447

P. K. Bajwa • N. K. Harner • T. L. Richardson • S. Sidhu • M. B. Habash • J. T. Trevors • H. Lee (*) School of Environmental Sciences, University of Guelph , 50 Stone Road East , Guelph , ON , Canada N1G 2W1 e-mail: hlee@uoguelph.ca

41

Abstract

This chapter presents the protocol for genome shuf fl ing based on recursive cross-mating in the pentose-fermenting yeast Scheffersomyces ( Pichia ) stipitis . Genome shuf fl ing involves two stages. In the fi rst 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 screen-ing after each mating cycle to select for improved phenotypes in the recombinants. A number of reports have described genome shuf fl ing based on recursive protoplast fusion in bacteria and yeasts. Recently, we devel-oped mating-based genome shuf fl ing 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 sul-phite liquor (HW SSL), a pulping waste liquor containing a complex mix-ture of inhibitory substances. This was achieved in the complete absence of knowledge as to the precise genetic modi fi cations needed to confer HW SSL tolerance. Here we describe the protocols for recursive UV mutagen-esis, cross-mating, sporulation and isolation of recombinants with improved phenotypic traits.

Keywords

Genome shuf fl ing • Random mutagenesis • Recursive cross-mating • Pentose fermentation • Scheffersomyces stipitis

Genome Shuf fl ing Protocol for the Pentose-Fermenting Yeast Scheffersomyces stipitis

Paramjit K. Bajwa , Nicole K. Harner , Terri L. Richardson , Sukhdeep Sidhu , Marc B. Habash , Jack T. Trevors, and Hung Lee

V.K. Gupta et al. (eds.), Laboratory Protocols in Fungal Biology: Current Methods in Fungal Biology, Fungal Biology, DOI 10.1007/978-1-4614-2356-0_41, © Springer Science+Business Media, LLC 2013

448 P.K. Bajwa et al.

Introduction

Genome shuf fl ing is a microbial strain improve-ment technology that involves the generation of a pool of mutants with improved phenotypes, fol-lowed by iterative recombination between their genomes. This approach offers a number of advan-tages compared to the classical and molecular methods of microbial strain improvement. Classical strain improvement using mutagenesis and selection is time-consuming, laborious and based on a single starting strain. Molecular approaches of strain improvement are only appli-cable to genes that can be isolated, along with some knowledge of what genetic modi fi cations are needed to produce the desired phenotypic effect. To improve a cell’s complex phenotype, such as stress tolerance, likely requires modi fi cation in a number of known and unknown genes; hence, the molecular methods are inadequate. Genome shuf fl ing is particularly suitable for the engineer-ing of complex multi-genic phenotypic traits that are dif fi cult to modify by either the classical or molecular strain improvement approaches, as it does not require a priori knowledge of the set of genes to be modi fi ed to confer bene fi cial changes.

Genome shuf fl ing was originally developed for bacteria and then extended to yeasts. In bacte-ria, genome shuf fl ing has been used successfully to increase the tylosin titer in Streptomyces fra-diae [ 1 ] , lactic acid tolerance by Lactobacillus [ 2 ] , and pentachlorophenol degradation and tol-erance by Sphingobium chlorophenolicum [ 3 ] . In yeasts, particularly Saccharomyces cerevisiae , genome shuf fl ing has been used to improve ther-motolerance, ethanol productivity, ethanol toler-ance and acetic acid tolerance [ 4– 7 ] .

In genome shuf fl ing, genome recombination is carried out using either recursive protoplast fusion or cross-mating. Most of the reports on genome shuf fl ing are based on protoplast fusion [ 1– 5 ] . We have developed a genome shuf fl ing protocol based on recursive mating in the pentose-ferment-ing yeast Scheffersomyces stipitis and isolated mutant strains with improved tolerance to hard-wood spent sulphite liquor (HW SSL) [ 8 ] . This approach led to a rapid improvement in tolerance

to HW SSL in the selected strains, which retained their growth and fermenting ability. In our approach, the haploid cells from a pool of S. stipi-tis mutants are mated to form diploid zygotes. In malt extract agar, the diploid zygotes undergo meiosis to form two-spored asci [ 8, 9 ] . The ascus wall dissolves on its own to release the hat-shaped ascospores. When transferred to a rich medium, the ascospores germinate to produce vegetative haploid cells. The recombinants with improved phenotypes are identi fi ed and selected using suit-able selection regimes (Fig. 41.1 ). The process of mating and sporulation is repeated several times to allow for recombination between multiple par-ents and the pooling of bene fi cial alleles from various genomes.

