Dissertation > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)

Genome and Functional Gene Research for Streptomyces Rimosus M4018

Author TangZhenYu
Tutor ZhangSiLiang; GuoMeiJin
School East China University of Science and Technology
Course Biochemical Engineering
Keywords Streptomyces rimosus genome secondary metabolites oxytetracycline redox sensor
Type PhD thesis
Year 2012
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Streptomyces are a group of soil-inhibating, aerobic and filamentous gram-positive bacteria, and it is the largest genus of Actinobacteria and the type genus of the family Streptomycetaceae belongs to the prokaryotes actinomycetales streptomycetaceae. Streptomyces play important roles in the life and activities of human beings. Over two-thirds of the clinically useful antibiotics are produced by Streptomyces. The aromatic polyketide antibiotic oxytetracycline (OTC) is produced by Streptomyces rimosus as an important secondary polyketide metabolite, which belongs to the kind of tetracycline antibiotics. OTC finds particularly heavy use in aquaculture and displays broad-spectrum activity against both Gram-positive and Gram-negative pathogens. So it is still wildly used in the antibiotic market. Since the S.rimosus genome has not been sequenced and published before, in this study, we first sequenced the whole genome and investigated some of the functional genes.1. Genome sequencing, annotation and assembly of S. rimosusThe nucleotide sequence was determined using a 454 GS FLX sequencer and Illumina Solexa sequencer. Assembly was performed using several methods, it produced 4 supercontigs. Finally we obtained the S. rimosus M4018 draft genome of 9.2 Mb distributed in 4 supercontgs with a GC content of 71.96%. The draft genome consists of one linear chromosome with 7744 protein-coding genes (CDSs). Among the CDSs,615 hypothetical proteins have no match to any known proteins in the datebases. The genome contains 6 rRNAs,66 tRNAs and 1 tmRNA gene.Meanwhile,8 genes encoded the gas vesicle proteins and two phage gene clusters (RP2 and RP3) were found in the S. rimosus chromosome. Furthermore, S. rimosus M4018 has an intact Embden-Meyerhof-Parnas (EMP) pathway, pentose phosphate pathway (PPP) and tricarboxylic acid cycle (TCA). The complete primary metabolite pathway can not only meet the needs of physiological activities but also provide the primary metabolites for the secondary metabolite pathway, and produce many kinds of bioactive secondary metabolites. Genome analysis revealed at least 23 genes related to secondary metabolites synthase/synthetase, including some involved in PKS and NRPS pathway. By the multilocus phylogenetic analysis, we found that S. rimosus might represent a distinct evolutionary lineage and the species was positioned at the edge of the Streptomyces clade.2. The revolutionary analysis of wild type strain G7, producer strain 23383 and M4018The wild type strain S. rimosus G7 and the producer strain S. rimosus 23383 were further sequenced by Solexa technique. By comparing with the S. rimosus M4018 data, there were 78 of single-nucleotide polymorphisms (SNPs) in S. rimosus G7 and 615 of SNPs in S. rimosus 23383. Among these SNPs, there were no SNPs, insertions or deletions found in the OTC biosynthesis cluster. While in S. rimosus 23383, some SNPs were identified in the primary metabolites related genes. These minor changes cound be responsible for the change of OTC production, since the primary metabolism can provide the energy, precursors and reducing power for the secondary metabolism.3. The effect of glucose-6-phosphate dehydrogenase (G6PDH) on the OTC productionGlucose-6-phosphate dehydrogenase (G6PDH) encoded by zwf1 and zwf2 is the first enzyme in oxiditative pentose phosphate pathway (PPP) and responsible for the NADPH generation. Disruption of zwf1 or zwf2 resulted in a higher producton of OTC (50 mg gDCW-1 day-1 and 40 mg gDCW-1 day-1, repectively). The disruption strain has an increased carbon flux through the glycolysis and a decreased carbon flux through PP pathway, as measured by the enzyme activities of G6PDH and phosphoglucose isomerase (PGI), also by the levels of ATP, establishing G6PDH as a key player in determining the carbon flux distribution. The increased production of OTC appeared largely because of the generation of more malonyl-CoA, one of OTC precursors, as experinmentally observed in the disruption mutants. In the zwf1 or zwf2 disruption mutants, the AATP/ANADPH ratio was 11.4 or 7.8 respectively, which was higher than that of the wild type strain (4.1). So, particular attention has been drawn to alter carbon flux by redirecting the central primary metabolic networks toward specific parts of the metabolism. In this case, we need further to find an optimal proportion between glycolysis (combined tricarboxylic acid cycle) and PP pathway for the maximum production. Furthermore, our research will have an impact on the antibiotics industry.4. rex gene cloning, Redox molecular sensor construction and applicationRex is a NADH/NAD+ redox sensor, which can be used to regulate the gene expression.In order to enable microorganisms to manifest their intracellular oxygen levels we constructed a genetic sensor circuitry which converts signals impinging on the cellular redox balance into a reporter gene expression readout. Based on the newly found Streptomyces rimosus redox control system, consisting of Rex modulating ROP-containing promoters in a NADH-dependent manner, we designed an Escherichia coli sensor transcription control system, which constitutes a Rex transactivator (REDOX) able to bind and activate promoters. When oxygen levels were high and resulted in depleted NADH pools, Rex-specific target promoter (cydPl) driven expression of secreted (Green fluorscent protein, GFP) reporter gene was low as a consequence of increased Rex-ROP affinity. Conversely, at hypoxic conditions leading to high intracellular NADH levels, strongly reduced Rex-ROP interaction and increased GFP expression in E. coli cells. The sensor capacity (oxygen levels) of redox system enabled monitoring of the population’s metabolic state in vivo. Our research will not only help to understand the molecular mechanism of the Rex family but also foster advances in biosensor development.

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