Application Of Gene Editing Technology In Re-analysis Of Key Genes Of Sterol Metabolic Pathway

Steroid drugs are the second largest class of drugs after antibiotics,which have important physiological activities and are widely used in clinic.Many mycobacteria can use low added value sterol as carbon source and energy source to produce steroid hormone components or precursors,therefore,modifying the metabolic pathway of mycobacteria to accumulate high value-added steroid intermediates is the most important step in the semisynthetic route of steroid pharmaceutical industry.Sterol oxidation process is highly conservative in mycobacteria,and many valuable pharmaceutical intermediates can be obtained by biotransformation of steroid rings or side chains,such as C19 steroid androst-4-ene-3,17-dione?AD?,androst-1,4-diene-3,17-dione?ADD?,9?-hydroxyandrost-4-ene-3,17-dione?9?-OH-AD?and C22 steroid 22-hydroxy-23,24-bisnorchol-4-ene-3-one?4-HBC?.In this thesis,a series of gene editing techniques were used to modify the genome of the strains in the laboratory accumulating AD and 9?-OH-AD in large quantities.For the first time,CRISPR-dCas9 was used to construct strains that inhibit the expression of hsd4A in Mn-AD,and the expression of hsd4A and kstD simultaneously in Mycobacterium neoaurum 2967?M.neoaurum 2967?.CRISPR-Cas12a was used to construct the inactivated strain of kstD1 in Mycobacterium smegmatis mc2155?M.smegmatis mc2155?.The CRISPR-dCas9 gene suppression method and CRISPR-Cas12a gene inactivation method were successfully introduced to this laboratory,which made up for the disadvantages of the homologous recombination method.Two aspects of work has been mainly carried out in this subject by the newly introduced gene editing methods:Firstly,the gene of kstD1?MSMEG₅941?,kstD2?MSMEG₂869?and kstD3?MSMEG₂867?in M.smegmatis mc2155 strain were studied,and the activities of 3-ketosteroid-?1-dehydrogenases?KstDs?encoded by these three genes were investigated.Secondly,Msm1,Msm 13 and Msml23 strains were constructed by sequentially inactivating kstD1,kstD2 and kstD3 with CRISPR-Cas12a.The Msml strain can mainly accumulate the product 9?-OH-AD,while the product of 9?-OH-AD can be stably accumulated in Msm123 strain and will not be degraded any more.When the strain was cultured with 5 g/L phytosterols as substrate for 14 days,the concentration of 9?-OH-AD was 2.86 g/L.The substrate conversion rate is increased by about 15%compared with that of Msml.At the same time,the roles of all of the three KstDs in the production and stability accumulation of 9?-OH-AD were revealed.In addition,mycolic acid is the main lipid component of mycobacterial cell membrane,therefore,the key enzymes in the process of mycolic acid synthesis,including the ?-ketoacyl-ACP synthetase?KasAB?and three kinds of SAM-dependent methyltransferases served as targets.A series of engineering strains were constructed to inhibit the genes' expression by using CRISPR-dCas9,or to knock out the genes directly The yields of main product of all engineering strains were analyzed by high performance liquid chromatography.Meanwhile,mycolic acids were extracted and detected by thin layer chromatography?TLC?,and the strain Mn-AD or Msm1 served as control.The results of this study indicate that partial inhibition or complete knockout of these genes has little effect on the biotransformation of phytosterols and the synthesis of mycolic acid.In this thesis,CRISPR gene editing technology was introduced into the laboratory for the first time.Then,a series of engineering strains were constructed in strains Mn-AD and M.smegmatis mc2155.The strain Msm123 was obtained,which can stably produce 9?-OH-AD within 14 days and has a yield of 57.2%?by mass?.At the last,the influence of key genes in mycolic acid synthesis process on phytosterols metabolization was explored.