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Objectives: The rapid dissemination of the mcr-1 gene via plasmid-mediated transfer has raised concerns regarding the efficacy of colistin as a last-resort treatment for multidrug-resistant Gram-negative bacterial infections. Current mcr-1 gene detection methods mainly focus on cultured bacteria, which is a complex and time-consuming process requiring skilled personnel, making it unsuitable for field analysis., Methods: A rapid detection technique combining recombinase polymerase amplification with a lateral flow dipstick targeting uncultured clinical samples was developed., Results: This new method targeting the mcr-1 gene region (23 232-23 642 bp, no. KP347127.1) achieved a low detection limit of 10 copies/μL. The whole process was carried out with high specificity and was completed within 20 min. The evaluation assay was conducted using 45 human faecal samples; 16 strains yielded a 98% accuracy, closely matching antimicrobial susceptibility outcomes., Conclusions: The novel method integrates nucleic acid extraction, isothermal amplification, and a test assay, suggesting the potential for timely colistin resistance surveillance in frontline disease control and healthcare settings, supporting future prevention and clinical standardization efforts., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

In this study, we report the characterization of three glycosyltransferases involved in the biosynthesis of ligupurpuroside B, a complex acylated phenolic glycoside in Ligustrum robustum . UGT85AF8 catalyzed the formation of salidroside from tyrosol. UGT79G7, an osmanthuside A 1,3-rhamnosyltransferase, and UGT79A19, an osmanthuside B 1,4-rhamnosyltransferase, sequentially converted osmanthuside A into ligupurpuroside B. Orthologs of UGT79G7 were also discovered from other plants producing verbascoside. These rhamnosyltransferases expand the toolbox for the biosynthesis of natural products with various sugar chains.

Actin cytoskeleton dynamic rearrangement is required for tumor cell metastasis and is a key characteristic of Helicobacter pylori ( H. pylori )-infected host cells. Actin cytoskeleton modulation is coordinated by multiple actin-binding proteins (ABP). Through Kyoto encyclopedia of gene and genomes database, GEPIA website, and real-time PCR data, we found that H. pylori infection significantly induced L-plastin, a key ABP, in gastric cancer cells. We further explored the regulation and function of L-plastin in H. pylori -associated gastric cancer and found that, mechanistically, H. pylori infection induced gastric cancer cells to express L-plastin via cagA -activated ERK signaling pathway to mediate SP1 binding to L-plastin promoter. Moreover, this increased L-plastin promoted gastric cancer cell proliferation and migration in vitro and facilitated the growth and metastasis of gastric cancer in vivo . Finally, we detected the expression pattern of L-plastin in gastric cancer tissues, and found that L-plastin was increased in gastric cancer tissues and that this increase of L-plastin positively correlated with cagA + H. pylori infection status. Overall, our results elucidate a novel mechanism of L-plastin expression induced by H. pylori , and a new function of L-plastin-facilitated growth and metastasis of gastric cancer, and thereby implicating L-plastin as a potential therapeutic target against gastric cancer. IMPLICATIONS: Our results elucidate a novel mechanism of L-plastin expression induced by H. pylori in gastric cancer, and a new function of L-plastin-facilitated gastric cancer growth and metastasis, implicating L-plastin as a potential therapeutic target against gastric cancer., (©2021 American Association for Cancer Research.)

The two-component system "AfsQ1/Q2" plays a crucial role to activate the production of antibiotics ACT, RED, and CDA through directly binding the promoters of pathway-specific activator genes actII-ORF4, redZ, and cdaR respectively when grown under glutamate-supplemented minimal medium in Streptomyces coelicolor. In this report, we demonstrated that the RspA1/A2 (a homologous protein of two-component system AfsQ1/Q2) plays a regulatory role in salinomycin biosynthesis in Streptomyces albus. Gene deletion and complementation experiments showed that the RspA1/A2 promoted salinomycin production but inhibited cell growth when cultured in YMG medium supplemented with 3% soybean oil. More importantly, RspA1/A2 strengthens salinomycin biosynthesis by directly affecting the transcription of the pathway-specific activator gene slnR. Meanwhile, RspA1/A2 plays a negative role in the regulation of nitrogen assimilation and urea decarboxylation by interacting with the promoters of genes gdhA, glnA, amtB, and SLNWT_1828/1829. Gene sigW is located downstream of rspA1/A2 and encodes an extracytoplasmic function sigma factor. Moreover, it negatively regulates the salinomycin biosynthesis and promotes cell growth, which antagonizes the function of RspA1/A2. In short, these useful findings are proved helpful to enrich the understanding of the regulatory pathways of antibiotic biosynthesis by an ECF σ factor-TCS signal transduction system in Streptomyces.

