Biosynthesis of steroidal intermediates using Mycobacteria: a review / 生物工程学报

Steroids are a class of medicines with important physiological and pharmacological effects. In pharmaceutical industry, steroidal intermediates are mainly prepared through Mycobacteria transformation, and then modified chemically or enzymatically into advanced steroidal compounds. Compared with the "diosgenin-dienolone" route, Mycobacteria transformation has the advantages of abundant raw materials, cost-effective, short reaction route, high yield and environmental friendliness. Based on genomics and metabolomics, the key enzymes in the phytosterol degradation pathway of Mycobacteria and their catalytic mechanisms are further revealed, which makes it possible for Mycobacteria to be used as chassis cells. This review summarizes the progress in the discovery of steroid-converting enzymes from different species, the modification of Mycobacteria genes and the overexpression of heterologous genes, and the optimization and modification of Mycobacteria as chassis cells.

Identification of a new C-23 metabolite in sterol degradation of Mycobacterium neoaurum HGMS2 and analysis of its metabolic pathways / 生物工程学报

Mycobacterium neoaurum has the ability to produce steroidal intermediates known as 22-hydroxy-23, 24-bisnorchol-4-en-3-one (BA) upon the knockout of the genes for either the hydroxyacyl-CoA dehydrogenase (Hsd4A) or acyl-CoA thiolase (FadA5). In a previous study, we discovered a novel metabolite in the fermentation products when the fadA5 gene was deleted. This research aims to elucidate the metabolic pathway of this metabolite through structural identification, homologous sequence analysis of the fadA5 gene, phylogenetic tree analysis of M. neoaurum HGMS2, and gene knockout. Our findings revealed that the metabolite is a C23 metabolic intermediate, named 24-norchol-4-ene-3, 22-dione (designated as 3-OPD). It is formed when a thioesterase (TE) catalyzes the formation of a β-ketonic acid by removing CoA from the side chain of 3, 22-dioxo-25, 26-bisnorchol-4-ene-24-oyl CoA (22-O-BNC-CoA), followed by spontaneously undergoing decarboxylation. These results have the potential to contribute to the development of novel steroid intermediates.

Complete genome sequence of a phthalic acid esters degrading Mycobacterium sp. YC-RL4

ABSTRACT Mycobacterium sp. YC-RL4 is capable of utilizing a broad range of phthalic acid esters (PAEs) as sole source of carbon and energy for growth. The preliminary studies demonstrated its high degrading efficiency and good performance during the bioprocess with environmental samples. Here, we present the complete genome of Mycobacterium sp. YC-RL4, which consists of one circular chromosome (5,801,417 bp) and one plasmid (252,568 bp). The genomic analysis and gene annotation were performed and many potential genes responsible for the biodegradation of PAEs were identified from the genome. These results may advance the investigation of bioremediation of PAEs-contaminated environments by strain YC-RL4.

Successful Treatment of Mycobacterium celatum Pulmonary Disease in an Immunocompetent Patient Using Antimicobacterial Chemotherapy and Combined Pulmonary Resection

Mycobacterium celatum is a nontuberculous mycobacterium that rarely causes pulmonary disease in immunocompetent subjects. We describe the successful treatment of M. celatum lung disease with antimicobacterial chemotherapy and combined pulmonary resection. A 33-year-old woman was referred to our hospital with a 3-month history of a productive cough. Her medical history included pulmonary tuberculosis 14 years earlier. Her chest X-ray revealed a large cavitary lesion in the left upper lobe. The sputum smear was positive for acid-fast bacilli, and M. celatum was subsequently identified in more than three sputum cultures, using molecular methods. After 1 year of therapy with clarithromycin, ethambutol, and ciprofloxacin, the patient underwent a pulmonary resection for a persistent cavitary lesion. The patient was considered cured after receiving 12 months of postoperative antimycobacterial chemotherapy. There has been no recurrence of disease for 18 months after treatment completion. In summary, M. celatum is an infrequent cause of potentially treatable pulmonary disease in immunocompetent subjects. Patients with M. celatum pulmonary disease who can tolerate resectional surgery might be considered for surgery, especially in cases of persistent cavitary lesions despite antimycobacterial chemotherapy.

