Efficient Synthesis of Steroidal Intermediates with a C17 Side Chain from Phytosterols by Genetically Modified Mycolicibacterium neoaurum NRRL B‐3805 Strain

Abstract

22‐Hydroxy‐23,24‐bisnorchol‐4‐ene‐3‐one (4‐HBC) and 3‐oxo‐4,17‐pregnadiene‐20‐carboxylic acid methyl ester (PDCE) are useful precursors for the synthesis of steroidal active pharmaceutical ingredients. In this study, we identify the sterol metabolism‐related genes, which encode the aldolases (Ltp2 and Thl) and carboxylic acid reductases (CAR) in Mycolicibacterium neoaurum NRRL B‐3805 (B3805), by analysis of the metabolites from phytosterols biotransformation. Based on these results, a genetically modified strain is constructed by disrupting the kstD, ltp2, and hsd4A genes and overexpressing the aldolase gene (thl) in the strain B3805. This recombinant strain (B3805V) is able to transform 5 g L⁻¹ phytosterols to 2.0 g L⁻¹ 4‐HBC without detectable AD by‐product. Additionally, by disrupting the ltp2 and car genes, a strain (strain B3805VI) is obtained to transform phytosterols to PDCE with 1.44 g L⁻¹ titer. The PDCE concentration is further increased by about 42% to 2.1 g L⁻¹ without 4‐HBC by‐product by deleting thl gene (strain B3805VII). On the preparative scale, the strain B3805VII transforms 10 g L⁻¹ of phytosterols into PDCE with 5.1 g L⁻¹. This study presents one‐step bioproduction of pharmaceutically important 4‐HBC and PDCE with high yield and purity from bio‐renewable phytosterols, which are readily available as a by‐product from the plant oil industry.

Two genetically modified strains are designed and constructed to produce 22‐hydroxy‐23,24‐bisnorchol‐4‐ene‐3‐one (4‐HBC) and 3‐oxo‐4,17‐pregnadiene‐20‐carboxylic acid methyl ester (PDCE) by disrupting and overexpressing the aldolase gene (thl) in the genetically modified strains of Mycolicibacterium neoaurum NRRL B‐3805, respectively, which are useful precursors for the synthesis of steroidal active pharmaceutical ingredients.

1. Introduction

The utilization of phytosterols as primary materials in the synthesis of steroidal medicines has gained popularity owing to their bio‐renewability and being readily available as a by‐product from vegetable oil industry.^{[ 1 ]} Microbial processes have been developed to efficiently break down the side chain of phytosterols, resulting in the industrial production of 19‐carbon steroidal intermediates such as 4‐androstene‐3,17‐dione (AD), androsta‐1,4‐en‐1,17‐dione (ADD), and 9α‐hydroxy‐androst‐4‐en‐1,17‐dione (9α‐OH‐AD).^{[ 2 ]} These intermediates serve as important building blocks for the commercial synthesis of various steroidal active pharmaceutical ingredients (APIs).

Over the past decades, a putative metabolic pathway has been proposed based on the intermediates identified in the sterol degradation. Phytosterols undergo degradation via two biochemical processes: elimination of alkyl side‐chain and catabolism of the core ring. The metabolism of the core rings is initiated by the transformation of 3β‐ol‐5‐en‐ to 3‐keto‐4‐en‐ moiety,^{[ 3 ]} and further mineralization of the steroidal core rings is proposed to employ a 9,10‐seco pathway, which was named due to the cleavage between C‐9 and C‐10 of the sterol ring.^{[ 4 ]} The initial terminal oxidation of alkyl side‐chain is believed to employ the steroid C27 monooxygenases (cyp125 and cyp142) of cytochrome P450s.^{[ 5 ]} A β‐oxidation‐like mechanism is suggested for the further degradation of the alkyl side‐chain. This process may involve several enzymes, including sterol‐CoA ligases such as FadD19 and FadD17,^{[ 6 ]} dehydrogenases including ChsE1‐5, FadE26‐29,34, or Scd1‐2,^{[ 7 ]} hydratases like ChsH1‐2 or Shy,^{[ 8 ]} acyl‐CoA thiolase such as FadA5,^{[ 9 ]} β‐hydroxyacyl‐CoA dehydrogenase (Hsd4A),^{[ 10 ]} and aldolase such as Ltp2‐4 or Sal1‐2.[ ⁸ , ¹¹ ] Manipulation of this degradation pathway has resulted in valuable strains for the production of C‐19 and C‐22 steroidal building blocks (Figure  1 ).[ ² , ¹⁰ , ¹² ]

Figure 1.

