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1.
Microb Cell Fact ; 21(1): 11, 2022 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-35033086

RESUMO

BACKGROUND: The bacteriocin nisin is naturally produced by Lactococcus lactis as an inactive prepeptide that is modified posttranslationally resulting in five (methyl-)lanthionine rings characteristic for class Ia bacteriocins. Export and proteolytic cleavage of the leader peptide results in release of active nisin. By targeting the universal peptidoglycan precursor lipid II, nisin has a broad target spectrum including important human pathogens such as Listeria monocytogenes and methicillin-resistant Staphylococcus aureus strains. Industrial nisin production is currently performed using natural producer strains resulting in rather low product purity and limiting its application to preservation of dairy food products. RESULTS: We established heterologous nisin production using the biotechnological workhorse organism Corynebacterium glutamicum in a two-step process. We demonstrate successful biosynthesis and export of fully modified prenisin and its activation to mature nisin by a purified, soluble variant of the nisin protease NisP (sNisP) produced in Escherichia coli. Active nisin was detected by a L. lactis sensor strain with strictly nisin-dependent expression of the fluorescent protein mCherry. Following activation by sNisP, supernatants of the recombinant C. glutamicum producer strain cultivated in standard batch fermentations contained at least 1.25 mg/l active nisin. CONCLUSIONS: We demonstrate successful implementation of a two-step process for recombinant production of active nisin with C. glutamicum. This extends the spectrum of bioactive compounds that may be produced using C. glutamicum to a bacteriocin harboring complex posttranslational modifications. Our results provide a basis for further studies to optimize product yields, transfer production to sustainable substrates and purification of pharmaceutical grade nisin.


Assuntos
Corynebacterium glutamicum/metabolismo , Nisina/biossíntese , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/crescimento & desenvolvimento , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Nisina/química , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Precursores de Proteínas/biossíntese , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismo , Tripsina/metabolismo
2.
Metab Eng ; 2021 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-34864214

RESUMO

Lignin is an important structural component of terrestrial plants and is readily generated during biomass fractionation in lignocellulose processing facilities. Due to lacking alternatives the majority of technical lignins is industrially simply burned into heat and energy. However, regarding its vast abundance and a chemically interesting richness in aromatics, lignin is presently regarded as the most under-utilized and promising feedstock for value-added applications. Notably, microbes have evolved powerful enzymes and pathways that break down lignin and metabolize its various aromatic components. This natural pathway atlas meanwhile serves as a guiding star for metabolic engineers to breed designed cell factories and efficiently upgrade this global waste stream. The metabolism of aromatic compounds, in combination with success stories from systems metabolic engineering, as reviewed here, promises a sustainable product portfolio from lignin, comprising bulk and specialty chemicals, biomaterials, and fuels.

3.
Metab Eng ; 68: 34-45, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34492380

RESUMO

Bacteriocins are antimicrobial peptides produced by bacteria to inhibit competitors in their natural environments. Some of these peptides have emerged as commercial food preservatives and, due to the rapid increase in antibiotic resistant bacteria, are also discussed as interesting alternatives to antibiotics for therapeutic purposes. Currently, commercial bacteriocins are produced exclusively with natural producer organisms on complex substrates and are sold as semi-purified preparations or crude fermentates. To allow clinical application, efficacy of production and purity of the product need to be improved. This can be achieved by shifting production to recombinant microorganisms. Here, we identify Corynebacterium glutamicum as a suitable production host for the bacteriocin pediocin PA-1. C. glutamicum CR099 shows resistance to high concentrations of pediocin PA-1 and the bacteriocin was not inactivated when spiked into growing cultures of this bacterium. Recombinant C. glutamicum expressing a synthetic pedACDCgl operon releases a compound that has potent antimicrobial activity against Listeria monocytogenes and Listeria innocua and matches size and mass:charge ratio of commercial pediocin PA-1. Fermentations in shake flasks and bioreactors suggest that low levels of dissolved oxygen are favorable for production of pediocin. Under these conditions, however, reduced activity of the TCA cycle resulted in decreased availability of the important pediocin precursor l-asparagine suggesting options for further improvement. Overall, we demonstrate that C. glutamicum is a suitable host for recombinant production of bacteriocins of the pediocin family.


