Involvement Of The AprC,aprD Genes Related Of Apramycin Biosynthesis In Streptomyces Tenebrarius H6

The 4.0 kb BglII-BamHI fragment, upstream fragment of the apramycin resistant gene, was subcloned and sequenced. Analysis of the nt sequence revealed that there was no ORP in the fragment. It perhaps was the end of the biosynthetic gene cluster of apramycin.The 4.8 kb fragment, downstream fragment of the apramycin resistant gene, was subcloned and sequenced. Analysis of the nt sequence revealed that it contained two complete ORFs: ORFC and ORFD; and an incomplete ORF: ORFF. It was found by BLAST analysis that ORFC designated as aprC was homologous to trehalose and maltose hydrolase or trehalose and maltose phosphrylase, and ORFD designated as aprD was homologous to UDP-glucose-4-epimerase. No homologous sequence of ORFF was found, so ORFF was supposed to be a new gene, designated as aprF.A fragment, containing 5. tenebrarius DNA, downstream fragment of the apramycin resistant gene, and reporting genes of ermE and xylE ,was inserted into plasmid pHZ132 (an E.coli-Streptomyces shuttleplasmid incorporating ori T from RK2) to construct disruption plasmidpCS304-2. The plasmid was transferred into E.coli ET12567 (pUZ8002) to construct recombinant E.coli ET12867 (pUZ8002, pCS304-2). Recombinant S. tenebrarius H6-42-4 was obtained by conjugation of E.coli ET12867(pUZ8002, pCS304-2) and S.tenebrarius H6 to screen strains whose plasmids had been integrated into S.tenebrarius H6 chromosomal DNA. The gene recombinant strain H6-42-4 couldn't generate apramycin, which indicated that the cloned genes aprC and aprDapramycin, which indicated that the cloned genes aprC and aprD were involved in apramycin biosynthesis in S.tenebrarius H6.The tobramycin producing strain H6-42-4 obtained by gene recombination was mutanted by NTG and NTG combined with tobramycin tolerance. A high-yielding mutant was screened out. The total titer was increased to 2095u/ml and the percentage of Carbamoyl-tobraycin (CTB) was increased to 91.9%.