Characterization of the depsidone gene cluster reveals etherification, decarboxylation and multiple halogenations as tailoring steps in depsidone assembly

Depsides and depsidones have attracted attention for biosynthetic studies due to their broad biological activities and structural diversity. Previous structure‒activity relationships indicated that triple halogenated depsidones display the best anti-pathogenic activity. However, the gene cluster and the tailoring steps responsible for halogenated depsidone nornidulin ( 3) remain enigmatic. In this study, we disclosed the complete biosynthetic pathway of the halogenated depsidone through in vivo gene disruption, heterologous expression and in vitro biochemical experiments. We demonstrated an unusual depside skeleton biosynthesis process mediated by both highly-reducing polyketide synthase and non-reducing polyketide synthase, which is distinct from the common depside skeleton biosynthesis. This skeleton was subsequently modified by two in-cluster enzymes DepG and DepF for the ether bond formation and decarboxylation, respectively. In addition, the decarboxylase DepF exhibited substrate promiscuity for different scaffold substrates. Finally, and interestingly, we discovered a halogenase encoded remotely from the biosynthetic gene cluster, which catalyzes triple-halogenation to produce the active end product nornidulin ( 3). These discoveries provide new insights for further understanding the biosynthesis of depsidones and their derivatives.

Cloning and characterization of phloroglucinol biosynthetic gene phlC from an Indian strain of Pseudomonas fluorescens

Phloroglucinol or 2,4-diacetyl phloroglucinol (DAPG) is a polyketide compound produced by gram negative soil bacteria Pseudomonas. It shows broad spectrum antibacterial and antifungal properties against soil-borne plant pathogens. In Pseudomonas spp., genes for biosynthesis of 2,4-DAPG are localized in phlABCD operon. All the four genes in phlABCD operon are indispensable and DAPG synthesis is attenuated even in the absence of one of the genes. In the present study, we identified and cloned phlC gene from an Indian strain of Pseudomonas and analyzed its sequence. The structural details ofthe PHLC protein was generated by three-dimensional homology modelling. Additionally, stereo-chemical properties of PHLC were analyzed by Ramachandran plot analysis and the generated model was validated by PDBsum. Our results demonstrate that the cloned PHLC protein contains structural features typical of a condensing enzyme involved inpolyketide synthesis.

Progress on pathogenicity research of pks-positive Enterobacteriaceae / 中华检验医学杂志

The pks genomic island encodes non-ribosomal peptide synthase (NRPS), polyketide synthases (PKS), and hybrid NRPS/PKS synthetase. This genomic island is mainly found in the members of the family Enterobacteriaceae, and is especially common in Escherichia coli of phylogroup B2 while frequently coexisting with other virulence factors. The pks-positive E. coli is able to synthesize colibactin, a genotoxic chemical compound. Thus, this pks-positive bacteria may induce the breaking of DNA double-strand and chromosomal instability, which lead to senescence and apoptosis of cells. As a result, pks-positive E. coli is positively associated with the occurrence of diseases such as colorectal neoplasms, neonatal meningitis, and septicemia. Epidemiological studies have also confirmed that pks-positive E. coli is associated with a variety of diseases. However, the exact pathogenic mechanism of pks-positive E. coli is still not understood. Despite its genotoxicity, the pks-positive E. coli also exhibits some positive effects including anti-inflammatory, analgesic, and antibiotic abilities. Therefore, the biological role of pks-positive E. coli is complicated. In this review, an overview of the pks genomic island and its prevalence in Enterobacteriaceae, as well as the biological function of pks-positive E. coli is described, aiming to provide references for further researches.

Overexpression of a fusion protein of 4-coumaroyl-CoA ligase and polyketide synthase for raspberry ketone production in Chlamydomonas reinhardtii / 生物工程学报

Raspberry ketones have important therapeutic properties such as anti-influenza and prevention of diabetes. In order to obtain raspberry ketone from Chlamydomonas reinhardtii, two enzymes catalyzing the last two steps of raspberry ketone synthesis, i.e. 4-coumaryl-CoA ligase (4CL) and polyketide synthase (PKS1), were fused using a glycine-serine-glycine (GSG) tripeptide linker to construct an expression vector pChla-4CL-PKS1. The fusion gene 4CL-PKS1 driven by a PSAD promoter was transformed into a wild-type (CC125) and a cell wall-deficient C. reinhardtii (CC425) by electroporation. The results showed the recombinant C. reinhardtii strain CC125 and CC425 with 4CL-PKS1 produced raspberry ketone at a level of 6.7 μg/g (fresh weight) and 5.9 μg/g (fresh weight), respectively, both were higher than that of the native raspberry ketone producing plants (2-4 μg/g).