Materials

Random Mutagenesis

1. A physical (ultraviolet light) or a chemical (e.g., N -methyl- N ¢ -nitro- N -nitrosoguanidine or ethylmethanesulphonate) mutagen.

2. S. stipitis culture grown for 48 h in minimal medium broth (0.67% [w/v] yeast nitrogen base (YNB) without amino acids and 2% [w/v] xylose).

3. Sterile Petri dishes, 15-mL centrifuge tubes and a tube rotator.

4. Selective agar (1.5% [w/v] medium). 5. Square plastic Petri dishes (120 × 120 × 17 mm).

Yeast Mating and Genome Shuf fl ing

1. Two auxotrophic yeast strains with different nutritional requirements.

2. YEPD broth (1% [w/v] yeast extract, 2% [w/v] peptone, 2% [w/v] glucose).

3. Minimal medium plates (0.67% [w/v] YNB without amino acids, 2% [w/v] xylose, 1.5% [w/v] agar).

4. Malt extract (ME) agar plates (3% [w/v] ME, 1.5% [w/v] agar).

5. Sterile Petri dishes, 15-mL centrifuge tubes and a tube rotator.

44941 Genome Shuf fl ing Protocol for the Pentose-Fermenting Yeast Scheffersomyces stipitis

Fig. 41.1 Schematic of genome shuf fl ing in yeasts

450 P.K. Bajwa et al.

Methods

Random Mutagenesis Procedure

Random mutagenesis can be carried out using a physical or chemical mutagen. Freshly grown yeast cells are exposed to the mutagen for a pre-determined time. The cells are then allowed to recover overnight in liquid or solid medium fol-lowed by screening and selection of the mutant colonies in a selective medium. In the following, we describe random mutagenesis using UV light. 1. Transfer a loopful of S. stipitis cells from an

isolated colony on YEPD agar plate to 20 mL of broth containing 0.67% [w/v] YNB without amino acids and 2% [w/v] xylose in a 125-mL Erlenmeyer fl ask at 28 ± 1 °C with shaking at 180 rpm for 48 h.

2. Aseptically transfer 1 mL of the 48-h grown yeast culture (OD600 between 8 and 10) to an empty sterile Petri dish.

3. Place the Petri dish under the UV light source at a distance of about 40 cm.

4. Remove the Petri dish cover and turn on the UV light. Prepare a yeast survival curve based on length of UV exposure. For S. stipitis wild-type (WT) cells, the length of UV exposure tested ranged from 10 to 60 s and 50% sur-vival was achieved at 20 s.

5. Transfer the UV-irradiated culture to a 15-mL sterile centrifuge tube covered with aluminium foil and incubate in a tube rotator (Fig. 41.2 ) for 24 h at 23 ± 1 °C. The tubes are rotated about their vertical axis at about 60° from hor-izontal at 90 cycles per min as previously described [ 10, 11 ] .

6. Spread 100 m L of the UV-exposed culture on several selective medium plates and incubate at 28 ± 1 °C for 4–5 days.

7. Isolate the mutant colonies appearing on the selective medium plates and maintain them on YEPD agar plates.

8. Individual mutants are further tested in liquid broth. Mutants that retain good ferment-ing ability in liquid broth were selected for further rounds of mutagenesis and genome shuf fl ing.

Fig. 41.2 Test tube rotator

45141 Genome Shuf fl ing Protocol for the Pentose-Fermenting Yeast Scheffersomyces stipitis

In our earlier study [ 12 ] , we used three consecu-tive rounds of UV mutagenesis followed by screening to obtain mutants of S. stipitis NRRL Y-7124 with improved tolerance to HW SSL. A schematic of the random mutagenesis procedure is presented in Fig. 41.3 . HW SSL is a waste liquor from the Tembec pulp and paper mill in Témiscaming, Québec, Canada. It contained, in [w/v]: 0.076% arabinose, 0.25% galactose, 0.33% glucose, 0.55% mannose, 2.2% xylose, 1% acetic acid, 0.18% furfural and 0.11% hydroxymethyl furfural. Prior to use, the pH of HW SSL was increased from 2.5 to 5.5 with 10 M NaOH. The liquor was then boiled for 5 min in a microwave oven, followed by gradual cooling to room temperature.

