Selectively improving nikkomycin Z production by blocking the imidazolone biosynthetic pathway of nikkomycin X and uracil feeding in Streptomyces ansochromogenes

<p>Abstract</p> <p>Background</p> <p>Nikkomycins are a group of peptidyl nucleoside antibiotics and act as potent inhibitors of chitin synthases in fungi and insects. Nikkomycin X and Z are the main components produced by <it>Streptomyces ansochromogenes</it>. Of them, nikkomycin Z is a promising antifungal agent with clinical significance. Since highly structural similarities between nikkomycin Z and X, separation of nikkomycin Z from the culture medium of <it>S. ansochromogenes </it>is difficult. Thus, generating a nikkomycin Z selectively producing strain is vital to scale up the nikkomycin Z yields for clinical trials.</p> <p>Results</p> <p>A nikkomycin Z producing strain (sanPDM) was constructed by blocking the imidazolone biosynthetic pathway of nikkomycin X via genetic manipulation and yielded 300 mg/L nikkomycin Z and abolished the nikkomycin X production. To further increase the yield of nikkomycin Z, the effects of different precursors on its production were investigated. Precursors of nucleoside moiety (uracil or uridine) had a stimulatory effect on nikkomycin Z production while precursors of peptidyl moiety (L-lysine and L-glutamate) had no effect. sanPDM produced the maximum yields of nikkomycin Z (800 mg/L) in the presence of uracil at the concentration of 2 g/L and it was approximately 2.6-fold higher than that of the parent strain.</p> <p>Conclusion</p> <p>A high nikkomycin Z selectively producing was obtained by genetic manipulation combined with precursors feeding. The strategy presented here might be applicable in other bacteria to selectively produce targeted antibiotics.</p

Cloning, reassembling and integration of the entire nikkomycin biosynthetic gene cluster into Streptomyces ansochromogenes lead to an improved nikkomycin production

<p>Abstract</p> <p>Background</p> <p>Nikkomycins are a group of peptidyl nucleoside antibiotics produced by <it>Streptomyces ansochromogenes</it>. They are competitive inhibitors of chitin synthase and show potent fungicidal, insecticidal, and acaricidal activities. Nikkomycin X and Z are the main components produced by <it>S. ansochromogenes</it>. Generation of a high-producing strain is crucial to scale up nikkomycins production for further clinical trials.</p> <p>Results</p> <p>To increase the yields of nikkomycins, an additional copy of nikkomycin biosynthetic gene cluster (35 kb) was introduced into nikkomycin producing strain, <it>S. ansochromogenes </it>7100. The gene cluster was first reassembled into an integrative plasmid by Red/ET technology combining with classic cloning methods and then the resulting plasmid(pNIK)was introduced into <it>S. ansochromogenes </it>by conjugal transfer. Introduction of pNIK led to enhanced production of nikkomycins (880 mg L^-1, 4 -fold nikkomycin X and 210 mg L^-1, 1.8-fold nikkomycin Z) in the resulting exconjugants comparing with the parent strain (220 mg L^-1nikkomycin X and 120 mg L^-1nikkomycin Z). The exconjugants are genetically stable in the absence of antibiotic resistance selection pressure.</p> <p>Conclusion</p> <p>A high nikkomycins producing strain (1100 mg L^-1nikkomycins) was obtained by introduction of an extra nikkomycin biosynthetic gene cluster into the genome of <it>S. ansochromogenes</it>. The strategies presented here could be applicable to other bacteria to improve the yields of secondary metabolites.</p

Characterization of EndoTT, a novel single-stranded DNA-specific endonuclease from Thermoanaerobacter tengcongensis

EndoTT encoded by tte0829 of Thermoanaerobacter tengcongensis binds and cleaves single-stranded (ss) and damaged double-stranded (ds) DNA in vitro as well as binding dsDNA. In the presence of a low concentration of NaCl, EndoTT cleaved ss regions of damaged dsDNA efficiently but did not cleave DNA that was entirely ss or ds. At high concentrations of NaCl or MgCl2 or ATP, there was also specific cleavage of ssDNA. This suggested a preference for ss/ds junctions to stimulate cleavage of the DNA substrates. EndoTT has six specific sites (a–f) in the oriC region (1–70 nt) of T. tengcongensis. Substitutions of nucleotides around site c prevented cleavage by EndoTT of both sites c and d, implying that the cleavage specificity may depend on both the nucleotide sequence and the secondary structure of the ssDNA. A C-terminal sub-fragment of EndoTT (residues 107–216) had both endonucleolytic and DNA-binding activity, whereas an N-terminal sub-fragment (residues 1–110) displayed only ssDNA-binding activity. Site-directed mutations showed that G170, R172 and G177 are required for the endonuclease activity of EndoTT, but not for DNA-binding, whereas D171, R178 and G189 are partially required for the DNA-binding activity

SabR enhances nikkomycin production via regulating the transcriptional level of sanG, a pathway-specific regulatory gene in Streptomyces ansochromogenes

