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1.
Microb Cell Fact ; 18(1): 163, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31581944

RESUMO

BACKGROUND: Sustainable production of microbial fatty acids derivatives has the potential to replace petroleum based equivalents in the chemical, cosmetic and pharmaceutical industry. Most fatty acid sources for production oleochemicals are currently plant derived. However, utilization of these crops are associated with land use change and food competition. Microbial oils could be an alternative source of fatty acids, which circumvents the issue with agricultural competition. RESULTS: In this study, we generated a chimeric microbial production system that features aspects of both prokaryotic and eukaryotic fatty acid biosynthetic pathways targeted towards the generation of long chain fatty acids. We redirected the type-II fatty acid biosynthetic pathway of Escherichia coli BL21 (DE3) strain by incorporating two homologues of the beta-ketoacyl-[acyl carrier protein] synthase I and II from the chloroplastic fatty acid biosynthetic pathway of Arabidopsis thaliana. The microbial clones harboring the heterologous pathway yielded 292 mg/g and 220 mg/g DCW for KAS I and KAS II harboring plasmids respectively. Surprisingly, beta-ketoacyl synthases KASI/II isolated from A. thaliana showed compatibility with the FAB pathway in E. coli. CONCLUSION: The efficiency of the heterologous plant enzymes supersedes the overexpression of the native enzyme in the E. coli production system, which leads to cell death in fabF overexpression and fabB deletion mutants. The utilization of our plasmid based system would allow generation of plant like fatty acids in E. coli and their subsequent chemical or enzymatic conversion to high end oleochemical products.


Assuntos
Arabidopsis/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ácido Graxo Sintases/metabolismo , Ácidos Graxos/biossíntese , Engenharia Metabólica , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/síntese química , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/síntese química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Vias Biossintéticas , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Ácido Graxo Sintases/genética , Ácidos Graxos/química , Isoenzimas/síntese química , Isoenzimas/genética , Isoenzimas/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo
3.
J Agric Food Chem ; 67(38): 10678-10684, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31475535

RESUMO

γ-Hydroxyvalerate (4HV) is an important monomer used to produce various valuable polymers and products. In this study, an engineered 3-hydroxybutyrate dehydrogenase that can convert levulinic acid (LA) into 4HV was co-expressed with a cofactor (NADH) regeneration system mediated by an NAD+-dependent formate dehydrogenase (CbFDH) in the Escherichia coli strain, MG1655. The resulting strain produced 23-fold more 4HV in a shake flask. The 4HV production was not dependent on ATP and required low aeration; all of these are considered beneficial characteristics for the production of target compounds, especially at an industrial scale. Under optimized conditions in a 5 L fermenter, the titer, productivity, and molar conversion efficiency for 4HV reached 100 g/L, 4.2 g/L/h, and 92%, respectively. Our system could prove to be a promising method for the large-scale production of 4HV from LA at low-cost and using a renewable biomass source.


Assuntos
Escherichia coli/metabolismo , Ácidos Levulínicos/metabolismo , Valeratos/metabolismo , Trifosfato de Adenosina/metabolismo , Biotransformação , Escherichia coli/genética , Fermentação , Engenharia Metabólica
4.
Microb Cell Fact ; 18(1): 160, 2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31547812

