Creation and Characterization of Mycolicibacterium Smegmatis mc2155 with Deletions in Genes Encoding Sterol Oxidation Enzymes

The fast-growing saprotrophic strain Mycolicibacterium smegmatis mc2155 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 3β-hydroxyl group and Δ5(6)→Δ4(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 3β-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 3β-hydroxysteroid dehydrogenase/Δ5(6)→Δ4(5) isomerase

Characterisation of Endo-Polygalacturonases Activities of Rice (<em>Oryza sativa</em>) Fungal Pathogens in Nigeria, West Africa

Rice (Oryza sativa) is cultivated in swampy geographical locations of tropical Nigeria, West Africa. Here it is infected by a host of fungal pathogens on the field or contaminated at postharvest. This has led to its loss and reduction in its production in both the national and global market. Lasiodiplodia theobromae and Rhizoctonia solani have recently been identified as the major fungal phytopathogens causing the deterioration of this grain on the field and at postharvest and affecting its production in Nigeria leading to gross capital loss. Hence the need to determine physiological control measures for the eradication of both phytopathogens on the field and at postharvest. In this study, tropical strains of Lasiodiplodia theobromae and Rhizoctonia solani obtained from deteriorated rice (Oryza sativa) were grown in a growth nutrient medium composed of MgSo4.7H20, K2HPO4, FeSO4.7H20, potassium nitrate and pectin at 30°C. Endo-Polygalacturonase activities were produced by the fungal isolates in the growth medium within ten days. The endo-polygalacturonases from both fungi were purified by a combination of ammonium sulphate precipitation, dialysis, gel filtration (on Sephadex G-100 column) and ion-exchange chromatography (on CM-Sephadex C-50 and CM-Sephadex C-25 columns). The molecular weight of endo-polygalacturonase from the Lasiodiplodia theobromae using Sephadex G-100 was estimated as 124,000 Daltons while that of the Rhizoctonia solani was estimated as 92,000 Daltons. The purified endo-polygalcuronase from the Lasiodiplodia theobromae exhibited optimum activity at 30°C and at pH 4.5 while that from the Rhizoctonia solani exhibited optimum activity at 32°C and at pH 5.0. The purified endo-polygalacturonases from both fungi exhibited optimum activities at 0.2% pectin concentration. They were stimulated by Ca2+ but inhibited by ethlylenediamine tetracetic acid (EDTA) and 2,4-dinitrophenol. The purified endo-polygalacturonase from the Lasiodiplodia theobromae lost 80% of its activity within 20 minutes of heat at 80°C. While the purified endo-polygalacturonase from the Rhizoctonia solani lost 82% of its activity within 20 minutes of heat at 80°C. Potassium nitrate as nitrogen source in the defined growth medium with pectin as carbon source supported highest activity of endo-polygalacturonase by the Lasiodiplodia theobromae while ammonium chloride as nitrogen source in the defined growth medium with pectin as carbon source supported highest activity of endo-polygalacturonase by the Rhizoctonia solani. In conclusion, the conditions inhibiting endo-polygalacturonases from Lasiodiplodia theobromae and Rhizoctonia solani capable of degrading the pectin portion of rice (Oryza sativa) can be adapted as feasible control measures limiting the infection and contamination of rice (Oryza sativa) by these phytopathogens on the field and at postharvest. Temperature and pH extreme from 30°C and pH 4.5 will be feasible inhibitory control measures for the growth of Lasiodiplodia theobromae on rice (Oryza sativa) in Nigeria while temperature and pH extreme from 32°C and pH 5.0 will inhibit growth of Rhizoctonia solani on the grain. These physiological conditions will preserve pectin in rice (Oryza sativa) from degradation by these two fungal phytopathogens

Microbial Steroid Production Technologies: Current Trends and Prospects

This Special Issue aims to collect articles and reviews on new methodologies, research, and achievements in the field of steroid microbial biotechnologies [...

Insight into Different Stages of Steroid Degradation in Thermophilic <i>Saccharopolyspora hirsuta</i> VKM Ac-666^T Strain

