Application Of Combinatorial Strategies In The Creation Of Superior Strains Of Arthrobacter Simplex

Arthrobacter simplex is a steroid C1,2 dehydrogenation strain which is widely used in industry.The anti-inflammatory activity of prednisone acetate（PA）produced by conversion of Cortisone acetate（CA）by Arthrobacter simplex was 3-4 times higher than that of CA.It was found that the transformation performance of the strain was not only related to key catalytic enzymes,but also positively regulated by multiple genes（pps,Amce G,irr E（M2）,rib A and rib E）.On this basis,we used the combinatorial strategies to create superior strains.Firstly,the optimal catalytic enzyme KsdD5_Asp and the optimal promoter P_M1210 were screened by using the overexpression technique,detecting the enzyme activity and substrate transformation ability.Then,ksd D5_Asp was combined with the excellent genes（pps,Amce G,irr E（M2））with promoter P_M1210.The strain A.simplex/p ART2AP_M1210-ksd D5_Asp-P_M1210irr E_{（M2）}-P_M1210pps-P_M1210Amce G（Asp 15）was obtained.The transformation process of the strain Asp 15 was optimized.The results showed that under the condition of 60 g/L CA and 8%ethanol using the growing cell as biocatalyst for 60 h,the PA content of the strain reached the maximum of 53.65 g/L,and the production intensity reached 0.89 g/L/h,compared with the control strain under initial conditions of 60 h（26.73 g/L;0.45 g/L/h）increased by 100.7%and 97.8%,respectively.Further analysis showed that Asp 15 showed higher KsdD activity（increased by 166.4%）,better transmembrane transport ability（increased by 72.9%）and stronger stress resistance than the control strain.On the other hand,the combined expression strains of genes related to cofactor synthesis and regeneration were constructed.The results showed that when the cofactor FAD-synthesis-related genes combination expression strain A.simplex/p ART2A-rib A-rib E（abbreviation:Asp 16）was transformed with 45 g/L CA and 8%ethanol for 48 h,the content of PA in the system was 29.08 g/L,which was 12.7%higher than that of the control strain p ART2A.The overexpression of cofactor regeneration-related genes fdh,nox1 and nox2improved the transformation ability compared with the control,fdh had the best effect,while nox1 and nox2 had no significant difference in effect.Further combined expression of cofactor regeneration-related genes Among them,the fdh-nox1 combination expressing strain A.simplex/p ART2A-fdh-nox1（abbreviation:Asp 20）had the best effect,The initial production rate was 1.35 g/L/h,and achieved the highest PA production（41.56 g/L）at 72 h with a productivity of 0.58 g/L/h,which was 53.2%and 52.6%higher than that of the control strain（27.12 g/L and 0.38 g/L/h）,respectively.This experiment showed that FAD regeneration positively regulated the transformation ability of Arthrobacter simplex,and confirmed the feasibility of FAD regeneration in steroid C1,2 dehydrogenation.The results provide a lot of basic data and modification direction for the subsequent creation of efficient strains.