Mycobacterial RNase E cleaves with a distinct sequence preference and controls the degradation rates of most Mycolicibacterium smegmatis mRNAs.

The mechanisms and regulation of RNA degradation in mycobacteria have been subject to increased interest following the identification of interplay between RNA metabolism and drug resistance. Mycobacteria encode multiple ribonucleases predicted to participate in mRNA degradation and/or processing of stable RNAs. RNase E is hypothesized to play a major role in mRNA degradation because of its essentiality in mycobacteria and its role in mRNA degradation in gram-negative bacteria. Here, we defined the impact of RNase E on mRNA degradation rates transcriptome-wide in the nonpathogenic model Mycolicibacterium smegmatis. RNase E played a rate-limiting role in degradation of the transcripts encoded by at least 89% of protein-coding genes, with leadered transcripts often being more affected by RNase E repression than leaderless transcripts. There was an apparent global slowing of transcription in response to knockdown of RNase E, suggesting that M. smegmatis regulates transcription in responses to changes in mRNA degradation. This compensation was incomplete, as the abundance of most transcripts increased upon RNase E knockdown. We assessed the sequence preferences for cleavage by RNase E transcriptome-wide in M. smegmatis and Mycobacterium tuberculosis and found a consistent bias for cleavage in C-rich regions. Purified RNase E had a clear preference for cleavage immediately upstream of cytidines, distinct from the sequence preferences of RNase E in gram-negative bacteria. We furthermore report a high-resolution map of mRNA cleavage sites in M. tuberculosis, which occur primarily within the RNase E-preferred sequence context, confirming that RNase E has a broad impact on the M. tuberculosis transcriptome.

Mycolicibacterium arseniciresistens sp. nov., isolated from lead-zinc mine tailing, and reclassification of two Mycobacterium species as Mycolicibacterium palauense comb. nov. and Mycolicibacterium grossiae comb. nov.

A Gram-positive, acid-fast, aerobic, rapidly growing and non-motile strain was isolated from lead-zinc mine tailing sampled in Lanping, Yunnan province, Southwest China. 16S rRNA gene sequence analysis showed that the most closely related species of strain KC 300T was Mycolicibacterium litorale CGMCC 4.5724T (98.47 %). Additionally, phylogenomic and specific conserved signature indel analysis revealed that strain KC 300T should be a member of genus Mycolicibacterium, and Mycobacterium palauense CECT 8779T and Mycobacterium grossiae DSM 104744T should also members of genus Mycolicibacterium. The genome size of strain KC 300T was 6.2 Mb with an in silico DNA G+C content of 69.2 mol%. Chemotaxonomic characteristics of strain KC 300T were also consistent with the genus Mycolicibacterium. The average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity values, as well as phenotypic, physiological and biochemical characteristics, support that strain KC 300T represents a new species within the genus Mycolicibacterium, for which the name Mycolicibacterium arseniciresistens sp. nov. is proposed, with the type strain KC 300T (=CGMCC 1.19494T=JCM 35915T). In addition, we reclassified Mycobacterium palauense and Mycobacterium grossiae as Mycolicibacterium palauense comb. nov. and Mycolicibacterium grossiae comb. nov., respectively.

Mycolicibacterium cyprinidarum sp. nov., a rapidly growing species isolated from diseased koi carp, Cyprinus carpio.

A rapidly growing nontuberculous mycobacterium was isolated from diseased koi carp in Niigata, Japan, which was identified as representing a novel Mycolicibacterium species through whole genome sequence analysis. The bacterial isolates (NGTWS0302, NGTWS1803T and NGTWSNA01) were found to belong to the genus Mycolicibacterium through phylogenetic analysis using whole genome sequences of mycobacteria species. The bacterial colony was smooth, moist and non-chromogenic on 1% Ogawa medium at 30 °C. In biochemical characteristic tests, the bacterial isolates showed positive reactions for catalase activity, Tween 80 hydrolysis and tellurite reduction. The isolates were sensitive to 2-4 µg ml-1 ampicillin, kanamycin and rifampicin. Based on these results, we propose a novel Mycolicibacterium species, Mycolicibacterium cyprinidarum sp. nov. The type strain is NGTWS1803T (=JCM 35117T=ATCC TSD-289T).

