Engineering the Steroid Hydroxylating System from Cochliobolus lunatus in Mycolicibacterium smegmatis.
ABSTRACT: 14α-hydroxylated steroids are starting materials for the synthesis of contraceptive and anti-inflammatory compounds in the steroid industry. A synthetic bacterial operon containing the cytochrome P450 CYP103168 and the reductase CPR64795 of the fungus Cochlioboluslunatus able to hydroxylate steroids has been engineered into a shuttle plasmid named pMVFAN. This plasmid was used to transform two mutants of Mycolicibacterium smegmatis named MS6039-5941 and MS6039 that accumulate 4-androstene-3,17-dione (AD), and 1,4-androstadiene-3,17-dione (ADD), respectively. The recombinant mutants MS6039-5941 (pMVFAN) and MS6039 (pMVFAN) were able to efficiently express the hydroxylating CYP system of C.lunatus and produced in high yields 14αOH-AD and 14αOH-ADD, respectively, directly from cholesterol and phytosterols in a single fermentation step. These results open a new avenue for producing at industrial scale these and other hydroxylated steroidal synthons by transforming with this synthetic operon other Mycolicibacterium strains currently used for the commercial production of steroidal synthons from phytosterols as feedstock.
Project description:11α-hydroxylated steroid synthons are one of the most important commercially pharmaceutical intermediates used for the production of contraceptive drugs and glucocorticoids. These compounds are currently produced by biotransformation using fungal strains in two sequential fermentation steps. In this work, we have developed by a rational design new recombinant bacteria able to produce 11α-hydroxylated synthons in a single fermentation step using cholesterol (CHO) or phytosterols (PHYTO) as feedstock. We have designed a synthetic operon expressing the 11α-hydroxylating enzymes from the fungus Rhizopus oryzae that was cloned into engineered mutant strains of Mycolicibacterium smegmatis that were previously created to produce 4-androstene-3,17-dione (AD), 1,4-androstadiene-3,17-dione (ADD) from sterols. The introduction of the fungal synthetic operon in these modified bacterial chassis has allowed producing for the first time 11αOH-AD and 11αOH-ADD with high yields directly from sterols in a single fermentation step. Remarkably, the enzymes of sterol catabolic pathway from M. smegmatis recognized the 11α-hydroxylated intermediates as alternative substrates and were able to efficiently funnel sterols to the desired hydroxylated end-products.
Project description:A number of pharmaceutical steroid synthons are currently produced through the microbial side-chain cleavage of natural sterols as an alternative to multi-step chemical synthesis. Industrially, these synthons have been usually produced through fermentative processes using environmental isolated microorganisms or their conventional mutants. Mycobacterium smegmatis mc<sup>2</sup> 155 is a model organism for tuberculosis studies which uses cholesterol as the sole carbon and energy source for growth, as other mycobacterial strains. Nevertheless, this property has not been exploited for the industrial production of steroidic synthons. Taking advantage of our knowledge on the cholesterol degradation pathway of M. smegmatis mc<sup>2</sup> 155 we have demonstrated that the MSMEG_6039 (kshB1) and MSMEG_5941 (kstD1) genes encoding a reductase component of the 3-ketosteroid 9?-hydroxylase (KshAB) and a ketosteroid ?<sup>1</sup> -dehydrogenase (KstD), respectively, are indispensable enzymes for the central metabolism of cholesterol. Therefore, we have constructed a MSMEG_6039 (kshB1) gene deletion mutant of M. smegmatis MS6039 that transforms efficiently natural sterols (e.g. cholesterol and phytosterols) into 1,4-androstadiene-3,17-dione. In addition, we have demonstrated that a double deletion mutant M. smegmatis MS6039-5941 [?MSMEG_6039 (?kshB1) and ?MSMEG_5941 (?kstD1)] transforms natural sterols into 4-androstene-3,17-dione with high yields. These findings suggest that the catabolism of cholesterol in M. smegmatis mc<sup>2</sup> 155 is easy to handle and equally efficient for sterol transformation than other industrial strains, paving the way for valuating this strain as a suitable industrial cell factory to develop à la carte metabolic engineering strategies for the industrial production of pharmaceutical steroids.