Overview of the cholesterol degradation pathway.,[ ² , ¹⁰ , ¹² ] Thl and Sal, aldolase; CAR, carboxylic acid reductases; MT, methyltransferase. 4‐HBC, 22‐hydroxy‐23,24‐bisnorchol‐4‐ene‐3‐one; PDCE, 3‐oxo‐4,17‐pregnadiene‐20‐carboxylic acid methyl ester; PECE, 3‐oxo‐4‐pregnaene‐20‐carboxylic acid methyl ester; PDCAL, 3‐oxo‐4,17‐pregadiene‐20‐carbaldehyde; BNC, 3‐oxo‐23,24‐bisnorchol‐4‐en‐22‐oate.

Many steroidal APIs, including progestational and adrenocortical hormones, possess a side‐chain at C17, which was typically constructed by introducing a nucleophile at C17 carbonyl group, followed by subsequent transformations.[ ¹² , ¹³ ] Recently, the transformations of phytosterols into 22‐hydroxy‐23,24‐bisnorchol‐4‐ene‐3‐one (4‐HBC, VI), 22‐hydroxy‐23,24‐bisnorchol‐1,4‐dien‐3‐one (1,4‐HBC), and 9,22‐dihydroxy‐23,24‐bisnorchol‐4‐ene‐3‐one (9‐OH‐HBC), wherein a 3‐carbon isopropanol side chain exists at C17‐position, have been studied.^{[ 10} , ¹² , ^{14 ]} The Hsd4A and a reductase (mnOpccr) might be involved in the formation of C22 steroids.^{[ 10} , ^{12 ]} The deletion of hsd4A gene in NwIB‐XII resulted in a 40% molar yield of 1,4‐HBC, while deletion of hsd4A gene in NwIB‐XIIΔkstD123 led to a 49% molar yield of 4‐HBC.^{[ 10 ]} Inactivation of the hsd4A and overexpression of mnOpccr in Mycolicibacterium neoaurum CCTCC AB2019054 achieved a 93% conversion of phytosterols to 4‐HBC. ^[12b] These C22 steroids offer new building blocks for the synthesis of steroidal APIs with a C17‐side chain. Recently, formation of 9‐OH‐PDC (9α‐hydroxy‐3‐oxo‐4,17‐pregnadiene‐20‐carboxylic acid) and 9‐OH‐PDCE (the methyl‐esterified 9‐OH‐PDC) as C22 steroid intermediates from phytosterols has also been reported.[ ¹¹ , ¹⁵ ] Arima et al. reported that the Nocardia corallina IFO 3338 degraded cholesterol, resulting in the accumulation of pregn‐1,4‐dien‐3‐one‐20‐carboxylic acid (1,4‐BNC, as Δ¹ BNC).^{[ 16 ]} Owen et al. confirmed Pseudomonas sp. NCIB 10590 possessed the ability to degrade cholesterol to form both 1.4‐BNC and BNC.^{[ 17 ]} A mutant strain of Rhodococcus rhodochrous RG32 has been reported to degrade cholesterol predominantly accumulating 1,4‐BNC.^{[ 6 ]} The genome‐modified strains of Mycobacterium neoaurum have been constructed to degrade phytosterols, generating 3‐oxo‐4,17‐pregnadiene‐20‐carboxylic acid methyl ester (PDCE) and PECE, or 9‐OH‐PDCE, respectively,[ ¹¹ , ¹² , ¹⁵ ] and 9α‐hydroxy‐3‐oxo‐4,17‐pregnadiene‐20‐carboxylic acid methyl ester (9‐OH‐PDCE) has been prepared at gram scale. ^[11d] 4‐HBC and PDCE would be useful intermediates for the synthesis of steroidal APIs because the C17‐C20 double bond, a side chain at C17, and the ester groups offer multiple sites for the construction of the molecular skeleton and functionalization (Figure  2 ).^{[ 13 ]} However, the production of 4‐HBC or PDCE encounters certain challenges. A variety of by‐products, such as 9‐OH‐PDC, were also formed during the degradation of phytosterols by the available microbial strains, thus leading to the difficulty in production isolation and low yield of the desired products. This is because the intricate and indistinct sterol biodegradation pathways present a barrier to constructing effective strains for the production of the target compounds with high purity.