Assuntos
Bacteriocinas , Corynebacterium glutamicum , Listeria , Bacteriocinas/genética , Corynebacterium glutamicum/genética , Pediocinas/genética
4.
Anal Chem ; 93(27): 9428-9436, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34197087

RESUMO

Stable-isotope labeling experiments are widely used to investigate the topology and functioning of metabolic networks. Label incorporation into metabolites can be quantified using a broad range of mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy methods, but in general, no single approach can completely cover isotopic space, even for small metabolites. The number of quantifiable isotopic species could be increased and the coverage of isotopic space improved by integrating measurements obtained by different methods; however, this approach has remained largely unexplored because no framework able to deal with partial, heterogeneous isotopic measurements has yet been developed. Here, we present a generic computational framework based on symbolic calculus that can integrate any isotopic data set by connecting measurements to the chemical structure of the molecules. As a test case, we apply this framework to isotopic analyses of amino acids, which are ubiquitous to life, central to many biological questions, and can be analyzed by a broad range of MS and NMR methods. We demonstrate how this integrative framework helps to (i) clarify and improve the coverage of isotopic space, (ii) evaluate the complementarity and redundancy of different techniques, (iii) consolidate isotopic data sets, (iv) design experiments, and (v) guide future analytical developments. This framework, which can be applied to any labeled element, isotopic tracer, metabolite, and analytical platform, has been implemented in IsoSolve (available at https://github.com/MetaSys-LISBP/IsoSolve and https://pypi.org/project/IsoSolve), an open-source software that can be readily integrated into data analysis pipelines.


Assuntos
Aminoácidos , Software , Isótopos de Carbono , Marcação por Isótopo , Espectroscopia de Ressonância Magnética , Espectrometria de Massas
5.
Metab Eng ; 67: 293-307, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34314893

RESUMO

Seaweeds emerge as promising third-generation renewable for sustainable bioproduction. In the present work, we valorized brown seaweed to produce l-lysine, the world's leading feed amino acid, using Corynebacterium glutamicum, which was streamlined by systems metabolic engineering. The mutant C. glutamicum SEA-1 served as a starting point for development because it produced small amounts of l-lysine from mannitol, a major seaweed sugar, because of the deletion of its arabitol repressor AtlR and its engineered l-lysine pathway. Starting from SEA-1, we systematically optimized the microbe to redirect excess NADH, formed on the sugar alcohol, towards NADPH, required for l-lysine synthesis. The mannitol dehydrogenase variant MtlD D75A, inspired by 3D protein homology modelling, partly generated NADPH during the oxidation of mannitol to fructose, leading to a 70% increased l-lysine yield in strain SEA-2C. Several rounds of strain engineering further increased NADPH supply and l-lysine production. The best strain, SEA-7, overexpressed the membrane-bound transhydrogenase pntAB together with codon-optimized gapN, encoding NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase, and mak, encoding fructokinase. In a fed-batch process, SEA-7 produced 76 g L-1l-lysine from mannitol at a yield of 0.26 mol mol-1 and a maximum productivity of 2.1 g L-1 h-1. Finally, SEA-7 was integrated into seaweed valorization cascades. Aqua-cultured Laminaria digitata, a major seaweed for commercial alginate, was extracted and hydrolyzed enzymatically, followed by recovery and clean-up of pure alginate gum. The residual sugar-based mixture was converted to l-lysine at a yield of 0.27 C-mol C-mol-1 using SEA-7. Second, stems of the wild-harvested seaweed Durvillaea antarctica, obtained as waste during commercial processing of the blades for human consumption, were extracted using acid treatment. Fermentation of the hydrolysate using SEA-7 provided l-lysine at a yield of 0.40 C-mol C-mol-1. Our findings enable improvement of the efficiency of seaweed biorefineries using tailor-made C. glutamicum strains.


Assuntos
Corynebacterium glutamicum , Alga Marinha , Corynebacterium glutamicum/genética , Humanos , Lisina/genética , Engenharia Metabólica , NADP
6.
Microb Cell Fact ; 20(1): 111, 2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34082758

