% of the genome was affected in expression by NifA in M. buryatense 5GB1. Besides genes associated with nitrogen assimilation such as nitrogenase structural genes, genes related to cofactor biosynthesis, electron transport, and post-transcriptional modification were significantly upregulated in the presence of NifA to enhance N2 fixation; other genes related to carbon metabolism, energy metabolism, membrane transport, and cell motility were strongly modulated by NifA to facilitate cell metabolisms. This study not only lays a comprehensive understanding of the physiological characteristics and nitrogen metabolism of methanotrophs, but also provides a potentially efficient strategy to achieve carbon and nitrogen co-utilization.
Key points
• N2 fixation ability of M. buryatense 5GB1 was demonstrated for the first time in experiments by regulating the supply of N2.
• NifA positively regulates nif-related genes to facilitate the uptake of N2 in M. buryatense 5GB1.
• NifA regulates a broad range of cellular functions beyond nif genes in M. buryatense 5GB1.
Methane is an abundant and low-cost gas with high global warming potential and its use as a feedstock can help mitigate climate change. Variety of valuable products can be produced from methane by methanotrophs in gas fermentation processes. By using methane as a sole carbon source, methanotrophic bacteria can produce bioplastics, biofuels, feed additives, ectoine and variety of other high-value chemical compounds. A lot of studies have been conducted through the years for natural methanotrophs and engineered strains as well as methanotrophic consortia. These have focused on increasing yields of native products as well as proof of concept for the synthesis of new range of chemicals by metabolic engineering. This review shows trends in the research on key methanotrophic bioproducts since 2015. Despite certain limitations of the known production strategies that makes commercialization of methane-based products challenging, there is currently much attention placed on the promising further development.
Microalgae can be used as a source of alternative food, animal feed, biofuel, fertilizer, cosmetics, nutraceuticals and for pharmaceutical purposes. The extraction of organic constituents from microalgae cultivated in the different nutrient compositions is influenced by microalgal growth rates, biomass yield and nutritional content in terms of lipid and fatty acid production. In this context, nutrient composition plays an important role in microalgae cultivation, and depletion and excessive sources of this nutrient might affect the quality of biomass. Investigation on the role of nitrogen and phosphorus, which are crucial for the growth of algae, has been addressed. However, there are challenges for enhancing nutrient utilization efficiently for large scale microalgae cultivation. Hence, this study aims to highlight the level of nitrogen and phosphorus required for microalgae cultivation and focuses on the benefits of nitrogen and phosphorus for increasing biomass productivity of microalgae for improved lipid and fatty acid quantities. Furthermore, the suitable extraction methods that can be used to utilize lipid and fatty acids from microalgae for biofuel have also been reviewed.
Some species belonging to Rhodococcus genus, such as R. opacus , R. jostii or R. wratislaviensis , are known to be oleaginous microorganisms, since they are able to accumulate more than 20% of triacylglycerols (TAG) of their cellular weight. Oleaginous rhodococci are promising microbial cell factories for the production of lipids to be used as fuels and chemicals. Cells could be engineered to create strains capable of producing high quantities of oils from industrial wastes and a variety of high-value lipids. The comprehensive understanding of carbon metabolism and its regulation will contribute to the design of a reliable process for bacterial oil production. Bacterial oleagenicity requires an integral configuration of metabolism and regulatory processes rather than the sole existence of an efficient lipid biosynthesis pathway. During the last years, several studies have been focused on basic aspects of TAG biosynthesis and accumulation using R. opacus PD630 and R. jostii RHA1 strains as models of oleaginous bacteria. The combination of results obtained in these studies allows us to propose a metabolic landscape for oleaginous rhodococci. In this context, this article provides a comprehensive and integrative view of different metabolic and regulatory attributes and innovations that explain the extraordinary ability of these bacteria to synthesize and accumulate TAG. We hope that the accessibility to such information in an integrated way will help researchers to rationally select new targets for further studies in the field.
Different microalgae species produce varying quantity and quality of the lipids. Fatty acid methyl ester composition, which comprises both saturated and unsaturated contents, critically affects biodiesel properties. Current study compares six locally isolated microalgae strains belonging to three classes (Trebouxiophyceae, Chlorophyceae, and Cyanophyceae) on the basis of lipid content and biodiesel properties. All the six species are grown in similar condition up to the late stationary phase, and their lipid content and fatty acid methyl ester composition are measured experimentally. Multi-criteria decision analysis (MCDA) tool has ranked Calothrix species (class Cyanophyceae) on the top, owing to better cetane number, density and oxidation stability; whereas Chlorococcum species (class Chlorophyceae) is ranked second because of its higher lipid content, better cold flow property, and low viscosity. Property analysis of these two species is extended in the enlarge temperature range for five properties, vapor pressure, latent heat of vaporization, liquid density, liquid viscosity and vapor diffusivity, which are important in spray and combustion modeling. It is found through detailed property estimation that Chlorococcum sp. is a more suitable species in comparison with Calothrix sp. as it is having better properties and its lipid content is much higher than that of Calothrix sp. Although the properties of microalgae biodiesel are poorer in comparison with conventional diesel fuel, a greater number of such studies will help in understanding the requisite changes as required for microalgae biodiesel–based engine and their properties as compared with conventional diesel.
