Accumulation of 9α-hydroxy derivatives
To eliminate Δ1-dehydrogenation and accumulate 9α-hydroxy derivatives from phytosterols, kstDs were identified and knocked out from the genome of the wild-type strain M. neoaurum DSM 44074, a steroid-degrading Mycobacterium that can completely degrade phytosterols to produce CO2 and H2O [7]. The genome of M. neoaurum DSM 44074 was sequenced as described in the Methods section. Three putative kstD genes (gene 5102 for kstd1, gene 5236 for kstd2, and gene 5233 for kstd3) were identified in M. neoaurum DSM 44704. kstDs was successfully knocked out from the genome of M. neoaurum DSM 44704 as described in the Methods section, and the mutant strain ΔKstD was obtained. The cell growth of the ΔkstD strain showed no significant difference from that of the wild-type strain (Fig. S1). The wild-type M. neoaurum DSM 44074 strain and the genetically modified strain ΔkstD were incubated with phytosterols for 168 h. The resulting metabolites were extracted from the culture supernatants and analysed by HPLC. Compared with the wild-type strain M. neoaurum DSM 44074, which showed no detectable product by HPLC analysis (Fig. 2a), the ΔkstD strain produced 9-OH-AD as the main product with a retention time of 4.2 min (Fig. 2a, peak A), along with 9-OH-4-HP as a by-product with a retention time of 7.1 min (Fig. 2a, peak B). No ADD was detected during phytosterol bioconversion by ΔkstD, which indicated that the phytosterol degradation pathway was interrupted because of the elimination of Δ1-dehydrogenation by kstDs knockout. When MP01 medium plus 1 g L-1 phytosterols was used for incubation of the ΔkstD strain, 0.62 g L-1 9-OH-AD and 0.04 g L-1 9-OH-4-HP were produced within 60 h (Fig. 3a and 3b). The molar yield of 9-OH-AD reached 84.9%. 9-OH-4-HP was the major by-product during- phytosterol bioconversion by ΔkstD, but the molar yield of 9-OH-4-HP was only 4.8%, and the selectivity of 9-OH-4-HP was 5.6% (Table 2). Thus, 9α -hydroxy derivatives successfully accumulated during phytosterol bioconversion by Mycobacterium, but the purity and yield of 9-OH-4-HP remained unsatisfactory.
Construction of a 9-OH-4-HP-producing strain
Dual pathways, the C19 steroid pathway and the C22 steroid pathway, competes during phytosterol side chain degradation. The C19 steroid pathway is the dominant pathway in M. neoaurum DSM 44074, which could be confirmed that ΔkstD produces 9-OH-AD as the main product along with 9-OH-4-HP as a by-product after culture with phytosterols. The two pathways diverge at 22-hydroxy-3,24-dioxo-4-ene-cholest-COA (22-OH-24-CDOE-COA), which could be Δ22-dehydrogenated by the β-hydroxyacyl-CoA dehydrogenase Hsd4A to generate 3,22,24-trioxo-4-ene-cholest-COA (24-CTOE-COA). 24-CTOE-COA could subsequently be catalysed by the thiolase FadA5, leading the phytosterol degradation pathway to the C19 pathway.
Thus, to construct a 9-OH-4-HP-producing strain, hsd4A and fadA5 were identified in the genome of M. neoaurum DSM 44074 and separately knocked out in ΔkstD, resulting in the strains ΔkstDΔhsd4A and ΔkstDΔfadA5.
The cell growth of ΔkstDΔhsd4A and ΔkstDΔfadA5 showed no significant difference from that of the wild-type strain M. neoaurum DSM 44074 (Fig. S1). The strains ΔkstDΔhsd4A and ΔkstDΔfadA5 were cultured with phytosterols for 168 h, and the metabolites were analysed by HPLC (Fig. 2b). As shown in Fig. 3b, 9-OH-4-HP successfully accumulated in both strains ΔkstDΔhsd4A and ΔkstDΔfadA5. The selectivity of 9-OH-4-HP from the ΔkstDΔhsd4A and ΔkstDΔfadA5 strains were 88.6% and 86.0%, respectively. Nevertheless, both strains still showed only a small amount of 9-OH-AD accumulation (Fig. 3a). The selectivity of 9-OH-AD from strains ΔkstDΔhsd4A and ΔkstDΔfadA5 were 7.2% and 8.0%, respectively. After culture with 1 g L-1 phytosterols in MP01 medium, the strain ΔkstDΔhsd4A accumulated 0.59 g L-1 9-OH-4-HP and 0.13 g L-1 9-OH-AD, while 0.47 g L-1 9-OH-4-HP and 0.08 g L-1 9-OH-AD were obtained from strain ΔkstDΔfadA5. The molar yields of 9-OH-4-HP and 9-OH-AD from strain ΔkstDΔfadA5 were both lower than those from strain ΔkstDΔhsd4A. The molar yield of 9-OH-4-HP from strain ΔkstDΔfadA5 was 20.3% lower than that from strain ΔkstDΔhsd4A, and the molar yield of 9-OH-AD from strain ΔkstDΔfadA5 which was 38.5% lower than that from strain ΔkstDΔhsd4A.
