Isolation and functional analysis of spy1 responsible for pristinamycin yield in Streptomyces pristinaespiralis

A gene related to high pristinamycin yield in Streptomyces pristinaespiralis was selected by amplified fragment length polymorphism (AFLP) and its functions were investigated by gene disruption. First, a 561 bp polymorphic sequence was acquired by AFLP from high-yield recombinants compared with the S. pristinaespiralis ancestor ATCC25486, indicating that this approach is an effective means of screening for valuable genes responsible for antibiotic yield. Then, a 2,127 bp open reading frame of a gene designated spy1 that overlaps with the above fragment was identified and its structure and biological functions were investigated. In silico analysis of spy1 encoding a deduced 708-amino-acid-long serine/threonine protein kinase showed that it only contains a catalytic domain in the N-terminal region, which is different from some known homologs. Gene inactivation of chromosomal spy1 indicated that it plays a pleiotropic regulatory function in pristinamycin production, with a positive correlation to pristinamycin I biosynthesis and a negative correlation to pristinamycin II biosynthesis.     

Genomic variability among high pristinamycin-producing recombinants of Streptomyces pristinaespiralis revealed by amplified fragment length polymorphism

Amplified fragment length polymorphism (AFLP) was used to analyze genomic variability between high pristinamycin-producing recombinants of Streptomyces pristinaespiralis produced by genome shuffling and their ancestral strain. The AFLP fingerprints obtained with two restriction enzyme combinations of ApaI/TaqI and PstI/SacII showed together that there was no major polymorphism (less than 10%) between these high yield recombinants and their ancestor. However, the unique polymorphic bands, which might be related to the yield increasing of pristinamycin, could be distinguished from all the recombinants. Clustering analysis further indicated that the recombinants with similar ability of pristinamycin production had similar genomic variability.     

Screening of multi-copy mannanase recombinants of Pichia pastoris based on colony size

Pichia pastoris pGAP (glyceraldehyde dehydrogenase promoter) expression system was widely used for the expression and production of heterologous proteins. Screening multi-copy recombinants was an effective strategy to improve the heterologous protein production in P. pastoris. Because multiple gene insertion events occurred with a low frequency, hundreds to thousands of antibiotic-resistance recombinants need to be screened. The common way of improving screening efficiency was to increase antibiotic concentration in screening plates. Here we developed a screening method by selecting small colonies from low-concentration antibiotic screening plates. This strategy greatly improved the probability of obtaining multi-copy mannanase gene (man) recombinants and it could replace the common strategy by increasing antibiotic concentration in screening plates. The further study in liquid shake flask cultures revealed that cell concentrations, growth rates and substrate consumption rates of recombinants gradually decreased with the increase in man copy number. This indicated that such a screening strategy was effective to screen multi-copy recombinants based on colony size.     

Genetic modification of a baculovirus vector for increased expression in insect cells

Generating large amounts of recombinant protein in transgenic animals is often challenging and has a number of drawbacks compared to cell culture systems. The baculovirus expression vector system (BEVS) uses virus-infected insect cells to produce recombinant proteins to high levels, and these are usually processed in a similar way to the native protein. Interestingly, since the development of the BEVS, the virus most often used (Autographa californica multi-nucleopolyhedovirus; AcMNPV) has been little altered genetically from its wild-type parental virus. In this study, we modified the AcMNPV genome in an attempt to improve recombinant protein yield, by deleting genes that are non-essential in cell culture. We deleted the p26, p10 and p74 genes from the virus genome, replacing them with an antibiotic selection cassette, allowing us to isolate recombinants. We screened and identified recombinant viruses by restriction enzyme analysis, PCR and Western blot. Cell viability analysis showed that the deletions did not improve the viability of infected cells, compared to non-deletion viruses. However, expression studies showed that recombinant protein levels for the deletion viruses were significantly higher than the expression levels of non-deletion viruses. These results confirm that there is still great potential for improving the BEVS, further increasing recombinant protein expression yields and stability in insect cells.     

