ABSTRACT
Introduction: Eggshell membranes offer beneficial properties for tissue regeneration and their biomedical applications are important. Objective: To demonstrate the effectiveness of hen unfertilized eggshell membranes in the treatment of superficial open wounds in mice compared to the conventional procedure. Materials and methods: 15-mm linear wounds were made on the back of 10 male CF-1 mice. The mice were divided into four groups. One group did not receive any treatment and the other three were given conventional treatment, eggshell membranes dressing, and eggshell membranes powder. The evolution of the wounds was registered by photographs. We measured wounds length and healing time and calculated the length reduction rate and the percentage of healing. The healing percentages were analyzed by ANOVA with a post hoc Dunnett test (p<0.05). Results: The treatments with eggshell membranes and membrane powder had a woundlength reduction rate of 1.009 and 1.020 mm/day, greater than the conventional treatment, of 0.852 mm/day, and a healing time of 12 days, i.e., less than the 16-day conventional treatment. The statistical analysis showed significant differences between eggshell membrane treatments and the conventional treatment. Conclusions: Eggshell membrane dressing and eggshell membrane powder applied to open superficial wounds in mice were more effective than the conventional treatment in regenerating injured tissue in mice.
Introducción. Las membranas de la cáscara de huevo presentan propiedades beneficiosas para la regeneración de tejidos y sus aplicaciones biomédicas son importantes. Objetivo. Demostrar la efectividad de las membranas de la cáscara de huevo no fecundado de gallina en el tratamiento de heridas abiertas superficiales en ratones, en comparación con el procedimiento convencional. Materiales y métodos. Se hizo una herida superficial lineal de 15 mm en la espalda de 10 ratones albinos machos. Los ratones se dividieron en cuatro grupos, uno no recibió ningún tratamiento y los otros tres sí: uno, tratamiento convencional, otro, con membranas de huevo directamente aplicadas a la herida y, el otro, con membranas en forma de polvo. La evolución de las heridas se registró en fotografías y se calculó la tasa de reducción de la longitud de la herida, así como el tiempo y el porcentaje de curación. Los porcentajes de curación se analizaron con ANOVA y la prueba de Dunnett (p<0,05). Resultados. Con los tratamientos con membranas de huevo y polvo de membrana, se logró una tasa de reducción de longitud de 1.009 y 1.020 mm/día, respectivamente, y un tiempo de curación de 12 días, en tanto que, con el tratamiento convencional, la tasa de reducción fue de 0,852 mm/día y la curación se dio en 16 días. El análisis estadístico mostró diferencias significativas entre los tratamientos con membrana de huevo y el tratamiento convencional. Conclusiones. Las membranas de la cáscara de huevo aplicadas de forma directa y en polvo resultaron más efectivas que la aplicación del procedimiento convencional en el tratamiento de heridas abiertas superficiales en ratones.
ABSTRACT
Caffeic acid (CA; 3,4-dihydroxycinnamic acid) is an aromatic compound obtained by the phenylpropanoid pathway. This natural product has antioxidant, antitumor, antiviral, and anti-inflammatory activities. It is also a precursor of CA phenethyl ester (CAPE), a compound with potential as an antidiabetic and liver-protective agent. CA can be found at low concentrations in plant tissues, and hence, its purification is difficult and expensive. Knowledge regarding the pathways, enzymes, and genes involved in CA biosynthesis has paved the way for enabling the design and construction of microbial strains with the capacity of synthesizing this metabolite. In this review, metabolic engineering strategies for the generation of Escherichia coli strains for the biotechnological production of CA are presented and discussed.
Subject(s)
Caffeic Acids/metabolism , Escherichia coli/metabolism , Metabolic Engineering/methods , Biological Products , Biotechnology , Coumaric AcidsABSTRACT
BACKGROUND: Escherichia coli W3110 and a group of six isogenic derivatives, each displaying distinct specific rates of glucose consumption were characterized to determine levels of GFP production and population heterogeneity. These strains have single or combinatory deletions in genes encoding phosphoenolpyruvate:sugar phosphotransferase system (PTS) permeases as PtsG and ManX, as well as common components EI, Hpr protein and EIIA, also the non-PTS Mgl galactose/glucose ABC transporter. They have been transformed for expressing GFP based on a lac-based expression vector, which is subject to bistability. RESULTS: These strains displayed specific glucose consumption and growth rates ranging from 1.75 to 0.45 g/g h and 0.54 to 0.16 h-1, respectively. The rate of acetate production was strongly reduced in all mutant strains when compared with W3110/pV21. In bioreactor cultures, wild type W3110/pV21 produced 50.51 mg/L GFP, whereas strains WG/pV21 with inactive PTS IICBGlc and WGM/pV21 with the additional inactivation of PTS IIABMan showed the highest titers of GFP, corresponding to 342 and 438 mg/L, respectively. Moreover, we showed experimentally that bistable expression systems, as lac-based ones, induce strong phenotypic segregation among microbial populations. CONCLUSIONS: We have demonstrated that reduction on glucose consumption rate in E. coli leads to an improvement of GFP production. Furthermore, from the perspective of phenotypic heterogeneity, we observed in this case that heterogeneous systems are also the ones leading to the highest performance. This observation suggests reconsidering the generally accepted proposition stating that phenotypic heterogeneity is generally unwanted in bioprocess applications.