The Incidence and Clinical Implication of Sputum with Positive Acid-Fast Bacilli Smear But Negative in Mycobacterial Culture in a Tertiary Referral Hospital in South Korea

Although it is not rare to find sputum that is positive acid-fast bacilli (AFB) smear but subsequent culture fails to isolate mycobacteria in clinical practice, the incidence and clinical implication of those sputa from new patients has not been clearly elucidated. The aim of this study was to determine the incidence and clinical implication of sputum with positive AFB smear but negative in mycobacterial culture. All sputa that were positive AFB smear requested during diagnostic work up for new patients visiting Seoul National University Hospital from 1 January 2005 through 31 December 2006 were included. Sputa producing a positive AFB smear but negative mycobacterial culture were classified into one of four categories: laboratory failure to isolate mycobacteria, false positive AFB smear, pathogen may show a positive AFB smear other than mycobacteria, and indeterminate results. Out of 447 sputa with a positive AFB smear, 29 (6.5%) failed to culture any organism. Among these 29 sputa, 18 were caused by laboratory failure to isolate mycobacteria, six were false positive smears, and five indeterminate. Although most sputum with a positive AFB smear but negative culture could be classified as a laboratory failure, clinicians should consider the possibility of false positive AFB smear.

Evaluation of the Broth Microdilution Method Using 2,3-Diphenyl-5-thienyl-(2)-tetrazolium Chloride for Rapidly Growing Mycobacteria Susceptibility Testing

As the incidence of nontuberculous mycobacterial infection has been increasing recently in Korea, the importance of drug susceptibility test for clinical isolates of mycobacteria has become larger. In this study we determined the antimicrobial susceptibility patterns of clinical isolates of M. fortuitum and M. abscessus in Korea, and evaluated the efficacy of a modified broth microdilution method using 2,3-diphenyl-5-thienyl-(2)-tetrazolium chloride (STC), in terms of its ability to provide accurate and easy-to-read minimal inhibitory concentration (MIC) endpoints for the susceptibility testing of rapidly growing mycobacteria. Most isolates of M. fortuitum and M. abscessus in Korea are susceptible or intermediately susceptible to amikacin, cefoxitin, ciprofloxacin, and clarithromycin. Many isolates of M. fortuitum are susceptible to doxycycline, sulfamethoxazole, and imipenem, while many M. abscessus isolates are resistant to these drugs. In the present study, the modified broth microdilution method using STC was found to be reliable, easy to read, and inexpensive for M. fortuitum and M. abscessus susceptibility testing. The modified colorimetric MIC testing method using STC was proven to be a useful surrogate for RGM antibiotic susceptibility testing.

Recombinant mycobacterial proteins future directions to improve protective efficacy.

A large number of subunit vaccine candidates have recently been developed as alternatives to Mycobacterium bovis Bacillus Calmette-Guerin (BCG), which gives unpredictable and highly variable protection against tuberculosis. Immunological potential of various recombinant proteins against mycobacterial infections has been discussed. Further, strategies have been suggested, which include development of constructs coexpressing cytokines or regimens utilizing recombinant proteins for further improving the protective efficacy.

Correlation between phospholipid biosynthesis and intracellular levels of cyclic adenosine monophosphate in Mycobacterium smegmatis.

The influence of intracellular levels of cAMP on phospholipid synthesis in Mycobacterium smegmatis has been examined under conditions of varying carbon source. A decreased phospholipid content was observed in glucose-grown cells, possibly due to decrease in intracellular cAMP levels caused by decreased/increased activity of adenylate cyclase and phosphodiesterase, respectively. The lowered phospholipid content was supported by decrease both in [14C]acetate incorporation and activities of key enzymes of phospholipid biosynthesis. These results in the light of our earlier observation of enhanced phospholipid synthesis in presence of increased levels of cAMP suggest a direct correlation between phospholipid biosynthesis and intracellular levels of cAMP in M. smegmatis.

Metabolism of silicon as a probable pathogenicity factor for Mycobacterium & Nocardia spp.

The role of silicon (Si) in metabolism and growth of 22 strains of mycobacteria and 3 strains of nocardiae, which were mostly pathogenic, was studied on Kirchner's medium solidified with sodium metasilicate (KSM) and the C-free solidified metasilicate minimal medium (SMM) consisting of mineral salts only. On KSM, initial growth of mycobacteria appeared to be better, compared with that on Lowenstein-Jensen medium (LJM), although subsequent growth on the former was slower. On SMM lacking C, growth of mycobacteria and nocardiae could be achieved, only after repeated passages. These findings indicate that the mycobacteria and nocardiae are able to utilise Si at least to a limited extent, possibly as an alternative to C, with greater chances of survival.

Calmodulin-like activity in mycobacteria.