Genetic and physical localization of the root-knot nematode resistance locus Mi in tomato

As part of a map-based cloning strategy designed to isolate the root-knot nematode resistance gene Mi, tomato F2 populations were analyzed in order to identify recombination points close to this economically important gene. A total of 21?089 F2 progeny plants were screened using morphological markers. An additional 1887 F2 were screened using PCR-based flanking markers. Fine-structure mapping of recombinants with newly developed AFLP markers, and RFLP markers derived from physically mapped cosmid subclones, localized Mi to a genomic region of about 550?kb. The low frequency of recombinants indicated that recombination was generally suppressed in these crosses and that crossovers were restricted to particular regions. To circumvent this problem, a population of Lycopersicon peruvianum, the species from which Mi was originally introgressed, that was segregating for resistance was developed. Screening of this population with PCR, RFLP and AFLP markers identified several plants with crossovers near Mi. Recombination frequency was approximately eight-fold higher in the Mi region of the L. peruvianum cross. However, even within the wild species cross, recombination sites were not uniformly distributed in the region. By combining data from the L.?esculentum and L. peruvianum recombinant analyses, it was possible to localize Mi to a region of the genome spanning less than 65?kb.     

Evolution of Streptomyces pristinaespiralis for resistance and production of pristinamycin by genome shuffling

Improvement of pristinamycin production by Streptomyces pristinaespiralis was performed by using recursive protoplast fusion and selection for improved resistance to the product antibiotic in a genome shuffling format. A 100-mug/ml pristinamycin resistant recombinant, G 4-17, was obtained after four rounds of protoplast fusion, and its production of pristinamycin reached 0.89 g/l, which was increased by 89.4% and 145.9% in comparison with that of the highest parent strain M-156 and the original strain CGMCC 0957, respectively. The subculture experiments indicated that the hereditary character of high producing S. pristinaespiralis G 4-17 was stable. It is concluded that genome shuffling improves the production of pristinamycin by enhancing product-resistance in a stepwise manner. Pristinamycin fermentation experiments by recombinant G 4-17 were carried out in a 5-l fermentor, and its production of pristinamycin reached 0.90 g/l after 60 h of fermentation.     

Development of a Novel Plasmid-Free Thymidine Producer by Reprogramming Nucleotide Metabolic Pathways

A novel thymidine-producing strain of Escherichia coli was prepared by genome recombineering. Eleven genes were deleted by replacement with an expression cassette, and 7 genes were integrated into the genome. The resulting strain, E. coli HLT013, showed a high thymidine yield with a low deoxyuridine content. DNA microarrays were then used to compare the gene expression profiles of HLT013 and its isogenic parent strain. Based on microarray analysis, the pyr biosynthesis genes and 10 additional genes were selected and then expressed in HLT013 to find reasonable candidates for enhancing thymidine yield. Among these, phage shock protein A (PspA) showed positive effects on thymidine production by diminishing redox stress. Thus, we integrated pspA into the HLT013 genome, resulting in E. coli strain HLT026, which produced 13.2 g/liter thymidine for 120 h with fed-batch fermentation. Here, we also provide a basis for new testable hypotheses regarding the enhancement of thymidine productivity and the attainment of a more complete understanding of nucleotide metabolism in bacteria.     

Cloning and Heterologous Expression of the Thioviridamide Biosynthesis Gene Cluster from Streptomyces olivoviridis

Thioviridamide is a unique peptide antibiotic containing five thioamide bonds from Streptomyces olivoviridis. Draft genome sequencing revealed a gene (the tvaA gene) encoding the thioviridamide precursor peptide. The thioviridamide biosynthesis gene cluster was identified by heterologous production of thioviridamide in Streptomyces lividans.     

LC-MS/MS-based metabolic profiling of Escherichia coli under heterologous gene expression stress

Escherichia coli is frequently exploited for genetic manipulations and heterologous gene expression studies. We have evaluated the metabolic profile of E. coli strain BL21 (DE3) RIL CodonPlus after genetic modifications and subjecting to the production of recombinant protein. Three genetically variable E. coli cell types were studied, normal cells (susceptible to antibiotics) cultured in simple LB medium, cells harboring ampicillin-resistant plasmid pET21a (+), grown under antibiotic stress, and cells having recombinant plasmid pET21a (+) ligated with bacterial lactate dehydrogenase gene grown under ampicillin and standard isopropyl thiogalactoside (IPTG)-induced gene expression conditions. A total of 592 metabolites were identified through liquid chromatography-mass spectrometry/mass spectrometry analysis, feature and peak detection using XCMS and CAMERA followed by precursor identification by METLIN-based procedures. Overall, 107 metabolites were found differentially regulated among genetically modified cells. Quantitative analysis has shown a significant modulation in DHNA-CoA, p-aminobenzoic acid, and citrulline levels, indicating an alteration in vitamin K, folic acid biosynthesis, and urea cycle of E. coli cells during heterologous gene expression. Modulations in energy metabolites including NADH, AMP, ADP, ATP, carbohydrate, terpenoids, fatty acid metabolites, diadenosine tetraphosphate (Ap4A), and l -carnitine advocate major metabolic rearrangements. Our study provides a broader insight into the metabolic adaptations of bacterial cells during gene manipulation experiments that can be prolonged to improve the yield of heterologous gene products and concomitant production of valuable biomolecules.     