RESUMO

BACKGROUND: Alpha-Terpineol (α-Terpineol), a C10 monoterpenoid alcohol, is widely used in the cosmetic and pharmaceutical industries. Construction Saccharomyces cerevisiae cell factories for producing monoterpenes offers a promising means to substitute chemical synthesis or phytoextraction. RESULTS: α-Terpineol was produced by expressing the truncated α-Terpineol synthase (tVvTS) from Vitis vinifera in S. cerevisiae. The α-Terpineol titer was increased to 0.83 mg/L with overexpression of the rate-limiting genes tHMG1, IDI1 and ERG20F96W-N127W. A GSGSGSGSGS linker was applied to fuse ERG20F96W-N127W with tVvTS, and expressing the fusion protein increased the α-Terpineol production by 2.87-fold to 2.39 mg/L when compared with the parental strain. In addition, we found that farnesyl diphosphate (FPP) accumulation by down-regulation of ERG9 expression and deletion of LPP1 and DPP1 did not improve α-Terpineol production. Therefore, ERG9 was overexpressed and the α-Terpineol titer was further increased to 3.32 mg/L. The best α-Terpineol producing strain LCB08 was then used for batch and fed-batch fermentation in a 5 L bioreactor, and the production of α-Terpineol was ultimately improved to 21.88 mg/L. CONCLUSIONS: An efficient α-Terpineol production cell factory was constructed by engineering the S. cerevisiae mevalonate pathway, and the metabolic engineering strategies could also be applied to produce other valuable monoterpene compounds in yeast.


Assuntos
Cicloexenos/metabolismo , Engenharia Metabólica , Monoterpenos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Sesquiterpenos/metabolismo , Vitis/enzimologia , Vitis/genética
5.
Bioresour Technol ; 293: 122054, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31487616

RESUMO

This work presents the exploitation of waste industrial by-products as raw materials for the production of microbial lipids in engineered strains of the filamentous fungus Ashbya gossypii. A lipogenic xylose-utilizing strain was used to apply a metabolic engineering approach aiming at relieving regulatory mechanisms to further increase the biosynthesis of lipids. Three genomic manipulations were applied: the overexpression of a feedback resistant form of the acetyl-CoA carboxylase enzyme; the expression of a truncated form of Mga2, a regulator of the main Δ9 desaturase gene; and the overexpression of an additional copy of DGA1 that codes for diacylglycerol acyltransferase. The performance of the engineered strain was evaluated in culture media containing mixed formulations of corn-cob hydrolysates, sugarcane molasses or crude glycerol. Our results demonstrate the efficiency of the engineered strains, which were able to accumulate about 40% of cell dry weight (CDW) in lipid content using organic industrial wastes as feedstocks.


Assuntos
Eremothecium , Xilose , Resíduos Industriais , Lipídeos , Engenharia Metabólica
6.
World J Microbiol Biotechnol ; 35(8): 129, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31376017

RESUMO

Metal whole-cell biosensors (WCBs) have been reported as very useful tools to detect and quantify the presence of bioavailable fractions of certain metals in water and soil samples. In the current work, two bacterial WCBs able to report Cr(VI) presence and plants growing on Cr(VI)-enriched soil/medium were used to assess the potential transfer of this metal to organisms of higher trophic levels, and the risk of transfer to the food chain. To do it, the functionality of the WCBs within tissues of inoculated plants in contact with Cr(VI)-contaminated soil and water was studied in vitro and in a controlled greenhouse environment. One WCB was the previously described Ochrobactrum tritici pCHRGFP2 and the second, Nitrospirillum amazonense pCHRGFP2, is a newly engineered naturally-occurring endophytic microorganism. Three rice varieties (IAC 4440, BRS 6 CHUÍ, IRGA 425) and one maize variety (1060) were tested as hosts and subjected to Cr(VI) treatments (25 µM), with different results obtained. Inoculation of each WCB into plants exposed to Cr(VI) showed GFP expression within plant tissues. WCBs penetrated the root tissues and later colonized the shoots and leaves. In general, a higher fluorescence signal was detected in roots, together with a higher Cr content and denser WCB colonization. Best fluorescence intensities per plant biomass of shoots were obtained for plant host IRGA 425. Therefore, by analyzing colonized tissues, both WCBs allowed the detection of Cr(VI) contamination in soils and its transfer to plants commonly used in crops for human diet.