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

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.

Immobilization of rough morphotype Mycolicibacterium neoaurum R for androstadienedione production.

OBJECTIVES: Enhance the androstadienedione (Androst-1,4-diene-3,17-dione, ADD) production of rough morphotype Mycolicibacterium neoaurum R by repeated-batch fermentation of immobilized cells. RESULTS: M. neoaurum R was a rough colony morphotype variant, obtained from the routine plating of smooth M. neoaurum strain CICC 21097. M. neoaurum R showed rougher cell surface and aggregated in broth. The ADD production of M. neoaurum R was notably lower than that of M. neoaurum CICC 21097 during the free cell fermentation, but the yield gap could be erased after proper cell immobilization. Subsequently, repeated-batch fermentation of immobilized M. neoaurum R was performed to shorten the production cycle and enhance the bio-production efficiency of ADD. Through the optimization of the immobilization carriers and the co-solvents for phytosterols, the ADD productivity of M. neoaurum R immobilized by semi-expanded perlite reached 0.075 g/L/h during the repeated-batch fermentation for 40 days. CONCLUSIONS: The ADD production of the rough-type M. neoaurum R was notably enhanced by the immobilization onto semi-expanded perlite. Moreover, the ADD batch yields of M. neoaurum R immobilized by semi-expanded perlite were maintained at high levels during the repeated-batch fermentation.

Co-augmentation of a transport gene mfsT1 in Mycolicibacterium neoaurum with genome engineering to enhance ergothioneine production.

Ergothioneine (EGT) is a rare thiohistidine derivative with exceptional antioxidant properties. The blood level of EGT is considered highly reliable predictors for cardiovascular diseases and mortality, yet animals lack the ability to synthesize this compound. Free plasmids have been previously used to overexpress genes involved in the EGT biosynthetic pathway of Mycolicibacterium neoaurum. Here, we tentatively introduced a putative transporter gene mfsT1 into high-copy plasmids and sharply increased the ratio of extracellular EGT concentration from 18.7% to 44.9%. Subsequently, an additional copy of egtABCDE, hisG, and mfsT1 was inserted into the genome with a site-specific genomic integration tool of M. neoaurum, leading a 2.7 times increase in EGT production. Co-enhancing the S-adenosyl-L-methionine regeneration pathway, or alternatively, the integration of three copies of egtABCDE, hisG and mfsT1 into the genome further increased the total EGT yield by 16.1% (64.6 mg/L) and 21.7% (67.7 mg/L), respectively. After 168-h cultivation, the highest titer reached 85.9 mg/L in the latter strain with three inserted copies. This study provided a solid foundation for genome engineering to increase the production of EGT in M. neoaurum.

Catheter-Related Bloodstream Infection Caused by Mycolicibacterium iranicum, California, USA.

We describe a case of catheter-related bacteremia caused by Mycolicibacterium iranicum in the United States. The case highlights the value of using next-generation sequencing to identify infrequent and emerging pathogens and the challenges associated with choosing appropriate treatments because of limited knowledge of drug resistance mechanisms in those emerging pathogens.

Healthcare-Associated Infections Caused by Mycolicibacterium neoaurum.

Mycolicibacterium neoaurum is a rapidly growing mycobacterium and an emerging cause of human infections. M. neoaurum infections are uncommon but likely underreported, and our understanding of the disease spectrum and optimum management is incomplete. We summarize demographic and clinical characteristics of a case of catheter-related M. neoaurum bacteremia in a child with leukemia and those of 36 previously reported episodes of M. neoaurum infection. Most infections occurred in young to middle-aged adults with serious underlying medical conditions and commonly involved medical devices. Overall, infections were not associated with severe illness or death. In contrast to other mycobacteria species, M. neoaurum was generally susceptible to multiple antimicrobial drugs and responded promptly to treatment, and infections were associated with good outcomes after relatively short therapy duration and device removal. Delays in identification and susceptibility testing were common. We recommend using combination antimicrobial drug therapy and removal of infected devices to eradicate infection.