RESUMO

BACKGROUND: Pamamycins are macrodiolides of polyketide origin which form a family of differently large homologues with molecular weights between 579 and 663. They offer promising biological activity against pathogenic fungi and gram-positive bacteria. Admittedly, production titers are very low, and pamamycins are typically formed as crude mixture of mainly smaller derivatives, leaving larger derivatives rather unexplored so far. Therefore, strategies that enable a more efficient production of pamamycins and provide increased fractions of the rare large derivatives are highly desired. Here we took a systems biology approach, integrating transcription profiling by RNA sequencing and intracellular metabolite analysis, to enhance pamamycin production in the heterologous host S. albus J1074/R2. RESULTS: Supplemented with L-valine, the recombinant producer S. albus J1074/R2 achieved a threefold increased pamamycin titer of 3.5 mg L-1 and elevated fractions of larger derivatives: Pam 649 was strongly increased, and Pam 663 was newly formed. These beneficial effects were driven by increased availability of intracellular CoA thioesters, the building blocks for the polyketide, resulting from L-valine catabolism. Unfavorably, L-valine impaired growth of the strain, repressed genes of mannitol uptake and glycolysis, and suppressed pamamycin formation, despite the biosynthetic gene cluster was transcriptionally activated, restricting production to the post L-valine phase. A deletion mutant of the transcriptional regulator bkdR, controlling a branched-chain amino acid dehydrogenase complex, revealed decoupled pamamycin biosynthesis. The regulator mutant accumulated the polyketide independent of the nutrient status. Supplemented with L-valine, the novel strain enabled the biosynthesis of pamamycin mixtures with up to 55% of the heavy derivatives Pam 635, Pam 649, and Pam 663: almost 20-fold more than the wild type. CONCLUSIONS: Our findings open the door to provide rare heavy pamamycins at markedly increased efficiency and facilitate studies to assess their specific biological activities and explore this important polyketide further.


Assuntos
Macrolídeos/metabolismo , Policetídeos/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Fatores de Transcrição/genética , Valina/metabolismo , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/genética , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/metabolismo , Proteínas de Bactérias/genética , Vias Biossintéticas , Perfilação da Expressão Gênica/métodos , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Microbiologia Industrial , Metaboloma , Família Multigênica , Mutação
7.
Essays Biochem ; 65(2): 197-212, 2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34096577

RESUMO

The soil microbe Corynebacterium glutamicum is a leading workhorse in industrial biotechnology and has become famous for its power to synthetise amino acids and a range of bulk chemicals at high titre and yield. The product portfolio of the microbe is continuously expanding. Moreover, metabolically engineered strains of C. glutamicum produce more than 30 high value active ingredients, including signature molecules of raspberry, savoury, and orange flavours, sun blockers, anti-ageing sugars, and polymers for regenerative medicine. Herein, we highlight recent advances in engineering of the microbe into novel cell factories that overproduce these precious molecules from pioneering proofs-of-concept up to industrial productivity.

8.
Biotechnol Bioeng ; 118(8): 3076-3093, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33974270

RESUMO

Actinobacteria provide a rich spectrum of bioactive natural products and therefore display an invaluable source towards commercially valuable pharmaceuticals and agrochemicals. Here, we studied the use of inorganic talc microparticles (hydrous magnesium silicate, 3MgO·4SiO2 ·H2 O, 10 µm) as a general supplement to enhance natural product formation in this important class of bacteria. Added to cultures of recombinant Streptomyces lividans, talc enhanced production of the macrocyclic peptide antibiotic bottromycin A2 and its methylated derivative Met-bottromycin A2 up to 109 mg L-1 , the highest titer reported so far. Hereby, the microparticles fundamentally affected metabolism. With 10 g L-1 talc, S. lividans grew to 40% smaller pellets and, using RNA sequencing, revealed accelerated morphogenesis and aging, indicated by early upregulation of developmental regulator genes such as ssgA, ssgB, wblA, sigN, and bldN. Furthermore, the microparticles re-balanced the expression of individual bottromycin cluster genes, resulting in a higher macrocyclization efficiency at the level of BotAH and correspondingly lower levels of non-cyclized shunt by-products, driving the production of mature bottromycin. Testing a variety of Streptomyces species, talc addition resulted in up to 13-fold higher titers for the RiPPs bottromycin and cinnamycin, the alkaloid undecylprodigiosin, the polyketide pamamycin, the tetracycline-type oxytetracycline, and the anthramycin-analogs usabamycins. Moreover, talc addition boosted production in other actinobacteria, outside of the genus of Streptomyces: vancomycin (Amycolatopsis japonicum DSM 44213), teicoplanin (Actinoplanes teichomyceticus ATCC 31121), and the angucyclinone-type antibiotic simocyclinone (Kitasatospora sp.). For teicoplanin, the microparticles were even crucial to activate production. Taken together, the use of talc was beneficial in 75% of all tested cases and optimized natural and heterologous hosts forming the substance of interest with clusters under native and synthetic control. Given its simplicity and broad benefits, microparticle-supplementation appears as an enabling technology in natural product research of these most important microbes.

