| Aspergillus oryzae is the most commonly used strain in kojic acid fermentation industry.In this paper,Aspergillus oryzae 3.042(A.oryzae 3.042),which is commonly used in soy sauce fermentation in China,was used as the original strain the to study the genetic modification method of A.oryzae and optimize the transformation conditions.In this study,the fungi global regulator LaeA was overexpressed.What’s more,the relationship between kojic acid production and oxidative stress during fermentation was explored.Details are as follows:A.oryzae has high native drug resistance,and the spores have serious polynuclear phenomenon.In this study,hygromycin resistant markers were used to optimize Agrobacterium tumefaciens mediated transformation of A.oryzae.By single factor experiment and orthogonal experiment,the optimal transformation conditions were as follows:A.oryzae 3.042 6-day spores,ultrasonic treatment for 45 s,infect spores with Agrobacterium tumefaciens AGL1,the ratio of Agrobacterium tumefaciens(A.tumefaciens)to spores is 150:1,co-cultivation at 25℃ for 48 h,the conversion efficiency is increased by 53.85%.In this study,a screening transformation system of A.oryzae uridine/uracil auxotrophic was establish.Firstly,pyrG knock-out plasmid pk5 was constructed and A.tumefaciens AGL1 was transferred to infect the spores of A.oryzae 3.042 and then got a stable hereditary auxotrophic strain A.oryzae g-1.After that,the msn2 knock-out plasmid pk1 with pyrG complementation was constructed,and the pyrG gene was complemented by A.oryzae g-1 as the starting strain.The success of the recovery experiment showed that pyrG marker was available,and the successful construction of this system laid the foundation for further transformation of A.oryzae.LaeA,a global regulator factor,can regulate secondary metabolism and morphological development of various fungi.LaeA is related to the production of kojic acid in A.oryzae.In this study,the overexpression of laeA gene was initiated by the manganese superoxide dismutase promoter PsodM,using pyrG knockout as a marker.Firstly,laeA overexpression plasmids pk6 and pk8 was constructed,and then was introduced into A.tumefaciens AGL1 to infect A.oryzae 3.042 spores.Finally,a uridine/uracil auxotrophic overexpression LaeA transformant A.oryzae k8320 was obtained by pk8 plasmids.A.oryzae k8320 as the original strain can be directly used to carry out subsequent genetic transformation with pyrG complementing gene marker.In this paper,the original strain A.oryzae 3.042,transformant A.oryzae g-1 and transformant A.oryzae k8320 were subjected to shake flask fermentation to produce kojic acid,and the relative expression of kojic transcriptional regulatory gene kojR and global regulatory gene laeA at 72th hour were compared.The expression levels of laeA and kojR of A.oryzae k8320 were higher than those of the other two strains.It was confirmed that over expression laeA was related to over expression kojR,but overexpression laeA did not directly increase kojic acid production.The effect of oxidative stress on kojic acid fermentation was studied.A.oryzae 3.042 was fermented with an inoculation amount of 1×104/mL,and adding less than 0.01%H2O2 at 24th hour had no significant effect on the yield of kojic acid.As for A.oryzae k8320,adding high concentration of H2O2 can significantly increase the yield of kojic acid.The kojic acid yield of A.oryzae k8320 was increased by 19.23%when 10 mM(0.03%)H2O2 was added in 24 h fermentation with 1 × 104/mL inoculation.In addition,the addition of H2O2 or menadione at 72 h of fermentation failed to increase the yield of A.oryzae 3.042 and A.oryzae k8320 kojic acid,but the addition of 2.5 mM menadione for 72 h of fermentation significantly increased the kojic acid yield of A.oryzae g-1 by 44.37%.This study provides ideas for further research on the relationship between secondary metabolism and environmental stress,and also lays a foundation for further targeted transformation to improve the yield of kojic acid. |
PDF Full Text Request