Polymyxins resistance has emerged worldwide and is threatening the treatment efficacy of multidrug resistant Klebsiella pneumoniae in humans and animals. In this research, we employed whole-genome sequencing (WGS) to investigate the polymyxin B resistance mechanism in selected polymyxin B-susceptible and polymyxin B-resistant K. pneumoniae , isolated from one patient of Huashan Hospital affiliated to Fudan University. The WGS results showed that the two K. pneumoniae all belong to ST11. The average nucleotide identity between the two K. pneumoniae was nearly 100%. No sense mutations of polymyxins resistance associated genes ( pmrA , pmrB , phoP , mgrB ) were observed in polymyxin B-resistant K. pneumonia (PRKP) compared to the polymyxin B-susceptible isolate. A 42 bp deletion was found in the sequence of phoQ in PRKP. The deletion of amino acid occurred on the periplasmic domain of PhoQ protein. We speculate that this is the domain that MgrB protein interact with the PhoQ protein and negatively regulate the PhoP/PhoQ system. qRT-PCR analysis revealed an overexpression of the pmrA (6.8-fold), pmrB (151.9-fold), pmrC (14.5-fold), pmrK (287.9-fold), phoP (14.5-fold), and phoQ (16.8-fold) genes in the polymyxin B-resistant isolate compared to the expression of the polymyxin B-susceptible K. pneumoniae isolate, suggesting that the phoQ deletion maybe responsible for the increased expression levels of those genes. In conclusion, this study identified a 42 bp deletion in the sequence of phoQ as being responsible for the overexpression of pmrCAB and pmrHFIJKLM operons, leading to resistance to polymyxin B., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Qingqing Xu et al.)

Candicidin is one of the frequent antibiotics for its high antifungal activity, but the productivity is still extremely low. Introduction of adpA into Streptomyces ZYJ-6 could improve candicidin productivity significantly and achieved 9338 μg/mL, which was the highest value ever reported in the literature. Combined analyses of transcriptional levels, metabolic flux and metabolomics indicate that para-aminobenzoic acid and the first step of shikimic acid metabolism were not the bottleneck for the candicidin production in the control. However, methylmalonyl-CoA played a central role in the candicidin production and the gene methB responsible for the biosynthesis of methylmalonyl-CoA might be the candidate gene target for further improving the production of candicidin.

A strategy for elucidating sequence determinants of function in the class of cytochrome P450 (CYP) enzymes that catalyze the first steps of terpene metabolism in wild microbiomes is described. Wild organisms that can use camphor, terpineol, pinene and limonene were isolated from soils rich in coniferous waste. Cell free extracts and growth beers were analyzed by gas chromatography/mass spectrometry to identify primary oxidative metabolites. For one organism, Pseudomonas nitroreducens TPJM, a cytochrome P450 (CYP108B1) isolated from cell free extracts was demonstrated to catalyze the oxidation of α-terpineol in assays combining the native ferredoxin and putidaredoxin reductase, and the resulting oxidation products identified by gas chromatography/mass spectrometry. Shotgun sequencing of PnTPJM identified four candidate P450 genes, including an apparently fragmentary gene with a high degree of homology with the known enzyme CYP108 (P450 terp )., (Copyright © 2018. Published by Elsevier Inc.)