Molecular evaluation of the metabolism of estrogenic di(2-ethylhexyl) phthalate in Mycolicibacterium sp.

BACKGROUND: Di(2-ethylhexyl) phthalate (DEHP) is a widely detected plasticizer and a priority pollutant of utmost concern for its adverse impact on humans, wildlife and the environment. To eliminate such toxic burden, biological processes are the most promising ways to combat rampant environmental insults under eco-friendly conditions. The present study investigated the biochemical and molecular assessment of the catabolic potential of Mycolicibacterium sp. strain MBM in the assimilation of estrogenic DEHP. RESULTS: A detailed biochemical study revealed an initial hydrolytic pathway of degradation for DEHP followed by the assimilation of hydrolyzed phthalic acid and 2-ethylhexanol to TCA cycle intermediates. Besides the inducible nature of DEHP-catabolic enzymes, strain MBM can efficiently utilize various low- and high-molecular-weight phthalate diesters and can grow under moderately halotolerant conditions. Whole genome sequence analysis exhibited a genome size of 6.2 Mb with a GC content of 66.51% containing 6,878 coding sequences, including multiple genes, annotated as relevant to the catabolism of phthalic acid esters (PAEs). Substantiating the annotated genes through transcriptome assessment followed by RT-qPCR analysis, the possible roles of upregulated genes/gene clusters in the metabolism of DEHP were revealed, reinforcing the biochemical pathway of degradation at the molecular level. CONCLUSIONS: A detailed co-relation of biochemical, genomic, transcriptomic and RT-qPCR analyses highlights the PAE-degrading catabolic machineries in strain MBM. Further, due to functional attributes in the salinity range of both freshwater and seawater, strain MBM may find use as a suitable candidate in the bioremediation of PAEs.

First Study Case of Microbial Biocontrol Agents Isolated from Aquaponics Through the Mining of High-Throughput Sequencing Data to Control Pythium aphanidermatum on Lettuce.

Aquaponics is defined as a sustainable and integrated system that combines fish aquaculture and hydroponic plant production in the same recirculated water loop. A recent study using high-throughput sequencing (HTS) technologies highlighted that microbial communities from an aquaponic system could control one of the most problematic pathogens in soilless lettuce culture, namely, Pythium aphanidermatum. Therefore, this study aims at isolating the microorganisms responsible for this biocontrol action. Based on the most promising genera identified by HTS, an innovative strategy for isolating and testing original biocontrol agents from aquaponic water was designed to control P. aphanidermatum. Eighty-two bacterial strains and 18 fungal strains were isolated, identified by Sanger sequencing, and screened in vivo to control damping-off of lettuce seeds caused by P. aphanidermatum. Out of these 100 isolates, the eight most efficacious ones were selected and further tested individually to control root rot disease caused by the same pathogen at a later stage of lettuce growth. Strains SHb30 (Sphingobium xenophagum), G2 (Aspergillus flavus), and Chito13 (Mycolicibacterium fortuitum) decreased seed damping-off at a better rate than a propamocarb fungicide and a Pseudomonas chlororaphis registered biocontrol agent did. In root rot bioassays, lettuce mortality was prevented by applying strains G2 and Chito13, which were at least as efficacious as the fungicide or biopesticide controls. Lettuce disease symptoms and mortality were eradicated by strain SHb30 in the first bioassay, but not in the second one. These results show that aquaponic systems are promising sources of original biocontrol agents, and that HTS-guided strategies could represent interesting approaches to identify new biocontrol agents.

Production of 21-hydroxy-20-methyl-pregna-1,4-dien-3-one by modifying multiple genes in Mycolicibacterium.

