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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/Δkstd1 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.


Steroids are active pharmaceutical ingredients (APIs) that are largely demanded in the clinic [1]. The most attractive and efficient strategy for producing sterol APIs is to utilize mycobacteria to transform phytosterols into androstane steroids as precursors that are used to synthesize various advanced steroid medicines through chemical and/or enzymatic modification [2,3,4,5,6,7]. Mycobacterial aerobic side chain degradation of phytosterols is fundamental to the production of androstane steroids as many mycobacteria can survive with sterols as the sole carbon source [3, 8,9,10]. Their sterol degradation pathways have been extensively explored and share a core pathway via 9,10-secosteroid intermediates that participate in the breakdown of steroid ring structures [11, 12].

Currently, only a few sterol APIs can be produced on an industrial scale, including 4-androstene-3,17-dione (4-AD), 1,4-androstadiene-3,17-dione (ADD) and 9α-hydroxy-4-androstenediol (9OH-AD) [2, 3], requiring efficient mycobacterial cell factories to produce new sterol API on an industrial scale. In addition to traditional mutation approaches, including ultraviolet light and chemical-mediated mutation, gene and metabolic engineering have been focused on manipulating mycobacterial sterol degradation pathways to improve the phytosterol biotransformation and construct new strains to accumulate desired sterol intermediates [13,14,15,16,17,18,19,20,21]. The degradation pathway and the accumulation of 4-AD in mycobacteria have been thoroughly explored (Scheme 1) and many key intermediates have been identified (Table 1). Knockout of the kstd, kshA and kshB genes from mycobacteria enhanced 4-AD, ADD and 9OH-AD accumulation [10, 22,23,24,25,26,27,28,29,30,31,32,33,34]. Depletion of the cho, hsd and kstd genes from M. smegmatis promoted 9OH-AD accumulation [35, 36]. Deletion of the kstd gene from the M. neoaurum NwIB-01 strain enhanced 4-AD and ADD production [37]. Knockout of multiple kstd genes from M. neoaurum ATCC25795 generated an efficient 9OH-AD-producing strain [26, 32].

Scheme 1
scheme 1

An overview of the phytosterol degradation pathway in Mycobacterium HGMS2. Each steroid intermediate is numbered. Open arrows refer to a known pathway for the accumulation of 4-AD (S5) and solid lines refer to the ring-broken reactions. Dashed lines refer to unknown pathways to accumulate BA (S11) and 3-OPCM (S13). The information on all steroid intermediates is summarized in Table 2

Table 1 List of steroid compounds

22-Hydroxy-23,24-bisnorchol-4-ene-3-one (BA or 4-HP or 4-HBC) is an important precursor for the synthesis of progesterone, adrenocortical hormones [3, 13], artificial ursodeoxycholic acid and vitamin D3 [38, 39]. To date, only a few strains have been explored for the potential industrial production of BA. The deletion of the hsd4A gene in the kshA-null strain of M. neoaurum ATCC 25795 resulted in BA accumulation [14]. Increasing cell permeability by deleting the MmpL3 gene, encoding a transmembrane transporter of trehalose monomycolate, was effective in enhancing BA productivity [40]. The productivity of these strains could be further improved by the use of mycobacterial resting cells [41,42,43,44]. Very recently, an engineered strain of M. neoaurum DSM 44074 could efficiently convert phytosterols to 9OH-BA [45]. These investigations have revealed that the dual competing pathways between the overwhelming C19 steroid pathway and the C22 steroid pathway are involved in phytosterol sidechain degradation [22, 24, 45,46,47,48]. Some key specific genes, such as hsd4A [14], fadA5 [13, 14], Sal [49], hsaG [50] and OpccR [13], were involved in controlling metabolic flux between the two pathways (Scheme 1). However, the detailed mechanism is still elusive.

Previously, we characterized a 4-AD producing strain, M. neoaurum HGMS2, using genomic and enzymatic analyses and found that it had a relatively simple pathway for phytosterol degradation [51]. After knocking out its active kstd211 and kshA395 and kshB122 genes, the HGMS2Δkstd1/ΔkshA395 and HGMS2Δkstd1/ΔkshB122 mutants significantly reduced the occurrence of the ADD and 9OH-AD impurities and increased the yield of the phytosterol to 4-AD conversion [52]. However, the mutants started to accumulate a small portion of the BA impurity. Thus, in this work, we dissected the two competing pathways in the HGMS2 strain, aiming to construct efficient sterol-producing strains. We bioinformatically identified two hsd4A genes (hsd4A1 and hsd4A2) and one opccR667 gene in the HGMS2 strain that were mainly responsible for tuning the C19-degradation pathway and the C22-degradatin pathway. Through gene knockout, we found that the roles of the hsd4A1, hsd4A2 and opccR667 genes in the HGMS2 strain and its kstd- and/or ksh-null mutants were significantly different in controlling BA accumulation. Moreover, blocking depletion of the BA pathway significantly reduced 4-AD productivity, suggesting that the BA pathway likely contributed to cell growth. Thus, our work provides useful guidance for engineering M. neoaurum HGMS2 to efficiently produce pharmaceutical sterols with tremendous potential for industrial applications.

