| Dethylchlortetracycline is a natural tetracyclic antibiotic produced by S.aureofaciens.It has a broad spectrum and mainly used as an intermediate to synthesize new tetracyclic antibiotics to solve the problem of bacterial resistance.This research studied the ctcK(structural gene),ctcB(regulatory gene)and ctcC(resistance gene)at the molecular level in S.aureofaciens.The gene deletion mutants and overexpression mutants were constructed by in-frame knockout and overexpression,and analyzed mutants on 6-DCT biosynthesis to reveal the gene function and construct engineering strains with high yield of 6-DCT.According to the structure difference of CTC and 6-DCT is C6 methyl group,we analyzed that ctcK of CTC synthesis gene cluster encodes C6 methylase.We constructed a new 6-DCT-producing mutant by knockouting ctcK in CT1.HPLC and LC-MS showed that the△ctcK mutant accumulated 376 μg/mL 6-DMT,432μg/mL 6-DCT,and little CTC compared with 3765 μg/mL CTC and 230 μg/mL TC produced by CT1.The CctcK mutant resumed the production of 328 μg/mL CTC and 421 μg/mL TC,which firmly demonstrated that the function of ctcK.The qRT-PCR showed that the ctcV expression of the △ctcK mutant was higher than CT1,we concluded the △ctcK mutant produced MP to result little antibiotics.According to ctcB is SARP family regulator of the ctc synthesis gene cluster,we constructed ctcB gene deletion mutant △ctcB and overexpression mutant ctcB+in DT1.Compared with the 6-DCT yield of DT1,the △ctcB mutant decreased from 3578 μg/mL to 1568 μg/mL,while the ctcB+mutant increased to 5068 μg/mL,we conclued that ctcB positively regulates the 6-DCT yield.The qRT-PCR showed that ctcB could improve the transcription level of ctcH,ctcN,ctcQ,ctcC,ctcG and ctcV of the synthetic gene cluster,which reveales the regulation mechanism of ctcB.Then the characteristics of DNA binding region of CtcB protein were analyzed by bioinformatics prediction.According to the functional prediction of ctcC resistance gene,we constructed ctcC overexpression strain ctcC+in DT1,whose 6-DCT was 9%higher than DT1 to 3818 μg/mL.The qRT-PCR showed that the expression of ctcC in ctcC+mutant is 2.2 times than DT1,which indicates that the increase of 6-DCT production in ctcC+ mutant may caused by overexpression of ctcC. |