Subject(s)
Escherichia coli/genetics , Glucose/metabolism , Metabolic Engineering/methods , Acetates/metabolism , Biological Transport , Bioreactors , Escherichia coli/growth & development , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Flow Cytometry , Glucose Transport Proteins, Facilitative/metabolism , Green Fluorescent Proteins/analysis , Kinetics , Microfluidic Analytical TechniquesABSTRACT
Background: The plant secondary metabolite pinosylvin is a polyphenol from the stilbene family, which have positive effects on human health. Biotechnological production is an attractive alternative for obtaining this stilbene. In Escherichia coli, malonyl-CoA is the precursor for both stilbene and fatty acid syntheses. In this study, with the aim of increasing pinosylvin production, we evaluated a novel approach that is based on reducing the expression of the gene fabI, which encodes the enzyme enoyl-acyl carrier protein reductase that is involved in fatty acid synthesis. Results: A recombineering method was employed to eliminate the chromosomal -35 promoter sequence and the upstream region of the gene fabI in E. coli strain W3110. Analysis, employing RT-qPCR, showed that such modification caused a 60% reduction in the fabI transcript level in the mutant strain W3110Δ-35fabI::Cm compared to the wild type W3110. Synthetic genes encoding a mutant version of 4-coumaroyl-CoA ligase from Streptomyces coelicolor A3 with improved catalytic activity employing cinnamic acid as substrate and a stilbene synthase from Vitis vinifera were cloned to generate the plasmid pTrc-Sc4CL(M)-VvSTS. The production performance of strains W3110Δ-35fabI::Cm/pTrc-Sc4CL(M)-VvSTS and W3110/pTrc-Sc4CL(M)- VvSTS was determined in shake flask cultures with Luria-Bertani medium supplemented with 10 g/L glycerol and 3 mM cinnamic acid. Under these conditions, the strain W3110Δ-35fabI::Cm/pTrc-Sc4CL(M)-VvSTS produced 52.67 mg/L pinosylvin, a level 1.5-fold higher than that observed with W3110/pTrc-Sc4CL(M)-VvSTS. Conclusion: A reduction in the transcript level of fabI caused by the elimination of the -35 and upstream promoter sequences is a successful strategy to improve pinosylvin production in E. coli.
Subject(s)
Stilbenes/metabolism , Escherichia coli/metabolism , Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)/genetics , Biological Products , Coenzyme A Ligases , Fatty Acids , Metabolic EngineeringABSTRACT
BACKGROUND: Resveratrol is a plant natural product with many health-protecting effects which makes it an attractive chemical both for academic studies and industrial purposes. However, the low quantities naturally produced by plants as well as the unsustainable procedures of extraction, purification and concentration have prompted many biotechnological approaches to produce this chemical in large quantities from renewable sources. None of these approaches have considered a microbial coculture strategy to produce this compound. The aim of this study was to prove the functionality of a microbial coculture for the biosynthesis of resveratrol. RESULTS: In this work, we have successfully applied a coculture system strategy comprised of two populations of Escherichia coli strains, each with a partial and complementary section of the pathway leading to the biosynthesis of the stilbene resveratrol. The first strain is a pheA knockout mutant previously engineered to excrete p-coumaric acid into the medium through the overexpression of genes encoding a tyrosine ammonia lyase from Rhodothorula glutinis, a feedback resistant 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and a transketolase. The second strain in the coculture was engineered to express the second part of the resveratrol biosynthetic pathway through the introduction of synthetic genes encoding the 4-coumaroyl-CoA ligase from Streptomyces coelicolor A2 and the stilbene synthase either from the peanut Arachis hypogaea or the grapevine Vitis vinifera, the latter synthesized employing a gene harmonization strategy and showing better resveratrol production performance. Batch cultures were performed in mineral medium with glycerol as the sole carbon source, where a final titer of 22.6 mg/L of resveratrol was produced in 30 h. CONCLUSIONS: To our knowledge, this is the first time that a coculture of bacterial strains is used for the biosynthesis of resveratrol from glycerol, having the potential for a greater improvement in the product yield and avoiding the use of precursors such as p-coumaric acid, yeast extract or an expensive inhibitor such as cerulenin.
