Engineering of Klebsiella oxytoca for simultaneous utilization of multiple sugars

Abstract

The processes that produce bio-chemicals from biomasses are sustainable and environmentally friendly methods

however, maximizing sugar utilization is needed for the processes to be more economical. Galactose or xylose is the second most prevalent carbon source present next to glucose according to the kind of biomass. To utilize the three sugars for the production of 2,3-butanediol, Klebsiella oxytoca was used because it can utilize many different substrates in its metabolism. Like other bacteria, the wild type of K.oxytoca assimilates glucose first before utilizing galactose and xylose because of carbon catabolite repression. The sequential utilization of the three sugars lowers the yield and productivity of 2,3-butanediol. In this study, K.oxytoca was genetically engineered to co-utilize the three sugars efficiently. The glucose phosphotransferase system ($PTS^{glc}$), which is the major cause for carbon catabolite repression, was disrupted, and a xylose/$H^+$ symporter was introduced to enhance the consumption rate of the slowest consumed-sugar, xylose. After that, the expression level of glucokinase was modulated, and adaptive laboratory evolution was carried out under a non-metabolizable sugar, 2-deoxy glucose, to recover the diminished glucose consumption rate by the disruption of $PTS^{glc}$. The disruption of $PTS^{glc}$ resulted in simultaneous utilization of 90% galactose and 30% xylose along with glucose, and the co-utilization ratio of xylose was increased to 80% by introducing the xylose/$H^+$ symporter. Adjustment of the glucokinase expression level under a constitutive promoter (J23115) and adaptive evolution led to a 49% recovery of the decreased glucose consumption rate by disruption of the $PTS^{glc}$. Finally, the developed strain showed a 35% and 40% higher overall consumption rate and 2,3-butanediol productivity, respectively, than that of the wild type strain in the three-sugar cultures. The engineered K.oxytoca could serve as a platform for strain development to co-utilize mixed sugars efficiently. In bio-refineries using the hydrolysates of biomasses, mixed sugars can be simultaneously used to produce high value-products such as enzymes and medicines through strain development based on the platform technique. The higher productivity will increase the competitiveness of the whole process.