C22 steroid drug intermediates are suitable for corticosteroids synthesis, and the production of C22 steroids is unsatisfactory due to the intricate steroid metabolism. Among the C22 steroids, 21-hydroxy-20-methyl-pregna-1,4-dien-3-one (1,4-HP) could be used for Δ1-steroid drug synthesis, such as prednisolone. Nevertheless, the production of 1,4-HP remains unsatisfactory. In this study, an ideal 1,4-HP producing strain was constructed. By the knockout of 3-ketosteroid-9-hydroxylase (KshA) genes and 17ß-hydroxysteroid dehydrogenase (Hsd4A) gene, the steroid nucleus degradation and the accumulation of C19 steroids in Mycolicibacterium neoaurum were blocked. The mutant strain could transform phytosterols into 1,4-HP as the main product and 21-hydroxy-20-methyl-pregna-4-ene-3-one as a by-product. Subsequently, the purity of 1,4-HP improved to 95.2% by the enhancement of 3-ketosteroid-Δ1-dehydrogenase (KSTD) activity, and the production of 1,4-HP was improved by overexpressing NADH oxidase (NOX) and catalase (KATE) genes. Consequently, the yield of 1,4-HP achieved 10.5 g/L. The molar yield and the purity of 1,4-HP were optimal so far, and the production of 1,4-HP provides a new intermediate for the pharmaceutical steroid industry. KEY POINTS: • A third 3-ketosteroid-9-hydroxylase was identified in Mycolicibacterium neoaurum. • An 1,4-HP producer was constructed by KshA and Hsd4A deficiency. • The production of 1,4-HP was improved by KSTD, NOX, and KATE overexpression.

Expression of Synthetic cyp102A1-LG23 Gene and Functional Analysis of Recombinant Cytochrome P450 BM3-LG23 in the Actinobacterium Mycolicibacterium smegmatis.

Cytochrome CYP102A1 (P450 BM3) of Priestia megaterium (bas. Bacillus megaterium) has several unique functional features and thus provides an ideal object for directed evolution and other synthetic applications. Previously, the CYP102A1-LG23 mutant with 14 mutations in the heme part was obtained that hydroxylates several androstanes at C7ß with the formation of products with the anti-inflammatory and neuroprotective activities. In this study, synthetic cyp102A1-LG23 gene encoding the P450 BM3 mutant was expressed as a component of either monocistronic operon or bicistronic operon containing the gdh (glucose dehydrogenase, GDH) or zwf2 (glucose 6-phosphate dehydrogenase, G6PD) gene in Mycolicibacterium smegmatis BD cells. The recombinant bacteria were able hydroxylate androst-4-ene-3,17-dione (AD) into 7ß-OH-AD. Their biocatalytic activity was increased twice by increasing the solubility of CYP102A1-LG23 protein in the cells and supplementing the cells with the additional cofactor regeneration system by introducing GDH and G6PD. The maximum 7ß-OH-AD yield (37.68 mol%) was achieved by co-expression of cyp102A1-LG23 and gdh genes in M. smegmatis. These results demonstrate the possibility of using synthetic genes to obtain recombinant enzymes and expand our understanding of the processes involved in steroid hydroxylation by bacterial cytochromes. The data obtained can be used to develop new approaches for microbiological production of 7ß-hydroxylated steroids in genetically modified Mycolicibacterium species.

A Keto Reductase Involved in Steroid Degradation in Mycolicibacterium neoaurum.

Phytosterols can be used by microorganisms as carbon and energy sources and completely degraded into CO2 and H2 O. The catabolic pathway of phytosterols was well characterized in many microorganisms. Blocking the steroid core ring degradation by deletions of fadE30 and fadD3 genes, two important steroid intermediates, 3aα-H-4α-(3'-Propionic acid)-5α-hydroxy-7aß-methylhexahydro-1-indanone-δ-lactone (sitolactone, or HIL) and 3aα-H-4α-(3'-propionic acid)-7aß-methylhexahydro-1,5-indanedione (HIP) can be accumulated. They are currently used to synthesize nor-steroid drugs with an α-methyl group or without the methyl group at the C10 -position, such as estrone and norethindrone. In this study, a key gene involved in the bioconversion of HIP to HIL was identified in Mycolicibacterium neoaurum. Through heterologous expression, gene hipR was found to be involved in the reduction of the C5 keto group of HIP to a hydroxy group, leading to spontaneously lactonization into HIL in vitro. Through gene complementation and knockout, HipR functions were verified and two HIP degradation pathways in vivo were elucidated. The finding of this research facilitated the understanding of the metabolic pathway of sterols, and was directly applied to engineering robust production strains by overexpression or knockout of related genes.

