Identification And Engineering Of Regulation-related Genes Toward Improved Kasugamycin Production

Kasugamycin,produced by Streptomyces kasugaensis and Streptomyces microaureus,is an important amino-glycoside antibiotic.Kasugamycin is widely used in Asia and South America for veterinary and agricultural applications.The basis of the selective toxicity of kasugamycin is its inhibitory activity on protein biosynthesis by binding to the 30 S ribosomal subunits.The gene cluster for kasugamycin biosynthesis had been identified and sequenced from the producer S.kasugaensis,and the putative biosynthetic pathway of kasugamycin had been proposed.However,the research on kasugamycin biosynthesis and its regulation remained very limited.In this work,we identified four additional regulation-related genes in the the left flanking region of the previously reported kasugamycin gene cluster,including two-component system kasW and kasX,MerR-family regulator kasV,and isoprenylcysteine carboxyl methyltransferase kasS.We identified the function of these genes by individual gene inactivation together with transcriptional analysis,both in the low-yielding S.kasugaensis BCRC12349(LY)and high-yielding S.microaureus XM301(HY).Previous research revealed that KasT is a putative regulatory protein and could directly interact with several putative promoter regions within the kas cluster,suggesting KasT might be a pathway-specific transcriptional regulator for kasugamycin biosynthesis.Sequence analysis revealed that kasT is a putative pathway-specific regulator and shares high homology with the strR transcriptional regulators from the streptomycin gene cluster.For the first time,we have established an efficient conjugation and gene replacement system in S.kasugaensis BCRC12349(LY)and obtain the kasT mutant strain CCZ1.HPLC analysis of the fermentation extract of CCZ1 clearly showed that kasugamycin production was abolished.Moreover,kasugamycin biosynthesis was restored in the kasT complementation strain CCZ6.Transcriptional analysis indicated that kas biosynthetic genes were not transcribed in CCZ1,whereas that of the four regulatory genes was increased.In addition,overexpressing kasT dramatically increased kasugamycin production by almost 3-folds.This result proved that KasT is essentially required to activate the transcription of kas genes and plays the role of the pathway-specific transcriptional regulator for kasugamycin biosynthesis.Sequence analysis showed that KasW/KasX probably form a two-component system.Deletion of kasW or kasX improved kasugamycin production by 12% or 19%.RT-PCR analysis demonstrated that the transcription of kas genes was significantly increased by 4-8 folds.These results suggested that kasW/X is negative transcriptional regulator for kasugamycin biosynthesis.Gene kasV shows 38% identity with TipA protein from Streptomyces lividans.The deletion mutant of kasV produced approximately 194% more kasugamycin,from 0.68 g/L to 2 g/L,with a dramatically enhanced transcription of kas genes for 4-8 folds,implying that kasV plays a pivotal negative regulatory role in kasugamycin biosynthesis in LY.Gene kasS is in the same operon as kasV and shows similarity with the isoprenylcysteine carboxyl methyltransferase(ICMT).Kasugamycin production was increased by 22% in kasS mutant strain,from 0.68 g/L of LY strain to 0.76 g/L.The transcription of kas genes were increased to 3-9 folds.These results suggested that kasS is negatively but indirectly involved in the regulation of kas genes and the production of kasugamycin.The results in low-yielding strain verified that engineering of the regulatory genes could promisingly improve kasugamycin production.Similar gene inactivation was performed in the high-yielding strain S.microaureus XM301(HY).As expected,the deletion of kasW/X resulted in a 58% increase of the yield from 6 to 9.5 g/L.However,the deletion of kasV and over-expression of kasT had no obvious effect,and the disruption of kasS surprisingly decreased kasugamycin production,which was opposite with the deletion effect in LY.In addition,trans-complementation of the kasS mutant with a TTA codon-mutated kasS increased the kasugamycin yield by 20%.Then we compared the transcription of genes in kasugamycin biosynthetic cluster in LY and HY strains.Indeed the transcription of regulation-related genes kasW,kasX,kasV,and kasS was increased by 40-100 folds,and the biosynthetic genes kasR-F was significantly increased by 37 folds in HY strain,which may partially account for the discrepancy of gene inactivation effects between them.This suggested that regulation may not be the main limiting element for titer increase and other factors alternatively become significant in HY strain.Our work not only generated engineered strains with improved kasugamycin yield,but also pointed out that different strategies on manipulating regulatory-related genes should be considered for low-yielding or high-yielding strains.