Figure 2.

The key C22 intermediates for the synthesis of various types of steroidal active pharmaceutical ingredients with a side‐chain at C17.[ ¹² , ¹³ ]

In this study, we performed an investigation on the biosynthesis of 4‐HBC and PDCE from phytosterols. Manipulation of β‐hydroxyacyl‐CoA dehydrogenase (Hsd4A) and aldolase (Ltp2 or Thl) in the M. neoaurum NRRL B‐3805 (B3805) resulted in 4‐HBC as the main product instead of AD. The PDCE‐producing strain was then constructed by knocking out both aldolase and carboxylic acid reductase (CAR), and PDCE was prepared from phytosterols in an eco‐friendly and efficient manner.

2. Results and Discussion

2.1. Identification and Classification of ltp2 in B3805

M. neoaurum NRRL B‐3805 (B3805) is capable of metabolizing phytosterols to produce AD as main product, as well as androst‐1,4‐dien‐3,17‐dione (ADD) and 4‐HBC as by‐products.^{[ 18 ]} In our previous studies, methyl 3‐oxo‐23,24‐bisnorchol‐4‐en‐22‐oate (PECE, XIII, Figure 1) was identified as a by‐product in the metabolism of phytosterols by strain B3805 and knockout of the 3‐ketosteroid‐Δ¹ ‐dehydrogenase gene (kstD, protein ID AMO08643.1) prevented the production of ADD. ^[5d] A protein complex of Ltp2 with a DUF35 domain derived from the C‐terminal domain of a hydratase (ChsH2_DUF35) was reported to be able to cleavage the C22 intermediate to generate AD through a reverse aldol mechanism. The strain of B3805 was sequenced (GenBank accession number CP011022^{[ 19 ]}), and a putative ltp2 gene (the accession number of AMO08652.1) was identified. The ltp2 gene was deleted in the kstD‐deletion B3805 strain (B3805ΔkstD, B3805I) by two‐step homologous recombination system.^{[ 20 ]} The genetically modified strain (B3805ΔkstDΔltp2, B3805II) was confirmed by polymerase chain reaction (PCR) using primers (ltp2 _3805del‐U‐Fand ltp2 _3805del‐D‐R) and sequencing (Figure S1, Supporting Information).

The transformations of phytosterols were carried out by the genome‐modified strains. The cultures were extracted with ethyl acetate and then analyzed by high‐performance liquid chromatography (HPLC) as reported previously.^{[ 21 ]} The strain B3805I produced 2.1 g L⁻¹ AD and 0.18 g L⁻¹ 4‐HBC when 5.0 g L⁻¹ phytosterols were served as substrate. However, no AD was detected, and only 0.3 g L⁻¹ 4‐HBC was found in the cultures of the B3805II strain (Figure  3B,C). This confirmed that putative ltp2 gene encoded an aldolase for the cleavage of C22 intermediate to form AD through an aldol cleavage mechanism in B3805 strain. In comparison to strain B3805I, strain B3805II produced several additional products (c, d, and f, as shown in Figure 3A), along with 4‐HBC (e) and product g. The product d was analyzed by liquid chromatography–mass spectrometry (LC–MS) with m/z of 327.2297 [M + H]⁺ (Figure S2A, Supporting Information) and was speculated as 3‐oxo‐4,17‐pregadiene‐20‐carbaldehyde (PDCAL, XV) (Figure 1). The product f was purified and characterized as 3‐oxo‐4,17‐pregadiene‐20‐carboxylic acid methyl ester (PDCE, XVI, Figure 1) through LC–MS with an m/z of 357.2401 [M + H]⁺ and ¹H, ¹³C NMR in chloroform‐d (Figure S2B, and S3, Supporting Information). Product g was determined as methyl 3‐oxo‐23,24‐bisnorchol‐4‐en‐22‐oate (PECE, XIII, Figure 1).^{[ 21 ]} The product c was not identified. These intermediates were possibly generated through successive thioester hydrolysis, carboxylic acid reduction, or carboxyl methylation of the CoA intermediates VIII or IX, respectively (Figure 1). The compound X should be accumulated after the knockout of ltp2. However, the compound X and AD were not detected after knocking out Ltp2 in strain B3805I. We speculate that the compound X was able to dehydrate to form compound IX when Ltp2 was inactivated. Then, compound IX was transformed into compound XIV by thiolase, and compound XIV was converted into PDCE by methyltransferase.