New Insights into the Modification of the Non-Core Metabolic Pathway of Steroids in Mycolicibacterium and the Application of Fermentation Biotechnology in C-19 Steroid Production.

Androsta-4-ene-3,17-dione (AD), androsta-1,4-diene-3,17-dione (ADD), and 9α-hydroxy-4-androstene-3,17-dione (9-OHAD), which belong to C-19 steroids, are critical steroid-based drug intermediates. The biotransformation of phytosterols into C-19 steroids by Mycolicibacterium cell factories is the core step in the synthesis of steroid-based drugs. The production performance of engineered mycolicibacterial strains has been effectively enhanced by sterol core metabolic modification. In recent years, research on the non-core metabolic pathway of steroids (NCMS) in mycolicibacterial strains has made significant progress. This review discusses the molecular mechanisms and metabolic modifications of NCMS for accelerating sterol uptake, regulating coenzyme I balance, promoting propionyl-CoA metabolism, reducing reactive oxygen species, and regulating energy metabolism. In addition, the recent applications of biotechnology in steroid intermediate production are summarized and compared, and the future development trend of NCMS research is discussed. This review provides powerful theoretical support for metabolic regulation in the biotransformation of phytosterols.

Bioconversion of Phytosterols to 9-Hydroxy-3-Oxo-4,17-Pregadiene-20-Carboxylic Acid Methyl Ester by Enoyl-CoA Deficiency and Modifying Multiple Genes in Mycolicibacterium neoaurum.

Steroid drug precursors, including C19 and C22 steroids, are crucial to steroid drug synthesis and development. However, C22 steroids are less developed due to the intricacy of the steroid metabolic pathway. In this study, a C22 steroid drug precursor, 9-hydroxy-3-oxo-4,17-pregadiene-20-carboxylic acid methyl ester (9-OH-PDCE), was successfully obtained from Mycolicibacterium neoaurum by 3-ketosteroid-Δ1-dehydrogenase and enoyl-CoA hydratase ChsH deficiency. The production of 9-OH-PDCE was improved by the overexpression of 17ß-hydroxysteroid dehydrogenase Hsd4A and acyl-CoA dehydrogenase ChsE1-ChsE2 to reduce the accumulation of by-products. The purity of 9-OH-PDCE in fermentation broth was improved from 71.7% to 89.7%. Hence, the molar yield of 9-OH-PDCE was improved from 66.7% to 86.7%, with a yield of 0.78 g/L. Furthermore, enoyl-CoA hydratase ChsH1-ChsH2 was identified to form an indispensable complex in Mycolicibacterium neoaurum DSM 44704. IMPORTANCE C22 steroids are valuable precursors for steroid drug synthesis, but the development of C22 steroids remains unsatisfactory. This study presented a strategy for the one-step bioconversion of phytosterols to a C22 steroid drug precursor, 9-hydroxy-3-oxo-4,17-pregadiene-20-carboxylic acid methyl ester (9-OH-PDCE), by 3-ketosteroid-Δ1-dehydrogenase and enoyl-CoA hydratase deficiency with overexpression of 17ß-hydroxysteroid dehydrogenase acyl-CoA dehydrogenase in Mycolicibacterium. The function of the enoyl-CoA hydratase ChsH in vivo was revealed. Construction of the novel C22 steroid drug precursor producer provided more potential for steroid drug synthesis, and the characterization of the function of ChsH and the transformation of steroids further revealed the steroid metabolic pathway.

Mycolicibacterium aurantiacum sp. nov. and Mycolicibacterium xanthum sp. nov., two novel actinobacteria isolated from mangrove sediments.