Assuntos
Técnicas Biossensoriais/métodos , Cromo/análise , Ochrobactrum/crescimento & desenvolvimento , Oryza/química , Rhodospirillaceae/crescimento & desenvolvimento , Zea mays/química , Disponibilidade Biológica , Engenharia Metabólica , Microscopia de Fluorescência , Ochrobactrum/genética , Ochrobactrum/metabolismo , Oryza/crescimento & desenvolvimento , Oryza/microbiologia , Rhodospirillaceae/genética , Rhodospirillaceae/metabolismo , Poluentes do Solo/análise , Poluentes Químicos da Água/análise , Zea mays/crescimento & desenvolvimento , Zea mays/microbiologia
7.
Sheng Wu Gong Cheng Xue Bao ; 35(8): 1411-1423, 2019 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-31441612

RESUMO

Biorefinery technologies provide promising solutions to achieve sustainable development facing energy and environment crisis, while abundant sugar feedstock is an essential basis for biorefinery industries. Photosynthetic production of sucrose with cyanobacteria is an alternative sugar feedstock supply route with great potentials. Driven by solar energy, cyanobacteria photosynthetic cell factory could directly convert carbon dioxide and water into sucrose, and such a process could simultaneously reduce carbon emissions and supply sugar feedstocks. Here we introduced the history and updated the state-of-the-art on development of cyanobacteria cell factories for photosynthetic production of sucrose, summarized the progress and problems on mechanisms of sucrose synthesis, metabolic engineering strategies and technology expansions, and finally forecasted the future development direction in this area.


Assuntos
Cianobactérias , Sacarose , Dióxido de Carbono , Engenharia Metabólica , Fotossíntese
8.
Bioresour Technol ; 291: 121812, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31376668

RESUMO

In this study, a signal peptide of AlkL was replaced with other signal peptides to improve the soluble expression and thereby facilitate the transport of dodecanoic acid methyl ester (DAME) substrate into the E. coli. Consequently, AlkL with signal peptide FadL (AlkLf) showed higher transport activity toward DAME. Furthermore, the promoter optimization for the efficient heterologous expression of the transporter AlkLf and alkane monooxygenase (AlkBGT) system was conducted and resulted in increased ω-oxygenation activity of AlkBGT system. Moreover, bioinformatic studies led to the identification of novel monooxygenase from Pseudomonas pelagia (Pel), which exhibited 20% higher activity towards DAME as substrate compared to AlkB. Finally, the construction of a chimeric transporter and the expression of newly identified monooxygenase enabled the production of 44.8 ±â€¯7.5 mM of 12-hydroxy dodecanoic acid methyl ester (HADME) and 31.8 ±â€¯1.7 mM of dodecanedioic acid monomethyl ester (DDAME) in a two-phase reaction system.


Assuntos
Proteínas de Membrana Transportadoras , Engenharia Metabólica , Escherichia coli , Oxigenases de Função Mista , Sinais Direcionadores de Proteínas
9.
Bioresour Technol ; 292: 121933, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31404755

RESUMO

Bio-production of 1,3-propanediol (1,3-PDO) from glycerol was studied using Pseudomonas denitrificans as host, which aerobically synthesizes coenzyme B12, an essential cofactor of glycerol dehydratase (GDHt). P. denitrificans was transformed with the 1,3-PDO synthesis pathway composed of GDHt and 1,3-PDO oxidoreductase (PDOR), and its putative 3-hydroxypropionaldehyde (3-HPA) dehydrogenase(s), leading to the production of 3-hydroxypropioninc acid form the intermediary 3-HPA, was identified and deleted. In addition, to improve the availability of NADH for PDOR, oxidation of NADH in the electron transport chain was disturbed by deletion of the nuo operon and/or ndh gene. Finally, acetate formation pathway was eliminated. One resulting strain could produce 68.95 mM 1,3-PDO with the yield of 0.92 mol 1,3-PDO/mol glycerol on flask scale and 440 mM with the yield of 0.89 mol 1,3-PDO/mol glycerol in a fed-batch bioreactor experiment. This study demonstrates that P. denitrificans is a promising recombinant host for the production of 1,3-PDO from glycerol.


