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1.
Nat Commun ; 11(1): 870, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054839

RESUMO

Land plants produce diverse flavonoids for growth, survival, and reproduction. Chalcone synthase is the first committed enzyme of the flavonoid biosynthetic pathway and catalyzes the production of 2',4,4',6'-tetrahydroxychalcone (THC). However, it also produces other polyketides, including p-coumaroyltriacetic acid lactone (CTAL), because of the derailment of the chalcone-producing pathway. This promiscuity of CHS catalysis adversely affects the efficiency of flavonoid biosynthesis, although it is also believed to have led to the evolution of stilbene synthase and p-coumaroyltriacetic acid synthase. In this study, we establish that chalcone isomerase-like proteins (CHILs), which are encoded by genes that are ubiquitous in land plant genomes, bind to CHS to enhance THC production and decrease CTAL formation, thereby rectifying the promiscuous CHS catalysis. This CHIL function has been confirmed in diverse land plant species, and represents a conserved strategy facilitating the efficient influx of substrates from the phenylpropanoid pathway to the flavonoid pathway.


Assuntos
Aciltransferases/metabolismo , Embriófitas/metabolismo , Liases Intramoleculares/metabolismo , Proteínas de Plantas/metabolismo , Aciltransferases/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Biocatálise , Vias Biossintéticas/genética , Chalconas/biossíntese , Embriófitas/genética , Evolução Molecular , Flavonoides/biossíntese , Genes de Plantas , Teste de Complementação Genética , Liases Intramoleculares/genética , Cinética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Policetídeos/metabolismo , Especificidade por Substrato
2.
Nat Biotechnol ; 38(1): 76-83, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31819261

RESUMO

Pharmaceutically important polyketides such as avermectin are mainly produced as secondary metabolites during the stationary phase of growth of Streptomyces species in fermenters. The source of intracellular metabolites that are funneled into polyketide biosynthesis has proven elusive. We applied multi-omics to reveal that intracellular triacylglycerols (TAGs), which accumulates in primary metabolism, are degraded during stationary phase. This process could channel carbon flux from both intracellular TAGs and extracellular substrates into polyketide biosynthesis. We devised a strategy named 'dynamic degradation of TAG' (ddTAG) to mobilize the TAG pool and increase polyketide biosynthesis. Using ddTAG we increased the titers of actinorhodin, jadomycin B, oxytetracycline and avermectin B1a in Streptomyces coelicolor, Streptomyces venezuelae, Streptomyces rimosus and Streptomyces avermitilis. Application of ddTAG increased the titer of avermectin B1a by 50% to 9.31 g l-1 in a 180-m3 industrial-scale fermentation, which is the highest titer ever reported. Our strategy could improve polyketide titers for pharmaceutical production.


Assuntos
Espaço Intracelular/metabolismo , Policetídeos/metabolismo , Streptomyces/metabolismo , Triglicerídeos/metabolismo , Carbono/metabolismo , Análise do Fluxo Metabólico , Redes e Vias Metabólicas , Metaboloma , Fatores de Tempo
3.
J Biotechnol ; 307: 69-76, 2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31689468

RESUMO

Branched chain fatty acids (BCFA) are an appealing biorefinery-driven target of fatty acid (FA) production. BCFAs typically have lower melting points compared to straight chain FAs, making them useful in lubricants and biofuels. Actinobacteria, especially Streptomyces species, have unique secondary metabolism that are capable of producing not only antibiotics, but also high percentage of BCFAs in their membrane lipids. Since biosynthesis of polyketide (PK) and FA partially share common pathways to generate acyl-CoA precursors, in theory, Streptomyces sp. with high levels of PK antibiotics production can be easily manipulated into strains producing high levels of BCFAs. To increase the percentage of the BCFA moieties in lipids, we redirected acyl-CoA precursor fluxes from PK into BCFAs using S. coelicolor M1146 (M1146) as a host strain. In addition, 3-ketoacyl acyl carrier protein synthase III and branched chain α-keto acid dehydrogenase were overexpressed to push fluxes of branched chain acyl-CoA precursors towards FA synthesis. The maximum titer of 354.1 mg/L BCFAs, 90.3% of the total FA moieties, was achieved using M1146dD-B, fadD deletion and bkdABC overexpression mutant of M1146 strain. Cell specific yield of 64.4 mg/L/gcell was also achieved. The production titer and specific yield are the highest ever reported in bacterial cells, which provides useful insights to develop an efficient host strain for BCFAs.


Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Ácidos Graxos/metabolismo , Streptomyces coelicolor/metabolismo , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , Acil Coenzima A/metabolismo , Antibacterianos/metabolismo , Biocombustíveis , Ácidos Graxos/análise , Expressão Gênica , Leucina/metabolismo , Mutação , Policetídeos/metabolismo , Metabolismo Secundário , Streptomyces coelicolor/genética
4.
J Toxicol Sci ; 44(12): 871-876, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31813906

RESUMO

Colibactin is a polyketide-peptide genotoxin produced by enteric bacteria such as E. coli, and is considered to contribute to the development of colorectal cancer. We previously isolated E. coli strains from Japanese colorectal cancer patients, and in the present study we investigated the genotoxic potency of the colibactin-producing (clb+) E. coli strains that carry the polyketide synthases "pks" gene cluster (pks+) and an isogenic clb- mutant in which the colibactin-producing ability is impaired. Measurement of phosphorylated histone H2AX indicated that DNA double strand breaks were induced in mammalian CHO AA8 cells infected with the clb+ E. coli strains. Induction of DNA damage response (SOS response) by crude extract of the clb+ strains was 1.7 times higher than that of the clb- E. coli in an umu assay with a Salmonella typhimurium TA1535/pSK1002 tester strain. Micronucleus test with CHO AA8 cells revealed that infection with the clb+ strains induced genotoxicity, i.e., the frequencies of micronucleated cells infected with clb+ strain were 4-6 times higher than with the clb- strain. Since the intestinal flora are affected by dietary habits that are strongly associated with ethnicity, these data may contribute to both risk evaluation and prevention of colorectal cancer in the Japanese population.


Assuntos
Colo/microbiologia , Neoplasias Colorretais/microbiologia , Escherichia coli/isolamento & purificação , Mutagênicos/toxicidade , Peptídeos/toxicidade , Policetídeos/toxicidade , Idoso , Animais , Células CHO , Cricetulus , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Masculino , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Mutagênicos/metabolismo , Peptídeos/metabolismo , Policetídeos/metabolismo , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética
5.
J Agric Food Chem ; 67(51): 14102-14109, 2019 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-31790231

RESUMO

Herbicidal activity-guided isolation from the fermentation extract of Penicillium viridicatum had obtained two herbicidal series of polyketides (1-7) and diketopiperazine derivatives (8-11), especially including three novel polyketides (1-3). The structures and absolute configurations of new polyketides 1-3 were elucidated by extensive spectroscopic analyses, as well as comparisons between measured and calculated ECD spectra. Novel polyketides 1-3 and known 4, all bearing the heptaketide skeleton with a trans-fused decalin ring of 8-CH3 substitution, could significantly inhibit the radicle growth of Echinochloa crusgalli seedlings with a dose-dependent relationship. Especially at the concentration of 10 µg/mL, 1-4 exhibited the inhibition rates with 81.5% ± 2.0, 76.4% ± 0.8, 79.6% ± 1.1, and 80.0 ± 1.8%, respectively, even better than the commonly used synthetic herbicide of acetochlor with 76.1 ± 1.4%. Further greenhouse bioassay revealed that 4 showed pre-emergence herbicidal activity against E. crusgalli with the fresh-weight inhibition rate of 74.1% at a dosage of 400 g ai/ha, also better than acetochlor, while the other isolated metabolites (5-11) exhibited moderate herbicidal activities. The structure-activity differences of isolated polyketides indicated that the heptaketide skeleton, characterized by a trans-fused decalin ring with 8-CH3 substitution, should be the key factor of their herbicidal activities, which could give new insights for the bioherbicide developments.