Advances in metabolic engineering of macrolide antibiotics / 生物工程学报

14- to 16-membered macrolide antibiotics (MA) are clinically important anti-infective drugs. With the rapid emergence of bacterial resistance, there is an urgent need to develop novel MA to counter drug-resistant bacteria. The targeted optimization of MA can be guided by analyzing the interaction between the MA and its ribosomal targets, and the desired MA derivatives can be obtained efficiently when combining with the rapidly developed metabolic engineering approaches. In the past 30 years, metabolic engineering approaches have shown great advantages in engineering the biosynthesis of MA to create new derivatives and to improve their production. These metabolic engineering approaches include modification of the structural domains of the polyketide synthase (PKS) and post-PKS modification enzymes as well as combinatorial biosynthesis. In addition, the R&D (including the evaluation of its antimicrobial activities and the optimization through metabolic engineering) of carrimycin, a new 16-membered macrolide drug, are described in details in this review.

Research advances in M. Tuberculosis polyketide synthase 13 inhibitors / 药学学报

Polyketide synthase 13 (Pks13) performs a critical role in the final assembly step of mycolic acid synthesis in Mycobacterium tuberculosis. The inhibition of Pks13 can influence the biosynthesis of mycolic acid, which leads to Mycobacterium tuberculosis cell death. Researchers have discovered Pks13 inhibitors with five chemical scaffolds as antituberculosis agents. Herein, we summarize recent advances in the study of Pks13 inhibitors including the process of discovery, the mechanism of action and structure-activity relationships.

Identification of polyketide synthase 13 inhibitor: Pharmacophore-based virtual screening and molecular dynamics simulation

Polyketide synthase 13 (Pks13) is one of prominent targets to treat Mycobacterium tuberculosis (Mtb). In the presentstudy, pharmacophore features for Pks13, including two hydrogen bond donors, one hydrogen bond acceptor, and onehydrophobic feature, were built using a novel Pks13 inhibitor, TAM16. The pharmacophore features were then usedto perform virtual screening on ZINC database to identify small molecules of Pks13 inhibitors. The obtained virtualhits of 107 small molecules were subjected to molecular docking studies employing iDock software to reveal theirbinding orientation to Pks13. Furthermore, four best hits, each bound to Pks13, were submitted to 40-ns moleculardynamics simulation to explore their conformational changes throughout simulation. The result showed that all hitcompounds, i.e., Lig79/ZINC09281113, Lig94/ZINC09584070, Lig95/ZINC09209668, and Lig97/ZINC09216165,have better stabilities than that of TAM16 as indicated by their lower values of root-mean-square-deviation and rootmean-square-fluctuation. In a similar way, prediction of binding free energy using molecular mechanics Poisson–Boltzmann Surface Area method showed that all hit compounds have lower binding free energies than that of TAM16,indicating their potential as novel compounds of Pks13 inhibitors.

Metabolites from Cercospora lagenariae MT-45, an endophytic fungus isolated from Huperzia serrata / 中草药

Objective: To investigate the chemical metabolites from Cercospora lagenariae MT-45, an endophytic fungus isolated from Huperzia serrata (Thunb.) Trev. Methods: The compounds were isolated and purified by using silica gel column chromatography and semi-preparative liquid chromatography. The structures were established using physicochemical properties and MS and NMR. The anti-inflammatory activities of all the isolates were also preliminarily investigated by using in vitro model. Results: Nine polyketide derivatives including cercolagenlic acid A (1), alternariol (2), alternariol 9-methyl ether (3), (+)-nigrosporaol A (4), alternarienonic acid B (5), 2-methyl-5-carboxymethyl-7-hydroxychromone (6), 2,5-dimethyl-7-hydroxychromone (7), 1-deoxy- rubralactone (8), and (-)-alternarlactam (9) were isolated from C. lagenariae MT-45 fermented on brown rice solids. Conclusion: Compound 1 is a new compound, and compound 6 can exhibit a certain inhibition on the nitric oxide production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells with an IC50 value of (57.5 ± 1.2) μmol/L.