For UV mutagenesis, the UV exposure time was optimized to achieve a survival rate of about 50%. We hypothesized that changes in many genes are needed to collectively confer tolerance to HW SSL in yeasts. Thus, we used a low UV dosage resulting in a high survival rate in order to maximize those surviving populations carrying a small number of mutations for screening. Higher UV doses resulting in a lower survival rate may result in many members of the surviving popula-

tions carrying multiple mutations, some of which may not be bene fi cial. The presence of nonbene fi cial mutations will likely mask the effect of the bene fi cial mutations, resulting in these mutants not being selected in the screen.

An increasing concentration of HW SSL was used as the selective agent for initial mutant screening on agar plates after UV mutagenesis. This was done by spreading 100 m L of the UV-exposed culture on HW SSL (pH 5.5) gradi-ent plates. The plates were incubated at 28 °C for 5–10 days. Colonies growing at a higher concen-tration of HW SSL as compared to the WT strain were isolated and maintained on YEPD plates. HW SSL gradient plates were prepared by succes-sively pouring two layers of agar into a square plastic Petri dish (120 × 120 × 17 mm). The bottom layer consisted of 25 mL of HW SSL agar medium (Fig. 41.4a ). The plate was allowed to solidify at a slightly inclined position. The Petri dish was then placed in a horizontal position and a second layer of plain agar was poured over the fi rst layer (Fig. 41.4b ). The Petri dish was incubated at 30 °C for 2 days before use. This allowed diffusion of the HW SSL components through the agar layer, thereby establishing a concentration gradient.

Fig. 41.3 Schematic of UV mutagenesis followed by screening on gradient plates to select for HW SSL tolerant mutants

452 P.K. Bajwa et al.

Because performance of the mutants on the plate screen may not translate to the broth, the mutants were further tested for growth in liquid HW SSL. Also, since our end goal is fermenta-tion of the sugars in the waste liquor, the ferment-ing ability of the mutants needs to be veri fi ed by screening in broth. Therefore, the mutants with improved HW SSL tolerance were assessed for the ability to ferment xylose, glucose, mannose, galactose and arabinose in de fi ned media as well as HW SSL. Six improved mutants obtained after several rounds of UV mutagenesis and screening were used as the starting strains for genome shuf fl ing.

Optimization of Yeast Mating and Genome Shuf fl ing

1. Grow each auxotrophic yeast strain in 5 mL of YEPD broth in a 15-mL centrifuge tube at 28 °C with shaking at 180 rpm for 24 h. Auxotrophic yeast strains can be obtained by random mutagenesis [ 13 ] .

2. Mix 2 × 10 8 cells of each auxotrophic strain together and spread the mixed cell suspension on ME plates. Also, spread 2 × 10 8 cells of

each auxotroph separately on ME plates as controls.

3. Incubate the ME plates at 28 °C for 7–10 days to allow mating and sporulation to occur. Examine the cells periodically under the microscope to follow spore formation. S. stip-itis forms two hat-shaped spores per ascus.

4. After sporulation, scoop out all the cells from each plate. Suspend them in sterile water, cen-trifuge and wash the cells several times with sterile water. The cells are then suspended in 5 mL of sterile water.

5. Spread the cells on minimal media plates. The auxotrophic strains cannot grow on these plates. Only the recombinant cells resulting from mating between the two auxotrophic strains can grow on minimal media plates.

6. Incubate the plates at 28 °C for 4–5 days. Enumerate the colonies appearing on minimal media plates and determine the mating frequency.

7. To check for the stability of recombinants, repeatedly streak the colonies derived from the mating on minimal media plates. In our lab, the mating and sporulation protocol

was optimized using two auxotrophic strains of S. stipitis (FPL Y14— ura3, ade2, met1 and FPL Y18— ura3, leu2 delta ). Each parent carried a common auxotrophic requirement for uracil in addition to the other different auxotrophic mark-ers. If mating and successful recombination have occurred, then the recombinants would require only uracil. Therefore, the sporulated cells were spread on uracil-containing minimal media plates (0.67% w/v YNB w/o amino acids, 2% w/v xylose and 0.005% w/v uracil). Only the recombinant cells could grow on these plates (Fig. 41.5 ). The mating ef fi ciency of the haploid S. stipitis aux-otrophs was estimated to be 0.05% after one round of mating and sporulation. The estimate was based on the number of recombinants obtained on uracil-containing plates after mating the two auxotrophic S. stipitis strains. The opti-mized protocol was used for genome shuf fl ing of UV-induced mutants of S. stipitis .

