Production of 9,21-dihydroxy-20-methyl-pregna-4-en-3-one from phytosterols in Mycobacterium neoaurum by modifying multiple genes and improving the intracellular environment

Background Steroid drugs are essential for disease prevention and clinical treatment. However, due to intricated steroid structure, traditional chemical methods are rarely implemented into the whole synthetic process for generating steroid intermediates. Novel steroid drug precursors and their ideal bacterial strains for industrial production have yet to be developed. Among these, 9,21-dihydroxy-20-methyl-pregna-4-en-3-one (9-OH-4-HP) is a novel steroid drug precursor, suitable for the synthesis of corticosteroids. In this study, a combined strategy of blocking Δ1-dehydrogenation and the C19 pathway as well as improving the intracellular environment was investigated to construct an effective 9-OH-4-HP-producing strain. Results The Δ1-dehydrogenation-deficient strain of wild-type Mycobacterium neoaurum DSM 44074 produces 9-OH-4-HP with a molar yield of 4.8%. Hsd4A, encoding a β-hydroxyacyl-CoA dehydrogenase, and fadA5, encoding an acyl-CoA thiolase, were separately knocked out to block the C19 pathway in the Δ1-dehydrogenation-deficient strain. The two engineered strains were able to accumulate 0.59 g L−1 and 0.47 g L−1 9-OH-4-HP from 1 g L−1 phytosterols, respectively. Furthermore, hsd4A and fadA5 were knocked out simultaneously in the Δ1-dehydrogenation-deficient strain. The 9-OH-4-HP production from the Hsd4A and FadA5 deficient strain was 11.9% higher than that of the Hsd4A deficient strain and 40.4% higher than that of the strain with FadA5 deficiency strain, respectively. The purity of 9-OH-4-HP obtained from the Hsd4A and FadA5 deficient strain has reached 94.9%. Subsequently, the catalase katE from Mycobacterium neoaurum and an NADH oxidase, nox, from Bacillus subtilis were overexpressed to improve the intracellular environment, leading to a higher 9-OH-4-HP production. Ultimately, 9-OH-4-HP production reached 3.58 g L−1 from 5 g L−1 phytosterols, and the purity of 9-OH-4-HP improved to 97%. The final 9-OH-4-HP production strain showed the best molar yield of 85.5%, compared with the previous reported strain with 30% molar yield of 9-OH-4-HP. Conclusion KstD, Hsd4A, and FadA5 are key enzymes for phytosterol side-chain degradation in the C19 pathway. Double deletion of hsd4A and fadA5 contributes to the blockage of the C19 pathway. Improving the intracellular environment of Mycobacterium neoaurum during phytosterol bioconversion could accelerate the conversion process and enhance the productivity of target sterol derivatives.

Efficient one-step biocatalytic multienzyme cascade strategy for direct conversion of phytosterol to C17-hydroxylated steroids