Two novel actinobacteria with the ability to degrade kerosene, designated as B3033T and Y57T, were isolated from mangrove sediments in Tieshan Harbour, South China Sea. Both strains are Gram-staining-positive, non-spore forming, slow-growing, oxidase-positive, non-motile and aerobic. Their major cellular fatty acids were C16 : 0 and C18 : 1ω9c. Analysis of 16S rRNA gene sequences revealed the close relationship of strain B3033T to Mycobacterium kyogaense DSM 107316T (99.4 % nucleotide identity) and strain Y57T to Mycolicibacterium chubuense ATCC 27278T (98.7 %) and Mycolicibacterium rufum JS14T (98.7 %). Whole genome average nucleotide blast identity (ANI) and the digital DNA-DNA hybridization (dDDH) values between the two isolates and the type strains of species of the genus Mycolicibacterium were lower than 94 and 45 %, respectively, which were below the threshold values of 95 % (for ANI) and 70 % (for dDDH) recommended for bacterial species differentiation. The genome sequence of B3033T comprised a circular 11.0 Mb chromosome with a DNA G+C content of 68.1 mol%. Y57T had a genome size of 5.6 Mb and a DNA G+C content of 68.7 mol%. Genes involved in degradation of aromatic compounds and copper resistance were identified in the genomes of both strains that could improve their adaptive capacity to the mangrove environment. These results combined with those of chemotaxonomic analyses, MALDI-TOF MS profiles and phenotypic analyses support the affiliation of these strains to two novel species within the genus Mycolicibacterium, for which we propose the names Mycolicibacterium aurantiacum sp. nov. B3033T (=KCTC 49712T=MCCC 1K04526T) and Mycolicibacterium xanthum sp. nov. Y57T (=KCTC 49711T=MCCC 1K04875T) as type strains.

Combined enhancement of the propionyl-CoA metabolic pathway for efficient androstenedione production in Mycolicibacterium neoaurum.

BACKGROUND: The production of androstenedione (AD) from phytosterols by Mycolicibacterium neoaurum is a multi-step biotransformation process, which requires degradation of sterol side chains, accompanied by the production of propionyl-CoA. However, the transient production of large amounts of propionyl-CoA can accumulate intracellularly to produce toxic effects and severely inhibit AD production. RESULTS: In the present study, the intracellular propionyl-CoA concentration was effectively reduced and the productivity of the strain was improved by enhancing the cytosolic methyl-branched lipid synthesis pathway and increasing the expression level of nat operator gene, respectively. Subsequently, the application of a pathway combination strategy, combined and the inducible regulation strategy, further improved AD productivity with a maximum AD conversion rate of 96.88%, an increase of 13.93% over the original strain. CONCLUSIONS: Overall, we provide a new strategy for reducing propionyl-CoA stress during biotransformation for the production of AD and other steroidal drugs using phytosterols.

One-pot biosynthesis of 7ß-hydroxyandrost-4-ene-3,17-dione from phytosterols by cofactor regeneration system in engineered mycolicibacterium neoaurum.

BACKGROUND: 7ß-hydroxylated steroids (7ß-OHSt) possess significant activities in anti-inflammatory and neuroprotection, and some of them have been widely used in clinics. However, the production of 7ß-OHSt is still a challenge due to the lack of cheap 7ß-hydroxy precursor and the difficulty in regio- and stereo-selectively hydroxylation at the inert C7 site of steroids in industry. The conversion of phytosterols by Mycolicibacterium species to the commercial precursor, androst-4-ene-3,17-dione (AD), is one of the basic ways to produce different steroids. This study presents a way to produce a basic 7ß-hydroxy precursor, 7ß-hydroxyandrost-4-ene-3,17-dione (7ß-OH-AD) in Mycolicibacterium, for 7ß-OHSt synthesis. RESULTS: A mutant of P450-BM3, mP450-BM3, was mutated and engineered into an AD producing strain for the efficient production of 7ß-OH-AD. The enzyme activity of mP450-BM3 was then increased by 1.38 times through protein engineering and the yield of 7ß-OH-AD was increased from 34.24 mg L- 1 to 66.25 mg L- 1. To further enhance the performance of 7ß-OH-AD producing strain, the regeneration of nicotinamide adenine dinucleotide phosphate (NADPH) for the activity of mP450-BM3-0 was optimized by introducing an NAD kinase (NADK) and a glucose-6-phosphate dehydrogenase (G6PDH). Finally, the engineered strain could produce 164.52 mg L- 1 7ß-OH-AD in the cofactor recycling and regeneration system. CONCLUSIONS: This was the first report on the one-pot biosynthesis of 7ß-OH-AD from the conversion of cheap phytosterols by an engineered microorganism, and the yield was significantly increased through the mutation of mP450-BM3 combined with overexpression of NADK and G6PDH. The present strategy may be developed as a basic industrial pathway for the commercial production of high value products from cheap raw materials.

