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1.
J Am Chem Soc ; 146(38): 26187-26197, 2024 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-39283600

RESUMO

Glycyl radical enzymes (GREs) catalyze mechanistically diverse radical-mediated reactions, playing important roles in the metabolism of anaerobic bacteria. The model bacterium Escherichia coli MG1655 contains two GREs of unknown function, YbiW and PflD, which are widespread among human intestinal bacteria. Here, we report that YbiW and PflD catalyze ring-opening C-O cleavage of 1,5-anhydroglucitol-6-phosphate (AG6P) and 1,5-anhydromannitol-6-phosphate (AM6P), respectively. The product of both enzymes, 1-deoxy-fructose-6-phosphate (DF6P), is then cleaved by the aldolases FsaA or FsaB to form glyceraldehyde-3-phosphate (G3P) and hydroxyacetone (HA), which are then reduced by the NADH-dependent dehydrogenase GldA to form 1,2-propanediol (1,2-PDO). Crystal structures of YbiW and PflD in complex with their substrates provided insights into the mechanism of radical-mediated C-O cleavage. This "anhydroglycolysis" pathway enables anaerobic growth of E. coli on 1,5-anhydroglucitol (AG) and 1,5-anhydromannitol (AM), and we probe the feasibility of harnessing this pathway for the production of 1,2-PDO, a highly demanded chiral chemical feedstock, from inexpensive starch. Discovery of the anhydroglycolysis pathway expands the known catalytic repertoire of GREs, clarifies the hitherto unknown physiological functions of the well-studied enzymes FsaA, FsaB, and GldA, and demonstrates how enzyme discovery efforts can cast light on prevalent yet overlooked metabolites in the microbiome.


Assuntos
Escherichia coli , Glicólise , Escherichia coli/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Radicais Livres/metabolismo , Radicais Livres/química , Modelos Moleculares
2.
Microb Cell Fact ; 23(1): 149, 2024 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-38790014

RESUMO

BACKGROUND: Streptomyces is renowned for its robust biosynthetic capacity in producing medically relevant natural products. However, the majority of natural products biosynthetic gene clusters (BGCs) either yield low amounts of natural products or remain cryptic under standard laboratory conditions. Various heterologous production hosts have been engineered to address these challenges, and yet the successful activation of BGCs has still been limited. In our search for a valuable addition to the heterologous host panel, we identified the strain Streptomyces sp. A4420, which exhibited rapid initial growth and a high metabolic capacity, prompting further exploration of its potential. RESULTS: We engineered a polyketide-focused chassis strain based on Streptomyces sp. A4420 (CH strain) by deleting 9 native polyketide BGCs. The resulting metabolically simplified organism exhibited consistent sporulation and growth, surpassing the performance of most existing Streptomyces based chassis strains in standard liquid growth media. Four distinct polyketide BGCs were chosen and expressed in various heterologous hosts, including the Streptomyces sp. A4420 wild-type and CH strains, alongside Streptomyces coelicolor M1152, Streptomyces lividans TK24, Streptomyces albus J1074, and Streptomyces venezuelae NRRL B-65442. Remarkably, only the Streptomyces sp. A4420 CH strain demonstrated the capability to produce all metabolites under every condition outperforming its parental strain and other tested organisms. To enhance visualization and comparison of the tested strains, we developed a matrix-like analysis involving 15 parameters. This comprehensive analysis unequivocally illustrated the significant potential of the new strain to become a popular heterologous host. CONCLUSION: Our engineered Streptomyces sp. A4420 CH strain exhibits promising attributes for the heterologous expression of natural products with a focus on polyketides, offering an alternative choice in the arsenal of heterologous production strains. As genomics and cloning strategies progress, establishment of a diverse panel of heterologous production hosts will be crucial for expediting the discovery and production of medically relevant natural products derived from Streptomyces.