ABSTRACT
BACKGROUND: Melanins comprise a chemically-diverse group of polymeric pigments whose function is related to protection against physical and chemical stress factors. These polymers have current and potential applications in the chemical, medical, electronics and materials industries. The biotechnological production of melanins offers the possibility of obtaining these pigments in pure form and relatively low cost. In this study, Escherichia coli strains were engineered to evaluate the production of melanin from supplemented catechol or from glycerol-derived catechol produced by an Escherichia coli strain generated by metabolic engineering. RESULTS: It was determined that an improved mutant version of the tyrosinase from Rhizobium etli (MutmelA), could employ catechol as a substrate to generate melanin. Strain E. coli W3110 expressing MutmelA was grown in bioreactor batch cultures with catechol supplemented in the medium. Under these conditions, 0.29 g/L of catechol melanin were produced. A strain with the capacity to synthesize catechol melanin from a simple carbon source was generated by integrating the gene MutmelA into the chromosome of E. coli W3110 trpD9923, that has been modified to produce catechol by the expression of genes encoding a feedback inhibition resistant version of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, transketolase and anthranilate 1,2-dioxygenase from Pseudomonas aeruginosa PAO1. In batch cultures with this strain employing complex medium with 40 g/L glycerol as a carbon source, 1.21 g/L of catechol melanin were produced. The melanin was analysed by employing Fourier transform infrared spectroscopy, revealing the expected characteristics for a catechol-derived polymer. CONCLUSIONS: This constitutes the first report of an engineered E. coli strain and a fermentation process for producing a catechol melanin from a simple carbon source (glycerol) at gram level, opening the possibility of generating a large quantity of this polymer for its detailed characterization and the development of novel applications.
ABSTRACT
BACKGROUND: The aromatic compounds cinnamic acid (CA) and p-hydroxycinnamic acid (pHCA) are used as flavoring agents as well as precursors of chemicals. These compounds are present in plants at low concentrations, therefore, complex purification processes are usually required to extract the product. An alternative production method for these aromatic acids is based on the use of microbial strains modified by metabolic engineering. These biotechnological processes are usually based on the use of simple sugars like glucose as a raw material. However, sustainable production processes should preferably be based on the use of waste material such as lignocellulosic hydrolysates. RESULTS: In this study, E. coli strains with active (W3110) and inactive phosphoenolpyruvate:sugar phosphotransferase system (PTS) (VH33) were engineered for CA and pHCA production by transforming them with plasmids expressing genes encoding phenylalanine/tyrosine ammonia lyase (PAL/TAL) enzymes from Rhodotorula glutinis or Arabidopsis thaliana as well as genes aroGfbr and tktA, encoding a feedback inhibition resistant version of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and transketolase, respectively. The generated strains were evaluated in cultures with glucose, xylose or arabinose, as well as a simulated lignocellulosic hydrolysate containing a mixture of these three sugars plus acetate. Production of CA was detected in strains expressing PAL/TAL from A. thaliana, whereas both CA and pHCA accumulated in strains expressing the enzyme from R. glutinis. These experiments identified arabinose and W3110 expressing PAL/TAL from A. thaliana, aroGfbr and tktA as the carbon source/strain combination resulting in the best CA specific productivity and titer. To improve pHCA production, a mutant with inactive pheA gene was generated, causing an 8-fold increase in the yield of this aromatic acid from the sugars in a simulated hydrolysate. CONCLUSIONS: In this study the quantitative contribution of active or inactive PTS as well as expression of PAL/TAL from R. glutinis or A. thaliana were determined for production performance of CA and pHCA when growing on carbon sources derived from lignocellulosic hydrolysates. These data will be a useful resource in efforts towards the development of sustainable technologies for the production of aromatic acids.
Subject(s)
Cinnamates/metabolism , Coumaric Acids/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , 3-Deoxy-7-Phosphoheptulonate Synthase/genetics , 3-Deoxy-7-Phosphoheptulonate Synthase/metabolism , Ammonia-Lyases/genetics , Ammonia-Lyases/metabolism , Arabidopsis/enzymology , Arabinose/metabolism , Cinnamates/chemistry , Coumaric Acids/chemistry , Glucose/metabolism , Kinetics , Lignin/chemistry , Lignin/metabolism , Metabolic Engineering , Plant Proteins/genetics , Plant Proteins/metabolism , Plasmids/genetics , Plasmids/metabolism , Propionates , Rhodotorula/enzymology , Transketolase/genetics , Transketolase/metabolism , Xylose/metabolismABSTRACT
BACKGROUND: The aromatic compound catechol is used as a precursor of chemical products having multiple applications. This compound is currently manufactured by chemical synthesis from petroleum-derived raw materials. The capacity to produce catechol is naturally present in several microbial species. This knowledge has been applied to the generation of recombinant Escherichia coli strains that can produce catechol from simple carbon sources. RESULTS: Several strains derived from E. coli W3110 trpD9923, a mutant that overproduces anthranilate, were modified by transforming them with an expression plasmid carrying genes encoding anthranilate 1,2-dioxygenase from Pseudomonas aeruginosa PAO1. The additional expression of genes encoding a feedback inhibition resistant version of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase and transketolase from E. coli, was also evaluated. Generated strains were characterized in complex or minimal medium in shake-flask and fed-batch bioreactor cultures and incubation temperatures ranging from 37 to 28°C. These experiments enabled the identification of culture conditions for the production of 4.47 g/L of catechol with strain W3110 trpD9923, expressing 1,2-dioxygenase, DAHP synthase and transketolase. When considering the amount of glucose consumed, a yield of 16% was calculated, corresponding to 42% of the theoretical maximum as determined by elementary node flux analysis. CONCLUSIONS: This work demonstrates the feasibility of applying metabolic engineering for generating E. coli strains for the production of catechol from glucose via anthranilate. These results are a starting point to further optimize environmentally-compatible production capacity for catechol and derived compounds.