Steroidal 17-carbonyl reduction is crucial to the production of natural bioactive steroid medicines, boldenone (BD) is one of the important C17-hydroxylated steroids. Although efforts have been made to produce BD through biotransformation, the challenge of complex transformation process, high substrates cost, and low catalytic efficiencies have yet to be mastered. Phytosterol (PS) is the most widely accepted substrate for the production of steroid medicines due to its similar foundational structure and ubiquitous sources. 17β-Hydroxysteroid dehydrogenase (17βHSD) and its native electron donor play significant roles in the 17β-carbonyl reduction reaction of steroids. In this study, we bridged 17βHSD with a cofactor regeneration strategy in Mycobacterium neoaurum to establish a one-step biocatalytic carbonyl reduction strategy for efficient biosynthesis of BD from PS for the first time. After investigating different intracellular electron transfer strategies, we rationally designed the engineered strain with co-expression of 17βhsd and glucose-6-phosphate dehydrogenase (G6PDH) gene in M. neoaurum . With establishment of an intracellular cofactor regeneration strategy, the ratio of [NADPH]/[NADP + ] was maintained at a relatively high level, the yield of BD increased from 17% (in MNR M3M- ayr1 S.c ) to 78% (in MNR M3M- ayr1 & g6p with glucose supplementation), and the productivity was increased by 6.5 times. Furthermore, under the optimal glucose supplementation condition, the yield of BD reached 82%, which is the highest yield reported by transformation from PS with one-step. This study demonstrated an excellent strategy for production of many other valuable carbonyl reduction steroidal products from natural cheap raw materials. Importance Steroid C17-carbonyl reduction is one of the important transformations for the production of valuable steroidal medicines or intermediates for further synthesis of steroidal medicines, but it remains a challenge through either chemical or biological synthesis. Phytosterol can be obtained from low-cost residue of waste natural materials, and it is preferred as the economical and applicable substrate for steroid medicines production by Mycobacterium . This study explored a green and efficient one-step biocatalytic carbonyl reduction strategy for direct conversion of phytosterol to C17-hydroxylated steroids by bridging 17β-Hydroxysteroid dehydrogenase with a cofactor regeneration strategy in Mycobacterium neoaurum . This work has practical value for the production of many valuable hydroxylated steroids from natural cheap raw materials.

Efficient conversion of phytosterols into 4-androstene-3,17-dione and its C1,2-dehydrogenized and 9α-hydroxylated derivatives by engineered Mycobacteria

Abstract4-Androstene-3,17-dione (4-AD), 1,4-androstadiene-3,17-dione (ADD) and 9α-hydroxyl-4-androstene-3,17-dione (9OH-AD), which are important starting compounds for the synthesis of steroidal medicines, can be biosynthetically transformed from phytosterols by Mycobacterium strains. Genomic and metabolic analyses have revealed that currently available 4-AD-producing strains maintain the ability to convert 4-AD to ADD and 9OH-AD via 3-ketosteroid-1,2-dehydrogenase (KstD) and 3-ketosteroid-9α-hydroxylase (Ksh), not only lowering the production yield of 4-AD but also hampering its purification refinement. Additionally, these 4-AD industrial strains are excellent model strains to construct ADD- and 9OH-AD-producing strains. We recently found that Mycobacterium neoaurum HGMS2, a 4-AD-producing strain, harbored fewer kstd and ksh genes through whole-genomic and enzymatic analyses, compared with other strains (Wang et al. in Microbial Cell Fact 19:187, 2020). In this study, we attempted to construct an efficient 4-AD-producing strain by knocking out the kstd and ksh genes from the M. neoaurum HGMS2 strain. Next, we used kstd- and ksh-default HGMS2 mutants as templates to construct ADD- and 9OH-AD-producing strains by knocking in active kstd and ksh genes, respectively. We found that after knocking out its endogenous kstd and ksh genes, one of these knockout mutants, HGMS2Δkstd211 + ΔkshB122, showed a 20% increase in the rate of phytosterol to 4-AD conversion, compared relative to the wild-type strain and an increase in 4-AD yield to 38.3 g/L in pilot-scale fermentation. Furthermore, we obtained the ADD- and 9OH-AD-producing strains, HGMS2kstd2 + Δkstd211+ΔkshB122 and HGMS2kshA51 + Δkstd211+ΔkshA226, by knocking in heterogenous active kstd and ksh genes to selected HGMS2 mutants, respectively. During pilot-scale fermentation, the conversion rates of the ADD- and 9OH-AD-producing mutants transforming phytosterol were 42.5 and 40.3%, respectively, and their yields reached 34.2 and 37.3 g/L, respectively. Overall, our study provides efficient strains for the production of 4-AD, ADD and 9OH-AD for the pharmaceutical industry and provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds.

