Improving autotrophic D-lactate production by heterologous expression of a pyruvate formate lyase in an LDHD-expressing Acetobacterium woodii strain

The anaerobic acetogen Acetobacterium woodii is a promising candidate producing chemicals while converting the greenhouse gas CO 2 . In this study, A. woodii was genetically engineered to express a lactate dehydrogenase (LDHD) from Leuconostoc mesenteroides , and a pyruvate formate lyase (PFL) as well as its activating enzyme (PFL-AE) originated from Clostridium pasteurianum to autotrophically produce D-lactate next to acetate, using H 2 + CO 2 . In this study, an improved D-lactate production was observed compared to the control strain without PFL and PFL-AE. Especially, during anabolism, positive effects were determined due to the recombinant expression of C. pasteurianum ’s PFL. During this work, the promoter system, used to control the expression of the recombinant ldhD gene, was exchanged to the lactate-inducible P lctA -promoter originated from A. woodii , which also caused an increase in D-lactate production. Cultivation of these P lctA promoter strains in a stirred-tank bioreactor (STR) resulted in elevated D-lactate and acetate concentrations compared to experiments in serum bottles and showed increased D-lactate accumulation during expression of the PFL compared to the strain carrying only the ldhD gene. A maximum D-lactate concentration of 21.0 ± 2.4 mM was reached in bottle-scale cultivation. Controlled STR batch cultivations with continuous gassing resulted in D-lactate concentrations of up to 34.9 mM. This finding demonstrates that A. woodii overexpressing LDHD, PFL, and PFL-AE is a robust strain for the autotrophic production of D-lactate.