New polyketides from chemical mutant strain of sponge-associated fungus emericella variecolor xsa-07-2 / 中国药学杂志

OBJECTIVE: To study the new bioactive secondary metabolites of diethyl sulfate chemical mutant strain of sponge-associated fungus Emericella variecolor XSA-07-2. METHODS: Diethyl sulfate was used to make chemical mutagenesis of strain XSA-07-2, and one mutant strain M8 was chosen for large-scale fermentation to generate new secondary metabolites. The compounds were isolated and purified by chromatography on silica gel and ODS reversed-phase column and semi-preparative HPLC techniques. And their structures were identified by their physicochemical properties and NMR, MS data analysis. RESULTS: Three new polyketides 1-3 were isolated from the extract of the solid fermentation culture of mutant strain M8. Compound 3 showed moderated antioxidant activity with IC50 of (13.58±0.14) μg·mL-1 by DPPH assay. CONCLUSION: Diethyl sulfate chemical mutagenesis can stimulate sponge-associated fungus Emericella variecolor XSA-07-2 mutant strain M8 to produce new antioxidant polyketides.

Cytotoxic and antibacterial polyketide-indole hybrids synthesized from indole-3-carbinol by

Two skeletally undescribed polyketide-indole hybrids (PIHs), named indolchromins A and B, were generated from indole-3-carbinol (I3C) in the fungal culture (). The indolchromin structures were elucidated mainly by their 1D and 2D NMR spectra with the former confirmed by the single-crystal X-ray crystallographic analysis. Each indolchromin alkaloid was chirally separated into four isomers, whose absolute configurations were assigned by comparing the recorded circular dichroism (CD) spectra with the electronic CD (ECD) curves computed for all optional stereoisomers. Furthermore, the indolchromin construction pathways in fungal culture were clarified through enzyme inhibition, precursor feeding experiment, and energy calculation. The cascade reactions, including decarboxylative Claisen condensation catalyzed by 8-amino-7-oxononanoate synthase (AONS), C()-H activation, double bond migration, and Michael addition, all undergone compatibly during the fungal cultivation. In an MIC range of 1.3-8.6 μmol/L, (2,4)- and (2,4)-indolchromin A and (2,4)-indolchromin B are inhibitory against , , sp., , and . (2,4)-Indolchromin A and (2,4)-indolchromin B were cytotoxic against the human breast cancer cell line MDA-MB-231 with IC values of 27.9 and 131.2 nmol/L, respectively, with the former additionally active against another human breast cancer cell line MCF-7 (IC 94.4 nmol/L).

Construction of a mutant of Actinoplanes sp. N902-109 that produces a new rapamycin analog / 中国天然药物

In the present study, we introduced point mutations into Ac_rapA which encodes a polyketide synthase responsible for rapamycin biosynthesis in Actinoplanes sp. N902-109, in order to construct a mutant with an inactivated enoylreductase (ER) domain, which was able to synthesize a new rapamycin analog. Based on the homologous recombination induced by double-strand breaks in chromosome mediated by endonuclease I-SceI, the site-directed mutation in the first ER domain of Ac_rapA was introduced using non-replicating plasmid pLYERIA combined with an I-SceI expression plasmid. Three amino acid residues of the active center, Ala-Gly-Gly, were converted to Ala-Ser-Pro. The broth of the mutant strain SIPI-027 was analyzed by HPLC and a new peak with the similar UV spectrum to that of rapamycin was found. The sample of the new peak was prepared by solvent extraction, column chromatography, and crystallization methods. The structure of new compound, named as SIPI-rapxin, was elucidated by determining and analyzing its MS and NMR spectra and its biological activity was assessed using mixed lymphocyte reaction (MLR). An ER domain-deficient mutant of Actinoplanes sp. N902-109, named as SIPI-027, was constructed, which produced a novel rapamycin analog SIPI-rapxin and its structure was elucidated to be 35, 36-didehydro-27-O-demethylrapamycin. The biological activity of SIPI-rapxin was better than that of rapamycin. In conclusion, inactivation of the first ER domain of rapA, one of the modular polyketide synthase responsible for macro-lactone synthesis of rapamycin, gave rise to a mutant capable of producing a novel rapamycin analog, 35, 36-didehydro-27-O-demethylrapamycin, demonstrating that the enoylreductase domain was responsible for the reduction of the double bond between C-35 and C-36 during rapamycin synthesis.

Advances in structure-function relation of plant type Ⅲ polyketide synthases by site-directed mutagenesis / 生物工程学报

Plant type Ⅲ polyketide synthases (PKSs), the pivotal enzymes in the biosynthesis of polyketides, produce backbones of many structurally diverse and functionally different polyketides. So far, a variety of functionally diverse plant type Ⅲ PKSs have been cloned and identified from plant origin. Site-directed mutagenesis is a useful technique to study the complex relationship between protein structure and function. This review summarized advances in the structure-function relation of plant type Ⅲ polyketide synthases by site-directed mutagenesis in recent years, including the modification of the amino acid residues influencing enzyme architectures (such as controlling the specificity of starter substrates, the number of condensation reactions, and the cyclization reactions of the intermediate product). This review provides information to study the structure-function relation of plant type Ⅲ polyketide synthases.