Assuntos
Dicetopiperazinas/farmacologia , Herbicidas/farmacologia , Penicillium/química , Policetídeos/farmacologia , Dicetopiperazinas/metabolismo , Echinochloa/efeitos dos fármacos , Echinochloa/crescimento & desenvolvimento , Herbicidas/metabolismo , Estrutura Molecular , Penicillium/metabolismo , Policetídeos/metabolismo
6.
Appl Microbiol Biotechnol ; 103(23-24): 9251-9262, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31686142

RESUMO

Microbial production of fuels and chemicals offers a means by which sustainable product manufacture can be achieved. In this regard, Yarrowia lipolytica is a unique microorganism suitable for a diverse array of biotechnological applications. As a robust oleaginous yeast, it has been well studied for production of fuels and chemicals derived from fatty acids. However, thanks in part to newfound genetic tools and metabolic understanding, Y. lipolytica has been explored for high-level production of a variety of non-lipid products. This mini-review will discuss some of the recent research surrounding the ability of Y. lipolytica to support bio-based chemical production outside the realm of fatty acid metabolism including polyketides, terpenes, carotenoids, pentose phosphate-derived products, polymers, and nanoparticles.


Assuntos
Microbiologia Industrial , Yarrowia/química , Yarrowia/metabolismo , Aminoácidos Aromáticos/biossíntese , Carotenoides/metabolismo , Ácidos Graxos/biossíntese , Engenharia Metabólica , Nanopartículas/metabolismo , Policetídeos/metabolismo , Polímeros/metabolismo , Terpenos/metabolismo
7.
Appl Microbiol Biotechnol ; 103(23-24): 9619-9631, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31686146

RESUMO

Type I polyketide synthases (PKSs) are large multi-domain proteins converting simple acyl-CoA thioesters such as acetyl-CoA and malonyl-CoA to a large diversity of biotechnologically interesting molecules. Such multi-step reaction cascades are of particular interest for applications in engineered microbial cell factories, as the introduction of a single protein with many enzymatic activities does not require balancing of several individual enzymatic activities. However, functional introduction of type I PKSs into heterologous hosts is very challenging as the large polypeptide chains often do not fold properly. In addition, PKS usually require post-translational activation by dedicated 4'-phosphopantetheinyl transferases (PPTases). Here, we introduce an engineered Corynebacterium glutamicum strain as a novel microbial cell factory for type I PKS-derived products. Suitability of C. glutamicum for polyketide synthesis could be demonstrated by the functional introduction of the 6-methylsalicylic acid synthase ChlB1 from Streptomyces antibioticus. Challenges related to protein folding could be overcome by translation fusion of ChlB1Sa to the C-terminus of the maltose-binding protein MalE from Escherichia coli. Surprisingly, ChlB1Sa was also active in the absence of a heterologous PPTase, which finally led to the discovery that the endogenous PPTase PptACg of C. glutamicum can also activate ChlB1Sa. The best strain, engineered to provide increased levels of acetyl-CoA and malonyl-CoA, accumulated up to 41 mg/L (0.27 mM) 6-methylsalicylic acid within 48 h of cultivation. Further experiments showed that PptACg of C. glutamicum can also activate nonribosomal peptide synthetases (NRPSs), rendering C. glutamicum a promising microbial cell factory for the production of several fine chemicals and medicinal drugs.


Assuntos
Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Policetídeo Sintases/metabolismo , Policetídeos/metabolismo , Salicilatos/metabolismo , Escherichia coli/metabolismo , Microbiologia Industrial , Engenharia Metabólica/métodos , Streptomyces antibioticus/enzimologia
8.
J Microbiol Biotechnol ; 29(12): 1931-1937, 2019 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-31693835

RESUMO

The heterologous expression of the Streptomyces natural product (NP) biosynthetic gene cluster (BGC) has become an attractive strategy for the activation, titer improvement, and refactoring of valuable and cryptic NP BGCs. Previously, a Streptomyces artificial chromosomal vector system, pSBAC, was applied successfully to the precise cloning of large-sized polyketide BGCs, including immunosuppressant tautomycetin and antibiotic pikromycin, which led to stable and comparable production in several heterologous hosts. To further validate the pSBAC system as a generally applicable heterologous expression system, the daptomycin BGC of S. roseosporus was cloned and expressed heterologously in a model Streptomyces cell factory. A 65-kb daptomycin BGC, which belongs to a non-ribosomal polypeptide synthetase (NRPS) family, was cloned precisely into the pSBAC which resulted in 28.9 mg/l of daptomycin and its derivatives in S. coelicolor M511(a daptomycin non-producing heterologous host). These results suggest that a pSBAC-driven heterologous expression strategy is an ideal approach for producing low and inconsistent Streptomyces NRPS-family NPs, such as daptomycin, which are produced low and inconsistent in native host.


