Potential production platform of n-butanol in Escherichia coli

Saini, Mukesh Kumar; Chen, Min Hong; Chiang, Chung-Jen; Chao, Yun-Peng

doi:10.1016/j.ymben.2014.11.001

Cited by 85 publications

(80 citation statements)

References 32 publications

(40 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A separate study adjusted the intracellular redox state by targeting central carbon metabolism to push flux of carbon through the pentose phosphate pathway (Saini et al, 2016). Additionally, other strains were engineered to produce 1-butanol from butanoate (Saini et al, 2015). 1-Butanol titers from E. coli and Clostridium species are comparable.…”

Section: Discussionmentioning

confidence: 99%

Metabolomics-driven approach to solving a CoA imbalance for improved 1-butanol production in Escherichia coli

Ohtake

Pontrelli

Laviña

et al. 2017

Metabolic Engineering

View full text Add to dashboard Cite

A B S T R A C THigh titer 1-butanol production in Escherichia coli has previously been achieved by overexpression of a modified clostridial 1-butanol production pathway and subsequent deletion of native fermentation pathways. This strategy couples growth with production as 1-butanol pathway offers the only available terminal electron acceptors required for growth in anaerobic conditions. With further inclusion of other well-established metabolic engineering principles, a titer of 15 g/L has been obtained. In achieving this titer, many currently existing strategies have been exhausted, and 1-butanol toxicity level has been surpassed. Therefore, continued engineering of the host strain for increased production requires implementation of alternative strategies that seek to identify non-obvious targets for improvement. In this study, a metabolomics-driven approach was used to reveal a CoA imbalance resulting from a pta deletion that caused undesirable accumulation of pyruvate, butanoate, and other CoA-derived compounds. Using metabolomics, the reduction of butanoyl-CoA to butanal catalyzed by alcohol dehydrogenase AdhE2 was determined as a rate-limiting step. Fine-tuning of this activity and subsequent release of free CoA restored the CoA balance that resulted in a titer of 18.3 g/L upon improvement of total free CoA levels using cysteine supplementation. By enhancing AdhE2 activity, carbon flux was directed towards 1-butanol production and undesirable accumulation of pyruvate and butanoate was diminished. This study represents the initial report describing the improvement of 1-butanol production in E. coli by resolving CoA imbalance, which was based on metabolome analysis and rational metabolic engineering strategies.

show abstract

Section: Discussionmentioning

confidence: 99%

Metabolomics-driven approach to solving a CoA imbalance for improved 1-butanol production in Escherichia coli

Ohtake

Pontrelli

Laviña

et al. 2017

Metabolic Engineering

View full text Add to dashboard Cite

show abstract

“…However, previous research on microbial consortia was primarily concerned with the study of mixed population stability and dynamic interactions (7)(8)(9)(10)(11), although a few recent studies have reported the engineering of microbial consortia for utilization of simple sugars to make small molecules of central carbon metabolism, such as ethanol and lactate (12,13). Progress was made recently, when a full n-butanol pathway was expressed in two separate E. coli cells to achieve higher production (14) and when a bacterium-yeast coculture was used to address the difficulties of functional reconstitution of a pathway involving prokaryotic and eukaryotic enzymes in a consortium, and thus improved production of complex pharmaceutical molecules (15). Here, we expand the generality of the coculture engineering by demonstrating that microbial cocultures can also be engineered to overcome more universal challenges in metabolic engineering, including high-level intermediate secretion and low-efficiency sugar mixture utilization.…”

Section: -Hydroxybenzoic Acidmentioning

confidence: 99%

Engineering Escherichia coli coculture systems for the production of biochemical products