Assuntos
Produtos Biológicos , Engenharia Metabólica , Família Multigênica , Policetídeos , Streptomyces , Streptomyces/genética , Streptomyces/metabolismo , Produtos Biológicos/metabolismo , Policetídeos/metabolismo , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Streptomyces lividans/genética , Streptomyces lividans/metabolismo , Vias Biossintéticas/genética
3.
Proc Natl Acad Sci U S A ; 117(27): 15599-15608, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571930

RESUMO

2(S)-dihydroxypropanesulfonate (DHPS) is a microbial degradation product of 6-deoxy-6-sulfo-d-glucopyranose (sulfoquinovose), a component of plant sulfolipid with an estimated annual production of 1010 tons. DHPS is also at millimolar levels in highly abundant marine phytoplankton. Its degradation and sulfur recycling by microbes, thus, play important roles in the biogeochemical sulfur cycle. However, DHPS degradative pathways in the anaerobic biosphere are not well understood. Here, we report the discovery and characterization of two O2-sensitive glycyl radical enzymes that use distinct mechanisms for DHPS degradation. DHPS-sulfolyase (HpsG) in sulfate- and sulfite-reducing bacteria catalyzes C-S cleavage to release sulfite for use as a terminal electron acceptor in respiration, producing H2S. DHPS-dehydratase (HpfG), in fermenting bacteria, catalyzes C-O cleavage to generate 3-sulfopropionaldehyde, subsequently reduced by the NADH-dependent sulfopropionaldehyde reductase (HpfD). Both enzymes are present in bacteria from diverse environments including human gut, suggesting the contribution of enzymatic radical chemistry to sulfur flux in various anaerobic niches.


Assuntos
Alcanossulfonatos/metabolismo , Anaerobiose , Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Microbioma Gastrointestinal/fisiologia , Biologia Computacional , Ensaios Enzimáticos , Sulfeto de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/toxicidade , Metilglucosídeos/metabolismo , Enxofre/metabolismo
4.
Biochemistry ; 61(24): 2861-2869, 2022 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-35414181

RESUMO

Capnine (2-amino-3-hydroxy-15-methylhexadecane-1-sulfonate) and capnoids (N-fatty acylated capnine derivatives) are sulfonolipids present in the outer membrane of gliding bacteria in the phylum Bacteroidetes and play a role in their unique gliding motility. They are structurally similar to sphingolipids and are thought to be biosynthesized via a similar pathway. Here we report the identification and biochemical characterization of the capnine biosynthetic enzymes cysteate synthase (CapA) and cysteate-C-fatty acyltransferase (CapB) from the pathogenic gliding bacterium Capnocytophaga ochracea and NAD(P)H-dependent dehydrocapnine reductase CapC from the avian pathogen Ornithobacterium rhinotracheale. CapA catalyzes the formation of cysteate from O-phospho-l-serine and sulfite, and CapB catalyzes the formation of dehydrocapnine from cysteate and 13-methyl-myristoyl-CoA, followed by reduction by CapC. CapA is closely related to cystathionine-ß-synthase but distantly related to the archaeal cysteate synthase. Close homologues of CapA, CapB, and the CapA isozyme archaeal cysteate synthase are present in many Bacteroidetes bacteria, including environmental, pathogenic, and human oral and intestinal microbiome bacteria, suggesting the widespread ability of these bacteria to biosynthesize capnine and related sulfonolipids.


Assuntos
Ácidos Alcanossulfônicos , Ácido Cisteico , Humanos , Ácido Cisteico/metabolismo , Vias Biossintéticas , Bactérias/metabolismo , Bacteroidetes
5.
J Am Chem Soc ; 144(22): 9715-9722, 2022 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-35611954

RESUMO

Hydroxyprolines are highly abundant in nature as they are components of many structural proteins and osmolytes. Anaerobic degradation of trans-4-hydroxy-l-proline (t4L-HP) was previously found to involve the glycyl radical enzyme (GRE) t4L-HP dehydratase (HypD). Here, we report a pathway for anaerobic hydroxyproline degradation that involves a new GRE, trans-4-hydroxy-d-proline (t4D-HP) C-N-lyase (HplG). In this pathway, cis-4-hydroxy-l-proline (c4L-HP) is first isomerized to t4D-HP, followed by radical-mediated ring opening by HplG to give 2-amino-4-ketopentanoate (AKP), the first example of a ring opening reaction catalyzed by a GRE 1,2-eliminase. Subsequent cleavage by AKP thiolase (OrtAB) yields acetyl-CoA and d-alanine. We report a crystal structure of HplG in complex with t4D-HP at a resolution of 2.7 Å, providing insights into its catalytic mechanism. Different from HypD commonly identified in proline-reducing Clostridia, HplG is present in other types of fermenting bacteria, including propionate-producing bacteria, underscoring the diversity of enzymatic radical chemistry in the anaerobic microbiome.


