Biofuel production in Escherichia coli: the role of metabolic engineering and synthetic biology

The microbial production of biofuels is a promising avenue for the development of viable processes for the generation of fuels from sustainable resources. In order to become cost and energy effective, these processes must utilize organisms that can be optimized to efficiently produce candidate fuels from a variety of feedstocks. Escherichia coli has become a promising host organism for the microbial production of biofuels in part due to the ease at which this organism can be manipulated. Advancements in metabolic engineering and synthetic biology have led to the ability to efficiently engineer E. coli as a biocatalyst for the production of a wide variety of potential biofuels from several biomass constituents. This review focuses on recent efforts devoted to engineering E. coli for the production of biofuels, with emphasis on the key aspects of both the utilization of a variety of substrates as well as the synthesis of several promising biofuels. Strategies for the efficient utilization of carbohydrates, carbohydrate mixtures, and noncarbohydrate carbon sources will be discussed along with engineering efforts for the exploitation of both fermentative and nonfermentative pathways for the production of candidate biofuels such as alcohols and higher carbon biofuels derived from fatty acid and isoprenoid pathways. Continued advancements in metabolic engineering and synthetic biology will help improve not only the titers, yields, and productivities of biofuels discussed herein, but also increase the potential range of compounds that can be produced.     

微藻三酰甘油合成途径及其最新研究进展

三酰甘油(triacylglycerols,TAGs)是动物、植物、微生物和微藻细胞主要的储藏性脂类,它可应用于食品、轻工业和生物燃料等方面,是一种新型可再生能源——生物柴油生产的重要原料。与高等油料作物相比,微藻具有光合作用效率高、生长速度快、油脂产量高、不占用农业耕地和适应多种生长环境等优势,是一种潜在的新型生物柴油生产原料。然而,目前人们对有机体,尤其是微藻细胞内TAG合成与积累的分子机制及细胞的代谢调控机制还知之甚少。对TAG合成的一系列重要过程,包括脂肪酸的合成,TAG生物合成的主要途径和旁路途径,以及与TAG合成相关的关键酶和重要基因等进行了综述,特别对微藻细胞中与TAG合成相关的关键基因的最新研究进展进行了总结,旨在更好地了解油脂代谢的调控途径,为最大限度地供应生物柴油的生产原料提供理论基础。     

Generation of an atlas for commodity chemical production in Escherichia coli and a novel pathway prediction algorithm,GEM-Path

The production of 75% of the current drug molecules and 35% of all chemicals could be achieved through bioprocessing (Arundel and Sawaya, 2009). To accelerate the transition from a petroleum-based chemical industry to a sustainable bio-based industry, systems metabolic engineering has emerged to computationally design metabolic pathways for chemical production. Although algorithms able to provide specific metabolic interventions and heterologous production pathways are available, a systematic analysis for all possible production routes to commodity chemicals in Escherichia coli is lacking. Furthermore, a pathway prediction algorithm that combines direct integration of genome-scale models at each step of the search to reduce the search space does not exist. Previous work (Feist et al., 2010) performed a model-driven evaluation of the growth-coupled production potential for E. coli to produce multiple native compounds from different feedstocks. In this study, we extended this analysis for non-native compounds by using an integrated approach through heterologous pathway integration and growth-coupled metabolite production design. In addition to integration with genome-scale model integration, the GEM-Path algorithm developed in this work also contains a novel approach to address reaction promiscuity. In total, 245 unique synthetic pathways for 20 large volume compounds were predicted. Host metabolism with these synthetic pathways was then analyzed for feasible growth-coupled production and designs could be identified for 1271 of the 6615 conditions evaluated. This study characterizes the potential for E. coli to produce commodity chemicals, and outlines a generic strain design workflow to design production strains.     

