Metabolic engineering of Clostridium kluyveri DSM555^T for increased CO tolerance

In biotechnology, microbial co-cultures offer the potential to combine metabolic properties from different organisms. As an example, the gas-fermenting Clostridium carboxidivorans supplies the carbon chain elongating Clostridium kluyveri with its substrates ethanol and acetate for the production of medium chain fatty acids, which in turn are finally reduced to the corresponding alcohols like butanol, hexanol and even octanol. The use of syngas as the initial substrate, which is derived from gasification of municipal or food waste or as a by-product from the steel industry, has the advantage of producing these bulk chemicals solely from waste materials as a sustainable alternative to petrochemical synthesis. Unfortunately, analysis of this co-culture revealed that at CO concentrations that are most favorable for good ethanol and acetate production by C. carboxidivorans, reversible growth-inhibition of C. kluyveri was observed, presumably by inhibition of the [FeFe]-hydrogenases.

In an effort to adapt C. kluyveri genetically to high CO partial pressures, a plasmid-encoded heterologous alcohol aldehyde dehydrogenase (AdhE2) was introduced in the organism as a potential alternative route for redox factor regeneration together with the organism"s Rnf complex. To this end, a protocol for triparental conjugation enabling reliable transfer of plasmids into the C. kluyveri DSM555^T wildtype strain was established. Protection against a dominant type II restriction-modification (RM) system was achieved by in vivo methylation of the plasmid in the E. coli donor strain. Furthermore, chromosomal insertion of the AdhE2 is attempted with a codAB-counterselection system and a highly efficient CRISPR-Cas9 system controlled by a theophylline riboswitch. For these engineering efforts the expression strength of different inducible and constitutive promoters from various Clostridium species, Bacillus subtilis as well as some synthetic promoters were characterized in C. kluyveri using FAST fusions as reporters. The repertoire of promoters is the basis for adjustments of the expression levels of the components of the gene editing systems and the introduced enzymes like AdhE2.