Heterologous expression of a glycosyl hydrolase and cellular reprogramming enable Zymomonas mobilis growth on cellobiose

Abstract

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, including Escherichia coli , Saccharomyces cerevisiae , and Zymomonas 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 for Z . mobilis , we engineered a strain ( Z . mobilis GH3) that expresses a glycosyl hydrolase (GH) with β-glucosidase activity from a related α-proteobacterial species, Caulobacter crescentus , and 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 extracellular release 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 h and use it for growth and ethanol production. Alternatively, pre-growth of Z . mobilis GH3 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 release or surface display of GHs, entry of cellobiose into the periplasm, or both. Our two key findings are that Z . mobilis can be reprogrammed to grow on cellobiose as a sole carbon source and that this reprogramming is related to a natural response of Z . mobilis to sucrose that promotes sucrase production.

Whole‐cell biocatalysis: Advancements toward the biosynthesis of fuels

“…mobilis AX101 that recombinantly expresses the arabinose and xylose utilization gene cascade 53 . To increase the substrate availability, heterologous expression of glycosyl hydrolases from Cellvibrio japonicus Cel3A and Caulobacter crescentus CC_0968 enabled Z. mobilis to use cellobiose for ethanol production 54 . As Z. mobilis uses the Entner Doudoroff pathway, producing only one ATP, it raises concern over cellular health if multiple genes are expressed at once.…”

“…53 To increase the substrate availability, heterologous expression of glycosyl hydrolases from Cellvibrio japonicus Cel3A and Caulobacter crescentus CC_0968 enabled Z. mobilis to use cellobiose for ethanol production. 54 As Z. mobilis uses the Entner Doudoroff pathway, producing only one ATP, it raises concern over cellular health if multiple genes are expressed at once. Thus, it is essential to introduce pathways that can satisfy the needs independently with minimal or no effect on cellular viability.…”

Zymomonas mobilis as an emerging biotechnological chassis for the production of industrially relevant compounds

“…However, these studies only demonstrated lignocellulosic materials conversion in resting cells. In order to overcome this issue, Kurumbang et al ( 2020 ) proposed the heterologous expression and secretion of a glycosyl hydrolase (GH) β-glucosidase from Caulobacter crescentus in Z. mobilis , that enables an efficient conversion of oligosaccharides. The engineered strain was further subjected to an adaptation in cellobiose medium, and growth on cellobiose was achieved.…”