Hemicellulose acts as one factor contributing to the recalcitrance of lignocellulose that prevents cellulases to degrade the cellulose efficiently even in low quantities. Supplement of hemicellulases can enhance the performance of commercial cellulases in the enzymatic hydrolyses of lignocellulose. Kluyveromyce marxianus is an attractive yeast for cellulosic ethanol fermentation, as well as a promising host for heterologous protein production, since it has remarkable thermotolerance, high growth rate, and broad substrate spectrum etc. In this study, we attempted to coordinately express multiple hemicellulases in K. marxianus through a 2A-mediated ribosome skipping to self-cleave polyproteins, and investigated their capabilities for saccharification and ethanol production from corncobs.

The FMDV P2A sequence showed high efficiency in self-cleavage of polyproteins in K. marxianus and could be used for secretory expression of multiple enzymes in the presence of their signal sequences. The IMPαXPαR strain coexpressed three hemicellulolytic enzymes improved the saccharification and ethanol production from corncobs, and could be used as a promising strain for ethanol production from lignocelluloses.

Two polycistronic genes IMPX and IMPαX were constructed to test the self-cleavage of P2A sequence from the Foot-and-Mouth Disease virus (FMDV) in K. marxianus. The IMPX gene consisted of a β-mannanase gene M330 (without the stop codon), a P2A sequence and a β-xylanase gene Xyn-CDBFV in turn. In the IMPαX gene, there was an additional α-factor signal sequence in frame with the N-terminus of Xyn-CDBFV. The extracellular β-mannanase activities of the IMPX and IMPαX strains were 21.34 and 15.50 U/mL, respectively, but the extracellular β-xylanase activity of IMPαX strain was much higher than that of the IMPX strain, which was 136.17 and 42.07 U/mL, respectively. Subsequently, two recombinant strains, the IXPαR and IMPαXPαR, were constructed to coordinately and secretorily express two xylantic enzymes, Xyn-CDBFV and β-D-xylosidase RuXyn1, or three hemicellulolytic enzymes including M330, Xyn-CDBFV and RuXyn1. In fed-batch fermentation, extracellular activities of β-xylanase and β-xylosidase in the IXPαR strain were 1664.2 and 0.90 U/mL. Similarly, the IMPαXPαR strain secreted the three enzymes, β-mannanase, β-xylanase, and β-xylosidase, with the activities of 159.8, 2210.5, and 1.25 U/mL, respectively. Hemicellulolases of both strains enhanced the yields of glucose and xylose from diluted acid pretreated (DAP) corncobs when acted synergistically with commercial cellulases. In hybrid saccharification and fermentation (HSF) of DAP corncobs, hemicellulases of the IMPαXPαR strain increased the ethanol yield by 8.7% at 144 h compared with the control. However, both ethanol and xylose yields were increased by 12.7 and 18.2%, respectively, at 120 h in HSF of aqueous ammonia pretreated (AAP) corncobs with this strain. Our results indicated that coordinate expression of hemicellulolytic enzymes in K. marxianus promoted the saccharification and ethanol production from corncobs. Conclusions: The FMDV P2A sequence showed high efficiency in self-cleavage of polyproteins in K. marxianus and could be used for secretory expression of multiple enzymes in the presence of their signal sequences. The IMPαXPαR strain coexpressed three hemicellulolytic enzymes improved the saccharification and ethanol production from corncobs, and could be used as a promising strain for ethanol production from lignocelluloses.