9.
Metab Eng ; 67: 11-18, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34051369

RESUMO

Pamamycins, a group of polyketides originally discovered in Streptomyces alboniger, induce sporulation in Streptomyces and inhibit the growth of Gram-positive bacteria, Mycobacterium tuberculosis and fungi. The pamamycin biosynthetic gene cluster encodes 6 ketosynthases that utilize a variety of three-carbon to five-carbon CoA thioesters as starter and extender units. This promiscuity in production results in an up to 18 different derivatives during fermentation. For more-selective production and simplified purification, we aimed to modify the precursor supply to narrow the spectrum of the produced derivatives. Eight genes potentially responsible for the supply of two major precursors, 2-S-methylmalonyl-CoA and 2-S-ethylmalonyl-CoA, were identified using the NCBI Basic Local Alignment Search Tool (BLAST) against the genome of the heterologous host S. albus J1074. Knockout mutants of the identified genes were constructed and their impact on intracellular CoA ester concentrations and on the production of pamamycins was determined. The created mutants enabled us to conclusively identify the ethylmalonyl-CoA supplying routes and their impact on the production of pamamycin. Furthermore, we gained significant information on the origin of the methylmalonyl-CoA supply in Streptomyces albus.


Assuntos
Streptomyces , Macrolídeos , Streptomyces/genética
10.
Microb Cell Fact ; 20(1): 109, 2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34049541

RESUMO

BACKGROUND: Plant-based milk alternatives are more popular than ever, and chickpea-based milks are among the most commercially relevant products. Unfortunately, limited nutritional value because of low levels of the essential amino acid L-lysine, low digestibility and unpleasant taste are challenges that must be addressed to improve product quality and meet consumer expectations. RESULTS: Using in-silico screening and food safety classifications, 31 strains were selected as potential L-lysine producers from approximately 2,500 potential candidates. Beneficially, 30% of the isolates significantly accumulated amino acids (up to 1.4 mM) during chickpea milk fermentation, increasing the natural level by up to 43%. The best-performing strains, B. amyloliquefaciens NCC 156 and L. paracasei subsp. paracasei NCC 2511, were tested further. De novo lysine biosynthesis was demonstrated in both strains by 13C metabolic pathway analysis. Spiking small amounts of citrate into the fermentation significantly activated L-lysine biosynthesis in NCC 156 and stimulated growth. Both microbes revealed additional benefits in eliminating indigestible sugars such as stachyose and raffinose and converting off-flavour aldehydes into the corresponding alcohols and acids with fruity and sweet notes. CONCLUSIONS: B. amyloliquefaciens NCC 156 and L. paracasei subsp. paracasei NCC 2511 emerged as multi-benefit microbes for chickpea milk fermentation with strong potential for industrial processing of the plant material. Given the high number of L-lysine-producing isolates identified in silico, this concept appears promising to support strain selection for food fermentation.


Assuntos
Vias Biossintéticas , Aromatizantes/metabolismo , Lactobacillales/genética , Lactobacillales/metabolismo , Lisina/biossíntese , Substitutos do Leite/metabolismo , Açúcares/metabolismo , Cicer/metabolismo , Fermentação , Microbiologia de Alimentos , Genoma Bacteriano , Lactobacillales/isolamento & purificação , Paladar
11.
Curr Opin Biotechnol ; 69: 199-211, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33540327

RESUMO

Polyunsaturated fatty acids (PUFAs), primarily docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), have received worldwide attention in recent years due to an increasing awareness of their uniqueness in improving diet and human health and their apparently inevitable shortage in global availability. Microbial cell factories are a major solution to supplying these precious molecules in sufficient amounts and providing PUFA-rich aquafeed, superfoods, and medical formulations. This review assesses the PUFA world markets and highlights recent advances in upgrading and streamlining microalgae, yeasts, fungi, and bacteria for high-level PUFA production and broadening of the PUFA spectrum.