The existence of a substrate-sensitive equilibrium between high spin (S=5/2) and low spin (S=1/2) ferric iron is a well-established phenomenon in the cytochrome P450 (CYP) superfamily, although its origins are still a subject of discussion. A series of mutations that strongly perturb the spin state equilibrium in the camphor hydroxylase CYP101A1 were recently described (Colthart et al., Sci. Rep. 6, 22035 (2016)). Wild type CYP101A1 as well as some CYP101A1 mutants are herein shown to be capable of catalyzing the reduction of nitroacetophenones by NADH to the corresponding anilino compounds (nitroreductase or NRase activity). The distinguishing characteristic between those mutants that catalyze the reduction and those that cannot appears to be the extent to which residual high spin form exists in the absence of the native substrate d-camphor, with those showing the largest spin state shifts upon camphor binding also exhibiting NRase activity. Optical and EPR spectroscopy was used to further examine these phenomena. These results suggest that reduction of nitroaromatics may provide a useful probe of residual high spin states in the CYP superfamily. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Cytotoxin-associated-gene A (CagA) of Helicobacter pylori ( H. pylori ) is a virulence factor that plays critical roles in H. pylori -induced gastric inflammation. In the present study, gastric biopsies were used for genotyping cagA and vacA genes, determining the autophagic activity, and the severity of gastric inflammation response. It was revealed that autophagy in gastric mucosal tissues infected with cagA + H. pylori strains was lower than the levels produced by cagA - H. pylori strains, accompanied with accumulation of SQSTM1 and decreased LAMP1 expression. In vitro , deletion mutant of cagA gene resulted in increased autophagic activity, and decreased expression of SQSTM1 and cytokines, whereas over-expression of CagA down-regulated the starvation-induced autophagy, and induced more production of the cytokines. Moreover, the production of the cytokines was increased by inhibition of autophagy, but decreased by enhancement of autophagy. Deletion of CagA decreased the ability to activate Akt kinase at Ser-473 site and increased autophagy. c-Met siRNA significantly affected CagA-mediated autophagy, and decreased the level of p-Akt, p-mTOR, and p-S6. Both c-Met siRNA and MK-2206 could reverse inflammatory response. H. pylori CagA protein negatively regulates autophagy and promotes the inflammation in H. pylori infection, which is regulated by c-Met-PI3K/Akt-mTOR signaling pathway activation.

A high erythromycin producing mutant strain Saccharopolyspora erythraea HL3168 E3-ΔmutB was constructed by deleting mutB (SACE_5639) gene encoding the beta subunit of methylmalonyl-CoA mutase of an industrial strain of S. erythraea HL3168 E3. Industrial media and process control strategies were adopted in a 5 L bioreactor for characterizing the physiological parameters. The total erythromycin titer and erythromycin A concentration in mutant were 46.9 (12740.5 μg/mL) and 64.9 % (8094.4 μg/mL) higher than those in original strain, respectively, which were comparable to industrial erythromycin production. The specific glucose and n-propanol consumption rates were increased by 52.4 and 39.8 %, respectively. During the rapid erythromycin synthesis phase, the yield of erythromycin on n-propanol also increased from 24.3 % in control group to 66.9 % in mutant group. Meanwhile, the specific formation rates of methylmalonyl-CoA and propionyl-CoA, two crucial precursors for erythromycin synthesis, were 1.89- and 2.02-folds higher in the mutant strain, respectively.

For strain improvement, robust and scalable high-throughput cultivation systems as well as simple and rapid high-throughput detection methods are crucial. However, most of the screening methods for lactic acid bacteria (LAB) strains were conducted in shake flasks and detected by high-performance liquid chromatography (HPLC), making the screening program laborious, time-consuming and costly. In this study, an integrated strategy for high-throughput screening of high L-lactic acid-productivity strains by Bacillus coagulans in deep-well microtiter plates (MTPs) was developed. The good agreement of fermentation results obtained in the MTPs platform with shake flasks confirmed that 24-well U-bottom MTPs could well alternate shake flasks for cell cultivation as a scale-down tool. The high-throughput pH indicator (bromocresol green) and L-lactate oxidase (LOD) assays were subsequently developed to qualitatively and quantitatively analyze L-lactic acid concentration. Together with the color halos method, the pH indicator assay and LOD assay, the newly developed three-step screening strategy has greatly accelerated the screening process for LAB strains with low cost. As a result, two high L-lactic acid-productivity mutants, IH6 and IIIB5, were successfully screened out, which presented, respectively, 42.75 and 46.10 % higher productivities than that of the parent strain in a 5-L bioreactor.

