Creation and Characterization of Mycolicibacterium Smegmatis mc 2 155 with Deletions in Genes Encoding Sterol Oxidation Enzymes

. The fast-growing saprotrophic strain Mycolicibacterium smegmatis mc 2 155 is capable of utilizing plant and animal sterols and can be used for creation of genetically engineered strains producing biologically active steroids. Oxidation of the 3 β -hydroxyl group and Δ 5(6) →Δ 4(5) double bond isomerization followed by formation of stenones from sterols are considered as the initial stage of steroid catabolism in some actinobacteria. The study of the mechanism of steroid nucleus 3β - hydroxyl group oxidation is relevant for the creation of a method of the microbiological production of valuable 3β -hydroxy-5-en-steroids. A mutant strain of M. smegmatis with deletions in three genes ( MSMEG_1604, MSMEG_5228 and MSMEG_5233 ) encoding known enzymes exhibiting 3 β -hydroxysteroid dehydrogenase activity was constructed by homologous recombination coupled with double selection. The resulting mutant retained macromorphological properties and the ability to convert cholesterol. 3-Keto-4-en-steroids were found among the sterol catabolism intermediates. Experimentally obtained data indicate the presence of a previously undetected intracellular enzyme that performs the function of 3 β -hydroxysteroid dehydrogenase/ Δ 5(6) →Δ 4(5) isomerase.


Introduction
The study of the mechanisms of oxidation of the steroid nucleus remains relevant in the development of technologies for the microbiological production of biologically active steroids with a 3β-hydroxy-5-ene structure.
The strain of actinobacteria Mycolicibacterium smegmatis mc 2 155 is characterized by rapid growth, absence of pathogenicity and high frequency of transformation, simplifying its genetic modification. The M.smegmatis strain is able to efficiently absorb and use sterols as a carbon source [1], has biotechnological potential for genetic engineering to create strains producing pharmaceutically valuable steroids [2][3][4]. The initial stage of sterol catabolism by actinobacteria starts with the 3β-hydroxyl group oxidation and the subsequent double bond Δ 5(6) → Δ 4 (5) isomerization resulted in the formation of 3-keto-4ene-steroids -stenones [5].
Three genes have been found in M. smegmatis that encode enzymes involved in the conversion of cholesterol into cholestenone. The constitutively expressed MSMEG_1604 gene encoding cholesterol oxidase (ChoD) is similar to the Rv3409c gene of Mycobacterium tuberculosis cholesterol oxidase. 3β-Hydroxysteroid dehydrogenase/Δ 5(6) →Δ 4(5) isomerase (HsdD) encoded by the MSMEG_5228 gene, on the contrary, is induced in the presence of cholesterol. Additional cholesterol-induced dehydrogenase/isomerase is a product of the MSMEG_5233 gene. It is known that mutant strains M. smegmatis mc 2 155 Δ(choD, hsdD) and M. smegmatis mc 2 155 Δ(hsdD, MSMEG_5233) retain the ability to grow in the presence of sterols as the only carbon source, but exhibit reduced 3β-hydroxysteroid dehydrogenase activity [6,7].
The aim of this study is to obtain a mutant strain of M. smegmatis with deletions in three genes encoding known enzymes of the initial stage of sterol catabolism, and to evaluate its cholesterol-transforming activity.

Bacterial strains and conditions for their maintenance
The bacterial strains used in the work are listed in Table 1. M. smegmatis cultures were maintained on M3 medium [8]. The growth of mycolicibacteria was evaluated on a minimal agar medium Middlebrook 7H9 Broth [9] in the presence of 0.5 g/l cholesterol and 8.5 g/l MCD. The strain of Escherichia coli DH5 was grown on LB medium [10].

Knockout of the MSMEG_5233 gene
Mutant cells were obtained using the suicide vector pJL020 encoding hygromycin resistance and sucrose sensitivity due to the levansucrase sucB gene [12]. The DNA regions flanking the sequence of the MSMEG_5233 gene upstream (A) and downstream (B) were amplified by PCR using oligonucleotide primers specified in Table 1 with the chromosomal DNA of M. smegmatis mc 2 155. Mutual splicing of flank amplicons MSMEG_5233_A and MSMEG_5233_B was performed by overlapping PCR. The resulting fragment MSMEG_5233_AB was cloned in the vector pJL020 by the restriction site BamHI. The pJE plasmid constructed in this way (Table 1) was used for directed mutagenesis. The competent M. smegmatis mc 2 155 Δ(choD, hsdD) (CH) cells were transformed by electrtoporation with pJE plasmid according to a known protocol [13]. Selection of transformants was carried out on an agar nutrient medium M3 containing hygromycin (75 µg/ml). Transformants were grown in 5 ml of liquid medium M3 for the second act of recombination. The bacteria were selected for their ability to grow on a dense agar medium M3 containing sucrose (50 g/l) [12]. The presence of the deletion was confirmed by PCR using external primers MSMEG_5233_A_F and MSMEG_5233_B_R. The resulting strain of M. smegmatis (CHE) with deletions in three genes simultaneously MSMEG_1604 (choD), MSMEG_5228 (hsdD) and MSMEG_5233 was used for further work.

