RESUMO
UNLABELLED: : Quantitative assessment of chemical reaction similarity aids database searches, classification of reactions and identification of candidate enzymes. Most methods evaluate reaction similarity based on chemical transformation patterns. We describe a tool, RxnSim, which computes reaction similarity based on the molecular signatures of participating molecules. The tool is able to compare reactions based on similarities of substrates and products in addition to their transformation. It allows masking of user-defined chemical moieties for weighted similarity computations. AVAILABILITY AND IMPLEMENTATION: RxnSim is implemented in R and is freely available from the Comprehensive R Archive Network, CRAN (http://cran.r-project.org/web/packages/RxnSim/). CONTACT: anirban.b@samsung.com or ty76.kim@samsung.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Fenômenos Bioquímicos , Software , Algoritmos , Bases de Dados de Compostos QuímicosRESUMO
Acid-tolerant Saccharomyces cerevisiae was engineered to produce lactic acid by expressing heterologous lactate dehydrogenase (LDH) genes, while attenuating several key pathway genes, including glycerol-3-phosphate dehydrogenase1 (GPD1) and cytochrome-c oxidoreductase2 (CYB2). In order to increase the yield of lactic acid further, the ethanol production pathway was attenuated by disrupting the pyruvate decarboxylase1 (PDC1) and alcohol dehydrogenase1 (ADH1) genes. Despite an increase in lactic acid yield, severe reduction of the growth rate and glucose consumption rate owing to the absence of ADH1 caused a considerable decrease in the overall productivity. In Δadh1 cells, the levels of acetyl-CoA, a key precursor for biologically applicable components, could be insufficient for normal cell growth. To increase the cellular supply of acetyl-CoA, we introduced bacterial acetylating acetaldehyde dehydrogenase (A-ALD) enzyme (EC 1.2.1.10) genes into the lactic acid-producing S. cerevisiae. Escherichia coli-derived A-ALD genes, mhpF and eutE, were expressed and effectively complemented the attenuated acetaldehyde dehydrogenase (ALD)/acetyl-CoA synthetase (ACS) pathway in the yeast. The engineered strain, possessing a heterologous acetyl-CoA synthetic pathway, showed an increased glucose consumption rate and higher productivity of lactic acid fermentation. The production of lactic acid was reached at 142g/L with production yield of 0.89g/g and productivity of 3.55gL(-1)h(-1) under fed-batch fermentation in bioreactor. This study demonstrates a novel approach that improves productivity of lactic acid by metabolic engineering of the acetyl-CoA biosynthetic pathway in yeast.
Assuntos
Acetilcoenzima A , Aldeído Oxirredutases , Proteínas de Escherichia coli , Escherichia coli/genética , Ácido Láctico/biossíntese , Saccharomyces cerevisiae , Acetilcoenzima A/biossíntese , Acetilcoenzima A/genética , Aldeído Oxirredutases/biossíntese , Aldeído Oxirredutases/genética , Escherichia coli/enzimologia , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Production of homo-organic acids without byproducts is an important challenge in bioprocess engineering to minimize operation cost for separation processes. In this study, we used multi-objective optimization to design Escherichia coli strains with the goals of maximally producing target organic acids, while maintaining sufficiently high growth rate and minimizing the secretion of undesired byproducts. Homo-productions of acetic, lactic and succinic acids were targeted as examples. Engineered E. coli strains capable of producing homo-acetic and homo-lactic acids could be developed by taking this systems approach for the minimal identification of gene knockout targets. Also, failure to predict effective gene knockout targets for the homo-succinic acid production suggests that the multi-objective optimization is useful in assessing the suitability of a microorganism as a host strain for the production of a homo-organic acid. The systems metabolic engineering-based approach reported here should be applicable to the production of other industrially important organic acids.
Assuntos
Ácidos Carboxílicos/metabolismo , Escherichia coli , Técnicas de Silenciamento de Genes , Engenharia Metabólica , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
There have been growing concerns regarding the limited fossil resources and global climate changes resulting from modern civilization. Currently, finding renewable alternatives to conventional petrochemical processes has become one of the major focus areas of the global chemical industry sector. Since over 4.2 million tons of acrylic acid (AA) is annually employed for the manufacture of various products via petrochemical processes, this chemical has been the target of efforts to replace the petrochemical route by ecofriendly processes. However, there has been limited success in developing an approach combining the biological production of 3-hydroxypropionic acid (3-HP) and its chemical conversion to AA. Here, we report the first direct fermentative route for producing 0.12 g/L of AA from glucose via 3-HP, 3-HP-CoA, and Acryloyl-CoA, leading to a strain of Escherichia coli capable of directly producing acrylic acid. This route was developed through extensive screening of key enzymes and designing a novel metabolic pathway for AA.
