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Journal ArticleDOI

Driving Forces Enable High-Titer Anaerobic 1-Butanol Synthesis in Escherichia coli

01 May 2011-Applied and Environmental Microbiology (American Society for Microbiology)-Vol. 77, Iss: 9, pp 2905-2915
TL;DR: A modified clostridial 1-butanol pathway is constructed in Escherichia coli to provide an irreversible reaction catalyzed by trans-enoyl-coenzyme A (CoA) reductase (Ter) and NADH and acetyl-CoA driving forces to direct the flux and demonstrate the importance of driving forces in the efficient production of nonnative products.
Abstract: 1-Butanol, an important chemical feedstock and advanced biofuel, is produced by Clostridium species. Various efforts have been made to transfer the clostridial 1-butanol pathway into other microorganisms. However, in contrast to similar compounds, only limited titers of 1-butanol were attained. In this work, we constructed a modified clostridial 1-butanol pathway in Escherichia coli to provide an irreversible reaction catalyzed by trans-enoyl-coenzyme A (CoA) reductase (Ter) and created NADH and acetyl-CoA driving forces to direct the flux. We achieved high-titer (30 g/liter) and high-yield (70 to 88% of the theoretical) production of 1-butanol anaerobically, comparable to or exceeding the levels demonstrated by native producers. Without the NADH and acetyl-CoA driving forces, the Ter reaction alone only achieved about 1/10 the level of production. The engineered host platform also enables the selection of essential enzymes with better catalytic efficiency or expression by anaerobic growth rescue. These results demonstrate the importance of driving forces in the efficient production of nonnative products.
Citations
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Journal ArticleDOI
16 Aug 2012-Nature
TL;DR: Data-driven and synthetic-biology approaches can be used to optimize both the host and pathways to maximize fuel production, and to compete with more conventional fuels.
Abstract: Advanced biofuels produced by microorganisms have similar properties to petroleum-based fuels, and can 'drop in' to the existing transportation infrastructure. However, producing these biofuels in yields high enough to be useful requires the engineering of the microorganism's metabolism. Such engineering is not based on just one specific feedstock or host organism. Data-driven and synthetic-biology approaches can be used to optimize both the host and pathways to maximize fuel production. Despite some success, challenges still need to be met to move advanced biofuels towards commercialization, and to compete with more conventional fuels.

986 citations

Journal ArticleDOI
Jeong Wook Lee1, Dokyun Na1, Jong Myoung Park1, Joungmin Lee1, Sol Choi1, Sang Yup Lee1 
TL;DR: The general strategies of systems metabolic engineering are discussed and examples of its application are offered and insights are offered as to when and how each of the different strategies should be used.
Abstract: Growing concerns over limited fossil resources and associated environmental problems are motivating the development of sustainable processes for the production of chemicals, fuels and materials from renewable resources. Metabolic engineering is a key enabling technology for transforming microorganisms into efficient cell factories for these compounds. Systems metabolic engineering, which incorporates the concepts and techniques of systems biology, synthetic biology and evolutionary engineering at the systems level, offers a conceptual and technological framework to speed the creation of new metabolic enzymes and pathways or the modification of existing pathways for the optimal production of desired products. Here we discuss the general strategies of systems metabolic engineering and examples of its application and offer insights as to when and how each of the different strategies should be used. Finally, we highlight the limitations and challenges to be overcome for the systems metabolic engineering of microorganisms at more advanced levels.

668 citations

Journal ArticleDOI
TL;DR: This work demonstrates the excellent capacity for lipid production by the oleaginous yeast Y. lipolytica and the effects of metabolic engineering of two important steps of the lipid synthesis pathway, which acts to divert flux towards the cholesterol synthesis and creates driving force for TAG synthesis.

