Analysis and functional expression of NPP pathway-specific regulatory genes in Pseudonocardia autotrophica.
Using the genomics-guided polyene screening method, a rare actinomycetes called Pseudonocardia autotrophica was previously identified to contain functionally clustered nystatin-like biosynthetic genes and to produce a presumably novel polyene compound named nystatin-like Pseudonocardia polyene (NPP) (Kim et al., J Ind Microbiol Biotechnol 36:1425-1434, 2009). Since very low NPP productivity was observed in most P. autotrophica culture conditions, its biosynthetic pathway was proposed to be tightly regulated. Herein we report in silico analysis of six putative NPP pathway-specific regulatory genes present in its biosynthetic gene cluster, followed by functional overexpression of these regulatory genes in P. autotrophica. Three pathway-specific regulatory genes (nppRI, RIII, and RV) were predicted to belong to a typical LAL-type transcriptional family. Each regulatory gene was cloned under the strong constitutive ermE* promoter in a Streptomyces integrative pSET152 plasmid, followed by direct intergeneric conjugation from a plasmid-containing E. coli donor cell to P. autotrophica. While all the P. autotrophica exconjugants exhibited improved NPP productivity, the one containing nppRIII showed the highest NPP productivity improvement. In addition, culture-time-dependent analysis revealed that the nppRIII-stimulated NPP biosynthesis was more significant in the late exponential growth stage than in the stationary stage. Moreover, the P. autotrophica nppRIII-disruption mutant failed to produce NPP, with significantly reduced transcription levels of most npp biosynthetic genes. The results described suggest that identification and overexpression of key pathway-specific regulatory gene, followed by optimum harvest timing, should be critical factors to maximize the productivity of an intrinsically low-level metabolite such as NPP produced by rare actinomycetes species.
Department of Biological Engineering, Inha University, Incheon 402-751, Korea.
Ho-Geun
HG
Jeon
Jiyoon
J
Seo
Mi-Jin
MJ
Lee
Kyuboem
K
Han
Eung-Soo
ES
Kim
D000192:Actinomycetales / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D053898:Biosynthetic Pathways / Q000235:genetics; D005809:Genes, Regulator; D018942:Macrolides / Q000378:metabolism*; D008969:Molecular Sequence Data; D009761:Nystatin / Q000378:metabolism; D011090:Polyenes / Q000378:metabolism; D016415:Sequence Alignment
D001426:Bacterial Proteins; D018942:Macrolides; D011090:Polyenes; D009761:Nystatin
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