Paramjit K. Bajwa

Strain improvement of the pentose-fermenting yeast Pichia stipitis by genome shuffling

Genome shuffling based on cross mating was used to improve the tolerance of the pentose-fermenting yeast Pichia stipitis towards hardwood spent sulphite liquor (HW SSL). Six UV-induced mutants of P. stipitis were used as the starting strains, and they were subjected to 4 rounds of genome shuffling. After each round, improved strains were selected based on their growth on HW SSL gradient plates. Mutant libraries were established after each round and these improved mutant strains served as the starting pool for the next round of shuffling. Apparent tolerance to HW SSL on the gradient plate increased progressively with each round of shuffling up to 4 rounds. Selected improved mutants were further tested for tolerance to liquid HW SSL. After 4 rounds of shuffling, 4 mutants, two from the third round (designated as GS301 and GS302) and two from the fourth round (designated as GS401 and GS402), were selected that could grow in 80% (v/v) HW SSL. GS301 and GS302 grew also in 85% (v/v) HW SSL. GS301 was viable in 90% (v/v) HW SSL, although no increase in cell number was seen. The P. stipitis wild type strain (WT) could not grow on HW SSL unless it was diluted to 65% (v/v) or lower. Genome-shuffled strains with improved tolerance to HW SSL retained their fermentation ability. Fermentation performance of GS301 and GS302, the 2 strains that exhibited the best tolerance to liquid HW SSL, was assessed in defined media and in HW SSL. Both strains utilized 4% (w/v) of xylose or glucose more efficiently and produced more ethanol than the WT. They also utilized 4% (w/v) of mannose or galactose and produced ethanol to the same extent as the WT. GS301 and GS302 were able to produce low levels of ethanol in undiluted HW SSL.

Genetic improvement of native xylose-fermenting yeasts for ethanol production.

Lignocellulosic substrates are the largest source of fermentable sugars for bioconversion to fuel ethanol and other valuable compounds. To improve the economics of biomass conversion, it is essential that all sugars in potential hydrolysates be converted efficiently into the desired product(s). While hexoses are fermented into ethanol and some high-value chemicals, the bioconversion of pentoses in hydrolysates remains inefficient. This remains one of the key challenges in lignocellulosic biomass conversion. Native pentose-fermenting yeasts can ferment both glucose and xylose in lignocellulosic biomass to ethanol. However, they perform poorly in the presence of hydrolysate inhibitors, exhibit low ethanol tolerance and glucose repression, and ferment pentoses less efficiently than the main hexoses glucose and mannose. This paper reviews classical and molecular strain improvement strategies applied to native pentose-fermenting yeasts for improved ethanol production from xylose and lignocellulosic substrates. We focus on Pachysolen tannophilus, Scheffersomyces (Candida) shehatae, Scheffersomyces (Pichia) stipitis, and Spathaspora passalidarum which are good ethanol producers among the native xylose-fermenting yeasts. Strains obtained thus far are not robust enough for efficient ethanol production from lignocellulosic hydrolysates and can benefit from further improvements.

Mutants of the pentose-fermenting yeast Pichia stipitis with improved tolerance to inhibitors in hardwood spent sulfite liquor

Mutants of Pichia stipitis NRRL Y‐7124 able to tolerate and produce ethanol from hardwood spent sulfite liquor (HW SSL) were obtained by UV mutagenesis. P. stipitis cells were subjected to three successive rounds of UV mutagenesis, each followed by screening first on HW SSL gradient plates and then in diluted liquid HW SSL. Six third generation mutants with greater tolerance to HW SSL as compared to the wild type (WT) were isolated. The WT strain could not grow in HW SSL unless it was diluted to 65% (v/v). In contrast, the third generation mutants were able to grow in HW SSL diluted to 75% (v/v). Mutants PS301 and PS302 survived even in 80% (v/v) HW SSL, although there was no increase in cell number. All the third generation mutants exhibited higher growth rates but significantly lower growth yields on xylose or glucose compared to the WT. The mutants fermented 4% (w/v) glucose as efficiently as the WT and fermented 4% (w/v) xylose more efficiently with a higher ethanol yield than the WT. In a medium containing 4% (w/v) each of xylose and glucose, all the third generation mutants utilized glucose as efficiently and xylose more efficiently than the WT. This resulted in higher ethanol yield by the mutants. The mutants retained the ability to utilize galactose and mannose and ferment them to ethanol. Arabinose was consumed slowly by both the mutants and WT with no ethanol production. In 60% (v/v) HW SSL, the mutants utilized and fermented glucose, mannose, galactose and xylose while the WT could not ferment any of these sugars. Biotechnol. Bioeng. 2009; 104: 892–900.

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

ABSTRACT 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.

Transcriptional profiling of Saccharomyces cerevisiae T2 cells upon exposure to hardwood spent sulphite liquor: comparison to acetic acid, furfural and hydroxymethylfurfural.

