Hello everyone, the article pushed today was published in Bioresource Technology in December 2023"
Novel cytochrome P450s for various hydroxylation of steroids from
filamentous fungi", the corresponding authors are Professor Long Mengfei of Southwest University and Professor Liao Guojian of Jiangnan University.
Hydroxylated steroids are value-added products with a variety of biological activities, but are rarely fully characterized in fungi. Through transcriptome and bioinformatics analysis, the authors introduced a rapid identification strategy for filamentous fungal P450 enzymes, identifying five unique P450 enzymes (CYP68J5, CYP68L10, CYP68J3, CYP68N1, and CYP68N3) at steroidal 6β, 7α, 7β, 11α, and 15α hydroxylation sites in Saccharomyces cerevisiae and Aspergillus oryzae, and explained the CYP68J5 by molecular self-docking and molecular dynamics simulations ( Hydroxylation preference mechanism between 11α and 7α bifunctional hydroxylases) and CYP68N1 (11α hydroxylases). In addition, the authors significantly increased the yield of (11α-OH-4AD) to 0.845 g⋅L-1 by constructing CYP68N1 with cytochrome P450 reductase (CPR), cytochrome b5 (Cytb5), and ABC transporter into Saccharomyces cerevisiae, which was 14-fold higher than the original strain. The authors' research provides a comprehensive approach for the identification and realization of novel cytochrome P450 enzymes, paving the way for the sustainable production of steroidal products.
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Filamentous fungi with multiple hydroxylation activities for steroids
The authors first performed hydroxylation activity assays for the basic precursor 4AD of steroidal drugs for Fusarium (F3-4268, F3-4488, and F3-6793), Beauveria bassiana ARSEF2860 and ARSEFrobertsiiARSEF23 in the laboratory. After 48 h of incubation with F3-4268, two main hydroxylation products appeared, which were identified by NMR spectroscopy as 7α-OH-4AD and 11α-OH-4AD. F3-4488 has 15α and 6β hydroxylation activity against 4AD, producing 15α-OH-4AD and 6β-oh-4AD. However, strain F3-6793 has poor specificity for substrate 4AD. Therefore, two hydroxylation products, 7α-oh-DHEA and 7β-oh-DHEA, were identified by replacing 4AD with DHEA, another steroidal drug-essential precursor. Beauveria bassiana cocci ARSEF23 incubated with 4AD, 11α-oh-4AD and a small amount of 11α,6β-diOH-4AD were identified. 11α-OH-4AD appears to be the main product of Beauveria bassiana ARSEF2860.
Figure 1.Biotransformation of steroids by different filamentous fungi
Characterization of candidate hydroxylases by Saccharomyces cerevisiae or Aspergillus oryzae
Steroidal hydroxylases are mainly induced by substrates, and the authors identified hydroxylases ARSEF2860 F3-4268, F3-6793, and Beauveria bassiana through transcriptome analysis by performing transcriptome sequencing on samples with and without substrates to mine candidate genes. Of the 43 upregulated genes of F3-4268, only one CYP gene (4268CYP-1) was upregulated by a factor of about 3. The authors used the reported c11α-hydroxylase (CYP68J5 as a probe to mine the homologous enzyme of F3-4268 using LocalBLAST. The 4268CYP-1, 4268cyp2, 4268CYP-3 and 4268cyp44 sequences with the highest homology ranking were selected as cytochrome P450 candidate genes through sequence alignment. The authors quantified their expression levels under both induced and non-induced conditions by real-time PCR (qRT-PCR). The results showed that both 4268CYP-1 and 4268CYP-3 genes were up-regulated. Therefore, 4268CYP-1 may be the gene responsible for steroidal hydroxylation in strain F3-4268. A total of 1017 genes in strain F3-6793 were relatively up-regulated, of which 15 were cytochrome P450 genes. CYP gene 6793CYP-1 (Cluster-371.5869) was the most significantly upregulated in the P450 gene, with a 10-fold increase in expression after induction, and was therefore screened for future analysis (Figure 2C).