Assuntos
Cromossomos Artificiais , Daptomicina/biossíntese , Família Multigênica , Streptomyces/genética , Streptomyces/metabolismo , Antibacterianos/metabolismo , Vias Biossintéticas/genética , Clonagem Molecular , Daptomicina/farmacologia , Furanos/metabolismo , Genes Bacterianos , Vetores Genéticos , Lipídeos , Macrolídeos/metabolismo , Peptídeo Sintases , Policetídeos/metabolismo , Staphylococcus aureus/efeitos dos fármacos
9.
Mar Drugs ; 17(10)2019 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-31635159

RESUMO

Marine-sourced actinomycete genus Streptomyces continues to be an important source of new natural products. Here we report the complete genome sequence of deep-sea-derived Streptomyces olivaceus SCSIO T05, harboring 37 putative biosynthetic gene clusters (BGCs). A cryptic BGC for type I polyketides was activated by metabolic engineering methods, enabling the discovery of a known compound, lobophorin CR4 (1). Genome mining yielded a putative lobophorin BGC (lbp) that missed the functional FAD-dependent oxidoreductase to generate the d-kijanose, leading to the production of lobophorin CR4 without the attachment of d-kijanose to C17-OH. Using the gene-disruption method, we confirmed that the lbp BGC accounts for lobophorin biosynthesis. We conclude that metabolic engineering and genome mining provide an effective approach to activate cryptic BGCs.


Assuntos
Antibacterianos/metabolismo , Genoma Bacteriano/genética , Streptomyces/genética , Sequência de Bases , Produtos Biológicos/metabolismo , Mapeamento Cromossômico/métodos , Engenharia Metabólica/métodos , Família Multigênica/genética , Policetídeos/metabolismo
10.
mSphere ; 4(5)2019 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-31578245

RESUMO

Colibactin is a polyketide/nonribosomal peptide produced by Escherichia coli strains that harbor the pks island. This toxin induces DNA double-strand breaks and DNA interstrand cross-links in infected eukaryotic cells. Colibactin-producing strains are found associated with colorectal cancer biopsy specimens and promote intestinal tumor progression in various murine models. Polyamines are small polycationic molecules produced by both microorganisms and eukaryotic cells. Their levels are increased in malignancies, where they contribute to disease progression and metastasis. In this study, we demonstrated that the endogenous spermidine synthase SpeE is required for full genotoxic activity of colibactin-producing E. coli Supplying spermidine in a ΔspeE pks + E. coli strain restored genotoxic activity. Spermidine is involved in the autotoxicity linked to colibactin and is required for direct damaging activity on DNA. The production of the colibactin prodrug motif is impaired in ΔspeE mutants. Therefore, we demonstrated that spermidine has a direct impact on colibactin synthesis.IMPORTANCE Colibactin-producing Escherichia coli strains are associated with cancerous and precancerous colorectal tissues and are suspected of promoting colorectal carcinogenesis. In this study, we describe a new interplay between the synthesis of the genotoxin colibactin and the polyamine spermidine. Polyamines are highly abundant in cancer tissue and are associated with cell proliferation. The need for spermidine in genotoxic activity provides a new perspective on the role of these metabolites in the pathogenicity of colibactin-producing E. coli strains in colorectal cancer.


