Improved xylose and arabinose utilization by an industrial recombinant Saccharomyces cerevisiae strain using evolutionary engineering

Department of Applied Microbiology, Lund University, PO Box 124, SE-22100 Lund, Sweden. .
Biotechnology for Biofuels (Impact Factor: 6.22). 06/2010; 3:13. DOI: 10.1186/1754-6834-3-13
Source: PubMed

ABSTRACT Cost-effective fermentation of lignocellulosic hydrolysate to ethanol by Saccharomyces cerevisiae requires efficient mixed sugar utilization. Notably, the rate and yield of xylose and arabinose co-fermentation to ethanol must be enhanced.
Evolutionary engineering was used to improve the simultaneous conversion of xylose and arabinose to ethanol in a recombinant industrial Saccharomyces cerevisiae strain carrying the heterologous genes for xylose and arabinose utilization pathways integrated in the genome. The evolved strain TMB3130 displayed an increased consumption rate of xylose and arabinose under aerobic and anaerobic conditions. Improved anaerobic ethanol production was achieved at the expense of xylitol and glycerol but arabinose was almost stoichiometrically converted to arabitol. Further characterization of the strain indicated that the selection pressure during prolonged continuous culture in xylose and arabinose medium resulted in the improved transport of xylose and arabinose as well as increased levels of the enzymes from the introduced fungal xylose pathway. No mutation was found in any of the genes from the pentose converting pathways.
To the best of our knowledge, this is the first report that characterizes the molecular mechanisms for improved mixed-pentose utilization obtained by evolutionary engineering of a recombinant S. cerevisiae strain. Increased transport of pentoses and increased activities of xylose converting enzymes contributed to the improved phenotype.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Lignocellulosic waste is a desirable biomass for use in second generation biorefineries. Up to 40 % of its sugar content consist of pentoses, which organisms either take up sequentially after glucose depletion, or not at all. A previously described Escherichia coli strain, PPA652ara, capable of simultaneous consumption of glucose, xylose and arabinose was in the present work utilized for production of (R)-3-hydroxybutyric acid (3HB) from a mixture of glucose, xylose and arabinose. The Halomonas boliviensis genes for 3HB production were for the first time cloned into E. coli PPA652ara, leading to product secretion directly into the medium. Process design was based on comparisons of batch, fed-batch and continuous cultivation, where both excess and limitation of the carbon mixture was studied. Carbon limitation resulted in low specific productivity of 3HB (<2 mg g(-1) h(-1)) compared to carbon excess (25 mg g(-1) h(-1)), but the yield of 3HB/cell dry weight (Y3HB/CDW) was very low (0.06 g g(-1)) during excess. Nitrogen-exhausted conditions could be used to sustain a high specific productivity (31 mg g(-1) h(-1)) and to increase the yield of 3HB/cell dry weight to 1.38 g g(-1). Nitrogen-limited fed-batch process design led to further increased specific productivity (38 mg g(-1) h(-1)) but also to additional cell growth (Y3HB/CDW = 0.16 g g(-1)). Strain PPA652ara did under all processing conditions simultaneously consume glucose, xylose and arabinose, which was not the case for a reference wild type E. coli, which also gave a higher carbon flux to acetic acid. It was demonstrated that by using E. coli PPA652ara, it was possible to design a production process for 3HB from a mixture of glucose, xylose and arabinose where all sugars were consumed. An industrial 3HB production process is proposed to be divided into a growth and a production phase, and nitrogen depletion/limitation is a potential strategy to maximize the yield of 3HB/CDW in the latter. The specific productivity of 3HB by reported here from glucose, xylose and arabinose by E. coli is further comparable to the current state of the art for production from glucose sources.
    Microbial Cell Factories 04/2015; 14(1):51. DOI:10.1186/s12934-015-0236-2 · 4.25 Impact Factor
  • Source
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Hardwood spent sulfite liquor (HSSL) is a by-product of acid sulfite pulping process that is rich in xylose, a monosaccharide that can be fermented to ethanol by Scheffersomyces stipitis. However, HSSL also contains acetic acid and lignosulfonates that are inhibitory compounds of yeast growth. The main objective of this study was the use of an evolutionary engineering strategy to obtain variants of S. stipitis with increased tolerance to HSSL inhibitors while maintaining the ability to ferment xylose to ethanol. A continuous reactor with gradually increasing HSSL concentrations, from 20% to 60% (v/v), was operated for 382 generations. From the final obtained population (POP), a stable clone (C4) was isolated and characterized in 60% undetoxified HSSL. C4 isolate was then compared with both the parental strain (PAR) and POP. Both POP and C4 were able to grow in 60% undetoxified HSSL, with a higher capability to withstand HSSL inhibitors than PAR. Higher substrate uptake rates, 7% higher ethanol efficiency and improved ethanol yield were obtained using C4. S. stipitis was successfully adapted to 60% (v/v) undetoxified eucalyptus HSSL. A stable isolate, C4, with an improved performance in undetoxified HSSL compared to PAR was successfully obtained from POP. Owing to its improved tolerance to inhibitors, C4 may represent a major advantage for the production of bioethanol using HSSL as substrate.
    Biotechnology for Biofuels 01/2015; 8:50. DOI:10.1186/s13068-015-0234-y · 6.22 Impact Factor

Full-text (4 Sources)

Available from
May 16, 2014