Project description:BACKGROUND:The bioconversion of phytosterols into high value-added steroidal intermediates, including the 9?-hydroxy-4-androstene-3,17-dione (9-OHAD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC), is the cornerstone in steroid pharmaceutical industry. However, the low transportation efficiency of hydrophobic substrates into mycobacterial cells severely limits the transformation. In this study, a robust and stable modification of the cell wall in M. neoaurum strain strikingly enhanced the cell permeability for the high production of steroids. RESULTS:The deletion of the nonessential kasB, encoding a ?-ketoacyl-acyl carrier protein synthase, led to a disturbed proportion of mycolic acids (MAs), which is one of the most important components in the cell wall of Mycobacterium neoaurum ATCC 25795. The determination of cell permeability displayed about two times improvement in the kasB-deficient strain than that of the wild type M. neoaurum. Thus, the deficiency of kasB in the 9-OHAD-producing strain resulted in a significant increase of 137.7% in the yield of 9?-hydroxy-4-androstene-3,17-dione (9-OHAD). Ultimately, the 9-OHAD productivity in an industrial used resting cell system was reached 0.1135 g/L/h (10.9 g/L 9-OHAD from 20 g/L phytosterol) and the conversion time was shortened by 33%. In addition, a similar self-enhancement effect (34.5%) was realized in the 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) producing strain. CONCLUSIONS:The modification of kasB resulted in a meaningful change in the cell wall mycolic acids. Deletion of the kasB gene remarkably improved the cell permeability, leading to a self-enhancement of the steroidal intermediate conversion. The results showed a high efficiency and feasibility of this construction strategy.
Project description:Nowadays steroid manufacturing occupies a prominent place in the pharmaceutical industry with an annual global market over $10 billion. The synthesis of steroidal active pharmaceutical ingredients (APIs) such as sex hormones (estrogens, androgens, and progestogens) and corticosteroids is currently performed by a combination of microbiological and chemical processes. Several mycobacterial strains capable of naturally metabolizing sterols (e.g., cholesterol, phytosterols) are used as biocatalysts to transform phytosterols into steroidal intermediates (synthons), which are subsequently used as key precursors to produce steroidal APIs in chemical processes. These synthons can also be modified by other microbial strains capable of introducing regio- and/or stereospecific modifications (functionalization) into steroidal molecules. Most of the industrial microbial strains currently available have been improved through traditional technologies based on physicochemical mutagenesis and selection processes. Surprisingly, Synthetic Biology and Systems Biology approaches have hardly been applied for this purpose. This review attempts to highlight the most relevant research on Steroid Biotechnology carried out in last decades, focusing specially on those works based on recombinant DNA technologies, as well as outlining trends and future perspectives. In addition, the need to construct new microbial cell factories (MCF) to design more robust and bio-sustainable bioprocesses with the ultimate aim of producing steroids à la carte is discussed.
Project description:Biotransformation of soybean phytosterols into 9<i>α</i>-hydroxy-4-androstene-3,17-dione (9-OHAD) by mycobacteria is the core step in the synthesis of adrenocortical hormone. However, the low permeability of the dense cell envelope largely inhibits the overall conversion efficiency of phytosterols. The antigen 85 (Ag85) complex encoded by <i>fbpA</i>, <i>fbpB</i>, and <i>fbpC</i> was proposed as the key factor in the combined catalysis of mycoloyl for producing mycolyl-arabinogalactan (m-AG) and trehalose dimycolate (TDM) in mycobacterial cell envelope. Herein, we confirmed that <i>fbpC3</i> was essential for the biotransformation of trehalose monomycolate (TMM) to TDM in <i>Mycolicibacterium neoaurum</i>. The deficiency of this gene raised the cell permeability, thereby enhancing the steroid uptake and utilization. The 9-OHAD yield in the <i>fbpC3</i>-deficient 9-OHAD-producing strain was increased by 21.3%. Moreover, the combined deletion of <i>fbpC3</i> and <i>embC</i> further increased the 9-OHAD yield compared to the single deletion of <i>fbpC3</i>. Finally, after 96 h of bioconversion in industrial resting cells, the 9-OHAD yield of 11.2 g/L was achieved from 20 g/L phytosterols and the productivity reached 0.116 g/L/h. In summary, this study suggested the critical role of the <i>fbpC3</i> gene in the synthesis of TDM in <i>M. neoaurum</i> and verified the feasibility of improving the bioconversion efficiency of phytosterols through the cell envelope engineering strategy.