Figure 3.

Metabolite analyses of phytosterols transformed by B3805, B3805I (B3805ΔkstD), and B3805II (B3805ΔkstDΔltp2) strains. (A) High‐performance liquid chromatography analysis of B3805, B3805I, and B3805II cultured with 5 g L⁻¹ phytosterol. (a) ADD; (b) AD (XI); (c) unknown product; (d) XV; (e) 4‐HBC; (f) XVI; (g) XIII. (B) Time course of AD accumulation by B3805, B3805I, and B3805II strains. (C) Time course of 4‐HBC accumulation by B3805, B3805I, and B3805II.

2.2. Enhancement of 4‐HBC Accumulation by Overexpressing thl and Deleting hsd4A gene

In Pseudomonas sp. strain Chol1, an aldehyde intermediate was discovered during the degradation of cholate, and a steroid aldolase enzyme called Sal was identified as the catalyst for the retro‐aldol cleavage reaction, which involves the aldolytic cleavage of the steroid compound.[ ⁸ , ²² ] In the M. neoaurum strains, an aldolytic cleavage and a reductive reaction were proposed to produce HBCs.^{[ 10 ]} An aldolase thl was predicted to remove the acyl side chain from C24 intermediate to generate 4‐HBC.[ ² , ¹² ] In the M. smegmatis strain, deletion of aldolase gene increased the production yield of AD by avoiding the formation of by‐product 4‐HBC. ^[2b] Moreover, introduction of thl into the genetically modified strains of Mycobacterium fortuitum resulted in generation of HIP‐IPA in the biotransformation of phytosterols.^{[ 23 ]} Based on these findings, introduction of thl gene into B3805II strain and deletion of hsd4A gene would be capable of enhancing the formation of 4‐HBC.

As such, both of thlA (AMO08280.1) and thlB (AMO05741.1) were overexpressed, and hsd4A gene (AMO07649.1) was knocked out in B3805II strain, resulting in the strains B3805III (B3805ΔkstDΔltp2Ωthl), B3805IV (B3805ΔkstDΔltp2Δhsd4A), and B3805V (B3805ΔkstDΔltp2Δhsd4AΩthl). The transformations of phytosterols were carried out using these strains, and the reaction mixtures were extracted and analyzed by HPLC.^{[ 21 ]} As shown in Figure  4 and Scheme  1 , the strains B3805III and B3805IV produced 1.22 and 1.79 g L⁻¹ of 4‐HBC, respectively, when 5.0 g L⁻¹ phytosterols served as the substrate. The B3805V strain yielded 2.00 g L⁻¹ 4‐HBC without detected by‐products, which was 64% and 12% higher than those of strains B3805III and B3805IV, respectively. Therefore, the yield of 4‐HBC was improved from 0.3 g L⁻¹ (strains B3805II) to 2.00 g L⁻¹ by overexpressing thl and inactivating hsd4A, and no AD was detected. The concentration of 4‐HBC was not increased by overexpressing of thlA or thlB alone (data not shown). Although it was reported that deletion of Ltp2 in genetically modified M. neoaurum ATCC 25795 was still able to generate AD, ^[2c] in our hands, the abrogation of aldolase Ltp2 in B3805 strain blocked the sterol side chain degradation to generate AD, while the aldolases (thl) are the key functional enzymes for the formation of 4‐HBC. This demonstrated an interesting strategy for improving the concentration and purity of 4‐HBC by manipulating the strain with different aldolase enzymes of distinct substrate specificity.

Figure 4.

Metabolite analyses of phytosterols transformed by B3805II (B3805ΔkstDΔltp2), B3805III (B3805ΔkstDΔltp2Ωthl), B3805IV (B3805ΔkstDΔltp2Δhsd4A), and B3805V (B3805ΔkstDΔltp2Δhsd4AΩthl) strains. (A) High‐performance liquid chromatography analysis of B3805II, B3805III, B3805IV, and B3805V strains cultured with 5 g L⁻¹ phytosterol. c, unknown product; d, XV; f, XVI; g, XIII. (B) Time course of 4‐HBC accumulation by B3805II, B3805III, B3805IV, and B3805V strains.

Scheme 1.

