| Validamycin A (abbreviated as VAL-A), a basic C7N aminocyclitol-containing antibiotic, hasbeen widely used as an antifungal agent against rice sheath blight disease. Meanwhile, itstransformation product valienamine is a precursor for the synthesis of voglibose, a highly effectivedrug for insulin-independent diabetes. VAL-A has been produced on large scale in China by thederivatives of Streptomyces hygroscopicus var. jinggangensis5008, which was isolated in1970sfrom Jinggang Mountain, China. Intriguingly, a higher fermentation temperature (37oC) for strain5008, rather than30oC for the normal Streptomyces growth, rendered remarkably increasedVAL-A yield. At the end of the fermentation,2g/L VAL-A was produced by5008. With recursivephysical and chemical mutagenesis, a derived producer TL01could produce18g/L VAL-A inflask and even30g/L in industrial fermentor. The significant yield difference between these twoclosely related strains provides a perfect system for the study on antibiotic overproduction ongenome-scale.Part One. Complete genome sequencing of validamycin-producing Streptomyceshygroscopicus var. jinggangensis5008The genome of strain5008was sequenced by454GS FLX sequencer. Plasmid library with6-8kb inserts and fosmid library were constructed, and end-sequenced to provide contig linkageinformation. Gaps were filled by primer walking, sub-cloning, or multiplex PCR. Also, the telomeresequences were individually determined. Except for the linear chromosome (10,145,833bp), strain5008also harbors a164,566-bp linear plasmid and a73,282-bp circular plasmid as detected bypulsed-field or conventional agarose gel electrophoresis.Compared with other Streptomyces genomes,5008chromosome has a smaller core region and shorter terminal inverted repeats (14bp), encodes more α/β hydrolases, major facilitatorsuperfamily transporters, and Mg2+/Mn2+-dependent phosphatases. It carries twenty-ninesecondary metabolite gene clusters putatively for the biosynthesis of polyketides (PKS),non-ribosomal peptides (NRPS), hybrid PKS-NRPSs, terpenoids, and lantibiotics, among whichthat of validamycin and cyclothiazomycin were experimentally confirmed. Also, we reconstructedthe primary metabolic pathways supplying precursors of carbon (sedoheptulose7-phosphate andUDP-glucose) and nitrogen (glutamate) for VAL-A biosynthesis. Interestingly, two genes encodeUDP-glucose-1-phosphate uridylyltransferases (Ugp) were found in the genome, whereas onlyone copy of ugp gene is present in other Streptomyces genomes.Part Two. Transcriptome analysis of thermo-regulated biosynthesis of validamycin inStreptomyces hygroscopicus var. jinggangensis5008We compared the transcriptome of strain5008cultured at30C or37C in a liquid mediumusing DNA microarray. Based on the statistical criteria of more than two-fold change and p<0.05,1,542differentially expressed genes (DEGs) were identified at37C compared with at30C.Additionally, the transcriptome of S. avermitilis NRRL8165cultivated at the same conditionswere analyzed in parallel to filter transcriptional changes common to Streptomyces genus. Filteredwith the DNA microarray dataset from NRRL8165, the number of DEGs in5008was reduced to1,405, and subsequently to359using more stringent criteria of more than four-fold change andp<0.01. The markedly down-regulated DEGs at37C are mostly assigned with functions for aminoacid transport and metabolism and inorganic ion transport and metabolism.Except for the glucosyltransferase gene valG, transporter gene valH, and two-componentregulatory genes valPQ, most of the VAL-A biosynthetic genes were transcriptionally enhanced at37C. Furthermore, other three gene clusters were also up-regulated at37C. In primarymetabolism, key enzymes for glycolysis were transcriptionally down-regulated at37C, including6-phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase. Also,gluconokinase and citrate lyase had notably enhanced transcription, which could in turn increasecarbon flux to pentose-phosphate pathway and decrease TCA cycle. Moreover, glutamine synthetase GlnA and its positive regulator GlnR were down-regulated at37C. However,glutamate dehydrogenase GdhA for converting2-oxoglutarate into L-glutamate was up-regulatedat37C, supplying more amino group for VAL-A biosynthesis. Using an amino acid analyzer,concentration of intracellular glutamate at30C was about10times of that at37C, while theconcentration of free glutamine was nearly the same. The dramatic drop of glutamateconcentration and synchronic accumulation of VAL-A suggested most of the glutamate wasconsumed for VAL-A biosynthesis at37C.Among22markedly differential expressed regulators at37C, a SARP-family regulator(SHJG0322) was most highly expressed. At30C, VAL-A yield of SHJG9322–deleted mutantJG27was similar to the amount produced by strain5008. At37C, the yield of VAL-A in mutantJG27was only20%of strain5008. Transcription of valA and valK of strain5008were increasedby100-fold and26-fold at37C than at30C, respectively. However, that of valA and valK at37Cwere dropped to6-fold in mutant JG27. Furthermore, we complemented mutant JG27withSHJG0322under the control of PermE*promoter or its native promoter. Similar amount ofVAL-A was produced in both complemented strains at37C, which accounted for88.6%and81.6%of