Assuntos
Ácidos Graxos Ômega-3 , Microalgas , Preparações Farmacêuticas , Ácidos Docosa-Hexaenoicos , Ácido Eicosapentaenoico , Ácidos Graxos , Ácidos Graxos Insaturados , Fungos , Humanos
12.
Microb Biotechnol ; 14(6): 2385-2402, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33171015

RESUMO

Lignin-based aromatics are attractive raw materials to derive medium-chain length poly(3-hydroxyalkanoates) (mcl-PHAs), biodegradable polymers of commercial value. So far, this conversion has exclusively used the ortho-cleavage route of Pseudomonas putida KT2440, which results in the secretion of toxic intermediates and limited performance. Pseudomonas putida H exhibits the ortho- and the meta-cleavage pathways where the latter appears promising because it stoichiometrically yields higher levels of acetyl-CoA. Here, we created a double-mutant H-ΔcatAΔA2 that utilizes the meta route exclusively and synthesized 30% more PHA on benzoate than the parental strain but suffered from catechol accumulation. The single deletion of the catA2 gene in the H strain provoked a slight attenuation on the enzymatic capacity of the ortho route (25%) and activation of the meta route by nearly 8-fold, producing twice as much mcl-PHAs compared to the wild type. Inline, the mutant H-ΔcatA2 showed a 2-fold increase in the intracellular malonyl-CoA abundance - the main precursor for mcl-PHAs synthesis. As inferred from flux simulation and enzyme activity assays, the superior performance of H-ΔcatA2 benefited from reduced flux through the TCA cycle and malic enzyme and diminished by-product formation. In a benzoate-based fed-batch, P. putida H-ΔcatA2 achieved a PHA titre of 6.1 g l-1 and a volumetric productivity of 1.8 g l-1 day-1 . Using Kraft lignin hydrolysate as feedstock, the engineered strain formed 1.4 g l- 1 PHA. The balancing of carbon flux between the parallel catechol-degrading routes emerges as an important strategy to prevent intermediate accumulation and elevate mcl-PHA production in P. putida H and, as shown here, sets the next level to derive this sustainable biopolymer from lignin hydrolysates and aromatics.

13.
Proc Natl Acad Sci U S A ; 117(42): 26374-26381, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-33020286

RESUMO

Mechanistic understanding of the factors that govern host tropism remains incompletely understood for most pathogens. Brucella species, which are capable of infecting a wide range of hosts, offer a useful avenue to address this question. We hypothesized that metabolic fine-tuning to intrahost niches is likely an underappreciated axis underlying pathogens' ability to infect new hosts and tropism. In this work, we compared the central metabolism of seven Brucella species by stable isotopic labeling and genetics. We identified two functionally distinct groups, one overlapping with the classical zoonotic species of domestic livestock that exclusively use the pentose phosphate pathway (PPP) for hexose catabolism, whereas species from the second group use mostly the Entner-Doudoroff pathway (EDP). We demonstrated that the metabolic dichotomy among Brucellae emerged after the acquisition of two independent EDP-inactivating mutations in all classical zoonotic species. We then examined the pathogenicity of key metabolic mutants in mice and confirmed that this trait is tied to virulence. Altogether, our data are consistent with the hypothesis that the PPP has been incrementally selected over the EDP in parallel to Brucella adaptation to domestic livestock.


Assuntos
Brucella/genética , Brucella/metabolismo , Via de Pentose Fosfato/genética , Adaptação Biológica/genética , Animais , Zoonoses Bacterianas/genética , Evolução Biológica , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Via de Pentose Fosfato/fisiologia , Fenótipo , Virulência
14.
Curr Opin Biotechnol ; 65: vi-vii, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32921551

Assuntos
Biotecnologia
15.
Appl Microbiol Biotechnol ; 104(18): 7745-7766, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32789744

RESUMO

Pseudomonas putida is a Gram-negative, rod-shaped bacterium that can be encountered in diverse ecological habitats. This ubiquity is traced to its remarkably versatile metabolism, adapted to withstand physicochemical stress, and the capacity to thrive in harsh environments. Owing to these characteristics, there is a growing interest in this microbe for industrial use, and the corresponding research has made rapid progress in recent years. Hereby, strong drivers are the exploitation of cheap renewable feedstocks and waste streams to produce value-added chemicals and the steady progress in genetic strain engineering and systems biology understanding of this bacterium. Here, we summarize the recent advances and prospects in genetic engineering, systems and synthetic biology, and applications of P. putida as a cell factory. KEY POINTS: • Pseudomonas putida advances to a global industrial cell factory. • Novel tools enable system-wide understanding and streamlined genomic engineering. • Applications of P. putida range from bioeconomy chemicals to biosynthetic drugs.