576 citations

Journal ArticleDOI
TL;DR: This Review discusses how microorganisms can be explored for the production of next-generation biofuels, based on the ability of bacteria and fungi to use lignocellulose; through direct CO2 conversion by microalgae; using lithoautotrophs driven by solar electricity; or through the capacity of microorganisms to use methane generated from landfill.
Abstract: Global climate change linked to the accumulation of greenhouse gases has caused concerns regarding the use of fossil fuels as the major energy source. To mitigate climate change while keeping energy supply sustainable, one solution is to rely on the ability of microorganisms to use renewable resources for biofuel synthesis. In this Review, we discuss how microorganisms can be explored for the production of next-generation biofuels, based on the ability of bacteria and fungi to use lignocellulose; through direct CO2 conversion by microalgae; using lithoautotrophs driven by solar electricity; or through the capacity of microorganisms to use methane generated from landfill. Furthermore, we discuss how to direct these substrates to the biosynthetic pathways of various fuel compounds and how to optimize biofuel production by engineering fuel pathways and central metabolism.

473 citations

Journal ArticleDOI
TL;DR: In this paper, the state of the art and future challenges in the recent development of biomass and associated transformation technologies for clean production of biofuels are reviewed, and a discussion of the synergistic integration of various biochemical and bioprocessing technologies is provided.

391 citations

References
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Journal ArticleDOI
TL;DR: A simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s), which should be widely useful, especially in genome analysis of E. coli and other bacteria.
Abstract: We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.

14,389 citations


"Driving Forces Enable High-Titer An..." refers background in this paper

  • ...Escherichia coli BW25113 (rrnBT14 lacZWJ16 hsdR514 araBADAH33 rhaBADLD78) was designated as the wild type (16)....

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Journal ArticleDOI
03 Jan 2008-Nature
TL;DR: This strategy uses the host’s highly active amino acid biosynthetic pathway and diverts its 2-keto acid intermediates for alcohol synthesis to achieve high-yield, high-specificity production of isobutanol from glucose.
Abstract: Global energy and environmental problems have stimulated increased efforts towards synthesizing biofuels from renewable resources. Compared to the traditional biofuel, ethanol, higher alcohols offer advantages as gasoline substitutes because of their higher energy density and lower hygroscopicity. In addition, branched-chain alcohols have higher octane numbers compared with their straight-chain counterparts. However, these alcohols cannot be synthesized economically using native organisms. Here we present a metabolic engineering approach using Escherichia coli to produce higher alcohols including isobutanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol and 2-phenylethanol from glucose, a renewable carbon source. This strategy uses the host's highly active amino acid biosynthetic pathway and diverts its 2-keto acid intermediates for alcohol synthesis. In particular, we have achieved high-yield, high-specificity production of isobutanol from glucose. The strategy enables the exploration of biofuels beyond those naturally accumulated to high quantities in microbial fermentation.

1,955 citations


"Driving Forces Enable High-Titer An..." refers background in this paper

  • ...These low titers of heterologous 1-butanol production demonstrate the difficulty in transferring this pathway to nonnative hosts and are in sharp contrast to the high titers of related compounds, such as ethanol (50 g/liter) (27, 45), isobutanol (20 to 50 g/liter) (3, 5), and isopropanol (40 to 140 g/liter) (24), produced by recombinant E....

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Journal ArticleDOI
TL;DR: This article reviews biotechnological production of butanol by clostridia and some relevant fermentation and downstream processes and the strategies for strain improvement by metabolic engineering and further requirements to make fermentative butanol production a successful industrial process.
Abstract: Butanol is an aliphatic saturated alcohol having the molecular formula of C4H9OH Butanol can be used as an intermediate in chemical synthesis and as a solvent for a wide variety of chemical and textile industry applications Moreover, butanol has been considered as a potential fuel or fuel additive Biological production of butanol (with acetone and ethanol) was one of the largest industrial fermentation processes early in the 20th century However, fermentative production of butanol had lost its competitiveness by 1960s due to increasing substrate costs and the advent of more efficient petrochemical processes Recently, increasing demand for the use of renewable resources as feedstock for the production of chemicals combined with advances in biotechnology through omics, systems biology, metabolic engineering and innovative process developments is generating a renewed interest in fermentative butanol production This article reviews biotechnological production of butanol by clostridia and some relevant fermentation and downstream processes The strategies for strain improvement by metabolic engineering and further requirements to make fermentative butanol production a successful industrial process are also discussed Biotechnol Bioeng 2008;101: 209-228 © 2008 Wiley Periodicals, Inc

