Loop pathways are responsible for tuning the accumulation of C19- and C22-sterol intermediates in the mycobacterial phytosterol degradation pathway.

4-Androstene-3,17-dione (4-AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA) are the most important and representative C19- and C22-steroidal materials. The optimalization of sterol production with mycobacterial phytosterol conversion has been investigated for decades. One of the major challenges is that current industrial mycobacterial strains accumulate unignorable impurities analogous to desired sterol intermediates, significantly hampering product extractions and refinements. Previously, we identified Mycobacterium neoaurum HGMS2 as an efficient 4-AD-producing strain (Wang et al. in Microb Cell Fact. 19:187, 2020). Recently, we have genetically modified the HGMS2 strain to remove its major impurities including ADD and 9OH-AD (Li et al. in Microb Cell Fact. 20:158, 2021). Unexpectedly, the modified mutants started to significantly accumulate BA compared with the HGMS2 strain. In this work, while we attempted to block BA occurrence during 4-AD accumulation in HGMS2 mutants, we identified a few loop pathways that regulated metabolic flux switching between 4-AD and BA accumulations and found that both the 4-AD and BA pathways shared a 9,10-secosteroidial route. One of the key enzymes in the loop pathways was Hsd4A1, which played an important role in determining 4-AD accumulation. The inactivation of the hsd4A1 gene significantly blocked the 4-AD metabolic pathway so that the phytosterol degradation pathway flowed to the BA metabolic pathway, suggesting that the BA metabolic pathway is a complementary pathway to the 4-AD pathway. Thus, knocking out the hsd4A1 gene essentially made the HGMS2 mutant (HGMS2Δhsd4A1) start to efficiently accumulate BA. After further knocking out the endogenous kstd and ksh genes, an HGMS2Δhsd4A1 mutant, HGMS2Δhsd4A1/Δkstd1, enhanced the phytosterol conversion rate to BA in 1.2-fold compared with the HGMS2Δhsd4A1 mutant in pilot-scale fermentation. The final BA yield increased to 38.3 g/L starting with 80 g/L of phytosterols. Furthermore, we knocked in exogenous active kstd or ksh genes to HGMS2Δhsd4A1/Δ kstd1 to construct DBA- and 9OH-BA-producing strains. The resultant DBA- and 9OH-BA-producing strains, HGMS2Δhsd4A1/kstd2 and HGMS2Δkstd1/Δhsd4A1/kshA1B1, efficiently converted phytosterols to DBA- and 9OH-BA with the rates of 42.5% and 40.3%, respectively, and their final yields reached 34.2 and 37.3 g/L, respectively, starting with 80 g/L phytosterols. Overall, our study not only provides efficient strains for the industrial production of BA, DBA and 9OH-BA but also provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds.

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

4-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.

Whole-genome and enzymatic analyses of an androstenedione-producing Mycobacterium strain with residual phytosterol-degrading pathways.

Mycobacterium neoaurum strains can transform phytosterols to 4-androstene-3,17-dione (4-AD), a key intermediate for the synthesis of advanced steroidal medicines. In this work, we presented the complete genome sequence of the M. neoaurum strain HGMS2, which transforms ß-sitosterol to 4-AD. Through genome annotation, a phytosterol-degrading pathway in HGMS2 was predicted and further shown to form a 9,10-secosteroid intermediate by five groups of enzymes. These five groups of enzymes included three cholesterol oxidases (ChoM; group 1: ChoM1, ChoM2 and Hsd), two monooxygenases (Mon; group 2: Mon164 and Mon197), a set of enzymes for side-chain degradation (group 3), one 3-ketosteroid-1,2-dehydrogenase (KstD; group 4: KstD211) and three 3-ketosteroid-9a-hydroxylases (Ksh; group 5: KshA226, KshA395 and KshB122). A gene cluster encoding Mon164, KstD211, KshA226, KshB122 and fatty acid ß-oxidoreductases constituted one integrated metabolic pathway, while genes encoding other key enzymes were sporadically distributed. All key enzymes except those from group 3 were prepared as recombinant proteins and their activities were evaluated, and the proteins exhibited distinct activities compared with enzymes identified from other bacterial species. Importantly, we found that the KstD211 and KshA395 enzymes in the HGMS2 strain retained weak activities and caused the occurrence of two major impurities, i.e., 1,4-androstene-3,17-dione (ADD) and 9-hydroxyl-4-androstene-3,17-dione (9OH-AD) during ß-sitosterol fermentation. The concurrence of these two 4-AD analogs not only lowered 4-AD production yield but also hampered 4-AD purification. HGMS2 has the least number of genes encoding KstD and Ksh enzymes compared with current industrial strains. Therefore, HGMS2 could be a potent strain by which the 4-AD production yield could be enhanced by disabling the KstD211 and KshA395 enzymes. Our work also provides new insight into the engineering of the HGMS2 strain to produce ADD and 9OH-AD for industrial application.