Assuntos
Acrilatos/metabolismo , Fermentação/genética , Coenzima A/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glucose/metabolismo , Química Verde , Ácido Láctico/análogos & derivados , Ácido Láctico/metabolismo , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genética , Saccharomyces cerevisiae/genéticaRESUMO
Owing to the growing market for the biodegradable and renewable polymer, polylactic acid, world demand for lactic acid is rapidly increasing. However, the very high concentrations desired for industrial production of the free lactic acid create toxicity and low pH concerns for manufacturers. Saccharomyces cerevisiae is the most well characterized eukaryote, a preferred microbial cell factory for the largest industrial biotechnology product (bioethanol), and a robust, commercially compatible workhorse to be exploited for the production of diverse chemicals. S. cerevisiae has also been explored as a host for lactic acid production because of its high acid tolerance. Here, we constructed an L-lactic acid-overproducing S. cerevisiae by redirecting cellular metabolic fluxes to the production of L-lactic acid. To this end, we deleted the S. cerevisiae genes encoding pyruvate decarboxylase 1 (PDC1), L-lactate cytochrome-c oxidoreductase (CYB2), and glycerol-3-phosphate dehydrogenase (GPD1), replacing them with a heterologous L-lactate dehydrogenase (LDH) gene. Two new target genes encoding isoenzymes of the external NADH dehydrogenase (NDE1 and NDE2), were also deleted from the genome to re-engineer the intracellular redox balance. The resulting strain was found to produce L-lactic acid more efficiently (32.6% increase in final L-lactic acid titer). When tested in a bioreactor in fed-batch mode, this engineered strain produced 117 g/L of L-lactic acid under low pH conditions. This result demonstrates that the redox balance engineering should be coupled with the metabolic engineering in the construction of L-lactic acid-overproducing S. cerevisiae.
Assuntos
Ácido Láctico/metabolismo , Engenharia Metabólica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Deleção de Genes , Expressão Gênica , Concentração de Íons de Hidrogênio , Redes e Vias Metabólicas/genética , Oxirredução , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
3-Hydroxypropionic acid (3-HP) can be produced in microorganisms as a versatile platform chemical. However, owing to the toxicity of the intermediate product 3-hydroxypropionaldehyde (3-HPA), the minimization of 3-HPA accumulation is critical for enhancing the productivity of 3-HP. In this study, we identified a novel aldehyde dehydrogenase, GabD4 from Cupriavidus necator, and found that it possessed the highest enzyme activity toward 3-HPA reported to date. To augment the activity of GabD4, several variants were obtained by site-directed and saturation mutagenesis based on homology modeling. Escherichia coli transformed with the mutant GabD4_E209Q/E269Q showed the highest enzyme activity, which was 1.4-fold higher than that of wild type GabD4, and produced up to 71.9 g L(-1) of 3-HP with a productivity of 1.8 g L(-1) h(-1) . To the best of our knowledge, these are the highest 3-HP titer and productivity values among those reported in the literature. Additionally, our study demonstrates that GabD4 can be a key enzyme for the development of industrial 3-HP-producing microbial strains, and provides further insight into the mechanism of aldehyde dehydrogenase activity.