Vaccination strategies for Staphylococcus aureus, particularly methicillin-resistant S. aureus (MRSA) infections have attracted much research attention. Recent efforts have been made to select manganese transport protein C, or manganese binding surface lipoprotein C (MntC), which is a metal ion associated with pathogen nutrition uptake, as potential candidates for an S. aureus vaccine. Although protective humoral immune responses to MntC are well-characterised, much less is known about detailed MntC-specific B cell epitope mapping and particularly epitope vaccines, which are less-time consuming and more convenient. In this study, we generated a recombinant protein rMntC which induced strong antibody response when used for immunisation with CFA/IFA adjuvant. On the basis of the results, linear B cell epitopes within MntC were finely mapped using a series of overlapping synthetic peptides. Further studies indicate that MntC113-136, MntC209-232, and MntC263-286 might be the original linear B-cell immune dominant epitope of MntC, furthermore, three-dimensional (3-d) crystal structure results indicate that the three immunodominant epitopes were displayed on the surface of the MntC antigen. On the basis of immunodominant MntC113-136, MntC209-232, and MntC263-286 peptides, the epitope vaccine for S. aureus induces a high antibody level which is biased to TH2 and provides effective immune protection and strong opsonophagocytic killing activity in vitro against MRSA infection. In summary, the study provides strong proof of the optimisation of MRSA B cell epitope vaccine designs and their use, which was based on the MntC antigen in the development of an MRSA vaccine.

We recently reported that an amide bond is unexpectedly formed by an acyl-CoA synthetase (which catalyzes the formation of a carbon-sulfur bond) when a suitable acid and l-cysteine are used as substrates. DltA, which is homologous to the adenylation domain of nonribosomal peptide synthetase, belongs to the same superfamily of adenylate-forming enzymes, which includes many kinds of enzymes, including the acyl-CoA synthetases. Here, we demonstrate that DltA synthesizes not only N-(d-alanyl)-l-cysteine (a dipeptide) but also various oligopeptides. We propose that this enzyme catalyzes peptide synthesis by the following unprecedented mechanism: (i) the formation of S-acyl-l-cysteine as an intermediate via its "enzymatic activity" and (ii) subsequent "chemical" S → N acyl transfer in the intermediate, resulting in peptide formation. Step ii is identical to the corresponding reaction in native chemical ligation, a method of chemical peptide synthesis, whereas step i is not. To the best of our knowledge, our discovery of this peptide synthesis mechanism involving an enzymatic reaction and a subsequent chemical reaction is the first such one to be reported. This new process yields peptides without the use of a thioesterified fragment, which is required in native chemical ligation. Together with these findings, the same mechanism-dependent formation of N-acyl compounds by other members of the above-mentioned superfamily demonstrated that all members most likely form peptide/amide compounds by using this novel mechanism. Each member enzyme acts on a specific substrate; thus, not only the corresponding peptides but also new types of amide compounds can be formed., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background/aims: Motility is a feature of many pathogens that contributes to the migration and dispersion of the infectious agent. Whether gentamycin has a post-antibiotic effect (PAE) on the swarming and swimming motility of Escherichia coli (E. coli) remains unknown. In this study, we aimed to examine whether short-term pretreatment of sub-inhibitory concentrations of gentamycin alter motility of E. coli and the mechanisms involved therein., Methods: After exposure to sub-inhibitory concentrations (0.8 μg/ml) of gentamicin, the swarming and swimming motility of E. coli was tested in semi-solid media. Real-time PCR was used to detect the gene expression of succinate dehydrogenase (SDH). The production of SDH and fumarate by E. coli pretreated with or without gentamycin was measured. Fumarate was added to swarming agar to determine whether fumarate could restore the swarming motility of E. coli., Results: After pretreatment of E. coli with sub-inhibitory concentrations of gentamycin, swarming motility was repressed in the absence of growth inhibition. The expression of all four subunits of SDH was down-regulated, and the intracellular concentration of SDH and fumarate, produced by E. coli, were both decreased. Supplementary fumarate could restore the swarming motility inhibited by gentamycin. A selective inhibitor of SDH (propanedioic acid) could strongly repress the swarming motility., Conclusion: Sub-inhibitory concentrations of gentamycin inhibits the swarming motility of E. coli. This effect is mediated by a reduction in cellular fumarate caused by down-regulation of SDH. Gentamycin may be advantageous for treatment of E. coli infections., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

1, 3-propanediol oxidoreductase (PDOR) is a key enzyme in glycerol bioconversion to 1,3-propanediol (1, 3-PD) which is a valuable chemical and one of the six new petrochemical products. We used error-prone PCR and activity screening to identify mutants of Klebsiella pneumoniae (K. pneumoniae) PDOR with improved activity. The activity of one of the identified mutants, PDOR'-24, which includes a single mutation, A199S, was 48 U/mg, 4.9 times that of the wild-type enzyme. Molecular docking was performed to analyze the identified mutants; and amino acids S103, H271, N366, D106, N262 and D364 were predicted to bond with NADH. The origins of the improved activity of PDOR'-24, as well as three other mutants were analyzed by simulating the interaction mechanism of the mutants with the substrate and coenzyme, respectively. This research provides useful information about the use of safranine O plate screening for the directed evolution of oxidoreductases, identifies interesting sites for improving PDOR activity, and demonstrates the utility of using molecular docking to analyze the interaction mechanism of the mutants with the substrate and coenzyme, respectively.