Background
Triacylglycerols (TAGs) rich in medium-chain fatty acids (MCFAs, C10–14 fatty acids) are valuable feedstocks for biofuels and chemicals. Natural sources of TAGs rich in MCFAs are restricted to a limited number of plant species, which are unsuitable for mass agronomic production. Instead, the modification of seed or non-seed tissue oils to increase MCFA content has been investigated. In addition, microbial oils are considered as promising sustainable feedstocks for providing TAGs, although little has been done to tailor the fatty acids in microbial TAGs. ResultsHere, we first assessed various wax synthase/acyl-coenzyme A:diacylglycerol acyltransferases, phosphatidic acid phosphatases, acyl-CoA synthetases as well as putative fatty acid metabolism regulators for producing high levels of TAGs in Escherichia coli. Activation of endogenous free fatty acids with tailored chain length via overexpression of the castor thioesterase RcFatB and the subsequent incorporation of such fatty acids into glycerol backbones shifted the TAG profile in the desired way. Metabolic and nutrient optimization of the engineered bacterial cells resulted in greatly elevated TAG levels (399.4 mg/L) with 43.8% MCFAs, representing the highest TAG levels in E. coli under shake flask conditions. Engineered cells were observed to contain membrane-bound yet robust lipid droplets. Conclusions
We introduced a complete Kennedy pathway into non-oleaginous E. coli towards developing a bacterial platform for the sustainable production of TAGs rich in MCFAs. Strategies reported here illustrate the possibility of prokaryotic cell factories for the efficient production of TAGs rich in MCFAs.
Methane, the primary component of natural gas, is the second most abundant greenhouse gas (GHG) and contributes significantly to climate change. The conversion of methane to industrial platform chemicals provides an attractive opportunity to decrease GHG emissions and utilize this inexpensive and abundantly available gas as a carbon feedstock. While technologies exist for chemical conversion of methane to liquid fuels, the technical complexity of these processes mandate high capital expenditure, large-scale commercial facilities to leverage economies of scale that cannot be efficiently scaled down. Alternatively, bioconversion technologies capable of efficient small-scale operation with high carbon and energy efficiency can enable deployment at remote methane resources inaccessible to current chemical technologies. Aerobic obligate methanotrophs, specifically Methylomicrobium buryatense 5GB1, have recently garnered increased research interest for development of such bio-technologies. In this study, we demonstrate production of C-4 carboxylic acids non-native to the host, specifically crotonic and butyric acids, from methane in an engineered M. buryatense 5GB1C by diversion of carbon flux through the acetyl-CoA node of central 'sugar' linked metabolic pathways using reverse β-oxidation pathway genes. The synthesis of short chain carboxylic acids through the acetyl-CoA node demonstrates the potential for engineering M. buryatense 5GB1 as a platform for bioconversion of methane to a number of value added industrial chemicals, and presents new opportunities for further diversifying the products obtainable from methane as the feedstock.
Background
Due to the success of shale gas development in the US, the production cost of natural gas has been reduced significantly, which in turn has made methane (CH4), the major component of natural gas, a potential alternative substrate for bioconversion processes compared with other high-price raw material sources or edible feedstocks. Therefore, exploring effective ways to use CH4 for the production of biofuels is attractive. Biological fixation of CH4 by methanotrophic bacteria capable of using CH4 as their sole carbon and energy source has obtained great attention for biofuel production from this resource. ResultsIn this study, a fast-growing and lipid-rich methanotroph, Methylomicrobium buryatense 5GB1 and its glycogen-knock-out mutant (AP18) were investigated for the production of lipids derived from intracellular membranes, which are key precursors for the production of green diesel. The effects of culture conditions on cell growth and lipid production were investigated in high cell density cultivation with continuous feeding of CH4 and O2. The highest dry cell weight observed was 21.4 g/L and the maximum lipid productivity observed was 45.4 mg/L/h obtained in batch cultures, which corresponds to a 2-fold enhancement in cell density and 3-fold improvement in lipid production, compared with previous reported data from cultures of 5GB1. A 90% enhancement of lipid content was achieved by limiting the biosynthesis of glycogen in strain AP18. Increased CH4/O2 uptake and CO2 evaluation rates were observed in AP18 cultures suggesting that more carbon substrate and energy are needed for AP18 growth while producing lipids. The lipid produced by M. buryatense was estimated to have a cetane number of 75, which is 50% higher than biofuel standards requested by US and EU. Conclusions
Cell growth and lipid production were significantly influenced by culture conditions for both 5GB1 and AP18. Enhanced lipid production in terms of titer, productivity, and content was achieved under high cell density culture conditions by blocking glycogen accumulation as a carbon sink in the strain AP18. Differences observed in CH4/O2 gas uptake and CO2 evolution rates as well as cell growth and glycogen accumulation between 5GB1 and AP18 suggest changes in the metabolic network between these strains. This bioconversion process provides a promising opportunity to transform CH4 into biofuel molecules and encourages further investigation to elucidate the remarkable CH4 biofixation mechanism used by these bacteria.
Oleaginous microorganisms represent possible platforms for the sustainable production of oleochemicals and biofuels due to their metabolic robustness and the possibility to be engineered. Streptomyces coelicolor is among the narrow group of prokaryotes capable of accumulating triacylglycerol (TAG) as carbon and energy reserve. Although the pathways for TAG biosynthesis in this organism have been widely addressed, the set of genes required for their breakdown have remained elusive so far. Here, we identified and characterized three gene clusters involved in the β-oxidation of fatty acids (FA). The role of each of the three different S. coelicolor FadAB proteins in FA catabolism was confirmed by complementation of an Escherichia coliΔfadBA mutant strain deficient in β-oxidation. In S. coelicolor, the expression profile of the three gene clusters showed variation related with the stage of growth and the presence of FA in media. Flux balance analyses using a corrected version of the current S. coelicolor metabolic model containing detailed TAG biosynthesis reactions suggested the relevance of the identified fadAB genes in the accumulation of TAG. Thus, through the construction and analysis of fadAB knockout mutant strains, we obtained an S. coelicolor mutant that showed a 4.3-fold increase in the TAG content compared to the wild type strain grown under the same culture conditions.