Biotransformation of phytosterols to 4‐HBC by B3805V strain

2.3. Enhancement of PDCE Accumulation by Deleting thl and Car

The methyl ester as C23 steroid intermediates has been identified as a by‐product in wild‐type strain B3805.^{[ 21 ]} Recently, the C23 steroid intermediates were successfully accumulated in M. neoaurum with chsH and/or ltp2 deficiency.[ ¹¹ , ¹² , ¹⁵ ] It has been reported that the strain was constructed to produce C22 steroid drug precursor 9‐OH‐PDCE by inactivating kstDs and ltp2, and overexpressing hsd4A and chsE1‐chsE2 at the same time in M. neoaurum ATCC 25 795. ^[11d] Similarly, a study with M. neoaurum DSM 44704 also achieved the formation of PDCE by knocking out kshA.[ ¹² , ¹⁵ ] Generally, disruption of ltp2 or chsH in vivo should generate C23 steroid intermediates as the major product, such as PDCE or 9‐OH‐PDCE. In our previous study, two carboxylic acid reductase genes (car1 and car2) were found to be involved in the degradation of HIP, and the strains ΔfadD3Δcar1,2 and ΔfadE30Δcar1,2 were able to efficiently transform phytosterols to HIP and HIL, respectively.^{[ 24 ]} Because 3‐oxo‐4,17‐pregadiene‐20‐carbaldehyde (PDCAL, XV) was detected in the transformation of phytosterols by strain B3805II (Figure 3A(d)), CAR was hypothesized to be involved in the metabolic pathways of compound XII and/or XIV.

Therefore, the CAR (ID: AMO03891.1), which had an identity of 71% and 69% with CAR1 and CAR2 of Mycobacterium fortuitum (ATCC 6841), respectively, was inactivated in strain B3805II to give the strain B3805VI (B3805ΔkstDΔltp2Δcar). As shown in Figure  5 , when the car gene was knocked out, the production of PDCAL (XV) was extremely declined, while the concentration of PDCE (XVI) was increased to 1.44 g L⁻¹. Additionally, a minimal amount of 4‐HBC was found from the transformation of phytosterols with strain B3805VI. To further enhance PDCE production and eliminate the formation of 4‐HBC, the thl gene clusters were knocked out to obtain strain B3805VII (B3805ΔkstDΔltp2ΔcarΔthl). As shown in Figure 5 and Scheme  2 , when the thl gene clusters were knocked out, the 4‐HBC production was blocked, and the accumulation of PDCE was enhanced to a concentration of 2.05 g L⁻¹, 42.4% higher than strain B3805VI (1.44 g L⁻¹).

Figure 5.

Metabolite analyses of phytosterols transformed by B3805II (B3805ΔkstDΔltp2), B3805VI (B3805ΔkstDΔltp2Δcar), and B3805VII (B3805ΔkstDΔltp2ΔcarΔthl). (A) High‐performance liquid chromatography analysis of metabolites by B3805II, B3805VI, and B3805VII strains cultured with 5 g L⁻¹ phytosterol. (B) Time course of PDCE accumulation by B3805II, B3805VI, and B3805VII strains.

Scheme 2.

Biotransformation of phytosterols to PDCE by B3805VII strain

The inactivation of car genes may eliminate the by‐products and improve the accumulation of the desired product PDCE or 3‐oxo‐4,17‐pregnadiene‐20‐carboxylic acid (PDCA, XIV, Figure 1). Considering that the carboxylic acid was the substrate for both carboxylic acid reductase and methyltransferases, inactivation of carboxylic acid reductase favored the formation of the methyl ester of PDCA, which can be generated by an O‐methyltransferase using SAM as the methyl group donor.^{[ 25 ]} Therefore, the strain with inactivation of ltp2, car, and thl (B3805VII) produced PDCE in high yield from phytosterols (Figure 5), a readily available renewable starting material.

Considering the unique structure of PDCE, which has different biological activities and applications from C19 steroids, it is worth further exploring its biological production. Therefore, the PDCE was prepared at a pilot scale by carrying out the transformation of 10 g L⁻¹ of phytosterols by B3805VII for 7 days in a 3 L fermentation. The fermentation bioconversion mixture was extracted with ethyl acetate and analyzed by HPLC. ≈5.1 g L⁻¹ of PDCE was achieved without PDCAL and 4‐HBC due to deletion of CAR and thl, although along with a small amount of PECE in the bioconversion mixture.

