Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

The overall goal of this adaptive evolution procedure is to derive robust strains of the native xylose fermenting yeast scheffersomyces stipitis NRRL Y-7124 that can more rapidly ferment the hexose and pentose sugars in undetoxified hydrolysates to economically recoverable ethanol. This method moves the lignocellulose to ethanol industry forward by providing a road map to strains with complex modifications, allowing fermentation of sugars in hydrolysates which contain microbial inhibitors. Here, the main advantage of targeted evolution is that robust strains are molded from a native yeast able to ferment xylose, eliminating the need to engineer industrial saccharomyces strains.

We realized that any single yeast strain was unlikely to be robust in different types of hydrolysates, such as AFEX pretreated corn stover or dilute acid pretreated switchgrass. The ability of scheffersomyces stipitis to undergo a sexual reproductive cycle is beneficial to further exploitation in the present work because the various strains developed can be combined into a single, more robust strain through mating of strains having these various traits. Demonstrating the procedure will be coauthors Stephanie Thomson and Maureen Shea-Andersh who are biological science technicians in my laboratory.

Prior to starting this enrichment procedure, prepare a preculture of strain NRRL Y-7124 in a 125 milliliter flask and a dilution series of the enzyme saccharified ammonia fiber expansion pretreated corn stover hydrolysate, or AFEX CSH. Use a repeating pipette to fill a 96 well microplate with 15 microliters per well, and eight wells per hydrolysate dilution. Inoculate with a few microliters of preculture per well to allow an initial absorbance at 620 nanometers, or A620 of greater than or equal to 0.1.

Place the microplate in a plastic box with a wet paper towel for humidity and incubate statically for 24 to 28 hours at 25 degrees Celsius. Next, use a microplate reading spectrophotometer to identify the cultures that physically grew to an A620 greater than 1.0 in the most concentrated hydrolysate dilution. Pool the identified wells in a microfuge tube, and distribute to a fill plate.

Transfer one to five microliters from the fill plate to each well of a new hydrolysate dilution series to achieve an initial A620 of greater than or equal to 0.1 and incubate the plate as before. Monitor the growth of the cultures in the microplate with a microplate reading spectrophotometer. Prepare glycerol stocks of adaptation cultures regularly.

Pool four wells of the greatest hydrolysate concentration colonized in a cryofile, and mix one to one with 20%sterile glycerol. Freeze cells at minus 80 degrees Celsius. Repeat this enrichment procedure until growth in 12%glucan AFEX CSH is consistently visible at 24 hours.

To begin this procedure, streak selected glycerol stocks of adaptation cultures to yeast malt peptone dextrose, or YM agar. Then after growth, use streaks to inoculate 50 microliters of 3%or 6%glucan AFEX CSH in each of three microplate wells. Incubate the microplate for 24 hours at 25 degrees Celsius.

Pool replicate colonized wells at the highest hydrolysate strength with strong growth. And dilution plate to YM or 6%glucan AFEX CSH agar. Incubate the plates in a 25 degree Celsius incubator for 24 to 48 hours.

Pick five single colonies from the highest dilution plate showing growth to freeze as glycerol stock cultures. Streak each colony to a YM plate and incubate for 24 hours. With a sterile loop, transfer a developed streak of cells to a small volume of 10%glycerol mixed to suspend the cells and distribute to two to three cryofiles for freezing at minus 80 degrees Celsius.

To test isolates in simple 6%glucan AFEX CSH batch cultures, transfer cells from 48 hour plates streaked from glycerol stocks to pH 7 potassium phosphate buffer to prepare cell suspensions. After measuring the optical density of each cell suspension, adjust the volume with additional buffer to reach an A620 of ten. Use one microliter of each suspension to inoculate each of four wells of 50 microliters of 3%glucan hydrolysate to an initial A620 of 0.2.

Incubate the microplate for 24 hours. Next, transfer two 24 hour microplate wells to inoculate 25 milliliter precultures of 6%glucan AFEX CSH at pH 5. Close the flasks with silicon sponge closures and incubate for 24 hours at 25 degrees Celsius and 150 RPM.

Use the 6%glucan AFEX CSH precultures to inoculate similar 25 milliliter growth cultures in duplicate to an initial A620 of 0.1. Incubate the growth cultures for 24 hours at 25 degrees Celsius and 150 RPM. To test dioxic lack of isolates in batch cultures of optimal defined medium, or ODM, with mixed sugars, inoculate precultures of 75 milliliters ODM with 150 grams per liter of xylose in 125 milliliter flasks with silicone sponge closures.

Incubate for 24 hours at 25 degrees Celsius and 150 RPM. Use the precultures to inoculate to an A620 of 0.1 similar duplicate 75 milliliter test cultures with ODM containing 75 grams per liter of glucose and 75 grams per liter of xylose at pH 6.5. Shake flasks at 25 degrees Celsius and 150 RPM.

