Оne of the main priorities today is to develop efficient technology for alternative energy sources. The composition of lignocellulose hydrolysates includes two main sugars: glucose, which ferments first, and xylose. Identification of new genes that may be targets in the regulation of alcoholic fermentation of xylose for further engineering is an urgent task. Saccharomyces cerevisiae fluorimetric and fluorescence analyzes showed that peroxisomes persisted longer during xylose fermentation than on glucose. To study the role not just individual peroxisomal enzymes but whole organelles, deletion of the PEX3 gene encoding a peroxisomal membrane protein was performed. For the first time it was shown that peroxisome deficiency leads to a decrease in the accumulation of ethanol from xylose in strain pex3Δ for 1,5 times as compared to the original strain.
The ROS level in GS010 strain cells during glucose and xylose fermentation was studied, and it was shown that xylose is more actively involved in the generation of ROS. Namely, 1,7-1,8 times higher ROS levels were recorded during the fermentation of xylose. For the first time, the role of peroxisomal catalase in the process of alcoholic fermentation of xylose in a recombinant strain of S. cerevisiae was revealed. The CTA1 gene deletion led to a limitation of biomass growth and a 2 fold decrease in ethanol production compared to the original strain. The catalase activity in the cta1∆ strain was 6 times lower compared to the recipient strain. It was found that the overexpression of the PEX34 gene, encoding a peroxisomal integral membrane protein, leads to the formation of larger peroxisomes and increases by 1,4 times the ethanol accumulation level from xylose under alcoholic fermentation conditions.
During this work, a collection of strains with deletion and enhanced expression of ADR1, CAT8, ASG1, HAP4, SIP4, TUP1, and ZNF1 genes encoding transcription factors was constructed based on the xylose-fermenting strain of S. cerevisiae and the mechanisms of regulation of alcoholic fermentation of xylose in the constructed strains were investigated. The transcription factor Znf1 S. cerevisiae belongs to the family of transcriptional activators of the zinc cluster and binds to the promoters of the genes whose products are involved in cellular respiration, gluconeogenesis, the tricarboxylic acid cycle and the glyoxylate shunt. It was found that Znf1 does not affect the alcoholic fermentation of xylose in S. cerevisiae, since both ZNF1 deletion and overexpression did not alter ethanol production during fermentation of this pentose. The transcription factor of the zinc cluster Sip4 is a substrate of the protein kinase Snf1, and interacts with CSREs elements and is an activator of genes encoding enzymes of gluconeogenesis. The production of ethanol by strain SIP4 was reduced more than twice compared to the parent strain and reached 2,8 g/l. Adr1 and Cat8 are transcriptional regulators that depend on Snf1-mediated induction and derepress a number of genes involved in gluconeogenesis and β-oxidation. We found that both deletion and overexpression of ADR1 led to a decrease in ethanol production from xylose, which was 4,3 g/l for adr1Δ and 4,25 g/l for ADR1, whereas strain GS010 showed 5,85 g/l of ethanol. The obtained cat8Δ mutant showed an increase in ethanol production from xylose by 9,5% compared to the parental strain GS010. Overexpression of CAT8, in contrast, led to a decrease in ethanol production by 7,3%. The transcription factor Asg1 also belongs to the Zn-cluster family and is a regulator of the genes of several metabolic pathways: β-oxidation, glyoxylate cycle, gluconeogenesis, and import of long-chain fatty acids to peroxisomes. Deletion of ASG1 led to a decrease in ethanol production from xylose by 1,23 times. Transcriptional repressor Tup1 forming a complex with Cyc8p, is involved in the creation of the repressive structure of chromatin through interaction with histones H3 and H4. During xylose fermentation ethanol production tup1Δ was 1,58 times lower, ethanol production from glucose was 1,26 times lower.
In S. cerevisiae transcriptional activator Hap4 was identified, which plays a key role in controlling mitochondrial respiration gene expression. Deletion of HAP4 was found to significantly improve ethanol production during xylose fermentation. The mutant produces 10,4 g/l of ethanol, while the yield of ethanol reaches 0,414 g/g. Instead, increased expression of HAP4 led to the opposite effect, namely a decrease in ethanol production during xylose fermentation.
As a result, new targets were identified to construct fuel ethanol producers based on xylose-fermenting S. cerevisiae yeast strains. The genetically engineered approaches used in the work can be extrapolated to other promising yeast producers of fuel ethanol. The engineered strains can serve as a basis for further genetic engineering manipulations.
