(597b) Genome Engineering of Lactococcus Lactis for Pyrolytic Sugar Usage By a Cryptic Native Transformation Pathway

Lactococcus lactis, a gram-positive lactic acid bacterium, is an important organism in the dairy, chemical, and health industries. We have engineered this organism to consume the sugar levoglucosan, produced from pyrolysis of lignocellulosic biomass. Expression of the levoglucosan kinase gene from Lipomyces starkeyi, allows L. lactis to consume pure levoglucosan and levoglucosan-rich bio-oil as a sugar source, producing optically pure L-lactic acid. However, lignocellulosic pyrolysate contains several inhibitory phenolic compounds which limit the growth of bacteria under fermentation conditions. We constructed a genome-scale library to identify genes and pathways which contribute to growth in bio-oil and confer resistance to inhibitors. In addition, we demonstrate a CRISPR-Cas9-assisted unique natural transformation technique to efficiently construct strains with the identified beneficial mutations on the genome level. Furthermore, we discuss metabolic engineering strategies for in vivo conversion of L-lactate to other high-value chemicals.