In light of the availability of low-cost methane (CH4) derived from natural gas and biogas along with increasing concerns of the greenhouse gas emissions, the production of alternative liquid biofuels directly from CH4 is a promising approach to capturing wasted energy. A novel biorefinery concept integrating biological conversion of CH4 to microbial lipids together with lipid extraction and generation of hydrocarbon fuels is demonstrated in this study for the first time. An aerobic methanotrophic bacterium, Methylomicrobium buryatense capable of using CH4 as the sole carbon source was selected on the basis of genetic tractability, cultivation robustness, and ability to accumulate phospholipids in membranes. A maximum fatty acid content of 10% of dry cell weight was obtained in batch cultures grown in a continuous gas sparging fermentation system. Although phospholipids are not typically considered as a good feedstock for upgrading to hydrocarbon fuels, we set out to demonstrate that using a combination of novel lipid extraction methodology with advanced catalyst design, we could prove the feasibility of this approach. Up to 95% of the total fatty acids from membrane-bound phospholipids were recovered by a two-stage pretreatment method followed by hexane extraction of the aqueous hydrolysate. The upgrading of extracted lipids was then demonstrated in a hydrodeoxygeation process using palladium on silica as a catalyst. Lipid conversion in excess of 99% was achieved, with a full selectivity to hydrocarbons. The final hydrocarbon mixture is dominated by 88% pentadecane (C15H32) based on decarbonylation/decarboxylation and hydrogenation of C16 fatty acids, indicating that a biological gas-to-liquid fuel (Bio-GTL) process is technically feasible.
Background
Construction of microbial biocatalysts for the production of biorenewables at economically viable yields and titers is frequently hampered by product toxicity. Membrane damage is often deemed as the principal mechanism of this toxicity, particularly in regards to decreased membrane integrity. Previous studies have attempted to engineer the membrane with the goal of increasing membrane integrity. However, most of these works focused on engineering of phospholipids and efforts to identify membrane proteins that can be targeted to improve fatty acid production have been unsuccessful. ResultsHere we show that deletion of outer membrane protein ompF significantly increased membrane integrity, fatty acid tolerance and fatty acid production, possibly due to prevention of re-entry of short chain fatty acids. In contrast, deletion of fadL resulted in significantly decreased membrane integrity and fatty acid production. Consistently, increased expression of fadL remarkably increased membrane integrity and fatty acid tolerance while also increasing the final fatty acid titer. This 34% increase in the final fatty acid titer was possibly due to increased membrane lipid biosynthesis. Tuning of fadL expression showed that there is a positive relationship between fadL abundance and fatty acid production. Combinatorial deletion of ompF and increased expression of fadL were found to have an additive role in increasing membrane integrity, and was associated with a 53% increase the fatty acid titer, to 2.3 g/L. Conclusions
These results emphasize the importance of membrane proteins for maintaining membrane integrity and production of biorenewables, such as fatty acids, which expands the targets for membrane engineering.
Methane utilization by methanotrophic bacteria is an attractive application for biotechnological conversion of natural or biogas into high-added-value products. Haloalcaliphilic methanotrophic bacteria belonging to the genus Methylomicrobium are among the most promising strains for methane-based biotechnology, providing easy and inexpensive cultivation, rapid growth, and the availability of established genetic tools. A number of methane bioconversions using these microbial cultures have been discussed, including the derivation of biodiesel, alkanes, and OMEGA-3 supplements. These compounds are derived from bacterial fatty acid pools. Here, we investigate fatty acid biosynthesis in Methylomicrobium buryatense 5G(B1). Most of the genes homologous to typical Type II fatty acid biosynthesis pathways could be annotated by bioinformatics analyses, with the exception of fatty acid transport and regulatory elements. Different approaches for improving fatty acid accumulation were investigated. These studies indicated that both fatty acid degradation and acetyl- and malonyl-CoA levels are bottlenecks for higher level fatty acid production. The best strain generated in this study synthesizes 111 ± 2 mg/gDCW of extractable fatty acids, which is ~20% more than the original strain. A candidate gene for fatty acid biosynthesis regulation, farE, was identified and studied. Its deletion resulted in drastic changes to the fatty acid profile, leading to an increased pool of C18-fatty acid methyl ester. The FarE-regulon was further investigated by RNA-seq analysis of gene expression in farE-knockout mutants and farE-overexpressing strains. These gene profiles highlighted a novel set of enzymes and regulators involved in fatty acid biosynthesis. The gene expression and fatty acid profiles of the different farE-strains support the hypothesis that metabolic fluxes upstream of fatty acid biosynthesis restrict fatty acid production in the methanotroph.
Microbially produced lipids like triacylglycerols or fatty acid ethyl esters are currently of great interest as fuel replacements or other industrially relevant compounds. They can even be produced by non-oleaginous microbes, like Escherichia coli, upon metabolic engineering. However, there is still much room for improvement regarding the yield for a competitive microbial production of lipids or biofuels. We genetically engineered E. coli by expressing fadD, fadR, pgpB, plsB and'tesA in combination with atfA from Acinetobacter baylyi. A total fatty acid contents of up to 16% (w/w) was obtained on complex media, corresponding to approximately 9% (w/w) triacylglycerols and representing the highest titers of fatty acids and triacylglycerols obtained in E. coli under comparable cultivation conditions, so far. To evaluate further possibilities for an optimization of lipid production, ten promising bacterial wax ester synthase/acyl-Coenzyme A:diacylglycerol acyltransferases were tested and compared. While highest triacylglycerol storage was achieved with AtfA, the mutated variant AtfA-G355I turned out to be most suitable for fatty acid ethyl ester biosynthesis and enabled an accumulation of approx. 500mg/L without external ethanol supplementation.
Microbial lipid production represents a potential alternative feedstock for the biofuel and oleochemical industries. Since Escherichia coli exhibits many genetic, technical, and biotechnological advantages over native oleaginous bacteria, we aimed to construct a metabolically engineered E. coli strain capable of accumulating high levels of triacylglycerol (TAG) and evaluate its neutral lipid productivity during high cell density fed-batch fermentations.