Objective: We studied the cloning and expression of lasB encoding solvent-resistant protease from Pseudomonas aeruginosa PT121. The recombinant protease was then characterized and applied in peptide synthesis., Methods: The PCR primers were designed to acquire the open read frame (ORF) of lasB according to similar protease gene (pseudolysin) reported in the literature. Inducible expression plasmid pET22b-lasB' was constructed and expressed in E. coli BL21 (DE3). The recombinant protease was then characterized and applied in peptide synthesis., Results: The protease PT121 was defined as metalloproteinase M4 family according to sequence blast. Gene sequence analysis shows that lasB encodes signal peptide, pro-peptide and mature peptide. Mature protein contains 301 residues with molecular weight of 33 kDa. One-step preparation of the recombinant proteases PT121 was optimized by breaking cell wall. The specific activity of protease PT121 reached up to 7700U/mg, and it was stable similar with wild type PT121 from P. aeruginosa PT121 in temperature, pH and organic solvent. The synthesis rate of various dipeptides in 50% DMSO was effective, especially productivity of aspartame precursor reached up to 91%., Conclusions: Successful hetero-expression of protease PT121 lays the foundation of studying mechanism of catalysis and molecular evolution.

Objective: To identify specific antigens related to streptomycin resistant (SMr) Mycobacterium tuberculosis., Methods: Cellular proteins were extracted from SMr clinical isolate 01108, SM-sensitive clinical isolate 01105 and H37Rv. Differential expression proteins were identified with isobaric tags for relative and absolute quantitation (iTRAQ) combined with Nano LC-MS/MS technology., Results: Approximately 194 and 146 differential expression proteins were identified in 01108 strain compared with the proteomic profiles of 01105 strain and H37Rv, respectively, and 121 proteins were identified in 01108 strain compared with the proteomic profiles of both 01105 strain and H37Rv. Identified proteins showed a pI (isoelectric point) variation between 3.74-12.48 and a molecular mass (M) range between 7.63 and 326.2 kDa. Differential expression proteins were mainly associated with metabolism (involved in intermediary metabolism, respiration, and lipid metabolism) and took part in catalysis and binding function. Seven ribosomal proteins (Rv0056, Rv0641, Rv0652, Rv0701, Rv1630, Rv2442c and Rv2785c) and seven proteins (the ratios > 1.20 or < 0.55) were commonly down-regulated in 01108 strain compared with both 01105 strain and H37Rv, i. e. the thiol peroxidase (Rv1932), acyl carrier protein dehydrogenase (Rv0824c), 30S ribosomal protein S15 (Rv2785c), acetone acid dehydrogenase E2 part (Rv2215), two-component transcriptional regulatory protein (Rv3133c) and Hypothetical protein (Rv2466e and Rv2626c)., Conclusion: Differential expression proteins were found in SMr strain compared with both SM-sensitive strain and H37Rv. Further studies are needed to assess the role of these differential expression proteins in SM resistance.

The neutral protease WQ from Bacillus cereus is stable in various aqueous organic mixtures, with the exception of those containing acetonitrile (ACN) and dimethylformamide (DMF). The stability of the enzyme in aqueous hydrophilic solvents was dramatically enhanced with the addition of calcium ions, with the degree of improvement in the half-life relative to different solutions ranging from fourfold to more than 70-fold. Studies of the kinetic constants showed that calcium ions induced slight conformational changes in the active site of the enzyme in aqueous ACN. We investigated the molecular mechanisms underlying this stabilizing effect by employing a combination of biophysical techniques and molecular dynamics simulation. In aqueous ACN, the intrinsic fluorescence and circular dichroism analysis demonstrated that the addition of calcium ions induced a relatively compact conformation and maintained both the native-like microenvironment near the tryptophan residues and the secondary structure. Alternatively, homology modeling confirmed the location of four calcium-ion-binding sites in the enzyme, and molecular dynamics simulation revealed that three other calcium ions were bound to the surface of the enzyme. Calcium ions, known as a type of kosmotrope, can strongly bond with water molecules, thus aiding in the formation of the regional hydration shell required for the maintenance of enzyme activity. In addition, the introduction of calcium ions resulted in the formation of additional ionic interactions, providing propitious means for protein stabilization. Thus, the stronger intramolecular interactions were also expected to contribute partially to the enhanced stability of the enzyme in an aqueous organic solvent.

