Efficient conversion of phytosterols into 4-androstene-3,17-dione and its C1,2-dehydrogenized and 9α-hydroxylated derivatives by engineered Mycobacteria
Bibliographic record
Abstract
Abstract 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, HGMS2 Δkstd211 + 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, HGMS2 kstd2 + Δkstd211+ΔkshB122 and HGMS2 kshA51 + 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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".