24 hours prior to inoculating a continuous culture, prepare a preculture of a representative AFEX CSH tolerant colony in ethanol-free ODM in a 125 milliliter flask. Use five to 10 milliliters of the AFEX CSH tolerant isolate preculture to inoculate 100 milliliters of ODM in a jacketed 100 milliliter spinner flask to obtain an initial A620 of 0.5. Maintain the culture holding volume at 25 degrees Celsius.

Stir it at 200 RPM and outfit it with a sterilizable pH electrode, a pH controller, and a temperature controlled circulating water bath. Initially, since cell growth will be slow due to the ethanol exposure, set up a pH actuated pump so that when the culture fermentation decreases the pH to 5.4, the pump will feed pH 6.3 ODM with 100 grams per liter of xylose and 50 grams per liter of ethanol to stop further pH decreases. Maintain the volume at 100 milliliters with an effluent pump continuously skimming the culture surface.

Measure the effluent volume collected and draw samples from continuous cultures every 48 to 72 hours for analysis of viable cell concentration, products, and substrates as described in the manuscript. Save glycerol stocks regularly by isolating colonies from viability plates of buffered sample dilutions forming 30 to 100 colonies, representing the most prevalent robust colonies at that time in enrichment. Flood the plates with five milliliters of 10%glycerol to allow preparation of duplicate cryofiles.

If cultures can grow steadily at specific rates of greater than 0.01 per hour on xylose in the presence of more than 25 grams per liter of ethanol, start the continuous culture feed at a low dilution rate of about 0.01 per hour to the 100 milliliter holding volume. Over time, raise the ethanol in the feed toward 50 grams per liter. Ultraviolet irradiation of inocula for restarting continuous cultures is an option to hasten the mutation rate.

Capture advanced populations in glycerol stocks. Prior to isolating single cell colonies, glycerol stock populations with improved xylose fermentation in the presence of ethanol, are identified as described in the text protocol. Use these superior populations to streak plates.

The streaked plates are used to prepare dense buffered cell suspensions of an A620 equal to five. Then cell suspensions are used to inoculate precultures in 96 deep well plates. Incubate for 48 hours at 25 degrees Celsius, 400 RPM and one inch orbit.

Next, enrich tolerant colonies by using each preculture to inoculate 16 one milliliter replicate cultures to an initial A620 of 0.5 in acid pretreated switchgrass hydrolysate liquor, mixed one to one with ODM plus 50 grams per liter of xylose, and enough ethanol to provide a 20, 30 or 40 grams per liter of ethanol challenge. Incubate for 48 hours at 25 degrees Celsius, 400 RPM and one inch orbit. For each different glycerol stock, harvest the culture wells with the highest ethanol concentration allowing growth and xylose use.

Plate each cell line to YM agar to obtain prevalent single colonies to preserve in glycerol. Pick 10 colonies per cell line and streak each to a YM agar plate for glycerol stock preparation. After adaptation on AFEX CSH, a comparison of fermentation performances in ODM between the parent strain and adapted populations reveal that the adapted populations use xylose more efficiently to make ethanol more rapidly and in higher amounts.

A hydrolysate tolerant colony was further developed for improved ethanol tolerance by continuous culture selection on ODM containing xylose and high levels of ethanol. All adapted populations surpassed the parent in the ability to induce xylose metabolism in the presence of 40 grams per liter of ethanol. These graphs summarize the performances of superior tolerant isolates assessed in terms of xylose uptake rates and ethanol yields relative to the parent strain for each formulation of PSGHL.

The best strains selected from the primary screen on xylose rich PSGHL were subsequently screened on three complete hydrolysates and the relative performance index, or RPI, was calculated for the fermentation of each isolate within each hydrolysate. Next, the combined overall performance indices for each isolate were calculated. Five isolates, three, 14, 27, 28 and 33, had overall RPIs above 60, which ranked them as the most robust to all of the variations of hydrolysate and nutrient conditions combined.

After watching this video, you should know how to challenge a xylose fermenting yeast like scheffersomyces stipitis NRRL Y-7124 in a targeted evolution process, enrich, isolate, and evaluate more robust derivatives which more effectively ferment mixed sugars in undetoxified hydrolysates. Once mastered, this technique can be used with a daily time investment averaging about an hour per day. Significant changes in the evolution process can be documented in as little as four months.

While attempting this procedure, it's important to save advancing populations regularly by preparing glycerol stocks to freeze at minus 80 degrees Celsius. This allows population recovery, periodic evaluation, isolation or restarting the evolution process. To manage various mutations, remember to regularly screen the yeast under process specific conditions for an array of phenotypes.

Calculation of overall dimensionless relative performance indices, or RPIs, based on kinetic parameters, allows assessment of strain rank and robustness to variations in fermentation conditions like hydrolysate type and nutrient supplementation. Superior yeasts arising from each phase of the evolution process are candidates for genome sequencing so that the nature of the improvements such as inhibitor tolerance are reduced and dioxic lag can be understood and used for design of next generation biocatalytic strains.