Assuntos
Escherichia coli/patogenicidade , Mutagênicos/metabolismo , Peptídeos/metabolismo , Policetídeos/metabolismo , Espermidina Sintase/metabolismo , Espermidina/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Células HeLa , Humanos , Mutação , Poliaminas/metabolismo , Espermidina Sintase/genética
12.
BMC Biotechnol ; 19(1): 70, 2019 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-31655589

RESUMO

BACKGROUND: Aspergillus oryzae, a useful industrial filamentous fungus, produces limited varieties of secondary metabolites, such as kojic acid. Thus, for the production of valuable secondary metabolites by genetic engineering, the species is considered a clean host, enabling easy purification from cultured cells. A. oryzae has been evaluated for secondary metabolite production utilizing strong constitutive promoters of genes responsible for primary metabolism. However, secondary metabolites are typically produced by residual nutrition after microbial cells grow to the stationary phase and primary metabolism slows. We focused on a promoter of the secondary metabolism gene kojA, a component of the kojic acid biosynthetic gene cluster, for the production of other secondary metabolites by A. oryzae. RESULTS: A kojA disruptant that does not produce kojic acid was utilized as a host strain for production. Using this host strain, a mutant that expressed a polyketide synthase gene involved in polyketide secondary metabolite production under the kojA gene promoter was constructed. Then, polyketide production and polyketide synthase gene expression were observed every 24 h in liquid culture. From days 0 to 10 of culture, the polyketide was continuously produced, and the synthase gene expression was maintained. Therefore, the kojA promoter was activated, and it enabled the continuous production of polyketide for 10 days. CONCLUSIONS: The combined use of the kojA gene promoter and a kojA disruptant proved useful for the continuous production of a polyketide secondary metabolite in A. oryzae. These findings suggest that this combination can be applied to other secondary metabolites for long-term production.


Assuntos
Aspergillus oryzae/genética , Proteínas Fúngicas/genética , Policetídeos/metabolismo , Regiões Promotoras Genéticas/genética
13.
Science ; 366(6471)2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31582523

RESUMO

Extensive progress has been made in determining the effects of the microbiome on human physiology and disease, but the underlying molecules and mechanisms governing these effects remain largely unexplored. Here, we combine a new computational algorithm with synthetic biology to access biologically active small molecules encoded directly in human microbiome-derived metagenomic sequencing data. We discover that members of a clinically used class of molecules are widely encoded in the human microbiome and that they exert potent antibacterial activities against neighboring microbes, implying a possible role in niche competition and host defense. Our approach paves the way toward a systematic unveiling of the chemical repertoire encoded by the human microbiome and provides a generalizable platform for discovering molecular mediators of microbiome-host and microbiome-microbiome interactions.


Assuntos
Interações entre Hospedeiro e Microrganismos/genética , Metagenoma , Metagenômica/métodos , Microbiota/genética , Policetídeos/metabolismo , Humanos , Família Multigênica , Policetídeos/química
14.
mBio ; 10(5)2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31575772

RESUMO

Intestinal inflammation is a risk factor for colorectal cancer formation, but the underlying mechanisms remain unknown. Here, we investigated whether colitis alters the colonic microbiota to enhance its cancer-inducing activity. Colitis increased epithelial oxygenation in the colon of mice and drove an expansion of Escherichia coli within the gut-associated microbial community through aerobic respiration. An aerobic expansion of colibactin-producing E. coli was required for the cancer-inducing activity of this pathobiont in a mouse model of colitis-associated colorectal cancer formation. We conclude that increased epithelial oxygenation in the colon is associated with an expansion of a prooncogenic driver species, thereby increasing the cancer-inducing activity of the microbiota.IMPORTANCE One of the environmental factors important for colorectal cancer formation is the gut microbiota, but the habitat filters that control its cancer-inducing activity remain unknown. Here, we show that chemically induced colitis elevates epithelial oxygenation in the colon, thereby driving an expansion of colibactin-producing Escherichia coli, a prooncogenic driver species. These data suggest that elevated epithelial oxygenation is a potential risk factor for colorectal cancer formation because the consequent changes in the gut habitat escalate the cancer-inducing activity of the microbiota.


