阅读说明：本技术 一种通过加强胞内代谢提高甾醇转化的方法 (Method for improving sterol conversion by enhancing intracellular metabolism ) 是由 王敏 张扬 申雁冰 周秀玲 骆健美 夏梦雷 张晓� 于 2020-06-30 设计创作，主要内容包括：本发明属于生物催化技术领域,具体涉及一种加强胞内代谢提高甾醇转化能力高效生产甾药前体的方法。本发明是通过向甾药前体生产菌株发酵培养基中添加甘蔗糖蜜和废菌体水解液,来提高甾药前体生产菌株胞内NAD<Sup>+</Sup>和辅酶A的水平,增强代谢丙酰辅酶A的甲基丙二酰途径中各关键酶的转录水平,从而提高菌株代谢丙酰辅酶A的能力,高效发酵生产甾药前体。(The invention belongs to the technical field of biocatalysis, and particularly relates to a method for efficiently producing a steroid precursor by enhancing intracellular metabolism and improving sterol conversion capacity. The invention improves the intracellular NAD (NAD) of the steroid precursor production strain by adding cane molasses and waste thallus hydrolysate into the fermentation culture medium of the steroid precursor production strain + And coenzyme A level, enhancing the transcription level of each key enzyme in the methylmalonyl pathway for metabolizing propionyl coenzyme A, thereby improving the propionyl coenzyme A metabolizing capability of the strainThe ability of the said method to produce steroid precursors by fermentation with high efficiency.)

1. The production method of the steroid precursor is characterized in that cane molasses and waste thallus hydrolysate are added into a fermentation culture medium of a steroid precursor production strain.

2. A method for producing an steroid precursor according to claim 1, wherein said sugar cane molasses is added in an amount such that the content of reducing sugars in the sugar cane molasses in the fermentation medium is 2 to 20 g/L.

3. The method for producing a steroid precursor according to claim 1, wherein the spent cell hydrolysate is obtained by centrifuging a fermentation broth obtained after the fermentation of the previous M.newcastle disease bacterium for producing androst-4-ene-3,17-dione using phytosterol as a substrate, collecting cells, washing the cells with water, adjusting the cell concentration to 20-200g/L, adjusting the pH to 6.8 with NaOH, and boiling the cells for 10-30 minutes.

4. The method for producing a steroid precursor according to claim 1, wherein the amount of the bacterial hydrolysate added to the culture medium is 20 to 100%.

5. A method of producing an steroid precursor according to claim 1, wherein said steroid precursor comprises androst-4-ene-3,17-dione, 9 α -hydroxyandrost-4-ene-3,17-dione, androst-1,4-diene-3,17-dione and a-ring degradants.

6. A process for the production of steroid precursors according to claim 1, wherein the fermentation medium consists of: k₂HPO₄0.1-3g/L，MgSO₄0.1-3g/L, 0.01-0.2g/L ferric ammonium citrate, 1-5g/L citric acid, 1-10g/L diammonium hydrogen phosphate, 1-50g/L phytosterol, 1-30mM cyclodextrin and the balance of water, and the pH value is 6.0-7.5.

7. A process for the production of a steroid precursor according to any one of claims 1 to 6, which comprises the steps of: inoculating seed liquid of steroid prodrug producing strain into fermentation culture medium according to the inoculum size of 2-10%, adding 2-40% cane molasses and 20-100% waste thallus hydrolysate, and culturing at 25-35 deg.C and 50-200rpm for 24-168 hr.

8. The method of producing a pro-steroid of claim 1, wherein the production strain is Mycobacterium neogold (Mycobacterium sp.) MNR M3.

9. The method of producing a pro-steroid of claim 1, wherein the production strain is Mycobacterium fortuitum ARL-91.

The technical field is as follows:

the invention belongs to the technical field of biocatalysis, and particularly relates to a method for efficiently producing a steroid precursor by enhancing intracellular metabolism and improving sterol conversion capacity.

