Screening For Steroid 14α-Hydroxylase And Construction Of Coenzyme-Coupled Mycolicibacterium Neoaurum Strains

14α-Hydroxy-androst-4-ene-3,17-dione(14α-OH-AD)is a key intermediate in the production of steroid drugs and has important anticancer and cardiac activities.In previous studies,fungi whole-cell catalysis has been used to produce 14α-OH-AD using the more expensive androstenedione as a substrate,which has problems such as expensive substrate,difficult product isolation and poor enzyme specificity.In this work,a one-step biotransformation strategy for 14α-OH-AD synthesis in Mycolicibacterium neoaurum was achieved by heterologously expressing eukaryotic-derived 14α-hydroxylase in prokaryotic Mycolicibacterium neoaurum using a cheaper phytosterol(PS)as a substrate.The amino acid sequence of the known active 14α-hydroxylase gene sequence reported in the literature was used as a template to run the BLAST comparison function of KEGG for screening nine genes with similar amino acid sequences to the known14α-hydroxylase gene.And these ten squences were synthesised for according to the codon preference of Mycolicibacterium neoaurum.The synthetic 14α-hydroxylase genes were transformed into the host bacterium MNR M3?ksd D by ligation to the shuttle plasmid p MV261.MNR M3?ksd D is a mycolicibacterium neoaurum from a pre-conserved laboratory knockout of the 3-steroid-?1-dehydrogenase(ksd D)gene.Thin layer chromatography(TLC)spot plates were used to screen strains that could produce unknown products other than AD as candidate strains for further HPLC analysis,and finally the14α-hydroxylase gene reported in the literature was screened for its best ability to produce unknown products by transformation.The steroid-transformed fermentation broth was purified by silica gel column separation and structurally identified.The results of TLC with sulphuric acid spraying as well as gas phase mass spectrometry and NMR spectroscopy indicated that the unknown product produced by the transformation of the recombinant strain was 14α-OH-AD.To improve the hydroxylation capacity of the recombinant strains,the recombinant strains MNR M3?ksd D/p MV261-14α-CPR and MNR M3?ksd D/p MV261-14α-FDX-FDXR and the engineered strains MNR M3?ksd D/p MV261-14α-G6 PDH and MNR M3?ksd D/ p MV261-14α-GDH were constructed.Steroid transformation experiments finally showed that the highest product molar yield of 50.7±2.2 % was achieved by the strain MNR M3Δksd D/p MV261-14α-G6 PDH.The molar productivity was increased by nearly 2 % compared to that of the mono-expressing strain MNR M3Δksd D/p MV261-14α.And the growth performance and coenzyme ratio of the tandem strains were further compared with that of the single-expressing strain,indicating that the strain MNR M3Δksd D/p MV261-14α-G6 PDH was more effective than the strain MNR M3Δksd D/p MV261-14α.Tandem expression of the G6 PDH gene had the least impact on the performance of the strain compared to tandem expression of other genetic elements.In summary,the engineered strain MNR M3Δksd D/p MV261-14α-G6 PDH was finally selected as the best fermentation strain.Subsequently,in order to improve the transformation efficiency of the recombinant strain,the type of co-solvent,the ratio of co-solvent addition,the concentration of sugar supplementation,the time of sugar supplementation and the state of strain were optimised for the transformation process.When a methyl-β-cyclodextrin co-solvent was selected,the molar ratio of co-solvent to substrate addition was 3∶1,the glucose supplementation time was day 4,the glucose supplementation concentration was 10 g/L,and growth cells were used,the highest molar conversion rate of 60.4 ± 2.3 % was achieved for 14α-OH-AD.This study provides a new idea for a one-step biotransformation of 14α-OH-AD production.