The Evolution of Oxygen As a Biosynthetic Reagent

The biosynthesis of certain cell constituents: monounsaturated fatty acids, tyrosine, and nicotinic acid, is oxygen-dependent in many higher organisms. The same compounds can be synthesized by different, oxygen-independent pathways in lower organisms. The general outlines of these pathways are described and the importance of the compounds synthesized is discussed. An examination of the distribution of these pathways among living organisms reveals that oxygen-dependent pathways replaced the "anaerobic" pathways at different branch points on the evolutionary tree. Other groups of compounds are discussed, which are not distributed as widely among living organisms, but are found in all higher organisms. These compounds have specialized functions and their biosynthesis requires molecular oxygen. The oxygen-dependent portions of the biosynthetic pathways leading to porphyrins, quinone coenzymes, carotenoids, sterols, and polyunsaturated fatty acids are summarized. The distribution and functions of these compounds are also considered and an attempt is made to place them in the framework of evolution. While sterols and polyunsaturated fatty acids are found exclusively in the higher Protista and multicellular organisms, carotenoids, porphyrins, and quinones are also found in bacteria. The possibility of oxygen-independent mechanisms for their biosynthesis is discussed.     

Metabolic engineering of edible plant oils]

Plant seed oil is the major source of many fatty acids for human nutrition, and also one of industrial feedstocks. Recent advances in understanding of the basic biochemistry of seed oil biosynthesis, coupled with cloning of the genes encoding the enzymes involved in fatty acid modification and oil accumulation, have set the stage for the metabolic engineering of oilseed crops that produce "designer" plant seed oils with the improved nutritional values for human being. In this review we provide an overview of seed oil biosynthesis/regulation and highlight the key enzymatic steps that are targets for gene manipulation. The strategies of metabolic engineering of fatty acids in oilseeds, including overexpression or suppression of genes encoding single or multi-step biosynthetic pathways and assembling the complete pathway for the synthesis of long-chain polyunsaturated fatty acids (e.g. arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid) are described in detail. The current "bottlenecks" in using common oilseeds as "bioreactors" for commercial production of high-value fatty acids are analyzed. It is also discussed that the future research focuses of oilseed metabolic engineering and the prospects in creating renewable sources and promoting the sustainable development of human society and economy.     

Yarrowia lipolytica as a biotechnological chassis to produce usual and unusual fatty acids

One of the most promising alternatives to petroleum for the production of fuels and chemicals is bio-oil based chemistry. Microbial oils are gaining importance because they can be engineered to accumulate lipids enriched in desired fatty acids. These specific lipids are closer to the commercialized product, therefore reducing pollutants and costly chemical steps. Yarrowia lipolytica is the most widely studied and engineered oleaginous yeast. Different molecular and bioinformatics tools permit systems metabolic engineering strategies in this yeast, which can produce usual and unusual fatty acids. Usual fatty acids, those usually found in triacylglycerol, accumulate through the action of several pathways, such as fatty acid/triacylglycerol synthesis, transport and degradation. Unusual fatty acids are enzymatic modifications of usual fatty acids to produce compounds that are not naturally synthetized in the host. Recently, the metabolic engineering of microorganisms has produced different unusual fatty acids, such as building block ricinoleic acid and nutraceuticals such as conjugated linoleic acid or polyunsaturated fatty acids. Additionally, microbial sources are preferred hosts for the production of fatty acid-derived compounds such as γ-decalactone, hexanal and dicarboxylic acids. The variety of lipids produced by oleaginous microorganisms is expected to rise in the coming years to cope with the increasing demand.     

Production of 1-octanol in Escherichia coli by a high flux thioesterase route

1-octanol is a valuable molecule in the chemical industry, where it is used as a plasticizer, as a precursor in the production of linear low-density polyethylene (LLDPE), and as a growth inhibitor of tobacco plant suckers. Due to the low availability of eight-carbon acyl chains in natural lipid feedstocks and the selectivity challenges in petrochemical routes to medium-chain fatty alcohols,1-octanol sells for the highest price among the fatty alcohol products. As an alternative, metabolic engineers have pursued sustainable 1-octanol production via engineered microbes. Here, we report demonstration of gram per liter titers in the model bacterium Escherichia coli via the development of a pathway composed of a thioesterase, an acyl-CoA synthetase, and an acyl-CoA reductase. In addition, the impact of deleting fermentative pathways was explored E. coli K12 MG1655 strain for production of octanoic acid, a key octanol precursor. In order to overcome metabolic flux barriers, bioprospecting experiments were performed to identify acyl-CoA synthetases with high activity towards octanoic acid and acyl-CoA reductases with high activity to produce 1-octanol from octanoyl-CoA. Titration of expression of key pathway enzymes was performed and a strain with the full pathway integrated on the chromosome was created. The final strain produced 1-octanol at 1.3 g/L titer and a >90% C₈ specificity from glycerol. In addition to the metabolic engineering efforts, this work addressed some of the technical challenges that arise when quantifying 1-octanol produced from cultures grown under fully aerobic conditions where evaporation and stripping are prevalent.     