Studies on the expression of linalool synthase using a promoter-β-glucuronidase fusion in transgenic Artemisia annua

Artemisinin, an antimalarial endoperoxide sesquiterpene, is synthesized in glandular trichomes of Artemisia annua L. A number of other enzymes of terpene metabolism utilize intermediates of artemisinin biosynthesis, such as isopentenyl and farnesyl diphosphate, and may thereby influence the yield of artemisinin. In order to study the expression of such enzymes, we have cloned the promoter regions of some enzymes and fused them to β-glucuronidase (GUS). In this study, we have investigated the expression of the monoterpene synthase linalool synthase (LIS) using transgenic A. annua carrying the GUS gene under the control of the LIS promoter. The 652 bp promoter region was cloned by the genome walker method. A number of putative cis-acting elements were predicted indicating that the LIS is driven by a complex regulation mechanism. Transgenic plants carrying the promoter-GUS fusion showed specific expression of GUS in T-shaped trichomes (TSTs) but not in glandular secretory trichomes, which is the site for artemisinin biosynthesis. GUS expression was observed at late stage of flower development in styles of florets and in TSTs and guard cells of basal bracts. GUS expression after wounding showed that LIS is involved in plant responsiveness to wounding. Furthermore, the LIS promoter responded to methyl jasmonate (MeJA). These results indicate that the promoter carries a number of cis-acting regulatory elements involved in the tissue-specific expression of LIS and in the response of the plant to wounding and MeJA treatment. Southern blot analysis indicated that the GUS gene was integrated in the A. annua genome as single or multi copies in different transgenic lines. Promoter activity analysis by qPCR showed that both the wild-type and the recombinant promoter are active in the aerial parts of the plant while only the recombinant promoter was active in roots. Due to the expression in TSTs but not in glandular trichomes, it may be concluded that LIS expression will most likely have little or no effect on artemisinin production.     

Molecular cloning of resistance genes and architecture of a linked gene cluster involved in biosynthesis of oxytetracycline by Streptomyces rimosus

Summary The isolation of mutants of Streptomyces rimosus which were blocked in oxytetracycline (OTC) production was described previously. The genes for the early steps of antibiotic biosynthesis mapped together. Genomic DNA fragments of S. rimosus which conferred resistance to OTC and complemented all of these non-producing mutants have been cloned. The cloned DNA is physically linked within approximately 30 kb of the genome of S. rimosus. The gene cluster is flanked at each end by a resistance gene each of which, independently, can confer resistance to the antibiotic. In OTC-sensitive strains of S. rimosus, the entire gene cluster including both resistance genes has been deleted. Complementation of blocked mutants by cloned DNA fragments in multi-copy vectors was often masked by a secondary effect of switching off antibiotic productions in strains othersise competent to produce OTC. This adverse effect on OTC production was not observed with recombinants using low copy-number vectors.     

Expression of various genes to enhance ubiquinone metabolic pathway in Agrobacterium tumefaciens