Project description:The possible presence of steroids in the tissue of induced hormone-dependent rat mammary tumours was investigated. The method used involves a preliminary extraction of tumours followed by chemical separation and thin-layer chromatography. The identified compounds were cholesterol, androst-4-ene-3,17-dione, 5beta-androst-1-ene-3,17-dione, androsta-1,4-diene-3,17-dione and oestrone. This is the first report of the presence of these steroids in the tissue of an experimental tumour of a non-endocrine organ. In particular 5beta-androst-1-ene-3,17-dione has not previously been identified from natural sources.
Project description:Androst-4-ene-3,17-dione (AD) and androst-1,4-diene-3,17-dione (ADD) are valuable steroid pharmaceutical intermediates obtained by soybean phytosterol biotransformation by <i>Mycobacterium</i> Cyclodextrins (CDs) are generally believed to be carriers for phytosterol delivery and can improve the production of AD and ADD due to their effects on steroid solubilization and alteration in cell wall permeability for steroids. To better understand the mechanisms of CD promotion, we performed proteomic quantification of the effects of hydroxypropyl-?-CD (HP-?-CD) on phytosterol metabolism in <i>Mycobacterium neoaurum</i> TCCC 11978 C2. Perturbations are observed in steroid catabolism and glucose metabolism by adding HP-?-CD in a phytosterol bioconversion system. AD and ADD, as metabolic products of phytosterol, are toxic to cells, with inhibited cell growth and biocatalytic activity. Treatment of mycobacteria with HP-?-CD relieves the inhibitory effect of AD(D) on the electron transfer chain and cell growth. These results demonstrate the positive relationship between HP-?-CD and phytosterol metabolism and give insight into the complex functions of CDs as mediators of the regulation of sterol metabolism.<b>IMPORTANCE</b> Phytosterols from soybean are low-cost by-products of soybean oil production and, owing to their good bioavailability in mycobacteria, are preferred as the substrates for steroid drug production via biotransformation by <i>Mycobacterium</i> However, the low level of production of steroid hormone drugs due to the low aqueous solubility (below 0.1?mmol/liter) of phytosterols limits the commercial use of sterol-transformed strains. To improve the bioconversion of steroids, cyclodextrins (CDs) are generally used as an effective carrier for the delivery of hydrophobic steroids to the bacterium. CDs improve the biotransformation of steroids due to their effects on steroid solubilization and alterations in cell wall permeability for steroids. However, studies have rarely reported the effects of CDs on cell metabolic pathways related to sterols. In this study, the effects of hydroxypropyl-?-CD (HP-?-CD) on the expression of enzymes related to steroid catabolic pathways in <i>Mycobacterium neoaurum</i> were systematically investigated. These findings will improve our understanding of the complex functions of CDs in the regulation of sterol metabolism and guide the application of CDs to sterol production.