Background: The otrC gene of Streptomyces rimosus was previously annotated as an oxytetracycline (OTC) resistance protein. However, the amino acid sequence analysis of OtrC shows that it is a putative ATP-binding cassette (ABC) transporter with multidrug resistance function. To our knowledge, none of the ABC transporters in S. rimosus have yet been characterized. In this study, we aimed to characterize the multidrug exporter function of OtrC and evaluate its relevancy to OTC production., Results: In order to investigate OtrC's function, otrC is cloned and expressed in E. coli The exporter function of OtrC was identified by ATPase activity determination and ethidium bromide efflux assays. Also, the susceptibilities of OtrC-overexpressing cells to several structurally unrelated drugs were compared with those of OtrC-non-expressing cells by minimal inhibitory concentration (MIC) assays, indicating that OtrC functions as a drug exporter with a broad range of drug specificities. The OTC production was enhanced by 1.6-fold in M4018 (P = 0.000877) and 1.4-fold in SR16 (P = 0.00973) duplication mutants, while it decreased to 80% in disruption mutants (P = 0.0182 and 0.0124 in M4018 and SR16, respectively)., Conclusions: The results suggest that OtrC is an ABC transporter with multidrug resistance function, and plays an important role in self-protection by drug efflux mechanisms. This is the first report of such a protein in S. rimosus, and otrC could be a valuable target for genetic manipulation to improve the production of industrial antibiotics.

Th17 cells represent a novel subset of CD4(+) T cells, which is associated with Helicobacter pylori infection. In the present study, we investigated the potential role of Urease subunit B (UreB) in the induction of Th17 cell response. Co-cultured splenic lymphocytes from H. pylori-infected mice with the recombinant UreB (rUreB) elevated IL-17 secretion and caused an increase in the number of Th17 cells. The expression of IL-6 and IL-23 p19 was significantly increased in rUreB-stimulated macrophages. Whole cell protein (WCP) of UreB-deficient strain (UreB(-) strain) induced less Th17 cell responses than that of wild-type strain. In addition, subcutaneous and intranasal immunization of rUreB elicited antigen-specific Th17 cell responses. Intranasal immunization of rUreB reduced H. pylori colonization in the stomach, which was closely related with the increased rUreB-specific Th17 cell responses. These results suggest that UreB is an important protein which is able to elicit Th17 cell responses against H. pylori both in vivo and in vitro., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Erythromycin A (Er-A) is the most potent and clinically important member in the Er family produced by Saccharopolyspora erythraea. Er-B and Er-C, which are biologically much less active and cause greater side effects than Er-A, serve as the intermediates for Er-A biosynthesis and impurities in fermentation processes of many industrial strains. In this study, systematical modulation of the amounts of tailoring enzymes EryK (a P450 hydroxylase) and EryG (an S-adenosylmethionine-dependent O-methyltransferase) was carried out by genetic engineering in S. erythraea, including alterations of gene copy number ratio and organization and integrating the locus on the chromosome by homologous recombination. Introduction of additional eryK and eryG genes into S. erythraea showed significant impacts on their transcription levels and enhanced the biotransformation process from Er-D to Er-A with gene dose effects. At the eryK/eryG copy number ratio of 3:2 as well as their resultant transcript ratio of around 2.5:1 to 3.0:1, Er-B and Er-C were nearly completely eliminated and accordingly converted to Er-A, and the Er titer was improved by around 25% in the recombinant strain ZL1004 (genotype PermK*-K-K-G + PermE*-K + PermA*-G) and ZL1007 (genotype PermK*-K-G-K + PermE*-K + PermA*-G). This study may contribute to the continuous efforts toward further evaluation of the Er-producing system, with the aims of improving Er-A purity and production at the fermentation stage and lowering the production costs and environmental concerns in industry.

