Improvement of Natamycin Production by Engineering of Phosphopantetheinyl Transferases in Streptomyces chattanoogensis L10

ABSTRACTPhosphopantetheinyl transferases (PPTases) are essential to the activities of type I/II polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) through converting acyl carrier proteins (ACPs) in PKSs and peptidyl carrier proteins (PCPs) in NRPSs from inactive apo-forms into active holo-forms, leading to biosynthesis of polyketides and nonribosomal peptides. The industrial natamycin (NTM) producer,Streptomyces chattanoogensisL10, contains two PPTases (SchPPT and SchACPS) and five PKSs. Biochemical characterization of these two PPTases shows that SchPPT catalyzes the phosphopantetheinylation of ACPs in both type I PKSs and type II PKSs, SchACPS catalyzes the phosphopantetheinylation of ACPs in type II PKSs and fatty acid synthases (FASs), and the specificity of SchPPT is possibly controlled by its C terminus. Inactivation of SchPPT inS. chattanoogensisL10 abolished production of NTM but not the spore pigment, while overexpression of the SchPPT gene not only increased NTM production by about 40% but also accelerated productions of both NTM and the spore pigment. Thus, we elucidated a comprehensive phosphopantetheinylation network of PKSs and improved polyketide production by engineering the cognate PPTase in bacteria.

CotA of Bacillus subtilis Is a Copper-Dependent Laccase

ABSTRACT The spore coat protein CotA of Bacillus subtilisdisplays similarities with multicopper oxidases, including manganese oxidases and laccases. B. subtilis is able to oxidize manganese, but neither CotA nor other sporulation proteins are involved. We demonstrate that CotA is a laccase. Syringaldazine, a specific substrate of laccases, reacted with wild-type spores but not with ΔcotA spores. CotA may participate in the biosynthesis of the brown spore pigment, which appears to be a melanin-like product and to protect against UV light.

New Sporulation Loci in Streptomyces coelicolor A3(2)

ABSTRACT Sporulation mutants of Streptomyces coelicolor appear white because they are defective in the synthesis of the grey polyketide spore pigment, and such white (whi) mutants had been used to define eight sporulation loci, whiA,whiB, whiD, whiE, whiG,whiH, whiI, and whiJ (K. F. Chater, J. Gen. Microbiol. 72:9–28, 1972; N. J. Ryding, Ph.D. thesis, University of East Anglia, 1995). In an attempt to identify new whi loci, we mutagenized S. coelicolor M145 spores with nitrosoguanidine and identified 770 mutants with colonies ranging from white to medium grey. After excluding unstable strains, we examined the isolates by phase-contrast microscopy and chose 115 whi mutants with clear morphological phenotypes for further study. To exclude mutants representing cloned whi genes, self-transmissible SCP2*-derived plasmids carrying whiA, whiB,whiG, whiH, or whiJ (but notwhiD, whiE, or whiI) were introduced into each mutant by conjugation, and strains in which the wild-type phenotype was restored either partially or completely by any of these plasmids were excluded from further analysis. In an attempt to complement some of the remaining 31 whi mutants, an SCP2* library of wild-type S. coelicolor chromosomal DNA was introduced into 19 of the mutants by conjugation. Clones restoring the wild-type phenotype to 12 of the 19 strains were isolated and found to represent five distinct loci, designated whiK,whiL, whiM, whiN, andwhiO. Each of the five loci was located on the ordered cosmid library: whiL, whiM, whiN, and whiO occupied positions distinct from previously clonedwhi genes; whiK was located on the same cosmid overlap as whiD, but the two loci were shown by complementation to be distinct. The phenotypes resulting from mutations at each of these new loci are described.

Developmental Regulation of Transcription ofwhiE, a Locus Specifying the Polyketide Spore Pigment in Streptomyces coelicolor A3(2)

ABSTRACT whiE is a complex locus that specifies the polyketide spore pigment in Streptomyces coelicolor A3(2). Two divergently oriented promoters, whiEP1 andwhiEP2, were identified in the whiE gene cluster, and their activities were analyzed during colony development in wild-type and sporulation-deficient strains. Both promoters were developmentally regulated; whiEP1 and whiEP2transcripts were detected transiently at approximately the time when sporulation septa were observed in the aerial hyphae, and transcription from both promoters depended on each of the six known “early”whi genes required for sporulation septum formation (whiA, -B, -G, -H, -I, and -J). Mutation of the late sporulation-specific sigma factor gene, sigF, had no effect on the activity of whiEP1 but blocked transcription fromwhiEP2. However, ςF-containing holoenzyme was not sufficient to direct transcription of whiEP2 in vitro. The whiEP2 promoter controls expression of whiEORFVIII, encoding a putative flavin adenine dinucleotide-dependent hydroxylase that catalyzes a late tailoring step in the spore pigment biosynthetic pathway. Disruption of whiE ORFVIII causes a change in spore color, from grey to greenish (T.-W. Yu and D. A. Hopwood, Microbiology 141:2779–2791, 1995). Consistent with these observations, construction of a sigF null mutant ofS. coelicolor M145 caused the same change in spore color, showing that disruption of sigF in S. coelicolor changes the nature of the spore pigment rather than preventing its synthesis altogether.

Influences of developmental genes on localized glycogen deposition in colonies of a mycelial prokaryote, Streptomyces coelicolor A3(2): a possible interface between metabolism and morphogenesis

Two spatially localized phases of glycogen accumulation were detected by electron microscopy after cytological staining of thin sections of Streptomyces coelicolor A3 (2) colonies. In phase I, glycogen granules were present in hyphae in the air—agar interface region of colonies that were undergoing aerial mycelium formation, though absent from aerial hyphae themselves. With one exception (a bldF mutant, which contained abundant glycogen), the absence of aerial mycelium caused by various developmental mutations ( bldA, bldB, bldC, bldD, bldG and bldH mutations) was associated with a virtual absence of detectable glycogen. Mutations that allow aerial hyphae to form but prevent or interfere with the septation needed for spore formation ( whiA,whiB, whiG, whiH and whil mutations) did not impair phase I deposition. In phase II, abundant glycogen granules were present in aerial hyphal tips during intermediate stages of sporulation, but disappeared as spores matured. Phase II glycogen accumulation was observed with bldA, bldC, bldD and bldG mutants grown with mannitol as carbon source — conditions that allowed normal aerial mycelium development and sporulation; but phase I deposition was still at a very low level in these colonies. Glycogen was also deposited in the coiling tips of aerial hyphae of whiA , whiB, whiH and whil mutants, and sporadic clusters of granules were present throughout whiG colonies. Significantly, glycogen was deposited in spore chains that developed ectopically in the normally sporeand glycogen-free substrate mycelium when multiple copies of whiG were present. Overall, the two phases of glycogen synthesis (and degradation) appear to be under separate developmental control rather than being mainly responsive to external growth conditions. Phase II glycogen levels were particularly high in a whiE mutant defective in spore pigment biosynthesis, and particularly low when hyper-pigmentation was induced by additional copies of the whiE genes. Spore pigment may therefore be a major sink for carbon stored as glycogen during sporulation. The possibility is discussed that, in addition to supplying carbon and energy at particular locations, glycogen synthesis and degradation may also play a part in morphogenesis by influencing turgor pressure.