Assuntos
Prolina , Proteínas , Anaerobiose , Hidroxiprolina/química , Prolina/metabolismo , Proteínas/metabolismo
6.
Chembiochem ; 23(21): e202200295, 2022 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-35959532

RESUMO

Naturally occurring DNA contains four canonical bases, forming two Watson-Crick base pairs (adenine-thymine, guanine-cytosine). Efforts over the past decades have led to the development of several unnatural base pairs, enabling the synthesis of unnatural DNA with an expanded genetic alphabet. The engineering of organisms capable of de novo biosynthesis of unnatural DNA would have significant technological and philosophical implications, but remains a challenge. Here we report the enzymatic conversion of 2'-deoxyxanthosine 5'-monophosphate (dXMP) into deoxyisoguanosine monophosphate (dBMP), a precursor of the unnatural isoguanine-isocytosine base pair. The reaction is catalyzed by the bacteriophage enzyme PurZ and bacterial PurB, and is a key addition to the toolbox for de novo biosynthesis of unnatural DNA.


Assuntos
Guanosina , Nucleotídeos , Pareamento de Bases , DNA
7.
J Biol Chem ; 294(43): 15662-15671, 2019 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-31455636

RESUMO

The reductive pyrimidine catabolic pathway is the most widespread pathway for pyrimidine degradation in bacteria, enabling assimilation of nitrogen for growth. This pathway, which has been studied in several bacteria including Escherichia coli B, releases only one utilizable nitrogen atom from each molecule of uracil, whereas the other nitrogen atom remains trapped in the end product ß-alanine. Here, we report the biochemical characterization of a ß-alanine:2-oxoglutarate aminotransferase (PydD) and an NAD(P)H-dependent malonic semialdehyde reductase (PydE) from a pyrimidine degradation gene cluster in the bacterium Lysinibacillus massiliensis Together, these two enzymes converted ß-alanine into 3-hydroxypropionate (3-HP) and generated glutamate, thereby making the second nitrogen from the pyrimidine ring available for assimilation. Using bioinformatics analyses, we found that PydDE homologs are associated with reductive pyrimidine pathway genes in many Gram-positive bacteria in the classes Bacilli and Clostridia. We demonstrate that Bacillus smithii grows in a defined medium with uracil or uridine as its sole nitrogen source and detected the accumulation of 3-HP as a waste product. Our findings extend the reductive pyrimidine catabolic pathway and expand the diversity of enzymes involved in bacterial pyrimidine degradation.


Assuntos
Bacillaceae/metabolismo , Redes e Vias Metabólicas , Nitrogênio/metabolismo , Pirimidinas/metabolismo , beta-Alanina/metabolismo , Bacillaceae/efeitos dos fármacos , Bacillaceae/crescimento & desenvolvimento , Proteínas de Bactérias/metabolismo , Biocatálise/efeitos dos fármacos , Cinética , Redes e Vias Metabólicas/efeitos dos fármacos , Família Multigênica , Nitrogênio/farmacologia , Proteínas Recombinantes/biossíntese , Uracila/metabolismo
8.
Biochem Biophys Res Commun ; 533(4): 1109-1114, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33036753

RESUMO

Sulfoquinovose (6-deoxy-6-sulfoglucose, SQ) is a component of sulfolipids found in the photosynthetic membranes of plants and other photosynthetic organisms, and is one of the most abundant organosulfur compounds in nature. Microbial degradation of SQ, termed sulfoglycolysis, constitutes an important component of the biogeochemical sulfur cycle. Two sulfoglycolysis pathways have been reported, with one resembling the Embden-Meyerhof-Parnas (sulfo-EMP) pathway, and the other resembling the Entner-Doudoroff (sulfo-ED) pathway. Here we report a third sulfoglycolysis pathway in the bacterium Bacillus megaterium DSM 1804, in which sulfosugar cleavage is catalyzed by the transaldolase SqvA, which converts 6-deoxy-6-sulfofructose and glyceraldehyde 3-phosphate into fructose -6-phosphate and (S)-sulfolactaldehyde. Variations of this transaldolase-dependent sulfoglycolysis (sulfo-TAL) pathway are present in diverse bacteria, and add to the diversity of mechanisms for the degradation of this abundant organosulfur compound.


