Dataset Information

Genes associated with 2-methylisoborneol biosynthesis in cyanobacteria: isolation, characterization, and expression in response to light.

ABSTRACT: The volatile microbial metabolite 2-methylisoborneol (2-MIB) is a root cause of taste and odor issues in freshwater. Although current evidence suggests that 2-MIB is not toxic, this compound degrades water quality and presents problems for water treatment. To address these issues, cyanobacteria and actinomycetes, the major producers of 2-MIB, have been investigated extensively. In this study, two 2-MIB producing strains, coded as Pseudanabaena sp. and Planktothricoids raciborskii, were used in order to elucidate the genetic background, light regulation, and biochemical mechanisms of 2-MIB biosynthesis in cyanobacteria. Genome walking and PCR methods revealed that two adjacent genes, SAM-dependent methyltransferanse gene and monoterpene cyclase gene, are responsible for GPP methylation and subsequent cyclization to 2-MIB in cyanobacteria. These two genes are located in between two homologous cyclic nucleotide-binding protein genes that may be members of the Crp-Fnr regulator family. Together, this sequence of genes forms a putative operon. The synthesis of 2-MIB is similar in cyanobacteria and actinomycetes. Comparison of the gene arrangement and functional sites between cyanobacteria and other organisms revealed that gene recombination and gene transfer probably occurred during the evolution of 2-MIB-associated genes. All the microorganisms examined have a common origin of 2-MIB biosynthesis capacity, but cyanobacteria represent a unique evolutionary lineage. Gene expression analysis suggested that light is a crucial, but not the only, active regulatory factor for the transcription of 2-MIB synthesis genes. This light-regulated process is immediate and transient. This study is the first to identify the genetic background and evolution of 2-MIB biosynthesis in cyanobacteria, thus enhancing current knowledge on 2-MIB contamination of freshwater.

SUBMITTER: Wang Z

PROVIDER: S-EPMC3072421 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Genes associated with 2-methylisoborneol biosynthesis in cyanobacteria: isolation, characterization, and expression in response to light.

Wang Zhongjie Z Xu Yao Y Shao Jihai J Wang Jie J Li Renhui R

PloS one 20110407 4

The volatile microbial metabolite 2-methylisoborneol (2-MIB) is a root cause of taste and odor issues in freshwater. Although current evidence suggests that 2-MIB is not toxic, this compound degrades water quality and presents problems for water treatment. To address these issues, cyanobacteria and actinomycetes, the major producers of 2-MIB, have been investigated extensively. In this study, two 2-MIB producing strains, coded as Pseudanabaena sp. and Planktothricoids raciborskii, were used in o ...[more]

PMID: 21490938

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Project description:To identify the genes for biosynthesis of the off-flavor terpenoid alcohol, 2-methylisoborneol (2-MIB), the key genes encoding monoterpene cyclase were located in bacterial genome databases by using a combination of hidden Markov models, protein-family search, and the sequence alignment of their gene products. Predicted terpene cyclases were classified into three groups: sesquiterpene, diterpene, and other terpene cyclases. Genes of the terpene cyclase group that form an operon with a gene encoding S-adenosyl-l-methionine (SAM)-dependent methyltransferase were found in genome data of seven microorganisms belonging to actinomycetes, Streptomyces ambofaciens ISP5053, Streptomyces coelicolor A3(2), Streptomyces griseus IFO13350, Streptomyces lasaliensis NRRL3382R, Streptomyces scabies 87.22, Saccharopolyspora erythraea NRRL2338, and Micromonospora olivasterospora KY11048. Among six microorganisms tested, S. ambofaciens, S. coelicolor A3(2), S. griseus, and S. lasaliensis produced 2-MIB but M. olivasterospora produced 2-methylenebornane (2-MB) instead. The regions containing monoterpene cyclase and methyltransferase genes were amplified by PCR from S. ambofaciens, S. lasaliensis, and Saccharopolyspora erythraea, respectively, and their genes were heterologously expressed in Streptomyces avermitilis, which was naturally deficient of 2-MIB biosynthesis by insertion and deletion. All exoconjugants of S. avermitilis produced 2-MIB. Full-length recombinant proteins, monoterpene cyclase and methyltransferase of S. lasaliensis were expressed at high level in Escherichia coli. The recombinant methyltransferase catalyzed methylation at the C2 position of geranyl diphosphate (GPP) in the presence of SAM. 2-MIB was generated by incubation with GPP, SAM, recombinant methyltransferase, and terpene cyclase. We concluded that the biosynthetic pathway involves the methylation of GPP by GPP methyltransferase and its subsequent cyclization by monoterpene cyclase to 2-MIB.

Project description:In cyanobacteria, alkanes are synthesized from a fatty acyl-ACP by two enzymes, acyl-acyl carrier protein reductase and aldehyde deformylating oxygenase. Despite the great interest in the exploitation for biofuel production, nothing is known about the transcriptional organization of their genes or the physiological function of alkane synthesis. The comparison of 115 microarray datasets indicates the relatively constitutive expression of aar and ado genes. The analysis of 181 available genomes showed that in 90% of the genomes both genes are present, likely indicating their physiological relevance. In 61% of them they cluster together with genes encoding acetyl-CoA carboxyl transferase and a short-chain dehydrogenase, strengthening the link to fatty acid metabolism and in 76% of the genomes they are located in tandem, suggesting constraints on the gene arrangement. However, contrary to the expectations for an operon, we found in Synechocystis sp. PCC 6803 specific promoters for the two genes, sll0208 (ado) and sll0209 (aar), which give rise to monocistronic transcripts. Moreover, the upstream located ado gene is driven by a proximal as well as a second, distal, promoter, from which a third transcript, the ~160 nt sRNA SyR9 is transcribed. Thus, the transcriptional organization of the alkane biosynthesis genes in Synechocystis sp. PCC 6803 is of substantial complexity. We verified all three promoters to function independently from each other and show a similar promoter arrangement also in the more distant Nodularia spumigena, Trichodesmium erythraeum, Anabaena sp. PCC 7120, Prochlorococcus MIT9313, and MED4. The presence of separate regulatory elements and the dominance of monocistronic mRNAs suggest the possible autonomous regulation of ado and aar. The complex transcriptional organization of the alkane synthesis gene cluster has possible metabolic implications and should be considered when manipulating the expression of these genes in cyanobacteria.

Dataset Information

Genes associated with 2-methylisoborneol biosynthesis in cyanobacteria: isolation, characterization, and expression in response to light.

Publications

Genes associated with 2-methylisoborneol biosynthesis in cyanobacteria: isolation, characterization, and expression in response to light.

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