| In recent years,the depletion of non-renewable energy sources such as coal and oil has become increasingly serious.Th low-cost lignocellulosic materials have become an alternative energy source to reduce dependence on traditional fossil fuels.As a renewable source of fermentable sugars,lignocellulosic biomass can be used for biofuels and chemicals through biotechnology.However,the presence of lignin and hemicellulose in lignocellulosic biomass makes it difficult for biomass to be hydrolyzed or digested during fermentation.The pretreatment is required to be used for fermentation.Many inhibitors are produced during a series of pretreatment and processing of lignocellulosic biomass.For example,furfural is the most common inhibitor in biomass pretreatment.After pretreatment of lignocellulose,the use of microbial fermentation hydrolysate is a key step.It was shown that C.tropicalis had strong adaptability to hydrolysates and was the ideal microorganism for xylitol production.It has high xylose hydrolysate yield and capacity productivity.In this study,C.tropicalis YB-3with higher tolerance to furfural concentration was screened by atmospheric and room temperature plasma mutagenesis(ARTP).The differentially expressed genes were analyzed from transcriptomics,and the key gene elements related to furfural tolerance in intracellular metabolic pathways and self-protection mechanisms were excavated.The tolerance mechanism of C.tropicalis YB-3 to furfural was explored,and the related gene components were verified by knocking out key genes.In this study,C.tropicalis was used as the starting strain.The C.tropicalis YB-3 that could tolerate 6.5 g/L furfural was screened by ARTP mutagenesis.Compared with the original strain,C.tropicalis YB-3 grew faster in the presence of the same concentration of furfural inhibitor.The tolerance of C.tropicalis YB-3 to furfural inhibitor was improved.The results of genetic stability of C.tropicalis YB-3 showed that it could be stably inherited for at least 10 generations.It also provided an important reference value for improving furfural tolerance of other strains.Through the analysis of transcriptome research,it was found that C.tropicalis played a major role in furfural tolerance mechanism under the furfural stress.These include oxidative stress-related genes,endoplasmic reticulum protein protection and damage clearance-related genes,and cell wall synthesis and degradation-related genes.Transcription levels are enhanced to remove misfolded proteins and maintain the balance of the intracellular redox environment with the integrity of the cell membrane for self-protection.A series of molecular mechanisms related to the activation of the cell,so that the normal life activities of the cell are restored.According to the results of transcriptome differential gene analysis,the sulfate adenosine transferase gene(MET3),glutathione reductase gene(GSH),and inositol phosphate synthase gene(MIPS)involved in the sulfur metabolism pathway in the tolerant strain C.tropicalis YB-3 were knocked out.Gene knockout vectors p MD20-MET3,p MD20-GSH and p MD20-MIPS were constructed.The highly up-regulated genes(MET3,GSH,MIPS)were knocked out by homologous recombination in C.tropicalis YB-3 strain.The gene knockout strains ΔMET3,ΔGSH and ΔMIPS were successfully obtained in turn,and gene complementation verification and exogenous expression verification were carried out.After that,the growth and tolerance characteristics of the gene knockout strains were analyzed and studied with the starting strain C.tropicalis YB-3 as a control.It provides a feasible method for the breeding of tolerant strains and the mining and verification of tolerant gene components.It provided a potential target for the transformation and breeding of more excellent industrial strains with high furfural tolerance,for a novel way to enhance the biological manufacturing capacity of bulk products and the comprehensive utilization level of biomass resources. |