Materials and methods

Strains and reagents

Mycobacterium neoaurum HGMS2 was maintained in our laboratory, and its genome sequence is available in GenBank (CP031414.1) [51]. The homology recombination vector p2NIL-Sac was constructed previously [52]. The plasmid pMV 261 was purchased from AddGene (Watertown, MA, USA). Restriction enzymes, dNTPs, and Taq and Pfu DNA polymerases were purchased from Takara Co. (Dalian, China). Other molecular biology reagents were of the highest grade and were obtained from New England Biotech (MA, USA). Genomic DNA extraction kits, plasmid purification kits and PCR purification kits were obtained from Tiangen (Beijing, China).

Standard samples of 4-androstene-3,17-dione (4-AD), 22-hydroxy-23,24-bisnorchol-1,4-diene-3-one (DBA), 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one (9OH-BA), and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA) were prepared by Amersino (Wuhan, China). Phytosterol (98%, 410.40 Da) [52] were obtained from Hubei Goto Pharmaceutical Co. (Xiangyang, China).

Bioinformatic prediction of phytosterol metabolic pathways

The phytosterol pathways in Mycobacterium HGMS2 were predicted with the KEGG pathway platform [53], integrating with previous exploration in other cholesterol-degrading strains [14, 40, 45], to query bacterial metabolic pathways, enzymes, and genes coding enzymes for steroid intermediates. A preliminary framework of the phytosterol metabolic pathways was constructed based on the genomic annotation of the Mycobacterium HGMS2 strain [51]. The framework includes all possible and putative enzymes, transporters and regulatory proteins that are involved in phytosterol metabolism.

Preparation of recombinant and complementary mutants

Recombinant HGMS2 mutants were prepared through gene knockout and knockin strategy [52]. The upstream sequence and the downstream sequence of each target gene approximately 1 kbp in length, including hsd4A1, hsd4A2, fadA5 and OpccR667, were amplified from the HGMS2 genome (Additional file 1: Tables S1 and S2) using two pairs of primers (Additional file 1: Table S3). The amplified upstream sequence and downstream sequence for each target gene were digested with two pairs of restriction enzymes, BamHI/XbaI (or NotI) and XbaI (or NotI)/HindIII, respectively. Digested fragments were ligated in one step into the p2NIL-Sac vector predigested with BamHI/HindIII. The ligated plasmids were amplified using E. coli DH5a competent cells and confirmed by DNA sequencing.

The recombination plasmid was electroporated into competent mycobacterial cells with a Scientz-2C electroporation System from ScienTZ (Ningbo, China) according to the procedure reported previously by Li et al. [52]. Positive recombinant colonies were transferred to 5 mL of LBT medium and cultured at 30 °C and 200 rpm for 2 days. Their genomic DNA was extracted for PCR verification. The verified mutant strains were stored at − 80 °C.

Transient expression of Kstd2 and KshA/B in the HGMS2 mutants were carried out with a modified pMV261 vector. The kstd2 gene from M. neoaurum DSM 1381 was amplified by PCR using the primers KstD2-F and KstD2-R (Additional file 1: Table S4), and then ligated into the plasmid pMV261 at the EcoRI site. The kshA/B gene was amplified from the HGMS2 genome using KshA395-F/R and KshB122-F/R primers (Additional file 1: Table S4), and then ligated into the plasmid pMV261 at the NdeI-HindIII site. The plasmids pMV261-KstD2 and pMV261-KshA/B were electroporated into the HGMS2hsd4A1 and HGMS2kstd1/hsd4A1 mutants, resulting in HGMS2hsd4A1/kstd2 and HGMS2kstd1/hsd4A1/kshA/B, respectively.

The complementation of each deleted gene by the homologous expression of the kshA226, OpccR667, hsd4A or fadA5 gene with pMV261 vector in corresponding mutant.

Phytosterol transformation in small-scale fermentation

Mycobacterium strains were initially cultured in 5 mL of LB medium containing 0.05% Tween-80 at 30 °C for two days until the OD600nm value reached 13–15. The culture was inoculated into 100 mL of fermentation medium that was composed of yeast extract (15 g/L), glucose (1 g/L), NaNO3 (5.4 g/L), (NH4)2HPO4 (0.6 g/L), β-cyclodextrin (3 g/L), TW-80 (0.05%, w/v), and phytosterol (10 g/L) and shaken at 30 °C and 200 rpm for 7 days unless otherwise mentioned. Then, 1 mL of culture broth was collected each day to extract metabolites for monitoring the process of phytosterol degradation. For comparison, only HPLC profiles on samples from the fermentation broth on the 7th day were presented when the substrate was completely consumed.

TLC and HPLC analyses of fermentation metabolites

The fermentation broth was thoroughly mixed with ethyl acetate at a ratio of 1:1. The mixture was centrifuged at 8000×g for 5 min and the supernatant was collected. An aliquot of the supernatant was directly used for the thin layer chromatographic (TLC) assay. The supernatant was collected and dried by heating using a hair drier. The dried sample was dissolved in 40% methanol solution for TLC and HPLC assays as described previously [52]. The identities of HPLC peaks were confirmed by comparison with the standards of 4-AD, BA, DBA, and 9OH-BA. Each peak area was integrated with software provided by Waters and used to evaluate the compound concentration.

