| Spiramycin is a 16-membered macrolide antibiotic produced by Streptomyces ambofaciens.It is a strong inhibitor of microbial growth and is widely used in the treatment of oral and otolaryngological infections.In this project,we established a strategy to select high spiramycin-producing strains under nutrient stress by adding N-acetylglucosamine(GlcNAc)to the solid medium,investigated the reasons for high spiramycin production of strain S.ambofaciens SA-10 through transcriptomic and metabolomic analysis,and explained the mechanism of GlcNAc’s effect on the regulation of secondary metabolism of the bacterium.Firstly,by simulating a nutrient-rich growth environment,different concentrations of GlcNAc(5-20 g/L)were added to the solid medium of S.ambofaciens,it was found that the spore production of S.ambofaciens was significantly hindered,and the morphological differences between colonies were analyzed by electron microscopy.Also,colonies sporulating under the inhibition of GlcNAc were selected,and these strains exhibited better spiramycin synthesis ability.The strains with higher spiramycin production were obtained with the addition of 10 g/L GlcNAc,and the average titer increased by 19.4%compared to the control(17070 U/mL),reaching 20383 U/mL.A high spiramycin-producing strain,S.ambofaciens SA-10,was obtained on the plates with addition of 10 g/L GlcNAc as the screening condition.By flask fermentation,the spiramycin titer reached to 21925 U/mL,which was 29.58%higher than that of the control.To investigate the mechanism of higher spiramycin yield of this strain,the fermentation parameters of strain SA-10 as well as the parent strain were analyzed,and the two strains showed significant differences in the rate of spiramycin synthesis,pH,bacterial concentration,sugar consumption rate,and amino nitrogen concentration.Metabolomic and transcriptomic studies were used to analyze the possible mechanism through the following three aspects;gene transcription levels,intracellular metabolite changes,and metabolic pathw-ays.The metabolomic analysis showed the levels of D-glucose-6-phosphate,succinate and D-ribose-5phosphate were higher in strain SA-10,while the levels of dihydroxyacetone phosphate,aspartate,isoleucine and O-succinyl-L-homoserine in the amino acid pathway;tetradecanoic acid and pentadecanoic acid in the fatty acid pathway were less in strain SA-10.The transcriptomic analysis showed up-regulation of genes related to glycolysis and pentose phosphate in the high yielding strain,down-regulation of genes related to TCA cycle,upregulation of genes related to oxidative phosphorylation pathway,down-regulation of transcriptional repressor dasR,and up-regulation of transcription of genes in spiramycin biosynthesis gene cluster.The above results indicated that the high-producing strain SA-10,obtained by GlcNAc screening strategy,had enhanced glycolytic pathway,pentose phosphate pathway,pyruvate metabolism,fatty acid degradation,and amino acid metabolic pathway,which provided more reducing power and acyl coenzyme A for spiramycin production and promoted the synthesis of spiramycin.To further determine that the increased spiramycin production of high yielding strains was associated with the down-regulation of dasR gene expression,targeted knockdown of dasR was constructed by CRISPR-Cas9 technology using the pCRISPomyces-2 plasmid to investigate the regulatory role of DasR in S.ambofaciens.The dasR deletion mutant strain was not obtained because the plasmid needed more optimization in various aspects,but the fermentation results showed that the empty plasmid pCRISPRomyces-2 had no effect on the production of spiramycin,which laid the foundation for the subsequent application of CRISPR-Cas9 technology in S.ambofaciens. |
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