Steroids are abundant molecules in nature, and various microorganisms evolved to utilize steroids. Thermophilic actinobacteria play an important role in such processes. However, very few thermophiles have so far been reported capable of degrading or modifying natural sterols. Recently, genes putatively involved in the sterol catabolic pathway have been revealed in the moderately thermophilic actinobacterium Saccharopolyspora hirsuta VKM Ac-666T, but peculiarities of strain activity toward sterols are still poorly understood. S. hirsuta catalyzed cholesterol bioconversion at a rate significantly inferior to that observed for mesophilic actinobacteria (mycobacteria and rhodococci). Several genes related to different stages of steroid catabolism increased their expression in response to cholesterol as was shown by transcriptomic studies and verified by RT–qPCR. Sequential activation of genes related to the initial step of cholesterol side chain oxidation (cyp125) and later steps of steroid core degradation (kstD3, kshA, ipdF, and fadE30) was demonstrated for the first time. The activation correlates with a low cholesterol conversion rate and intermediate accumulation by the strain. The transcriptomic analyses revealed that the genes involved in sterol catabolism are linked functionally, but not transcriptionally. The results contribute to the knowledge on steroid catabolism in thermophilic actinobacteria and could be used at the engineering of microbial catalysts

Steroid Metabolism in Thermophilic Actinobacterium Saccharopolyspora hirsuta VKM Ac-666T

The application of thermophilic microorganisms opens new prospects in steroid biotechnology, but little is known to date on steroid catabolism by thermophilic strains. The thermophilic strain Saccharopolyspora hirsuta VKM Ac-666T has been shown to convert various steroids and to fully degrade cholesterol. Cholest-4-en-3-one, cholesta-1,4-dien-3-one, 26-hydroxycholest-4-en-3-one, 3-oxo-cholest-4-en-26-oic acid, 3-oxo-cholesta-1,4-dien-26-oic acid, 26-hydroxycholesterol, 3&beta;-hydroxy-cholest-5-en-26-oic acid were identified as intermediates in cholesterol oxidation. The structures were confirmed by 1H and 13C-NMR analyses. Aliphatic side chain hydroxylation at C26 and the A-ring modification at C3, which are putatively catalyzed by cytochrome P450 monooxygenase CYP125 and cholesterol oxidase, respectively, occur simultaneously in the strain and are followed by cascade reactions of aliphatic sidechain degradation and steroid core destruction via the known 9(10)-seco-pathway. The genes putatively related to the sterol and bile acid degradation pathways form three major clusters in the S. hirsuta genome. The sets of the genes include the orthologs of those involved in steroid catabolism in Mycobacterium tuberculosis H37Rv and Rhodococcus jostii RHA1 and related actinobacteria. Bioinformatics analysis of 52 publicly available genomes of thermophilic bacteria revealed only seven candidate strains that possess the key genes related to the 9(10)-seco pathway of steroid degradation, thus demonstrating that the ability to degrade steroids is not widespread among thermophilic bacteria

Reconstruction of the Steroid 1(2)-Dehydrogenation System from <i>Nocardioides simplex</i> VKM Ac-2033D in <i>Mycolicibacterium</i> Hosts

Microbial 1(2)-dehydrogenation of 3-ketosteroids is an important basis for the production of many steroid pharmaceuticals and synthons. When using the wild-type strains for whole cell catalysis, the undesirable reduction of the 20-carbonyl group, or 1(2)-hydrogenation, was observed. In this work, the recombinant strains of Mycolicibacterium neoaurum and Mycolicibacterium smegmatis were constructed with blocked endogenous activity of 3-ketosteroid-9α-hydroxylase, 3-ketosteroid-1(2)-dehydrogenase (3-KSD), and expressing 3-KSD encoded by the gene KR76_27125 (kstD2NS) from Nocardioides simplex VKM Ac-2033D. The in vivo activity of the obtained recombinant strains against phytosterol, 6α-methyl-hydrocortisone, and hydrocortisone was studied. When using M. smegmatis as the host strain, the 1(2)-dehydrogenation activity of the constructed recombinant cells towards hydrocortisone was noticeably higher compared to those on the platform of M. neoaurum. A comparison of the strengths of inducible acetamidase and constitutive hsp60 promoters in M. smegmatis provided comparable results. Hydrocortisone biotransformation by M. smegmatis BD/pMhsp_k expressing kstD2NS resulted in 95.4% prednisolone yield, and the selectivity preferred that for N. simplex. Mycolicibacteria showed increased hydrocortisone degradation at 35 °C compared to 30 °C. The presence of endogenous steroid catabolism in Mycolicibacterium hosts does not seem to confer an advantage for the functioning of KstD2NS. The results allow for the evaluation of the prospects for the development of simple technological methods for the selective 1(2)-dehydrogenation of 3-ketosteroids by growing bacterial cells

Pregnenolone and progesterone production from natural sterols using recombinant strain of Mycolicibacterium smegmatis mc2 155 expressing mammalian steroidogenesis system

