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.

Figure 1

Valienamine-containing C₇N Aminocyclitols and Proposed Biosynthetic Pathways to VAL-And Acarbose. (A) Chemical structures of VAL-A, acarbose and pyralomicin 1a. Dashed-boxed regions show the cyclitol moiety shared by all three compounds. (B) Previously proposed biosynthetic pathways to VAL-A [9] and acarbose [14, 15].

Figure 2

Cloning Strategy for Sequencing and Genetic Organization of val Gene Cluster. (A) Cloning strategy for sequencing of the 45 kb region. B: BamHI; C: ClaI; E: EcoRI. (B) Genetic organization of the val gene cluster. Genes proposed to be involved in validamycin biosynthesis are indicated as black arrows. The previously sequenced 6.0 kb BamHI fragment was indicated by double-arrowed line.

Figure 3

Heterologous Production of Validoxylamine A and VAL-A (A) Reassembly of valABCKLMN and MS analysis (MS₁). PermE*, the un-mutated promoter of the erythromycin resistance gene; E, EcoRI; Fragmentation of validoxylamine A (m/z 336.1) yields validamine (m/z 178.1) (MS₂). (B) Co-expression of valABCKLMN and valG and MS detection (MS₁). Fragmentation of VAL-A (m/z 498.2) generates validoxylamine A (m/z 336.1) and validamine (m/z 178.1) (MS₂).

Figure 4

Inactivation and complementation of glycosyltransferase valG. (A) Schematic representation of the replacement of an 806 bp internal fragment of valG with the 1.4 kb aac(3)IV. In shuttle plasmid pJTU609, aac(3)IV was inserted between the 2.4 kb and 1.0 kb genomic fragments originally flanking the deleted 806 bp region. While wild-type S. hygroscopicus should give a 2.1 kb PCR-amplified product, mutant LL-1 should yield a 1.5 kb product using a pair of primers, ValG2-F and ValG2-R. (B) PCR analysis of wild-type S. hygroscopicus and mutant LL-1. (C) Bioassay comparison between the wild-type (left), LL-1 (middle) and LL-101 (right). LL-101 is the derivative of LL-1 harboring shuttle plasmid pJTU612 with valG. (D) HPLC profiles of the standards, wild-type, LL-1 and LL-101. The retention time of VAL-A is 9.7 min and that of validoxylamine A is 6.5 min.

Figure 5

Heterologous Expression of valG and Characterization of the Glycosyltransferase. (A) SDS-PAGE analysis of ValG protein: 1. molecular weight marker, 2. soluble protein of extract of E. coli BL21Gold(DE3)pLysS/valG before induction, 3. total protein of extract of E. coli BL21Gold(DE3)pLysS/valG after induction with IPTG, 4. soluble protein of line 3, 5. purified his-tagged ValG. (B) TLC analysis of ValG reaction (silica gel, solvent system: nPrOH:AcOH:H₂O = 4:1:1); R: validoxylamine A standard, U: ValG reaction using UDP-glucose as glucosyl donor, G: ValG reaction using GDP-glucose as glucosyl donor, A: ValG reaction using ADP-glucose as glucosyl donor, T: ValG reaction using TDP-glucose as glucosyl donor. (C) Conversion scheme of validoxylamine A to VAL-A catalyzed by ValG.

Figure 6

Proposed Biosynthetic Pathway to VAL-A.