Enhanced heterologous expression of Trichoderma reesei Cel5A/Cel6A in Pichia pastoris with extracellular co-expression of Vitreoscilla hemoglobin

BACKGROUNDThe deficiency of family 5 endoglucanase (Cel5A) and family 6 cellobiohydrolase (Cel6A) has become a key limiting factor on cellulase enzymatic hydrolysis in bioprocessing of cellulosic biomass. To improve the production of Trichoderma reesei Cel5A / Cel6A, a Vitreoscilla hemoglobin (VHb) gene was tried to co-express extracellularly for the first time with Cel5A / Cel6A in Pichia pastoris GS115. RESULTSNewly constructed recombinant of co-expressing Cel5A / Cel6A extracellularly with VHb was consistent with the single expression at some key variables of culture condition, i.e. inoculum size, initial pH, culture temperature and methanol concentration. Comparing their single expression, the CMCase activity of co-expressed Cel5A and Cel6A enzymes enhanced by 40% and 30%, respectively. With high-cell-density fed-batch (HCDFB) fermentation, the co-expressed Cel5A enzyme activity was 366.8U mL(-1) with 4.3g L-1 protein content and the Cel6A enzyme activity reached 1.3U mL(-1) with 2.23g L-1 protein content. The two co-expressed enzyme activities were enhanced by 35% and 20%, respectively, compared with the single expression. CONCLUSIONVHb protein capable of binding oxygen can be successfully co-expressed extracellularly with other target proteins. The co-expression of VHb with recombinant Cel5A / Cel6A is efficient at improving oxygen-limited condition and thus enzyme production in both shake-flask and HCDFB fermentation. (c) 2017 Society of Chemical Industry