There are 383 CYP genes in the ARSEF2860 upregulated genes of Beauveria bassiana. After 4AD induction, the transcriptional abundance of the CYP68N1 gene (gene pool ID: 19886090) increased by 11.32 times, which was the gene with the largest up-regulation of the CYP gene (Fig. 2D), followed by the CYP561D2P gene with a 7.34-fold up-regulation amplitude. Both CYP genes are thought to be candidate steroidal hydroxylases for Beauveria bassiana ARSEF2860. For the hydroxylase of F3-4488, the C15α hydroxylation product was slightly higher than the C6β hydroxylation product, indicating that C15 hydroxylase may be the initial hydroxylation process. The authors took C15α-hy-hydroxylase (P450pra) as the reference gene, mined the homologous enzymes of strain F3-4488 by LocalBLAST, and selected the 4488CYP-1, 4488CYP-2, 4488CYP-3 and 4488CYP-44 sequences with the highest homology ranking as CYP candidate genes to quantify the expression levels under induced and non-induced conditions. The 4488CYP-1 gene was found to be upregulated (Figure 2E) and was therefore selected as a candidate hydroxylase for analysis. The authors further excavated steroidal hydroxylase in M. robertsiARSEF23 by constructing a phylogenetic tree (Figure 2F). Among them CYP68N3 CYP68N1 and 4268CYP-1 ARSEF2860 had the highest homology with Coccidiomonas, which were 43.95% and 38.93%, respectively. Therefore, CYP68N3 is considered a candidate P450 enzyme responsible for steroid hydroxylation in M. robertsiARSEF23.
Figure 2.Mining of CYP genes by transcriptome sequencing and bioinformatics analysis
Characterization of candidate hydroxylases by Saccharomyces cerevisiae or Aspergillus oryzae
The authors heterologously expressed the candidate genes 4268CYP-1 and 4268CYP-3 by Saccharomyces cerevisiae and found that the 4268CYP-1 product was identical to the original strain (F3-4268), suggesting that 4268CYP-1 (named CYP68J5 by the International Committee on CYP Nomenclature) is the hydroxylase responsible for the catalytic process of the F3-4268 strain. The results are consistent with the above hypothesis and demonstrate the feasibility of transcriptome sequencing and LocalBLAST mining steroidal hydroxylases. The authors heterologously expressed the 6793CYP-1 gene in yeast and verified its hydroxylation activity with DHEA as substrate, but no expected product was detected. The authors speculate that Saccharomyces cerevisiae BY4741ayr1Δ is not a suitable host for expressing the 6793CYP-1 gene. Therefore, a heterologous expression system of a. moryzaeNSAR1 was designed to verify the function of 6793CYP-1. After 24 h of DHEA (0.1 g⋅L-1), 7α-OH-DHEA and 7β-oh-DHEA appeared in A. oryzaeNSAR1 carrying 6793CYP-1, indicating that 6793CYP-1 (named CYP68L10) was indeed an hydroxylase with 7α and 7β hydroxylation activity for DHEA in F3-6793. Using 4AD as substrate, the hydroxylation ability of the gene CYP68N1 and CYP561D2P was verified by the Saccharomyces cerevisiae BY4741ayr1Δ expression system. Compared with the standard control, the gene with the highest regulatory level of C11α hydroxylase was CYP68N1. In strain F3-4488, the efficiency of 4488CYP-1 was quite low, although the 4488CYP-1 gene was successfully expressed in Saccharomyces cerevisiae BY4741ayr1Δ. Therefore, the authors used the a. oryzaeNSAR1 expression system for expression. The resulting strain is then fed with substrate 4AD to confirm its hydroxylation ability. As expected, two target hydroxylation products appeared, and the substrate 4AD was also completely converted within 24 h, suggesting that the P450 enzyme 4488CYP-1 (named CYP68J3) worked well in A. oryzaeNSAR1. In addition, candidate gene CYP68N3 was introduced into Saccharomyces cerevisiae BY4741ayr1Δ to verify its hydroxylation function. The addition of 4AD yields products 11α-oh-4AD and 6β,11α-diOH-4AD. In summary, a total of 5 novel steroidal hydroxylases were identified, 4 of which were bifunctional, and CYP68N1 had high C11α hydroxylation specificity. In the identification of the putative hydroxylase in strain F3-6793, the most upregulated CYP gene CYP68L10 proved to be unable to exert its hydroxylating function in Saccharomyces cerevisiae. However, CYP68L10 has been shown to be able to convert DHEA to 7α-OH-DHEA and 7β-OH-DHEA in Oryza oryzae. These results suggest that expression system replacement can be a major consideration when identifying P450 hydroxylated steroidases.
CYP68J5和CYP68N1催化甾体羟基化的机理研究
In order to explore the mechanism of hydroxylation of 4AD at C11 and C7 positions by CYP68J5 and CYP68N1, the authors used AutoDock to molecularly dock these two enzymes with heme and 4AD, respectively, and found that the residues bound to 4AD in the CYP68J5 were more conserved and the substrates were more mixed. In CYP68J5, 85W, 298L, 299V, 302H, 303T, and 480I form hydrophobic interactions with substrates, of which 50% (302H, 303T, and 480I) are conserved, while in CYP68N1, only 304T is conserved, with a proportion of less than 10%. In addition, 211S and 219R have additional hydrogen bonding with 4AD in the CYP68J5. Thus, the approximate plane of the B-C ring in 4AD is more parallel to the heme plane, while the B-C ring of 4AD in the CYP68N1 is more perpendicular to the heme plane. Therefore, the close proximity between the Fe atom in the CYP68J5 and C11 or C7 (5.7Å and 5.9Å) may be the reason why CYP68J5 have hydroxylation ability at both C11 and C7 positions in 4AD. In the CYP68N1, the distance between Fe and C11 is 4.9Å, and there is only 11α-hydroxylation product.
