Journal of Microbiology

Journal Articles

Identification and heterologous reconstitution of a 5-alk(en)ylresorcinol synthase from endophytic fungus Shiraia sp. Slf14: Huiwen Yan , Lei Sun , Jinge Huang , Yixing Qiu , Fuchao Xu , Riming Yan , Du Zhu , Wei Wang , Jixun Zhan; J. Microbiol. 2018;56(11):805-812. Published online October 24, 2018; DOI: https://doi.org/10.1007/s12275-018-8278-x

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Abstract: A new type III polyketide synthase gene (Ssars) was discovered from the genome of Shiraia sp. Slf14, an endophytic fungal strain from Huperzia serrata. The intron-free gene was cloned from the cDNA and ligated to two expression vectors pET28a and YEpADH2p-URA3 for expression in Escherichia coli BL21(DE3) and Saccharomyces cerevisiae BJ5464, respectively. SsARS was efficiently expressed in E. coli BL21(DE3), leading to the synthesis of a series of polyketide products. Six major products were isolated from the engineered E. coli and characterized as 1,3-dihydroxyphenyl- 5-undecane, 1,3-dihydroxyphenyl-5-cis-6􍿁-tridecene,1,3-dihydroxyphenyl- 5-tridecane, 1,3-dihydroxyphenyl-5-cis-8􍿁- pentadecene, 1,3-dihydroxyphenyl-5-pentadecane, and 1,3- dihydroxyphenyl-5-cis-10􍿁-heptadecene, respectively, based on the spectral data and biosynthetic origin. Expression of SsARS in the yeast also led to the synthesis of the same polyketide products, indicating that this enzyme can be reconstituted in both heterologous hosts. Supplementation of soybean oil into the culture of E. coli BL21(DE3)/SsARS increased the production titers of 1–6 and led to the synthesis of an additional product, which was identified as 5-(8􍿁Z,11􍿁Z-heptadecadienyl) resorcinol. This work thus allowed the identification of SsARS as a 5-alk(en)ylresorcinol synthase with flexible substrate specificity toward endogenous and exogenous fatty acids. Desired resorcinol derivatives may be synthesized by supplying corresponding fatty acids into the culture medium.

A computationally simplistic poly-phasic approach to explore microbial communities from the Yucatan aquifer as a potential sources of novel natural products: Marfil-Santana Miguel David , O’Connor-Sánchez Aileen , Ramírez-Prado Jorge Humberto , De los Santos-Briones Cesar , López- Aguiar , Lluvia Korynthia , Rojas-Herrera Rafael , Lago-Lestón Asunción , Prieto-Davó Alejandra; J. Microbiol. 2016;54(11):774-781. Published online October 29, 2016; DOI: https://doi.org/10.1007/s12275-016-6092-x

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Abstract: The need for new antibiotics has sparked a search for the microbes that might potentially produce them. Current sequencing technologies allow us to explore the biotechnological potential of microbial communities in diverse environments without the need for cultivation, benefitting natural product discovery in diverse ways. A relatively recent method to search for the possible production of novel compounds includes studying the diverse genes belonging to polyketide synthase pathways (PKS), as these complex enzymes are an important source of novel therapeutics. In order to explore the biotechnological potential of the microbial community from the largest underground aquifer in the world located in the Yucatan, we used a polyphasic approach in which a simple, non-computationally intensive method was coupled with direct amplification of environmental DNA to assess the diversity and novelty of PKS type I ketosynthase (KS) domains. Our results suggest that the bioinformatic method proposed can indeed be used to assess the novelty of KS enzymes; nevertheless, this in silico study did not identify some of the KS diversity due to primer bias and stringency criteria outlined by the metagenomics pipeline. Therefore, additionally implementing a method involving the direct cloning of KS domains enhanced our results. Compared to other freshwater environments, the aquifer was characterized by considerably less diversity in relation to known ketosynthase domains; however, the metagenome included a family of KS type I domains phylogenetically related, but not identical, to those found in the curamycin pathway, as well as an outstanding number of thiolases. Over all, this first look into the microbial community found in this large Yucatan aquifer and other fresh water free living microbial communities highlights the potential of these previously overlooked environments as a source of novel natural products.