工程大肠杆菌合成游离脂肪酸的研究进展

游离脂肪酸作为一种重要的平台化合物,其衍生产品被广泛应用到能源、化学工业中。作为更加可持续、绿色的生产策略,利用工程微生物合成游离脂肪酸是以石油基和动植物为原料生产脂肪酸类产品的重要补充。大肠杆菌作为经典的模式微生物,通过对其进行代谢工程改造,脂肪酸的积累已经从痕量提高到了约9g/L,展示了其作为脂肪酸合成菌株的巨大应用潜力。随着合成生物学技术的涌现,“感应-调控器”、体外重构、β氧化逆循环、异源合成途径的整合等思路的引入极大地加快了工程大肠杆菌脂肪酸合成的进化速率,并赋予大肠杆菌合成多种脂肪酸产品的能力。对近年来通过代谢工程和合成生物学手段改造大肠杆菌合成游离脂肪酸的研究进展进行综述,对其发展前景进行展望。     

脂肪醇制备研究进展

脂肪醇是合成表面活性剂、洗涤剂、增塑剂及其他多种精细化学用品的基础化工原料,广泛应用于纺织、日化、造纸、食品、医药、皮革等领域。本文介绍了脂肪醇的市场现状,综述了工业制备脂肪醇的传统方法,阐述了以可再生非粮生物质为原料,利用生物法制备生物基脂肪醇的方法,并对生物基脂肪醇的新合成路线的发展方向进行展望。     

A single-host fermentation process for the production of flavor lactones from non-hydroxylated fatty acids

Lactone flavors with fruity, milky, coconut, and other aromas are widely used in the food and fragrance industries. Lactones are produced by chemical synthesis or by biotransformation of plant-sourced hydroxy fatty acids. We established a novel method to produce flavor lactones from abundant non-hydroxylated fatty acids using yeast cell factories. Oleaginous yeast Yarrowia lipolytica was engineered to perform hydroxylation of fatty acids and chain-shortening via β-oxidation to preferentially twelve or ten carbons. The strains could produce γ-dodecalactone from oleic acid and δ-decalactone from linoleic acid. Through metabolic engineering, the titer was improved 4-fold, and the final strain produced 282 mg/L γ-dodecalactone in a fed-batch bioreactor. The study paves the way for the production of lactones by fermentation of abundant fatty feedstocks.     

Biosynthesis of butyric acid by Clostridium tyrobutyricum

Butyric acid (C₃H₇COOH) is an important chemical that is widely used in foodstuffs along with in the chemical and pharmaceutical industries. The bioproduction of butyric acid through large-scale fermentation has the potential to be more economical and efficient than petrochemical synthesis. In this paper, the metabolic pathways involved in the production of butyric acid from Clostridium tyrobutyricum using hexose and pentose as substrates are investigated, and approaches to enhance butyric acid production through genetic modification are discussed. Finally, bioreactor modifications (including fibrous bed bioreactor, inner disk-shaped matrix bioreactor, fibrous matrix packed in porous levitated sphere carriers), low-cost feedstocks, and special treatments (including continuous fermentation with cell recycling, extractive fermentation with solvent, using different artificial electron carriers) intended to improve the feasibility of commercial butyric acid bioproduction are summarized.     