Ubiquinone (UQ), a lipid-soluble component, acts as a mobile component of the respiratory chain by playing an essential role in the electron transport system in many organisms, and has been widely used in pharmaceuticals due to its antioxidant property. The biosynthesis of UQ involves 10 sequential reactions brought about by various enzymes. In this study, dps gene, which encodes decaprenyl diphosphate synthase, involved in ubiquinone biosynthesis from Agrobacterium tumefaciens, and coq2 gene of Saccharomyces cerevisiae, ppt1 gene of Schizosaccahromyces pombe and ubiA gene of Escherichia coli, all of them encoding 4-hydroxybenzoate:polyprenyl diphosphate (4-HB:PPP) transferase, were reconfigured into an operon under the control of a single promoter to yield various plasmids including pBIV-dps, pBIV-dpsq, pBIV-dpsp and pBIV-dpsca. The recombinant A. tumefaciens containing dps-ubiC-ubiA gene showed the highest level ubiquinone production than that of the other recombinants and the nonrecombinant bacterium. In an aerobic fed-batch fermentation, A. tumefaciens containing the pBIV-dpsca plasmid produced 25.2 mg of ubiquinone-10 per liter which was 1.68 times higher than that of nonrecombinant type. While in microaerobic fed-batch fermentation, recombinant cell pBIV-dpsca produced 30.8 mg L⁻¹ of ubiquinone-10. Compared to the original A. tumefaciens, the ubiquinone-10 yield and productivities of the recombinant bacterium pBIV-dpsca increased 88.9% and 77.7%, respectively, under microaerobic fed-batch conditions.     

Detecting positive selection from genome scans of linkage disequilibrium

Background

During the lifetime of a fermenter culture, the soil bacterium S. coelicolor undergoes a major metabolic switch from exponential growth to antibiotic production. We have studied gene expression patterns during this switch, using a specifically designed Affymetrix genechip and a high-resolution time-series of fermenter-grown samples.

Results

Surprisingly, we find that the metabolic switch actually consists of multiple finely orchestrated switching events. Strongly coherent clusters of genes show drastic changes in gene expression already many hours before the classically defined transition phase where the switch from primary to secondary metabolism was expected. The main switch in gene expression takes only 2 hours, and changes in antibiotic biosynthesis genes are delayed relative to the metabolic rearrangements. Furthermore, global variation in morphogenesis genes indicates an involvement of cell differentiation pathways in the decision phase leading up to the commitment to antibiotic biosynthesis.

Conclusions

Our study provides the first detailed insights into the complex sequence of early regulatory events during and preceding the major metabolic switch in S. coelicolor, which will form the starting point for future attempts at engineering antibiotic production in a biotechnological setting.     

Pleiotropic regulatory genes bldA,adpA and absB are implicated in production of phosphoglycolipid antibiotic moenomycin

Unlike the majority of actinomycete secondary metabolic pathways, the biosynthesis of peptidoglycan glycosyltransferase inhibitor moenomycin in Streptomyces ghanaensis does not involve any cluster-situated regulators (CSRs). This raises questions about the regulatory signals that initiate and sustain moenomycin production. We now show that three pleiotropic regulatory genes for Streptomyces morphogenesis and antibiotic production—bldA, adpA and absB—exert multi-layered control over moenomycin biosynthesis in native and heterologous producers. The bldA gene for tRNA^Leu_UAA is required for the translation of rare UUA codons within two key moenomycin biosynthetic genes (moe), moeO5 and moeE5. It also indirectly influences moenomycin production by controlling the translation of the UUA-containing adpA and, probably, other as-yet-unknown repressor gene(s). AdpA binds key moe promoters and activates them. Furthermore, AdpA interacts with the bldA promoter, thus impacting translation of bldA-dependent mRNAs—that of adpA and several moe genes. Both adpA expression and moenomycin production are increased in an absB-deficient background, most probably because AbsB normally limits adpA mRNA abundance through ribonucleolytic cleavage. Our work highlights an underappreciated strategy for secondary metabolism regulation, in which the interaction between structural genes and pleiotropic regulators is not mediated by CSRs. This strategy might be relevant for a growing number of CSR-free gene clusters unearthed during actinomycete genome mining.     

Identification and functional characterization of phenylglycine biosynthetic genes involved in pristinamycin biosynthesis in Streptomyces pristinaespiralis

Pristinamycin I (PI), a streptogramin type B antibiotic produced by Streptomyces pristinaespiralis, contains the aproteinogenic amino acid l-phenylglycine. Recent sequence analysis led to the identification of a set of putative phenylglycine biosynthetic genes. Successive inactivation of the individual genes resulted in a loss of PI production. Production was restored by supplementation with externally added l-phenylglycine, which demonstrates that these genes are involved in phenylglycine biosynthesis and thus probably disclosing the last essential pristinamycin biosynthetic genes. Finally, a putative pathway for phenylglycine synthesis is proposed.