To further investigate the mechanism of hydroxylation preference, the authors performed a molecular dynamics simulation of the CYP68J5 and CYP68N1 complexes with Gromacs for 100 ns, after which the two systems reached stability (Figure 3C). Interestingly, there is 38.41% homology between the two enzymes, but they exhibit similar carryover fluctuations except for the main regions of 140-150, 180-190, 360-370, 420-430, and 460-470. These major regions may have an impact on the substrate conformation of specific hydroxylations. To verify this, the authors measured the distance of Fe atoms to C11 and C7, respectively (Figure 3E). In the CYP68N1, the average distance between Fe and C11 is 5.2Å, which is significantly shorter than the distance between Fe and C7. However, within the first 30ns of the CYP68J5, the distance between Fe-C7 is smaller than that between Fe-C11. However, in the subsequent process, the distances between Fe-C7 and Fe-C11 become equal at some points, suggesting that from the perspective of spatial distance, CYP68J5 can hydroxylate both 11 and 7 positions. In the CYP68J5, the B-C ring of sterols is nearly parallel to that of heme, which promotes the activation of C-H at multiple positions and contributes to the production of multiple hydroxylation products. Conversely, in CYP68N1 with a pronounced preference for site-specific hydroxylation, the B-C ring of 4AD underwent orientation bias due to increased interaction with residues close to the C11 side. Further enzymatic engineering approaches are needed to elucidate the specific amino acid residue changes that lead to these changes in hydroxylation positions.
Figure 3.4 Molecular docking and MD simulation of CYP68N1/CYP68J5 during AD hydroxylation
工程酿酒酵母11α-OH-4AD的生物合成
Among the five P450 enzymes, the authors selected CYP68N1 for the construction of microbial cell factories to facilitate the biosynthesis of their single product (11α-OH-4AD). 11α-OH-4AD is an important pharmaceutical intermediate for the production of the diuretic eplerenone, which was the first selective aldosterone receptor antagonist approved for the treatment of hypertension and left ventricular dysfunction after acute myocardial infarction. The authors found that Staphylococcus cerevisiae Z-1, which carried only CYP68N1, showed only low levels of 11α-OH-4AD yield, approximately 0.059 g⋅L-1 (Table 1).
In order to increase the yield of 11α-OH-4AD, the authors further introduced electron transport chain element reductase, ABC transporter, and related fusion proteins. New pr-1, CPR-2 and Cytb5 were identified in Beauveria bassiana ARSEF2860 by BLASTp analysis. The reported CPR homology between CPR-2 and orchid Absidia was 37.98% and 39.70%, respectively, and the homology of Cytb5 reported with Cytb5 and orchid Absidia was 56.96%, respectively. It was found that CPRs and Cytb5 could promote the enhancement of CYP68N1 activity through efficient electron transfer. Fusion proteins have recently been classified as a unique class of novel biomolecules with a variety of functions, including enhancing protein folding and stability, promoting protein expression, and enhancing intrinsic biological activity. The authors further studied the fusion expression of CYP68N1 and CPRs, and found that the fusion of CYP68N1 and CPR-1/CPR-2 promoted the biosynthesis of 11α-OH-4AD. After that, the authors further explored the optimal fusion conditions in terms of the assembly order of the expression elements, the ABC transporter, and the length of the fusion link, and finally, the yield of 11α-OH-4AD of 0.845 g⋅L-1 was obtained within 72 h using the Z-11 strain under the condition of feeding 4AD at 1 g/L-1 (Fig. 4B), which was more than 14 times that of the original strain Z-1 and was one of the strains with the highest 11α-OH-4AD biosynthetic performance among yeasts to date.
Fig.4 Modified CYP68N1 hydroxylase to increase the yield of 11α-OH-4AD
Summary:
In this study, five new P450 enzymes were identified, and more P450 enzymes need to be identified from fungi in the future to enrich the P450 enzyme library for the synthesis of high-value natural products. (ii) The mechanism of 4AD hydroxylation site bias (11α and 7α) also requires more experimental evidence rather than computer-aided analysis. (iii) Novel enabling technologies for synthetic biology, such as modular metabolic engineering, reconstitution of metabolic networks and design of substrate channel systems to improve the catalytic efficiency of P450 enzymes, which can be further used for efficient biosynthesis of steroid-based natural products.