A synthetic pathway for the production of 2-hydroxyisovaleric acid in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Synthetic biology, encompassing the design and construction of novel artificial biological pathways and organisms and the redesign of existing natural biological systems, is rapidly expanding the number of applications for which biological systems can play an integral role. In the context of chemical production, the combination of synthetic biology and metabolic engineering approaches continues to unlock the ability to biologically produce novel and complex molecules from a variety of feedstocks. Here, we utilize a synthetic approach to design and build a pathway to produce 2-hydroxyisovaleric acid in Escherichia coli and demonstrate how pathway design can be supplemented with metabolic engineering approaches to improve pathway performance from various carbon sources. Drawing inspiration from the native pathway for the synthesis of the 5-carbon amino acid l-valine, we exploit the decarboxylative condensation of two molecules of pyruvate, with subsequent reduction and dehydration reactions enabling the synthesis of 2-hydroxyisovaleric acid. Key to our approach was the utilization of an acetolactate synthase which minimized kinetic and regulatory constraints to ensure sufficient flux entering the pathway. Critical host modifications enabling maximum product synthesis from either glycerol or glucose were then examined, with the varying degree of reduction of these carbons sources playing a major role in the required host background. Through these engineering efforts, the designed pathway produced 6.2 g/L 2-hydroxyisovaleric acid from glycerol at 58% of maximum theoretical yield and 7.8 g/L 2-hydroxyisovaleric acid from glucose at 73% of maximum theoretical yield. These results demonstrate how the combination of synthetic biology and metabolic engineering approaches can facilitate bio-based chemical production.     

Lipid and fatty acid metabolism in Ralstonia eutropha: relevance for the biotechnological production of value-added products

Lipid and fatty acid metabolism has been well studied in model microbial organisms like Escherichia coli and Bacillus subtilis. The major precursor of fatty acid biosynthesis is also the major product of fatty acid degradation (β-oxidation), acetyl-CoA, which is a key metabolite for all organisms. Controlling carbon flux to fatty acid biosynthesis and from β-oxidation allows for the biosynthesis of natural products of biotechnological importance. Ralstonia eutropha can utilize acetyl-CoA from fatty acid metabolism to produce intracellular polyhydroxyalkanoate (PHA). R. eutropha can also be engineered to utilize fatty acid metabolism intermediates to produce different PHA precursors. Metabolism of lipids and fatty acids can be rerouted to convert carbon into other value-added compounds like biofuels. This review discusses the lipid and fatty acid metabolic pathways in R. eutropha and how they can be used to construct reagents for the biosynthesis of products of industrial importance. Specifically, how the use of lipids or fatty acids as the sole carbon source in R. eutropha cultures adds value to these biotechnological products will be discussed here.     

Recent advances in metabolic engineering of Corynebacterium glutamicum for bioproduction of value-added aromatic chemicals and natural products

Recent progress in synthetic and systems metabolic engineering technologies has explored the potential of microbial cell factories for the production of industrially relevant bulk and fine chemicals from renewable biomass resources in an eco-friendly manner. Corynebacterium glutamicum, a workhorse for industrial amino acid production, has currently evolved into a promising microbial platform for bioproduction of various natural and non-natural chemicals from renewable feedstocks. Notably, it has been recently demonstrated that metabolically engineered C. glutamicum can overproduce several commercially valuable aromatic and related chemicals such as shikimate, 4-hydroxybenzoate, and 4-aminobenzoate from sugars at remarkably high titer suitable to commercial application. On the other hand, overexpression and/or extension of its endogenous metabolic pathways by integrating heterologous metabolic pathways enabled production of structurally intricate and valuable natural chemicals like plant polyphenols, carotenoids, and fatty acids. In this review, we summarize recent advances in metabolic engineering of C. glutamicum for production of those value-added aromatics and other natural products, which highlights high potential and the versatility of this microbe for bioproduction of diverse chemicals.