3. Conclusion

In summary, we developed genetically modified strains capable of efficiently producing 4‐HBC and PDCE. By eliminating the genes of ltp2 and hsd4A and enhancing the expression of the thl gene, 2.0 g L⁻¹ 4‐HBC was generated from 5 g L⁻¹ of phytosterols without the unwanted by‐product AD. Furthermore, the generation of PDCE was systematically studied. The strain B3805VI with the deletions of ltp2 and car showed a 30% increase in PDCE production with minimal 4‐HBC by‐product. Knocking out thl in strain B3805VI further increased the PDCE production to about 2.1 g L⁻¹ without any detectable 4‐HBC by‐product in the extraction of the culture. These findings have led to the development of highly efficient processes for the production of 4‐HBC and PDCE with exceptional yield and purity. Furthermore, these results provide valuable insights for the molecular engineering of industrial strains for the production of new intermediates, which may be used in the design of environmentally friendly and commercially viable routes for steroidal drug synthesis.

4. Experimental Section

4.1.

4.1.1.

Chemicals and Strains

The phytosterols (95%), AD, 4‐HBC, and other chemical reagents and solvents were purchased from chemical companies with reagent grade or the highest purity available. Phusion high‐fidelity DNA polymerase and FastDigest restriction enzymes were purchased from Fermentas (ThermoFisher, USA). The plasmid extraction kit and gel extraction kit were supplied by SIGMA (Beijing, China). The T5 Direct PCR kit (plant) and the CloneExpress II/MultiS One Step Cloning Kit were from Tsingke (Beijing, China) and Vazyme (Nanjing, China), respectively.

The strains used in this study are listed in Table  1 . Strain B3805ΔkstD (B3805I) was a genetically modified strain of B3805 with the unmarked deletions of one 3‐ketosteroid‐Δ¹‐dehydrogenase gene (kstD).

Table 1.

Strains and plasmids used in this study.

Straina)	Genotype and/or description	Source or reference
Mycolicibacterium neoaurum B‐3805		[19]
B3805I	kstD‐deleted strain of B3805	[5d]
	AD as the main product, none of ADD
B3805II	ltp2 deleted in B3805I strain none of AD	This study
B3805III	B3805II strain harboring plasmid pMV261‐thl	This study
B3805IV	hsd4A deleted in B3805II strain	This study
B3805V	B3805IV strain harboring plasmid pMV261‐thl	This study
B3805VI	car deleted in B3805II strain	This study
B3805VII	thl deleted in B3805VI strain	This study
Plasmids
p2NIL	Vector of two homologous arms for allelic recombination in mycobacteria, Kan R	[20]
pGOAL19	Hyg R , Pag85‐lacZ, P hsp60 ‐sacB, PacI cassette vector, Amp R	[20]
pKHdel‐ltp2 3805	p2NIL Harboring two homologous arms of ltp2 with selection cassette of pGOAL19 for deletion in mycobacteria	This study
pKHdel‐car 3805	p2NIL Harboring two homologous arms of car with selection cassette of pGOAL19 for deletion in mycobacteria	This study
pKHdel‐thl 3805	p2NIL Harboring two homologous arms of thl with selection cassette of pGOAL19 for deletion in mycobacteria	This study
pKHdel‐hsd4A 3805	p2NIL Harboring two homologous arms of hsd4A with selection cassette of pGOAL19 for deletion in mycobacteria	This study
pMV261	Mycobacterium/Escherichia. coli shuttle vector harboring hsp60 promoter, Kan R	[26]
pMV261‐thl	pMV261 contain thl from M. neoaurum B‐3805	This study

^a) Kan ^R , kanamycin‐resistant; Amp ^R , ampicillin‐resistant; Hyg ^R , hygromycin‐resistant.