Improving the Production of 9,21-di​hydroxy-​20-​methyl-pregna-​4-​en-​3-​one from Phytosterols in Mycobacterium Neoaurum by Modifying Multiple Genes and Improving the Intracellular Environment

BackgroundSteroid drugs are particularly important for disease prevention and clinical treatment. However, traditional chemical methods are rarely implemented during the whole synthetic process to generate steroid intermediates due to the intricate steroid structure. Novel steroid drug precursors and their ideal bacterial strains for industrial production have yet to be developed. Among these, 9-OH-4-HP is a potential steroid drug precursor for the synthesis of corticosteroids. In this study, a combined strategy of blocking Δ1-dehydrogenation and the C19 pathway as well as improving the intracellular environment was investigated to construct an effective 9-OH-4-HP-producing strain.ResultsA Δ1-dehydrogenation-deficient strain of wild-type Mycobacterium neoaurum DSM 44074 produces 9-OH-4-HP with a molar yield of 4.8%. hsd4A, encoding a β-hydroxyacyl-CoA dehydrogenase, and fadA5 encoding an acyl-COA thiolase, were separately knocked out to block the C19 pathway in the Δ1-dehydrogenation-deficient strain. The two engineered strains could accumulate 0.59 g L-1 and 0.47 g L-1 9-OH-4-HP from 1 g L-1 phytosterols. Furthermore, hsd4A and fadA5 were knocked out simultaneously in the Δ1-dehydrogenation-deficient strain. The 9-OH-4-HP production from the Hsd4A and FadA5 double-deficient strain was 11.9% higher than that of the Hsd4A -deficient strain and 40.4% higher than that of the strain with FadA5 deficiency, and its selectivity reached 94.9%. Subsequently, the catalase katE from Mycobacterium and an NADH oxidase, nox, from Bacillus subtilis were overexpressed to improve the intracellular environment. Ultimately, 9-OH-4-HP production reached 3.58 g L-1 from 5 g L-1 phytosterols, and the selectivity of 9-OH-4-HP improved to 97%.Conclusionhsd4A and fadA5 are key enzymes in the C19 pathway for phytosterol side chain degradation. Deletion of hsd4A and fadA5 could almost entirely block the C19 pathway. Improving the intracellular environment of Mycobacterium during phytosterol bioconversion could accelerate the conversion process and enhance the productivity of target sterol derivatives.

Mycobacterium neoaurum Bloodstream Infection Associated with a Totally Implanted Subclavian Port in an Adult with Diabetes and History of Colon Cancer

Background. Mycobacterium neoaurum is a rapidly growing nontuberculosis mycobacterium (NTM) that was first isolated from soil in 1972 and is ubiquitous in soil, water, and dust. The first reported case of human infection by M. neoaurum was published in 1988, presenting as a Hickman catheter-related bacteremia in a patient with ovarian cancer. M. neoaurum has since been recognized as a source of predominantly opportunistic bloodstream infections in immunocompromised hosts. We report the case of an adult diabetic male with M. neoaurum bloodstream infection secondary to an infected venous-access port that had been implanted nearly six years prior for temporary chemotherapy. Case Presentation. A 66-year-old male with schizophrenia, type 2 diabetes mellitus, and a history of excision and chemotherapy to treat adenocarcinoma of the colon 6 years prior, presented with fever and behavioral changes. He was found to have a M. neoaurum bloodstream infection secondary to his implanted subclavian port. Multiple preoperative blood cultures, as well as the removed catheter tip culture, were positive for M. neoaurum. The patient’s condition improved to near premorbid levels after port removal and 6 weeks of targeted antimicrobial therapy. Discussion and Conclusions. Bloodstream infections due to rapidly growing NTM, such as M. neoaurum, have been infrequently reported; however, improved isolation and identification techniques based on genomic testing are resulting in a more in-depth recognition of these widely scattered environmental microbes in human infections. Nonetheless, lengthy identification and susceptibility processes remain a diagnostic and treatment barrier. Patients such as ours who have a history of malignancy and an indwelling foreign body have most often been reported as acquiring M. neoaurum bacteremia. Fortunately, device removal and appropriate antimicrobial therapy guided by susceptibility data is often enough to manage these atypical mycobacterial infections.