Subject(s)
Bacterial Proteins , Catechols/metabolism , Escherichia coli , Gene Expression , Glucose/metabolism , Mixed Function Oxygenases , Pseudomonas aeruginosa/genetics , ortho-Aminobenzoates/metabolism , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Mixed Function Oxygenases/biosynthesis , Mixed Function Oxygenases/genetics , Pseudomonas aeruginosa/enzymologyABSTRACT
Phosphoenolpyruvate (PEP) is a precursor involved in the biosynthesis of aromatics and other valuable compounds in Escherichia coli. The PEP:carbohydrate phosphotransferase system (PTS) is the major glucose transport system and the largest PEP consumer. To increase intracellular PEP availability for aromatics production purposes, mutant strains of E. coli JM101 devoid of the ptsHIcrr operon (PB11 strain) have been previously generated. In this derivative, transport and growth rate on glucose decreased significantly. A laboratory evolved strain derived from PB11 that partially recovered its growth capacity on glucose was named PB12. In the present study, we blocked carbon skeletons interchange between PEP and pyruvate (PYR) in these ptsHIcrr(-) strains by deleting the pykA, pykF, and ppsA genes. The PB11 pykAF(-) ppsA(-) strain exhibited no growth on glucose or acetate alone, but it was viable when both substrates were consumed simultaneously. In contrast, the PB12 pykAF(-) ppsA(-) strain displayed a low growth rate on glucose or acetate alone, but in the mixture, growth was significantly improved. RT-qPCR expression analysis of PB11 pykAF(-) ppsA(-) growing with both carbon sources showed a downregulation of all central metabolic pathways compared with its parental PB11 strain. Under the same conditions, transcription of most of the genes in PB12 pykAF(-) ppsA(-) did not change, and few like aceBAK, sfcA, and poxB were overexpressed compared with PB12. We explored the aromatics production capabilities of both ptsHIcrr(-) pykAF(-) ppsA(-) strains and the engineered PB12 pykAF(-) ppsA(-) tyrR(-) pheA(ev2+) /pJLBaroG(fbr) tktA enhanced the yield of aromatic compounds when coutilizing glucose and acetate compared with the control strain PB12 tyrR(-) pheA(ev2+) /pJLBaroG(fbr) tktA.
Subject(s)
Acetates/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Profiling , Glucose/metabolism , Phosphoenolpyruvate/metabolism , Pyruvic Acid/metabolism , Escherichia coli/growth & development , Gene Deletion , Metabolic Networks and Pathways , Transcription, GeneticABSTRACT
The glycolytic intermediate phosphoenolpyruvate (PEP) is a precursor of several cellular components, including various aromatic compounds. Modifications to the PEP node such as PEP:sugar phosphotransferase system (PTS) or pyruvate kinase inactivation have been shown to have a positive effect on aromatics production capacity in Escherichia coli and Bacillus subtilis. In this study, pyruvate kinase and PTS-deficient B. subtilis strains were employed for the construction of derivatives lacking shikimate kinase activity that accumulate two industrially valuable chemicals, the intermediates of the common aromatic pathway, shikimic and dehydroshikimic acids. The pyruvate kinase-deficient strain (CLC6-PYKA) showed the best production parameters under resting-cell conditions. Compared to the PTS-deficient strain, the shikimic and dehydroshikimic acids specific production rates for CLC6-PYKA were 1.8- and 1.7-fold higher, respectively. A batch fermentor culture using complex media supplemented with 83 g/l of glucose was developed with strain CLC6-PYKA, where final titers of 4.67 g/l (shikimic acid) and 6.2 g/l (dehydroshikimic acid) were produced after 42 h.
