Surface tethered xylosidase activity improved xylan conversion in engineered strains of Saccharomyces cerevisiae

Abstract

BACKGROUND Second‐generation biofuel production strategies require utilization of the largest possible fraction of the available sugars in renewable biomass. Xylose can make up as much as 35% of plant dry cell weight (DCW), primarily in the form of the hemicellulose polymer xylan. Xylo‐oligosaccharides (XOS) are breakdown products of xylan released during pre‐treatment and hydrolysis that may inhibit the fermentation process. To enable growth on xylan and the removal of inhibitory XOS, CRISPR‐Cas9 was used to confer xylanase and GH43 xylosidase activity to xylose‐assimilating Saccharomyces cerevisiae strains. Xylosidase activity was engineered to be cell free or tethered to the yeast cell surface. RESULTS Gene‐integration and expression of both xylanolytic enzyme‐encoding genes was successful. Secretion yielded higher overall xylosidase activity when strains were cultivated on glucose, but cell‐associated activity was higher when strains were cultivated in the presence of xylose. Growth trials on both xylan and XOS showed that a combination of both enzymes, along with cell‐associated xylosidase production, resulted in the best growth on both substrates. The increased growth on xylan also translated to substantial improvements in ethanol production from polymeric xylan as sole carbon source. CONCLUSION Increased enzyme production, growth capabilities, and hemicellulosic substrate conversion due to cell‐tethered activity over secreted activity were shown for the first time. The use of a GH43 xylosidase to help avoid problematic transglycosylation for this purpose is also novel. The development of S. cerevisiae strains capable of xylan utilization and fermentation brings the industry a step closer to the ideal of utilizing the full range of sugars in biomass feedstocks for large scale ethanol production. © 2022 Society of Chemical Industry (SCI).

Engineering natural isolates of Saccharomyces cerevisiae for consolidated bioprocessing of cellulosic feedstocks

“…To create yeast strains expressing a core set of cellulases in robust strain backgrounds, we initially transformed natural and reference S. cerevisiae isolates with pCas9-NAT, and with each respective gRNA plasmid and homology repair cassette, corresponding to T.r.eg2 , S.f.bgl1 , and T.e.cbh1 , in successive rounds of transformation. Through the CRISPR/Cas9 system, these gene cassettes could be integrated into targeted intergenic regions on Chromosome 10, -11, or in the repeated -sequences, as previously described (Jacob et al 2022 ; Kruger and Den Haan 2022 ). For each successive round of transformation, a homology repair cassette with its respective gRNA plasmid was transformed into the individual strain isolates (Table 2 ).…”

“…BGL 1 - PGK1 T gene cassette Van Rooyen et al ( 2005 ) pMU784 Contains PGK1 p - C.l. CBH 2 - PGK1 T gene cassette Ilmén et al ( 2011 ) pIBG-SSAD Contains SED1 p - A.a. BGL 1 - DIT1 T gene cassette Inokuma et al ( 2021 ) pCas9-Nat Plasmid with cas9 expression cassette ADDGENE pRS42-G-ChX gRNA scaffold plasmid that targets chromosome 10 intergenic region Jacob et al ( 2022 ) pRS42-G-ChXI gRNA scaffold plasmid that targets chromosome 11 intergenic region Kruger and Den Haan ( 2022 ) pRS42-G-Delta gRNA scaffold plasmid that targets delta sequences within the yeast chromosome Jacob et al ( 2022 ) …”

CRISPR-Based Multi-Gene Integration Strategies to Create Saccharomyces cerevisiae Strains for Consolidated Bioprocessing

“…terminator for gRNA expression [21] pRS42-G-DELTA Similar to pRS42G_ChX, but targeting the yeast DELTA sequences This study pRSCG_ChXI pRS423-cas9-gRNA-G418 targeting Chromosome XI (Ch11) intergenic region protospacer; G418 resistance; this plasmid also contains the S.p.Cas9 under TEF1 promoter and CYC1 terminator -1-plasmid system.…”

Co-Fermentation of Glucose–Xylose–Cellobiose–XOS Mixtures Using a Synthetic Consortium of Recombinant Saccharomyces cerevisiae Strains

“…Various origins of β-glucosidase genes have been isolated and heterologously expressed in S. cerevisiae to enable the growth and fermentation of cellobiose [12,22,23]. The β-xylosidase coding genes were also expressed in S. cerevisiae single or together with the xylanase/β-glucosidase gene to utilize XOS or xylan as a carbohydrate source [13,21,24] or to co-ferment cellulose and xylan [25].…”