1,017 citations


"Driving Forces Enable High-Titer An..." refers background in this paper

  • ...1-Butanol, a potential fuel substitute and an important C4 chemical feedstock (30), is naturally synthesized by Clostridium species using a pathway that involves multiple coenzyme A (CoA)-activated intermediates (hereinafter called the CoAdependent pathway)....

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  • ...Glycerol had no observable contribution, while yeast extract appeared to be an important nitrogen source that enhanced cell growth and helped lead to higher titers of 1-butanol (30)....

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Journal ArticleDOI
TL;DR: A synthetic pathway is engineered in Escherichia coli and the production of 1-butanol is demonstrated from this non-native user-friendly host, showing promise for using E. coli for 1- butanol production.

900 citations


"Driving Forces Enable High-Titer An..." refers background or methods or result in this paper

  • ...The crt-hbd fragment was created by individually amplifying crt with primers crtfxba and crtrSOE and hbd with hbdfSOE and hbdrxba using pJCL60 (2) as a...

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  • ...Detailed procedures were described previously (2)....

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  • ...The construction of strains JCL16 (BW25113 with lacI provided on F ), JCL166, and JCL299 was described previously (2)....

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  • ...In contrast, our previous work (2) used the BcdEtfAB complex instead of Ter....

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  • ...Contrary to what was reported previously using the Bcd-EtfAB complex (2), where a small amount of oxygen was necessary to achieve higher 1-butanol productivity, the highest accumulation of 1-butanol was observed under fully anaerobic conditions in this case (Fig....

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Journal ArticleDOI
TL;DR: Saccharomyces cerevisiae was engineered with an n-butanol biosynthetic pathway, in which isozymes from a number of different organisms were substituted for Clostridial enzymes and their effect on n- butanol production was compared.
Abstract: Increasing energy costs and environmental concerns have motivated engineering microbes for the production of "second generation" biofuels that have better properties than ethanol. Saccharomyces cerevisiae was engineered with an n-butanol biosynthetic pathway, in which isozymes from a number of different organisms (S. cerevisiae, Escherichia coli, Clostridium beijerinckii, and Ralstonia eutropha) were substituted for the Clostridial enzymes and their effect on n-butanol production was compared. By choosing the appropriate isozymes, we were able to improve production of n-butanol ten-fold to 2.5 mg/L. The most productive strains harbored the C. beijerinckii 3-hydroxybutyryl-CoA dehydrogenase, which uses NADH as a co-factor, rather than the R. eutropha isozyme, which uses NADPH, and the acetoacetyl-CoA transferase from S. cerevisiae or E. coli rather than that from R. eutropha. Surprisingly, expression of the genes encoding the butyryl-CoA dehydrogenase from C. beijerinckii (bcd and etfAB) did not improve butanol production significantly as previously reported in E. coli. Using metabolite analysis, we were able to determine which steps in the n-butanol biosynthetic pathway were the most problematic and ripe for future improvement.

536 citations


"Driving Forces Enable High-Titer An..." refers background in this paper

  • ...5 mg/liter) (38), Lactobacillus brevis (300 mg/liter) (7), Pseudomonas putida (580 mg/liter), and Bacillus subtilis (120 mg/liter) (32)....

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  • ...This general scheme of alleviating the anaerobic redox imbalance (22, 25, 38, 40, 42) caused by the inactivation of NADH-consuming reactions with target production pathways is applicable to systems where directed evolution is desired....

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