title: 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.
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.
authors.affiliation: United States Department of Agriculture (USDA), Agricultural Research Service (ARS), National Center for Agricultural Utilization Research (NCAUR), Renewable Product Technology Research Unit, Peoria, IL, USA stephen.hughes@ars.usda.gov.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Hughes
authors.affiliation: United States Department of Agriculture (USDA), Agricultural Research Service (ARS), National Center for Agricultural Utilization Research (NCAUR), Renewable Product Technology Research Unit, Peoria, IL, USA Department of Chemistry and Biochemistry, Bradley University, Peoria, IL, USA.
authors.forename: Elby J
authors.initials: EJ
authors.lastname: Cox
authors.affiliation: South Dakota School of Mines & Technology, Chemical and Biological Engineering, Rapid City, SD, USA.
authors.forename: Sookie S
authors.initials: SS
authors.lastname: Bang
authors.affiliation: South Dakota School of Mines & Technology, Chemical and Biological Engineering, Rapid City, SD, USA.
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Pinkelman
authors.affiliation: National Coffee Research Centre (Cenicafe), National Federation of Coffee Growers of Colombia (FNC), Manizales, Caldas, Colombia.
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: López-Núñez
authors.affiliation: USDA, ARS, NCAUR, Bioenergy Research Unit, Peoria, IL, USA.
authors.forename: Badal C
authors.initials: BC
authors.lastname: Saha
authors.affiliation: USDA, ARS, NCAUR, Bioenergy Research Unit, Peoria, IL, USA.
authors.forename: Nasib
authors.initials: N
authors.lastname: Qureshi
authors.affiliation: Department of Biology and Microbiology, South Dakota State University, Brookings, SD, USA.
authors.forename: William R
authors.initials: WR
authors.lastname: Gibbons
authors.affiliation: Department of Chemistry and Biochemistry, Bradley University, Peoria, IL, USA.
authors.forename: Michelle R
authors.initials: MR
authors.lastname: Fry
authors.affiliation: USDA, ARS, NCAUR, Bio-Oils Research Unit, Peoria, IL, USA.
authors.forename: Bryan R
authors.initials: BR
authors.lastname: Moser
authors.affiliation: United States Department of Agriculture (USDA), Agricultural Research Service (ARS), National Center for Agricultural Utilization Research (NCAUR), Renewable Product Technology Research Unit, Peoria, IL, USA.
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Bischoff
authors.affiliation: United States Department of Agriculture (USDA), Agricultural Research Service (ARS), National Center for Agricultural Utilization Research (NCAUR), Renewable Product Technology Research Unit, Peoria, IL, USA.
authors.forename: Siqing
authors.initials: S
authors.lastname: Liu
authors.affiliation: Progenra, Inc., Malvern, PA, USA.
authors.forename: David E
authors.initials: DE
authors.lastname: Sterner
authors.affiliation: LifeSensors, Inc., Malvern, PA, USA.
authors.forename: Tauseef R
authors.initials: TR
authors.lastname: Butt
authors.affiliation: Hudson Control Group, Inc., Springfield, NJ, USA.
authors.forename: Steven B
authors.initials: SB
authors.lastname: Riedmuller
authors.affiliation: Department of Chemistry, Illinois State University, Normal, IL, USA.
authors.forename: Marjorie A
authors.initials: MA
authors.lastname: Jones
authors.affiliation: National Coffee Research Centre (Cenicafe), National Federation of Coffee Growers of Colombia (FNC), Manizales, Caldas, Colombia.
authors.forename: Néstor M
authors.initials: NM
authors.lastname: Riaño-Herrera
mesh_terms: D018244:Chromosomes, Artificial, Yeast; D003069:Coffee; D003470:Culture Media / Q000737:chemistry; D004798:Enzymes / Q000235:genetics*; D000431:Ethanol / Q000378:metabolism; D005285:Fermentation; D015870:Gene Expression; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D014170:Transformation, Genetic; D014994:Xylose / Q000378:metabolism*; D003313:Zea mays
chemical_list: D003069:Coffee; D003470:Culture Media; D004798:Enzymes; D000431:Ethanol; D014994:Xylose
keywords: multigene expression cassette; stable cellulosic yeast; synthetic biology platform; synthetic yeast chromosome assembly; yeast artificial expression chromosome