A recently evolved diflavin-containing monomeric nitrate reductase is responsible for highly efficient bacterial nitrate assimilation.

Nitrate is one of the major inorganic nitrogen sources for microbes. Many bacterial and archaeal lineages have the capacity to express assimilatory nitrate reductase (NAS), which catalyzes the rate-limiting reduction of nitrate to nitrite. Although a nitrate assimilatory pathway in mycobacteria has been proposed and validated physiologically and genetically, the putative NAS enzyme has yet to be identified. Here, we report the characterization of a novel NAS encoded by Mycolicibacterium smegmatis Msmeg_4206, designated NasN, which differs from the canonical NASs in its structure, electron transfer mechanism, enzymatic properties, and phylogenetic distribution. Using sequence analysis and biochemical characterization, we found that NasN is an NADPH-dependent, diflavin-containing monomeric enzyme composed of a canonical molybdopterin cofactor-binding catalytic domain and an FMN-FAD/NAD-binding, electron-receiving/transferring domain, making it unique among all previously reported hetero-oligomeric NASs. Genetic studies revealed that NasN is essential for aerobic M. smegmatis growth on nitrate as the sole nitrogen source and that the global transcriptional regulator GlnR regulates nasN expression. Moreover, unlike the NADH-dependent heterodimeric NAS enzyme, NasN efficiently supports bacterial growth under nitrate-limiting conditions, likely due to its significantly greater catalytic activity and oxygen tolerance. Results from a phylogenetic analysis suggested that the nasN gene is more recently evolved than those encoding other NASs and that its distribution is limited mainly to Actinobacteria and Proteobacteria. We observed that among mycobacterial species, most fast-growing environmental mycobacteria carry nasN, but that it is largely lacking in slow-growing pathogenic mycobacteria because of multiple independent genomic deletion events along their evolution.

Mycolicibacterium baixiangningiae sp. nov. and Mycolicibacterium mengxianglii sp. nov., two new rapidly growing mycobacterial species.

Four bacterial strains (LJ126T/S18 and Z-34T/S20) recovered from faecal samples of Tibetan antelopes on the Qinghai-Tibet Plateau of China were analysed using a polyphasic approach. All four isolates were aerobic, short rod-shaped, non-motile, Gram-stain-positive, acid-fast and fast-growing. Phylogenetic analyses based upon 16S rRNA and whole-genome sequences showed that the two pair of strains formed two distinct branches within the evolutionary radiation of the genus Mycolicibacterium. Strains LJ126T/S18 and Z-34T/S20 were most closely related to Mycolicibacterium austroafricanum CCUG 37667T, Mycobacterium aurum NCTC 10437T, Mycobacterium pyrenivorans DSM 44605T, Mycobacterium monacense JCM 15658T, Mycolicibacterium sarraceniae JCM 30395T, Mycolicibacterium tokaiense JCM 6373T and Mycobacterium murale JCM 13392T, but readily distinguished from the known species by a combination of chemotaxonomic and phenotypic features and by low average nucleotide identity values (74.4-84.9 %). Consequently, the two strain pairs are considered to represent different novel species of Mycolicibacterium for which the names Mycolicibacterium baixiangningiae sp. nov. and Mycolicibacterium mengxianglii sp. nov. are proposed, with LJ126T (=CGMCC 1.1992T=KCTC 49535T) and Z-34T (=CGMCC 1.1993T=DSM 106172T) as the respective type strains.