Process for assembly and transformation into Saccharomyces cerevisiae of a synthetic yeast artificial chromosome containing a multigene cassette to express enzymes that enhance xylose utilization designed for an automated pla

Authors: Hughes, Stephen; Cox, Elby; BANG, SOOKIE - South Dakota School Of Mines And Technology; PINKELMAN, REBECCA - South Dakota State University; LOPEZ-NUNEZ, JUAN CARLOS - Cenicafe; Saha, Badal; Qureshi, Nasib; GIBBONS, WILLIAM R - South Dakota State University; FRY, MICHELLE - Bradley University; Moser, Bryan; Bischoff, Kenneth; Liu, Siqing; JONES, MARJORIE - Illinois State University; RIANO-HERRERA, NESTOR - Cenicafe

Submitted to: Journal of Laboratory Automation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/27/2015
Publication Date: 2/1/2015
Publication URL: http://handle.nal.usda.gov/10113/62180
Citation: Hughes, S.R., Cox, E.J., Bang, S.S., Pinkelman, R.J., Lopez-Nunez, J.C., Saha, B.C., Qureshi, N., Gibbons, W.R., Fry, M.R., Moser, B.R., Bischoff, K.M., Liu, S., Sterner, D.E., Butt, T.R., Reidmuller, S.B., Jones, M.A., Riano-Herrera, N.M. 2015. Process for assembly and transformation into Saccharomyces cerevisiae of a synthetic yeast artificial chromosome containing a multigene cassette to express enzymes that enhance xylose utilization designed for an automated platform. Journal of Laboratory Automation. 20(6):621-635. doi: 10.1177/2211068215573188.

Interpretive Summary: Converting renewable cellulosic biomass resources into commercially viable biofuels and bioproducts requires utilization of all sugars in biomass of which glucose and xylose are the most abundant. Saccharomyces cerevisiae, currently the most-used industrial microbial catalyst, ferments glucose but not xylose. To enable xylose utilization in S. cerevisiae, we constructed an artificial chromosome containing a gene for simultaneous expression of xylose isomerase and xylulokinase and stably introduced it into S. cerevisiae without affecting the integrity of the strain. The transformed strain produced high levels of ethanol from whole-corn biomass. This research will benefit scientists developing microbial catalysts capable of utilizing all sugars found in cellulosic biomass. The stable artificial chromosome system also allows introduction of genes expressing high-value bioproducts or expressing enzymes for entire metabolic pathways to improve profitability of biomass utilization.

Technical Abstract: A yeast artificial chromosome (YAC) containing a multigene cassette for expression of enzymes that enhance xylose utilization (xylose isomerase [XI] and xylulokinase [XKS]) was constructed and transformed into Saccharomyces cerevisiae to demonstrate feasibility as a stable protein expression system in yeast and to design an assembly process suitable for an automated platform. Expression of XI and XKS from the YAC was confirmed by Western blot and PCR analyses. The recombinant and wild-type strains showed similar growth on plates containing hexose sugars, but only recombinant grew on D-xylose and L-arabinose plates. In glucose fermentation, doubling time (4.6 h) and ethanol yield (0.44 g ethanol/g glucose) of recombinant were comparable to wild type (4.9 h and 0.44 g/g). In whole-corn hydrolysate, ethanol yield (0.55 g ethanol/g [glucose + xylose]) and xylose utilization (38%) for recombinant were higher than for wild type (0.47 g/g and 12%). In hydrolysate from spent coffee grounds, yield was 0.46 g ethanol/g (glucose + xylose), and xylose utilization was 93% for recombinant. These results indicate introducing a YAC expressing XI and XKS enhanced xylose utilization without affecting integrity of the host strain, and the process provides a potential platform for automated synthesis of a YAC for expression of multiple optimized genes to improve yeast strains.