Metabolic engineering of Saccharomyces cerevisiae for second-generation ethanol production from xylo-oligosaccharides and acetate

Procopio, D. P.; Lee, J. W.; Shin, J.; Tramontina, R.; Avila, P. F.; Brenelli, L. B.; Squina, F. M.; Damasio, A.; Rabelo, S. C.; Goldbeck, R.; Franco, T. T.; Leak, D.; Jin, Y.-S.; Basso, T. O.

doi:10.1101/2023.02.04.527128

bioRxiv · 10.1101/2023.02.04.527128

Metabolic engineering of Saccharomyces cerevisiae for second-generation ethanol production from xylo-oligosaccharides and acetate

Procopio, D. P.·Lee, J. W.·Shin, J.·Tramontina, R.·Avila, P. F.·Brenelli, L. B.·Squina, F. M.·Damasio, A.·Rabelo, S. C.·Goldbeck, R.·Franco, T. T.·Leak, D.·Jin, Y.-S.·Basso, T. O.

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Abstract

AO_SCPLOWBSTRACTC_SCPLOWSimultaneous intracellular depolymerization of xylo-oligosaccharides (XOS) and acetate fermentation by engineered Saccharomyces cerevisiae offers an advance towards more cost-effective second-generation (2G) ethanol production. As xylan is one of the most abundant polysaccharides present in lignocellulosic residues, the transport and breakdown of XOS in an intracellular environment might bring a competitive advantage for recombinant strains in competition with contaminating microbes, which are always present in fermentation tanks; furthermore, acetic acid is a ubiquitous toxic component in lignocellulosic hydrolysates, deriving from hemicellulose and lignin breakdown. In the present work, the previously engineered S. cerevisiae strain, SR8A6S3, expressing NADPH-linked xylose reductase (XR), NAD+-linked xylitol dehydrogenase (XDH) (for xylose assimilation), as well as NADH-linked acetylating acetaldehyde dehydrogenase (AADH) and acetyl-CoA synthetase (ACS) (for an NADH-dependent acetate reduction pathway), was used as the host for expressing of two {beta}-xylosidases, GH43-2 and GH43-7, and a xylodextrin transporter, CDT-2, from Neurospora crassa, yielding the engineered strain SR8A6S3-CDT2-GH432/7. Both {beta}-xylosidases and the transporter were introduced by replacing two endogenous genes, GRE3 and SOR1, that encode aldose reductase and sorbitol (xylitol) dehydrogenase, respectively, which catalyse steps in xylitol production. Xylitol accumulation during xylose fermentation is a problem for 2G ethanol production since it reduces final ethanol yield. The engineered strain, SR8A6S3-CDT2-GH432/7, produced ethanol through simultaneous co-utilization of XOS, xylose, and acetate. The mutant strain produced 60% more ethanol and 12% less xylitol than the control strain when a hemicellulosic hydrolysate was used as a mono- and oligosaccharide source. Similarly, the ethanol yield was 84% higher for the engineered strain using hydrolysed xylan compared with the parental strain. The consumption of XOS, xylose, and acetate expands the capabilities of S. cerevisiae for utilization of all of the carbohydrate in lignocellulose, potentially increasing the efficiency of 2G biofuel production. HighlightsO_LIIntegration of XOS pathway in an acetate-xylose-consuming S. cerevisiae strain; C_LIO_LIIntracellular fermentation of XOS, acetate and xylose improved ethanol production; C_LIO_LIDeletion of both sor1{Delta} and gre3{Delta} reduced xylitol production. C_LI

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Procopio, D. P., Lee, J. W., Shin, J., Tramontina, R., Avila, P. F., Brenelli, L. B., Squina, F. M., Damasio, A., Rabelo, S. C., Goldbeck, R., Franco, T. T., Leak, D., Jin, Y.-S., Basso, T. O.. 2023-02-04. Metabolic engineering of Saccharomyces cerevisiae for second-generation ethanol production from xylo-oligosaccharides and acetate. https://doi.org/10.1101/2023.02.04.527128

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