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Lactate has various uses as industrial platform chemical, poly-lactic acid precursor or feedstock for anaerobic co-cultivations. The aim of this study was to construct and characterise  Acetobacterium woodii  strains capable of autotrophic lactate production. Therefore, the  lctBCD  genes, encoding the native Lct dehydrogenase complex, responsible for lactate consumption, were knocked out. Subsequently, a gene encoding a  d -lactate dehydrogenase (LDHD) originating from  Leuconostoc mesenteroides  was expressed in  A. woodii , either under the control of the anhydrotetracycline-inducible promoter P  tet   or under the lactose-inducible promoter P  bgaL  . Moreover, LDHD was N-terminally fused to the oxygen-independent fluorescence-activating and absorption-shifting tag (FAST) and expressed in respective  A. woodii  strains. Cells that produced the LDHD fusion protein were capable of lactate production of up to 18.8 mM in autotrophic batch experiments using H 2  + CO 2  as energy and carbon source. Furthermore, cells showed a clear and bright fluorescence during exponential growth, as well as in the stationary phase after induction, mediated by the N-terminal FAST. Flow cytometry at the single-cell level revealed phenotypic heterogeneities for cells expressing the FAST-tagged LDHD fusion protein. This study shows that FAST provides a new reporter tool to quickly analyze gene expression over the course of growth experiments of  A. woodii . Consequently, fluorescence-based reporters allow for faster and more targeted optimization of production strains.   Key points  • Autotrophic lactate production was achieved with A. woodii . • FAST functions as fluorescent marker protein in A. woodii . • Fluorescence measurements on single-cell level revealed population heterogeneity.

Autotrophic lactate production from H2 + CO2 using recombinant and fluorescent FAST-tagged Acetobacterium woodii strains