Assuntos
Glicerol , Engenharia Metabólica , Propilenoglicóis , Pseudomonas
10.
Bioresour Technol ; 292: 121953, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31405625

RESUMO

Over the past decade, the number of original articles and reviews presenting microalgae as a promising feedstock for biodiesel has increased tremendously. Many improvements of microalgae have been achieved through selection and strain development for industrial applications. However, the large-scale production of lipids for commercialization is not yet realistic because the production is still much more expensive than that of agricultural products. This review summarizes recent research on the induction of lipid biosynthesis in microalgae and the various strategies of genetic and metabolic engineering for enhancing lipid production. Strain engineering targets are proposed based on these strategies. To address current limitations of strain engineering for lipid production, this review provides insights on recent engineering strategies based on molecular tools and methods, and also discusses further perspectives.


Assuntos
Microalgas , Biocombustíveis , Biotecnologia , Lipídeos , Engenharia Metabólica
11.
Gene ; 718: 144073, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31446096

RESUMO

Cell morphology of the oleaginous fungus, Aspergillus oryzae BCC7051, was genetically engineered by disruption of non-essential genes involved in cell wall biosynthesis. Comparative phenotypic analysis of two disruptant strains defective either in α-1,3-glucan synthase 1 (ΔAoAgs1) or chitin synthase B (ΔAoChsB), and the wild type showed that the ΔAoAgs1 strain had no alterations in colonial growth and sporulation when grown on agar medium whereas the ΔAoChsB disruptant showed growth retardation and lower sporulation. However, tiny and loose pellets were found in the ΔAoAgs1 culture grown in liquid medium, where fungal pellet size was decreased by 35-50% of the wild type size. Further investigation of the ΔAoAgs1 mutant grown under stress-induced conditions, including high salt concentration, ionic strength and osmolarity, showed that its growth and development remained similar to that of the wild type. When cultivating the ΔAoAgs1 strain in a stirred-tank bioreactor, lipid production in terms of titer and productivity was significantly improved. As compared to the wild type, an increase of triacylglycerol and ergosterol contents with a proportional decrease in steryl ester content was observed in the ΔAoAgs1 strain. These results suggest that the morphologically engineered strain of A. oryzae is a robust cell chassis useful for exploitation in further production development of functional lipids with industrial significance.


Assuntos
Aspergillus oryzae/metabolismo , Ergosterol/biossíntese , Engenharia Metabólica , Microrganismos Geneticamente Modificados/metabolismo , Triglicerídeos/biossíntese , Aspergillus oryzae/genética , Quitina Sintase/genética , Quitina Sintase/metabolismo , Ergosterol/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Genes Fúngicos , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Microrganismos Geneticamente Modificados/genética , Triglicerídeos/genética
12.
J Agric Food Chem ; 67(35): 9858-9867, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31389230

RESUMO

Farnesene is an important chemical platform for many industrial products, such as biofuels and polymers. We performed high-efficiency utilization of corncobs for ß-farnesene production by separate hydrolysis and fermentation with an optimized Escherichia coli strain. First, we developed a recycling strategy for both corncob pretreatment and cellulose hydrolysis, which saved great amounts of pretreatment reagents and presented a 96.83% cellulose conversion rate into glucose. However, the corncob hydrolysate strongly repressed cell growth and ß-farnesene production, being caused by high-concentrated citrate. Through expressing a heterologous ATP citrate lyase and screening for a suitable expression host, an optimized strain was constructed that produced ß-farnesene at 4.06 g/L after 48 h in a 5 L fermenter, representing an approximately 2.3-fold increase over the initial strain. Therefore, the proposed strategy about the recycling process and repression elimination was successful and suitable for the production of lignocellulosic-based ß-farnesene, which can be further studied to scale up for industrialization.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Lignina/metabolismo , Sesquiterpenos/metabolismo , Fermentação , Glucose/metabolismo , Hidrólise , Engenharia Metabólica , Reciclagem , Resíduos/análise , Zea mays/química , Zea mays/microbiologia
13.
J Agric Food Chem ; 67(35): 9851-9857, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31418561