Background art:

steroid drugs, abbreviated as steroid drugs, are widely used clinically, and are important production products in pharmaceutical industry as the second major class of drugs next to antibiotics. At present, pharmaceutical enterprises mainly adopt a microbial fermentation conversion method to produce various core precursors of steroids by taking phytosterol as a raw material, such as 4-androstene-3, 17-dione (AD), 1, 4-androstene-3, 17-dione (ADD), 9 alpha-hydroxyandrost-4-ene-3, 17-dione (9 alpha-OH-AD) and the like. The vast majority of steroids can be synthesized from these core precursors. Currently, the main microorganisms used for steroid precursor production are fast-growing microorganisms such as Mycobacterium Agrobacterium, Arthrobacter simplex, and Rhodococcus. As an alternative to chemical synthesis, bioconversion has become the primary production method in the pharmaceutical industry for the production of steroid precursors. However, both the original strain and the engineered strain have long transformation period (more than or equal to 120 hours), and the transformation efficiency of the strain is reduced sharply in the middle and later transformation period and is accompanied with product degradation, which is also one of the reasons for high production cost of the steroid precursor.

When sterol is used as a substrate for biotransformation to produce a steroid core precursor, the sterol can be degraded in side chains under the action of microorganisms. NAD is required in this process⁺And coenzyme A to produce large amounts of NADH, acetyl-CoA and propionyl-CoA. NAD (nicotinamide adenine dinucleotide)⁺And increased intracellular levels of coenzyme a contribute to the degradation of the sterol side chains. propionyl-CoA, produced by degradation of the sterol side chain, can enter the tricarboxylic acid cycle via the methylmalonyl pathway. In this pathway propionyl-coa is catalyzed by biotin-dependent propionyl-coa carboxylase (PCC) to synthesize (S) -methylmalonyl-coa by its carboxyl group, followed by methylmalonyl-coa epimerase (MCEE) to racemize it into the (R) -enantiomer, (R) -methylmalonyl-coa is finally isomerized into succinyl-coa by methylmalonyl-coa mutase into the tricarboxylic acid cycle, cyanocobalamin (vitamin B12) is the coenzyme for methylmalonyl-coa mutase. Excess accumulation of propionyl-CoA occurs when propionyl-CoA is produced in large quantities beyond the metabolic capacity of the methylmalonyl pathway or when individual enzymes in the methylmalonyl pathway are mutated to cause a reduction or blockade in the metabolic capacity of the metabolic pathway. Large amounts of propionyl-coa can be toxic to cells and even lethal. The propionyl coenzyme A generated by the steroid precursor production strain with stronger production capacity is more, and the accumulation of the propionyl coenzyme A is one of the main reasons for influencing the vitality of the bacteria, reducing the conversion efficiency and prolonging the conversion period.

At present, methods for improving the production capacity of steroid precursor production strains mostly aim at the modification of key enzymes in a sterol metabolic pathway, and the methods usually only focus on the function enhancement of a single enzyme or a few enzymes, and neglect the global understanding of a metabolic pathway. The disadvantages of these methods are the insufficient supply of cofactors and the accumulation of toxic intermediates in the processes that trigger the sterol metabolism. There is therefore a need for a process that increases intracellular cofactor levels, reduces the accumulation of intermediate metabolites and increases sterol conversion capacity of a sterol precursor producing strain.

The invention content is as follows:

aiming at the problems, the invention improves intracellular coenzyme I (NADH and NAD) by utilizing cane molasses and waste thallus hydrolysate⁺) The level of the microbial method is that the degradation of the sterol side chain is enhanced, the methylmalonyl metabolic pathway of propionyl coenzyme A is enhanced, the toxic action on strains caused by the accumulation of the propionyl coenzyme A process is reduced, the problems of long fermentation period and low production efficiency in the production of the microbial method steroid precursor are solved, and the microbial method is used as the steroidReduction of precursor production costs provides a new approach.

The technical scheme for realizing the aim is that cane molasses and waste thallus hydrolysate are added into a fermentation culture medium of a steroid precursor production strain to improve the intracellular coenzyme I level of the steroid precursor production strain and enhance the transcription level of each key enzyme in a methylmalonyl pathway for metabolizing propionyl coenzyme A, so that the propionyl coenzyme A metabolizing capacity of the strain is improved, and the steroid precursor is efficiently fermented and produced;

the steroid precursors include, but are not limited to, Androst-4-ene-3,17-dione (Androst-4-ene-3,17-dione, AD), 9 α -hydroxyandrost-4-ene-3,17-dione (9 α -hydroxyandrost-4-ene-3,17-dione, 9 α -OH-AD), Androst-1,4-diene-3,17-dione (Androst-1,4-diene-3,17-dione, ADD), a-ring degradant, and the like;

