Evaluation of Streptomyces coelicolor A3(2) as a heterologous expression host for the cyanobacterial protein kinase C activator lyngbyatoxin A.
Filamentous marine cyanobacteria are extremely rich sources of bioactive natural products and often employ highly unusual biosynthetic enzymes in their assembly. However, the current lack of techniques for stable DNA transfer into these filamentous organisms, combined with the absence of heterologous expression strategies for nonribosomal cyanobacterial gene clusters, prohibit the creation of mutant strains or the heterologous production of these cyanobacterial compounds in other bacteria. In this study, we evaluated the capability of a derivative of the model actinomycete Streptomyces coelicolor A3(2) to express enzymes involved in the biosynthesis of the protein kinase C activator lyngbyatoxin A from a Hawaiian strain of Moorea producta (previously classified as Lyngbya majuscula). Despite large differences in GC content between these two bacteria and the presence of rare TTA/UUA leucine codons in lyngbyatoxin ORFs we were able to achieve expression of the cytochrome P450 monooxygenase LtxB and reverse prenyltransferase LtxC in S. coelicolor M512 and confirmed the in vitro functionality of S. coelicolor overexpressed LtxC. Attempts to express the entire lyngbyatoxin A gene cluster in S. coelicolor M512 were not successful because of transcript termination observed for the ltxA gene, which encodes a large nonribosomal peptide synthetase. However, these attempts did show a detectable level of cyanobacterial promoter recognition in Streptomyces. Successful expression of lyngbyatoxin A proteins in Streptomyces provides a new platform for biochemical investigation of natural product enzymes from Moorea strains.
Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA 92093-0212, USA.
Adam C
AC
Jones
Sabine
S
Ottilie
Alessandra S
AS
Eustáquio
Daniel J
DJ
Edwards
Lena
L
Gerwick
Bradley S
BS
Moore
William H
WH
Gerwick
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001688:Biological Products / Q000737:chemistry / Q000378:metabolism; D000458:Cyanobacteria / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008235:Lyngbya Toxins / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D005810:Multigene Family; D016366:Open Reading Frames; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D048372:Streptomyces coelicolor / Q000235:genetics* / Q000378:metabolism*
D001426:Bacterial Proteins; D001688:Biological Products; D008235:Lyngbya Toxins; D011493:Protein Kinase C; C019457:lyngbyatoxin A