Cholesterol biotransformation
Cultures of mutant M. smegmatis CH and CHE were inoculated to 50 ml of M3 medium containing 5 g/l cholesterol and 25.4 g/l MCD and grown under aerobic conditions at 37°C and 200 rpm. Products of cholesterol bioconversion were analysed by thin-layer chromatography (TLC).

Bioconversion of cholesterol by the cell-free culture liquid M. smegmatis
M. smegmatis CHE was cultured in 50 ml of medium M3. After 15 hours of growth, cholesterol (20 mg/l) was added as an inducer, cultures continued to grow for 9 hours. In the control, the inducer was not added. Bacterial cells were pelleted by centrifugation (40 min, 4200 g, 4°C). The supernatant was filtered; cholesterol was introduced into the filtrate to a final concentration of 0.05 g/l. Steroids were controlled by TLC as described earlier [8]. For mutagenesis, plasmid pJE containing variant of the target gene with an internal deletion (ΔMSMEG_5233) was created. This plasmid was transferred to M. smegmatis mc 2 155 Δ(choD, hsdD) cells. Hygromycin-resistant transformants were selected, as the products of the first crossing-over resulting in the integration of the pJE plasmid into the cell chromosome. Due to the presence of a counter-selection marker, the sacB levansucrase gene cassette, bacterial clones growing in the presence of sucrose and losing antibiotic resistance were selected.
The presence of the mutant allele ΔMSMEG_5233 in the genome of double recombinants was confirmed by PCR ( Figure 1) by detecting ~2.18 kb DNA fragment, whereas in the case of a wild-type gene, amplicons of ~2.92 kb are detected. Thus, a strain of M. smegmatis (CHE) was constructed with simultaneous deletions in the genes encoding enzymes catalysing oxidation of steroid nucleus 3β-hydroxyl group.

Oxidation of sterols by mutant mycolicibacteria
As can be seen from Fig.2, M. smegmatis cultures in the presence of cholesterol show more active growth regardless of the number of mutations. The triple mutant CHE has a similar colony macromorphology with both the precursor strain CH and the parent M. smegmatis mc 2 155 (WT). Like a parental strain, the triple deletion mutant CHE demonstrated the preservation of the ability to utilize sterols ( Figure 3) with a complete loss of the substrate within 72 hours of growth. Intermediates with 3-keto-4-ene structure, such as androstenedione and androstadienedione, typical for sterol catabolism by actinobacteria, were found among the conversion products [1,14]. Thus, inactivation of all three known enzymes that catalyze the conversion of cholesterol into cholestenone does not lead to block this activity in cultures of mycolicibacteria. This fact is consistent with the conclusions of other authors [6,7] about the presence in M. smegmatis cells of at least one additional protein that oxidizes the 3βhydroxyl group of the steroid nucleus.
It was previously reported that the oxidation of sterols can be catalysed by extracellular bacterial oxidases [5,15,16]. The study of the cell-free culture liquid of the M. smegmatis CHE strain did not reveal cholesterol oxidase activity ( Figure 4). This suggests the intracellular localization of an unknown enzyme functioning as 3β-hydroxysteroid dehydrogenase/Δ 5(6) →Δ 4(5) isomerase. A mutant M. smegmatis strain with deletions in three genes encoding known enzymes of the initial stage of sterol catabolism was obtained. Gene knockout did not affect the strain growth and its ability to convert 3β-hydroxy-5-ene steroids to 3-keto-4-ene derivatives. In vitro analysis of the culture liquid did not reveal the formation of cholestenone from cholesterol. The experimental data obtained indicate the presence of a previously undetected intracellular enzyme 3β-hydroxysteroid dehydrogenase/Δ 5(6) →Δ 4(5) isomerase.