Considering that 9-OH-AD still accumulated in both the ΔkstDΔhsd4A and ΔkstDΔfadA5 strains, the C19 steroid pathway of the phytosterol degradation pathway was not completely blocked in either strain. Thus, to enhance the purity and productivity of 9-OH-4-HP and obstruct the yield of 9-OH-AD, hsd4A and fadA5 were both knocked out in strain ΔkstD, and strain ΔkstDΔhsd4AΔfadA5 was obtained. The cell growth of the ΔkstDΔhsd4AΔfadA5 strain showed a trend similar to that of the wild-type strain M. neoaurum DSM 44074 (Fig. S1). As shown in Fig. 2b, after culture with phytosterols and metabolites being analysed by HPLC, the strain ΔkstDΔhsd4AΔfadA5 accumulated 9-OH-4-HP as the main product, while the accumulation of 9-OH-AD was significantly decreased compared with the strains ΔkstDΔhsd4A and ΔkstDΔfadA5. The selectivity of 9-OH-4-HP and 9-OH-AD from strain ΔkstDΔhsd4AΔfadA5 were 94.9% and 2.0%, respectively. The purity of 9-OH-4-HP from strain ΔkstDΔhsd4AΔfadA5 was higher than those from strains ΔkstdΔhsd4A and ΔkstDΔfadA5. After culture with 1 g L-1 phytosterols, 0.66 g L-1 9-OH-4-HP was obtained from strain ΔkstDΔhsd4AΔfadA5, which is 11.9% more than that from strain ΔkstDΔhsd4A and 40.4% more than that from strain ΔkstDΔfadA5 (Fig. 3b). The selectivity and production of 9-OH-4-HP from strain ΔkstDΔhsd4AΔfadA5 were both higher than those from strains ΔkstDΔhsd4A and ΔkstDΔfadA5, indicating that double knockout of hsd4A and fadA5 could effectively block the accumulation of AD homologues.
Moreover, to verify the functions of hsd4A and fadA5 during phytosterol degradation, ΔkstDΔhsd4A-hsd4A, the hsd4A complementation strain, and ΔkstDΔfadA5-fadA5, the fadA5 complementation strain, were also constructed. As shown in Figure 2c, when the two complementation strains were cultured with phytosterols and metabolites being analysed by HPLC, the accumulation of 9-OH-AD was recovered. The purities of 9-OH-AD from ΔkstDΔhsd4A-hsd4A and ΔkstDΔfadA5-fadA5 were 90.0% and 88.5%, respectively, which is nearly consistent with those of strain ΔkstD. These results indicated that Hsd4A and FadA5 were key enzymes in the C19 steroid pathway during phytosterol degradation. Phylogenetic trees of Hsd4A and FadA5 were constructed to elucidate evolutionary relationship of the two enzymes (Fig. S2).
Evaluation of the 9-OH-4-HP producer
After culture with 1 g L-1 phytosterols, the molar yield of 9-OH-4-HP from ΔkstDΔhsd4AΔfadA5 was 78.9%. To evaluate the ability of ΔkstDΔhsd4AΔfadA5 to transform phytosterols into 9-OH-4-HP, different concentrations of phytosterols were incubated with ΔkstDΔhsd4AΔfadA5.
As shown in Fig. 4b, the yields of 9-OH-4-HP from the bioconversion of 2 g L-1, 5 g L-1, 8 g L-1, and 10 g L-1 phytosterols by ΔkstDΔhsd4AΔfadA5 were 1.43 g L-1, 2.78 g L-1, 1.98 g L-1, and 1.73 g L-1, respectively. 9-OH-AD was also obtained during the incubation, showing yields of 0.06 g L-1, 0.10 g L-1, 0.03 g L-1, and 0.04 g L-1, respectively (Fig. 4a). The productivity of 9-OH-4-HP was enhanced as the concentration of phytosterols increased up to 5 g L-1. However, at phytosterol concentrations of 8 g L-1 and 10 g L-1, the productivity of 9-OH-4-HP decreased, showing results only slightly higher than that with 2 g L-1 phytosterols, and was obviously lower than that with 5 g L-1 phytosterols. The molar yields of 9-OH-4-HP from different concentrations of phytosterols are listed in Table 3. A downward trend in the molar yield of 9-OH-4-HP appeared as the concentration of phytosterols increased from 2 g L-1 to 10 g L-1. However, the purity of 9-OH-4-HP remained stable. Previous research has reported that phytosterols and their metabolites could be noxious to cells during bioconversion [16, 20-22], which might account for the poor performance of ΔkstDΔhsd4AΔfadA5 during phytosterol bioconversion as the concentration of phytosterols increased.
Intracellular environmental balance contributes to higher 9-OH-4-HP production
The 9-OH-4-HP-producing strain ΔkstDΔhsd4AΔfadA5 did not perform well during the bioconversion of phytosterols when the concentration of phytosterols was higher than 2 g L-1. This might be due to multiple factors that influence the bioconversion of phytosterols.