Zhang

Pereira

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

269

199

View full text Add to dashboard Cite

Engineering microbial consortia to express complex biosynthetic pathways efficiently for the production of valuable compounds is a promising approach for metabolic engineering and synthetic biology. Here, we report the design, optimization, and scale-up of an Escherichia coli-E. coli coculture that successfully overcomes fundamental microbial production limitations, such as high-level intermediate secretion and low-efficiency sugar mixture utilization. For the production of the important chemical cis,cis-muconic acid, we show that the coculture approach achieves a production yield of 0.35 g/g from a glucose/xylose mixture, which is significantly higher than reported in previous reports. By efficiently producing another compound, 4-hydroxybenzoic acid, we also demonstrate that the approach is generally applicable for biosynthesis of other important industrial products.metabolic engineering | microbial coculture | muconic acid | 4-hydroxybenzoic acidM etabolic engineering and synthetic biology have made great strides in constructing and optimizing metabolic pathways for biochemical product synthesis in a pure culture (1, 2). There are, however, situations where this approach may be limited, as in the cases where (i) a single host cell cannot provide an optimal environment for the functioning of all pathway enzymes, (ii) biosynthetic efficiency is reduced due to overwhelming metabolic stress from the overexpression of long and complex pathways (3, 4), or (iii) pathway intermediates are secreted yielding undesired byproducts and reducing substrate utilization (5, 6). The above limitations can potentially be overcome through the use of rationally designed microbial coculture systems. Different from previous modular engineering approaches, such coculturebased systems completely modularize and segregate a biosynthetic pathway into two separate cells, each of which carries a portion of the pathway, and thus can be engineered independently to achieve optimal functioning of the combined pathway.The concept of microbial cocultures is not new. However, previous research on microbial consortia was primarily concerned with the study of mixed population stability and dynamic interactions (7-11), although a few recent studies have reported the engineering of microbial consortia for utilization of simple sugars to make small molecules of central carbon metabolism, such as ethanol and lactate (12, 13). Progress was made recently, when a full n-butanol pathway was expressed in two separate E. coli cells to achieve higher production (14) and when a bacterium-yeast coculture was used to address the difficulties of functional reconstitution of a pathway involving prokaryotic and eukaryotic enzymes in a consortium, and thus improved production of complex pharmaceutical molecules (15). Here, we expand the generality of the coculture engineering by demonstrating that microbial cocultures can also be engineered to overcome more universal challenges in metabolic engineering, including high-level intermediate secretion and low-efficiency s...

show abstract

“…While the native producer Clostridium remains as the major workhorse for the production of 1-butanol on the industrial scale [3–8], engineering and characterization of the clostridial CoA-dependent pathway in various heterologous hosts have been extensively performed to decipher pathway bottleneck and address its limitation in recombinant systems [9–13]. Production titer and industrial practicality of heterologous 1-butanol synthesis have been significantly improved by many metabolic engineering approaches, such as replacement of inefficient enzymes [13, 14], creation of synthetic driving forces [15, 16], development of co-culturing system [17], utilization of inducer-free promoter [18–20], and analysis of system-level pathway inefficiency [18, 20, 21], reaching the highest productivity of 5–6 g/L/d so far using Escherichia coli in bench-scale flasks. In addition to the CoA-dependent pathway, other synthetic pathways based on amino acid biosynthesis [22] and reverse β-oxidation [23] were also explored as the alternative 1-butanol production system.…”

Section: Introductionmentioning

confidence: 99%

Self-regulated 1-butanol production in Escherichia coli based on the endogenous fermentative control

Wen

Shen

2016

Biotechnol Biofuels

View full text Add to dashboard Cite

BackgroundAs a natural fermentation product secreted by Clostridium species, bio-based 1-butanol has attracted great attention for its potential as alternative fuel and chemical feedstock. Feasibility of microbial 1-butanol production has also been demonstrated in various recombinant hosts.ResultsIn this work, we constructed a self-regulated 1-butanol production system in Escherichia coli by borrowing its endogenous fermentation regulatory elements (FRE) to automatically drive the 1-butanol biosynthetic genes in response to its natural fermentation need. Four different cassette of 5′ upstream transcription and translation regulatory regions controlling the expression of the major fermentative genes ldhA, frdABCD, adhE, and ackA were cloned individually to drive the 1-butanol pathway genes distributed among three plasmids, resulting in 64 combinations that were tested for 1-butanol production efficiency. Fermentation of 1-butanol was triggered by anaerobicity in all cases. In the growth-decoupled production screening, only combinations with formate dehydrogenase (Fdh) overexpressed under FREadhE demonstrated higher titer of 1-butanol anaerobically. In vitro assay revealed that 1-butanol productivity was directly correlated with Fdh activity under such condition. Switching cells to oxygen-limiting condition prior to significant accumulation of biomass appeared to be crucial for the induction of enzyme synthesis and the efficiency of 1-butanol fermentation. With the selection pressure of anaerobic NADH balance, the engineered strain demonstrated stable production of 1-butanol anaerobically without the addition of inducer or antibiotics, reaching a titer of 10 g/L in 24 h and a yield of 0.25 g/g glucose under high-density fermentation.ConclusionsHere, we successfully engineered a self-regulated 1-butanol fermentation system in E. coli based on the natural regulation of fermentation reactions. This work also demonstrated the effectiveness of selection pressure based on redox balance anaerobically. Results obtained from this study may help enhance the industrial relevance of 1-butanol synthesis using E. coli and solidifies the possibility of strain improvement by directed evolution.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0680-1) contains supplementary material, which is available to authorized users.

show abstract

Potential production platform of n-butanol in Escherichia coli

Cited by 85 publications

References 32 publications

Metabolomics-driven approach to solving a CoA imbalance for improved 1-butanol production in Escherichia coli

Metabolomics-driven approach to solving a CoA imbalance for improved 1-butanol production in Escherichia coli

Engineering Escherichia coli coculture systems for the production of biochemical products

Self-regulated 1-butanol production in Escherichia coli based on the endogenous fermentative control

Contact Info

Product

Resources

About