Study On Co-fermentation To Improve Riboflavin Production Of Ashbya Gossypii In Adaptive Laboratory Evolution

Riboflavin is an essential vitamin for human body.It mainly exists in the form of flavin adenine dinucleotide（FAD）and flavin mononucleotide（FMN）in organisms.As a coenzyme of flavinases,riboflavin participates in the metabolism of hydrogen transmission in cells.Although the fermentation technology of riboflavin by Ashbya gossypii is relatively mature,the production cost is still the main limiting factor for the application of riboflavin.Therefore,seeking low-cost raw materials for microbial fermentation has become an important issue.In this study,adaptive laboratory evolution（ALE）was used to evaluate a A.gossypii was selected and its fermentation performance,antioxidant activity,cell membrane permeability and integrity were verified;Secondly,by means of transcriptomic analysis,it is determined that it is related to a A.gossypii is closely related to molasses resistance,and explore the mechanism that affects the molasses resistance of bacteria;Finally,the obtained strains were used for substrate co fermentation to produce riboflavin,and the co-fermentation system was studied.This study provides theoretical basis and method guidance for riboflavin fermentation.The main research results are as follows:（1）A.gossypii was used for the starting strain,the evolutionary strain AG-24suitable for fermentation in cane molasses was screened by ALE method.After 8generations of continuous culture,it was confirmed that AG-24 has good genetic stability.Compared with the parent strain,the riboflavin yield of the AG-24 in cane molasses medium increased from 149.66 mg/L to 298.39 mg/L,an increase of 97.5%,and the cell biomass increased from 4.82 g/L to 8.48 g/L,an increase of 96%.The problems of poor riboflavin yield,low substrate utilization and low cell survival rate of A.gossypii in cane molasses medium were solved.In addition,the antioxidant activity,cell membrane permeability,cell membrane integrity and fermentation kinetic parameters of the AG-24 further proved that it had excellent fermentation performance in cane molasses medium and could be used as a potential strain for riboflavin production.（2）Through transcriptome analysis of A.gossypii,it was found that a total of3625 differentially expressed genes were detected,including 1807 up-regulated genes and 1818 down regulated genes.The expression of genes related to furfural stress in sugarcane molasses in AG-24 changed significantly,including the down-regulation of ABC transporter related genes,and the composition and structure of cell membrane were changed or modified to minimize furfural induced damage;The expression of genes encoding ribosomal protein（encoding mitochondrial ribosomal protein）,amino acids and oxidoreductase were up-regulated,and 5-hydroxymethyl furfural and furfural were transformed into less toxic substances.These results also showed that adaptive evolution led to the improvement of mitochondrial function at the translation level;The up regulation of transcription level of DNA replication,recombination and transcription reduced DNA damage.Compared with the parent strain,the transcription level of riboflavin synthesis pathway was up-regulated,which was consistent with the increase of riboflavin production of the strain.It is speculated that the above genes play a very important role in the molasses tolerance of AG-24,and provide more references for further optimization and construction of model strains.（3）The effects of different ratios of cane molasses and glucose on riboflavin fermentation were studied with the total sugar of 40 g/L.The single fermentation of cane molasses and the mixed fermentation of cane molasses and glucose according to the total sugar ratio of 80%:20%,60%:40%,40%:60%and 20%:80%for 7 days.The results showed that the fermentation of monosaccharide molasses was the worst in terms of cell biomass and riboflavin yield.The riboflavin yield and cell biomass of the fermentation system were increased after adding glucose,The riboflavin yield of 80%:20%,60%:40%,40%:60%,20%:80%glucose and cane molasses increased by 270%,99.08%,4.03%and 2.28%respectively.At the same time,the changes of organic acid content and p H value were compared.It was found that for the co fermentation of glucose and cane molasses,the addition of glucose would increase the organic acid content in glycolysis and tricarboxylic acid cycle,and improve the productivity,growth rate and maximum biomass of the strain.The synergistic effect analysis showed that the combined fermentation had a synergistic effect on the increase of riboflavin production from cane molasses.（4）Through the kinetic analysis and study of the co-fermentation glucose and cane molasses,the kinetic models of cell growth,riboflavin production and substrate consumption were established.The results of analysis of variance showed that R² was greater than 0.8,so it can well predict the dynamic changes of fermentation process.By comparing the kinetic parameters of co-fermentation with different proportions,it was found that the production of riboflavin by A.gossypii was a partially growth related process.The results showed that when the ratio of cane molasses and glucose was 20%:80%,it had the largestμ_m(4.39h^-1)andα（77.66 mg/g）,which is more conducive to riboflavin production.