Subject(s)
Bacillus subtilis/enzymology , Bacillus subtilis/metabolism , Metabolic Engineering , Phosphoenolpyruvate Sugar Phosphotransferase System/antagonists & inhibitors , Pyruvate Kinase/antagonists & inhibitors , Shikimic Acid/analogs & derivatives , Shikimic Acid/metabolism , Bacillus subtilis/genetics , Fermentation , Metabolic Networks and Pathways/genetics , Phosphoenolpyruvate Sugar Phosphotransferase System/genetics , Pyruvate Kinase/geneticsABSTRACT
BACKGROUND: During the last two decades many efforts have been directed towards obtaining efficient microbial processes for the production of shikimic acid (SA); however, feeding high amounts of substrate to increase the titer of this compound has invariably rendered low conversion yields, leaving room for improvement of the producing strains. In this work we report an alternative platform to overproduce SA in a laboratory-evolved Escherichia coli strain, based on plasmid-driven constitutive expression of six genes selected from the pentose phosphate and aromatic amino acid pathways, artificially arranged as an operon. Production strains also carried inactivated genes coding for phosphotransferase system components (ptsHIcrr), shikimate kinases I and II (aroK and aroL), pyruvate kinase I (pykF) and the lactose operon repressor (lacI). RESULTS: The strong and constitutive expression of the constructed operon permitted SA production from the beginning of the cultures, as evidenced in 1 L batch-mode fermentors starting with high concentrations of glucose and yeast extract. Inactivation of the pykF gene improved SA production under the evaluated conditions by increasing the titer, yield and productivity of this metabolite compared to the isogenic pykF+ strain. The best producing strain accumulated up to 43 g/L of SA in 30 h and relatively low concentrations of acetate and aromatic byproducts were detected, with SA accounting for 80% of the produced aromatic compounds. These results were consistent with high expression levels of the glycolytic pathway and synthetic operon genes from the beginning of fermentations, as revealed by transcriptomic analysis. Despite the consumption of 100 g/L of glucose, the yields on glucose of SA and of total aromatic compounds were about 50% and 60% of the theoretical maximum, respectively. The obtained yields and specific production and consumption rates proved to be constant with three different substrate concentrations. CONCLUSIONS: The developed production system allowed continuous SA accumulation until glucose exhaustion and eliminated the requirement for culture inducers. The obtained SA titers and yields represent the highest reported values for a high-substrate batch process, postulating the strategy described in this report as an interesting alternative to the traditionally employed fed-batch processes for SA production.
Subject(s)
Escherichia coli/genetics , Escherichia coli/metabolism , Glucose/metabolism , Pentose Phosphate Pathway/genetics , Phosphotransferases/metabolism , Pyruvate Kinase/metabolism , Shikimic Acid/metabolism , Bioreactors , Fermentation , Phosphotransferases/genetics , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Pyruvate Kinase/geneticsABSTRACT
El potencial de las cepas fúngicas en biorremediación, que han sido aisladas en el Instituto de Investigaciones Fármaco Bioquímicas (IIFB), es bastante amplio, se han encontrado cepas que han sido usadas en investigaciones a nivel internacional, tal es el caso de Bjerkandera sp BOL 13, un hongo aislado en un efluente contaminado con aceite en Oruro, con capacidad de degradar varios compuestos hidrocarbonados aromáticos policíclicos. En otro extremo de la nación se logró aislar Galerina sp., un hongo productor de lacasas, enzima importante en los procesos de decoloración.
The potential of fungi strains in biorremdiation that have been isolated in the Intiture of Research Pharmaceutical and Buiochemical (IIFB) is broad, it have been found strains that have been used in research at international level, as Bjerkandera sp BOL 13, a fungi isolated in an oil contaminated effluent, able to degrade polycycloic aromatic hydrocarbons. In other side of the country Galerina sp. was isolated, a laccase producing fungi, an enzyme important in decolorizing processes. contaminado con aceite en Oruro, con capacidad de degradar varios compuestos hidrocarbonados aromáticos policíclicos.
Subject(s)
Laccase , Research , Fungi , Hydrocarbons, AromaticABSTRACT
The phosphoenolpyruvate (PEP) node is an important carbon distribution point in the central metabolic networks; therefore, its modification is a common strategy employed for developing microbial production strains. In this study, mutants of Bacillus subtilis 168 were generated with deletions of pykA (which encodes pyruvate kinase), ptsG (which encodes the glucose-specific IICBA(Glc) component) or the ptsGHI operon [which encodes IICBA(Glc), HPr protein and enzyme I from the PEP:sugar phosphotransferase system (PTS)]. These modifications caused a reduction in the initial rate of [(14)C]-glucose import, corresponding to 10.99, 2.83 and 0.50% of that found in B. subtilis 168 for strains with inactive pykA, ptsG or ptsGHI genes, respectively. Characterization of derivative strains lacking 3-dehydroquinate synthase activity showed that inactivation of pykA leads to an 8-fold increase in carbon flow to the common aromatic pathway. Quantitative real-time PCR analyses of 76 genes from several functional classes revealed a carbon starvation transcriptional pattern that includes a partial gluconeogenic response and overexpression of genes encoding non-PTS glucose importers in the strains lacking functional pykA, ptsG or ptsGHI genes. A transcriptional response consistent with pyruvate limitation was also detected, which includes upregulation of genes encoding malic enzymes that generate pyruvate from malate.