Assuntos
Pseudomonas putida , Biotecnologia , Genômica , Pseudomonas putida/genética , Biologia Sintética , Biologia de Sistemas
16.
Microb Cell Fact ; 19(1): 160, 2020 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-32778124

RESUMO

BACKGROUND: Thioesters of coenzyme A participate in 5% of all enzymatic reactions. In microbial cell factories, they function as building blocks for products of recognized commercial value, including natural products such as polyketides, polyunsaturated fatty acids, biofuels, and biopolymers. A core spectrum of approximately 5-10 short chain thioesters is present in many microbes, as inferred from their genomic repertoire. The relevance of these metabolites explains the high interest to trace and quantify them in microbial cells. RESULTS: Here, we describe a common workflow for extraction and absolute quantification of short chain CoA thioesters in different gram-positive and gram-negative bacteria and eukaryotic yeast, i.e. Corynebacterium glutamicum, Streptomyces albus, Pseudomonas putida, and Yarrowia lipolytica. The approach assessed intracellular CoA thioesters down to the picomolar level and exhibited high precision and reproducibility for all microbes, as shown by principal component analysis. Furthermore, it provided interesting insights into microbial CoA metabolism. A succinyl-CoA synthase defective mutant of C. glutamicum exhibited an unaffected level of succinyl-CoA that indicated a complete compensation by the L-lysine pathway to bypass the disrupted TCA cycle. Methylmalonyl-CoA, an important building block of high-value polyketides, was identified as dominant CoA thioester in the actinomycete S. albus. The microbe revealed a more than 10,000-fold difference in the abundance of intracellular CoA thioesters. A recombinant strain of S. albus, which produced different derivatives of the antituberculosis polyketide pamamycin, revealed a significant depletion of CoA thioesters of the ethylmalonyl CoA pathway, influencing product level and spectrum. CONCLUSIONS: The high relevance of short chain CoA thioesters to synthetize industrial products and the interesting insights gained from the examples shown in this work, suggest analyzing these metabolites in microbial cell factories more routinely than done so far. Due to its broad application range, the developed approach appears useful to be applied this purpose. Hereby, the possibility to use one single protocol promises to facilitate automatized efforts, which rely on standardized workflows.


Assuntos
Coenzima A/análise , Ésteres/análise , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/metabolismo , Engenharia Metabólica/métodos , Yarrowia/metabolismo , Técnicas de Cultura Celular por Lotes , Bactérias Gram-Negativas/genética , Bactérias Gram-Positivas/genética , Espaço Intracelular/química , Reprodutibilidade dos Testes , Yarrowia/genética
17.
Biotechnol Bioeng ; 117(12): 3858-3875, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32808679

RESUMO

Streptomyces spp. are a rich source for natural products with recognized industrial value, explaining the high interest to improve and streamline the performance of in these microbes. Here, we studied the production of pamamycins, macrodiolide homologs with a high activity against multiresistant pathogenic microbes, using recombinant Streptomyces albus J1074/R2. Talc particles (hydrous magnesium silicate, 3MgO·4SiO2 ·H2 O) of micrometer size, added to submerged cultures of the recombinant strain, tripled pamamycin production up to 50 mg/L. Furthermore, they strongly affected morphology, reduced the size of cell pellets formed by the filamentous microbe during the process up to sixfold, and shifted the pamamycin spectrum to larger derivatives. Integrated analysis of transcriptome and precursor (CoA thioester) supply of particle-enhanced and control cultures provided detailed insights into the underlying molecular changes. The microparticles affected the expression of 3,341 genes (56% of all genes), revealing a global and fundamental impact on metabolism. Morphology-associated genes, encoding major regulators such as SsgA, RelA, EshA, Factor C, as well as chaplins and rodlins, were found massively upregulated, indicating that the particles caused a substantially accelerated morphogenesis. In line, the pamamycin cluster was strongly upregulated (up to 1,024-fold). Furthermore, the microparticles perturbed genes encoding for CoA-ester metabolism, which were mainly activated. The altered expression resulted in changes in the availability of intracellular CoA-esters, the building blocks of pamamycin. Notably, the ratio between methylmalonyl CoA and malonyl-CoA was increased fourfold. Both metabolites compete for incorporation into pamamycin so that the altered availability explained the pronounced preference for larger derivatives in the microparticle-enhanced process. The novel insights into the behavior of S. albus in response to talc appears of general relevance to further explore and upgrade the concept of microparticle enhanced cultivation, widely used for filamentous microbes.

