Metabolic engineering of Escherichia coli for 1-butanol production

Compared to ethanol, butanol offers many advantages as a substitute for gasoline because of higher energy content and higher hydrophobicity. Typically, 1-butanol is produced by Clostridium in a mixed-product fermentation. To facilitate strain improvement for specificity and productivity, we engineered a synthetic pathway in Escherichia coli and demonstrated the production of 1-butanol from this non-native user-friendly host. Alternative genes and competing pathway deletions were evaluated for 1-butanol production. Results show promise for using E. coli for 1-butanol production.     

Metabolic engineering of Escherichia coli for the production of 1-propanol

An engineered Escherichia coli strain that produces 1-propanol under aerobic condition was developed based on an l-threonine-overproducing E. coli strain. First, a feedback resistant ilvA gene encoding threonine dehydratase was introduced and the competing metabolic pathway genes were deleted. Further engineering was performed by overexpressing the cimA gene encoding citramalate synthase and the ackA gene encoding acetate kinase A/propionate kinase II, introducing a modified adhE gene encoding an aerobically functional AdhE, and by deleting the rpoS gene encoding the stationary phase sigma factor. Fed-batch culture of the final engineered strain harboring pBRthrABC-tac-cimA-tac-ackA and pTacDA-tac-adhE(mut) allowed production of 10.8gL(-1) of 1-propanol with the yield and productivity of 0.107gg(-1) and 0.144gL(-1)h(-1), respectively, from 100gL(-1) of glucose, and 10.3gL(-1) of 1-propanol with the yield and productivity of 0.259gg(-1) and 0.083gL(-1)h(-1), respectively, from 40gL(-1) glycerol.     

代谢工程方法改造大肠杆菌生产胸苷

胸苷是抗艾滋病药物司他夫定(3′-脱氧-2′,3′-双脱氢胸苷)和叠氮胸苷的重要前体物质。应用代谢工程方法对大肠杆菌Escherichia coli BL21(DE3)生物合成胸苷进行了研究。通过敲除E.coli BL21嘧啶回补途径的deo A、tdk和udp三个基因,BS03工程菌株能够积累21.6 mg/L胸苷。为了增加合成胸苷前体物核糖-5-磷酸和NADPH的供给,进一步敲除pgi和pyr L使工程菌BS05胸苷的产量提高到90.5 mg/L。而通过过表达胸苷合成途径的ush A、thy A、dut、ndk、nrd A和nrd B六个基因,菌株BS08胸苷的产量能达到272 mg/L。通过分批补料发酵,BS08最终可以积累1 248.8 mg/L的胸苷。本研究结果表明经过代谢工程改造的E.coli BL21具有良好的胸苷合成能力和应用潜力。     

代谢工程改造大肠杆菌生产L-高丝氨酸

L-高丝氨酸是一种非必需氨基酸,在工业生产中常被用作重要的平台化合物和添加剂.为提高产物合成效率,本文以实验室构建的L-高丝氨酸高产工程菌大肠杆菌Escherichia coli H0-0作为底盘菌株进行代谢改造.首先对ppc和pyccgP458S基因进行过表达来优化柠檬酸循环,然后通过thrAC1034T和lysCc...     

Metabolic engineering of Escherichia coli for agmatine production

Agmatine is a kind of important biogenic amine. The chemical synthesis route is not a desirable choice for industrial production of agmatine. To date, there are no reports on the fermentative production of agmatine by microorganism. In this study, the base Escherichia coli strain AUX4 (JM109 ?speC ?speF ?speB ?argR) capable of excreting agmatine into the culture medium was first constructed by sequential deletions of the speC and speF genes encoding the ornithine decarboxylase isoenzymes, the speB gene encoding agmatine ureohydrolase and the regulation gene argR responsible for the negative control of the arg regulon. The speA gene encoding arginine decarboxylase harboured by the pKK223‐3 plasmid was overexpressed in AUX4, resulting in the engineered strain AUX5. The batch and fed‐batch fermentations of the AUX5 strain were conducted in a 3‐L bioreactor, and the results showed that the AUX5 strain was able to produce 1.13 g agmatine L^?1 with the yield of 0.11 g agmatine g^?1 glucose in the batch fermentation and the fed‐batch fermentation of AUX5 allowed the production of 15.32 g agmatine L^?1 with the productivity of 0.48 g agmatine L^?1 h^?1, demonstrating the potential of E. coli as an industrial producer of agmatine.     

