Climate change progresses by increasing amounts of greenhouse gases such as CO2 in our atmosphere and is intensified by the production and disposal of e.g. plastics. A. woodii is capable of autotrophic growth using H2 + CO2, producing acetate. By plasmid-borne expression of a D-lactate dehydrogenase (ldhD) from Leuconostoc mesenteriodes and deleting the native genes encoding the bifurcating lactate dehydrogenase complex, a recombinant A. woodii strain was constructed, capable of D-lactate production from, using H2 + CO2 (Mook et al., 2022, doi: 10.1007/s00253-022-11770-z). D-lactate is the monomer for the bioplastic polylactic acid (Vaidya et al., 2005, doi: 10.1080/10643380590966181).

The lactate production shall be increased in A. woodii by expressing the pyruvate-formate-lyase (PFL) to bypass the methyl branch of the Wood-Ljungdahl pathway. PFL functions in a reversible manner in many anaerobic bacteria, converting acetyl-CoA and formate to HS-CoA and pyruvate, depending on a PFL-activating enzyme (ACT). A plasmid containing the genes pfl (controlled by the theophylline-inducible PackA-theo promoter (Beck et al., 2020, doi: 10.1007/s00253-019-10248-9)) and act (controlled by the constitutive Ppta-ack promoter (Clostridium ljungdahlii) from C. pasteurianum was constructed.
A. woodii"s endogenous type 1-B CRISPR-Cas system was used to delete its pheA gene, using a plasmid containing the CRISPR array, spacer and homology arms needed for homologous recombination (Poulalier-Delavelle et al., 2023 doi: 10.3389/fbioe.2023.1213236). Currently the endogenous CRISPR-Cas system was successfully applied to implement a pheA deletion in A. woodii, verified via PCR. Next, pheA will be restored together with the ldhD gene regulated by the lactose inducible promoter system PbgaL from C. perfringens as well as by the lactate inducible promoter system PlctA from A. woodii. Thus, two plasmids containing the respective CRISPR array, spacer, pheA locus, ldhD and promoter systems were constructed.

These plasmids will be used to construct respective A. woodii strain and growth as well as production kinetics will be analyzed.