Synthetic Formatotrophs for One-Carbon Biorefinery

Abstract

The use of CO₂ as a carbon source in biorefinery is of great interest, but the low solubility of CO₂ in water and the lack of efficient CO₂ assimilation pathways are challenges to overcome. Formic acid (FA), which can be easily produced from CO₂ and more conveniently stored and transported than CO₂, is an attractive CO₂-equivalent carbon source as it can be assimilated more efficiently than CO₂ by microorganisms and also provides reducing power. Although there are native formatotrophs, they grow slowly and are difficult to metabolically engineer due to the lack of genetic manipulation tools. Thus, much effort is exerted to develop efficient FA assimilation pathways and synthetic microorganisms capable of growing solely on FA (and CO₂). Several innovative strategies are suggested to develop synthetic formatotrophs through rational metabolic engineering involving new enzymes and reconstructed FA assimilation pathways, and/or adaptive laboratory evolution (ALE). In this paper, recent advances in development of synthetic formatotrophs are reviewed, focusing on biological FA and CO₂ utilization pathways, enzymes involved and newly developed, and metabolic engineering and ALE strategies employed. Also, future challenges in cultivating formatotrophs to higher cell densities and producing chemicals from FA and CO₂ are discussed.

Keywords: formatotroph; formic acid assimilation; one‐carbon biorefinery; systems metabolic engineering.

Figure 1

Overall strategies of systems metabolic engineering for the bio‐based production of chemicals from FA and CO₂. Synthetic formatotrophs are developed by constructing synthetic FA assimilation pathways, introducing Fdhs to supply ATP and reducing powers from FA, and employing ALE and/or rational metabolic engineering strategies, such as redistribution of carbon flux, optimization of gene expression, enhancement of ATP conversion, and optimization of culture condition. Then, fermentation processes are developed to facilitate enhanced formatotrophic growth. Finally, the synthetic formatotrophs are metabolically engineered for fermentative production of chemicals. Abbreviations are: Ac‐CoA, acetyl‐CoA; Cyd, cytochrome bd‐I ubiquinol oxidase; Cyo, cytochrome bo3 ubiquinol oxidase; F6P, fructose 6‐phosphate; FA, formic acid; G6P, glucose 6‐phosphate; GAP, glyceraldehyde 3‐phosphate; Glu, glucose; GOI, gene of interest; LPS, lipopolysaccharide; PEP, phosphoenolpyruvate; PYR, pyruvate; R5P, ribose 5‐phosphate; SA, succinic acid; LA, lactate; Fdh, formate dehydrogenase.

Figure 2

Synthetic pathways for FA or FA and CO₂ assimilations. a) Fls pathway, b) SACA pathway, c) rTHF‐rgcv pathway, d) modified serine cycle, and e) synthetic homoserine cycle. All of the synthetic FA assimilation pathways reported to date are shown. Pathways in blue‐colored boxes are developed using de novo enzymes. Pathways in green‐colored boxes are developed by reconstruction of FA assimilation pathways. Enzymes involved in the synthetic pathways are indicated in blue. Abbreviations are: 2‐OG, 2‐oxoglutarate; 5,10‐CH₂—THF, 5,10‐methylenetetrahydrofolate; 5,10‐CH=THF, 5,10‐methenyltetrahydrofolate; 10‐CHO—THF, 10‐formyltetrahydrofolate; Acdh, acetaldehyde dehydrogenase; Acetyl‐P, acetyl‐phosphate; Acps, acetyl‐phosphate synthase; Acs, acetyl‐CoA synthase; Agt, alanine‐glyoxylate transaminase; Dhak, dihydroxyacetone kinase; FA, formic acid; FALD, formaldehyde; DHA, dihydroxyacetone; DHAP, dihydroxyacetone‐phosphate; Fch, 5,10‐CH=THF cyclohydrolase; Fls, formolase; Ftl, formate‐tetrahydrofolate ligase; GALD, glycolaldehyde; Gals, glycolaldehyde synthase; GcvTHP, gcv complex; Gldh, glutamate dehydrogenase; GlyA, serine hydroxymethyltransferase; Gpt, glutamate‐pyruvate transaminase; Hal, 4‐hydroxy‐2‐oxobutanoate aldolase; Hat, 4‐hydroxy‐2‐oxobutanoate aminotransferase; Hsk, homoserine kinase; Lpd, lipoamide dehydrogenase; Lta, threonine aldolase; Mcl, malyl‐CoA lyase; Mdh, malate dehydrogenase; MeOH, methanol; Mtd, 5,10‐CH₂—THF dehydrogenase; Mtk, malate thiokinase; OAA, oxaloacetate; PEP, phosphoenolpyruvate; Pfl, pyruvate formate lyase; Ppc, phosphoenolpyruvate carboxylase; Pps, phosphoenolpyruvate synthase; Pta, phosphate acetyltransferase; Sal, serine aldolase; Sda, serine deaminase; THF, tetrahydrofolate; Ts, threonine synthase.