Calmodulin-like activity has been reported for the first time in mycobacterial species, namely Mycobacterium tuberculosis BCG and M. smegmatis ATCC 14468. The activity was mainly located in the soluble fraction of the mycobacterial cells, Radioimmunoassay revealed maximum levels of calmodulin in young growing cells (early logarithmic phase of growth). Calmodulin-dependent phosphodiesterase activation assay revealed low activity (22%) of partially purified calmodulin either due to insufficient amount of calmodulin to activate phosphodiesterase or due to the presence of some factors interfering with the assay. Calmodulin antagonists, viz. trifluoperazine and phenothiazine, significantly inhibited the 32Pi incorporation into mycobacterial phospholipids. Similar inhibition was observed when EGTA (which removes calcium) was added to the medium. Significant inhibition of 32Pi incorporation in the presence of calmodulin antagonists suggested the involvement of calmodulin in mycobacterial phospholipid metabolism.

Transformation of sitosterol to androsta-1, 4-diene-3, 17-dione by immobilized Mycobacterium cells.

Transformation of sitosterol to androsta-1, 4-diene-3, 17-dione was studied with Mycobacterium cells entrapped in various polymeric matrices. Of the three supports viz. alginate, carageenan, agarose and polyacrylamide, studied, the polyacrylamide immobilized cells showed optimum catalytic stability and reusability.

Radiometric studies on the oxidation of (U-14C) L-amino acids by drug-susceptible and drug-resistant mycobacteria

Um sistema radiométrico foi utilizado para estudar os padröes de oxidaçäo dos (U-C)L-aminoácidos por micobactérias sensíveis e resistentes a drogas. Foram usadas duas cepas do M. tuberculosis sensíveis a todas as drogas, H Rv e Erdman. As micobactérias resistentes foram M. tuberculosis H Rv resistente a 5 ug/ml de hidrazida, M. bovis, M. avium, M. intracellulare, M. kansasii e M. chelonei. As micobactérias foram inoculadas em frascos estéreis contendo o meio líquido 7H9 e um dos (U-C)L-aminoácidos. Cada micobactéria apresentou um padräo de oxidaçäo de aminoácidos, mas estes padröes näo foram suficientemente diferentes para identificá-la. Aminoácidos complexos como a prolina, fenilalanina e tirosina näo tiveram utilidade na identificaçäo das micobactérias, pois praticamente todos os microorganismos foram incapazes de oxidá-los. Nenhuma combinaçäo de aminoácidos foi capaz de separar as micobacterias sensíveis das resistentes a drogas

Radiometric studies on the oxidation of (1-14C) fatty acids by drug-susceptible and drug-resistant mycobacteria

Um sistema radiométrico foi utilizado para estudar os padröes de oxidaçäo dos (1-14C) ácidos graxos por microorganismos do gênero Mycobacterium sensíveis e resistentes a drogas. Foram usadas duas cepas do M. tuberculosis sensíveis a todas as drogas, H37Rv e Erdman. As micobactérias resistentes foram M. tuberculosis H37Rv resistente a 5 ug/ml de hidrazida, M. bovis, M. avium, M. intracellulare, M. kansasii e M. chelonei. As micobactérias foram inoculadas em frascos estéreis contendo o meio líquido 7H9 com 10% do complexo albumina-dextrose-catalase e 1,0 uCi de um dos (1-14C) ácidos graxos (butírico, hexanoico, octanoico, decanóico, láurico, mirístico, plamítico, esteárico, olêico, linolêico, linolênico). Os frascos foram incubados a 37-C e o 14CO2 produzido pelas micobactérias foi medido durante 3 dias, com uma máquina Bactec-R-301. Embora cada micobactéria apresentasse um padräo distinto de oxidaçäo de ácido graxo, estes padröes näo foram suficientemente diferentes para identificá-la. Nenhuma combinaçäo de ácidos graxos nem a oxidaçäo preferencial de ácidos graxos de cadeias longas ou curtas foi capaz de separar as micobactérias resistentes das sensíveis. Outras experiências com um maior número de micobactérias sensíveis, incluindo estudo da assimilaçäo de substâncias marcadas, säo necessárias para se tentar a diferenciaçäo entre as micobactérias sensíveis e as resistentes a drogas

Comparison of biochemical characterisation of ICRC bacilli with M. leprae: effect of substrate alteration in the medium.

ICRC-bacilli strain C-44 when grown in Dubos medium of its equivalent, express M. avium taxonomic biochemical characters. Assuming that difference in characters of M. leprae and ICRC bacilli, could be due to 'in vivo' and 'in vitro' milieu, we altered the substrates in the medium. The bacilli grow well in the new medium containing selenium, ferric nitrate, magnesium chloride and deleting Tween 80. The ICRC strain C-44 grown in new medium expressed characters: 9/10 similarity with M. leprae. The 10 day tween hydrolysis reaction in weak but positive. It is probable that 'M. leprae culture isolate', may have acquired 'in vitro' growth potential by recombination with M. avium, an ubiquitous mycobacterium. The M. leprae culture isolate thus may express some characters of both M. leprae and M. avium.