RESUMO

Arachidonic acid (ARA, C20:4) is a typical ω-6 polyunsaturated fatty acid with special functions. Using Yarrowia lipolytica as an unconventional chassis, we previously showed the performance of the Δ-6 pathway in ARA production. However, a significant increase in the Δ-9 pathway has rarely been reported. Herein, the Δ-9 pathway from Isochrysis galbana was constructed via pathway engineering, allowing us to synthesize ARA at 91.5 mg L-1. To further improve the ARA titer, novel enzyme fusions of Δ-9 elongase and Δ-8 desaturase were redesigned in special combinations containing different linkers. Finally, with the integrated pathway engineering and synthetic enzyme fusion, a 29% increase in the ARA titer, up to 118.1 mg/L, was achieved using the reconstructed strain RH-4 that harbors the rigid linker (GGGGS). The results show that the combined pathway and protein engineering can significantly facilitate applications of Y. lipolytica.


Assuntos
Ácido Araquidônico/biossíntese , Engenharia Metabólica , Yarrowia/genética , Yarrowia/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Glucose/metabolismo , Haptófitas/enzimologia
14.
World J Microbiol Biotechnol ; 35(7): 109, 2019 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-31280382

RESUMO

Echinocandin B (ECB) is an important lipohexapeptide used for chemical manufacture of the antifungal agent anidulafungin. Sterigmatocystin (ST) is a polyketide mycotoxin produced by certain species of Aspergillus such as Aspergillus delacroxii SIPIW15, which could produce both ECB and ST. However, the presence of the potent carcinogen ST will greatly affect the quality and safety of ECB production. Therefore, it is essential to eliminate the ST biosynthesis and increase ECB titers in Asp. delacroxii SIPIW15. In this study, the polyketide synthase gene (stcA) required for biosynthesis of ST and its flanking region in Asp. delacroxii SIPIW15 were cloned, sequenced and analyzed firstly. Based on Agrobacterium-mediated transformation, the ΔstcA mutant AMT-1 was obtained and its yield of ECB was increased by 40% without ST detected at the same time as compared to the original strain. The results of the fed-batch experiments showed that the ECB yield of the ΔstcA strain AMT-1 was increased to 2163 ± 31 mg/l and no ST was detected in the 50 l bioreactor. This work suggested that the ΔstcA strain AMT-1 has the potential for application in ECB production improvement, and more importantly, to eliminate ST-related environmental pollution in ECB fermentation industry.


Assuntos
Aspergillus/genética , Aspergillus/metabolismo , Equinocandinas/biossíntese , Equinocandinas/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Genes Fúngicos/genética , Policetídeo Sintases/genética , Esterigmatocistina/biossíntese , Agrobacterium/genética , Anidulafungina , Antifúngicos , Sequência de Bases , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , DNA Fúngico/isolamento & purificação , Fermentação , Engenharia Metabólica , Redes e Vias Metabólicas/genética , Metabolismo Secundário/genética , Transformação Genética
15.
World J Microbiol Biotechnol ; 35(7): 111, 2019 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-31280424