18.
Curr Opin Biotechnol ; 65: 118-128, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32199140

RESUMO

Extremolytes are small organic molecules, which protect cells under extreme, virtually inhabitable conditions. Their exceptional properties can be translated into health-promoting and therapeutic activities, which open an avenue of opportunities for the cosmetic, medical, and food industries. Supported by powerful approaches from systems and synthetic biology and systems metabolic engineering, the bio-industry becomes more and more attracted to exploit this 'goldmine'. In addition to the well-established flagship ectoine, several novel extremolytes have emerged in the past years and high-efficiency cell factories have been created for bio-based extremolyte production. Here, we review recent prominent examples and success stories in the field.


Assuntos
Engenharia Metabólica , Biologia Sintética , Atenção à Saúde
19.
mBio ; 11(2)2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32184246

RESUMO

Pseudomonas aeruginosa is an opportunistic human pathogen, particularly noted for causing infections in the lungs of people with cystic fibrosis (CF). Previous studies have shown that the gene expression profile of P. aeruginosa appears to converge toward a common metabolic program as the organism adapts to the CF airway environment. However, we still have only a limited understanding of how these transcriptional changes impact metabolic flux at the systems level. To address this, we analyzed the transcriptome, proteome, and fluxome of P. aeruginosa grown on glycerol or acetate. These carbon sources were chosen because they are the primary breakdown products of an airway surfactant, phosphatidylcholine, which is known to be a major carbon source for P. aeruginosa in CF airways. We show that the fluxes of carbon throughout central metabolism are radically different among carbon sources. For example, the newly recognized "EDEMP cycle" (which incorporates elements of the Entner-Doudoroff [ED] pathway, the Embden-Meyerhof-Parnas [EMP] pathway, and the pentose phosphate [PP] pathway) plays an important role in supplying NADPH during growth on glycerol. In contrast, the EDEMP cycle is attenuated during growth on acetate, and instead, NADPH is primarily supplied by the reaction catalyzed by isocitrate dehydrogenase(s). Perhaps more importantly, our proteomic and transcriptomic analyses revealed a global remodeling of gene expression during growth on the different carbon sources, with unanticipated impacts on aerobic denitrification, electron transport chain architecture, and the redox economy of the cell. Collectively, these data highlight the remarkable metabolic plasticity of P. aeruginosa; that plasticity allows the organism to seamlessly segue between different carbon sources, maximizing the energetic yield from each.IMPORTANCE Pseudomonas aeruginosa is an opportunistic human pathogen that is well known for causing infections in the airways of people with cystic fibrosis. Although it is clear that P. aeruginosa is metabolically well adapted to life in the CF lung, little is currently known about how the organism metabolizes the nutrients available in the airways. In this work, we used a combination of gene expression and isotope tracer ("fluxomic") analyses to find out exactly where the input carbon goes during growth on two CF-relevant carbon sources, acetate and glycerol (derived from the breakdown of lung surfactant). We found that carbon is routed ("fluxed") through very different pathways during growth on these substrates and that this is accompanied by an unexpected remodeling of the cell's electron transfer pathways. Having access to this "blueprint" is important because the metabolism of P. aeruginosa is increasingly being recognized as a target for the development of much-needed antimicrobial agents.


Assuntos
Adaptação Fisiológica , Carbono/metabolismo , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Acetatos/metabolismo , Perfilação da Expressão Gênica , Glucose/metabolismo , Glicólise , Via de Pentose Fosfato , Proteômica
20.
Curr Opin Biotechnol ; 65: 102-113, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32171887

RESUMO

Sustainable production from seaweed has grown into an area of intense research and development. Meanwhile, more than 30 million tonnes of seaweed are produced, of which 70% are used as food and 30% have other applications such as feed, fertilizer, chemicals, and energy. Towards biorefining seaweed in an environmentally friendly and economically viable manner, we need efficient approaches that convert its biomass and residuals into added value products. Smart cell factories and fermentation strategies which can be integrated into future seaweed biorefineries are at the heart of the development and therefore receive increasing attention. Here, we review advances in the field including novel fermentation routes from seaweed to chemicals, materials, pharmaceuticals, fuels and energy, and discuss challenges and opportunities.


Assuntos
Alga Marinha , Biomassa , Fermentação
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