The Streptomyces coelicolor TAG biosynthesis pathway, defined by the acyl-CoA:diacylglycerol acyltransferase (DGAT) Sco0958 and the phosphatidic acid phosphatase (PAP) Lppβ, was successfully reconstructed in an E. coli diacylglycerol kinase (dgkA) mutant strain. TAG production in this genetic background was optimized by increasing the levels of the TAG precursors, diacylglycerol and long-chain acyl-CoAs. For this we carried out a series of stepwise optimizations of the chassis by 1) fine-tuning the expression of the heterologous SCO0958 and lppβ genes, 2) overexpression of the S. coelicolor acetyl-CoA carboxylase complex, and 3) mutation of fadE, the gene encoding for the acyl-CoA dehydrogenase that catalyzes the first step of the β-oxidation cycle in E. coli. The best producing strain, MPS13/pET28-0958-ACC/pBAD-LPPβ rendered a cellular content of 4.85% cell dry weight (CDW) TAG in batch cultivation. Process optimization of fed-batch fermentation in a 1-L stirred-tank bioreactor resulted in cultures with an OD600nm of 80 and a product titer of 722.1 mg TAG L(-1) at the end of the process.
This study represents the highest reported fed-batch productivity of TAG reached by a model non-oleaginous bacterium. The organism used as a platform was an E. coli BL21 derivative strain containing a deletion in the dgkA gene and containing the TAG biosynthesis genes from S. coelicolor. The genetic studies carried out with this strain indicate that diacylglycerol (DAG) availability appears to be one of the main limiting factors to achieve higher yields of the storage compound. Therefore, in order to develop a competitive process for neutral lipid production in E. coli, it is still necessary to better understand the native regulation of the carbon flow metabolism of this organism, and in particular, to improve the levels of DAG biosynthesis.
Aerobic methanotrophs oxidize methane at ambient temperatures and pressures, and are therefore attractive systems for methane-based bioconversions. In this work we developed and validated genetic tools for Methylomicrobium buryatense, a haloalkaliphilic gammaproteobacterial (type I) methanotroph. M. buryatense was isolated directly on natural gas and grows robustly in pure culture with a three hour doubling time, enabling rapid genetic manipulation compared to many other methanotrophic species. As a proof of concept we used a sucrose counterselection system to eliminate glycogen production in M. buryatense by constructing unmarked deletions in two redundant glycogen synthase genes. We also selected for a more genetically tractable variant strain that can be conjugated with small IncP-based broad host range vectors, and determined that this capability is due to loss of the native plasmid. These tools make M. buryatense a promising model system for studying aerobic methanotroph physiology and enable metabolic engineering in this bacterium for industrial biocatalysis of methane.
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Robust growth of the gammaproteobacterium Methylomicrobium buryatense strain 5G on methane makes it an attractive system for CH4-based biocatalysis. Here we present a draft genome sequence of the strain that will provide a valuable framework for metabolic
engineering of the core pathways for the production of valuable chemicals from methane.
From the view of a circular economy, the bioconversion of methane into cell protein and carbohydrates could provide alternative food resources while cutting greenhouse gases, considering renewable gases from anaerobic...
The development of biorefineries for a sustainable bioeconomy has been driven by the concept of utilizing environmentally friendly and cost-effective renewable energy sources. Methanotrophic bacteria with a unique capacity to utilize methane as a carbon and energy source can serve as outstanding biocatalysts to develop C1 bioconversion technology. By establishing the utilization of diverse multi-carbon sources, integrated biorefinery platforms can be created for the concept of the circular bioeconomy. An understanding of physiology and metabolism could help to overcome challenges for biomanufacturing. This review summaries fundamental gaps for methane oxidation and the capability to utilize multi-carbon sources in methanotrophic bacteria. Subsequently, breakthroughs and challenges in harnessing methanotrophs as robust microbial chassis for industrial biotechnology were compiled and overviewed. Finally, capabilities to exploit the inherent advantages of methanotrophs to synthesize various target products in higher titers are proposed.
Metabolic engineering aims to improve the production of economically valuable molecules through the genetic manipulation of microbial metabolism. While the discipline is a little over 30 years old, advancements in metabolic engineering have given way to industrial-level molecule production benefitting multiple industries such as chemical, agriculture, food, pharmaceutical, and energy industries. This review describes the design, build, test, and learn steps necessary for leading a successful metabolic engineering campaign. Moreover, we highlight major applications of metabolic engineering, including synthesizing chemicals and fuels, broadening substrate utilization, and improving host robustness with a focus on specific case studies. Finally, we conclude with a discussion on perspectives and future challenges related to metabolic engineering.
C1 gaseous substrates (CH4, CO2, and CO) derived from natural gas, biogas, and syngas, are of interest due to their threats to the environment or inefficient utilization. Benefiting from advanced genetic editing tools and bioconversion strategies, metabolically engineered C1-gas-utilizing microorganisms (CGUM), such as methanotrophs, cyanobacteria, and acetogens, are capable of utilizing C1 gaseous feedstocks as the sole substrates for cell growth and synthesis of chemicals and biofuels. In this paper, we critically review metabolic pathways related to the assimilation of C1 gaseous substrates for alcohol biosynthesis in several model CGUM. Metabolic engineering approaches utilized to enhance the carbon conversion efficiency, microbial growth and biosynthesis of desired alcohols are summarized, including the regulation of C1 gaseous substrates activation and electron and energy supply, the accumulation of key intermediates, and the manipulation of target gene expression to optimize carbon flux to bioalcohols. In addition, challenges in the efficient microbial conversion of C1 gaseous substrates are explored and discussed. The strategies of bioalcohol biosynthesis presented here could guide the development of a variety of efficient biological routes for CH4, CO2, and CO utilization in the future.