Assuntos
Escherichia coli/metabolismo , Ácido Láctico/análogos & derivados , Engenharia Metabólica/métodos , Aldeído Desidrogenase/química , Aldeído Desidrogenase/genética , Aldeído Desidrogenase/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cupriavidus necator/enzimologia , Cupriavidus necator/genética , Escherichia coli/genética , Glicerol/metabolismo , Ácido Láctico/análise , Ácido Láctico/metabolismo , Modelos MolecularesRESUMO
3-Hydroxypropionic acid (3-HP) is a renewable-based platform chemical which may be used to produce a wide range of chemicals including acrylic acid, 1,3-propanediol, and acrylamide. Commercialization of microbial 3-HP production from glycerol, which is produced inexpensively as a by-product of biodiesel production, could be expedited when global biodiesel production increases significantly. For enhancing 3-HP production, this study aimed to investigate metabolic engineering strategies towards eliminating by-products of 3-HP as well as optimizing the glycerol metabolism. The removal of genes involved in the generation of major by-products of 3-HP including acetate and 1,3-propanediol increased both 3-HP production level (28.1g/L) and its average yield (0.217g/g). Optimization of l-arabinose inducible expression of glycerol kinase GlpK, which catalyzes the conversion of glycerol to glycerol-3-phosphate, was also made to increase the metabolic flow from glycerol to 3-HP. To activate the whole glycerol metabolism towards 3-HP, the regulatory factor repressing the utilization of glycerol in Escherichia coli, encoded by glpR was eliminated by knocking-out in its chromosomal DNA. The resulting strain showed a significant improvement in the glycerol utilization rate as well as 3-HP titer (40.5g/L). The transcriptional analysis of glpR deletion mutant revealed the poor expression of glycerol facilitator GlpF, which is involved in glycerol transport in the cell. Additional expression of glpF in the glpR deletion mutant successfully led to an increase in 3-HP production (42.1g/L) and an average yield (0.268g/g).
Assuntos
Escherichia coli/metabolismo , Glicerol/metabolismo , Ácido Láctico/análogos & derivados , Engenharia Metabólica/métodos , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Ácido Láctico/biossínteseRESUMO
1,4-Butanediol (1,4-BDO) is currently produced from succinate via six enzymatic reactions in an engineered Escherichia coli strain. Butyraldehyde dehydrogenase (Bld) and butanol dehydrogenase of Clostridium saccharoperbutylacetonicum were selected based on their activities of catalyzing the final two reactions in the 1,4-BDO pathway. To fit Bld into the non-natural 1,4-BDO pathway, we engineered it through random mutagenesis. Five Bld mutants were then isolated using a colorimetric Schiff's reagent-based method. Subsequent site-directed mutagenesis of Bld generated the two best Bld mutants, L273I and L273T, which produced 1,4-BDO titers fourfold greater than those of wild-type Bld. The enhanced 1,4-BDO titers obtained using L273I and L273T clearly correlated with their enhanced activities, which were caused by amino acid mutations at position 273 of Bld. The highest titer of 1,4-BDO (660 ± 40 mg/L) was obtained in a knock-out E. coli strain [ΔldhA ΔpflB ΔadhE ΔlpdA::K. lpd(E354K) Δmdh ΔarcA gltA(R164L)] coexpressing Bld273T+Bdh.
Assuntos
Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Butileno Glicóis/metabolismo , Clostridium/enzimologia , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genética , Clostridium/genética , Escherichia coli/genética , Mutagênese , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismoRESUMO
The dramatic increase in healthcare cost has become a significant burden to the world. Many patients are denied the accessibility of medication because of the high price of drugs. Total biosynthesis of chiral drug intermediates is an environmentally friendly approach that helps provide more affordable pharmaceuticals. Here we have expanded the natural metabolic capability to biosynthesize a nonnatural amino acid L-homoalanine, which is a chiral precursor of levetiracetam, brivaracetam, and ethambutol. We developed a selection strategy and altered the substrate specificity of ammonium-assimilating enzyme glutamate dehydrogenase. The specificity constant k(cat)/K(m) of the best mutant towards 2-ketobutyrate is 50-fold higher than that towards the natural substrate 2-ketoglutarate. Compared to transaminase IlvE and NADH-dependent valine dehydrogenases, the evolved glutamate dehydrogenase increased the conversion yield of 2-ketobutyrate to L-homoalanine by over 300% in aerobic condition. As a result of overexpressing the mutant glutamate dehydrogenase and Bacillus subtilis threonine dehydratase in a modified threonine-hyperproducing Escherichia coli strain (ATCC98082, DeltarhtA), 5.4 g/L L-homoalanine was produced from 30 g/L glucose (0.18 g/g glucose yield, 26% of the theoretical maximum). This work opens the possibility of total biosynthesis of other nonnatural chiral compounds that could be useful pharmaceutical intermediates.