The mass conversion rate (Conv) of phytosterol to BA, DBA and 9OH-BA was estimated using the following equation:

$$Conv=\frac{Mst}{Mp}\mathrm{ \%},$$

where Mst and Mp are the weights of steroid and phytosterol, respectively.

Phytosterol transformation in pilot-scale culture

Pilot-scale culture was conducted in a 5 L steel fermenter containing 3 L of fermentation medium. The fermentation medium was composed of yeast extract (15 g/L), glucose (8 g/L), NaNO3 (5.4 g/L), (NH4)2HPO4 (0.6 g/L), phytosterol (80 g/L), Tween-80 (0.5%, w/v), antifoam (0.3%, w/v) and lectin (3 g/L). Phytosterol was premixed with glycerol and β-cyclodextrin, and thoroughly emulsified before being transferred to the fermenter. Other materials were added to the fermenter, and water was added until the total volume of the medium reached 3 L. The medium was autoclaved in situ at 121 ℃ for 30 min, followed by cooling down to 30 ℃ with stirring at 500 rpm. The fermentation medium was inoculated with 1% (v/v) of the seed which OD600nm was about 13–15. The pilot-scale fermentation was conducted at 30 ℃ with stirring at 500 rpm. The dissolved oxygen concentration and the pH value of the fermentation medium were constantly monitored in-line and automatically adjusted to maintain 50–60% and 7.5, respectively. Samples were collected every 6 h to determine the concentration of 4-AD, BA, DBA, and 9OH-BA.


Predicted loop pathways caused residual BA in the 4-AD producing strain

Mycobacterium HGMS2 is an efficient industrial 4-AD producing strain, but its major shortcoming is that the HGMS2 strain generated certain percentage of ADD and 9OH-AD. As shown in Fig. 1a (curve 1), these impurities appeared in front of 4-AD (S5) in the HPLC profile. After the deletion of active kstd211 and kshA395 genes, the resultant mutant, HGMS2Δkstd1/ΔkshA395, almost abolished the occurrence of ADD (S6) while significantly reducing the content of 9OH-AD (S7). However, the contents of BA (S11) and other impurities appeared to accumulate (Fig. 1a, curve 2). To completely deplete the formation of any 9α-hydroxyl sterol intermediates, the kshB122 gene, which encodes a reductase complementary to KshAs, was further knocked out from the HGMS2Δkstd1/ΔkshA395 strain, and the resultant triple mutant, named HGMS6 or HGMS2Δkstd1/Δksh395/ΔkshB122 (Table 2), indeed reduced many impurities (Fig. 1a, curve 3). Contrary to our expectation, this triple mutant significantly accumulated BA (S11), although 4-AD (S5) remained the major component, which were confirmed by mass spectrometry (Fig. 1b). The percentages of 4-AD (S5) and BA (S11) were 63.5% and 23.9%, respectively. Although the content of ADD (S6) in the fermentation extract was undetectable, the trace amount of 9OH-AD (S7) was still generated (Fig. 1a, curve 3). Thus, we further knocked out the kshA226 genes encoding a 3-ketosteroid-9α-hydroxylase, which was less active for 4-AD (S5) [51]. However, as examined by HPLC profiling (Fig. 1a, curve 4), this tetraplex mutant, named HGMS6ΔkshA226 (Table 2), increased the contents of 9OH-AD (S7) while the BA (S11) content was significantly reduced. These observations suggested that 4-AD (S5) and BA (S11) accumulated in parallel pathways in the HGMS2 strain, and that the two pathways shared the KstD211 and KshA/KshB-mediated ring-opening mechanism, consistent with previous investigations on other mycobacteria [14, 40, 45].

Fig. 1
figure 1

Depleting 3-ketosteroid-9α-hydroxylases caused the accumulation of BA (S11) in the HGMS2 strain. a HPLC profiling of the extracts from the fermentation broths of different HGMS2 mutants in comparison with that of standard BA (S15). 1: HMGS2; 2: HMGS2Δkstd1/ΔkshA395; 3: HMGS6; 4: HMGS6ΔkshA226 and 5: BA (S15). b Mass spectra of the 4-AD (S5) and BA (S11) peaks collected from HPLC analysis