Genes of zwf1 and zwf2 encode two isozymes of glucose-6-phosphate dehydrogenase (G6PDH) of Streptomyces, respectively. G6PDH is the first enzyme in the oxidative pentose phosphate pathway (PPP) and the key enzyme for NADPH generation. Based on thermal sensitive plasmid pKC1139, a recombinant plasmid pKC1139-zwf2' was constructed and verified with restriction enzyme digestion. The plasmid pKC1139-zwf2' was electropolated into competent Streptomyces rimosus M4018 cells after it was demethylated by E. coli GM2929. Transformants grown on Tryptone Soya Agar (TSA) plate containing 500 ug/mL apramycin were selected, and identified using dot hybridization analysis and PCR amplification with apramycin resistant gene as primers. A positive clone was then selected and designated M4018-delta zwf2. With parent strain S. rimosus M4018 as control, mutant M4018-delta zwf2 was cultured in shaking flask. Specific acitivity of G6PDH of M4018-delta zwf2 was only half of that of parent strain whereas yield of oxytetracycline (OTC) of mutant was 27% higher to the mutant had a similar biomass profileto that of the control biosynthesis started when the growth entered stationary phase on the 4th day. However, specific oxytetracycline production of mutant was 31% higher than that of the parent strain, indicating that zwf2 disruption could enhance oxytetracycline biosynthesis in S. rimosus M4018-delta zwf2.

To develop an efficient way to produce S-adenosylmethionine (SAM), methionine adenosyltransferase gene (mat) from Streptomyces spectabilis and Vitreoscilla hemoglobin gene (vgb) were coexpressed intracellularly in Pichia pastoris, both under control of methanol-inducible promoter. Expression of mat in P. pastoris resulted in about 27 times higher specific activity of methionine adenosyltransferase (SMAT) and about 19 times higher SAM production relative to their respective control, suggesting that overexpression of mat could be used as an efficient method for constructing SAM-accumulating strain. Under induction concentration of 0.8 and 2.4% methanol, coexpression of vgb improved, though to different extent, cell growth, SAM production, and respiratory rate. However, the effects of VHb on SAM content (specific yield of SAM production) and SMAT seemed to be methanol concentration-dependent. When cells were induced with 0.8% methanol, no significant effects of VHb expression on SAM content and specific SMAT could be detected. When the cells were induced with 2.4% methanol, vgb expression increased SAM content significantly and depressed SMAT remarkably. We suggested that under our experimental scheme, the presence of VHb might improve ATP synthesis rate and thus improve cell growth and SAM production in the recombinant P. pastoris.

The specific activities of key enzymes relating to the pathway of inosine synthesis of three different bacterial strains including high-yield, low-yield and wild strains were determined and compared systematically. A close relationship between inosine production and the specific activities of key enzymes was found. According to the enzyme characteristics of high-yield strain, suggestions on further strain improvement by modification of genetic engineering were proposed. Enzymology study is believed to be an effective way to make screening of high-yield strains more efficient.

To provide a basis of molecular genetic analysis of M. tubereulosis, the proteomic profiling was prompted. M. tuberculosis H37RV was cultured in Sauton medium at 37 degrees C for 3 weeks, harvested and fractionated into three portions: suspension filtration proteins(A), cytosol proteins(B) and membrane proteins(C). These fractions were analyzed by pH3-10 IPG gradient and SDS-PAGE. The silver-stained technigue and gel images were used. Then the image was transfered into 2-DE gel analysis Software. A part of protein sports of expression level higher from the culture filtrate fraction were identified by peptide mass fingerprinting. A total of 907, 884 and 681 protein sports were observed for A. B. C fractions in M. tuberculosis H37RV, respectively. Distribution of proteins mass for 3 fractions were principally similar, About 70.5-74.4 per cent were distributed in the ranges of Mr 10-49 kD.pI of the proteins for A, B fractions were pricipally similar, About 80.9-83.5 per cent were distributed in the ranges of pH 3.0-6.4, But the number of protein sports for C fraction distributed in the ranges of pH 7.6-10.0 were more than A, B fractions. The number of protein sports of expression level higher for A, B and C fractions were 71(7.8%), 242(27.4%), 19 (2.8%), respectively. 90 pen cent from them, pH of the proteins were distributed in the ranges of pH 3.0-6.4. 73.1 per cent for proteins mass of C fraction were distributed in the ranges of 10 kD-49 kD, which were more than A, B fractions. Nine of the proteins identified ih this study appeared to be homology or putative fanction proteins, but another five proteins were unknown. The proteomic profiling of different fractions in M. tuberculosis obtained in here will be provide a basis for detailed analysis of biology functions of the proteins.