Abstract Background Pregnenolone and progesterone are the life-important steroid hormones regulating essential vital functions in mammals, and widely used in different fields of medicine. Microbiological production of these compounds from sterols is based on the use of recombinant strains expressing the enzyme system cholesterol hydroxylase/C20-C22 lyase (CH/L) of mammalian steroidogenesis. However, the efficiency of the known recombinant strains is still low. New recombinant strains and combination approaches are now needed to produce these steroid hormones. Results Based on Mycolicibacterium smegmatis, a recombinant strain was created that expresses the steroidogenesis system (CYP11A1, adrenodoxin reductase, adrenodoxin) of the bovine adrenal cortex. The recombinant strain transformed cholesterol and phytosterol to form progesterone among the metabolites. When 3-methoxymethyl ethers of sterols were applied as bioconversion substrates, the corresponding 3-ethers of pregnenolone and dehydroepiandrosterone (DHEA) were identified as major metabolites. Under optimized conditions, the recombinant strain produced 85.2 ± 4.7 mol % 3-methoxymethyl-pregnenolone within 48 h, while production of 3-substituted DHEA was not detected. After the 3-methoxymethyl function was deprotected by acid hydrolysis, crystalline pregnenolone was isolated in high purity (over 98%, w/w). The structures of steroids were confirmed using TLC, HPLC, MS and 1H- and 13C-NMR analyses. Conclusion The use of mycolicybacteria as a microbial platform for the expression of systems at the initial stage of mammalian steroidogenesis ensures the production of valuable steroid hormones—progesterone and pregnenolone from cholesterol. Selective production of pregnenolone from cholesterol is ensured by the use of 3-substituted cholesterol as a substrate and optimization of the conditions for its bioconversion. The results open the prospects for the generation of the new microbial biocatalysts capable of effectively producing value-added steroid hormones

Complete genome sequence of ‘Mycobacterium neoaurum’ NRRL B-3805, an androstenedione (AD) producer for industrial biotransformation of sterols

[EN] Microbial bioconversion of sterols into high value steroid precursors, such as 4-androstene-3,17-dione (AD), is an industrial challenge. Genes and enzymes involved in sterol degradation have been proposed, although the complete pathway is not yet known. The genome sequencing of the AD producer strain ‘Mycobacterium neoaurum’ NRRL B-3805 (formerly Mycobacterium sp. NRRL B-3805) will serve to elucidate the critical steps for industrial processes and will provide the basis for further genetic engineering. The genome comprises a circular chromosome (5 421 338 bp), is devoid of plasmids and contains 4844 protein-coding genesSIThis work was fully supported by a grant of the European Union program ERA-IB [MySterI (EIB.12.010) through the APCIN call of the Spanish Ministry of Economy and Competitively (MINECO, Spain) (PCIN-2013-024-C02-01)]. Dmitry Dovbnya and Marina Donova acknowledge Russian Science Foundation (Grant No. 14-24-00169) for the supporting of their work. The authors want to thanks the European Union program ERA-IB; the Spanish Ministry of Economy and Competitiveness (MINECO, Spain); the Biotechnology and Biological Sciences Research Council (BBSRC, United Kingdom); the Foundation for Assistance to Small Innovative Enterprises (FASIE, Russia) and the MySterI Consortium (INBIOTEC, Pharmins Ltd., University of York, SINTEF, Technische Universität Dortmund, Gadea Biopharma S.L.)

Genome-Wide Transcriptome Profiling Provides Insight on Cholesterol and Lithocholate Degradation Mechanisms in Nocardioides simplex VKM Ac-2033D

Steroid microbial degradation plays a significant ecological role for biomass decomposition and removal/detoxification of steroid pollutants. In this study, the initial steps of cholesterol degradation and lithocholate bioconversion by a strain with enhanced 3-ketosteroid dehydrogenase (3-KSD) activity, Nocardioides simplex VKM Ac-2033D, were studied. Biochemical, transcriptomic, and bioinformatic approaches were used. Among the intermediates of sterol sidechain oxidation cholest-5-en-26-oic acid and 3-oxo-cholesta-1,4-dien-26-oic acid were identified as those that have not been earlier reported for N. simplex and related species. The transcriptomic approach revealed candidate genes of cholesterol and lithocholic acid (LCA) catabolism by the strain. A separate set of genes combined in cluster and additional 3-ketosteroid &Delta;1-dehydrogenase and 3-ketosteroid 9&alpha;-hydroxylases that might be involved in LCA catabolism were predicted. Bioinformatic calculations based on transcriptomic data showed the existence of a previously unknown transcription factor, which regulates cholate catabolism gene orthologs. The results contribute to the knowledge on diversity of steroid catabolism regulation in actinobacteria and might be used at the engineering of microbial catalysts for ecological and industrial biotechnology