In vivo synthesis of csypyrone derivatives by exploring the substrate diversity of start units of type Ⅲ polyketide synthase CsyB / 生物工程学报

As a novel fungal type Ⅲ polyketide synthase, CsyB from Aspergillus oryzae can sequentially accept one molecular short chain fatty acyl CoA as start unit, one molecular malonyl-CoA and one molecular acetoacetyl-CoA as extend unit to produce the short chain csypyrone B1-3. On the basis of crystal structure of CsyB, a fatty acyl CoA binding tunnel of a length of about 16 Å is located in its active center that is proposed to accept diversified start units. In order to examine the substrate diversity of CsyB, CsyB gene was introduced and expressed in Escherichia coli that contained a number of precursors of long chain fatty acyl CoA in vivo. The results of HPLC revealed that a series of long chain csypyrone derivatives were detected in the recombinant strain in comparison with the control strain. These new csypyrone compounds were preliminarily analyzed by UV-visible spectroscopy and LC-HRMS. Three hydroxylated csypyrones were intensively determined by 1D and 2D NMR experiments, especially the position of the hydroxyl group in these compounds. These results demonstrate that CsyB exhibits a broad substrate specificity, which not only can accept the long chain saturated or unsaturated fatty acyl CoA as substrate, but also accept hydroxylated long chain fatty acyl CoA.

Advances in study of plant type Ⅲ polyketide synthases / 中国中药杂志

Polyketides are a large class of natural products with notable structural diversity and different biological activities. They have essential pharmacological value for human health. In plants, the enzymes responsible for the formation of phenolic metabolites backbone structures are collectively known as type Ⅲ polyketide synthases (PKSs), which are the key enzymes for the polyketides biosynthesis. The PKSs catalyze a series of condensation reactions of two-carbon acetate units with an acyl starter. A brief overview of this group of enzymes, including their reaction mechanisms, function modification, expression regulation, molecular evolution, and recent interesting findings are presented here.

Progress in fungal polyketide biosynthesis / 生物工程学报

Fungal polyketides display complex structures and variously biological activities. Their biosynthetic pathways generally contain novel enzyme-catalyzed reactions. This review provides a summary of recent research advances in molecular mechanism of the biosynthesis of fungal polyketides including highly-reducing polyketide synthases (HR-PKSs), non-reducing polyketide synthases (NR-PKSs), as well as polyketide-nonribosomal peptide synthase (PKS-NRPSs) and reducingnon- reducing polyketide synthase (HR-NR PKSs) hybrids. The elucidation of biosynthetic mechanism of many fungal polyketides provides guidance on the discovery of new biosynthetic gene cluster of fungal polyketide natural products and compounds with novel structures as well as their analogue.

Construction of a mutant of Actinoplanes sp. N902-109 that produces a new rapamycin analog / 中国天然药物

In the present study, we introduced point mutations into Ac_rapA which encodes a polyketide synthase responsible for rapamycin biosynthesis in Actinoplanes sp. N902-109, in order to construct a mutant with an inactivated enoylreductase (ER) domain, which was able to synthesize a new rapamycin analog. Based on the homologous recombination induced by double-strand breaks in chromosome mediated by endonuclease I-SceI, the site-directed mutation in the first ER domain of Ac_rapA was introduced using non-replicating plasmid pLYERIA combined with an I-SceI expression plasmid. Three amino acid residues of the active center, Ala-Gly-Gly, were converted to Ala-Ser-Pro. The broth of the mutant strain SIPI-027 was analyzed by HPLC and a new peak with the similar UV spectrum to that of rapamycin was found. The sample of the new peak was prepared by solvent extraction, column chromatography, and crystallization methods. The structure of new compound, named as SIPI-rapxin, was elucidated by determining and analyzing its MS and NMR spectra and its biological activity was assessed using mixed lymphocyte reaction (MLR). An ER domain-deficient mutant of Actinoplanes sp. N902-109, named as SIPI-027, was constructed, which produced a novel rapamycin analog SIPI-rapxin and its structure was elucidated to be 35, 36-didehydro-27-O-demethylrapamycin. The biological activity of SIPI-rapxin was better than that of rapamycin. In conclusion, inactivation of the first ER domain of rapA, one of the modular polyketide synthase responsible for macro-lactone synthesis of rapamycin, gave rise to a mutant capable of producing a novel rapamycin analog, 35, 36-didehydro-27-O-demethylrapamycin, demonstrating that the enoylreductase domain was responsible for the reduction of the double bond between C-35 and C-36 during rapamycin synthesis.