Cetane number (CN) is one of the most important parameters affecting diesel fuel behavior. It is related to the time that elapses between fuel injection and beginning of combustion. To determine it, a combustion standardized test, under ASTM D613 regulation, needs to be carried out. As this process is limiting, since it requires a specific engine, CN can be approximated from the distillation temperatures of diesel fuels, providing another dimensionless parameter, named calculated cetane index (CCI). It also provides an approach to the explosion delay. ASTM D4737 standard is the regulation that allows its calculation. It is based on a four-term equation that may be applied to oil-derived products. Chemical composition of diesel fuel is composed exclusively of hydrocarbons of different nature. However, biodiesel is mainly composed of alkyl esters and, to a lesser extent, glycerides and glycerol impurities. This difference makes convenient to adapt the equation to biodiesel distillation curve. The aim of this work is to provide an alternative to CN calculation for biodiesel, based on CCI prediction. This manuscript presents distillation curves and density values of 14 different biodiesel types, from both animal and vegetable origin. This data, together with data from literature, have been used with non-linear equation modeling techniques. As a result, a modification of ASTM D4737 regulation with experimental data from biodiesel sources has been proposed. The proposed equation exhibits an error with respect to experimental data below 3.5% for most evaluated cases. Subsequently, it may be concluded that the proposed methodology may be applied to accurately calculate biodiesel CCI, as a substitute of CN.
Nowadays, biological mitigation of carbon dioxide (CO2) by means of microalgae is one of the promising approaches, as biomass can be used to produce renewable fuels such as biodiesel and crude bio-oil. In this study, the growth of Desmodesmus sp. in response to different CO2 levels (ambient air, 5%, 10%, 15%, 20%, 25%, 30%, and 35% CO2) was studied, and then the suitability of the biomass for bio-oil and biodiesel production was evaluated. The studied strain was able to grow efficiently in a broad range of elevated CO2 (5–30%, v/v), suggesting its suitability for CO2 mitigation. The highest specific growth rate (0.769 d⁻¹) and biomass productivity (0.033 g L⁻¹ d⁻¹) were achieved at 15% CO2; therefore, the concentration of 15% CO2 was selected as the optimum. Alpha-Linolenic acid (ALA) was the dominant polyunsaturated fatty acids (PUFAs) in studied Desmodesmus strain, and its content increased from 31.82% under ambient air to 41.94% and 50.11% under 15% CO and 30% CO2, respectively, suggesting a key role of ALA in response to CO2 elevation. Using fatty acid methyl ester (FAME) profiles of cultures in different CO2 conditions, biodiesel parameters such as cetane number (CN), saponification value (SV), iodine value (IV), long chain saturation factor (LCSF), oxidative stability (OS), higher heating value (HHV) and degree of unsaturation (DU) were estimated. Overall, the lipid extract was not suitable for biodiesel production due to high PUFA content ranging between 52.91% and 71.14%. Under optimum CO2 (15%), saturated fatty acids (SFAs) content increased to 31.91% and PUFA content reduced to 52.91%. This could significantly improve the CN to 38.07, which was 11% and 13% higher than the air and 30% CO2. Pyrolysis gas chromatography-mass spectrometry (Py-GC-MS) was used to analyze the potential of microalgal biomass for bio-oil production upon CO2 changes. The pyrolysate contained 40–47% acid content due to the high content of fatty acids. The pyrolysate quality declined under 15% CO2 due to the higher accumulation of acidic and nitrogen-containing compounds and reduced hydrocarbon proportion. In conclusion, the optimum CO2 could enhance biodiesel parameters such as CN, but demonstrated negative effects on bio-oil quality.
The present nitrogen fixation industry is usually energy-intensive and environmentally detrimental. Therefore, it is appealing to find alternatives. Here, we achieved both a synchronized biological nitrogen fixation and electric energy production by using Geobacter sulfurreducens in a microbial electrochemical system. The results showed that G. sulfurreducens was able to fix nitrogen depending on anode respiration, producing a maximum current density of 0.17 ± 0.015 mA cm⁻² and a nitrogen-fixing activity of ca. 0.78 μmol C2H4 mg protein⁻¹ h⁻¹, thereby achieving a net total nitrogen-fixing rate of ca. 5.6 mg L⁻¹ day⁻¹. Specifically, nitrogen fixation did not impair coulombic efficiency. Transcriptomic and metabolic analyses demonstrated that anode respiration provided sufficient energy to drive nitrogen fixation, and in turn nitrogen fixation promoted anode respiration of the cell by increasing acetate catabolism but reducing acetate anabolism. Furthermore, we showed that G. sulfurreducens could be supplied in a bioelectrochemical system for N-deficient wastewater treatment to relieve N-deficiency stress contributing to the formation of an electroactive biofilm, thereby simultaneously achieving nitrogen fixation, current generation and dissoluble organic carbon removal. Our study revealed a synergistic effect between biological nitrogen fixation and current generation by G. sulfurreducens, providing a green nitrogen fixation alternative through shifting the nitrogen fixation field from energy consumption to energy production and having implications for N-deficient wastewater treatment.
Microbial oils have become a research hotspot in alleviating energy challenges and environmental problems because of their potential to be green alternatives of traditional fossil fuels. Yarrowia lipolytica is a promising oleaginous yeast that can utilize various, especially low-cost carbon sources to synthesize considerable lipids more than 30% of dry cell weight, which is attracting researchers’ attention. Based on well understanding of its lipid synthesis and metabolism mechanism, various optimization approaches have been studied dispersedly to improve the lipid synthesis and realize the industrial-scale application of Y. lipolytica. Some approaches are focused on improving and optimizing culture conditions, such as temperature, pH value, and rotating speed, etc. Other approaches are dedicated to the optimization of nutrient elements, such as carbon source, nitrogen source type and/or concentration, and C/N ratio, etc. Some adjust the cultivation mode to facilitate nutrient assimilation and transformation, and others use genetic engineering to modify this yeast. This review focuses on the comprehensive and detailed analyses of feasible enhancement approaches for lipid synthesis. Some prospects will also be introduced as references for further study.