Subject(s)
Bacillus subtilis/physiology , Bacterial Proteins/metabolism , Glucose/metabolism , Phosphoenolpyruvate/metabolism , Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Biological Transport , DNA, Bacterial/genetics , Drug Resistance, Bacterial , Gene Expression Regulation, Bacterial , Genes, Bacterial , Genetic Markers , Metabolic Networks and Pathways , Operon , Phosphoenolpyruvate Sugar Phosphotransferase System/genetics , Phosphoenolpyruvate Sugar Phosphotransferase System/metabolism , Phosphorus-Oxygen Lyases/genetics , Phosphorus-Oxygen Lyases/metabolism , Pyruvate Kinase/genetics , Pyruvate Kinase/metabolism , Real-Time Polymerase Chain Reaction , Sequence Analysis, DNA , Spectinomycin/pharmacologyABSTRACT
BACKGROUND: Glycerol has enhanced its biotechnological importance since it is a byproduct of biodiesel synthesis. A study of Escherichia coli physiology during growth on glycerol was performed combining transcriptional-proteomic analysis as well as kinetic and stoichiometric evaluations in the strain JM101 and certain derivatives with important inactivated genes. RESULTS: Transcriptional and proteomic analysis of metabolic central genes of strain JM101 growing on glycerol, revealed important changes not only in the synthesis of MglB, LamB and MalE proteins, but also in the overexpression of carbon scavenging genes: lamB, malE, mglB, mglC, galP and glk and some members of the RpoS regulon (pfkA, pfkB, fbaA, fbaB, pgi, poxB, acs, actP and acnA). Inactivation of rpoS had an important effect on stoichiometric parameters and growth adaptation on glycerol. The observed overexpression of poxB, pta, acs genes, glyoxylate shunt genes (aceA, aceB, glcB and glcC) and actP, suggested a possible carbon flux deviation into the PoxB, Acs and glyoxylate shunt. In this scenario acetate synthesized from pyruvate with PoxB was apparently reutilized via Acs and the glyoxylate shunt enzymes. In agreement, no acetate was detected when growing on glycerol, this strain was also capable of glycerol and acetate coutilization when growing in mineral media and derivatives carrying inactivated poxB or pckA genes, accumulated acetate. Tryptophanase A (TnaA) was synthesized at high levels and indole was produced by this enzyme, in strain JM101 growing on glycerol. Additionally, in the isogenic derivative with the inactivated tnaA gene, no indole was detected and acetate and lactate were accumulated. A high efficiency aromatic compounds production capability was detected in JM101 carrying pJLBaroG(fbr)tktA, when growing on glycerol, as compared to glucose. CONCLUSIONS: The overexpression of several carbon scavenging, acetate metabolism genes and the absence of acetate accumulation occurred in JM101 cultures growing on glycerol. To explain these results it is proposed that in addition to the glycolytic metabolism, a gluconeogenic carbon recycling process that involves acetate is occurring simultaneously in this strain when growing on glycerol. Carbon flux from glycerol can be efficiently redirected in JM101 strain into the aromatic pathway using appropriate tools.
Subject(s)
Acetates/metabolism , Carbon/metabolism , Escherichia coli Proteins/genetics , Escherichia coli/metabolism , Glycerol/metabolism , Escherichia coli/enzymology , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial , ProteomicsABSTRACT
L-3,4-dihydroxyphenylalanine (L-DOPA) is an aromatic compound employed for the treatment of Parkinson's disease. Metabolic engineering was applied to generate Escherichia coli strains for the production of L-DOPA from glucose by modifying the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and aromatic biosynthetic pathways. Carbon flow was directed to the biosynthesis of L-tyrosine (L-Tyr), an L-DOPA precursor, by transforming strains with compatible plasmids carrying genes encoding a feedback-inhibition resistant version of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, transketolase, the chorismate mutase domain from chorismate mutase-prephenate dehydratase from E. coli and cyclohexadienyl dehydrogenase from Zymomonas mobilis. The effects on L-Tyr production of PTS inactivation (PTS(-) gluc(+) phenotype), as well as inactivation of the regulatory protein TyrR, were evaluated. PTS inactivation caused a threefold increase in the specific rate of L-Tyr production (q( L-Tyr)), whereas inactivation of TyrR caused 1.7- and 1.9-fold increases in q( L-Tyr) in the PTS(+) and the PTS(-) gluc(+) strains, respectively. An 8.6-fold increase in L-Tyr yield from glucose was observed in the PTS(-) gluc(+) tyrR (-) strain. Expression of hpaBC genes encoding the enzyme 4-hydroxyphenylacetate 3-hydroxylase from E. coli W in the strains modified for L-Tyr production caused the synthesis of L-DOPA. One of such strains, having the PTS(-) gluc(+) tyrR (-) phenotype, displayed the best production parameters in minimal medium, with a specific rate of L-DOPA production of 13.6 mg/g/h, L-DOPA yield from glucose of 51.7 mg/g and a final L-DOPA titer of 320 mg/l. In a batch fermentor culture in rich medium this strain produced 1.51 g/l of L-DOPA in 50 h.