Gene Deletion and Expression

The unmarked gene deletion strains of B3805 were constructed using homologous recombination with the plasmids pGOAL19 and p2NIL, as described previously.^{[ 20} , ^{23 ]} Two 1 kbp fragments were amplified from B3805 genomic DNA with pairs of primers (Table S1, Supporting Information) ltp2 ₃₈₀₅‐U‐F, ltp2 ₃₈₀₅‐U‐R, ltp2 ₃₈₀₅‐D‐F, ltp2 ₃₈₀₅‐D‐R, car ₃₈₀₅‐U‐F, car ₃₈₀₅‐U‐R, car ₃₈₀₅‐D‐F, car ₃₈₀₅‐D‐R, thl ₃₈₀₅‐U‐F, thl ₃₈₀₅‐U‐R, thl ₃₈₀₅‐D‐F, thl ₃₈₀₅‐D‐R, hsd4A ₃₈₀₅‐U‐F, hsd4A ₃₈₀₅‐U‐R, hsd4A ₃₈₀₅‐D‐F, and hsd4A ₃₈₀₅‐D‐R, respectively. These fragments were then ligated with linear p2NIL (digest with KpnI and PstI) and pGOAL19 (digest with PacI) to produce the recombinant plasmid pKH_del‐ltp2 ₃₈₀₅, pKH_del‐car ₃₈₀₅, pKH_del‐thl _3805, and pKH_del‐hsd4A ₃₈₀₅ using the CloneExpress MultiS One Step Cloning Kit. The plasmids were introduced into the strains through electroporation, respectively. Then, the gene deletion strains were selected using a two‐step selection process and confirmed through PCR and gene sequencing.

The thiolase gene was cloned from B3805 by PCR using the primers thl ₃₈₀₅‐F, thl ₃₈₀₅‐R (Table S1, Supporting Information). These fragments and the plasmid pMV261^{[ 26 ]} were digested with EcoRI and HindIII and then ligated using a ClonExperss II One Step Cloning Kit to produce expression plasmid pMV261‐thl (Table 1). The plasmid was then transferred into strains by electroporation, and the strains hosting the expression plasmid were selected with 50 μg mL⁻¹ kanamycin.

Biotransformation of Phytosterols and Product Analysis

For the biotransformation of phytosterols, all Mycolicibacteria strains were cultured as previously described.^{[ 21} , ^{24 ]} Briefly, the strains were cultured in Luria‐Bertani (LB) broth with 0.5% Tween 80 at 30 °C for 2 days. Then, they were inoculated into a transformation culture containing defatted soy flour (10 g L⁻¹), corn steep power (5 g L⁻¹), (NH₄)₂HPO₄ (2 g L⁻¹), and emulsified phytosterols (5 g L⁻¹), adjusted to pH 7.5 using NaOH.^{[ 24 ]} These strains were initially cultured at 30 °C for 20 h, followed by at 42 °C for 30 min for inducing the expression of thiolases. Subsequently, the strains were cultured at 30 °C for an additional 6 days. Culture samples (1 mL) were taken every 24 h (24, 48, 72, 96, 120, 144, and 168 h) to monitor the concentration of products (AD, 4‐HBC and PDCE) using HPLC.

For the preparative scale biotransformation, the B3805VII strains were cultured in seed medium with glucose (6 g L⁻¹), yeast powder (15 g L⁻¹), NaNO₃ (5.4 g L⁻¹), glycerol (2 g L⁻¹), and NH₄H₂PO₄ (0.6 g L⁻¹), adjusted to pH 7.5, at 30 °C for 3 days. Then, the strain was inoculated into a transformation culture containing emulsified phytosterols (10 g L⁻¹) at 30 °C for 7 days.

For the product analysis, the bioconversion mixture was extracted with 3 volumes of ethyl acetate, and analyzed by HPLC as previously described.^{[ 21 ]} The HPLC was equipped with a C18‐column (250 mm × 4.6 mm × 0.5 μm, Agilent, USA), and a mixture of methanol and water (80:20, v/v) was used as mobile phase at a flow rate of 0.8 mL min⁻¹. Signals were detected with a diode array detector at wavelengths of 210, 230, and 254 nm.

Isolation and Identification of the Products by B3805II Strain

The culture of ltp2 deletion strain (B3805II strain) was extracted with ethyl acetate, and the organic phase was concentrated under reduced pressure. The product was isolated and purified using a silica gel column with petroleum ether and ethyl acetate (6:1, v/v) as eluents. Preparative reverse phase recycling HPLC (Agilent 1260) with C18‐column was used for the final purification, using a mixture of methanol and water (70:30, v/v) at flow rate of 11 mL min⁻¹. The detecting wavelengths were set at 254 and 210 nm. The ¹H and ¹³C NMR spectra of PDCE were recorded at 400 MHz using Bruker Avance III devices with solvent CDCl₃.

Conflict of Interest

The authors declare no conflict of interest.

Supporting information

Supplementary Material

Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article.