Aviation sector discharges approximately 2% of the global anthropogenic CO 2, and the proportion is growing. The search for cost-effective and environmental-friendly bio-jet fuels derived from natural resources is gaining momentum. The microalgae cultivation conditions including temperature, pH, light intensity and nutrients have shown significant influence on the microalgae growth rate and chemical composition, which create the opportunities to enhance the yield and quality of microalgae bio-jet fuel. This review is focused on the hydroprocessing method for converting microalgae oil into bio-jet fuel, as well as the novel conceptual approaches for bio-jet fuel production such as gasification with Fischer-Tropsch and sugar-to-jet. Fischer-Tropsch synthesis of biomass is one of the best alternative ways to replace natural aviation fuel due to the high maximum energy efficiency and low emission of greenhouse gas. In addition, hydroprocessing with the aid of Ni and zeolites catalysts has successfully converted the microalgae biodiesel to bio-jet fuel with high yield and alkane selectivity. Among these techniques, hydroprocessing used the lowest production cost with the longest duration, whereas the bio-jet fuel with high selectivity (C 8-C 16) could be produced by using gasification with the Fischer-Tropsch process. Consequently, gasification and Fischer-Tropsch and sugar-to-jet can become the future alternative process to convert microalgae to bio-jet fuel. The development of microalgae bio-jet fuel will increase the security of energy supply and reduce the fuel expenses in aviation industry.
Nowadays, concerns about rising emissions and climate change have raised the issue of decarbonization. Several approaches have been promoted in the aeronautical sector to reduce CO2 emissions. The present work provides quantitative data to support decision-making for the first pillar of International Air Transport Association (IATA) strategy to mitigate aviation climate impact. This strategy comprises improving aircraft technology and deploying sustainable low-carbon fuels. The most promising technologies for an imminent application are new engine architecture and natural laminar flow. On the other hand, efforts have been put to produce Sustainable Aviation Fuel (SAF) reaching the point where some methods for the production of alternative jet fuel are already approved by ASTM. Therefore, the present work quantifies the future reduction of CO2 emissions by 2050 in the aeronautical sector with these strategies. For this purpose, two methodologies are used, a numerical model to calculate fuel consumption and CO2 emissions from the global air transport fleet. For the SAF analysis, it is developed an approach that considers, besides the SAF production, the feedstocks, and the production pathway. Two cases and three scenarios represent the technological improvements and quantify the effects of new aircraft concepts and technologies on future CO2 emissions. For the SAF analysis, four scenarios and two conditions assess the different production capacities and feedstocks. The combined effect of technologies with SAF is considered verifying if the goals proposed by IATA, carbon-neutral growth from 2020, and a reduction of 50% in net emissions by 2050 compared to 2005 levels are achieved. The assessment results reveal that the goals cannot be met only with the combined action of imminent aircraft technologies and the use of alternative fuels. Carbon-neutral growth is only reached when it is considered the combined effect of technologies with the scenario where the amount of SAF introduced is higher (an increase of 15% annually between 2030 and 2050). However, this carbon-neutral growth is only possible to start in 2040. Imminent aircraft technologies can reduce up to 15% in CO2 emissions when compared to the Business as Usual scenario. The different feedstocks used in each process to produce SAF do not have a considerable impact on reducing CO2 emissions, the maximum difference registered between each condition was 1.47%.
Microalgal lipids are highly promising feedstocks for biofuel production. Microalgal lipids, especially triacylglycerol, and practical applications of these compounds have received increasing attention in recent years. For the commercial use of microalgal lipids to be feasible, many fundamental biological questions must be addressed based on detailed studies of algal biology, including how lipid biosynthesis occurs and is regulated. Here, we review the current understanding of microalgal lipid biosynthesis, with a focus on the underlying regulatory mechanisms. We also present possible solutions for overcoming various obstacles to understanding the basic biology of microalgal lipid biosynthesis and the practical application of microalgae-based lipids. This review will provide a theoretical reference for both algal researchers and decision makers regarding the future directions of microalgal research, particularly pertaining to microalgal-based lipid biosynthesis.
Microalgae are potential sources of high-value lipids, with essential fatty acids that provide health benefits, as the omega-3 polyunsaturated fatty acids. However, its cultivation and downstream processing is still not commercially viable for some applications due to high-water consumption and high costs mainly regarding energy demands and nutrients as nitrogen. Therefore, using waste streams in existing industries as carbon and nutrient sources, as well as evaluating the best methodologies for growth and lipid extraction are essential to viabilize this process. This review focused on the study of scenario the of using different microalgae species, integrating their cultivation into biorefineries using their wastewater and carbon dioxide combating water and air pollution, aiming lipid productivity and fatty acid profile with specific composition. It was found that culture medium conditions and cultivation systems are key elements in understanding the lipid production and can decisively affect the process performance. For example, closed photobioreactors with CO 2 supply and light can provide higher photosynthetic efficiency and lipid accumulation, coupled with polyunsaturated fatty acid production. Wastewater use can reduce productivity and affect lipid composition, but CO 2 injection can promote both higher biomass and lipid productivities; being Chlorella a potential candidate for implementation in industrial facilities once it showed high PUFA (around 1/3) and lipid content, up to 27%, grown in wastewater. Moreover, it is crucial to seek biomass fractioning to obtain different high-value products that will compensate for high capital and operating costs. Further evaluation of possible effects in the final product quality is required.
Deoxygenation and molecular structural modification is a critical step before the practical application of triglycerides in aviation biofuels. For low quality waste triglycerides with high viscosity, traditional way of direct hydrogenation usually has problems such as catalyst deactivation and pipe clogging. Herein, we demonstrate a facile catalytic biorefinery process for high viscosity waste triglycerides for the production of aviation biofuels via a two-step catalytic conversion. Several typical plant oils (soybean and rubber seed oils) and waste triglycerides (waste cooking and acidified plant oils) are tested, which indicate that this approach is widely applicable, and can be used to produce aviation range products, linear hydrocarbons, at high yields (55%–60%), accompanied by a diesel range fuel of 15%–40% by adjusting the operation parameters. The aviation range hydrocarbons are further catalyzed by an Ni/ZSM-5 catalyst to achieve aromatization and hydrogenation in a one-pot synthesis. Three dominant components existing in aviation fuels, including chain hydrocarbons, aromatics and cycloalkanes, can be obtained, which present the same physical-chemical properties and components as petroleum-based aviation fuels. The proportion of the three main components can be modified by the amount of molecular sieve catalyst and the initial pressure of H 2 , in order to meet the requirements of variable-grade aviation fuels. Overall, a catalytic refining process of decarboxylation and molecular structural modification of low quality waste triglycerides for the entire/total substitution of petroleum aviation fuels from waste triglyceride is reported.
