Genome-Wide Screening and Identification of Factors Affecting the Biosynthesis of Prodigiosin by Hahella chejuensis , Using Escherichia coli as a Surrogate Host

Abstract

A marine bacterium, Hahella chejuensis, recently has attracted attention due to its lytic activity against a red-tide dinoflagellate. The algicidal function originates from its red pigment, prodigiosin, which also exhibits immunosuppressive or anticancer activity. Genome sequencing and functional analysis revealed a gene set contained in the hap gene cluster that is responsible for the biosynthesis of prodigiosin. To screen for the factors affecting the prodigiosin biosynthesis, we constructed a plasmid library of the H. chejuensis genomic DNA, introduced it into Escherichia coli strains harboring the hap cluster, and observed changes in production of the red pigment. Among the screened clones, hapXY genes whose products constitute a two-component signal transduction system were elucidated as positive regulators of the pigment production. In addition, an Hfqdependent, noncoding region located at one end of the hap cluster was confirmed to play roles in regulation. Identification of factors involved in the regulation of prodigiosin biosynthesis should help in understanding how the prodigiosin-biosynthetic pathway is organized and controlled and also aid in modulating the overexpression of prodigiosin in a heterologous host, such as E. coli, or in the natural producer, H. chejuensis.Harmful algal blooms (HABs), commonly called red tide, are a phenomenon in which toxin-producing marine algae rapidly proliferate in the offshore area. The HAB-causing phytoplanktons are reported to interact with other organisms such as bacteria and fungi. Among them, the marine bacteria are known to play important roles in decreasing or developing HABs (3,5,14). For instance, Hahella chejuensis, isolated from the coastal area of Marado in South Korea (15), is capable of killing Cochlodinium polykrikoides (12). C. polykrikoides is a major microalga that causes HABs, especially in the Northeast Pacific coastal area (8). The bacterial determinant that kills C. polykrikoides was further characterized as a red pigment referred to as prodigiosin (12). Prodigiosin belongs to a family of tripyrrole antibiotic molecules called prodiginines, which have potential as anticancer agents or immunosuppressants (24). The prodigiosin congener isolated from H. chejuensis also exerts an immunosuppressive effect (11). Through completed genome sequencing of H. chejuensis and its functional analysis, the genomic region involved in biosynthesis of prodigiosin was elucidated (12). This complete set of prodigiosin-biosynthetic genes was named the hap gene cluster. The red pigment prodigiosin was further characterized structurally, and the biosynthetic pathway was proposed by Kim and colleagues (13,14). Genes of the hap cluster share homology with those in the pig cluster and the red cluster which are involved in prodiginine-biosynthetic intermediates of Serratia marcescens and Streptomyces coelicolor, respectively (7,23,25). Enzymes encoded by the genes in the pig and red clusters have been characterized (24). However, gene expression of the hap cluster can be...

Asymmetric Stetter reactions catalyzed by thiamine diphosphate-dependent enzymes

“…The characteristic red color of prodigiosin has also been used for the identification and optimization of a heterologous production strain. The H. chejuensis hap cluster (see above and Kim et al 2007Kim et al , 2008 has been expressed in a recombinant E. coli strain (Kwon et al 2010). Drepper and coworkers have successfully expressed the S. marcescens pig cluster (in total 15 genes) heterologously in Pseudomonas putida using the TREX technology (Loeschcke et al 2013).…”

Efficient recombinant production of prodigiosin in Pseudomonas putida

“…2.4 g/L ( Su et al, 2011 ), and 15.6 g/L ( Chen et al, 2013 ). Successful heterologous prodigiosin production has so far been shown in Erwinia carotovora ( Thomson et al, 2000 ) and E. coli ( Kwon et al, 2010 ). Although absolute yields were not in the focus of these studies prodigiosin production in E. coli can be estimated by the given data (A 534 /OD 600 ) to be in the range of 10 mg/gDCW.…”

“…So far, heterologous prodigiosin production at mg-scale could only be established in Escherichia coli by expressing the biosynthetic genes from Hahella chejuensis ( Kwon et al, 2010 ). In addition, we have recently identified the GRAS (generally recognized as safe) certified strain Pseudomonas putida KT2440 as a promising prodigiosin producer ( Loeschcke et al, 2013 ) in the context of validating a newly developed system for the transfer and expression of clustered genes (TREX).…”

Utilizing the algicidal activity of aminoclay as a practical treatment for toxic red tides

“…Other management strategies include the application of zooplankton to graze on bloom species – a form of top-down control. This biological approach also faces obstacles: It is complicated by the need for mass culturing of zooplankton and the substantial cost of field trials1011121314, and so far large-scaled applicability of this method has been limited1516.…”