title: Metabolic engineering of a haploid strain derived from a triploid industrial yeast for producing cellulosic ethanol.
abstract: Many desired phenotypes for producing cellulosic biofuels are often observed in industrial Saccharomyces cerevisiae strains. However, many industrial yeast strains are polyploid and have low spore viability, making it difficult to use these strains for metabolic engineering applications. We selected the polyploid industrial strain S. cerevisiae ATCC 4124 exhibiting rapid glucose fermentation capability, high ethanol productivity, strong heat and inhibitor tolerance in order to construct an optimal yeast strain for producing cellulosic ethanol. Here, we focused on developing a general approach and high-throughput screening method to isolate stable haploid segregants derived from a polyploid parent, such as triploid ATCC 4124 with a poor spore viability. Specifically, we deleted the HO genes, performed random sporulation, and screened the resulting segregants based on growth rate, mating type, and ploidy. Only one stable haploid derivative (4124-S60) was isolated, while 14 other segregants with a stable mating type were aneuploid. The 4124-S60 strain inherited only a subset of desirable traits present in the parent strain, same as other aneuploids, suggesting that glucose fermentation and specific ethanol productivity are likely to be genetically complex traits and/or they might depend on ploidy. Nonetheless, the 4124-60 strain did inherit the ability to tolerate fermentation inhibitors. When additional genetic perturbations known to improve xylose fermentation were introduced into the 4124-60 strain, the resulting engineered strain (IIK1) was able to ferment a Miscanthus hydrolysate better than a previously engineered laboratory strain (SR8), built by making the same genetic changes. However, the IIK1 strain showed higher glycerol and xylitol yields than the SR8 strain. In order to decrease glycerol and xylitol production, an NADH-dependent acetate reduction pathway was introduced into the IIK1 strain. By consuming 2.4g/L of acetate, the resulting strain (IIK1A) exhibited a 14% higher ethanol yield and 46% lower byproduct yield than the IIK1 strain from anaerobic fermentation of the Miscanthus hydrolysate. Our results demonstrate that industrial yeast strains can be engineered via haploid isolation. The isolated haploid strain (4124-S60) can be used for metabolic engineering to produce fuels and chemicals.
authors.affiliation: Energy Biosciences Institute, University of Illinois at Urbana-Champaign, Urbana, IL, USA; School of Food Science and Biotechnology, Kyungpook National University, Daegu, Korea.
authors.forename: Soo Rin
authors.initials: SR
authors.lastname: Kim
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, CA, USA; Department of Bioengineering, University of California, Berkeley, CA, USA; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Skerker
authors.affiliation: Energy Biosciences Institute, University of Illinois at Urbana-Champaign, Urbana, IL, USA; Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
authors.forename: In Iok
authors.initials: II
authors.lastname: Kong
authors.affiliation: Energy Biosciences Institute, University of Illinois at Urbana-Champaign, Urbana, IL, USA; Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
authors.forename: Heejin
authors.initials: H
authors.lastname: Kim
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, CA, USA.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Maurer
authors.affiliation: Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
authors.forename: Guo-Chang
authors.initials: GC
authors.lastname: Zhang
authors.affiliation: Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
authors.forename: Dairong
authors.initials: D
authors.lastname: Peng
authors.affiliation: Energy Biosciences Institute, University of Illinois at Urbana-Champaign, Urbana, IL, USA; Department of Civil and Environmental Engineering and Earth Sciences, University of Notre Dame,, South Bend, IN, USA.
authors.forename: Na
authors.initials: N
authors.lastname: Wei
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, CA, USA; Department of Bioengineering, University of California, Berkeley, CA, USA; Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. Electronic address: aparkin@lbl.gov.
authors.forename: Adam P
authors.initials: AP
authors.lastname: Arkin
authors.affiliation: Energy Biosciences Institute, University of Illinois at Urbana-Champaign, Urbana, IL, USA; Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL, USA; Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA. Electronic address: ysjin@illinois.edu.
authors.forename: Yong-Su
authors.initials: YS
authors.lastname: Jin
mesh_terms: D000085:Acetates / Q000378:metabolism; D053898:Biosynthetic Pathways / Q000235:genetics; D002482:Cellulose / Q000378:metabolism*; D000431:Ethanol / Q000302:isolation & purification / Q000378:metabolism*; D024861:Genetic Enhancement / Q000379:methods*; D006238:Haploidy; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000145:classification* / Q000502:physiology*; D013045:Species Specificity
chemical_list: D000085:Acetates; D000431:Ethanol; D002482:Cellulose
keywords: Cellulosic ethanol; Haploid isolation; Industrial strain; Inhibitor tolerance; Miscanthus; Polyploid; Xylose fermentation