Assuntos
Bacillus megaterium/metabolismo , Glicólise , Redes e Vias Metabólicas , Metilglucosídeos/metabolismo , Transaldolase/metabolismo , Bacillus megaterium/enzimologia , Cromatografia Líquida , Biologia Computacional , Expressão Gênica , Glicólise/genética , Espectrometria de Massas , Redes e Vias Metabólicas/genética , Família Multigênica , Filogenia
9.
Chembiochem ; 21(6): 797-800, 2020 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-31587437

RESUMO

Uronic acid-rich plant materials such as pectin are potential renewable feedstocks for the chemical industry. Uronic acid oxidase activity was first reported in extracts of citrus leaves, and was subsequently found to be widely distributed in plants, with the highest activity detected in extracts of the pectin-rich citrus peel. Herein we report the identification of the primary sequence of uronic acid oxidase from extracts of the peel of Citrus sinensis, by partial purification and protein mass spectrometry. Activity of the enzyme, a member of the berberine bridge family, was confirmed by recombinant expression in Pichia pastoris. Biochemical characterization of the recombinant enzyme is reported. Our findings facilitate further investigation of the biological function of uronic acid oxidation in the economically important orange fruit, and also provide a basis for the development of a catalyst for bioconversion of uronic acids.


Assuntos
Citrus sinensis/enzimologia , Oxirredutases/análise , Ácidos Urônicos/análise , Oxirredução , Oxirredutases/metabolismo , Ácidos Urônicos/metabolismo
10.
Appl Environ Microbiol ; 86(7)2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31953335

RESUMO

Bacteria utilize diverse biochemical pathways for the degradation of the pyrimidine ring. The function of the pathways studied to date has been the release of nitrogen for assimilation. The most widespread of these pathways is the reductive pyrimidine catabolic pathway, which converts uracil into ammonia, carbon dioxide, and ß-alanine. Here, we report the characterization of a ß-alanine:pyruvate aminotransferase (PydD2) and an NAD+-dependent malonic semialdehyde dehydrogenase (MSDH) from a reductive pyrimidine catabolism gene cluster in Bacillus megaterium Together, these enzymes convert ß-alanine into acetyl coenzyme A (acetyl-CoA), a key intermediate in carbon and energy metabolism. We demonstrate the growth of B. megaterium in defined medium with uracil as its sole carbon and energy source. Homologs of PydD2 and MSDH are found in association with reductive pyrimidine pathway genes in many Gram-positive bacteria in the order Bacillales Our study provides a basis for further investigations of the utilization of pyrimidines as a carbon and energy source by bacteria.IMPORTANCE Pyrimidine has wide occurrence in natural environments, where bacteria use it as a nitrogen and carbon source for growth. Detailed biochemical pathways have been investigated with focus mainly on nitrogen assimilation in the past decades. Here, we report the discovery and characterization of two important enzymes, PydD2 and MSDH, which constitute an extension for the reductive pyrimidine catabolic pathway. These two enzymes, prevalent in Bacillales based on our bioinformatics studies, allow stepwise conversion of ß-alanine, a previous "end product" of the reductive pyrimidine degradation pathway, to acetyl-CoA as carbon and energy source.


Assuntos
Acetilcoenzima A/metabolismo , Bacillus megaterium/metabolismo , Redes e Vias Metabólicas , Uracila/metabolismo , Malonato-Semialdeído Desidrogenase (Acetilante)/metabolismo , beta-Alanina-Piruvato Transaminase/metabolismo
11.
Microb Cell Fact ; 19(1): 3, 2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31906943

RESUMO

Using an established CRISPR-Cas mediated genome editing technique for streptomycetes, we explored the combinatorial biosynthesis potential of the auroramycin biosynthetic gene cluster in Streptomyces roseosporous. Auroramycin is a potent anti-MRSA polyene macrolactam. In addition, auroramycin has antifungal activities, which is unique among structurally similar polyene macrolactams, such as incednine and silvalactam. In this work, we employed different engineering strategies to target glycosylation and acylation biosynthetic machineries within its recently elucidated biosynthetic pathway. Auroramycin analogs with variations in C-, N- methylation, hydroxylation and extender units incorporation were produced and characterized. By comparing the bioactivity profiles of five of these analogs, we determined that unique disaccharide motif of auroramycin is essential for its antimicrobial bioactivity. We further demonstrated that C-methylation of the 3, 5-epi-lemonose unit, which is unique among structurally similar polyene macrolactams, is key to its antifungal activity.


