Protein expression analysis of a high-demeclocycline producing strain of Streptomyces aureofaciens and the roles of CtcH and CtcJ in demeclocycline biosynthesis

Abstract

Background: Streptomyces aureofaciens strain A6-9, obtained with traditional mutagenesis, produces elevated levels of 6-DCT. The increased formation of 6-DCT may be attributable to the changes in the expression of some proteins in the 6-DCT biosynthetic pathway. For this reason, we explored the differences in protein expression between A6-9 and wild-type (WT) strains of Streptomyces aureofaciens, and based on the differences (CtcH and CtcJ were overexpressed in A6-9), investigated the roles of CtcH and CtcJ in biosynthesis.Results: Two-dimensional gel electrophoresis and a Mascot search indicated that some enzymes (including CtcH and CtcJ) involved in the primary and secondary metabolism were more strongly expressed in the high-6-DCT-yielding strain A6-9 than in the WT strain DT1. To examine the roles of CtcH and CtcJ in 6-DCT biosynthesis, ctcH-deleted, ctcJdeleted, ctcH-overexpressing, and ctcJ-overexpressing mutants and a mutant overexpressing both ctcH and ctcJ were constructed. Compared with WT, 6-DCT production was 50 and 37 % higher in the ctcH-overexpressing and ctcJ-overexpressing strains, respectively, and increased by 60 % in the ctcH-ctcJ-overexpressing strain. The ctcH-deleted and ctcJ-deleted strains produced almost no 6-DCT. Analysis of the metabolic flux distribution indicated that ctcH encodes a hydroxyacyl-CoA dehydrogenase and ctcJ encodes a monooxygenase that are essential for 6-DCT biosynthesis. Conclusion:Protein expression differs between high-6-DCT-yielding and WT strains, and the enzymes increased in the high-6-DCT-yielding strain explain the increased 6-DCT production. ctcH encodes a hydroxyacyl-CoA dehydrogenase and ctcJ encodes a monooxygenase that are essential for 6-DCT biosynthesis.

Increasing Demeclocycline Production in Streptomyces aureofaciens by Manipulating the Expression of a Novel SARP Family Regulator and Its Genes

“…DMCTC has become an essential antibiotic against a wide range of gram-positive and gram-negative bacteria and an industrial material for the production of semisynthetic TCs for use in cases of clinical resistance. Although some methods have been developed to improve DMCTC production, such as "random mutagenesis and screening" procedures and overexpression of key pathway genes [6] or the 6-demethylchlortetracycline biosynthetic gene cluster [34], most approaches have limitations. However, selection of an appropriate starting strain is crucial.…”

“…The two fragments were ligated into HindIII and BamHI/EcoRI-digested pOJ260-neo to generate the ctcK deletion vector pOJ260-ctcK, which was introduced into S. aureofaciens CT1. Kanamycin-sensitive strains were selected as described previously [6], and the deletion strain ∆ctcK generated by double crossover recombination was confirmed by PCR analysis using primers Kan-F and Kan-R.…”

“…After incubation, the broth was adjusted to pH 1.8-2.0 with oxalic acid to release the antibiotics from the cells. HPLC was performed as previously described [6]. For high-resolution mass measurements, an Agilent 1200 series LC/MSD trap system was used in tandem with a 6530 accurate-mass quadrupole time-of-flight (Q-TOF) mass spectrometer with an electrospray ionization source (100-1000 m/z mass range, positive mode).…”

“…Yu Lan reported that overexpression of the mini-PKS gene in an S. rimosus strain (M4018) and in an industrial overproducer (SR16) resulted in 51.2% and 32.9% improvement in OTC production [28]. Another study showed that overexpression of ctcH, which encodes cyclase in the core structure of TC, increased the production of DMCTC by 50% [6]. However, the transcriptional levels of structural genes (ctcX and ctcM) were lower in the mutant strain than in the WT strain.…”

“…It is currently used clinically as a prescription medication to treat susceptible bacterial infections and sustained hyponatremia in patients with syndrome of inappropriate antidiuretic hormone secretion (SIADH). Although DMCTC has been the subject of several biochemical studies, reports of DMCTC biosynthesis are limited [6]. Based on the understanding of the regulatory network of secondary metabolites and the molecular mechanism of biosynthesis in Streptomyces, genetic engineering may be an effective tool to increase the yield of target products, including DMCTC.…”