Metabolic engineering of cyanobacteria for 1-butanol production from carbon dioxide

Production of chemicals and fuels directly from CO(2) is an attractive approach to solving the energy and environmental problems. 1-Butanol, a chemical feedstock and potential fuel, has been produced by fermentation of carbohydrates, both in native Clostridium species and various engineered hosts. To produce 1-butanol from CO(2), we transferred a modified CoA-dependent 1-butanol production pathway into a cyanobacterium, Synechococcus elongatus PCC 7942. We demonstrated the activity of each enzyme in the pathway by chromosomal integration and expression of the genes. In particular, Treponema denticola trans-enoyl-CoA reductase (Ter), which utilizes NADH as the reducing power, was used for the reduction of crotonyl-CoA to butyryl-CoA instead of Clostridium acetobutylicum butyryl-CoA dehydrogenase to by-pass the need of Clostridial ferredoxins. Addition of polyhistidine-tag increased the overall activity of Ter and resulted in higher 1-butanol production. Removal of oxygen is an important factor in the synthesis of 1-butanol in this organism. This result represents the first autotrophic 1-butanol production.     

代谢工程改造大肠杆菌合成羟基酪醇

羟基酪醇是重要精细化学品,作为天然抗氧化剂被广泛应用于食品、医药领域.利用合成生物学技术生产羟基酪醇具有重要意义.本文克隆并功能鉴定了来源于大肠杆菌Escherichia coli BL21的羟化酶编码基因HpaBC,结果表明该酶的两个亚基均能成功表达并能催化酪醇生成羟基酪醇.通过CRISPR-Cas9技术将由tac启...     

Metabolic engineering strategies for butanol production in Escherichia coli

The global market of butanol is increasing due to its growing applications as solvent, flavoring agent, and chemical precursor of several other compounds. Recently, the superior properties of n-butanol as a biofuel over ethanol have stimulated even more interest. (Bio)butanol is natively produced together with ethanol and acetone by Clostridium species through acetone-butanol-ethanol fermentation, at noncompetitive, low titers compared to petrochemical production. Different butanol production pathways have been expressed in Escherichia coli, a more accessible host compared to Clostridium species, to improve butanol titers and rates. The bioproduction of butanol is here reviewed from a historical and theoretical perspective. All tested rational metabolic engineering strategies in E. coli to increase butanol titers are reviewed: manipulation of central carbon metabolism, elimination of competing pathways, cofactor balancing, development of new pathways, expression of homologous enzymes, consumption of different substrates, and molecular biology strategies. The progress in the field of metabolic modeling and pathway generation algorithms and their potential application to butanol production are also summarized here. The main goals are to gather all the strategies, evaluate the respective progress obtained, identify, and exploit the outstanding challenges.     

Metabolic engineering for improving l-tryptophan production in Escherichia coli

Journal of Industrial Microbiology & Biotechnology - l-Tryptophan is an important aromatic amino acid that is used widely in the food, chemical, and pharmaceutical industries. Compared with the...     

Metabolic engineering for advanced biofuels production from Escherichia coli

Global energy and environmental problems have stimulated increasing efforts toward synthesizing liquid biofuels as transportation energy. Compared to the traditional biofuel, ethanol, advanced biofuels should offer advantages such as higher energy density, lower hygroscopicity, lower vapor pressure, and compatibility with existing transportation infrastructure. However, these fuels are not synthesized economically using native organisms. Metabolic engineering offers an alternative approach in which synthetic pathways are engineered into user-friendly hosts for the production of these fuel molecules. These hosts could be readily manipulated to improve the production efficiency. This review summarizes recent progress in the engineering of Escherichia coli to produce advanced biofuels.     