Assuntos
Antibacterianos/biossíntese , Antifúngicos/química , Vias Biossintéticas/genética , Engenharia Metabólica/métodos , Streptomyces/genética , Antibacterianos/química , Antibacterianos/farmacologia , Antifúngicos/farmacologia , Sistemas CRISPR-Cas , Edição de Genes/métodos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Polienos/química , Streptomyces/metabolismo
12.
Biochem J ; 476(4): 733-746, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30718306

RESUMO

Sulfoacetaldehyde reductase (IsfD) is a member of the short-chain dehydrogenase/reductase (SDR) family, involved in nitrogen assimilation from aminoethylsulfonate (taurine) in certain environmental and human commensal bacteria. IsfD catalyzes the reversible NADPH-dependent reduction of sulfoacetaldehyde, which is generated by transamination of taurine, forming hydroxyethylsulfonate (isethionate) as a waste product. In the present study, the crystal structure of Klebsiella oxytoca IsfD in a ternary complex with NADPH and isethionate was solved at 2.8 Å, revealing residues important for substrate binding. IsfD forms a homotetramer in both crystal and solution states, with the C-terminal tail of each subunit interacting with the C-terminal tail of the diagonally opposite subunit, forming an antiparallel ß sheet that constitutes part of the substrate-binding site. The sulfonate group of isethionate is stabilized by a hydrogen bond network formed by the residues Y148, R195, Q244 and a water molecule. In addition, F249 from the diagonal subunit restrains the conformation of Y148 to further stabilize the orientation of the sulfonate group. Mutation of any of these four residues into alanine resulted in a complete loss of catalytic activity for isethionate oxidation. Biochemical investigations of the substrate scope of IsfD, and bioinformatics analysis of IsfD homologs, suggest that IsfD is related to the promiscuous 3-hydroxyacid dehydrogenases with diverse metabolic functions.


Assuntos
Acetaldeído/análogos & derivados , Oxirredutases do Álcool/química , Proteínas de Bactérias/química , Klebsiella oxytoca/enzimologia , NADP/química , Multimerização Proteica , Acetaldeído/química , Acetaldeído/metabolismo , Oxirredutases do Álcool/metabolismo , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , NADP/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína
13.
Biochem J ; 476(11): 1605-1619, 2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31088892

RESUMO

Taurine aminotransferases catalyze the first step in taurine catabolism in many taurine-degrading bacteria and play an important role in bacterial taurine metabolism in the mammalian gut. Here, we report the biochemical and structural characterization of a new taurine:2-oxoglutarate aminotransferase from the human gut bacterium Bifidobacterium kashiwanohense (BkToa). Biochemical assays revealed high specificity of BkToa for 2-oxoglutarate as the amine acceptor. The crystal structure of BkToa in complex with pyridoxal 5'-phosphate (PLP) and glutamate was determined at 2.7 Šresolution. The enzyme forms a homodimer, with each monomer exhibiting a typical type I PLP-enzyme fold and conserved PLP-coordinating residues interacting with the PLP molecule. Two glutamate molecules are bound in sites near the predicted active site and they may occupy a path for substrate entry and product release. Molecular docking reveals a role for active site residues Trp21 and Arg156, conserved in Toa enzymes studied to date, in interacting with the sulfonate group of taurine. Bioinformatics analysis shows that the close homologs of BkToa are also present in other anaerobic gut bacteria.


