Construction Of A 22-Hydroxy-23,24-Bisnorchol-1,4-Dien-3-One Engineered Strain Of Phytosterol Degradation

Steroidal drugs are widely used in the prevention and treatment of various diseases and are the second class of drugs next to antibiotics.As an important new steroid drug intermediates,22-hydroxy-23,24-bisnorchol-1,4-dien-3-one(HPD)is one of the important raw materials for the synthesis of some cortical drugs.At present,the application of microbial transformation technology to produce steroid drug intermediates has been widely used in steroid drug industry because of its advantages such as environmental friendliness,fewer production steps and simple reaction conditions.It is an important way to construct an engineered Mycobacterium neoaurum to degrade phytosterol and produce HPD as a new steroid drug intermediate.3-sterone?~1 dehydrogenase plays an important role in the process of microbial degradation of phytosterols,which can catalyze steroid22-hydroxy-23,24-bisnorchol-4-ene-3-one(4-HP)A ring C1,2 dehydrogenation to form double bond HPD.In this dissertation,a recombinant strain was constructed by overexpressing Kstd gene in a mutant strain Mycobacterium neoaurum DSM 1381preserved in the laboratory,and its fermentation medium was optimized to obtain a strain with high HPD yield.Then,in order to build a better engineered strain,the genes and degradation products related to sterol side chain metabolism pathway in the model strain Mycobacterium neoaurum DSM 44074 were analyzed,and the negative regulation genes related to HPD accumulation and genes related to cell wall permeability were knocked out,then by regulating the stability of the intracellular environment.A better engineered strain of plant sterol to produce HPD was obtained.The main results are as follows:1.Mycobacterium neoaurum DSM 1381 was used as the initial strain to construct a recombinant strain overexpressing Kst D.With phytosterol as substrate,the purity of HPD in fermentation broth was increased from 71%to 84%.Then,Firstly,each nutrient component in the fermentation medium was screened out,and then the response surface optimization experiment on the influence of interaction between each component on HPD yield was conducted to fit the response surface map,and a quadratic regression equation was established for the three-level interaction of four factors,corn steep liquor,anhydrous dipotassium hydrogen phosphate,glucose and sodium nitrate.The most suitable fermentation medium formula is:9 g/L corn steep liquor,1.8 g/L sodium nitrate,6 g/L glucose,and 2 g/L dipotassium hydrogen phosphate anhydrous.Under these conditions,phytosterol are used as substrates.The purity of HPD in fermentation broth was increased from 84%to 92%,and the yield was increased from 1.51 g/L to 3.73 g/L.2.To further construct a better HPD producing engineering strain,we used CRISPR/cpf1 system to knock out three hydroxylase genes ksh A1&2&3 in Mycobacterium neoaurum DSM 44074 to prevent the complete degradation of phytosterol.On this basis,we further knocked out the negative regulation genes fad A5 and hsd4A related to the accumulation of HPD,and found that the main products of single knockout hsd4A and double knockout fad A5 and hsd4A genes were both HPD.To further accelerate the phytosterol transformation,we also knocked out the mmpk3 and kas D genes related to cell wall permeability.Finally,by stabilizing the internal environment in the fermentation process,the recombinant strain Mycobacterium neoaurum DSM 44074?AHM/p38-kata-nadh was constructed by overexpression of NADH dehydrogenase and catalase.When the substrate sterol concentration was 20 g/L,the yield of HPD reached 12.7 g/L,and the purity of the main product reached 93%.3.Through the above research,we obtained a recombinant strain Mycobacterium neoaurum DSM 1381/P38-Kstd that efficiently transforms phytosterol to produce HPD.The production of HPD was significantly increased by optimizing the fermentation medium,and then a series of experiments were performed in the model strain Mycobacterium neoaurum DSM 44074 gene knockout and plasmid introduction,constructed a high-producing HPD strain Mycobacterium neoaurum DSM 44074?AHM/P38-kata-nadh.The results provided a theoretical basis for microbial transformation of phytosterol to HPD.