A series of redox reactions occur during phytosterol degradation, which use oxygen as an electron acceptor, and cholesterol dehydrogenases/isomerases require intracellular nicotinamide adenine dinucleotides (NAD+ and NADH) as cofactors [23, 24]. NAD+ and NADH play important roles during phytosterol transformation. They act in many oxidation-reduction reactions and regulate various enzymatic activities and genetic processes. The intracellular NAD+ concentration decreased due to its consumption. Therefore, NAD+ and NADH have critical effects on the maintenance of the intracellular redox balance. Regeneration of NAD+ and enhancement of the NAD+/NADH ratio may be of great assistance during phytosterol transformation.
In addition, hydrogen peroxide (H2O2) is produced due to incomplete oxidation during aerobic metabolism and the regeneration of flavin adenine dinucleotide (FAD) during the phytosterol transformation process [17]. A high level of H2O2 can damage proteins, DNA, and lipids in cells, resulting in inhibition of cell growth and metabolite yield [25].
To enhance the ability of strain ΔkstDΔhsd4AΔfadA5 to transform phytosterols into 9-OH-4-HP, the catalase katE from DSM 44074 and the NADH oxidase nox from Bacillus subtilis [17] were co-expressed in strain ΔkstDΔhsd4AΔfadA5 to construct strain ΔkstDΔhsd4AΔfadA5-NK.
The extracellular H2O2 concentrations of the two strains ΔkstDΔhsd4AΔfadA5 and ΔkstDΔhsd4AΔfadA5-NK were measured when they were cultured with 5 g L-1 phytosterols for 168 h. As shown in Figure 5b, the extracellular H2O2 concentration of strain ΔkstDΔhsd4AΔfadA5 showed an upward trend during the bioconversion process. The extracellular H2O2 concentration increased from 0.59 μmol L-1 at the beginning to 1.05 μmol L-1 after 168 h and reached a peak of 1.10 μmol L-1 at 120 h. In contrast, the extracellular H2O2 concentration of strain ΔkstDΔhsd4AΔfadA5-NK remained nearly stable during the bioconversion process at approximately 0.51 μmol L-1. Therefore, the overexpression of katE eliminated excessive extracellular H2O2. Moreover, to verify the toxicity of H2O2, cell growth of the strains ΔkstDΔhsd4AΔfadA5 and ΔkstDΔhsd4AΔfadA5-NK was also measured. As shown in Figure 5a, the biomass of strain ΔkstDΔhsd4AΔfadA5-NK was higher than that of strain ΔkstDΔhsd4AΔfadA5, indicating that the elimination of extracellular H2O2 could help with cell growth.
Likewise, the NAD+/NADH ratios of the strains ΔkstDΔhsd4AΔfadA and ΔkstDΔhsd4AΔfadA-NK were also measured after they were cultured with 5 g L-1 phytosterols for 168 h. As shown in Figure 5c, the NAD+/NADH ratio of strain ΔkstDΔhsd4AΔfadA-NK was consistently higher than that of strain ΔkstDΔhsd4AΔfadA by at least 10.9%. At 96 h, the NAD+/NADH ratio was enhanced by 25.4% in strain ΔkstDΔhsd4AΔfadA-NK compared with strain ΔkstDΔhsd4AΔfadA. The overexpression of nox could significantly influence the NAD+/NADH ratio during phytosterol bioconversion.
9-OH-4-HP productivity was also measured to test whether overexpression of katE and nox could enhance the ability of ΔkstDΔhsd4AΔfadA to transform phytosterols into 9-OH-4-HP. The recombinant strain ΔkstDΔhsd4AΔfadA-NK was cultured with 1 g L-1, 2 g L-1, 5 g L-1, 8 g L-1, and 10 g L-1 phytosterols for 168 h, and the productivity of 9-OH-4-HP was measured every 24 h. As shown in Figure 5d, the final productivities of 9-OH-4-HP from 1 g L-1, 2 g L-1, 5 g L-1, 8 g L-1, and 10 g L-1 phytosterols were 0.68 g L-1, 1.53 g L-1, 3.58 g L-1, 2.51 g L-1, and 2.73 g L-1, respectively. Compared with ΔkstDΔhsd4AΔfadA cultured with the same concentrations of phytosterols, the productivities of 9-OH-4-HP were enhanced by 3.03%, 6.99%, 28.7%, 26.8%, and 57.8%. The highest yield of 9-OH-4-HP was obtained when strain ΔkstDΔhsd4AΔfadA-NK was cultured with 5 g L-1 phytosterols, with a molar yield that reached 85.5%, which was 28.8% higher than that of ΔkstDΔhsd4AΔfadA. Moreover, no significant difference in the productivity of 9-OH-AD was observed between the two strains ΔkstDΔhsd4AΔfadA5 and ΔkstDΔhsd4AΔfadA5-NK (Fig. 5e), indicating that the purity of 9-OH-4-HP was also enhanced during phytosterol bioconversion by the strain ΔkstDΔhsd4AΔfadA5-NK. All of the results above confirm that regulation of the intracellular NAD+/NADH ratio and H2O2 levels could be an effective way to improve sterol transformation efficiency and the production of steroid intermediates.