Project description:The retro steroids 17beta-hydroxy-5beta,9beta,10alpha-androstan-3-one and 5beta,9beta,10alpha-androstane-3,17-dione were good substrates for cortisone reductase in the presence of NADH, and the products corresponded to the respective 3beta-hydroxy compounds, in which the 3beta-hydroxyl group is axial and the absolute configuration is 3S. The analogous natural steroids 17beta-hydroxy-5beta,9alpha,10beta-androstan-3-one and 5beta,9alpha,10beta-androstane-3,17-dione were very poor substrates, and gave the corresponding 3alpha(equatorial,3R)-hydroxy compounds, and, in the latter case, also an appreciable amount of 3beta(axial, 3S)-hydroxy-5beta,9alpha,10beta-androstan-17-one. The natural steroids 17beta-hydroxy-5alpha,9alpha,10beta-androstan-3-one and 5alpha,9alpha,10beta-androstane-3,17-dione were better substrates than the retro steroid 17beta-hydroxy-5alpha,9beta,10alpha-androstan-3-one, but were not such good substrates as the retro steroids 17beta-hydroxy-5beta,9beta,10alpha-androstan-3-one and 5beta,9beta,10alpha-androstane-3,17-dione. Unlike these retro steroid 5beta,9beta,10alpha-androstan-3-ones, the natural steroids 17beta-hydroxy-5alpha,9alpha,10beta-androstan-3-one and 5alpha,9alpha,10beta-androstane-3,17-dione gave the corresponding 3alpha(axial,3R)-hydroxy compounds. The retro steroid 17beta-hydroxy-5alpha,9beta,10alpha-androstan-3-one was not a good substrate, and the product of reaction corresponded to the 3alpha(axial,3R)-hydroxy compound. The nature of substrate recognition by this enzyme is discussed in the light of these structure-activity relationships.
Project description:<label>BACKGROUND</label>3-Ketosteroid-?1-dehydrogenase (KstD) is a key enzyme in the metabolic pathway for chemical modifications of steroid hormones. Only a few KstDs have thus far been characterized biochemically and applied for the production of steroidal pharmaceutical intermediates. Three KstDs, KstD1, KstD2, and KstD3, were identified in Mycobacterium neoaurum DSM 1381, and they shared up to 99, 85 and 97% amino acid identity with previously reported KstDs, respectively. In this paper, KstDs from M. neoaurum DSM 1381 were investigated and exemplified their potential application for industrial steroid transformation.<label>RESULTS</label>The recombinant KstD2 from Bacillus subtilis exhibited higher enzymatic activity when 4-androstene-3,17-dione (AD) and 22-hydroxy-23, 24-bisnorchol-4-ene-3-one (4HP) were used as the substrates, and resulted in specific activities of 22.40 and 19.19 U mg-1, respectively. However, the specific activities of recombinant KstD2 from Escherichia coli, recombinant KstD1 from B. subtilis and E. coli, and recombinant KstD3, also fed with AD and 4HP, had significantly lower specific activities. We achieved up to 99% bioconversion rate of 1,4-androstadiene-3,17-dione (ADD) from 8 g L-1 AD after 15 h of fermentation using E. coli transformant BL21-kstD2. And in vivo transcriptional analysis revealed that the expression of kstD1 in M. neoaurum DSM 1381 increased by 60.5-fold with phytosterols as the substrate, while the mRNA levels of kstD2 and kstD3 were bearly affected by the phytosterols. Therefore, we attempted to create a 4HP producing strain without kstD1, which could covert 20 g L-1 phytosterols to 14.18 g L-1 4HP.<label>CONCLUSIONS</label>In vitro assay employing the recombinant enzymes revealed that KstD2 was the most promising candidate for biocatalysis in biotransformation of AD. However, in vivo analysis showed that the cellular regulation of kstD1 was much more active than those of the other kstDs in response to the presence of phytosterols. Based on the findings above, we successfully constructed E. coli transformant BL21-kstD2 for ADD production from AD and M. neoaurum DSM 1381 ?kstD1 strain for 4HP production using phytosterols as the substrate.
Project description:3-Ketosteroid-Delta(1)-dehydrogenase, KsdD(M), was identified by targeted gene disruption and augmentation from Mycobacterium neoaurum NwIB-01, a newly isolated strain. The difficulty of separating 4-androstene-3,17-dione (AD) from 1,4-androstadiene-3,17-dione (ADD) is a key bottleneck to the microbial transformation of phytosterols in industry. This problem was tackled via genetic manipulation of the KsdD-encoding gene. Mutants in which KsdD(M) was inactivated or augmented proved to be good AD(D)-producing strains.