Table 2 Strains used in this study

To ascertain how BA (S11) was generated in the HGMS2 strain, we predicted few possible loops in phytosterol degradation pathway, turning the phytosterol degradation pathway away from the 4-AD (S5) pathway and switching to the BA (S11) metabolic pathway (Fig. 2). Both pathways eventually converged into 9,10-secosteroid intermediates to completely degrade to CO2 and H2O. Both pathways require a series of key enzymes, including cholesterol oxidases (ChoM), 3β-hydroxyl-dehydrogenase (Hsd), 3-ketosteroid-1,2-dehydrogenase (KstD) and 3-ketosteroid-9α-hydroxylases (Ksh) and side-chain degrading enzymes, to catabolite phytosterols [22, 51, 54, 55]. As shown in Fig. 2, the phytosterol degradation pathway started to derive branch pathways from one key intermediate, i.e., 22-OH-BNC-CoA (S2). Few key enzymes were predicted to control the switching between the 4-AD (S5)_and BA (S11) pathways. The Hsd4A1 and Hsd4A2 enzymes reduced 22-OH-BNC-CoA (S2) to 22-O-BNC-CoA (S3), which was lysed to generate 4-AD (S5) by the FadA5 enzyme. 22-OH-BNC-CoA (S3) could be hydrolyzed into 3-OPA (S10) either through one step by Sal enzyme, or two steps by the Lpt1 and DmpG enzymes, via 22-OH-BNC (S10). 3-OPA (S10) is converted into BA (S11) and 3-OPC-CoA (S4) by OpccR667 and HsaG or DmpF enzymes, respectively. BA could be reversibly converted back to 3-OPA (S10) by the AdhE and Aldo enzymes, while 3-OPA (S10) could be converted into 3-OPC-CoA by the HsaG/DmpF enzyme and returns to the 4-AD pathway.

Fig. 2
figure 2

Predicted loop pathways for differentiating the 4-AD (S5) and BA (S11) accumulations during phytosterol degradation by the Mycobacterium HGMS2 strain. choM: cholesterol oxidase gene; hsd: 3β-hydroxyl steroid dehydrogenase/isomerase gene; cyp125: cytochrome P450 gene; fad19: acyl-CoA synthetase gene; chsE3-E5: long chain acyl-CoA dehydrogenase gene; hsd4A: β-hydroxyacyl-CoA dehydrogenase gene; fadA5: acyl-CoA thiolase gene; chsH1-H2: acyl-CoA hydratase gene; lpt: thioesterase gene; dmpG/Sal: aldolase genes; hsaG: aldehyde dehydrogenase gene; opccR667/adhE/aldo: aldehyde reductase genes; kstd: 3-ketosteroid-1-dehydrogenase gene and ksh: 3-ketosteroid-9α-hydroxylase gene. The information on all steroid intermediates is summarized in Table 2

The OpccR enzyme in the HGMS2 strain inhibited BA-accumulation

Since BA (S11) significantly accumulated during phytosterol fermentation with the HGMS6 and HGMS6ΔkshA226 mutants, we would like to block its occurrence during 4-AD (S5) accumulation. As indicated in Fig. 2, the key step before BA (S11) accumulation was the transformations of 3-OPA (S10) to BA (S11), which was catalyzed by an OpccR enzyme [13]. Through DNA blast analysis against GenBank, we identified an OpccR homologous gene in the HGMS2 strain, named OpccR667 (GenBank accession No. AXK76639.1), which has 100% identity to M. neoaurum CCTCC AB2019054 (Additional file 1: Fig. S1).

We knocked out the OpccR667 gene from the HGMS6ΔkshA226 mutant and the resultant mutant was named HGMS6ΔkshA226/ΔopccR667 (Additional file 1: Table S1, Fig. S2). This mutant was evaluated for its efficiency of phytosterol conversion in small-scale fermentation. The extracts from the two- and six-day fermentation broths were evaluated by HPLC assays (Fig. 3, curves 3 & 4). Unexpectedly, we found that the content of BA (S11) in the extract was not reduced, but significantly increased within two days of fermentation (Fig. 3, curve 3). After six days of fermentation, its 4-AD (S5) productivity remained unchanged in the extract, while the BA (S11) content significantly decreased (Fig. 3, curve 4). However, we observed that a new compound was significantly accumulated (Cpd1 in Fig. 3, curve 4). Cpd1 was not DBA as it had a different retention time from that of DBA on HPLC profiling (Fig. 3, curve 5). Thus, it was likely that OpccR667 reversibly catalyzed the 3-OPA/BA reaction in the HGMS2 strain. Although we could not eliminate BA (S11) from the 4-AD-producing strains, the HGMS6, HGMS6ΔkshA226 and HGMS6ΔkshA226/ΔOpccR667 mutants were good 4-AD-producing strains, compared with HGMS2, because BA (S11) was easily separated from 4-AD (S5) during solvent extraction. Thus, we were more interested in constructing a BA-producing strain using the HGMS2 strain to accumulate BA (S11).

Fig. 3
figure 3

Inactivation of the OpccR667 enzyme in the HGMS strain enhanced the accumulation of BA (S11). The extracts from the fermentation broths of different HGMS6 mutants in comparison with DBA standard sample by HPLC profiling. 1: HMGS6; 2: HMGS6ΔkshA226 (6 days); 3: HMGS6ΔkshA226/ΔopccR667 (2 days); 4: HMGS6ΔkshA226/ΔOpccR667 (6 days) and 5: DBA. Cpd1 represents an unidentified intermediate

The Hsd4A1 enzyme functions as a switch for tuning the phytosterol-degrading metabolic flux between 4-AD (S5) and BA (S11) accumulation

As suggested by Fig. 2, the Hsd4A1-A2 enzymes should determine 4-AD (S5) accumulation via the transformation of 22-OH-BNC-CoA (S2) to 22-O-BNC-CoA (S3). Another enzyme should be HsaG, which transforms 3-OPA (S10) or 3-OPC (S8) to 3-OPC-CoA (S4). Deleting these three enzymes should benefit BA (S11) accumulation.

