Development Of A High Efficiency Biosynthetic Pathway For 9α-hydroxy-4-androstene-3,17-dione

Steroids are a series of compounds derived from the basic framework of cyclopentane and phenanthrene,also known as steroid compounds.As an important branch of the steroid drug family,fluorosteroid compounds,such as dexamethasone,betamethasone and their derivatives,are widely used in the treatment of inflammation,allergy and autoimmune diseases.Due to the introduction of fluorine atoms,their molecules are more fully bound to target proteins,and their biological and pharmacological activities have been further improved 9α-hydroxy-4-androstene-3,17-dione is an important precursor for the production of 9 halogenated corticosteroids.At present,the shortcomings of poor substrate solubility,low conversion efficiency and large proportion of by-products in the industrial production process restrict the improvement of its production.Therefore,it is of great practical significance to develop an efficient synthesis pathway of 9α-hydroxy-4-androstene-3,17-dione.We obtained an industrial strain with steroid 9α hydroxylation function from the factory.After species identification by 16 S r RNA,we determined the hydroxylase-Ksh A2B1,which has the strongest hydroxylation ability in this strain,through biological means such as gene mining,enzyme activity test,heterologous expression and kinetic test.After substrate spectrum test,it was found that this enzyme has substrate universality.Many compounds can be identified.After the comparison of protein expression and co-expression,we found that Ksh A2 was different from other reported oxidation components,and it could still maintain its activity under aerobic purification conditions.In the previous study,we knocked out the kst D and mn Opcc R genes in the sterol degradation pathway of Mycobacterium aureus,and obtained a high quality strain with high sterol production and no by-product production.On this basis,different construction methods were adopted to transfer ksh A2 and ksh B1 into this chassis,and the best construction method was to add its own RBS sequence and introduce exogenous ksh B1 gene through a small amount of fermentation screening.Finally,the engineering strain AJTU-1 with the best transformation effect was obtained,which was fed 20 g/L sterols in shaker and fermentor for fermentation,and the highest conversion rate was 9.8 g/L9α-hydroxy-4-androstene-3,17-dione,and the byproducts only account for2.8%.This study successfully discovered a unique steroid C9 hydroxylase,Ksh A2B1,which adds a new member to the hydroxylase family and provides a powerful enzyme tool for in vitro hydroxylation reactions.At the same time,the prediction of ksh A4 and ksh A5 in previous literatures is corrected,and the reason why Ksh A2 oxidase can remain active without coexpression is explained.On this basis,a highly efficient biosynthesis of9α-hydroxy-4-androstene-3,17-dione was developed,using cheaper phytosterol as raw material,with high utilization of substrate sterols,high conversion rate and few by-products,greatly saved the production cost,which provided theoretical basis and technical guidance for the industrial production of 9α-hydroxy-4-androstene-3,17-dione.