| The transformation of sterols to androstenone by microbiologies, such as Mycobaceria, is one of the important technological lines to produce steroidal medicines in the pharmaceutical industry. Androstenone, including androst-4-ene-3,17-dione (AD), androst-1,4-diene-3,17-dione (ADD),9a-hydroxy androst-4-ene-3,17-dione (9a-OHAD) and their derivatives, can be used to synthesize almost all of the clinical steroids. However, due to the high technical threshold, only a few pharmaceutical companies, such as Pfizer, can apply this kind of technological line to produce steroidal medicines in industrial scale. Many studies indicated that poor solubility of sterols, limited mass transfer, substrate toxicity, product inhibition, and product degradation are bottlenecks that seriously limit the industrial application of the transformation process of sterols to androstenone. In view of these bottlenecks, the paper is intended to enhance the productivity of androstenone from sterols converted by Mycobacterium neoaurum by means of reaction process designs, strain evolution and genetic engineering, focusing on the strain toleration to high concentration of substrates, the transportation of sterols, and the adsorption separation of androstenone in situ.1. The effect of macroporous resins to promote the productivityTo alleviate the effects of product inhibition and degradation, we introduced macroporous resins in the transformation process of sterols to separate the androstenone from broths in the expectation of alleviating the product inhibition and product degradation with resins adsorption separation in situ, to improve the conversion efficiency and product yield. Firstly, we selected the XAD-7(Rohm&Haas) resin to carry out related experiments. The results showed that:when the resin was added, it’s obivious to relieve product inhibition, reduce product degradation as well as to ease the toxicity of high concentration of steroids. Due to it, the conversion capacity performanced fairly good, for example, the conversion rates of product AD and9a-OH AD had improved significantly in the level of the shake flask. The highest AD accumulation of the strain R10could be increased from0.493g/1to1.599g/l, and9a-OH AD of the strain HK86raised from2.069g/l to4.478g/l, or even higher.7L fermenter larger experimental data showed that the maximum accumulation amount of product9a-OH AD could be reached7.559g/l, in the case of the flow of substrate,20g/1of the sterol feeding. With the optimization analysis on the resin addition, we found that the best addition amount is the saturated adsorption amount, the most appropriate time is48-72hours, and the best desorption solution is ethyl acetate.Taking industrial practicality into considerations, ethanol can be used as a good alternative solvent, which can greatly improve the security of the process, reduce the cost of production and investment, to enhance the economic benefits. In summary, the introduction of the resin adsorption separation of the product in situ can obiviously increases yields and makes the downstream extraction of steroidal products more economical, simple and efficient at the same time. To further reduce costs, we also selected a best alternative resin HZ807to substitute XAD-7, which is a cheaper domestic resin, performancing excellent in absorption, and exhibits good traits such as low adsorption of defoamers.2. To enhance the transporter system of steroidsThe sterol uptaking of Mycobacteria and other microorganisms is realized by sterol crystal particles with the cell wall in close contact. It has now been found that the sterol transformed microorganism had a steroidal active transporter system, i.e. Mce4. Mechanism speaking, enhancing the activity of this system will help to improve the conversion efficiency of the substrate in the microorganisms. Found by analysis on related papers:Mce4gene cluster containing two steroid transporters is sup4A and B coding systems, respectively. We tried to raise gene copy numbers of sup4A and B, to enhance the corresponding transporter function, but the final effect is not significant. We speculate that sup4A and B genes may work in the form of a protein complex with other proteins coded by other genes in Mce4pHytosterols transit system. So it’s quite difficult to significantly strengthen the function of the entire transit system through a single form. This part would be further analyzed and optimized by a newly-constructed gene knockout tool.3. To screen strains of toleration to high concentrations of substratesHigh concentrations of sterol substrate feeding have a signifcient toxicity effect on transformation ability of strains. That is to say, when the substrate concentration exceeds the optimum concentration, the amount of product accumulation is inversely proportionalto substrate concentration. In view of this, we uninterrupted adapted Mycobacterium to high concentration of substrate medium to screen for strains resistant to high concentrations of sterols by means ofadaptive evolution. We had screened four strains of toleration to high concentrations of substrates from thousands of potential strains, two of which are the strains RIO-Kal and R10-Ka2of producingAD, and the other two are strains HK86-Kal and HK86-Ka2of producing9a-OHAD. Under the10g/1of substrate feeding, their transformation ability were significantly enhanced, and the yield of AD was improved to about2times, while the9a-OHAD yield can be elevated to3times approximately.4. To select the conversion acceleratorsThe steroids of non-polar compound are a very low solubility in water, which severely restrict strains from uptaking of the steroid substrate. Based on this, we screened some high-quality conversion accelerator from a variety of silicone oils and PPG2000. The experimental data showed that:the conversion ability of strains could be effectively improved by the addition of polyether-modified silicone oil or PPG2000, with which, the accumulated amount of products could be increased by about300%and200%, respectively. Meanwhile, we optimized corresponding concentrations and the addition account of various silicone oils. We found that the optimum accelerator is5%of the polyether-modified silicone oil, and the best time is to add at0day.5. To build a un-marked gene knockout system in MycobacteriumIn the study of enhancing the Mce4sterol transporter system, we found that it’s quite difficult to significantly strengthen the function of the entire transporter system through a single enhancement model of sup4A/B. As a result, we planted to analyze various genes in Mce4cluster systematically and meticulously, to enhance the system more effectively, by method of gene knock-out tools. However, to traditional means of gene knockout, we will introduce a resistance gene to screen for target strains with each gene knockout, which can not meet analysis on a gene cluster of multiple genes. Therefore, we were committed to build a new unmarked gene-knockout system in mycobaterium. This newly-constructed gene knockout tool has many advantages such as simple operation, time-saving and high efficiency, with no introduction of resistance genes, which is qualified for knockouting various genes in a operon for several times, to anlyze a gene cluster function of multiple genes, respectively. |
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