further, the content of reducing sugar in the cane molasses in a fermentation culture medium is 2-20 g/L;

preferably, the cane molasses is diluted by double distilled water until the content of reducing sugar is 20%, sterilized at 115 ℃ and added into a culture medium;

further, the waste thallus hydrolysate is prepared by centrifugally collecting thallus from fermentation liquor of last round of new mycobacterium aurum after the fermentation of androstane-4-ene-3, 17-dione produced by taking phytosterol as a substrate, washing twice with water, adjusting the thallus concentration to 20-200g/L, adjusting the pH to 6.8 with NaOH, and boiling for 10-30 minutes;

preferably, the addition amount of the thallus hydrolysate in the culture medium is 20-100% (volume ratio; v/v);

further, the above method for improving the production of steroid precursors is as follows:

transferring seed liquid of steroid prodrug producing strain into fermentation culture medium according to the inoculum size of 2-10% (volume ratio of seed liquid to fermentation culture medium), adding cane molasses (the content of reducing sugar in cane molasses is 2-20g/L in fermentation culture medium) and 20-100% waste thallus hydrolysate, and culturing at 25-35 deg.C and 50-200rpm for 24-168 hr;

preferably, when the production strain is mycobacterium neogold and the substrate phytosterol addition amount is 1-50g/L, cane molasses and waste thallus hydrolysate culture medium are used, the AD yield reaches 0.35-34g/L after fermentation is finished, and the molar conversion rate can reach 50% -99%; compared with a culture medium without cane molasses and waste thallus hydrolysate, the conversion rate and the yield are improved by 10-35 percent;

preferably, when the production strain is mycobacterium fortuitum and the substrate phytosterol dosage is 1-50g/L, the 9 alpha-OH-AD yield reaches 0.45-36g/L after fermentation is finished, and the molar conversion rate can reach 60% -99%; compared with a culture medium without cane molasses and waste thallus hydrolysate, the yield and the conversion rate are improved by 5-16%;

the fermentation medium comprises the following components: k₂HPO₄0.1-3g/L，MgSO₄0.1-3g/L, 0.01-0.2g/L ferric ammonium citrate, 1-5g/L citric acid, 1-10g/L diammonium hydrogen phosphate, 1-50g/L phytosterol, 1-30mM cyclodextrin and the balance of water, and the pH value is 6.0-7.5.

Has the advantages that:

under the condition of not carrying out any genetic engineering modification on a production strain, the method uses a culture medium containing cane molasses and waste thallus hydrolysate to ferment for 144 hours, so that the level of intracellular coenzyme I is improved by 98 percent; it was unexpectedly found that the use of a culture medium containing cane molasses and waste microbial hydrolysate can promote the transcription levels of propionyl-CoA carboxylase, methylmalonyl-CoA epimerase and methylmalonyl-CoA mutase in the methylmalonyl pathway, and the transcription levels of the three enzymes are 4.58 times, 2.19 times and 1.74 times of the glucose culture medium after fermentation for 60 hours; meanwhile, the use of the cane molasses and the waste thallus hydrolysate culture medium improves the yield of androstane-4-ene-3, 17-dione of new mycobacterium aurum by 26 percent and improves the conversion rate of 9 alpha-hydroxyandrost-4-ene-3, 17-dione of mycobacterium fortuitum by 18 percent; meanwhile, the method has the advantage of reducing the production cost. The invention has the advantages of stable performance, simple and convenient process operation and the like, can be effectively used for strengthening the propionyl coenzyme A metabolism of other steroid prodrug production strains, has wide application value and provides a new method for reducing the production cost of the steroid prodrug.

Description of the drawings:

FIG. 1 changes in intracellular coenzyme I levels using cane molasses and spent thallus hydrolysate;

FIG. 2 shows the change of transcription levels of propionyl-CoA carboxylase, methylmalonyl-CoA epimerase and methylmalonyl-CoA mutase after the use of cane molasses and waste cell hydrolysate;

FIG. 3 is a process diagram of androst-4-ene-3,17-dione production using cane molasses and waste microbial hydrolysate;

FIG. 4 is a diagram of a process for producing 9 alpha-hydroxyandrost-4-ene-3, 17-dione from cane molasses and waste microbial hydrolysate.

The specific implementation mode is as follows:

in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present patent and are not intended to limit the present invention.