Evaluation of Streptomyces coelicolor A3(2) as a heterologous expression host for the cyanobacterial protein kinase C activator lyngbyatoxin A

Abstract

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.

Engineering cyanobacteria for industrial products

“…Recent advances have shown that Streptomyces might serve as a suitable expression host for cyanobacterial PKS/NRPS genes (Jones et al, 2012) and, very recently, there has been a report that heterologous expression of the barbamide biosynthetic gene cluster from Moorea producens in Streptomyces venezuelae results in the production of a novel barbamide congener (Kim et al, 2012). Now that this milestone has been reached, it can be expected that research on heterologous expression of cyanobacterial metabolites will gain momentum.…”

Rapid construction of a Bacterial Artificial Chromosomal (BAC) expression vector using designer DNA fragments

“…Streptomyces are well known for their capability to produce a large variety of secondary metabolites, and the availability of abundant precursors needed for natural products biosynthesis in these strains is a great advantage of Streptomyces hosts. Furthermore, many Streptomyces strains are easy to be genetically manipulated, and genomic sequences of various Streptomyces strains are available, making Streptomyces the optimal hosts for heterologous expression (Baltz, 2010;Gomez-Escribano and Bibb, 2014;Jones et al, 2012;Zhang et al, 2008).…”

“…Streptomyces strains have been employed as one of the most commonly used hosts for heterologous expression of natural bioactive compounds (Jones et al, 2012;Zhang et al, 2008). Streptomyces are well known for their capability to produce a large variety of secondary metabolites, and the availability of abundant precursors needed for natural products biosynthesis in these strains is a great advantage of Streptomyces hosts.…”

Cyanobacteria: Promising biocatalysts for sustainable chemical production

“…Because of their metabolic similarity to plants and capacity for production of NPs, cyanobacteria are attractive production platforms for highvalue NPs including isoprenoids and those synthesized by non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS). In contrast, heterotrophic expression of NRPS, PKS, or cytochrome P450s from plants or cyanobacteria is challenging due to genetic and biochemical incompatibilities (93)(94)(95)(96). Furthermore, the biosynthesis of plant terpenoids often relies on cytochrome P450 monooxygenases that use NADPH, a cofactor that is in abundance in cyanobacteria but not in heterotrophs (97).…”