Functional Analysis of the Validamycin Biosynthetic Gene Cluster and Engineered Production of Validoxylamine A

Abstract

A 45 kb DNA sequencing analysis from Streptomyces hygroscopicus 5008 involved in validamycin A (VAL-A) biosynthesis revealed 16 structural genes, 2 regulatory genes, 5 genes related transport, transposition/integration or tellurium resistance; another 4 genes had no obvious identity. The VAL-A biosynthetic pathway was proposed, with assignment of the required genetic functions confined to the sequenced region. A cluster of eight reassembled genes was found to support VAL-A synthesis in a heterologous host, S. lividans 1326. In vivo inactivation of the putative glycosyltransferase gene (valG) abolished the final attachment of glucose for VAL production and resulted in accumulation of the VAL-A precursor, validoxylamine, while the normal production of VAL-A could be restored by complementation with valG. The role of valG in the glycosylation of validoxylamine to VAL-A was demonstrated in vitro by enzymatic assay.

Characterization of the tunicamycin gene cluster unveiling unique steps involved in its biosynthesis

“…Interestingly, the tunL homolog was missing in the potential gene cluster of A. mirum DSM 43827, which may partly account for the lack of tunicamycin production by this strain. In addition, the sequence homologies among the three gene clusters were relatively low (Table S3) compared with the gene clusters of validamycin, which was produced by several Streptomyces, for example (Yu et al, 2005;Bai et al, 2006;Jian et al, 2006;Singh et al, 2006). Remarkably, there are no discernable nucleotide homologies in two tunicamycin gene clusters of S. chartreusis and S. clavuligerus (Table S3).…”

Deciphering Pactamycin Biosynthesis and Engineered Production of New Pactamycin Analogues

“…[15,16] This vector contains a thiostrepton resistance (Thio R ) gene and an OriT transfer element required for conjugation. Since S. pactum has much greater sensitivity to apramycin than to thiostrepton, a 1 kb aac(3)IV apramycin resistance gene along with a multiple cloning site isolated from pTMN002 (see Table S1 in the Supporting Information) was A C H T U N G T R E N N U N G introduced into the BamHI site of pHZ1358 to generate pTMW018.…”

Enhanced validamycin production and gene expression at elevated temperature in Streptomyces hygroscopicus subsp. jingangensis 5008

“…Two putative regulatory genes, valP and valQ, are located downstream of valG encoding a glycosyltransferase. They are transcribed in the same direction and overlap for 4 bp (Figure 1(b)), suggesting functional correlation and forming an operon [13] . The 201-aa ValQ shows 38% identity/51% similarity with the N-terminal 186 bp of SCO3723 from S. coelicolor, a segment possibly acting as signal transduction histidine kinase.…”

“…The 201-aa ValQ shows 38% identity/51% similarity with the N-terminal 186 bp of SCO3723 from S. coelicolor, a segment possibly acting as signal transduction histidine kinase. Interestingly, the 293-aa ValP has the highest identity (49%) with the C-terminal 292 bp of SCO3723, which putatively has σ B -related phosphatase activity, a sigma factor responding to external stresses such as osmosis and temperature [13] . The presence of a GAF domain at the N-terminus of ValP suggests that it may respond to the cGMP level of the cell at elevated temperature [14] .…”