Accelerated Electro-Fermentation of Acetoin in Escherichia coli by Identifying Physiological Limitations of the Electron Transfer Kinetics and the Central Metabolism

Anode-assisted fermentations offer the benefit of an anoxic fermentation routine that can be applied to produce end-products with an oxidation state independent from the substrate. The whole cell biocatalyst transfers the surplus of electrons to an electrode that can be used as a non-depletable electron acceptor. So far, anode-assisted fermentations were shown to provide high carbon efficiencies but low space-time yields. This study aimed at increasing space-time yields of an Escherichia coli-based anode-assisted fermentation of glucose to acetoin. The experiments build on an obligate respiratory strain, that was advanced using selective adaptation and targeted strain development. Several transfers under respiratory conditions led to point mutations in the pfl, aceF and rpoC gene. These mutations increased anoxic growth by three-fold. Furthermore, overexpression of genes encoding a synthetic electron transport chain to methylene blue increased the electron transfer rate by 2.45-fold. Overall, these measures and a medium optimization increased the space-time yield in an electrode-assisted fermentation by 3.6-fold.

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A Tailor-Made Self-Sufficient Whole-Cell Biocatalyst Enables Scalable Enantioselective Synthesis of (R)-3-Quinuclidinol in a High Space-Time Yield

Organic Process Research & Development ◽

10.1021/acs.oprd.9b00004 ◽

2019 ◽

Vol 23 (9) ◽

pp. 1813-1821 ◽

Cited By ~ 2

Author(s):

Qian Chen ◽

Baogang Xie ◽

Liping Zhou ◽

Lili Sun ◽

Shanshan Li ◽

...

Keyword(s):

Space Time ◽

Enantioselective Synthesis ◽

Whole Cell ◽

Whole Cell Biocatalyst ◽

High Space ◽

Space Time Yield

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Faculty Opinions recommendation of IscR, an Fe-S cluster-containing transcription factor, represses expression of Escherichia coli genes encoding Fe-S cluster assembly proteins.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1002791.46105 ◽

2002 ◽

Author(s):

Regine Hengge

Keyword(s):

Escherichia Coli ◽

Transcription Factor ◽

Cluster Assembly ◽

Genes Encoding ◽

Assembly Proteins

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Faculty Opinions recommendation of Genetic screen yields mutations in genes encoding all known components of the Escherichia coli signal recognition particle pathway.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1003128.30060 ◽

2001 ◽

Author(s):

Koreaki Ito

Keyword(s):

Escherichia Coli ◽

Signal Recognition Particle ◽

Genetic Screen ◽

Signal Recognition ◽

Genes Encoding

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Faculty Opinions recommendation of Selection for functional diversity drives accumulation of point mutations in Dr adhesins of Escherichia coli.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1088684.542790 ◽

2007 ◽

Author(s):

Antonio Juarez Gimenez

Keyword(s):

Escherichia Coli ◽

Functional Diversity ◽

Point Mutations ◽

Selection For

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Tuning a bi-enzymatic cascade reaction in Escherichia coli to facilitate NADPH regeneration for ε-caprolactone production

Bioresources and Bioprocessing ◽

10.1186/s40643-021-00370-w ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Jinghui Xiong ◽

Hefeng Chen ◽

Ran Liu ◽

Hao Yu ◽

Min Zhuo ◽

...

Keyword(s):

Escherichia Coli ◽

Binding Sites ◽

Regeneration System ◽

Nadph Regeneration ◽

Whole Cell ◽

Cyclohexanone Monooxygenase ◽

Ribosome Binding ◽

Ribosome Binding Sites ◽

Whole Cell Biocatalyst ◽

Substrate Tolerance

Abstractε-Caprolactone is a monomer of poly(ε-caprolactone) which has been widely used in tissue engineering due to its biodegradability and biocompatibility. To meet the massive demand for this monomer, an efficient whole-cell biocatalytic approach was constructed to boost the ε-caprolactone production using cyclohexanol as substrate. Combining an alcohol dehydrogenase (ADH) with a cyclohexanone monooxygenase (CHMO) in Escherichia coli, a self-sufficient NADPH-cofactor regeneration system was obtained. Furthermore, some improved variants with the better substrate tolerance and higher catalytic ability to ε-caprolactone production were designed by regulating the ribosome binding sites. The best mutant strain exhibited an ε-caprolactone yield of 0.80 mol/mol using 60 mM cyclohexanol as substrate, while the starting strain only got a conversion of 0.38 mol/mol when 20 mM cyclohexanol was supplemented. The engineered whole-cell biocatalyst was used in four sequential batches to achieve a production of 126 mM ε-caprolactone with a high molar yield of 0.78 mol/mol.

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Glutamate Mediates Proton-Coupled Electron Transfer Between Tyrosines 730 and 731 in Escherichia coli Ribonucleotide Reductase

Journal of the American Chemical Society ◽

10.1021/jacs.1c02152 ◽

2021 ◽

Author(s):

Clorice R. Reinhardt ◽

Elvira R. Sayfutyarova ◽

Jiayun Zhong ◽

Sharon Hammes-Schiffer

Keyword(s):

Escherichia Coli ◽

Electron Transfer ◽

Ribonucleotide Reductase ◽

Proton Coupled Electron Transfer

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L-Alanine Prototrophic Suppressors Emerge from L-Alanine Auxotroph through Stress-Induced Mutagenesis in Escherichia coli

Microorganisms ◽

10.3390/microorganisms9030472 ◽

2021 ◽

Vol 9 (3) ◽

pp. 472

Author(s):

Harutaka Mishima ◽

Hirokazu Watanabe ◽

Kei Uchigasaki ◽

So Shimoda ◽

Shota Seki ◽

...

Keyword(s):

Escherichia Coli ◽

Pyruvate Dehydrogenase ◽

Pyruvate Dehydrogenase Complex ◽

Spontaneous Mutation ◽

Point Mutations ◽

Whole Genome Analysis ◽

E Coli ◽

Clone Pair ◽

Induced Mutagenesis ◽

Isogenic Mutants

In Escherichia coli, L-alanine is synthesized by three isozymes: YfbQ, YfdZ, and AvtA. When an E. coli L-alanine auxotrophic isogenic mutant lacking the three isozymes was grown on L-alanine-deficient minimal agar medium, L-alanine prototrophic mutants emerged considerably more frequently than by spontaneous mutation; the emergence frequency increased over time, and, in an L-alanine-supplemented minimal medium, correlated inversely with L-alanine concentration, indicating that the mutants were derived through stress-induced mutagenesis. Whole-genome analysis of 40 independent L-alanine prototrophic mutants identified 16 and 18 clones harboring point mutation(s) in pyruvate dehydrogenase complex and phosphotransacetylase-acetate kinase pathway, which respectively produce acetyl coenzyme A and acetate from pyruvate. When two point mutations identified in L-alanine prototrophic mutants, in pta (D656A) and aceE (G147D), were individually introduced into the original L-alanine auxotroph, the isogenic mutants exhibited almost identical growth recovery as the respective cognate mutants. Each original- and isogenic-clone pair carrying the pta or aceE mutation showed extremely low phosphotransacetylase or pyruvate dehydrogenase activity, respectively. Lastly, extracellularly-added pyruvate, which dose-dependently supported L-alanine auxotroph growth, relieved the L-alanine starvation stress, preventing the emergence of L-alanine prototrophic mutants. Thus, L-alanine starvation-provoked stress-induced mutagenesis in the L-alanine auxotroph could lead to intracellular pyruvate increase, which eventually induces L-alanine prototrophy.

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