[P006] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P03-368] Bioproduction from formate in an engineered Escherichia coli via the tetrahydrofolate cycle

○Tatsumi Imada¹, Keitaro Tatsumi¹, Kyoka Mizuta¹, Kinuka Isshiki¹, Hiroshi Shimizu¹, Yoshihiro Toya¹ (1. The University of Osaka (Japan))

Keywords:

formate assimilation,metabolic engineering,tetrahydrofolate cycle,acetoin production,L-serine production

Carbon dioxide (CO₂) is considered as a main reason for climate change. Recently, many chemical technologies converting CO₂ to other C1 compounds have been developed, and metabolic engineering constructing microorganisms to possible to assimilate C1 compounds is attracting attention. Particularly, one of the C1 compounds, formate has a higher reduction power. Several methylotrophic microorganisms assimilate formate by tetrahydrofolate (THF) cycle. Although these microorganisms assimilate C1 compounds rapidly, the genetic engineering of these strains is still challenging, and the use of these microorganisms is limited for bioproduction. Recently, a model organism, Escherichia coli, has been extensively studied by introducing this pathway for formate fixation. However, its efficiency remains low compared to native methylotrophic bacteria and only few studies have been shown that the practical bioproduction from formate. Here, we developed engineered E. coli strains for formate assimilation by introducing four genes involved in the THF cycle and evaluated their bioproduction capability by producing L-serine and acetoin as a model compounds.The THF cycle generally consists of four enzymes that catalyze the formate assimilation and are encoded ftfL, fch, mtdA, and glyA. First, we introduced ftfL, fch, and mtdA from Methylobacterium extorquens AM1 into E. coli strain, MG1655 (DE3) (FCM strain) and evaluated formate assimilation efficiency by measuring the specific formate uptake rate and ¹³C-fraction of L-serine. The engineered E. coli was cultivated in M9 medium supplemented with 5 g L^-1 glucose, 2 g L^-1 glycine, and 10 mM ¹³C-labeled formate. The ¹³C-enrichment of [Ser-85]⁺ increased to approximately 35% in the engineered strain. However, no significant change in the extracellular formate concentration was observed in either the engineered or control strain. We then overexpressed the endogenous glyA gene to enhance formate assimilation (FCMG strain). The FCMG strain assimilated 7.31 mM formate, whereas FCM strain assimilated 0.61 mM formate. This indicates that glyA overexpression is important for formate assimilation. Finally, we developed L-serine and acetoin producing strain as benchmarks. In L-serine-producing strain, sdaA, which encodes serine deaminase, was knocked out to prevent L-serine degradation. The L-serine-producing strain produced up to 9.2 mM L-serine from 8.3 mM formate and 26.4 mM glycine whereas the control strain did not produce any L-serine. In acetoin-producing strain, alsS and alsD from Bacillus subtilis were introduced into the FCMG strain. The constructed strain produced up to 3.83 mM acetoin and, which was 1.49-fold higher than that of the control strain. These results suggest that the engineered strains are capable of converting formate into other valuable compounds.