Thus, we started with the wild-type strain HGMS2 to explore how these enzymes affected BA (S11) accumulation. As examined by HPLC profiling the HGMS2Δhsd4A1 mutant that deleted the hsd4A1 gene from HGMS2 (Table 2, Additional file 1: Fig. S2) almost deplete 4-AD (S5), and significantly accumulated BA (Fig. 4a, curve 2 and inset). Since we used the wild-type strain as the starting template, we expected to observe a few impurities such as 9OH-BA, 9OH-AD and ADD (Fig. 4a, Curve 2). A mutant generated by knocking out the hsd4A2 gene, an hsd4A1 homolog named HGMS2Δhsd4A2, exhibited no significant effect on the conversion profile of phytosterol to 4-AD (S5) (Fig. 4a, curve 3). The mutant generated by knocking out the hsaG gene, named HGMS2ΔhsaG, exhibited no significant effect on the conversion profile of phytosterol to 4-AD (S5) (Fig. 4a, curve 4). These data indicated that the activity of the hsd4A2 and HsaG enzymes was much weaker than that of the hsd4A1 enzyme in HGMS2 and that hsd4A1 played an important role in switching between 4-AD (S5) and BA (S11) accumulation. Interestingly, as unexpected from Fig. 2, the mutant obtained by knocking out the fadA5 gene, named HGMS2ΔfadA5, promoted the accumulation of a new compound that appeared behind BA in the HPLC profile (Cpd2, Fig. 4a, curve 5). Taken together, HGMS2Δhsd4A1 was a good BA-producing strain, although it still generated small amounts of impurities including DBA, 4-AD (S5), ADD (S6) and 9OH-AD (S7), as shown in Fig. 4a (curve 5).

Fig. 4
figure 4

Comparison of the hsd4A-, hsaG-, fadA5- and kstd-knockout mutants for phytosterol fermentation in small-scale fermentation. a HPLC profiles of the extracts from phytosterol fermentation for 144 h in comparison with that of the HGMS2 strain. 1: HGMS2; 2: HGMS2Δhsd4A1; 3: HGMS2Δhsd4A2; 4: HGMS2ΔhsaG; 5: HGMS2ΔfadA5; 6: DBA and 7: 9OH-BA. Cpd2: an unidentified intermediate. Inset: TLC assay of the extracts from the fermentation broths with the HGMS2Δhsd4A1mutant. Δhsd4A1#1 and Δhsd4A1#2 refer to two different colonies. b) Effect of kstd211-knockout on BA (S11) accumulation. 1. HGMS2; 2. HGMS2Δkstd1/Δhsd4A1; 3. HGMS2Δkstd1/Δhsd4A2; 4. HGMS2Δkstd1/ΔfadA5 and 5. HGMS2Δkstd1/hsdA1/ΔhsaG

The Hsd4A2 enzyme benefited BA (S11) accumulation in a complementary manner to the KshA395/B122 enzymes

As DBA was the major impurity during phytosterol conversion by HGMS2Δhsd4A1, HGMS2Δhsd4A2 and HGMS2ΔfadA5 strains, we would like to remove the sole 3-ketosteroid-1,2-dehydrogenase (KstD211) from these three strains. Knockout of its kstd211 gene from the three HGMS2 mutants exhibited the expected effects on BA (S11) accumulation.

After knocking out the kstd211 gene from the HGMS2Δhsd4A1 strain (Additional file 1: Fig. S2), HPLC profiling of its fermentation broth indicated that DBA was almost disappeared in the resultant mutant, i.e., HGMS2Δkstd1/Δhsd4A1 (Table 2), as shown in Fig. 4b (curve 2), except that a small portion of 4-AD remained. Contrary to the HGMS2Δkstd1/Δhsd4A1 mutant, the HGMS2Δkstd1/Δhsd4A2 mutant in which the kstd211 gene was knocked out from HGMS2Δhsd4A2 (Table 2) accumulated less BA (S11) (Fig. 4b, curve 3). Nevertheless, DBA occurrence was significantly reduced in both HGMS2Δkstd1/Δhsd4A1 and HGMS2Δkstd1/Δhsd4A2 mutants.

When the ktdst211 gene was knocked out from HGMS2ΔfadA5 mutant (Additional file 1: Fig. S2), the resultant double mutant, HGMS2Δkstd1/ΔfadA5 (Table 2), was also able to significantly accumulate BA, as profiled by HPLC analysis (Fig. 4b, curve 4). Compared with HGMS2ΔfadA5, this double mutant showed enhanced BA (S11) accumulation and reduced 4-AD (S5) accumulation. It was notable that similar to HGMS2ΔfadA5, this double mutant continues to accumulate a high concentration of the Cpd2 compound (Fig. 4b, curve 4). Furthermore, we knocked out the hsaG gene from the HGMS2Δkstd1/Δhsd4A1 strain. The resultant triple mutant, HGMS2Δkstd1/Δhsd4A1/ΔhsaG (Table 1), exhibited no significant difference from HGMS2Δkstd1/Δhsd4A1, by HPLC profiling of its fermentation broth, confirming that the hsaG gene was inactive in the HGMS2 strain (Fig. 4b, curve 5). Nevertheless, the HGMS2Δkstd1/Δhsd4A1, HGMS2Δkstd1/Δhsd4A2 and HGMS2Δkstd1/ΔfadA5 mutants could demolish the occurrence of DBA. Taken together, the Hsd4A1, Hsd4A2 and FadA5 enzymes played an important role in controlling the switching of the 4-AD pathway and the BA pathway (Fig. 2) The deletion of KstD211 not only removed DBA and ADD impurities, but also rebalanced the profiles of 4-AD and BA accumulation.