We previously published a comprehensive review paper reviewing the Raman spectroscopy of biological molecules. This research area has expanded rapidly, which warranted an update to the existing review paper by adding the recently reported studies in literature. This article reviews some of the recent advances of Raman spectroscopy in relation to biomedical applications starting from natural tissues to cancer biology. Raman spectroscopy, an optical molecular detective, is a vibrational spectroscopic technique that has potential not only in cancer diagnosis but also in understanding progression of the disease. This article summarizes some of the most widely observed peak frequencies and their assignments. The aim of this review is to develop a database of molecular fingerprints, which will facilitate researchers in identifying the chemical structure of the biological tissues including most of the significant peaks reported both in the normal and cancerous tissues. It has covered a variety of Raman approaches and its quantitative and qualitative biochemical information. In addition, it covers the use of Raman spectroscopy to analyse a variety of different malignancies including breast, brain, cervical, gastrointestinal, lung, oral, and skin cancer. Multivariate analysis approaches used in these studies have also been covered.
Keywords: Raman spectroscopy, biological tissues, analysis of cancer tissues, characteristic peak assignments
The U.S. Energy Information Administration (EIA) projects that U.S. natural gas prices will provide a substantial cost advantage over petroleum products as a transportation fuel during the next 30+ years. Although U.S. natural gas prices closely tracked those of world oil prices from the mid-1990s to early 2009, U.S. natural gas prices have been much lower relative to those for oil since early 2009. The break owes to technological change that substantially increased the supply of U.S. shale gas resources.
Although recent weakness in world oil prices has restored at least some temporary comparability between U.S. natural gas prices and those for petroleum products, EIA projects U.S. natural gas prices will rise more slowly as world oil prices recover. Relatively weaker gains in U.S. natural gas prices would support the substitution of natural gas for other fuels—such as petroleum products in transportation—whether such substitution is driven by business or policy decisions. Will U.S. natural gas prices be sustainably lower relative to those for petroleum products so as to support the increased use of natural gas in the transportation sector? An interpretative review of current market developments and recent research finds the answer is yes.
The bioconversion of one-carbon compounds into high-value products is importance in the energy and chemical fields. In this study, two one-pot multi-enzyme reaction systems were constructed using known formolase and aldolases to synthesise stereodefined functional sugars directly from prebiotic compound formaldehyde. A stepwise cascade biocatalysis approach was employed, and it decreased byproduct formation and increased the conversion rate of L-erythrulose from 85% to 91% and that of L-sorbose form 0 to 68%. This approach was also employed in whole-cell transformation of low-carbon units, such as formaldehyde, glycolaldehyde, and dihydroxyacetone, and successfully produced 7.56 g/L L-sorbose. Whole-cell transformation of formaldehyde and dihydroxyacetone synthesised 252 g/L (2.21 M) L-erythrulose with a productivity of 126 g.L−1.h−1, which is the highest level reported to date.
The aviation sector contributes with 2% of the total anthropogenic CO2 emissions, and predictions estimate that air traffic will double in the next 20 years, doubling fuel requirements and CO2 emissions. The International Air Transport Association (IATA) has identified the development of renewable aviation fuel, known as biojet fuel, as the most promising strategy to reduce the environmental impact of the aviation sector. The renewable hydrocarbons that constitute biojet fuel are also known as synthetic paraffinic kerosene (SPK), and their properties are almost identical to those of jet fuel. SPK has also the advantage of containing very little sulfur, producing lower CO2 emissions than jet fuel. The focus of this paper is to review the scientific and technological advances related to the existing pathways to produce biojet fuel, and to identify those that could lead to the future implementation of a sustainable production chain for renewable aviation fuel. The production process of biojet fuel is the key to satisfying both technical and economic goals required to obtain a more competitive biofuel, and allow the sustainable development of aviation sector.
Microdiesel obtained from microbes using renewable materials as carbon sources is an important alternative to petroleum diesel. This review provides information related to microdiesel production using various carbon sources; i.e. carbon dioxide, C2, saccharides, and lignocellulose. Microbes can accumulate different contents of fatty acids in the form of triacylglycerol (TAG). Not all microbes store fatty acids and utilize a broad range of substrates as carbon sources, and vice versa. Microbes can be engineered to consume various carbon sources, and accumulate increased amounts of fatty acids with different composition. The properties of microdiesel depend on its fatty acid profile, which in turn determines its efficacy. The structural features of the fatty acids, such as carbon chain length, branching and degree of unsaturation, affect the physiochemical properties of the biodiesel (cetane number (CN), oxidation stability (OS), iodine value (IV), cold flow properties, density and kinematic viscosity). Fatty acid methyl ester (FAME) profiles can be used to evaluate the key properties of biodiesel, i.e. the stability of the oil used. The overview presented herein concludes that microdiesel production using non-feed carbon sources and genetically engineered microbes shows much promise.
Over the last decade, there has been a huge upsurge of interest in sustainable production of biomass-based biofuels to fulfill the existing energy demand and simultaneously reducing the environmental deterioration. Earlier, vegetable oils and animal fats were utilized for biodiesel production, but due to food crisis and environmental sustainability, renewable sources such as neutral lipid derived from microbes are gaining much attention for budding biodiesel industries. Among various types of microorganisms, oleaginous yeasts are more promising feedstock to accomplish the current demand of biodiesel production and utilize a large number of cost-effective renewable substrates for their growth and lipid accumulation. However, biodiesel obtained from oleaginous yeasts have certain restrictions regarding their commercial utilization due to their unstable fuel properties such as oxidative stability, cetane number, viscosity and low-temperature performance etc. Numerous articles have been published in the public domain describing the fatty acid profiles of oleaginous yeast as feedstock for biodiesel production. However, the evaluation of quality parameters of biodiesel obtained from oleaginous yeasts is still in infancy. Although there is a huge disparity in a number of papers published for biodiesel production yet the reporting performance on diesel engines need to be verified in details. In this review article, attempt has been made to assess the important biofuel properties on the basis of the fatty acid profile of oleaginous yeast. Thus this evaluation would provide a guideline to the biodiesel producer to improve the production plans related to feedstocks for oleaginous yeast, culture conditions and biodiesel blending.
