Characterisation of acetogen formatotrophic potential using E. limosum

Formate is a promising energy carrier that could be used to transport renewable electricity. Some acetogenic bacteria, such as Eubacterium limosum , have the native ability to utilise formate as a sole substrate for growth, which has sparked interest in the biotechnology industry. However, formatotrophic metabolism in acetogens is poorly understood, and a systems-level characterization in continuous cultures is yet to be reported. Here we present the first steady-state dataset for E. limosum formatotrophic growth. At a defined dilution rate of 0.4 d-1, there was a high specific uptake rate of formate (280±56 mmol/gDCW/d), however, most carbon went to CO2 (150±11 mmol/gDCW/d). Compared to methylotrophic growth, protein differential expression data and intracellular metabolomics revealed several key features of formate metabolism. Upregulation of pta appears to be a futile attempt of cells to produce acetate as the major product. Instead, a cellular energy limitation resulted in the accumulation of intracellular pyruvate and upregulation of Pfl to convert formate to pyruvate. Therefore, metabolism is controlled, at least partially, at the protein expression level, an unusual feature for an acetogen. We anticipate that formate could be an important one-carbon substrate for acetogens to produce chemicals rich in pyruvate, a metabolite generally in low abundance during syngas growth. ### Competing Interest Statement The authors have declared no competing interest. * D : Dilution rate LLOQ : Lower Limit of Quantification OD : Optical Density WLP : Wood-Ljungdahl Pathway 2PG : 2-phosphoglycerate 3DHQ : 3-dehydroquinate 3DHSA : 3-dehydroshikimate 3PG : 3-phosphoglycerate Ac-CoA : acetyl-CoA BPG : 1,3-biphosphoglycerate DAHP : 3-deoxy-arabino-heptulonate 7-phosphate DHAP : Dihydroxy acetone phosphate E4P : Erythrose 4-phosphate EPSP : 3-enolpyruvyl-shikimate 5-phosphate F6P : Fructose 6-hosphate FBP : Fructose 1,6-biphosphate G3P : Glyceraldehyde 3-phosphate G6P : Glucose 6-phosphate Glc : Glucose PEP : Phosphoenolpyruvate PRPP : 5-phosphoribosyl diphosphate PYR : Pyruvate R5P : Ribose 5-phosphate RL5P : Ribulose 5-phosphate S3P : Shikimate 3-phosphate S7P : Sedoheptulose 7-phosphate THF : tetrahydrofolate X5P : Xylulose 5-phosphate ALD : aldehyde dehydrogenase ACK : acetate kinase ACN : anonitase ACS : acetyl-CoA synthase ADH : alcohol dehydrogenase AOR : aldehyde:ferredoxin oxidoreductase aroA : DAHP synthase aroA : 3-phosphoshikimate 1-carboxyvinyltransferase aroB : 3-dehydroquinate synthase aroC : chorismate synthase aroD : 3-dehydroquinate dehydratase aroKL : shikimate kinase BK : butyrate kinase cimA : citramalate synthase CODH : CO dehydrogenase crt : crotonase CS : citrate synthase ENO : enolase Etf-Bcd : butyryl-CoA dehydrogenase FBA : Fructose biphosphate aldose FBP : fructose 1 FDH : formate dehydrogenase FTHFS : formyl-THF synthetase FUM : fumarase GAD : glutamate decarboxylase GAPDH : glyceraldehyde 3-phosphate dehydrogenase GDH : glutamate dehydrogenase GLK : Glucokinase hbd : 3-hydroxybutyryl-CoA dehydrogenase IDH : isocitrate dehydrogenase ilvBN : pyruvate:2-oxobutanoate acetaldehydetransferase ilvC : 2-aceto-2hydroxy-butanoate:NADP+ oxidoreductase ilvD : dihydroxy acid dehydratase ilvE : branched chain amino acid aminotransferase KOR : oxoglutarate ferredoxin oxidoreductase leuB : methylmalate dehydrogenase leuCD : methylmalate hydrolase LeuDH : leucine dehydrogenase MDH : malate dehydrogenase MtaABC : methanol dependent methyl transferase MTHFC : methenyl-THF cylcohydrolase MTHFD : methylene-THF dehydrogenase MTHFR : methyltransferase/methylene-THF reductase PC : pyruvate carboxylase PD : pyruvate dehydrogenase PFKA : Phosphofructose kinase PFL : pyruvate formate ligase PFOR : pyruvate ferredoxin oxidoreductase PGAM : phosphoglycerate mutase PGI : Glucose 6-phosphate isomerase PGK : phosphoglycerate kinase PK : pyruvate kinase PPDK : phosphoenolpyruvate synthetase PRPS : Ribose phosphat epyrophosphokinase PTA : phosphotransacetylase PTB : phosphotransbutyrylase PTS : phosphotransferase system RPE : Ribulose phosphate epimerase RPI : Ribose phosphate isomerase Stn : Sporomusa type Nfn transhydrogenase TAL : Transaldolase thl : thiolase TKT : Transketolase TPI : Trisephosphate isomerase trpE : anthranilate synthase ydiB : shikimate dehydrogenase