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Assimilation of formic acid and CO₂ by engineered Escherichia coli equipped with reconstructed one-carbon assimilation pathways.

ABSTRACT: Gaseous one-carbon (C1) compounds or formic acid (FA) converted from CO₂ can be an attractive raw material for bio-based chemicals. Here, we report the development of Escherichia coli strains assimilating FA and CO₂ through the reconstructed tetrahydrofolate (THF) cycle and reverse glycine cleavage (gcv) pathway. The Methylobacterium extorquens formate-THF ligase, methenyl-THF cyclohydrolase, and methylene-THF dehydrogenase genes were expressed to allow FA assimilation. The gcv reaction was reversed by knocking out the repressor gene (gcvR) and overexpressing the gcvTHP genes. This engineered strain synthesized 96% and 86% of proteinogenic glycine and serine, respectively, from FA and CO₂ in a glucose-containing medium. Native serine deaminase converted serine to pyruvate, showing 4.5% of pyruvate-forming flux comes from FA and CO₂ The pyruvate-forming flux from FA and CO₂ could be increased to 14.9% by knocking out gcvR, pflB, and serA, chromosomally expressing gcvTHP under trc, and overexpressing the reconstructed THF cycle, gcvTHP, and lpd genes in one vector. To reduce glucose usage required for energy and redox generation, the Candida boidinii formate dehydrogenase (Fdh) gene was expressed. The resulting strain showed specific glucose, FA, and CO₂ consumption rates of 370.2, 145.6, and 14.9 mg?g dry cell weight (DCW)^-1?h^-1, respectively. The C1 assimilation pathway consumed 21.3 wt% of FA. Furthermore, cells sustained slight growth using only FA and CO₂ after glucose depletion, suggesting that combined use of the C1 assimilation pathway and C. boidinii Fdh will be useful for eventually developing a strain capable of utilizing FA and CO₂ without an additional carbon source such as glucose.

SUBMITTER: Bang J

PROVIDER: S-EPMC6176599 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Assimilation of formic acid and CO2 by engineered Escherichia coli equipped with reconstructed one-carbon assimilation pathways.

Bang Junho J Lee Sang Yup SY

Proceedings of the National Academy of Sciences of the United States of America 20180917 40

Gaseous one-carbon (C1) compounds or formic acid (FA) converted from CO2 can be an attractive raw material for bio-based chemicals. Here, we report the development of Escherichia coli strains assimilating FA and CO2 through the reconstructed tetrahydrofolate (THF) cycle and reverse glycine cleavage (gcv) pathway. The Methylobacterium extorquens formate-THF ligase, methenyl-THF cyclohydrolase, and methylene-THF dehydrogenase genes were expressed to allow FA ass ...[more]

PMID: 30224468

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Project description:BACKGROUND:Hydroxycinnamoyl anthranilates, also known as avenanthramides (avns), are a group of phenolic alkaloids with anti-inflammatory, antioxidant, anti-itch, anti-irritant, and antiatherogenic activities. Some avenanthramides (avn A-H and avn K) are conjugates of hydroxycinnamic acids (HC), including p-coumaric acid, caffeic acid, and ferulic acid, and anthranilate derivatives, including anthranilate, 4-hydroxyanthranilate, and 5-hydroxyanthranilate. Avns are primarily found in oat grain, in which they were originally designated as phytoalexins. Knowledge of the avns biosynthesis pathway has now made it possible to synthesize avns through a genetic engineering strategy, which would help to further elucidate their properties and exploit their beneficial biological activities. The aim of the present study was to synthesize natural avns in Escherichia coli to serve as a valuable resource. RESULTS:We synthesized nine avns in E. coli. We first synthesized avn D from glucose in E. coli harboring tyrosine ammonia lyase (TAL), 4-coumarate:coenzyme A ligase (4CL), anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT), and anthranilate synthase (trpEG). A trpD deletion mutant was used to increase the amount of anthranilate in E. coli. After optimizing the incubation temperature and cell density, approximately 317.2 mg/L of avn D was synthesized. Avn E and avn F were then synthesized from avn D, using either E. coli harboring HpaBC and SOMT9 or E. coli harboring HapBC alone, respectively. Avn A and avn G were synthesized by feeding 5-hydroxyanthranilate or 4-hydroxyanthranilate to E. coli harboring TAL, 4CL, and HCBT. Avn B, avn C, avn H, and avn K were synthesized from avn A or avn G, using the same approach employed for the synthesis of avn E and avn F from avn D. CONCLUSIONS:Using different HCs, nine avns were synthesized, three of which (avn D, avn E, and avn F) were synthesized from glucose in E. coli. These diverse avns provide a strategy to synthesize both natural and unnatural avns, setting a foundation for exploring the biological activities of diverse avns.

Dataset Information

Assimilation of formic acid and CO₂ by engineered Escherichia coli equipped with reconstructed one-carbon assimilation pathways.

Publications

Assimilation of formic acid and CO<sub>2</sub> by engineered <i>Escherichia coli</i> equipped with reconstructed one-carbon assimilation pathways.

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