     

Role of essential fatty acids in trophometabolic interactions in the freshwater ecosystems (a review)

Polyunsaturated fatty acids (PUFA) of omega3 family are of crucial physiological importance for the most animals and they are an essential and deficient component of human nutrition. These compounds are most effectively synthesized by some groups of algae, hence, aquatic ecosystems are considered to be the main source of these PUFA for human nutrition. Factors controlling the content and distribution of omega3 PUFA in freshwater organisms of basic trophic levels and determined PUFA final production in freshwater ecosystems are considered in the review. PUFA biosynthesis is known to be tightly related to basic fatty acid metabolic pathways. Hence, fatty composition and the PUFA content of major freshwater hydrobiont groups, including bacteria, algae, invertebrates and vertebrates, and environmental and population age effects are described. The peculiarities of PUFA transfer between organisms of various trophic levels are discussed. The essential omega3 PUFA is one of the important parameter of food quality of aquatic consumers and they can determine the rate of energy and matter transfer between producers and primary consumers and, as a result, in a whole freshwater food chain. Analysis of PUFA content and its regulation in biomass of various fish populations indicates that freshwater ecosystems are of the same value in respect of PUFA sources as marine ecosystems. Despite the great practical importance, the studies focused on production and whole pools of omega3 PUFA in different freshwater ecosystems are still scarce and need to be continued.     

Advancing oleaginous microorganisms to produce lipid via metabolic engineering technology

With the depletion of global petroleum and its increasing price, biodiesel has been becoming one of the most promising biofuels for global fuels market. Researchers exploit oleaginous microorganisms for biodiesel production due to their short life cycle, less labor required, less affection by venue, and easier to scale up. Many oleaginous microorganisms can accumulate lipids, especially triacylglycerols (TAGs), which are the main materials for biodiesel production. This review is covering the related researches on different oleaginous microorganisms, such as yeast, mold, bacteria and microalgae, which might become the potential oil feedstocks for biodiesel production in the future, showing that biodiesel from oleaginous microorganisms has a great prospect in the development of biomass energy. Microbial oils biosynthesis process includes fatty acid synthesis approach and TAG synthesis approach. In addition, the strategies to increase lipids accumulation via metabolic engineering technology, involving the enhancement of fatty acid synthesis approach, the enhancement of TAG synthesis approach, the regulation of related TAG biosynthesis bypass approaches, the blocking of competing pathways and the multi-gene approach, are discussed in detail. It is suggested that DGAT and ME are the most promising targets for gene transformation, and reducing PEPC activity is observed to be beneficial for lipid production.     

Functional expression of prokaryotic and eukaryotic genes in Escherichia coli for conversion of glucose to p-hydroxystyrene

The chemical monomer p-hydroxystyrene (pHS) is used for producing a number of important industrial polymers from petroleum-based feedstocks. In an alternative approach, the microbial production of pHS can be envisioned by linking together a number of different metabolic pathways, of which those based on using glucose for carbon and energy are currently the most economical. The biological process conserves petroleum when glucose is converted to the aromatic amino acid L-tyrosine, which is deaminated by a tyrosine/phenylalanine ammonia-lyase (PAL/TAL) enzyme to yield p-hydroxycinnamic acid (pHCA). Subsequent decarboxylation of pHCA gives rise to pHS. Bacteria able to efficiently decarboxylate pHCA to pHS using a pHCA decarboxylase (PDC) include Bacillus subtilis, Pseudomonas fluorescens and Lactobacillus plantarum. Both B. subtilis and L. plantarum possess high levels of pHCA-inducible decarboxylase activity and were chosen for further studies. The genes encoding PDC in these organisms were cloned and the pHCA decarboxylase expressed in Escherichia coli strains co-transformed with a plasmid encoding a bifunctional PAL/TAL enzyme from the yeast Rhodotorula glutinis. Production of pHS from glucose was ten-fold greater for the expressed L. plantarum pdc gene (0.11mM), compared to that obtained when the B. subtilis PDC gene (padC) was used. An E. coli strain (WWQ51.1) expressing both tyrosine ammonia-lyase(PAL) and pHCA decarboxylase (pdc), when grown in a 14L fermentor and under phosphate limited conditions, produced 0.4g/L of pHS from glucose. We, therefore, demonstrate pHS production from an inexpensive carbohydrate feedstock by fermentation using a novel metabolic pathway comprising genes from E. coli, L. plantarum and R. glutinis.