Physiological characteristics of EAL domain mutants of Zymomonas mobilis for ethanol and bacterial cellulose production

Abstract

The physiological characteristics of cell flocculation has been widely studied in various industrial microorganisms to provide several advantages including the cost-efficient cell recovery, high cell density culture as well as relatively high resistance to various external environmental stresses such as toxic antimicrobial chemicals. To identify the major responsible genes involved in cell flocculation behaviors of an ethanol producing bacterium Zymomonas mobilis, the genes ZMO_1055 and ZMO_0401 coding for GGDEF-EAL hybrid domain proteins were targeted for the specific gene deletion. The GGDEF domain has a putative diguanylate cyclase (DGC) activity converting guanosine monophosphate (GMP) to cyclic di-GMP (c-di GMP) and the EAL domain has a putative phosphodiesterase (PDE) catalyzing c-di GMP to GMP or pGpG. The cellular concentration of the nucleotide c-di GMP known as bacterial specific signal molecule has been known to be governed by the two enzymes DGC and PDE according to their facing environments for the transition lifestyle between single cell behaviors and multicellular behaviors. Therefore to maintain cell flocculation behaviors in Z. mobilis with the relatively high cellular concentration of c-di-GMP, two ZMO_1055ΔEAL mutants, ZAM1 and ZAM2 (respectively derived from ZM4 (a planktonic stage strain) and ZM401 (an auto-flocculating strain)), the other two ZMO_0401ΔEAL mutants, ZAM3 and ZAM4 (respectively derived from ZM4 and ZM401) were characterized for their phenotypic properties including bacterial cellulose iv synthesis, cell motilities and concentration of intracellular c-di-GMP, and potential ethanol production capacities. The result indicated that among the four EAL mutant strains, the ZAM4 strain with ZMO_0401ΔEAL display a highest cell flocculation with highest content of bacterial cellulose and lowest cell motility. The quantification of the cellular concentration of c-di-GMP by LC-MS/MS analysis indicated that the degree of cell flocculation and the increased intracellular c-di-GMP level were positively correlated with the EAL mutant strains ZAM2 and ZAM4 derived from the ZM401 however the EAL mutant strains ZAM1 with ZMO_1055ΔEAL and ZAM3 with ZMO_0401ΔEAL derived from ZM4 showed a low concentration of c-di-GMP with non-flocculent cell stage. This result indicated other major responsible genes involved in the cell flocculation behavior in this bacterium. Lastly the ethanol production capacities for the four EAL mutants were investigated using synthetic lignocellulose media containing toxic chemical compounds such as acetic acid, furfural, and vanillin derived during the pretreatment step for lignocellulosic ethanol production process. Among the EAL mutant strains tested, ZAM1 and ZAM2 with ZMO_1055ΔEAL derived from ZM4 and ZM401 respectively showed positive effects on ethanol fermentation kinetics in the presence of such toxic inhibitors. Therefore, these cell flocculant strains could improve ethanol productivity and cost-effective cell recovery in ethanol fermentation. Moreover, in terms of bacterial cellulose production, ZAM4 with ZMO_0401ΔEAL derived from ZM401 showed highest bacterial cellulose production, although ZAM4 strain showed lower ethanol production kinetic parameters than those from ZM401. In conclusion, this study demonstrated that cell flocculant strains with EAL domain mutation could be useful strains in industrial application related with enhanced ethanol productivity as well as the bacterial cellulose production. Also, the cell flocculation behavior of Z. mobilis suggested the possibility of economical ethanol production by cost-efficient cell recovery characteristics.