Subject(s)
Escherichia coli/metabolism , Glucose/metabolism , Levodopa/biosynthesis , 3-Deoxy-7-Phosphoheptulonate Synthase/genetics , 3-Deoxy-7-Phosphoheptulonate Synthase/metabolism , Chorismate Mutase/genetics , Chorismate Mutase/metabolism , Escherichia coli/genetics , Metabolic Engineering , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , Phosphoenolpyruvate Sugar Phosphotransferase System/genetics , Phosphoenolpyruvate Sugar Phosphotransferase System/metabolism , Plasmids , Prephenate Dehydratase/genetics , Prephenate Dehydratase/metabolism , Prephenate Dehydrogenase/genetics , Prephenate Dehydrogenase/metabolism , Transketolase/genetics , Transketolase/metabolism , Tyrosine/biosynthesis , Zymomonas/enzymologyABSTRACT
BACKGROUND: Anthranilate is an aromatic amine used industrially as an intermediate for the synthesis of dyes, perfumes, pharmaceuticals and other classes of products. Chemical synthesis of anthranilate is an unsustainable process since it implies the use of nonrenewable benzene and the generation of toxic by-products. In Escherichia coli anthranilate is synthesized from chorismate by anthranilate synthase (TrpED) and then converted to phosphoribosyl anthranilate by anthranilate phosphoribosyl transferase to continue the tryptophan biosynthetic pathway. With the purpose of generating a microbial strain for anthranilate production from glucose, E. coli W3110 trpD9923, a mutant in the trpD gene that displays low anthranilate producing capacity, was characterized and modified using metabolic engineering strategies. RESULTS: Sequencing of the trpED genes from E. coli W3110 trpD9923 revealed a nonsense mutation in the trpD gene, causing the loss of anthranilate phosphoribosyl transferase activity, but maintaining anthranilate synthase activity, thus causing anthranilate accumulation. The effects of expressing genes encoding a feedback inhibition resistant version of the enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (aroGfbr), transketolase (tktA), glucokinase (glk) and galactose permease (galP), as well as phosphoenolpyruvate:sugar phosphotransferase system (PTS) inactivation on anthranilate production capacity, were evaluated. In shake flask experiments with minimal medium, strains W3110 trpD9923 PTS- and W3110 trpD9923/pJLBaroGfbrtktA displayed the best production parameters, accumulating 0.70-0.75 g/L of anthranilate, with glucose-yields corresponding to 28-46% of the theoretical maximum. To study the effects of extending the growth phase on anthranilate production a fed-batch fermentation process was developed using complex medium, where strain W3110 trpD9923/pJLBaroGfbrtktA produced 14 g/L of anthranilate in 34 hours. CONCLUSION: This work constitutes the first example of a microbial system for the environmentally-compatible synthesis of anthranilate generated by metabolic engineering. The results presented here, including the characterization of mutation in the trpD gene from strain W3110 trpD9923 and the development of a fermentation strategy, establish a step forward towards the future improvement of a sustainable process for anthranilate production. In addition, the present work provides very useful data regarding the positive and negative consequences of the evaluated metabolic engineering strategies.
ABSTRACT
BACKGROUND: A metabolic regulation study was performed, based upon measurements of enzymatic activities, fermentation performance, and RT-PCR analysis of pathways related to central carbon metabolism, in an ethanologenic Escherichia coli strain (CCE14) derived from lineage C. In comparison with previous engineered strains, this E coli derivative has a higher ethanol production rate in mineral medium, as a result of the elevated heterologous expression of the chromosomally integrated genes encoding PDCZm and ADHZm (pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis). It is suggested that this behavior might be due to lineage differences between E. coli W and C. RESULTS: This study demonstrated that the glycolytic flux is controlled, in this case, by reactions outside glycolysis, i.e., the fermentative pathways. Changes in ethanol production rate in this ethanologenic strain result in low organic acid production rates, and high glycolytic and ethanologenic fluxes, that correlate with enhanced transcription and enzymatic activity levels of PDCZm and ADHZm. Furthermore, a higher ethanol yield (90% of the theoretical) in glucose-mineral media was obtained with CCE14 in comparison with previous engineered E. coli strains, such as KO11, that produces a 70% yield under the same conditions. CONCLUSION: Results suggest that a higher ethanol formation rate, caused by ahigher PDCZm and ADHZm activities induces a metabolic state that cells compensate through enhanced glucose transport, ATP synthesis, and NAD-NADH+H turnover rates. These results show that glycolytic enzymatic activities, present in E. coli W and C under fermentative conditions, are sufficient to contend with increases in glucose consumption and product formation rates.