RESUMO

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) immune systems in bacteria have been used as tools for genome engineering. Thus far, the CRISPR-Cas system has been used in various yeast, bacterial, and mammalian cells. Saccharomyces cerevisiae is a nonpathogenic yeast, classified under "generally recognized as safe", and has long been used to produce consumables such as alcohol or bread. Additionally, recombinant cells of S. cerevisiae have been constructed and used to produce various bio-based chemicals. Some types of CRISPR-Cas system for genetic manipulation have been constructed during the early developmental stages of the CRISPR-Cas system and have been mainly used for gene knock-in and knock-out manipulations. Thereafter, these systems have been used for various novel purposes such as metabolic engineering and tolerance engineering. In this review, we have summarized different aspects of the CRISPR-Cas in the yeast S. cerevisiae, from its basic principles to various applications. This review describes the CRISPR system in S. cerevisiae based on the differences in its origin and efficiency followed by its basic applications; for example, its involvement in gene knock-in and knock-out has been outlined. Finally, advanced applications of the CRISPR system in the bioproduction of useful chemicals have been summarized.


Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/metabolismo , Edição de Genes/métodos , Regulação Fúngica da Expressão Gênica , Técnicas de Introdução de Genes/métodos , Técnicas de Inativação de Genes/métodos , Saccharomyces cerevisiae/genética
16.
World J Microbiol Biotechnol ; 35(7): 112, 2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31286266

RESUMO

Microorganisms have evolved permeases to incorporate various essential nutrients and exclude harmful products, which assists in adaptation to different environmental conditions for survival. As permeases are directly involved in the utilization of and regulatory response to nutrient sources, metabolic engineering of microbial permeases can predictably influence nutrient metabolism and regulation. In this mini-review, we have summarized the mechanisms underlying the general regulation of permeases, and the current advancements and future prospects of metabolic engineering strategies targeting the permeases in Saccharomyces cerevisiae. The different types of permeases and their regulatory mechanisms have been discussed. Furthermore, methods for metabolic engineering of permeases have been highlighted. Understanding the mechanisms via which permeases are meticulously regulated and engineered will not only facilitate research on regulation of global nutrition and yeast metabolic engineering, but can also provide important insights for future studies on the synthesis of valuable products and elimination of harmful substances in S. cerevisiae.


Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Carbono/metabolismo , Glucose/metabolismo , Proteínas de Membrana Transportadoras/genética , Nitrogênio/metabolismo , Saccharomyces cerevisiae/genética
17.
Bioengineered ; 10(1): 335-344, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31322471

RESUMO

Selenium-enriched yeast can transform toxic inorganic selenium into absorbable organic selenium, which is of great significance for human health and pharmaceutical industry. A yeast Rhodotorula glutinis X-20 we obtained before has good selenium-enriched ability, but its selenium content is still low for industrial application. In this study, strategies of process optimization and transport regulation of selenium were thus employed to further improve the cell growth and selenium enrichment. Through engineering phosphate transporters from Saccharomyces cerevisiae into R. glutinis X-20, the selenium content was increased by 21.1%. Through using mixed carbon culture (20 g L-1, glycerol: glucose 3:7), both biomass and selenium content were finally increased to 5.3 g L-1 and 5349.6 µg g-1 (cell dry weight, DWC), which were 1.14 folds and 6.77 folds compared to their original values, respectively. Our results indicate that high selenium-enrichment ability and biomass production can be achieved through combining process optimization and regulation of selenium transport.


Assuntos
Engenharia Metabólica/métodos , Fosfatos/metabolismo , Rhodotorula/genética , Saccharomyces cerevisiae/genética , Selênio/metabolismo , Transgenes , Transporte Biológico , Biomassa , Meios de Cultura/química , Meios de Cultura/farmacologia , Fermentação , Expressão Gênica , Glucose/química , Glucose/metabolismo , Glicerol/química , Glicerol/metabolismo , Proteínas de Transporte de Fosfato/genética , Proteínas de Transporte de Fosfato/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Simportadores de Próton-Fosfato/genética , Simportadores de Próton-Fosfato/metabolismo , Rhodotorula/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/metabolismo
18.
J Agric Food Chem ; 67(31): 8590-8598, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31287301