<p>Abstract</p> <p>Background</p> <p><it>sabR </it>is a pleiotropic regulatory gene which has been shown to positively regulate the nikkomycin biosynthesis and negatively affect the sporulation of <it>Streptomyces ansochromogenes</it>. In this study, we investigate the mechanism of SabR on modulating nikkomycin production in <it>Streptomyces ansochromogenes</it>.</p> <p>Results</p> <p>The transcription start point of <it>sabR </it>was determined by high-resolution S1 nuclease mapping and localized at the nucleotide T at position 37 bp upstream of the potential <it>sabR </it>translation start codon (GTG). Disruption of <it>sabR </it>enhanced its own transcription, but retarded the nikkomycin production. Over-expression of <it>sabR </it>enhanced nikkomycin biosynthesis in <it>Streptomyces ansochromogenes</it>. EMSA analysis showed that SabR bound to the upstream region of <it>sanG</it>, but it did not bind to the upstream region of its encoding gene (<it>sabR</it>), <it>sanF </it>and the intergenic region between <it>sanN </it>and <it>sanO</it>. DNase 1 footprinting assays showed that the SabR-binding site upstream of <it>sanG </it>was 5'-CTTTAAGTCACCTGGCTCATTCGCGTTCGCCCAGCT-3' which was designated as SARE. Deletion of SARE resulted in the delay of nikkomycin production that was similar to that of <it>sabR </it>disruption mutant.</p> <p>Conclusions</p> <p>These results indicated that SabR modulated nikkomycin biosynthesis as an enhancer via interaction with the promoter region of <it>sanG</it>, and expanded our understanding about regulatory cascade in nikkomycin biosynthesis.</p

Patterns of Gene Expression in Western Corn Rootworm (\u3ci\u3eDiabrotica virgifera virgifera\u3c/i\u3e) Neonates, Challenged with Cry34Ab1, Cry35Ab1 and Cry34/35Ab1, Based on Next-Generation Sequencing

With Next Generation Sequencing technologies, high-throughput RNA sequencing (RNAseq) was conducted to examine gene expression in neonates of Diabrotica virgifera virgifera (LeConte) (Western Corn Rootworm, WCR) challenged with individual proteins of the binary Bacillus thuringiensis insecticidal proteins, Cry34Ab1 and Cry35Ab1, and the combination of Cry34/Cry35Ab1, which together are active against rootworm larvae. Integrated results of three different statistical comparisons identified 114 and 1300 differentially expressed transcripts (DETs) in the Cry34Ab1 and Cry34/35Ab1 treatment, respectively, as compared to the control. No DETs were identified in the Cry35Ab1 treatment. Putative Bt binding receptors previously identified in other insect species were not identified in DETs in this study. The majority of DETs (75% with Cry34Ab1 and 68.3% with Cry34/35Ab1 treatments) had no significant hits in the NCBI nr database. In addition, 92 DETs were shared between Cry34Ab1 and Cry34/35Ab1 treatments. Further analysis revealed that the most abundant DETs in both Cry34Ab1 and Cry34/35Ab1 treatments were associated with binding and catalytic activity. Results from this study confirmed the nature of these binary toxins against WCR larvae and provide a fundamental profile of expression pattern of genes in response to challenge of the Cry34/35Ab1 toxin, which may provide insight into potential resistance mechanisms

Cloning, expression and characterization of a gene encoding nitroalkane-oxidizing enzyme from Streptomyces ansochromogenes

A nitroalkane-oxidizing enzyme gene (naoA) was cloned from a genomic DNA library of Streptomyces ansochromogenes 7100. The deduced protein (NaoA) of this gene contains 363 amino acids and has high similarity to several nitroalkane-oxidizing enzymes from various micro-organisms. The naoA gene was subcloned into an expression vector pET23b and overexpressed in Escherichia coli BL21(DE3). The protein was then purified, and its characteristics were studied. Experimental results showed that NaoA can convert 1-nitropropane, 2-nitropropane and nitroethane into the corresponding carbonyl compounds. The optimal pH and temperature for NaoA was found to be pH 7-8 and 48-56°C, respectively. The Km of NaoA for nitroethane is ≈26.8 mM. NADH and nitro blue tetrazolium are strong inhibitors of NaoA, and thiol compounds and superoxide dismutase partially inhibit the enzyme activity. Therefore, superoxide may be an essential intermediate in the oxidation of nitroalkane by NaoA

The Moderating Effect of R&amp;D Investment on Income and Carbon Emissions in China: Direct and Spatial Spillover Insights

R&amp;D investment plays a great role in achieving China&#8217;s low-carbon economy goals, which has a moderating effect on the relationship between income and carbon emissions. Furthermore, such a moderating effect may have spatial differences, given the possible spatial dependence of carbon emissions. Therefore, this paper explores the direct and spatial spillover moderating effects of R&amp;D investment by adopting the panel spatial Durbin model and data of 30 provinces in China during 1998&#8315;2015. The empirical results firstly indicate that R&amp;D investment moderates the positive impact of income on local carbon emissions for both the non-spatial and spatial model, and that more R&amp;D investment can make carbon emissions reach the turning point earlier. Secondly, R&amp;D investment in the local province increases the positive influence of local income on neighboring carbon emissions, which mainly results from the transfer effect of carbon emissions rather than the knowledge spillovers effect. The results are indicated to be robust by three types of robustness analyses. Finally, FDI and patents are the main constrained forces of local and neighboring carbon emissions; coal consumption is the main driver of local carbon emissions