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Enhancing the stress tolerance and virulence of an entomopathogen by metabolic engineering of dihydroxynaphthalene melanin biosynthesis genes.

ABSTRACT: Entomopathogenic fungi have been used for biocontrol of insect pests for many decades. However, the efficacy of such fungi in field trials is often inconsistent, mainly due to environmental stresses, such as UV radiation, temperature extremes, and desiccation. To circumvent these hurdles, metabolic engineering of dihydroxynaphthalene (DHN) melanin biosynthetic genes (polyketide synthase, scytalone dehydratase, and 1,3,8-trihydroxynaphthalene reductase genes) cloned from Alternaria alternata were transformed into the amelanotic entomopathogenic fungus Metarhizium anisopliae via Agrobacterium-mediated transformation. Melanin expression in the transformant of M. anisopliae was verified by spectrophotometric methods, liquid chromatography/mass spectrometry (LC/MS), and confocal microscopy. The transformant, especially under stresses, showed notably enhanced antistress capacity and virulence, in terms of germination and survival rate, infectivity, and reduced median time to death (LT50) in killing diamondback moth (Plutella xylostella) larvae compared with the wild type. The possible mechanisms in enhancing the stress tolerance and virulence, and the significance and potential for engineering melanin biosynthesis genes in other biocontrol agents and crops to improve antistress fitness are discussed.

SUBMITTER: Tseng MN

PROVIDER: S-EPMC3127726 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Enhancing the stress tolerance and virulence of an entomopathogen by metabolic engineering of dihydroxynaphthalene melanin biosynthesis genes.

Tseng Min N MN Chung Pei C PC Tzean Shean S SS

Applied and environmental microbiology 20110513 13

Entomopathogenic fungi have been used for biocontrol of insect pests for many decades. However, the efficacy of such fungi in field trials is often inconsistent, mainly due to environmental stresses, such as UV radiation, temperature extremes, and desiccation. To circumvent these hurdles, metabolic engineering of dihydroxynaphthalene (DHN) melanin biosynthetic genes (polyketide synthase, scytalone dehydratase, and 1,3,8-trihydroxynaphthalene reductase genes) cloned from Alternaria alternata were ...[more]

PMID: 21571888

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Project description:1,8-Dihydroxynaphthalene (1,8-DHN) is a key intermediate in the biosynthesis of DHN melanin, which is specific to fungi. In this study, we characterized the enzymatic properties of the gene products of an operon consisting of soceCHS1, bdsA, and bdsB from the Gram-negative bacterium Sorangium cellulosum Heterologous expression of soceCHS1, bdsA, and bdsB in Streptomyces coelicolor caused secretion of a dark-brown pigment into the broth. High-performance liquid chromatography (HPLC) analysis of the broth revealed that the recombinant strain produced 1,8-DHN, indicating that the operon encoded a novel enzymatic system for the synthesis of 1,8-DHN. Simultaneous incubation of the recombinant SoceCHS1, BdsA, and BdsB with malonyl-coenzyme A (malonyl-CoA) and NADPH resulted in the synthesis of 1,8-DHN. SoceCHS1, a type III polyketide synthase (PKS), catalyzed the synthesis of 1,3,6,8-tetrahydroxynaphthalene (T4HN) in vitro T4HN was in turn converted to 1,8-DHN by successive steps of reduction and dehydration, which were catalyzed by BdsA and BdsB. BdsA, which is a member of the aldo-keto reductase (AKR) superfamily, catalyzed the reduction of T4HN and 1,3,8-tetrahydroxynaphthalene (T3HN) to scytalone and vermelone, respectively. The stereoselectivity of T4HN reduction by BdsA occurred on the si-face to give (R)-scytalone with more than 99% optical purity. BdsB, a SnoaL2-like protein, catalyzed the dehydration of scytalone and vermelone to T3HN and 1,8-DHN, respectively. The fungal pathway for the synthesis of 1,8-DHN is composed of a type I PKS, naphthol reductases of the short-chain dehydrogenase/reductase (SDR) superfamily, and scytalone dehydratase (SD). These findings demonstrated 1,8-DHN synthesis by novel enzymes of bacterial origin.IMPORTANCE Although the DHN biosynthetic pathway was thought to be specific to fungi, we discovered novel DHN synthesis enzymes of bacterial origin. The biosynthesis of bacterial DHN utilized a type III PKS for polyketide synthesis, an AKR superfamily for reduction, and a SnoaL2-like NTF2 superfamily for dehydration, whereas the biosynthesis of fungal DHN utilized a type I PKS, SDR superfamily enzyme, and SD-like NTF2 superfamily. Surprisingly, the enzyme systems comprising the pathway were significantly different from each other, suggesting independent, parallel evolution leading to the same biosynthesis. DHN melanin plays roles in host invasion and adaptation to stress in pathogenic fungi and is therefore important to study. However, it is unclear whether DHN biosynthesis occurs in bacteria. Importantly, we did find that bacterial DHN biosynthetic enzymes were conserved among pathogenic bacteria.

Project description:Alternaria brassicicola is a successful saprophyte and necrotrophic plant pathogen. Several A. brassicicola genes have been characterized as virulence factors affecting pathogenesis of Brassica species. To study regulatory mechanisms of pathogenesis, we mined 421 genes in silico encoding putative transcription factors in a machine-annotated, draft genome sequence of A. brassicicola. Targeted gene disruption mutants for 114 of the genes with proteins predicted to contain at least one putative zinc-finger domain were produced and functionally analyzed. Six of these genes were associated with pathogenesis. Disruption mutants corresponding to five of the genes were ≥50% less virulent than the wild type. Unexpectedly, the mutants of one gene (designated Amr1) were 100% more virulent. Amr1 is a homolog of Cmr1, a transcription factor previously found to regulate melanin biosynthesis in several fungi. Gene deletion mutants (Δamr1) were created and their phenotypes characterized. The Δamr1 mutants utilized pectin as a carbon source more efficiently than the wild type, were melanin-deficient, and more sensitive to UV light and glucanase digestion. The Amr1 protein was localized in the nuclei of hyphae and in highly melanized conidia during the late stage of plant pathogenesis. RNA-seq analysis revealed that three genes in the melanin biosynthesis pathway, along with the deleted Amr1 gene, were not expressed in the mutants. In contrast, many hydrolytic enzyme-coding genes were expressed at much higher levels in the mutants than in the wild type during pathogenesis. The results of this study suggested that only a small number of transcription factors with zinc finger domains are needed to maintain strong virulence. Furthermore, a gene important for survival in nature negatively affected virulence, probably by less efficient use of pectin. We speculate that the functions of the Amr1 gene are important to the success of A. brassicicola as a competitive saprophyte and plant parasite gene expression profile comparisons between wild type and a transcription factor mutant during the late stage of host infection

Dataset Information

Enhancing the stress tolerance and virulence of an entomopathogen by metabolic engineering of dihydroxynaphthalene melanin biosynthesis genes.

Publications

Enhancing the stress tolerance and virulence of an entomopathogen by metabolic engineering of dihydroxynaphthalene melanin biosynthesis genes.

Similar Datasets

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