strain5008, respectively. It can be concluded that SHJG0322is a positive regulatorinvolved in the thermo-regulation of VAL-A biosynthesis.Part Three. Comparative ‘Omics’ for validamycin overproductionWe further determined the genomic sequence of industrial producer TL01. Compared withstrain5008, TL01has302.6-kb deletion of three large regions at its chromosomal left arm. Thesethree regions are about79kb,144kb,80kb in length, together coding for234CDSs including18regulators,16transport/membrane proteins,28hydrolases,61transposases, and56hypotheticalproteins. Each region was deleted in strain5008to investigate its involvement in validamycinproduction. Differently enhanced VAL-A yield was obtained in each mutant (7.5g/L,7.6g/L, or13.9g/L). Furthermore, a mutant with all three regions deleted reached the highest productivity(14.9g/L), corresponding to80%of the yield in TL01, revealing critical contribution on thevalidamycin overproduction. In addition,33insertions/deletions and90single nucleotide variations (SNVs) were identifiedbetween the two genomes, affecting78functional genes,24hypothetical genes, and7transposaseseither in CDSs or upstream regions. The78functional genes encode2key enzymes forvalidamycin biosynthesis,18transcriptional factors,39enzymes for related primary metabolism,8transport/membrane proteins, and so on. Firstly, the valK-valA intergenic region is1174bp in TL01,shorter than that (1311bp) in5008, accounting for a137-bp deletion. Secondly, seven regulatorygenes were selectively deleted in5008. VAL-A yield of the mutants was increased to differentextent (5.6-13.3g/L), indicating that genetic variations of key transcriptional factors probably alterthe metabolic network in strain TL01. Thirdly, several key enzymes (citrate synthase,homogentisate1,2-dioxygenase, and acetyl/propionyl-CoA carboxylase) for primary metabolismwere mutated in TL01probably leading to a reduced metabolic rate of TCA cycle.Furthermore, we compared the transcriptome of strain5008and TL01cultivated in yeastextract-malt extract-glucose (YMG) medium and rice-peanut cake based industrial medium (IND).Pair-wise comparisons were performed, respectively identifying980,1,754,5,336and4,774differential expressed genes (DEGs). Irrespective of medium change,282genes consistentlyshowed significant expression in strain TL01than in strain5008, which merely contains terpeneand cyclothiazomycin biosynthetic gene clusters. Primary metabolic genes encodingfructose-1,6-bisphosphatase and phosphoenolpyruvate carboxyl kinase, key enzymes involved ingluconeogenesis pathway, were up-regulated by5.1-fold and16.8-fold in average. As expected,most of131significantly down-regulated genes were encoded by the three large-deletion regionsand linear plasmid. Intriguingly, we found a regional organization of gene expression in thechromosome of strain TL01relative to strain5008only cultured in IND medium. Approximately1.9-Mb subtelomeric segment spanning the region from the chromosomal left end to Det-III leftflanking sequence was transcriptionally down-regulated in TL01except for the gene clusters forVAL-A and a type II PKS biosynthesis. Moreover, all genes encoded by the linear plasmid weretranscribed at very low levels in TL01. Independent of strain variation,3749common DEGs wereidentified in IND medium compared with in YMG medium. Validamycin biosynthetic genes showed much more significantly increased transcripts in IND cultures of both strains. Intriguing,the majority of genes encoded by linear plasmid and circular plasmid were significantlyup-regulated in IND medium.It was implicated that TL01has certain differential gene expressions to influence carbohydrateand energy metabolisms for validamycin overproduction. Alternatively, when culturing the strainsusing the IND medium rather than YMG, more energy was supplied not only for the activation ofvalidamycin biosynthesis, but also for efficient replication of indigenous plasmid.Part Four.‘Omics’-aided forward engineering towards validamycin overproductionBased on these Omic analyses, we carried out an initial experiment to targeted engineering ofstrain TL01. On one hand, we deleted a1.2-Mb region using Cre-loxP site-specific recombinationsystem, and the mutant had no improved yield. However, it produced more biomass, exhibiting amore vigorous metabolism. On the other hand, curing of the linear plasmid was performed instrain5008cultivated at high temperature with SDS. VAL-A titer of the plasmid-cured mutantswas significantly improved. Likewise, we carry out the curing of linear plasmid in TL01, and theVAL-A titer was increased by20.5%from18.4g/L in TL01to22.2g/L in the mutant.In summary, a comprehensive illustration of the molecular basis for validamycinoverproduction was obtained through genome sequencing, comparative genomics, andtranscriptome analysis as well as genetic manipulation. Based on these analyses, we performedinitial attempts for targeted engineering for validamycin overproduction, also defined as ‘forwardengineering’. These studies will pave the way for validamycin productivity improvement andindustrial bioprocess optimization. The strategy applied here is helpful for improving theproduction of many other antibiotics. |
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