Improving Ethanol Yield Of Saccharomyces Cerevisiae Strains For Very High Gravity Fermentation

Fuel ethanol, which is commonly produced by anaerobic fermentations withSaccharomyces cerevisiae, is one of the most important products originating from thebiotechnological industry with respect to both value and amount. However, thebioethanol business is operating on tight profit margins due to the high cost of rawmaterials. High gravity fermentation (VHG fermentation) technology is widelyapplied in fuel ethanol industry to reduce energy consumption and improve equipmentutilization, but it also expose fermentation strains to high osmotic pressure. The keyissue in S. cerevisiae improvement for ethanol fermentation is how to improve itsVHG fermentation capacity including growth rate, ethanol yield and ethanolconversion.To determine the least glycerol production to maintain the rate of fermentation, aserial nucleotide sequences were truncated form the3’end of GPD1promoter forreducing the GPD1gene expression. Investigation shows with the GPD1geneexpression decreased gradually with the increase in the length of GPD1promotertruncated, and it will significantly reduce osmotic resistance when the truncationlength is no less than100bp. LE34U and LE35U, with GPD1promoter truncated60and80bp respectively, ferment3.89%and4.49%more ethanol and27.13%and40.90%less glycerol than the original strain KAM-2U, and the ethanol conversion is0.466and0.470g/g respectively, which were better than FTG2. Besides, GPD1promoter truncation does not affect the regulation of GPD expression by osmoticpressure.Further researches have been conducted to improve the ability of LE34U andLE35U in VHG fermentation:1. knock out the ADH2gene, encoding alcoholdehydrogenase, which results in rising of ethanol yield by0.72%and0.61%respectively to LE34U and LE35U, and the ethanol conversion is0.469and0.477g/grespectively;2. convert LE34U and LE35U to diploid to improve the strains ability toadapt to adverse environment, but the results show that this method is invalid;3. addglycerol into the fermentation medium won’t improve the fermentation rate or theethanol yield;4.3g/L or less acetic acid can improve the VHG fermentation, however,4g/L or more acetic acid inhibits LE34U and LE35U strongly, and the inhibition is stronger than that to KAM-2;5. acclimate the strain FTG2G1U in the medium withhigh glucose concentration. And after the growth is improved remarkably, partiallyrestore the expression of GPD1gene. As a sequence, the growth of strains with GPD1promoter truncated is not improved as expected.In this study, a new evolutionary engineering method, based on chemicalmutagenesis and chromosomal sexual shuffling in Saccharomyces cerevisiae wasdeveloped. In a diploid S. cerevisiae strain W303D, two ste2alleles were knocked outto block the mating process. After EMS mutagenesis, the diploid cells were applied tohigh efficiency sporation process to achieve genome shuffling. Two mutants, weregained through a screen process. WS1and WS5produced more ethanol thanW303-1A by15.91%and16.67%respectively, and their tolerance to high sugar orhigh ethanol concentration was significantly enhanced, which demonstrates theeffectiveness of this new method. Besides, recovering the STE2gene and convertingWS1and WS5didn’t improve the fermentation performance.