Thus, the HGMS2Δkstd1/Δhsd4A1 mutant could be considered to construct BA-producing strains. To maximumally reduce impurities, we further removed the 3-ketosteroid-9α-hydroxylase genes from the HGMS2Δkstd1/Δhsd4A1 mutant. Since HGMS6 or HGMS2Δkstd1/ΔkshA395/ΔkshB122 is a kstd- and ksh-null mutant (Table 2), we simply used HGMS6 as starting strain to knock out the hshA1 gene. A resultant mutant, HGMS6Δhsd4A1, did not reduce the residual 4-AD, but increased its content as indicated by HPLC profiling (Fig. 5, curve 5), compared with the HPLC profile of the fermentation broth of the HGMS2kstd1/hsd4A1 mutant. We further knocked out the hsd4A2 gene from the HGMS6Δhsd4A1 mutant, and found that the resultant mutant, HGMS6Δhsd4A1/Δhsd4A2 (Table 2), recovered its high productivity of BA, as indicated by HPLC profiling (Fig. 5, curve 6), although a tiny portion of 4-AD remained. Thus, it was highly likely that the roles of Hsd4A1, Hsd4A2 and FadA5 enzymes in switching the 4-AD and BA pathways were dependent on the KstD and KshA/B enzymes. Thus, we selected the HGMS6Δhsd4A1/Δhsd4A2 strain as a BA-producing strain for further investigation in pilot-scale fermentation.

Fig. 5
figure 5

Effect of 3-ketosteroid-9α-hydroxylases on BA (S11) accumulation in HGMS2 examined by HPLC profiling. 1: HMGS2; 2: HGMS2Δhsd4A1; 3: HGMS2Δkstd1/Δhsd4A1; 4: HMGS6; 5: HGMS6Δhsd4A1 and 6: HMGS6ΔhsdA1/ΔhsdA2. Inset: TLC assay of the extracts from the fermentation broth with the HGMS6Δhsd4A1/ΔhsdA2 mutant. #1: 3 days and #2: 5 days

Comparison of 4-AD- and BA-producing strains in pilot‑scale fermentation

Pilot-scale phytosterol fermentations were conducted in a 5 L fermenter supplied with 3 L fermentation media for HGMS6, HGMS6ΔkshA226, HGMS6Δhsd4A1/Δhsd4A2 and HGMS2Δkstd1/Δhsd4A1 (see “Materials and methods”).

As shown in Fig. 6a, the rate of phytosterol conversion to 4-AD by the HGMS6 strain increased dramatically within the first 5 days and reached 44.6% after 7 days of fermentation with 80 g/L of phytosterol. On average, the final yield of 4-AD was 37.5 ± 3.2 g/L (Fig. 6a). Accompanying the efficient production of 4-AD, the BA accumulation was also observed with yields of 6.45 g/L on average after 7 days of fermentation (Fig. 6a), resulting in the total BA contents of 8.7% on average. Compared with HGMS6, the production of BA in the metabolites of HGMS6ΔkshA226 decreased relatively, and the production of 4-AD increased slightly from 44.6% to 46.8% (Fig. 6b).

Fig. 6
figure 6

Comparison of phytosterol conversion to 4-AD (S5) and BA (S11) in pilot-scale fermentation. a 4-AD (S5) production by the HGMS6 strain. b 4-AD (S5) production by the HGMS6ΔkshA226 strain; c BA (S11) production by the HGMS6Δhsd4A1/Δhsd4A2 strain and d BA production by the HGMS2Δkstd1/Δhsd4A1 strain

As expected, the HGMS6Δhsd4A1/Δhsd4A2 mutant exhibited an enhanced conversion rate of phytosterol to BA. As shown in Fig. 6c, the conversion rate of phytosterol to BA increased to 49.2% after 7 days of fermentation. The final yield of BA in the fermentation broth was estimated to be 39.4 ± 2.8 g/L on average (Table 3) with 80 g/L of phytosterol. Notably, 4-AD almost completely disappeared. Moreover, the HGMS6Δhsd4A1/Δhsd4A2 mutant completely catabolized the substrate with the same efficiency as the HGMS6 strain after 7 days of fermentation (Fig. 6c). Simmiar to the HGMS6Δhsd4A1/Δhsd4A2 mutant, the HGMS2Δkstd1/Δhsd4A1 mutant was also an efficient strain with high BA production (Fig. 6d, Table 3). Trace amounts of 9-hydroxyal products, such as 9OH-BA, 9OH-AD (Fig. 5, curve 2), were observed due to the existence of KshA/B enzymes in the strain. Nevertheless, both the HGMS6Δhsd4A1/Δhsd4A2 and HGMS2Δkstd1/Δhsd4A1 mutants were efficient BA-producing strains.