Six HW SSL-tolerant mutants obtained after the third round of UV mutagenesis served as the parental strains for genome shuf fl ing. The

The first layer of agar with HWSSL is poured into the Petri dishand allowed to solidify while theplate is held in an inclinedposition

The second layer of plain agaris poured into the Petri dishand allowed to solidify whilethe plate is held in a leveledposition

Fig. 41.4 Preparation of HW SSL gradient agar plate

45341 Genome Shuf fl ing Protocol for the Pentose-Fermenting Yeast Scheffersomyces stipitis

selected mutants were individually grown over-night in YEPD broth followed by mixing of 1 × 10 8 cells of each mutant on ME plates. The plates were incubated for 7 to 10 days to allow mating and sporulation to occur in the same way as described above for the auxotrophic strains. All the cells were scooped out from the ME plates and suspended in 5 mL of sterile water. After centrifugation and several washings with sterile water, the spore suspension was transferred to 20 mL of fresh YEPD broth in 125-mL Erlenmeyer fl ask and incubated at 28 °C with shaking (180 rpm). After 48 h of growth, 100 m l of cell suspension was spread on several HW SSL (pH 5.5) gradient plates. Colonies appearing at a higher concentration of HW SSL as compared to the best UV-induced mutant were isolated. Three consecutive rounds of genome shuf fl ing involv-ing mating, sporulation and selection on HW SSL plates were done. Improved mutants obtained from one round served as the starting strains for the next round of genome shuf fl ing. Since we started with six UV-induced mutants, three rounds of genome shuf fl ing were deemed suf fi cient to recombine all the bene fi cial mutations together.

A greater number of rounds would be desirable if we had started with a larger mutant pool. Figure 41.6 illustrates the growth of S. stipitis WT, UV-induced mutant and a genome shuf fl ed mutant on the HW SSL gradient plate. The genome shuf fl ed strain (GS401) was clearly more tolerant than the UV-induced mutant (PS302), which in turn was more tolerant than the WT, on HW SSL gradient plate. The isolated mutants were further tested for growth in liquid HW SSL to con fi rm their HW SSL tolerant character. The WT was unable to grow in HW SSL unless diluted to 65% (v/v) or lower. The UV-induced mutants grew in 75% (v/v) HW SSL. Two mutants obtained after three rounds of genome shuf fl ing grew in 85% (v/v) HW SSL and one of these could survive in 90% (v/v) HW SSL, although no increase in cell number was seen [ 8 ] . The genome shuf fl ed mutants consumed 4% [w/v] xylose or glucose in de fi ned media more ef fi ciently and produced more ethanol as compared to the S. stipitis WT strain. These mutants also utilized mannose and galactose and produced the same amount of ethanol as the WT.

Fig. 41.5 Growth of the sporulated cell suspensions obtained from individual auxotrophs and mated popula-tions on minimal media plates + uracil. Sectors 1 and 2 represent the growth of individual auxotrophs FPL Y14— ura3, ade2, met1 and P. stipitis FPL Y1— ura3, leu2 delta , respectively. Sector 3 represents the growth of sporulated cells obtained after mating of the two auxotrophs

Fig. 41.6 Growth of S. stipitis WT ( left ), UV-induced mutant (PS302, middle ) and genome shuf fl ed strain (GS401, right ) on HW SSL (pH 5.5) gradient plate

454 P.K. Bajwa et al.

Summary

The results from our research demonstrated the utility of genome shuf fl ing via cross-mating as a means for industrial strain improvement in S. stipitis . The protocol is easy, inexpensive and convenient. The key requirement is the availabil-ity of a screening method that is speci fi c and sen-sitive. The improved haploid recombinant(s) are genetically stable and amenable to be further improved (i.e., they are not dead-end strains). For example, the selected genome shuf fl ed strains in our study can be subjected to further genome shuf fl ing again to select for mutants with improved tolerance to other stresses such as ace-tic acid and other pretreatment-derived inhibi-tors. Bene fi cial mutations in different mutant lines can be easily recombined into one strain. The same method can be applied to other yeasts, including native pentose-fermenting yeasts such as Pachysolen tannophilus, for which a genetic mating system has been described [ 14 ] .

Acknowledgments We thank Juraj Strmen and Frank Giust of Tembec Inc. (Témiscaming, Québec, Canada) for providing the HW SSL, and Tom Jeffries (USDA, Madison, Wisconsin, USA) for providing the auxotrophic S. stipitis strains.

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