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Protein engineering of the transcriptional activator FhlA To enhance hydrogen production in Escherichia coli.

ABSTRACT: Escherichia coli produces H(2) from formate via the formate hydrogenlyase (FHL) complex during mixed acid fermentation; the FHL complex consists of formate dehydrogenase H (encoded by fdhF) for forming 2H(+), 2e(-), and CO(2) from formate and hydrogenase 3 (encoded by hycGE) for synthesizing H(2) from 2H(+) and 2e(-). FHL protein production is activated by the sigma(54) transcriptional activator FhlA, which activates transcription of fdhF and the hyc, hyp, and hydN-hypF operons. Here, through random mutagenesis using error-prone PCR over the whole gene, as well as over the fhlA region encoding the first 388 amino acids of the 692-amino-acid protein, we evolved FhlA to increase H(2) production. The amino acid replacements in FhlA133 (Q11H, L14V, Y177F, K245R, M288K, and I342F) increased hydrogen production ninefold, and the replacements in FhlA1157 (M6T, S35T, L113P, S146C, and E363K) increased hydrogen production fourfold. Saturation mutagenesis at the codons corresponding to the amino acid replacements in FhlA133 and at position E363 identified the importance of position L14 and of E363 for the increased activity; FhlA with replacements L14G and E363G increased hydrogen production (fourfold and sixfold, respectively) compared to FhlA. Whole-transcriptome and promoter reporter constructs revealed that the mechanism by which the FhlA133 changes increase hydrogen production is by increasing transcription of all of the genes activated by FhlA (the FHL complex). With FhlA133, transcription of P(fdhF) and P(hyc) is less sensitive to formate regulation, and with FhlA363 (E363G), P(hyc) transcription increases but P(hyp) transcription decreases and hydrogen production is less affected by the repressor HycA.

SUBMITTER: Sanchez-Torres V

PROVIDER: S-EPMC2737898 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Protein engineering of the transcriptional activator FhlA To enhance hydrogen production in Escherichia coli.

Sanchez-Torres Viviana V Maeda Toshinari T Wood Thomas K TK

Applied and environmental microbiology 20090706 17

Escherichia coli produces H(2) from formate via the formate hydrogenlyase (FHL) complex during mixed acid fermentation; the FHL complex consists of formate dehydrogenase H (encoded by fdhF) for forming 2H(+), 2e(-), and CO(2) from formate and hydrogenase 3 (encoded by hycGE) for synthesizing H(2) from 2H(+) and 2e(-). FHL protein production is activated by the sigma(54) transcriptional activator FhlA, which activates transcription of fdhF and the hyc, hyp, and hydN-hypF operons. Here, through ra ...[more]

PMID: 19581479

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Project description:BackgroundEthyl acetate (C4H8O2) and hydrogen (H2) are industrially relevant compounds that preferably are produced via sustainable, non-petrochemical production processes. Both compounds are volatile and can be produced by Escherichia coli before. However, relatively low yields for hydrogen are obtained and a mix of by-products renders the sole production of hydrogen by micro-organisms unfeasible. High yields for ethyl acetate have been achieved, but accumulation of formate remained an undesired but inevitable obstacle. Coupling ethyl acetate production to the conversion of formate into H2 may offer an interesting solution to both drawbacks. Ethyl acetate production requires equimolar amounts of ethanol and acetyl-CoA, which enables a redox neutral fermentation, without the need for production of by-products, other than hydrogen and CO2.ResultsWe engineered Escherichia coli towards improved conversion of formate into H2 and CO2 by inactivating the formate hydrogen lyase repressor (hycA), both uptake hydrogenases (hyaAB, hybBC) and/or overexpressing the hydrogen formate lyase activator (fhlA), in an acetate kinase (ackA) and lactate dehydrogenase (ldhA)-deficient background strain. Initially 10 strains, with increasing number of modifications were evaluated in anaerobic serum bottles with respect to growth. Four reference strains ΔldhAΔackA, ΔldhAΔackA p3-fhlA, ΔldhAΔackAΔhycAΔhyaABΔhybBC and ΔldhAΔackAΔhycAΔhyaABΔhybBC p3-fhlA were further equipped with a plasmid carrying the heterologous ethanol acyltransferase (Eat1) from Wickerhamomyces anomalus and analyzed with respect to their ethyl acetate and hydrogen co-production capacity. Anaerobic co-production of hydrogen and ethyl acetate via Eat1 was achieved in 1.5-L pH-controlled bioreactors. The cultivation was performed at 30 °C in modified M9 medium with glucose as the sole carbon source. Anaerobic conditions and gas stripping were established by supplying N2 gas.ConclusionsWe showed that the engineered strains co-produced ethyl acetate and hydrogen to yields exceeding 70% of the pathway maximum for ethyl acetate and hydrogen, and propose in situ product removal via gas stripping as efficient technique to isolate the products of interest.

Project description:BACKGROUND: Riboflavin (vitamin B2), the precursor of the flavin cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), is used commercially as an animal feed supplement and food colorant. E. coli is a robust host for various genetic manipulations and has been employed for efficient production of biofuels, polymers, amino acids, and bulk chemicals. Thus, the aim of this study was to understand the metabolic capacity of E. coli for the riboflavin production by modification of central metabolism, riboflavin biosynthesis pathway and optimization of the fermentation conditions. RESULTS: The basic producer RF01S, in which the riboflavin biosynthesis genes ribABDEC from E. coli were overexpressed under the control of the inducible trc promoter, could accumulate 229.1 mg/L of riboflavin. Further engineering was performed by examining the impact of expression of zwf (encodes glucose 6-phosphate dehydrogenase) and gnd (encodes 6-phosphogluconate dehydrogenase) from Corynebacterium glutamicum and pgl (encodes 6-phosphogluconolactonase) from E. coli on riboflavin production. Deleting pgi (encodes glucose-6-phosphate isomerase) and genes of Entner-Doudoroff (ED) pathway successfully redirected the carbon flux into the oxidative pentose phosphate pathway, and overexpressing the acs (encodes acetyl-CoA synthetase) reduced the acetate accumulation. These modifications increased riboflavin production to 585.2 mg/L. By further modulating the expression of ribF (encodes riboflavin kinase) for reducing the conversion of riboflavin to FMN in RF05S, the final engineering strain RF05S-M40 could produce 1036.1 mg/L riboflavin in LB medium at 37°C. After optimizing the fermentation conditions, strain RF05S-M40 produced 2702.8 mg/L riboflavin in the optimized semi-defined medium, which was a value nearly 12-fold higher than that of RF01S, with a yield of 137.5 mg riboflavin/g glucose. CONCLUSIONS: The engineered strain RF05S-M40 has the highest yield among all reported riboflavin production strains in shake flask culture. This work collectively demonstrates that E. coli has a potential to be a microbial cell factory for riboflavin bioproduction.

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Protein engineering of the transcriptional activator FhlA To enhance hydrogen production in Escherichia coli.

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Protein engineering of the transcriptional activator FhlA To enhance hydrogen production in Escherichia coli.

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