Table 3 Comparison of the BA yields by a variety of Mycobacterium strains

Construction of DBA-producing and 9OH-BA-producing strains

Encouraged by our successful work on construct ADD- and 9OH-AD-producing strains based on the overexpression of the kstd2 gene and kshA395/kshB122 genes in HGMS3 and HGMS2Δkstd1, respectively [52], we would like to employ the same strategy to construct DBA-producing strain and 9OH-BA-producing strain using the HGMS2Δhsd4A1 and HGMS2Δkstd1/Δhsd4A1 mutant as templates.

Therefore, to develop a DBA-producing strain, KstD2 was overexpressed in HGMS2Δhsd4A1 with an expression plasmid pMV261-kstD2 [52]. The resultant mutant, named HGMS2Δhsd4A1/kstd2, tested in a small-scale fermentation system using phytosterol as substrate, efficiently accumulated DBA within 7 days with a conversion rate of 68.2% and a yield of 6.17 g/L starting with 10 g/L of phytosterol (Table 2). Upon HPLC analysis (Fig. 7a), small amouint of BA and ADD were remained, no detectable 4-AD and 9OH-AD peaks were observed (Fig. 7a, curve 4). During pilot-scale fermentation, the conversion rate of phytosterol to DBA was 46.2% on average after 7 days of fermentation with 80 g/L of phytosterol. The final DBA yield in the fermentation broth was estimated to be 37.9 ± 3.6 g/L on average (Fig. 7b). As shown in Fig. 7b, this DBA-producing mutant completely catabolized the substrate after 5.5 days during pilot-scale fermentation.

Fig. 7
figure 7

Characterization of DBA- and 9OH-BA-producing mutants for phytosterol fermentation. a HPLC profiles of the extracts from the phytosterol fermentation by DBA- and 9-OH-BA-producing mutants for 144 h compared with those of HGMS2hsd4A1/kstd2 and the HGMS2kstd1/hsd4A1/kshA/B mutants. 1: HGMS2; 2: HGMS2hsd4A1; 3: HGMS2kstd1/hsdA1; 4: HGMS2hsd4A1/kstd2; 5: HGMS2kstd1/hsd4A1/kshA/B. b, c. Time course of DBA and 9OH-BA yields by the HGMS2hsd4A1/kstd2 and HGMS2kstd1/hsd4A1/kshA/B strains

To construct a 9OH-BA-producing strain, a previously constructed KshA1 and KshB1-expression vector, i.e., pMV261-KshA1B1 [52], was transformed into HGMS2Δkstd1/Δhsd4A1 to express cellular KshA1/KshB1, resulting in the HGMS2Δkstd1/Δhsd4A1/kshA1B1 mutant. We examined the HGMS2Δkstd1/Δhsd4A1/kshA1B1 mutant for phytosterol transformation in small-scale fermentation for 7 days, and the fermentation broth was extracted with ether acetate and evaluated by HPLC. As shown in Fig. 7a (curve 5), 9OH-BA significantly accumulated in this mutant, although a small portion of BA remained. At the end of 7 days of fermentation. The final conversion rate and the yield were 67.3% and of 6.3 g/L, respectively, when starting from 10 g/L of phytosterol. With pilot-scale fermentation the conversion rate of phytosterol to 9OH-BA was 39.8% on average after 7 days of fermentation (Fig. 7c). This 9OH-BA-producing mutant completely catabolized the phytosterol substrate (80 g/L) after 7 days, with an estimated final 9OH-BA yield in the fermentation broth of 42.8 ± 3.1 g/L on average. During phytosterol transformation, we found that no BA significantly accumulated as an intermediate.


In this work, we dissected loop pathways that tuned the 4-AD (S5) and BA (S11) accumulations in an industrial 4-AD producing strain, Mycobacterium neoaurum HGMS2 [51]. These loop pathways (Fig. 2) were predicted based on the genomic DNA sequencing data of the HGMS2 strain in combination with previous metabolic engineering investigations of key specific genes including hsd4A [14], opccR [13], fadA5 [14], and hsaG [50]. Our mutation assays on HGMS2 confirmed that different Mycobacteria exhibited distinct branched pathways in phytosterol-degradation, as outlined in Fig. 2. Furthermore, our gene complementation experiments confirmed that the knockout of these genes did not cause any polar effects on the phytosterol degradation pathways in the HGMS2 strain (Additional file 1: Table S5, Fig S3), indicating that these key genes play important roles in the BA (S11) accumulation.

