Heterologous glycosyl hydrolase expression and cellular reprogramming resembling sucrose-induction enableZymomonas mobilisgrowth on cellobiose

Plant derived fuels and chemicals from renewable biomass have significant potential to replace reliance on petroleum and improve global carbon balance. However, plant biomass contains significant fractions of oligosaccharides that are not usable natively by many industrial microorganisms, includingEscherichia coli,Saccharomyces cerevisiae,andZymomonas mobilis. Even after chemical or enzymatic hydrolysis, some carbohydrate remains as non-metabolizable oligosaccharides (e.g., cellobiose or longer cellulose-derived oligomers), thus reducing the efficiency of conversion to useful products. To begin to address this problem forZ. mobilis, we engineered a strain (Z. mobilisGH3) that expresses a glycosyl hydrolase (GH) with β-glucosidase activity fromCaulobacter crescentusand subjected it to an adaptation in cellobiose medium. Growth on cellobiose was achieved after a prolonged lag phase in cellobiose medium that induced changes in gene expression and cell composition, including increased expression and secretion of GH. These changes were reversible upon growth in glucose-containing medium, meaning they did not result from genetic mutation but could be retained upon transfer of cells to fresh cellobiose medium. After adaptation to cellobiose, our GH-expressing strain was able to convert about 50% of cellobiose to glucose within 24 hours and use it for growth and ethanol production. Alternatively, pre-growth ofZ. mobilisGH3 in sucrose medium enabled immediate growth on cellobiose. Proteomic analysis of cellobiose- and sucrose-adapted strains revealed upregulation of secretion-, transport-, and outer membrane-related proteins, which may aid secretion or surface display of GHs, entry of cellobiose into the periplasm, or both. Our two key findings are thatZ. mobiliscan be reprogrammed to grow on cellobiose as a sole carbon source and that this reprogramming is related to a natural responseof Z. mobilisto sucrose that enables sucrose secretion.