Assuntos
Proteínas de Bactérias/química , Bifidobacterium/enzimologia , Transaminases/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bifidobacterium/genética , Bifidobacterium/isolamento & purificação , Domínio Catalítico/genética , Sequência Conservada , Cristalografia por Raios X , Trato Gastrointestinal/microbiologia , Humanos , Cinética , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Filogenia , Fosfato de Piridoxal/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Transaminases/genética , Transaminases/metabolismo
14.
Biochem J ; 476(15): 2271-2279, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31350331

RESUMO

Aminoethylsulfonate (taurine) is widespread in the environment and highly abundant in the human body. Taurine and other aliphatic sulfonates serve as sulfur sources for diverse aerobic bacteria, which carry out cleavage of the inert sulfonate C-S bond through various O2-dependent mechanisms. Taurine also serves as a sulfur source for certain strict anaerobic fermenting bacteria. However, the mechanism of C-S cleavage by these bacteria has long been a mystery. Here we report the biochemical characterization of an anaerobic pathway for taurine sulfur assimilation in a strain of Clostridium butyricum from the human gut. In this pathway, taurine is first converted to hydroxyethylsulfonate (isethionate), followed by C-S cleavage by the O2-sensitive isethionate sulfo-lyase IseG, recently identified in sulfate- and sulfite-reducing bacteria. Homologs of the enzymes described in this study have a sporadic distribution in diverse strict and facultative anaerobic bacteria, from both the environment and the taurine-rich human gut, and may enable sulfonate sulfur acquisition in certain nutrient limiting conditions.


Assuntos
Proteínas de Bactérias , Clostridium butyricum , Microbioma Gastrointestinal , Intestinos/microbiologia , Família Multigênica , Taurina , Anaerobiose , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clostridium butyricum/genética , Clostridium butyricum/metabolismo , Humanos , Ácido Isetiônico/metabolismo , Sulfatos/metabolismo , Taurina/biossíntese , Taurina/genética
15.
Appl Environ Microbiol ; 85(15)2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31126948

RESUMO

Hydroxyethyl sulfonate (isethionate) is widely distributed in the environment as an industrial pollutant and as a product of microbial metabolism. It is used as a substrate for growth by metabolically diverse environmental bacteria. Aerobic pathways for isethionate dissimilation in Gram-negative bacteria involve the cytochrome c-dependent oxidation of isethionate to sulfoacetaldehyde by a membrane-bound flavoenzyme (IseJ), followed by C-S cleavage by the thiamine pyrophosphate (TPP)-dependent enzyme sulfoacetaldehyde acetyltransferase (Xsc). Here, we report a bioinformatics analysis of Xsc-containing gene clusters in Gram-positive bacteria, which revealed the presence of an alternative isethionate dissimilation pathway involving the NAD+-dependent oxidation of isethionate by a cytosolic metal-dependent alcohol dehydrogenase (IseD). We describe the biochemical characterization of recombinant IseD from the haloalkaliphilic environmental bacterium Bacillus krulwichiae AM31DT and demonstrate the growth of this bacterium using isethionate as its sole carbon source, with the excretion of sulfite as a waste product. The IseD-dependent pathway provides the only mechanism for isethionate dissimilation in Gram-positive species to date and suggests a role of the metabolically versatile Bacilli in the mineralization of this ubiquitous organosulfur compound.IMPORTANCE Isethionate of biotic and industrial sources is prevalent. Dissimilation of isethionate under aerobic conditions is thus far only known in Gram-negative bacteria. Here, we report the discovery of a new pathway in Gram-positive Bacillus krulwichiae Isethionate is oxidized by a cytosolic metal-dependent alcohol dehydrogenase (which we named IseD), with NAD+ as the electron acceptor, generating sulfoacetaldehyde for subsequent cleavage by Xsc. This work highlights the diversity of organisms and pathways involved in the degradation of this ubiquitous organosulfonate. The new pathway that we discovered may play an important role in organosulfur mineralization and in the sulfur cycle in certain environments.


Assuntos
Acetaldeído/análogos & derivados , Acetiltransferases/genética , Bacillus/metabolismo , Proteínas de Bactérias/genética , Ácido Isetiônico/metabolismo , Acetaldeído/metabolismo , Família Multigênica
16.
Nat Chem Biol ; 2017 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-28398287

RESUMO

Here we report an efficient CRISPR-Cas9 knock-in strategy to activate silent biosynthetic gene clusters (BGCs) in streptomycetes. We applied this one-step strategy to activate multiple BGCs of different classes in five Streptomyces species and triggered the production of unique metabolites, including a novel pentangular type II polyketide in Streptomyces viridochromogenes. This potentially scalable strategy complements existing activation approaches and facilitates discovery efforts to uncover new compounds with interesting bioactivities.