Previously, Wei and coworkers found that inactivation of the hsd4A gene in the kshA-null strain of M. neoaurum ATCC 25795 enabled BA (S11) accumulation [14]. In the HGMS2 strain, we identified two homologous hsd4A genes, i.e., hsd4A1 and hsd4A2. Similar to the sole Hsd4A enzyme in the ATCC 25795 strain, the Hsd4A1 enzyme in the HGMS2 strain determined phytosterol-degrading metabolic flux to 4-AD (S5) accumulation (Fig. 4a). Although the Hsd4A2 enzyme was much less active than the Hsd4A1 enzyme, the Hsd4A2 enzyme could also benefit BA (S11) accumulation complementary to the KshA395/B122 enzymes (Fig. 4b). Recently, Qu and coworkers found that the OpccR enzyme in M. neoaurum CCTCC AB2019054 functioned as a dual-role reductase for phytosterol catabolism, enabling efficient BA (S11) accumulation. Although the OpccR enzyme from the HGMS2 strain was identical to that of the CCTCC AB2019054 strain (Additional file 1: Fig. S1), we found that the inactivation of the opccR gene in the HGMS6 strain increased BA (S11) accumulation within 2 days of fermentation (Fig. 3). It is likely that BA (S11) pathways in these mycobacterial strains are different. After 6 days of fermentation, the HGMS6 strain started to accumulate a novel intermediate (Cpd1 in Fig. 3). It was likely that the phytosterol metabolic pathway in the HGMS2 strain was more complicated than that in the CCTCC AB2019054 strain and other isoenzymes should still exist in the HGMS2 strain, needing further investigation to verify their function. Furthermore, knockout of the fadA5 gene from the HGMS2 strain promoted the accumulation of another new intermediate (Cpd2, in Fig. 4), accompanied by an increase in BA (S11) content. On the other hand, it was likely that the knockout of the hsaG gene from the HGMS2 strain did not affect 4-AD (S5) and BA (S11) accumulation. However, the inactivation of the hsd4A, fadA5 and hsaG genes in the kstd- and ksh-deficient HGMS2 strains significantly enhanced BA (S11) accumulation (Figs. 4, 5). Thus, multiple and complicated phytosterol degradation pathways exist in mycobacteria. In terms of two new compounds, Cpd1 and Cpd2, we are currently working on their structures by nuclear magnetic resonance (NMR) spectrometry. Cpd2 has been identified as a novel intermediate that accumulates in the phytosterol degradation pathway. The molecular formula of Cpd2 is C23H34O2 and its structure is shown in Additional file 1: Fig S4. However, the characterization of Cpd1 is still under investigation.

Nevertheless, in this work, we not only reduced the BA content in our previously constructed 4-AD producing mutant, i.e., HGMS2Δkstd1/Δksh395/ΔkshB122 [52] or named HGMS6 in this study (Table 2), but also have significantly transformed this 4-AD-producing strain to a few efficient BA-producing strains, such as HGMS2kstd1/hsd4A1 and HGMS6hsd4A1/hsd4A2. As examined in pilot-scale fermentation, the HGMS6Δhsd4A1/Δhsd4A2 mutant converted phytosterols to BA with a rate of 49.2% and a final yield of 39.4 ± 2.8 g/L within 7 days, ready for industrial application, comparable to other strains (Table 3). Furthermore, we have not observed transiently released intermediates during phytosterol degradation by those mutants created in this work (Additional file 1: Fig S5). Although the strains remain to accumulate trace amounts of 4-AD (S5), it should not be an issue for industrial application as 4-AD (S5) and BA (S11) are easily separated with solvent extraction. Because both 4-AD (S5) and BA (S11) are two important starting materials for the synthesis of sterol medicine, many mutants generated from this work can be useful tools to produce 4-AD (S5) and BA (S11) simultaneously. Furthermore, we transformed the BA-producing strains into DBA- and 9OH-BA-producing strains by overexpressing KstD2 and KshA/B enzymes, respectively.

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We thank Xin Li, Tian Chen and Fei Peng for their technical assistance. We also thank National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Hubei University of Technology, Wuhan, 430068, China, for their research facility cores.


The Hubei Provincial Ministry of Technology (ZDS, No: 2016ACA128), Natural Science Foundation of Hubei Province (Grant Number 2019CFB713) and National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Hubei University of Technology (grant XBTK-2018001).

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ZDS conceived and designed the research. SKS, JXH and XYC conducted the experiments. SKS, MG and YQH analyzed the data. ZDS, SKS and XYC wrote and edited the manuscript. All authors read and approved the manuscript.

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Correspondence to Xiyao Cheng or Zhengding Su.

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Additional file 1

: Table S1. Homologous recombinant sequences for knocking out targeted genes from the HGMS2 mutants. Table S2. DNA sequence of key genes. Table S3. Primers for constructing knockout vectors. Table S3. Primers for constructing knockout vectors. Table S4. Primers used for gene overexpression. Table S5. Summary of gene complementation. Fig. S1. Amino acid alignment of OpccR667 with MnOpccR enzyme. MnOpccR: Mycobacterium sp. CCTCC AB2019054. Fig. S2. Generation of the opccR667-, hsdA1-, hsdA2- and fadA5-deficient mutants. Fig. S3. The complementation assays of the deleted OpccR667, kshA226, hsd4A and fadA5 genes by homologous expression. Fig. S4. Structure characterization of Cpd2. Fig. S5. HPLC profiles of samples extracted from the fermentation broth during 5-day fermentation.

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Song, S., He, J., Gao, M. et al. Loop pathways are responsible for tuning the accumulation of C19- and C22-sterol intermediates in the mycobacterial phytosterol degradation pathway. Microb Cell Fact 22, 19 (2023).

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