Research Support, Non-U.S. Gov'ts

Note] Analysis of a draft genome sequence of Kitasatospora cheerisanensis KCTC 2395 producing bafilomycin antibiotics: Jae Yoon Hwang , Soo Hee Kim , Hye Ryeung Oh , Eunju Kwon , Doo Hyun Nam; J. Microbiol. 2015;53(1):84-89. Published online December 4, 2014; DOI: https://doi.org/10.1007/s12275-015-4340-0

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Abstract: Kitasatospora cheerisanensis KCTC 2395, producing bafilomycin antibiotics belonging to plecomacrolide group, was isolated from a soil sample at Mt. Jiri, Korea. The draft genome sequence contains 8.04 Mb with 73.6% G+C content and 7,810 open reading frames. All the genes for aerial mycelium and spore formations were confirmed in this draft genome. In phylogenetic analysis of MurE proteins (UDPN- acetylmuramyl-L-alanyl-D-glutamate:DAP ligase) in a conserved dcw (division of cell wall) locus, MurE proteins of Kitasatospora species were placed in a separate clade between MurEs of Streptomyces species incorporating LL-diaminopimelic acid (DAP) and MurEs of Saccharopolyspora erythraea as well as Mycobacterium tuberculosis ligating meso- DAP. From this finding, it was assumed that Kitasatospora MurEs exhibit the substrate specificity for both LL-DAP and meso-DAP. The bafilomycin biosynthetic gene cluster was located in the left subtelomeric region. In 71.3 kb-long gene cluster, 17 genes probably involved in the biosynthesis of bafilomycin derivatives were deduced, including 5 polyketide synthase (PKS) genes comprised of 12 PKS modules.

Isolation and Characterization of a Reducing Polyketide Synthase Gene from the Lichen-Forming Fungus Usnea longissima: Yi Wang , Jung A Kim , Yong Hwa Cheong , Yogesh Joshi , Young Jin Koh , Jae-Seoun Hur; J. Microbiol. 2011;49(3):473-480. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-0362-4

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Abstract: The reducing polyketide synthases found in filamentous fungi are involved in the biosynthesis of many drugs and toxins. Lichens produce bioactive polyketides, but the roles of reducing polyketide synthases in lichens remain to be clearly elucidated. In this study, a reducing polyketide synthase gene (UlPKS3) was isolated and characterized from a cultured mycobiont of Usnea longissima. Complete sequence information regarding UlPKS3 (6,519 bp) was obtained by screening a fosmid genomic library. A UlPKS3 sequence analysis suggested that it contains features of a reducing fungal type I polyketide synthase with β-ketoacyl synthase (KS), acyltransferase (AT), dehydratase (DH), enoyl reductase (ER), ketoacyl reducatse (KR), and acyl carrier protein (ACP) domains. This domain structure was similar to the structure of ccRads1, which is known to be involved in resorcylic acid lactone biosynthesis in Chaetomium chiversii. The results of phylogenetic analysis located UlPKS3 in the clade of reducing polyketide synthases. RT-PCR analysis results demonstrated that UlPKS3 had six intervening introns and that UlPKS3 expression was upregulated by glucose, sorbitol, inositol, and mannitol.

Cloning and Analysis of a Type II Polyketide Synthase Gene Cluster from Streptomyces toxytricini NRRL 15,443: Anna Yoo , Atanas V. Demirev , Ji Seon Lee , Sang Dal Kim , Doo Hyun Nam; J. Microbiol. 2006;44(6):649-654.; DOI: https://doi.org/2462 [pii]

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Abstract: A standard type II polyketide synthase (PKS) gene cluster was isolated while attempting to clone the biosynthetic gene for lipstatin from Streptomyces toxytricini NRRL 15,443. This result was observed using a Southern blot of a PstI-digested S. toxytricini chromosomal DNA library with a 444 bp amplified probe of a ketosynthase (KS) gene fragment. Four open reading frames [thioesterase (TE), β-ketoacyl systhase (KAS), chain length factor (CLF), and acyl carrier protein (ACP)], were identified through the nucleotide sequence determination and analysis of a 4.5 kb cloned DNA fragment. In order to confirm the involvement of a cloned gene in lipstatin biosynthesis, a gene disruption experiment for the KS gene was performed. However, the resulting gene disruptant did not show any significant difference in lipstatin production when compared to wild-type S. toxytricini. This result suggests that lipstatin may not be synthesized by a type II PKS.