17.
Chembiochem ; 2018 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-29799651

RESUMO

Silent biosynthetic gene clusters represent a potentially rich source of new bioactive compounds. We report the discovery, characterization, and biosynthesis of a novel doubly glycosylated 24-membered polyene macrolactam from a silent biosynthetic gene cluster in Streptomyces roseosporus by using the CRISPR-Cas9 gene cluster activation strategy. Structural characterization of this polyketide, named auroramycin, revealed a rare isobutyrylmalonyl extender unit and a unique pair of amino sugars. Relative and absolute stereochemistry were determined by using a combination of spectroscopic analyses, chemical derivatization, and computational analysis. The activated gene cluster for auroramycin production was also verified by transcriptional analyses and gene deletions. Finally, auroramycin exhibited potent anti-methicillin-resistant Staphylococcus aureus (anti-MRSA) activity towards clinical drug-resistant isolates.

18.
Bioorg Med Chem ; 26(7): 1275-1284, 2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28709846

RESUMO

Biocatalysis has been increasingly used for pharmaceutical synthesis in an effort to make manufacturing processes greener and more sustainable. Biocatalysts that possess excellent activity, specificity, thermostability and solvent-tolerance are highly sought after to meet the requirements of practical applications. Generating biocatalysts with these specific properties can be achieved by either discovery of novel biocatalysts or protein engineering. Meanwhile, chemoenzymatic routes have also been designed and developed for pharmaceutical synthesis on an industrial scale. This review discusses the recent discoveries, engineering, and applications of biocatalysts for the synthesis of pharmaceuticals and pharmaceutical intermediates. Key classes of biocatalysts include reductases, oxidases, hydrolases, lyases, isomerases, and transaminases.


Assuntos
Hidrolases/metabolismo , Isomerases/metabolismo , Liases/metabolismo , Oxirredutases/metabolismo , Preparações Farmacêuticas/metabolismo , Transaminases/metabolismo , Biocatálise , Humanos , Preparações Farmacêuticas/química , Engenharia de Proteínas
19.
J Ind Microbiol Biotechnol ; 45(7): 491-516, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29380152

RESUMO

In vivo biosensors can recognize and respond to specific cellular stimuli. In recent years, biosensors have been increasingly used in metabolic engineering and synthetic biology, because they can be implemented in synthetic circuits to control the expression of reporter genes in response to specific cellular stimuli, such as a certain metabolite or a change in pH. There are many types of natural sensing devices, which can be generally divided into two main categories: protein-based and nucleic acid-based. Both can be obtained either by directly mining from natural genetic components or by engineering the existing genetic components for novel specificity or improved characteristics. A wide range of new technologies have enabled rapid engineering and discovery of new biosensors, which are paving the way for a new era of biotechnological progress. Here, we review recent advances in the design, optimization, and applications of in vivo biosensors in the field of metabolic engineering and synthetic biology.


Assuntos
Técnicas Biossensoriais , Engenharia Metabólica/métodos , Proteínas/metabolismo , Biologia Sintética/métodos , Técnicas Biossensoriais/métodos , Genes Reporter
20.
Nat Prod Rep ; 33(8): 963-87, 2016 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-27072804

RESUMO

Covering up to end 2015Microbial fermentation provides an attractive alternative to chemical synthesis for the production of structurally complex natural products. In most cases, however, production titers are low and need to be improved for compound characterization and/or commercial production. Owing to advances in functional genomics and genetic engineering technologies, microbial hosts can be engineered to overproduce a desired natural product, greatly accelerating the traditionally time-consuming strain improvement process. This review covers recent developments and challenges in the engineering of native and heterologous microbial hosts for the production of bacterial natural products, focusing on the genetic tools and strategies for strain improvement. Special emphasis is placed on bioactive secondary metabolites from actinomycetes. The considerations for the choice of host systems will also be discussed in this review.


Assuntos
Actinomyces/química , Bactérias/química , Produtos Biológicos/metabolismo , Engenharia Genética , Estrutura Molecular
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