Metabolic engineering of Escherichia coli for the production of fumaric acid

Fumaric acid is a naturally occurring organic acid that is an intermediate of the tricarboxylic acid cycle. Fungal species belonging to Rhizopus have traditionally been employed for the production of fumaric acid. In this study, Escherichia coli was metabolically engineered for the production of fumaric acid under aerobic condition. For the aerobic production of fumaric acid, the iclR gene was deleted to redirect the carbon flux through the glyoxylate shunt. In addition, the fumA, fumB, and fumC genes were also deleted to enhance fumaric acid formation. The resulting strain was able to produce 1.45 g/L of fumaric acid from 15 g/L of glucose in flask culture. Based on in silico flux response analysis, this base strain was further engineered by plasmid‐based overexpression of the native ppc gene, encoding phosphoenolpyruvate carboxylase (PPC), from the strong tac promoter, which resulted in the production of 4.09 g/L of fumaric acid. Additionally, the arcA and ptsG genes were deleted to reinforce the oxidative TCA cycle flux, and the aspA gene was deleted to block the conversion of fumaric acid into L ‐aspartic acid. Since it is desirable to avoid the use of inducer, the lacI gene was also deleted. To increase glucose uptake rate and fumaric acid productivity, the native promoter of the galP gene was replaced with the strong trc promoter. Fed‐batch culture of the final strain CWF812 allowed production of 28.2 g/L fumaric acid in 63 h with the overall yield and productivity of 0.389 g fumaric acid/g glucose and 0.448 g/L/h, respectively. This study demonstrates the possibility for the efficient production of fumaric acid by metabolically engineered E. coli. Biotechnol. Bioeng. 2013; 110: 2025–2034. © 2013 Wiley Periodicals, Inc.     

代谢工程改造大肠杆菌合成丙二酸

丙二酸是一种重要的有机二元羧酸,其应用价值遍及化工、医药、食品等领域。本文以大肠杆菌为底盘细胞,过表达了ppc、aspC、panD、pa0132、yneI和pyc基因,成功构建了丙二酸合成重组菌株大肠杆菌BL21(TPP)。该菌株在摇瓶发酵条件下,丙二酸产量达到0.61 g/L。在5 L发酵罐水平,采用间歇补料的方式丙二酸的积累量达3.32 g/L。本研究应用了融合蛋白技术,将ppc和aspC、pa0132和yneI分别进行融合表达,构建了工程菌BL21(SCR)。在摇瓶发酵水平,该菌株丙二酸的积累量达到了0.83 g/L,较出发菌株BL21(TPP)提高了36%。在5 L发酵罐中,工程菌BL21(SCR)的丙二酸产量最高达5.61 g/L,较出发菌株BL21(TPP)提高了69%。本研究实现了丙二酸在大肠杆菌中的生物合成,为构建丙二酸合成的细胞工厂提供了理论依据和技术基础,同时也对其他二元羧酸的生物合成具有启发和指导意义。     

Metabolic engineering of Escherichia coli for high production of 1,5-pentanediol via a cadaverine-derived pathway

1,5-Pentanediol (1,5-PDO) is a high value-added chemical which is widely used as a monomer in the polymer industry. There are no natural organisms that could directly produce 1,5-PDO from renewable carbon sources. In this study, we report metabolic engineering of Escherichia coli for high-level production of 1,5-PDO from glucose via a cadaverine-derived pathway. In the newly proposed pathway, cadaverine can be converted to 1,5-PDO via 5-hydroxyvalerate (5-HV) by introducing only one heterologous enzyme in E. coli. Different endogenous genes of E. coli were screened and heterologous carboxylic acid reductase genes were tested to build a functional pathway. Compared to the previously reported pathways, the engineered cadaverine-based pathway has a higher theoretical yield (0.70 mol/mol glucose) and higher catalytic efficiency. By further combining strategies of pathway engineering and process engineering, we constructed an engineered E. coli strain that could produce 2.62 g/L 1,5-PDO in shake-flask and 9.25 g/L 1,5-PDO with a yield of 0.28 mol/mol glucose in fed-batch fermentation. The proposed new pathway and engineering strategies reported here should be useful for developing biological routes to produce 1,5-PDO for real application.     

Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production

Limonene is a valuable monoterpene used in the production of several commodity chemicals and medicinal compounds. Among them, perillyl alcohol (POH) is a promising anti-cancer agent that can be produced by hydroxylation of limonene. We engineered E. coli with a heterologous mevalonate pathway and limonene synthase for production of limonene followed by coupling with a cytochrome P450, which specifically hydroxylates limonene to produce POH. A strain containing all mevalonate pathway genes in a single plasmid produced limonene at titers over 400 mg/L from glucose, substantially higher than has been achieved in the past. Incorporation of a cytochrome P450 to hydroxylate limonene yielded approximately 100 mg/L of POH. Further metabolic engineering of the pathway and in situ product recovery using anion exchange resins would make this engineered E. coli a potential production platform for any valuable limonene derivative.     

Metabolic engineering of Escherichia coli for the production of succinate from glycerol

Glycerol has become an ideal feedstock for the microbial production of bio-based chemicals due to its abundance, low cost, and high degree of reduction. We have previously reported the pathways and mechanisms for the utilization of glycerol by Escherichia coli in minimal salts medium under microaerobic conditions. Here we capitalize on such results to engineer E. coli for the production of value-added succinate from glycerol. Through metabolic engineering of E. coli metabolism, succinate production was greatly elevated by (1) blocking pathways for the synthesis of competing by-products lactate, ethanol, and acetate and (2) expressing Lactococcus lactis pyruvate carboxylase to drive the generation of succinate from the pyruvate node (as opposed to that of phosphoenolpyruvate). As such, these metabolic engineering strategies coupled cell growth to succinate production because the synthesis of succinate remained as the primary route of NAD+ regeneration. This feature enabled the operation of the succinate pathway in the absence of selective pressure (e.g. antibiotics). Our biocatalysts demonstrated a maximum specific productivity of ～400 mg succinate/gcell/h and a yield of 0.69 g succinate/g glycerol, on par with the use of glucose as a feedstock.     

Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol

1,4-Butanediol (BDO) is an important commodity chemical used to manufacture over 2.5 million tons annually of valuable polymers, and it is currently produced exclusively through feedstocks derived from oil and natural gas. Herein we report what are to our knowledge the first direct biocatalytic routes to BDO from renewable carbohydrate feedstocks, leading to a strain of Escherichia coli capable of producing 18 g l(-1) of this highly reduced, non-natural chemical. A pathway-identification algorithm elucidated multiple pathways for the biosynthesis of BDO from common metabolic intermediates. Guided by a genome-scale metabolic model, we engineered the E. coli host to enhance anaerobic operation of the oxidative tricarboxylic acid cycle, thereby generating reducing power to drive the BDO pathway. The organism produced BDO from glucose, xylose, sucrose and biomass-derived mixed sugar streams. This work demonstrates a systems-based metabolic engineering approach to strain design and development that can enable new bioprocesses for commodity chemicals that are not naturally produced by living cells.     

Metabolic engineering of Escherichia coli for production of salvianic acid A via an artificial biosynthetic pathway

Salvianic acid A, a valuable derivative from L-tyrosine biosynthetic pathway of the herbal plant Salvia miltiorrhiza, is well known for its antioxidant activities and efficacious therapeutic potential on cardiovascular diseases. Salvianic acid A was traditionally isolated from plant root or synthesized by chemical methods, both of which had low efficiency. Herein, we developed an unprecedented artificial biosynthetic pathway of salvianic acid A in E. coli, enabling its production from glucose directly. In this pathway, 4-hydroxyphenylpyruvate was converted to salvianic acid A via D-lactate dehydrogenase (encoding by d-ldh from Lactobacillus pentosus) and hydroxylase complex (encoding by hpaBC from E. coli). Furthermore, we optimized the pathway by a modular engineering approach and deleting genes involved in the regulatory and competing pathways. The metabolically engineered E. coli strain achieved high productivity of salvianic acid A (7.1 g/L) with a yield of 0.47 mol/mol glucose.