Figure 3

Strategies to enhance FA and CO₂ assimilation efficiency of the rTHF‐rgcv pathway. Metabolic engineering strategies employed in three recent studies (a) Tashiro et al.;^{[ 29 ]} b) Yishai et al.;^{[ 30 ]} c) Bang and Lee^{[ 31 ]}) to enhance FA and CO₂ assimilation efficiency of the rTHF‐rgcv pathway are shown. Similar engineering strategies employed in three recent studies are grouped together in colored boxes. Red X marks represent gene deletion. Abbreviations are: 5‐10‐CH=THF, 5,10‐methenyl THF; 5,10‐CH₂—THF, 5,10‐methlylene THF; 10‐CHO—THF, 10‐formyl THF; FA, formic acid; fch, 5,10‐CH=THF cyclohydrolase; fdh, formate dehydrogenase; folD, bifunctional 5,10‐CH₂—THF dehydrogenase/5,10‐CH₂—THF cyclohydrolase; ftl, formate‐tetrahydrofolate ligase; gcvR, transcriptional regulator of glycine cleavage complex; gcvTHP, gcv complex; glyA, serine hydroxymethyltransferase; lpd, lipoamide dehydrogenase; mtd, 5,10‐CH₂—THF dehydrogenase; sda, serine deaminase; serA, phosphoglycerate dehydrogenase; THF, tetrahydrofolate.

Figure 4

Metabolic pathways for the production of chemicals from FA and CO₂. The metabolic pathways for the production of four chemicals (LA, L‐alanine, L‐serine, and SA), which can be produced from FA and CO₂, using the formatotrophic E. coli strain employing the rTHF‐rgcv pathway are shown. The yellow circles represent the chemicals that can be produced from FA and CO₂. The values provided in the yellow circle represent the maximum theoretical yield (mol mol FA⁻¹) of the target chemical in the formatotrophic E. coli strain employing the rTHF‐rgcv pathway calculated by GEM simulation. Abbreviations are: SA, succinic acid; LA, lactate; 5‐10‐CH=THF, 5,10‐methenyl THF; 5,10‐CH₂—THF, 5,10‐methlylene THF; 10‐CHO—THF, 10‐formyl THF; aceA, isocitrate lyase; aceB, malate synthase; Acetyl‐P, acetyl phosphate; ack, acetate kinase; acn, aconitate hydratase; AKG, α‐ketoglutarate; ald, alanine dehydrogenase; alr, alanine racemase I; dadA, D‐amino acid dehydrogenase; dadX, alanine racemase II; FA, formic acid; fch, 5,10‐CH=THF cyclohydrolase; fdh, formate dehydrogenase; frd, fumarate reductase; ftl, formate‐tetrahydrofolate ligase; fum, fumarase; gcvTHP, gcv complex; gltA, citrate synthase; glyA, serine hydroxymethyltransferase; icd, isocitrate dehydrogenase; ldhA, lactate dehydrogenase; lpd, lipoamide dehydrogenase; mdh, malate dehydrogenase; mtd, 5,10‐CH₂—THF dehydrogenase; OAA oxaloacetate; PEP, phosphoenolpyruvate; pfl, pyruvate formate lyase; poxB, pyruvate dehydrogenase; ppc, phosphoenolpyruvate carboxylase; pps, phosphoenolpyruvate synthase; pta, phosphate acetyltransferase; ptsG, PTS system glucose‐specific EIICB component; sda, serine deaminase; sdh, succinate dehydrogenase; sucAB, α‐ketoglutarate dehydrogenase; sucCD, succinyl‐CoA synthetase; THF, tetrahydrofolate.