Improving Production of Tacrolimus In Streptomyces Tacrolimicus (ATCC 55098) Through Development of Novel Mutant by Dual Mutagenesis

ABSTRACT Tacrolimus is a polyketide macrolide produced by Streptomyces species which is widely used as anti-fibrotic agent and potent immunosuppressant. In this article dual mutagenesis approach using mutagens (NTG+EMS+UV) was used to develop a mutant strain of Streptomyces tacrolimicus (ATCC 55098) for higher tacrolimus production and this strain showed higher tacrolimus production at 82.5 mg/l. Interestingly; addition of L-Lysine (0.2 g/l) into the production medium further enhanced the tacrolimus production to ~102 mg/l at 7-L fed-batch bioreactor. To the best of our knowledge this is the first report mentioning efficient strain development for higher production of tacrolimus using dual mutagenesis. The obtained data presents an impressive model for higher production of tacrolimus and enhanced our understanding regarding improvement in production capacity of tacrolimus in Streptomyces tacrolimicus.

Isolation and expression of two polyketide synthase genes from Trichoderma harzianum 88 during mycoparasitism

Abstract Metabolites of mycoparasitic fungal species such as Trichoderma harzianum 88 have important biological roles. In this study, two new ketoacyl synthase (KS) fragments were isolated from cultured Trichoderma harzianum 88 mycelia using degenerate primers and analysed using a phylogenetic tree. The gene fragments were determined to be present as single copies in Trichoderma harzianum 88 through southern blot analysis using digoxigenin-labelled KS gene fragments as probes. The complete sequence analysis in formation of pksT-1 (5669 bp) and pksT-2 (7901 bp) suggests that pksT-1 exhibited features of a non-reducing type I fungal PKS, whereas pksT-2 exhibited features of a highly reducing type I fungal PKS. Reverse transcription polymerase chain reaction indicated that the isolated genes are differentially regulated in Trichoderma harzianum 88 during challenge with three fungal plant pathogens, which suggests that they participate in the response of Trichoderma harzianum 88 to fungal plant pathogens. Furthermore, disruption of the pksT-2 encoding ketosynthase–acyltransferase domains through Agrobacterium -mediated gene transformation indicated that pksT-2 is a key factor for conidial pigmentation in Trichoderma harzianum 88.

Pteridic acid hydrate and pteridic acid C produced by StreStreptomyces pseudoverticillus YN17707 induce cell cycle arrest / 中国天然药物

The present study aimed at identifying cell cycle inhibitors from the fermentation broth of Streptomyces pseudoverticillus YN17707. Activity-guided isolation was performed on tsFT210 cells. Compounds were isolated through various chromatographic methods and elucidated by spectroscopic analyses. Flow cytometry was used to evaluate the cell cycle inhibitory activities of the fractions and compounds. Two compounds were obtained and identified as pteridic acid hydrate (1) and pteridic acid C (2), which arrested the tsFT210 cells at the G0/G1 phase with the MIC values being 32.8 and 68.9 μmol·L(-1), respectively. These results provide a basis for future development of Compounds 1 and 2 as novel cell cycle inhibitors for cancer therapy.

Three New Non-reducing Polyketide Synthase Genes from the Lichen-Forming Fungus Usnea longissima

Usnea longissima has a long history of use as a traditional medicine. Several bioactive compounds, primarily belonging to the polyketide family, have been isolated from U. longissima. However, the genes for the biosynthesis of these compounds are yet to be identified. In the present study, three different types of non-reducing polyketide synthases (UlPKS2, UlPKS4, and UlPKS6) were identified from a cultured lichen-forming fungus of U. longissima. Phylogenetic analysis of product template domains showed that UlPKS2 and UlPKS4 belong to group IV, which includes the non-reducing polyketide synthases with an methyltransferase (MeT) domain that are involved in methylorcinol-based compound synthesis; UlPKS6 was found to belong to group I, which includes the non-reducing polyketide synthases that synthesize single aromatic ring polyketides, such as orsellinic acid. Reverse transcriptase-PCR analysis demonstrated that UlPKS2 and UlPKS4 were upregulated by sucrose; UlPKS6 was downregulated by asparagine, glycine, and alanine.