Mechanisms And Engineering Of Validamycin And Salinomycin Overproduction

Secondary metabolites are chemical compounds produced by microorganisms?especially Streptomyces?in late exponential growth stage,which can be widely used in healthcare,agriculture,and food industry.Secondary metabolites are synthesized via secondary metabolism,which shows a coupled relationship with primary metabolism.The metabolic pathways of primary metabolism are not only essentially supporting cellular growth,but also supply precursors and energy for secondary metabolism.Herein,the primary metabolism in actinobacteria was engineered for the titer improvement of antibiotics.Part One.Attenuating primary metabolism for the enhancement of antibiotic productionValidamycin A,a basic C₇N aminocyclitol antibiotics produced by Streptomyces hygroscopicus 5008,has been widely used as an antifungal agent against rice sheath blight disease in China and other Eastern Asian countries.In addition,validamycin A?VAL-A?can also be used as the source material for the chemical synthesis of voglibose,an important antidiabetic drug.The previously isolated 5008 strain produces 3-5 g/L VAL-A in a rice-peanut based fermentation medium.However,multiple rounds of random mutagenesis and selection resilted in a high-yielding industrial strain called TL01,producing 20-30 g/L VAL-A with the same fermentation condition.The genomes of VAL-A producers,5008 and TL01,had been recently sequenced.Although TL01 genome was highly similar to that of 5008 with respect to gene content and organization,it is 302.6 kb smaller with three large regions deleted?Det-I,Det-?,and Det-??near the left chromosome termini.Subsequently,the mutants with individual or combined deletions of the three large regions in 5008 were obtained.Compared with wild-type 5008?4.7g/L?,the production of VAL-A in mutants was enhanced,while the cellular growth was decreased.Therefore,we supposed that the attenuation of primary metabolism could lead to the enhancement of antibiotic production in actinobacteria.Based on this hypothesis,genes shjg115,shjg1601,shjg1609,and shjg1617 within Det-I and Det-? regions are selected as the candidates,which are putatively involved in primary metabolism.The deletion of these genes in5008 led to an expected enhanced VAL-A titer and reduced cellular growth.The metabolomic technique was used to compare the biochemical profiles between the wild-type 5008 and its derived mutants.Compared with the wild-type,the concentrations of pyruvate and?-oxoglutarate were decreased,while the concentration of heptulose 7-phosphate was enhanced in these deletion mutants.The results of metabolomics indicated that the large deletions redirected the carbon flux from EMP to PPP,and the attenuated primary metabolism lead to the reduced cellular growth and increased VAL-A production.To examine the applicability of this hypothesis,we further applied the engineering strategy to erythromycin A producer Saccharopolyspora erythraea NRRL2338,lincomycin A producer Streptomyces lincolnensis LC-G,gentamicin producer Micromonospora echinospora ATCC15838,and avermectin producer Streptomyces avermitilis 76-5.The productions of erythromycin A,lincomycin A,and gentamicin were respectively increased by 68%,50%and 49%via gene deletion.The titer of avermectin B1a was successfully increased to 1.48 g/L via tripledeletion of genes gltA,sdh,and suc in S.avermitilis 76-5.However,the avermectin B1a titer and the cellular growth were both decreased by quadrupledeletions of genes gltA,sdh,suc,and pyk,indicating that certain amount of biomass is an important prerequisite for the maintainance of high yield of antibiotics.These two strains both harbor a linear plasmid pSHJG1 and a circular plasmid pSHJG2.Intriguingly,the linear plasmid pSHJG1 in TL01 shows a lower expression compared with that in 5008 via the microarray assay,and the relationship between the expression of pSHJG1 and the overproduction of VAL-A remained unknown.Therefore,we developed a strategy to cure the linear plasmid in 5008 and TL01,and the titer of VAL-A was correspondingly increased by 43.7%and 12.5%,while the cellular growth of these two strains was both reduced.Subsequently,the plasmid-cured mutant 5008?pSHJG1 was complemented with four structure genes from pSHJG1,putatively involved in primary metabolism.Among them,a putative hydrolase gene pshjg1.69 and an ATPase gene pshjg1.72 restored the cellular growth and VAL-A titer.Part Two.Functional genomics for salinomycin overproductionSalinomycin,a member of polyether antibiotics,has been widely used in veterinary medicine and animal husbandry as food additive and growth promoter for30 years.In addition,salinomycin has recently been identified as a potential agent to inhibit leukemia stem cell and epithelial cancer stem cells.It is produced by the Streptomyces albus DSM 41398 and its derivatives.The complete genome sequence of DSM 41398 was determined by 454 GS-FLX sequencing and pair-end library sequencing.It is particularly interesting to note that,in the genome of DSM 41398,the percentage of genes involved in lipid metabolism was the highest among the sequenced Streptomyces genomes,and there are 48 genes involved in bean-oil catabolism pathways.Moreover,bean-oil addition significantly induced the transcription of genes fadD,acd,paa,fadJ and fadA.However,the percentage of genes related to carbohydrate transport and metabolism was the lowest among the sequenced Streptomyces genomes,which agreed well with the high salinomycin productivity in oil-rich media.Additionally,there are at least 35 putative gene clusters in the genome of DSM41398 as predicted by antiSMASH,and seven of them are actively expressed type-I PKS/PKS-NRPS gene clusters.The deletion of PKS-NRPS-2 or PKS-6 significantly enhanced salinomycin production.However,a combined deletion of them showed no further improvement.Whereas the concentrations of malonyl-CoA and methylmalonyl-CoA were increased in the mutants,the concentration of ethylmalonyl-CoA remained low.A native crotonyl-CoA reductase gene?ccr?was introduced into the combined deletion mutant,and the concentration of ethylmalonyl-CoA was increased,resulted in an improved salinomycin production by15.80%.Therefore,the combined deletion of PKS-NRP-2/PKS-6 and the over-expression of ccr gene led to an overall titer improvement of salinomycin from0.60 to 6.60 g/L.When the ccr gene was introduced into a high-yielding strain BK3-25,the production of salinomycin was enhanced from 11.5 to 14.3 g/L.In conclusion,we clarified the mechanism for validamycin and salinomycin overproduction via functional genomics,transcriptomics,and metabonomics.Based on these“-omics”analysis,we carried out experiments for targeted engineering of validamycin and salinomycin overproduction.The strategies described here are helpful for improving the production of many other antibiotics.