As oleaginous microorganisms represent an upcoming novel feedstock for the biotechnological production of lipids or lipid-derived biofuels, we searched for novel, lipid-producing strains in desert soil. This was encouraged by the hypothesis that neutral lipids represent an ideal storage compound, especially under arid conditions, as several animals are known to outlast long periods in absence of drinking water by metabolizing their body fat. Ten lipid-accumulating bacterial strains, affiliated to the genera Bacillus, Cupriavidus, Nocardia, Rhodococcus and Streptomyces, were isolated from arid desert soil due to their ability to synthesize poly(β-hydroxybutyrate), triacylglycerols or wax esters. Particularly two Streptomyces sp. strains and one Rhodococcus sp. strain accumulate significant amounts of TAG under storage conditions under optimized cultivation conditions. Rhodococcus sp. A27 and Streptomyces sp. G49 synthesized approx. 30% (w/w) fatty acids from fructose or cellobiose, respectively, while Streptomyces isolate G25 reached a cellular fatty acid content of nearly 50% (w/w) when cultivated with cellobiose. The stored triacylglycerols were composed of 30-40% branched fatty acids, such as anteiso-pentadecanoic or iso-hexadecanoic acid. To date, this represents by far the highest lipid content described for streptomycetes. A biotechnological production of such lipids using (hemi)cellulose-derived raw material could be used to obtain sustainable biodiesel with a high proportion of branched-chain fatty acids to improve its cold-flow properties and oxidative stability.
In this study, we metabolically engineered C. glutamicum to produce triacylglycerols (TAGs) by completing and constraining a de novo TAG biosynthesis pathway. First, the plasmid pZ8_TAG4 was constructed which allows the heterologous expression of four genes: three (atf1 and atf2, encoding the diacylglycerol acyltransferase; pgpB, encoding the phosphatidic acid phosphatase) to complete the TAG biosynthesis pathway, and one gene (tadA) for lipid body assembly. Second, we applied three metabolic strategies to increase TAGs accumulation: (i) reduction of TAG degradation and precursor consumption by deleting four cellular lipases (cg0109, cg0110, cg1676 and cg1320) and the diacylglycerol kinase (cg2849), (ii) enhancement of fatty acid biosynthesis by deletion of fasR (cg2737, TetR-type transcriptional regulator of genes for the fatty acid biosynthesis) as well as heterologous expression of tesA (encoding thioesterase to form free fatty acid to reduce the feedback inhibition by acyl-ACP) and fadD (encoding acyl-CoA synthetase to enhance acyl-CoA supply), and (iii) elimination of the observed by-product formation of organic acids by blocking the acetic acid (pqo) and lactic acid production (ldh) pathways. The final strain (CgTesRtcEfasEbp/pZ8_TAG4) achieved a 7.5% yield of total fatty acids (2.38±0.05g/L intracellular fatty acids and 0.64±0.09g/L extracellular fatty acids) from 4% glucose in shake flasks after process optimization. This corresponds to maximum intracellular lipids content of 17.8±0.5% of the dry cell.
For rapid analysis of microbial metabolisms, (13) C-fingerprinting employs a set of tracers to generate unique labeling patterns in key amino acids that can highlight active pathways. In contrast to rigorous (13) C-metabolic flux analysis ((13) C-MFA), this method aims to provide metabolic insights without expensive flux measurements. Using (13) C-fingerprinting, we investigated the metabolic pathways in Rhodococcus opacus PD630, a promising biocatalyst for the conversion of lignocellulosic feedstocks into value-added chemicals. Specifically, seven metabolic insights were gathered: 1) glucose metabolism mainly via the Entner-Doudoroff (ED) pathway, 2) lack of glucose catabolite repression during phenol co-utilization, 3) simultaneous operation of gluconeogenesis and the ED pathway for the co-metabolism of glucose and phenol, 4) an active glyoxylate shunt in acetate-fed culture, 5) high flux through anaplerotic pathways (e.g., malic enzyme and phosphoenolpyruvate carboxylase), 6) presence of alternative glycine synthesis pathway via glycine dehydrogenase, and 7) utilization of preferred exogenous amino acids (e.g., phenylalanine). Additionally, a (13) C-fingerprinting kit was developed for studying the central metabolism of non-model microbial species. This low-cost kit can be used to characterize microbial metabolisms and facilitate the design-build-test-learn cycle during the development of microbial cell factories. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
An engineered strain MITXM-61 of Rhodococcus opacus PD630 capable of utilizing xylose was used to produce biofuel precursor-triacylglycerols (TAG) from mixed glucose–xylose substrates. Optimal nitrogen source and carbon source concentrations were investigated for cell growth and microbial lipid production by MITXM-61 in flask cultures. In a two-stage batch culture, the maximum lipid yield (0.152 g TAG per g consumed carbon source) was achieved by utilizing a mixture of glucose and xylose. The fed-batch culture featured intermittent feeding of refined xylose solution during the lipid accumulation stage provided 45 g L−1 of cell dry weight, 54% (g TAG per g cell dry weight) of lipid content, 0.179 g g−1 (g TAG per g consumed carbon source) of lipid yield, and a 6.9 g L−1 day−1 of lipid productivity. Fatty acids extracted from microbial lipids produced by MITXM-61 were predominately palmitic and oleic acids, which are the major components in TAG-derived biofuels.