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bioRxiv · 10.1101/2025.09.12.675768

Heterologous expression of pyruvate formate lyase enhances cell growth of Clostridium ljungdahlii during microbial electrosynthesis

Im, C.·Krige, A.·Valgepea, K.·Modin, O.·Nygard, Y.·Franzen, C. J.
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Abstract

Slow cell growth and low biomass yields are hurdles for gas fermentation by acetogens. Microbial electrosynthesis (MES) utilizes acetogens as biocatalysts to reduce CO2 and produce commodity chemicals using electricity. However, limited electron supply in a bioelectrochemical system (BES) aggravates the poor cell growth of acetogens resulting in low productivities. Formate is a soluble C1 feedstock that can be produced by CO2 reduction (1). Thus, assimilation of formate can unburden the amount of electrons required for MES. Acetobacterium woodii posseses multiple pyruvate formate lyase (PFL) genes that are upregulated during formatotrophic growth. In this study, Clostridium ljugdahlii was engineered to heterologously express a pfl geneset of A. woodii to increase the cell growth of C. ljungdahlii during microbial electrosynthesis. Different combinations of pfl A and pfl B from A. woodii were tested in C. ljungdahlii to find the best working combination under control of Pfdx-riboswitch expression system. Expression of pfl B1 and pflA of A. woodii showed higher maximum OD of C. ljugdahlii under H2:CO2 condition with supplementation of 80 mM sodium formate. More than 40 mM of sodium formate caused significantly longer lag phase but the lag phase could be shortened after adaptation in 80 mM of sodium formate. At the end, the engineered strain showed improved cell growth (ODmax 0.22 {+/-} 0.05) and acetate production (21.8 {+/-} 5.6 mM) during microbial electrosynthesis compared to the control strain (ODmax 0.10 {+/-}0.06 and 10.2 {+/-}2.5 mM acetate). These results will be useful for strain development for microbial electrosynthesis, as well as gas fermentation. HighlightsO_LIDifferent combinations of pyruvate formate lyases (pfl Bs) and pyruvate formate lyase acting enzymes (pfl As) from Acetobacterium woodii were tested to improve the cell growth of Clostridium ljungdahlii during gas fermentation and microbial electrosynthesis C_LIO_LIHeterologous expression of pfl B1 and pfl A using riboswitch expression system improved cell growth of C. ljungdahlii under H2:CO2 condition, even without inducer and formate supplementation. C_LIO_LIHigh concentraton of sodium formate caused longer lag phase, which was shortened by adaptation when pfl from A. woodii was heterologously expressed. C_LIO_LICell growth and acetate production of the engineered C. ljungdahlii strain improved during microbial electrosynthesis C_LI

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Im, C., Krige, A., Valgepea, K., Modin, O., Nygard, Y., Franzen, C. J.. 2025-09-13. Heterologous expression of pyruvate formate lyase enhances cell growth of Clostridium ljungdahlii during microbial electrosynthesis. https://doi.org/10.1101/2025.09.12.675768

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