Lactobacillus casei as a biocatalyst for the production of isobutanol

Isobutanol has advantages over ethanol, which include higher energy density and lower hygroscopicity. L. casei presents advantages over common biocatalysts, such as high tolerance for alcohols, availability of genetic tools, utilization of plant carbohydrates of interest, and high tolerance to inhibitors present in pretreated lignocellulosic biomass. To demonstrate this, L. casei strains were screened for alcohol tolerance. L. casei ATCC 334 and 12A exhibited the highest isobutanol tolerance (1.96 and 1.89%). L. casei 12A had the most advance genetic tools; hence it was selected for use in all subsequent studies. L. casei 12A was compared to E. coli and Z. mobilis, in terms of tolerance to alcohols and stressors present in corn stover hydrolysate. Results indicate L. casei 12A is more robust.

To performed metabolic flux analysis of L. casei 12A and ATCC 334, in silico genome-scale metabolic models were constructed and validated against physiological data. We compared the metabolic networks of these two strains to identify metabolic, genetic and ortholog differences that may lead to different phenotypic behaviors. 

 To enable production of isobutanol in L. casei 12A an isobutanol cassette was synthesized and introduced into the 12A genome. Analysis of metabolic flux through central metabolism in L. casei 12A was performed with the aid of the L. casei 12A model and the isobutanol pathway was placed in context with competing pathways. Enzymes, responsible for the production of lactate were deleted. The expected outcome of the project is the optimization of L. casei 12A for the production of isobutanol.