Assuntos
Carcinogênese , Colite/microbiologia , Neoplasias Colorretais/microbiologia , Infecções por Escherichia coli/complicações , Microbioma Gastrointestinal , Oxigênio/metabolismo , Aerobiose , Animais , Colite/induzido quimicamente , Colite/complicações , Sulfato de Dextrana , Escherichia coli , Infecções por Escherichia coli/microbiologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/metabolismo , Policetídeos/metabolismo
15.
Molecules ; 24(18)2019 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-31533358

RESUMO

Drug-like molecules are known to contain many different building blocks with great potential as pharmacophores for drug discovery. The continued search for unique scaffolds in our laboratory led to the isolation of a novel Ghanaian soil bacterium, Streptomyces sp. MA37. This strain produces many bioactive molecules, most of which belong to carbazoles, pyrrolizidines, and fluorinated metabolites. Further probing of the metabolites of MA37 has led to the discovery of a new naphthacene-type aromatic natural product, which we have named accramycin A 1. This molecule was isolated using an HPLC-photodiode array (PDA) guided isolation process and MS/MS molecular networking. The structure of 1 was characterized by detailed analysis of LC-MS, UV, 1D, and 2D NMR data. Preliminary studies on the antibacterial properties of 1 using Group B Streptococcus (GBS) produced a minimum inhibitory concentration (MIC) of 27 µg/mL. This represents the first report of such bioactivity amongst the naphthacene-type aromatic polyketides, and also suggests the possibility for the further development of potent molecules against GBS based on the accramycin scaffold. A putative acc biosynthetic pathway for accramycin, featuring a tridecaketide-specific type II polyketide synthase, was proposed.


Assuntos
Policetídeos/química , Policetídeos/isolamento & purificação , Microbiologia do Solo , Streptomyces/química , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Vias Biossintéticas , Genes Bacterianos , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Estrutura Molecular , Família Multigênica , Policetídeos/metabolismo , Policetídeos/farmacologia , Streptomyces/genética , Streptomyces/metabolismo
17.
Nat Chem ; 11(10): 890-898, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31548676

RESUMO

The clb gene cluster encodes the biosynthesis of metabolites known as precolibactins and colibactins. The clb pathway is found in gut commensal Escherichia coli, and clb metabolites are thought to initiate colorectal cancer via DNA crosslinking. Here we report confirmation of the structural assignment of the complex clb product precolibactin 886 via a biomimetic synthetic pathway. We show that an α-ketoimine linear precursor undergoes spontaneous cyclization to precolibactin 886 on HPLC purification. Studies of this α-ketoimine and the related α-dicarbonyl revealed that these compounds are unexpectedly susceptible to nucleophilic cleavage under mildly basic conditions. This cleavage pathway forms other known clb metabolites or biosynthetic intermediates and explains the difficulties in isolating fully mature biosynthetic products. This cleavage also accounts for a recently identified colibactin-adenine adduct. The colibactin peptidase ClbP deacylates synthetic precolibactin 886 to form a non-genotoxic pyridone, which suggests precolibactin 886 lies off the path of the major biosynthetic route.


Assuntos
Peptídeos/metabolismo , Policetídeos/metabolismo , Ciclização , Escherichia coli/genética , Escherichia coli/metabolismo , Iminas/química , Iminas/metabolismo , Conformação Molecular , Peptídeos/química , Policetídeos/química
18.
Nat Commun ; 10(1): 3918, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477708

RESUMO

Polyketides produced by modular type I polyketide synthases (PKSs) play eminent roles in the development of medicines. Yet, the production of structural analogs by genetic engineering poses a major challenge. We report an evolution-guided morphing of modular PKSs inspired by recombination processes that lead to structural diversity in nature. By deletion and insertion of PKS modules we interconvert the assembly lines for related antibiotic and antifungal agents, aureothin (aur) and neoaureothin (nor) (aka spectinabilin), in both directions. Mutational and functional analyses of the polyketide-tailoring cytochrome P450 monooxygenases, and PKS phylogenies give contradictory clues on potential evolutionary scenarios (generalist-to-specialist enzyme evolution vs. most parsimonious ancestor). The KS-AT linker proves to be well suited as fusion site for both excision and insertion of modules, which supports a model for alternative module boundaries in some PKS systems. This study teaches important lessons on the evolution of PKSs, which may guide future engineering approaches.