RESUMO

Patchoulol, a natural sesquiterpene compound, is widely used in perfumes and cosmetics. Several strategies were adopted to enhance patchoulol production in Saccharomyces cerevisiae: (i) farnesyl pyrophosphate (FPP) synthase and patchoulol synthase were fused to increase the utilization of FPP precursor; (ii) expression of the limiting genes of the mevalonate pathway was enhanced; (iii) squalene synthase was weakened by a glucose-inducible promoter of HXT1 (promoter for hexose transporter) to reduce metabolic flux from FPP to ergosterol; and (iv) farnesol biosynthesis was inhibited to decrease the consumption of FPP. Glucose was used to balance the trade-off between the competitive squalene and patchoulol pathways. The patchoulol production was 59.2 ± 0.7 mg/L in a shaken flask with a final production of 466.8 ± 12.3 mg/L (20.5 ± 0.5 mg/g dry cell weight) combined with fermentation optimization, which was 7.8-fold higher than the reported maximum production. The work significantly promoted the industrialization process of patchoulol production using biobased microbial platforms.


Assuntos
Engenharia Metabólica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sesquiterpenos/metabolismo , Fermentação , Ácido Mevalônico/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Esqualeno/metabolismo
19.
J Agric Food Chem ; 67(31): 8581-8589, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31321975

RESUMO

Intermediates in aromatic amino acid biosynthesis can serve as substrates for the synthesis of bioactive compounds. In this study we used two intermediates in the shikimate pathway of Escherichia coli, chorismate and anthranilate, to synthesize three bioactive compounds: 4-hydroxycoumarin (4-HC), 2,4-dihydroxyquinoline (DHQ), and 4-hydroxy-1-methyl-2(1H)-quinolone (NMQ). We introduced genes for the synthesis of salicylic acid from chorismate to supply the substrate for 4-HC and the gene encoding N-methyltransferase for the synthesis of N-methylanthranilate from anthranilate. Polyketide synthases and coenzyme (Co)A ligases were tested to determine the optimal combination of genes for the synthesis of each compound. We also tested several constructs and identified the best one for increasing levels of endogenous substrates for chorismate, anthranilate, and malonyl-CoA. With the use of these strategies, 255.4 mg/L 4-HC, 753.7 mg/L DHQ, and 17.5 mg/L NMQ were synthesized. This work provides a basis for the synthesis of diverse coumarin and quinoline derivatives with potential medical applications.


Assuntos
4-Hidroxicumarinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica , Policetídeo Sintases/genética , Quinolinas/metabolismo , 4-Hidroxicumarinas/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ácido Corísmico/metabolismo , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Photorhabdus/enzimologia , Photorhabdus/genética , Policetídeo Sintases/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Quinolinas/química , ortoaminobenzoatos/metabolismo
20.
J Agric Food Chem ; 67(33): 9314-9324, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31352776

RESUMO

Trehalose, a stable nonreducing disaccharide, protects biomolecules against environmental stress. However, trehalose production using secretory trehalose synthase (TreS) by Bacillus subtilis has not been well studied. In this study, a mutant TreS was successfully secreted and expressed in B. subtilis WB800N. The extracellular enzyme activity of TreS regulated by the P43 promoter and SPPhoD signal peptide in recombinant B. subtilis WB800N reached 23080.6 ± 1119.4 U/L in a 5-L fermenter after optimizing the culture medium, while xpF, skfA, lytC, and sdpC were knocked out. To reduce maltose consumption, malP and amyE corresponding to maltose transporters were further deleted. To simplify the trehalose production process, we invented a fermentation-coupling biocatalysis process involving recombinant bacteria fermentation to secrete TreS and simultaneous conversion of maltose to trehalose by TreS and found that the conversion rate of maltose to trehalose reached 75.5%, suggesting that this is an efficient strategy for large-scale trehalose production using recombinant B. subtilis.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Trealose/biossíntese , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Biocatálise , Fermentação , Maltose/metabolismo , Engenharia Metabólica
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