Metabolic Analysis And Genetic Operation Of Meyerozyma Guilliermondii And Pichia Kudriavzevii

Natural microorganisms play an important role in industrial and agricultural production.In the light of the different characteristics of microorganisms,they are widely used in the production of different compounds,purification of polluted environment,metallurgy and mining.It is important to understand the biological characteristics and metabolic information of these microorganisms and to establish the genetic operation system to further explore their application potential and expand the application field.For this purpose,the characteristics of two nonconventional yeast used in citric acid synthesis and desulphurization of industrial wastewater were investigated.In the first part of the study,the industrial citric acid producing strain was identified as M.guilliermondii by ITS amplicon sequencing,and the strain was identified as a haploid strain that was easy to operate in the laboratory.The biosynthesis ability of riboflavin and citric acid was verified under laboratory fermentation conditions.It was found that the strain could not synthesize riboflavin,but showed a strong ability to accumulate citric acid,and 65.33 g/L citric acid could be accumulated.In order to understand the mechanism of citric acid metabolic pathway,the whole genome of the strain was sequenced and various amino acid mutations were found in several enzymes that catalytic critical pathway metabolic pathways.In order to facilitate metabolic engineering of the strain to increase the yield of citric acid and the production of various metabolites,the genetic operation system was explored.The transformation rate of the optimized chemical transformation method and electroporation transformation method reached 70 transformants per μg of DNA.Subsequently,the free expression vector of the strain was developed,and the strength of the promoters was characterized and verified,and the Cre-LoxP system was established.After expression of heterogenous mevalonate synthesis pathway and citrate lyase,the yield of mevalonate was 0.60 g/L,indicating that the strain could be developed as a production platform for Acetyl-CoA derivatives.In the second part of the study,the industrial desulphurization strain was identified as P.kudriavzevii by ITS amplicon sequencing,and diploid strain was identified by flow cytometry.Subsequently,the desulfurization capacity of the strain was analyzed under laboratory conditions.When cultured for 40 h,the consumption of hydrogen sulfide under aerobic condition was 350 mg/L,and that under anaerobic condition was 200 mg/L.The whole genome sequencing and sulfide metabolic pathways are analyzed in detail,Thus it can be proved that may affect the desulfurization effect of genes including hydrogen sulfide generates related cysH,cysJ,as well as the specific consumption of hydrogen sulfide in cysteine cysK and met17 synthetic gene.Because the strain is diploid,it is difficult and tedious to establish genetic manipulation system.The genetic manipulation system was different from those of M.guilliermondii.Chemical transformation method and electroporation transformation method were also explored for this strain.In the end,P.kudriavzevii could obtain up to 20 transformants per μg of DNA transformed by these two methods.Subsequently,a gene knockout method was developed for this strain,and a single-copy ura3 gene knockout strain was obtained through homologous recombination.In this paper,the metabolic characteristics of two nonconventional industrial strains were verified and the related metabolic pathways were analyzed by genome sequencing.The available transformation methods of the two strains were explored.The free expression vector,Cre-LoxP system and mevalonate production platform of M.guilliermondii were established.A gene knockout method have developed for P.kudriavzevii.Finally,it proposes a new design and theoretical basis for the two strains to be widely used in various fields.