Assuntos
Cromonas/metabolismo , Oxigenases/metabolismo , Policetídeo Sintases/metabolismo , Policetídeos/metabolismo , Streptomyces/metabolismo , Sequência de Aminoácidos , Antibacterianos/química , Antibacterianos/metabolismo , Cromonas/química , Engenharia Genética/métodos , Modelos Químicos , Estrutura Molecular , Mutação , Filogenia , Policetídeo Sintases/classificação , Policetídeo Sintases/genética , Policetídeos/química , Streptomyces/genética
19.
J Microbiol Biotechnol ; 29(10): 1570-1579, 2019 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-31474098

RESUMO

The fungal products dibenzodioxocinones promise a novel class of inhibitors against cholesterol ester transfer protein (CEPT). Knowledge as to their biosynthesis is scarce. In this report, we characterized four more dibenzodioxocinones, which along with a previously described member pestalotiollide B, delimit the dominant spectrum of secondary metabolites in P. microspora. Through mRNA-seq profiling in gα1Δ, a process that halts the production of the dibenzodioxocinones, a gene cluster harboring 21 genes including a polyketide synthase, designated as pks8, was defined. Disruption of genes in the cluster led to loss of the compounds, concluding the anticipated role in the biosynthesis of the chemicals. The biosynthetic route to dibenzodioxocinones was temporarily speculated. This study reveals the genetic basis underlying the biosynthesis of dibenzodioxocinone in fungi, and may facilitate the practice for yield improvement in the drug development arena.


Assuntos
Família Multigênica , Policetídeos/metabolismo , Xylariales/genética , Vias Biossintéticas , Proteínas de Transferência de Ésteres de Colesterol/antagonistas & inibidores , Endófitos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Família Multigênica/genética , Mutação , Paclitaxel/biossíntese , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Policetídeos/química , Xylariales/química , Xylariales/metabolismo
20.
Metab Eng ; 56: 60-68, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31470116

RESUMO

Acetyl-CoA is the central metabolic node connecting glycolysis, Krebs cycle and fatty acids synthase. Plant-derived polyketides, are assembled from acetyl-CoA and malonyl-CoA, represent a large family of biological compounds with diversified bioactivity. Harnessing microbial bioconversion is considered as a feasible approach to large-scale production of polyketides from renewable feedstocks. Most of the current polyketide production platform relied on the lengthy glycolytic steps to provide acetyl-CoA, which inherently suffers from complex regulation with metabolically-costly cofactor/ATP requirements. Using the simplest polyketide triacetic acid lactone (TAL) as a testbed molecule, we demonstrate that acetate uptake pathway in oleaginous yeast (Yarrowia lipolytica) could function as an acetyl-CoA shortcut to achieve metabolic optimality in producing polyketides. We identified the metabolic bottlenecks to rewire acetate utilization for efficient TAL production in Y. lipolytica, including generation of the driving force for acetyl-CoA, malonyl-CoA and NADPH. The engineered strain, with the overexpression of endogenous acetyl-CoA carboxylase (ACC1), malic enzyme (MAE1) and a bacteria-derived cytosolic pyruvate dehydrogenase (PDH), affords robust TAL production with titer up to 4.76 g/L from industrial glacier acetic acid in shake flasks, representing 8.5-times improvement over the parental strain. The acetate-to-TAL conversion ratio (0.149 g/g) reaches 31.9% of the theoretical maximum yield. The carbon flux through this acetyl-CoA metabolic shortcut exceeds the carbon flux afforded by the native glycolytic pathways. Potentially, acetic acid could be manufactured in large-quantity at low-cost from Syngas fermentation or heterogenous catalysis (methanol carbonylation). This alternative carbon sources present a metabolic advantage over glucose to unleash intrinsic pathway limitations and achieve high carbon conversion efficiency and cost-efficiency. This work also highlights that low-cost acetic acid could be sustainably upgraded to high-value polyketides by oleaginous yeast species in an eco-friendly and cost-efficient manner.


Assuntos
Acetilcoenzima A , Engenharia Metabólica , Policetídeos/metabolismo , Pironas/metabolismo , Yarrowia , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Yarrowia/genética , Yarrowia/metabolismo
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