Study On High Yield Kojic Acid And Fermentation Technology Of Aspergillus Oryzae By Genetic Engineering

Kojic acid has antibacterial,antioxidant and inhibition of tyrosinase activity,which has important application value in brewing,cosmetics and medicine.Natural kojic acid is a secondary metabolite produced by saccharide fermentation by Aspergillus oryzae.Aspergillus oryzae is regarded as the main microorganism of kojic acid fermentation because of its high yield and no toxin production.Previous studies have shown that pressure response factor msn2,koj R of kojic acid synthesis gene cluster and global regulatory transcription factor laeA all have certain effects on kojic acid synthesis.In this study,Aspergillus oryzae 3.042 was used as the starting strain,and pyrG nutritional deficiency was used as the screening marker to construct msn2 knockout plasmid pk1.The pyrG-deficient aspergillus oryzae g-1 strain was obtained through agrobacterium AGL1 mediated transformation.On this basis,msn2 was knocked out and pyrG gene was restored to obtain g-5 strain.Compared with the original strain Aspergillus oryzae 3.042,the growth of g-5 strain was inhibited and the spore production was significantly reduced by 60%.The strain did not sporulate in the environment containing10%or more glucose,but it could tolerate 30 mmol/L H₂O₂.The yield of kojic acid of g-5strain reached 21.55 g/L,which was increased by 81%compared with 11.86 g/L of Aspergillus oryzae 3.042.Compared with Aspergillus oryzae 3.042,msn2 gene was not expressed in strain g-5 by q RT-PCR.The expression levels of koj R and laeA genes related to kojic acid synthesis were up-regulated by Aspergillus oryzae after 6 days of fermentation.The expression levels of mycelial growth and sporulation related genes brl A and tps1 were significantly down-regulated in g-5 strain,while the expression levels of age B gene were significantly up-regulated.In this study,koj R overexpressed plasmid pk7 was constructed and transformed into pyrG-deficient Aspergillus oryzae g-1 strain,and an overexpressing koj R strain supplemented by pyrG-deficient mutant no.58 was obtained.The yield of kojic acid of strain 58 was 26.9 g/L,which was 127%higher than 3.042.QRT-PCR results showed that the relative expression level of koj R gene in strain 58 was significantly up-regulated at 3 d and 6 d.The expression levels of koj A and koj T gene clusters were also significantly increased on day 3,and msn2 and laeA were also increased compared with 3.042.The yield of kojic acid of A-1 strain reached 31 g/L,which was increased by 15%compared with 26.9 g/L of 58 strain.The laeA overexpressed plasmid pk8 was transferred into Agrobacterium AGL1 to infect Aspergillus oryzae A-1.Finally,A uridine/uracil nutritional deficiency overexpressed laeA strain No.5 was obtained.The result of shaking flask fermentation was 29.42 g/L,which was 9%higher than that of koj R 58 26.9 g/L.Compared with 11.86 g/L of 3.042,it increased by 148%.QRT-PCR results showed that the expression levels of laeA and koj R were significantly higher than 3.042,and the two had the same expression trend.Overexpression of laeA led to the increase of koj A expression at the early stage of fermentation,and contributed to the increase of koj T expression at the later stage.Meanwhile,the expression levels of GOD（glucose dehydrogenase）and Cat B（catalase）genes in HMP pathway were investigated in all strains.The results showed that the overexpression of koj R increased most significantly on day 6,indicating that GOD was still highly expressed at the late fermentation stage,which promoted the conversion of glucose to kojic acid and inhibited the expression of Cat B while msn2 inhibited the expression of Cat B.However,overexpression of laeA only promoted the transformation of GOD pair in the early stage of fermentation,and had a slight positive regulation of Cat B in the whole fermentation stage.Single factor test was used to determine the optimal carbon and nitrogen sources of the fermentation medium for A-1 strain kojic acid were maltose and soybean meal,respectively.On the basis of single factor test,orthogonal optimization of the medium components was carried out,and the optimized medium was as follows:maltose 12%,soybean meal 0.4%,KH₂PO₄0.1%,Mg SO₄0.15%,Na₂Mo O₄0.05%The single factor experiment was used to determine the fermentation conditions of seed liquor as follows:seed age 33 h,inoculation amount 3.3×10⁵seeds/m L.The yield of kojic acid was 38.2 g/L.