Yeast Strain Improvement for Lignocellulosic Biomass Conversion

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Barry Nelson
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1 Yeast Strain Improvement for Lignocellulosic Biomass Conversion Hung Lee University of Guelph BIO World Congress on Industrial Biotechnology Montreal, Québec, CANADA July 22, 2015

2 Challenges faced by yeasts in fermenting the sugars in lignocellulosic hydrolysates Metabolic and regulatory problems from the presence of mixed hexose and pentose sugars S. cerevisiae WT strains cannot ferment xylose Yeasts ferment xylose less well than glucose Yeasts are subject to glucose repression & inactivation. This leads to preferential utilization of glucose over xylose when present in a mixture Toxicity problems from the presence of pretreatment-derived inhibitors They act synergistically to inhibit yeast growth, viability and fermentation

3 Native pentose-fermenting yeasts Main advantage Can ferment the dominant hexose and pentose sugars in hydrolysates to ethanol Three categories based on the main fermentation products Ethanol: Scheffersomyces (Pichia) stipitis and Scheffersomyces (Candida) shehatae Xylitol: Candida guilliermondii and Candida tropicalis Ethanol and xylitol: Pachysolen tannophilus

4 Our research on pentose-fermenting yeasts 1. Develop yeasts able to co-utilize a mixture of glucose and xylose and ferment them efficiently to ethanol 2. Develop yeasts able to tolerate the inhibitors in lignocellulosic hydrolysates 3. Produce other high-value compound(s) from xylose, eg., xylitol and triglycerides.

5 Addressing glucose repression in S. stipitis Approach: Isolate glucose-derepressed yeasts S. stipitis contains 4 hexo- or gluco-kinases. We hypothesized that one of these is involved in glucose repression Experimental o Disrupted hxk1 gene encoding hexokinase 1 in S. stipitis. o Assessed sugar utilization and fermentation performance of mutant on individual and mixed sugars

6 [Sugar] (%w/v) Ethanol & Xylitol Concentrations (%w/v) [Sugar] (%w/v) Ethanol & Xylitol Concentrations (%w/v) Glucose fermentation by S. stipitis hxk1 strain High initial cell density; in YNB + 4% (w/v) glucose Inocula were raised in YNB + 2% (w/v) galactose 5.0 WT 5.0 SS Time (h) Time (h) Glucose Xylitol EtOH Glucose EtOH Xylitol

7 [Sugar] (%w/v) Ethanol & Xylitol Concentrations (%w/v) [Sugar] (%w/v) Ethanol & Xylitol Concentrations (%w/v) Xylose fermentation by S. stipitis hxk1 strain High initial cell density; in YNB + 4% (w/v) xylose Inocula were raised in YNB + 2% (w/v) galactose 5.0 WT 5.0 SS Time (h) Time (h) Xylose EtOH Xylitol Xylose EtOH Xylitol

8 [Sugar] (% w/v) Mixed xylose and glucose fermentation by S. Ethanol & Xylitol Concentrations (% w/v) [Sugar] (% w/v) Ethanol & Xylitol Concentrations (% w/v) stipitis hxk1 strain High cell density; in 4% (w/v) each of glucose & xylose Inocula were raised in YNB + 2% (w/v) galactose 5.0 WT SS Time (h) Time (h) Glucose Xylose EtOH Xylitol Glucose Xylose EtOH Xylitol

9 Addressing inhibitor toxicity in S. stipitis and P. tannophilus Approach: Isolate inhibitor tolerant yeasts We hypothesized that Classical random mutagenesis + screening is the best way to obtain such yeasts Yeast mutants exhibiting tolerance to one hydrolysate should exhibit cross tolerance to other hydrolysates. Availability of inhibitor tolerant yeasts would eliminate the need for detoxification of hydrolysates. This reduces processing cost

10 Isolating inhibitor tolerant yeast mutants Yeasts were subjected to several rounds of (a) random mutagenesis and (b) genome shuffling Putative mutants were screened on gradient plates of hardwood spent sulfite liquor (HW SSL) Those showing improved tolerance were selected Genome shuffling was done by cross mating of putative improved mutants

11 Mutant screening 4 levels 1. Growth on HW SSL gradient plates 2. Growth in diluted liquid HW SSL 3. Fermentation testing in defined media 4. Fermentation testing in lignocellulosic hydrolysates

12 [HW SSL] Screening of UV-induced mutants on HW SSL gradient plates

13 [HW SSL] Improved HW SSL tolerance in genome shuffled S. stipitis strains

14 Wood hydrolysates tested for fermentability 1. Hardwood spent sulfite liquor (HW SSL) from Tembec 2. Steam pretreated poplar hydrolysate from UBC 3. Steam pretreated enzymatically hydrolysed poplar from SunOpta (Mascoma Canada) 4. S0 2 treated mixed wood hydrolysate (65% maple, 15% aspen and 20% birch) from FPInnovations

15 Fermentation of hydrolysate from SunOpta Glucose: 3.3 Xylose: 3.0 Mannose: 0.12 Arabinose: 8 Composition (% w/v) Cellobiose: 0.23 Total sugars: 6.73 Acetic acid: 0.85 ph: 4.8

16 Determinants of inhibitor tolerance in Pachysolen tannophilus Genomes of 3 inhibitor tolerant P. tannophilus strains were sequenced and analysed Identified 60 single nucleotide variations common to the 3 strains 40 mutations occurred in gene-coding sequences 33 were in genes of known functions. Of these 22 led to amino acid changes 7 were in genes of unknown function 20 mutations occurred in intragenic regions

17 Summary Deletion of hxk1 gene led to derepression of xylose utilization Isolated genetically stable mutants of S. stipitis and P. tannophilus with improved tolerance to inhibitors Mutants are amenable to be further improved. Inhibitor tolerance conferring mutations identified in P. tannophilus

18 Acknowledgements Funded by NSERC Bioconversion Network, BioFuelNet, OMAFRA Juraj Strmen and Frank Giust (Tembec) Richard Chandra, Pablo Chung, Jack Saddler (UBC) Chris Hlynialuk (SunOpta/Mascoma Canada) Mike Paice & Xiao Zhang (FPInnovations) Tom Jeffries (USDA)

19 Contributors Paramjit Bajwa Mehdi Dashtban Nicola Grant Marc Habash Nicole Harner Yip Ho Spencer Horemans Jackie Luu Ana Caroline De Oliveira Junqueira Vince Martin Evan Mayer Mike Paice Chetsada Phaenark Dominic Pinel Terri Richardson Tasnina Shireen Sukhdeep Sidhu Najjapak Sooksawat Jack Trevors Judith Vandeven Xin Wen Stephen Woo Xiao Zhang

20 Cellular functions that might be affected by inhibitor tolerance mutations Nutrient transport & metabolism Cell division and ribosome biogenesis Transcription Cell trafficking & cytoskeleton function Q. What are the roles of mutations in inhibitor tolerance? 36

21 Spent sulfite liquor (SSL)

Comparison of Laboratory and Industrial Saccharomyces cerevisiae Strains for Their Inhibitor Resistance and Xylose Utilization

Comparison of Laboratory and Industrial Saccharomyces cerevisiae Strains for Their Inhibitor Resistance and Xylose Utilization Geng Anli*, Wang Zhankun, Lai Kok Soon and Tan Wei Yi Mark, Goh Kiow Leng