Global gene expression was analyzed in Saccharomyces cerevisiae T2 cells grown in the presence of hardwood spent sulphite liquor (HW SSL) and each of the three main inhibitors in HW SSL, acetic acid, hydroxymethyfurfural (HMF) and furfural, using a S. cerevisiae DNA oligonucleotide microarray. The objective was to compare the gene expression profiles of T2 cells in response to the individual inhibitors against that elicited in response to HW SSL. Acetic acid mainly affected the expression of genes related to the uptake systems of the yeast as well as energy generation and metabolism. Furfural and HMF mainly affected the transcription of genes involved in the redox balance of the cell. On the other hand, the effect of HW SSL on S. cerevisiae T2 cells was distinct and considerably more diverse as compared to the effect of individual inhibitors found in lignocellulosic hydrolysates. This is not surprising as HW SSL contains a complex mixture of inhibitors which may act synergistically. HW SSL elicited significant changes in expression of genes involved in diverse and multiple effects on several aspects of the cellular structure and function. A notable response to HW SSL was decreased expression of the ribosomal protein genes in T2 cells. In addition, HW SSL decreased the expression of genes functioning in the synthesis and transport of proteins as well as metabolism of carbohydrates, lipids, vitamins and vacuolar proteins. Furthermore, the expression of genes involved in multidrug resistance, iron transport and pheromone response was increased, suggesting that T2 cells grown in the presence of HW SSL may have activated pheromone response and/or activated pleiotropic drug response. Some of the largest changes in gene expression were observed in the presence of HW SSL and the affected genes are involved in mating, iron transport, stress response and phospholipid metabolism. A total of 59 out of the 400 genes differentially expressed in the presence of HW SSL, acetic acid, HMF and furfural, belonged to the category of poorly characterized genes. The results indicate that transcriptional responses to individual lignocellulosic inhibitors gave a different picture and may not be representative of how the cells would respond to the presence of all the inhibitors in lignocellulosic hydrolysates such as HW SSL.

Ethanol production from selected lignocellulosic hydrolysates by genome shuffled strains of Scheffersomyces stipitis

Two genome-shuffled Scheffersomyces stipitis strains, GS301 and GS302, exhibiting improved tolerance to hardwood spent sulphite liquor, were tested for growth and fermentation performance on three wood hydrolysates: (a) steam-pretreated enzymatically hydrolyzed poplar hydrolysate from Mascoma Canada, (b) steam pretreated poplar hydrolysate from University of British Columbia Forest Products Biotechnology Laboratory, and (c) mixed hardwoods pre-hydrolysate from FPInnovations (FPI). In the FPI hydrolysate, the wild type (WT) died off within 25 h, while GS301 and GS302 survived beyond 100 h. In fermentation tests, GS301 and GS302 completely utilized glucose and xylose in each hydrolysate and produced 0.39-1.4% (w/v) ethanol. In contrast, the WT did not utilize or poorly utilized glucose and xylose and produced non-detectable to trace amounts of ethanol. The results demonstrated cross tolerance of the mutants to inhibitors in three different wood hydrolysates and reinforced the utility of mating-based genome shuffling approach in industrial yeast strain improvement.

Genome Shuffling Protocol for the Pentose-Fermenting Yeast Scheffersomyces stipitis

This chapter presents the protocol for genome shuffling based on recursive cross-mating in the pentose-fermenting yeast Scheffersomyces (Pichia) stipitis. Genome shuffling involves two stages. In the first stage, a pool of mutants with improved phenotypes is selected. Several rounds of random mutagenesis can be done using different mutagens, and mutant selection can be based on different criteria to generate different mutant cell lines. In the second stage, the genomes of mutants derived from different lines are mated recursively to allow for genetic recombination, followed by screening after each mating cycle to select for improved phenotypes in the recombinants. A number of reports have described genome shuffling based on recursive protoplast fusion in bacteria and yeasts. Recently, we developed mating-based genome shuffling in the pentose-fermenting yeast S. stipitis. We have used this approach to obtain genetically stable mutants of S. stipitis with considerably improved tolerance to hardwood spent sulphite liquor (HW SSL), a pulping waste liquor containing a complex mixture of inhibitory substances. This was achieved in the complete absence of knowledge as to the precise genetic modifications needed to confer HW SSL tolerance. Here we describe the protocols for recursive UV mutagenesis, cross-mating, sporulation and isolation of recombinants with improved phenotypic traits.

Transfer of plasmid into the pentose-fermenting yeast Pachysolen tannophilus

The pentose-fermenting yeast Pachysolen tannophilus can convert glucose and xylose in lignocellulosic hydrolysates to ethanol. However, it performs poorly in industrially relevant lignocellulosic hydrolysates containing mixed sugars and inhibitors. Efforts have been directed at improving the performance of this yeast to enable efficient lignocellulosic biomass conversion. While some successes have been reported using random mutagenesis and/or hybridization-based approaches, further genetic improvement of this yeast is hampered by the lack of efficient gene transfer methods as well as limited genetic information to guide further construction of robust strains of P. tannophilus. In this study, we aimed to address this short-coming by establishing the optimal conditions needed for efficient gene transfer into P. tannophilus. We ascertained that plasmids can be transferred into P. tannophilus through trans-kingdom conjugation or lithium acetate (LiAc) transformation. The efficiency of plasmid YEp13 (2-micron, LEU2) transferred into a P. tannophilus leucine auxotroph (Leu-) reached as high as 1.93 × 10-2 transconjugants per input recipient and 3.25 × 104 transformants per μg plasmid DNA through trans-kingdom conjugation and transformation, respectively. In trans-kingdom conjugation, the number of recipient P. tannophilus cells played an important role, while the ratio of donor (Escherichia coli) to recipient cells was less important. For efficient transformation in P. tannophilus, the use of PEG 3350 was essential, as no transformants were obtained in its absence. The transformation efficiency increased with the addition of single-stranded carrier DNA and incubation at 30 °C for >60 min. Plasmids with different replication origins or 2-micron plasmids with different CUG codon-optimized antibiotic resistance markers were unable to transform P. tannophilus under our experimental conditions. The results are of interest in the genetic manipulation and improvement of P. tannophilus.