ABSTRACT
Modification of ethanol productivity and yield, using mineral medium supplemented with glucose or xylose as carbon sources, was studied in ethanologenic Escherichia coli KO11 by increasing the activity of five key carbon metabolism enzymes. KO11 efficiently converted glucose or xylose to ethanol with a yield close to 100% of the theoretical maximum when growing in rich medium. However, when KO11 ferments glucose or xylose in mineral medium, the ethanol yields decreased to only 70 and 60%, respectively. An increase in GALP(Ec) (permease of galactose-glucose-xylose) or PGK(Ec) (phosphoglycerate kinase) activities did not change xylose or glucose and ethanol flux. However, when PDC(Zm) (pyruvate decarboxylase from Zymomonas mobilis) activity was increased 7-fold, the yields of ethanol from glucose or xylose were increased to 85 and 75%, respectively, and organic acid formation rates were reduced. Furthermore, as a response to a reduction in acetate and ATP yield, and a limited PDC(Zm) activity, an increase in PFK(Ec) (phosphofructokinase) or PYK(Bs) (pyruvate kinase from Bacillus stearothermophilus) activity drastically reduced glucose or xylose consumption and ethanol formation flux. This experimental metabolic control analysis showed that ethanol flux in KO11 is negatively controlled by phosphofructokinase and pyruvate kinase, and positively influenced by the PDC(Zm) activity level.
Subject(s)
Escherichia coli/metabolism , Ethanol/metabolism , Pyruvate Decarboxylase/metabolism , Xylose/metabolism , Culture Media , Escherichia coli/enzymology , Escherichia coli/growth & development , Fermentation , Glucose/metabolism , Industrial Microbiology , Phosphofructokinase-1/genetics , Phosphofructokinase-1/metabolism , Pyruvate Decarboxylase/genetics , Recombinant Proteins/metabolismABSTRACT
The expression of the feedback inhibition-insensitive enzyme cyclohexadienyl dehydrogenase (TyrC) from Zymomonas mobilis and the chorismate mutase domain from native chorismate mutase-prephenate dehydratase (PheA(CM)) from Escherichia coli was compared to the expression of native feedback inhibition-sensitive chorismate mutase-prephenate dehydrogenase (CM-TyrA(p)) with regard to the capacity to produce l-tyrosine in E. coli strains modified to increase the carbon flow to chorismate. Shake flask experiments showed that TyrC increased the yield of l-tyrosine from glucose (Y(l-Tyr/Glc)) by 6.8-fold compared to the yield obtained with CM-TyrA(p). In bioreactor experiments, a strain expressing both TyrC and PheA(CM) produced 3 g/liter of l-tyrosine with a Y(l-Tyr/Glc) of 66 mg/g. These values are 46 and 48% higher than the values for a strain expressing only TyrC. The results show that the feedback inhibition-insensitive enzymes can be employed for strain development as part of a metabolic engineering strategy for l-tyrosine production.
Subject(s)
Escherichia coli/genetics , Metabolic Networks and Pathways , Prephenate Dehydratase/genetics , Prephenate Dehydrogenase/genetics , Tyrosine/biosynthesis , Zymomonas/enzymology , Biomass , Bioreactors , Cloning, Molecular , Escherichia coli/metabolism , Gene Expression , Glucose/metabolism , Models, Biological , Plasmids , Zymomonas/geneticsABSTRACT
Las Bacterias Sulfato Reductoras (BSR) son responsables de la producción de sulfuro de hidrógeno (H2S) en anaerobiosis a partir de sulfatos y un donador de electrones. El sulfuro producido podría utilizarse para precipitar metales catiónicos divalentes,permitiendo la biorremediación de efluentes contaminados. El presente estudio forma parte del proyecto: Biodiversidad Microbiana del programa UMSA-SAREC con cooperación del departamento deBiotecnología de la Universidad de Lund y establece la posibilidad de montar biorreactores en el altiplano Boliviano para generar H2S. En el trabajo, seutilizó paja de trigo (donador de electrones) y consorcio bacteriano A10 (BSR) obtenido de la laguna ChalviriPotosí. Bajo condiciones de cultivo batch, se obtuvo una concentración de H2S entre 5 y 8 mM, y en condiciones de cultivo continuo una concentración de25 mM a los 100 días, concentraciones similares se sostuvieron por 90 días, lo que hace este modelo prometedor para su aprovechamiento a escala industrial.