Batch and continuous culture-based selection strategies for acetic acid tolerance in xylose-fermenting Saccharomyces cerevisiae

Spent sulfite liquor (SSL) is a waste effluent from sulfite pulping that contains monomeric sugars which can be fermented to ethanol. However, fermentative yeasts used for the fermentation of the sugars in SSL are adversely affected by the inhibitory substances in this complex feedstock. To overcome this limitation, evolutionary engineering of Saccharomyces cerevisiae was carried out using genome-shuffling technology based on large-scale population cross mating. Populations of UV-light-induced yeast mutants more tolerant than the wild type to hardwood spent sulfite liquor (HWSSL) were first isolated and then recursively mated and enriched for more-tolerant populations. After five rounds of genome shuffling, three strains were isolated that were able to grow on undiluted HWSSL and to support efficient ethanol production from the sugars therein for prolonged fermentation of HWSSL. Analyses showed that greater HWSSL tolerance is associated with improved viability in the presence of salt, sorbitol, peroxide, and acetic acid. Our results showed that evolutionary engineering through genome shuffling will yield robust yeasts capable of fermenting the sugars present in HWSSL, which is a complex substrate containing multiple sources of inhibitors. These strains may not be obtainable through classical evolutionary engineering and can serve as a model for further understanding of the mechanism behind simultaneous tolerance to multiple inhibitors.

Section: Characterization Of Strains For Enhanced Viability and Growtmentioning

confidence: 99%

Saccharomyces cerevisiae Genome Shuffling through Recursive Population Mating Leads to Improved Tolerance to Spent Sulfite Liquor

Pinel

D'Aoust

Cardayré³

et al. 2011

“…Complex phenotypes, such as enhanced resistance to biofuels (3,4,12), lignocellulosic hydrolysates (2,13), antibiotics (14,15), and environmental conditions (16), have all been successfully characterized using this approach. In this study, sodium chloride (NaCl) was selected as the osmotic inhibitor.…”

mentioning

confidence: 99%

Evolved Osmotolerant Escherichia coli Mutants Frequently Exhibit Defective N -Acetylglucosamine Catabolism and Point Mutations in Cell Shape-Regulating Protein MreB

Winkler

Garcı́a

Olson

et al. 2014

c Biocatalyst robustness toward stresses imposed during fermentation is important for efficient bio-based production. Osmotic stress, imposed by high osmolyte concentrations or dense populations, can significantly impact growth and productivity. In order to better understand the osmotic stress tolerance phenotype, we evolved sexual (capable of in situ DNA exchange) and asexual Escherichia coli strains under sodium chloride (NaCl) stress. All isolates had significantly improved growth under selection and could grow in up to 0.80 M (47 g/liter) NaCl, a concentration that completely inhibits the growth of the unevolved parental strains. Whole genome resequencing revealed frequent mutations in genes controlling N-acetylglucosamine catabolism (nagC, nagA), cell shape (mrdA, mreB), osmoprotectant uptake (proV), and motility (fimA). Possible epistatic interactions between nagC, nagA, fimA, and proV deletions were also detected when reconstructed as defined mutations. Biofilm formation under osmotic stress was found to be decreased in most mutant isolates, coupled with perturbations in indole secretion. Transcriptional analysis also revealed significant changes in ompACGL porin expression and increased transcription of sulfonate uptake systems in the evolved mutants. These findings expand our current knowledge of the osmotic stress phenotype and will be useful for the rational engineering of osmotic tolerance into industrial strains in the future. Escherichia coli, an important industrial microorganism for the production of a wide variety of fine chemicals, fuels, and proteins, has been extensively targeted to improve its suitability as a biofactory. Strain development efforts have focused on improving tolerance of feedstocks containing toxic compounds (1, 2) or products (3, 4). Many environmental variables, including osmotic pressure, can negatively impact biocatalyst performance (5). Use of nonconventional waste streams, such as waste glycerol or brackish water sources, to support microbial growth can also reduce process costs (6, 7) while reducing pressure on fresh water resources; however, these carbon and water sources generally contain high concentrations of salt that may be inhibitory to microbial growth. In addition to osmotic stresses, excess Na ϩ can disrupt the ion homeostasis in E. coli as well (8). Previous studies have attempted to engineer improved osmotic tolerance in E. coli (9, 10), but overall, knowledge of the genetic mechanisms that confer tolerance of osmotic stress in general or to specific osmolytes remains limited. A detailed analysis of E. coli osmotolerance to osmolytes would therefore provide new insight into the molecular mechanisms underlying this complex phenotype.Adaptive laboratory evolution (11) is a promising approach to identify potentially novel osmotic tolerance mechanisms, as this technique requires no assumptions about the underlying genotype-phenotype relationship. Complex phenotypes, such as enhanced resistance to biofuels (3,4,12), lignocellulosic hydrolysates (2, 13), antibiot...

“…In addition, a variety of inhibitory compounds are released, such as furfural, hydroxymethylfurfural, methylglyoxal, and acetate (4)(5)(6). It is therefore desirable not only to expand the substrate range of S. cerevisiae (3,(7)(8)(9) but also to increase its inhibitor tolerance (10)(11)(12)(13).…”

mentioning

confidence: 99%

Increasing Anaerobic Acetate Consumption and Ethanol Yields in Saccharomyces cerevisiae with NADPH-Specific Alcohol Dehydrogenase

Henningsen

Hon

Covalla

et al. 2015

Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter ؊1 acetate during fermentation of 114 g liter ؊1 glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter ؊1 , this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter ؊1and raised the ethanol yield to 7% above the wild-type level. Saccharomyces cerevisiae is the principal microorganism used to produce ethanol, of which 93 billion liters were globally produced in 2014 (1). However, new strains are needed for the production of second-generation biofuels. Pretreatment and monomerization of lignocellulosic substrates produce complex sugar mixtures, including the C 5 sugars xylose and arabinose that are poorly fermented by most wild-type S. cerevisiae strains (2, 3). In addition, a variety of inhibitory compounds are released, such as furfural, hydroxymethylfurfural, methylglyoxal, and acetate (4-6). It is therefore desirable not only to expand the substrate range of S. cerevisiae (3, 7-9) but also to increase its inhibitor tolerance (10-13).Acetate is released during deacylation of hemicellulose and lignin and can be present in concentrations of Ͼ10 g liter Ϫ1 in cellulosic hydrolysates (4,14). At low pH particularly, this weak organic acid is a potent inhibitor of S. cerevisiae metabolism (15-17). Unfortunately, wild-type S. cerevisiae strains are poorly equipped to eliminate acetate from the medium under anoxic conditions. S. cerevisiae can grow on acetate as the sole carbon source under oxic conditions by converting acetate to acetyl coenzyme A (acetyl-CoA) with acetyl-CoA synthetase (ACS) (EC 6.2.1.1) and by either respiring acetyl-CoA in the tricarboxylic acid (TCA) cycle or upgrading acetyl-CoA to four-...