Review

Genomics Reveals Traces of Fungal Phenylpropanoid-flavonoid Metabolic Pathway in the F ilamentous Fungus Aspergillus oryzae: Praveen Rao Juvvadi , Yasuyo Seshime , Katsuhiko Kitamoto; J. Microbiol. 2005;43(6):475-486.; DOI: https://doi.org/2302 [pii]

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Abstract: Fungal secondary metabolites constitute a wide variety of compounds which either play a vital role in agricultural, pharmaceutical and industrial contexts, or have devastating effects on agriculture, animal and human affairs by virtue of their toxigenicity. Owing to their beneficial and deleterious characteristics, these complex compounds and the genes responsible for their synthesis have been the subjects of extensive investigation by microbiologists and pharmacologists. A majority of the fungal secondary metabolic genes are classified as type I polyketide synthases (PKS) which are often clustered with other secondary metabolism related genes. In this review we discuss on the significance of our recent discovery of chalcone synthase (CHS) genes belonging to the type III PKS superfamily in an industrially important fungus, Aspergillus oryzae. CHS genes are known to play a vital role in the biosynthesis of flavonoids in plants. A comparative genome analyses revealed the unique character of A. oryzae with four CHS-like genes (csyA, csyB, csyC and csyD) amongst other Aspergilli (Aspergillus nidulans and Aspergillus fumigatus) which contained none of the CHS-like genes. Some other fungi such as Neurospora crassa, Fusarium graminearum, Magnaporthe grisea, Podospora anserina and Phanerochaete chrysosporium also contained putative type III PKSs, with a phylogenic distinction from bacteria and plants. The enzymatically active nature of these newly discovered homologues is expected owing to the conservation in the catalytic residues across the different species of plants and fungi, and also by the fact that a majority of these genes (csyA, csyB and csyD) were expressed in A. oryzae. While this finding brings filamentous fungi closer to plants and bacteria which until recently were the only ones considered to possess the type III PKSs, the presence of putative genes encoding other principal enzymes involved in the phenylpropanoid and flavonoid biosynthesis (viz., phenylalanine ammonia-lyase, cinnamic acid hydroxylase and p-coumarate CoA ligase) in the A. oryzae genome undoubtedly prove the extent of its metabolic diversity. Since many of these genes have not been identified earlier, knowledge on their corresponding products or activities remain undeciphered. In future, it is anticipated that these enzymes may be reasonable targets for metabolic engineering in fungi to produce agriculturally and nutritionally important metabolites.

Journal Article

Alternative Production of Avermectin Components in Streptomyces avermitilis by Gene Replacement: Joon-Hyoung Yong , Woo-Hyeon Byeon; J. Microbiol. 2005;43(3):277-284.; DOI: https://doi.org/2212 [pii]

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Abstract: The avermectins are composed of eight compounds, which exhibit structural differences at three positions. A family of four closely-related major components, A1a, A2a, B1a and B2a, has been identified. Of these components, B1a exhibits the most potent antihelminthic activity. The coexistence of the "1" components and "2" components has been accounted for by the defective dehydratase of aveAI module 2, which appears to be responsible for C22-23 dehydration. Therefore, we have attempted to replace the dehydratase of aveAI module 2 with the functional dehydratase from the erythromycin eryAII module 4, via homologous recombination. Erythromycin polyketide synthetase should contain the sole dehydratase domain, thus generating a saturated chain at the C6-7 of erythromycin. We constructed replacement plasmids with PCR products, by using primers which had been derived from the sequences of avermectin aveAI and the erythromycin eryAII biosynthetic gene cluster. If the original dehydratase of Streptomyces avermitilis were exchanged with the corresponding erythromycin gene located on the replacement plasmid, it would be expected to result in the formation of precursors which contain alkene at C22-23, formed by the dehydratase of erythromycin module 4, and further processed by avermectin polyketide synthase. Consequently, the resulting recombinant strain JW3105, which harbors the dehydratase gene derived from erythromycin, was shown to produce only C22,23-unsaturated avermectin compounds. Our research indicates that the desired compound may be produced via polyketide gene replacement.

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