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Screening and Engineering Yeast Transporters to Improve Cellobiose Fermentation by Recombinant Saccharomyces cerevisiae

Publication . Kretzer, Leonardo; Knychala, Marília; Silva, Lucca C.; Fontoura, Isadora C.C.; Leandro, Maria José; Fonseca, César; Verstrepen, Kevin J.; Stambuk, Boris

ABSTRACT: Developing recombinant Saccharomyces cerevisiae strains capable of transporting and fermenting cellobiose directly is a promising strategy for second-generation ethanol production from lignocellulosic biomass. In this study, we cloned and expressed in the S. cerevisiae CEN.PK2-1C strain an intracellular beta-glucosidase (SpBGL7) from Spathaspora passalidarum and co-expressed the cellobiose transporter SiHXT2.4 from Scheffersomyces illinoinensis, and two putative transporters, one from Candida tropicalis (CtCBT1 gene), and one from Meyerozyma guilliermondii (MgCBT2 gene). While all three transporters allowed cell growth on cellobiose, only the MgCBT2 permease allowed cellobiose fermentation, although cellobiose consumption was incomplete. The analysis of the beta-glucosidase and transport activities revealed that the cells stopped consuming cellobiose due to a drop in the transport activity. Since ubiquitinylation of lysine residues at the N- or C-terminal domains of the permease are involved in the endocytosis and degradation of sugar transporters, we constructed truncated versions of the permease lacking lysine residues at the C-terminal domain (MgCBT2 Delta C), and at both the C- and N-terminal domain (MgCBT2 Delta N Delta C) and co-expressed these permeases with the SpBGL7 beta-glucosidase in an industrial strain. While the strain harboring the MgCBT2 Delta C transporter continued to produce incomplete cellobiose fermentations as the wild-type MgCBT2 permease, the strain with the MgCBT2 Delta N Delta C permease was able to consume and ferment all the cellobiose present in the medium. Thus, our results highlight the importance of expressing cellobiose transporters lacking lysine at the N- and C-terminal domains for efficient cellobiose fermentation by recombinant S. cerevisiae.

2024-09Journal article

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