No abstract

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

There has been much effort exerted to reduce one carbon (C1) gas emission to address climate change. As one promising way to more conveniently utilize C1 gas, several technologies have been developed to convert C1 gas into useful chemicals such as formic acid (FA). In this study, systems metabolic engineering was utilized to engineer Mannheimia succiniciproducens to efficiently utilize FA. C isotope analysis of M. succiniciproducens showed that FA could be utilized through formate dehydrogenase (FDH) reaction and/or the reverse reaction of pyruvate formate lyase (PFL). However, the naturally favored forward reaction of PFL was found to lower the SA yield from FA. In addition, FA assimilation via FDH was found to be more efficient than the reverse reaction of PFL. Thus, the M. succiniciproducens LPK7 strain, which lacks in pfl, ldh, pta, and ack genes, was selected as a base strain. In silico metabolic analysis confirmed that utilization of FA would be beneficial for the enhanced production of SA and suggested FDH as an amplification target. To find a suitable FDH, four different FDHs from M. succiniciproducens, Methylobacterium extorquens, and Candida boidinii were amplified in LPK7 strain to enhance FA assimilation. High-inoculum density cultivation using C labeled sodium formate was performed to evaluate FA assimilation efficiency. Fed-batch fermentations of the LPK7 (pMS3-fdh2 meq) strain was carried out using glucose, sucrose, or glycerol as a primary carbon source and FA as a secondary carbon source. As a result, this strain produced 76.11 g/L SA with the yield and productivity of 1.28 mol/mol and 4.08 g/L/h, respectively, using sucrose and FA as dual carbon sources. The strategy employed here will be similarly applicable in developing microorganisms to utilize FA and to produce valuable chemicals and materials from FA.

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.

Engineering of microorganisms to produce desired bio-products with high titer, yield, and productivity is often limited by product toxicity. This is also true for succinic acid (SA), a four carbon dicarboxylic acid of industrial importance. Acid products often cause product toxicity to cells through several different factors, membrane damage being one of the primary factors. In this study, cis-trans isomerase from Pseudomonas aeruginosa was expressed in Mannheimia succiniciproducens to produce trans-unsaturated fatty acid (TUFA) and to reinforce the cell membrane of M. succiniciproducens. The engineered strain showed significant decrease in membrane fluidity as production of TUFA enabled tight packing of fatty acids, which made cells to possess more rigid cell membrane. As a result, the membrane-engineered M. succiniciproducens strain showed higher tolerance toward SA and increased production of SA compared with the control strain without membrane engineering. The membrane engineering approach employed in this study will be useful for increasing tolerance to, and consequently enhancing production of acid products.