Assuntos
Aminobutiratos/metabolismo , Escherichia coli/metabolismo , Aminação , Aminobutiratos/química , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Butiratos/metabolismo , Escherichia coli/genética , Glutamato Desidrogenase/química , Glutamato Desidrogenase/genética , Glutamato Desidrogenase/metabolismo , Modelos Moleculares , Estrutura Molecular , Mutação/genética , Estrutura Terciária de Proteína , Streptomyces/enzimologia , Streptomyces/genética , Especificidade por SubstratoRESUMO
In this study, we developed recombinant Escherichia coli strains expressing Lactococcus lactis subsp. lactis Il1403 glutamate decarboxylase (GadB) for the production of GABA from glutamate monosodium salt (MSG). Syntheses of GABA from MSG were examined by employing recombinant E. coli XL1-Blue as a whole cell biocatalyst in buffer solution. By increasing the concentration of E. coli XL1-Blue expressing GadB from the OD600 of 2-10, the concentration and conversion yield of GABA produced from 10 g/L of MSG could be increased from 4.3 to 4.8 g/L and from 70 to 78 %, respectively. Furthermore, E. coli XL1-Blue expressing GadB highly concentrated to the OD600 of 100 produced 76.2 g/L of GABA from 200 g/L of MSG with 62.4 % of GABA yield. Finally, nylon 4 could be synthesized by the bulk polymerization using 2-pyrrolidone that was prepared from microbially synthesized GABA by the reaction with Al2O3 as catalyst in toluene with the yield of 96 %.
Assuntos
Escherichia coli/metabolismo , Nylons/metabolismo , Recombinação Genética , Ácido gama-Aminobutírico/metabolismo , Sequência de Bases , Primers do DNA , Escherichia coli/genética , Glutamato Descarboxilase/metabolismoRESUMO
An Escherichia coli strain was engineered to synthesize 1-hexanol from glucose by extending the coenzyme A (CoA)-dependent 1-butanol synthesis reaction sequence catalyzed by exogenous enzymes. The C4-acyl-CoA intermediates were first synthesized via acetyl-CoA acetyltransferase (AtoB), 3-hydroxybutyryl-CoA dehydrogenase (Hbd), crotonase (Crt), and trans-enoyl-CoA reductase (Ter) from various organisms. The butyryl-CoA synthesized was further extended to hexanoyl-CoA via ß-ketothiolase (BktB), Hbd, Crt, and Ter. Finally, hexanoyl-CoA was reduced to yield 1-hexanol by aldehyde/alcohol dehydrogenase (AdhE2). Enzyme activities for the C6 intermediates were confirmed by assays using HPLC and GC. 1-Hexanol was secreted to the fermentation medium under anaerobic conditions. Furthermore, co-expressing formate dehydrogenase (Fdh) from Candida boidinii increased the 1-hexanol titer. This demonstration of 1-hexanol production by extending the 1-butanol pathway provides the possibility to produce other medium chain length alcohols using the same strategy.
Assuntos
1-Butanol/metabolismo , Enzimas/metabolismo , Escherichia coli/enzimologia , Engenharia Genética , Glucose/metabolismo , Hexanóis/metabolismo , 1-Butanol/química , Biocatálise , Escherichia coli/genética , Escherichia coli/metabolismo , Glucose/química , Hexanóis/químicaRESUMO
1-Butanol, an important chemical feedstock and advanced biofuel, is produced by Clostridium species. Various efforts have been made to transfer the clostridial 1-butanol pathway into other microorganisms. However, in contrast to similar compounds, only limited titers of 1-butanol were attained. In this work, we constructed a modified clostridial 1-butanol pathway in Escherichia coli to provide an irreversible reaction catalyzed by trans-enoyl-coenzyme A (CoA) reductase (Ter) and created NADH and acetyl-CoA driving forces to direct the flux. We achieved high-titer (30 g/liter) and high-yield (70 to 88% of the theoretical) production of 1-butanol anaerobically, comparable to or exceeding the levels demonstrated by native producers. Without the NADH and acetyl-CoA driving forces, the Ter reaction alone only achieved about 1/10 the level of production. The engineered host platform also enables the selection of essential enzymes with better catalytic efficiency or expression by anaerobic growth rescue. These results demonstrate the importance of driving forces in the efficient production of nonnative products.
Assuntos
1-Butanol/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Redes e Vias Metabólicas/genética , Acetilcoenzima A/metabolismo , Acil-CoA Desidrogenases/genética , Acil-CoA Desidrogenases/metabolismo , Anaerobiose , Clostridium/genética , Clostridium/metabolismo , NAD/metabolismoRESUMO
Promising approaches to produce higher alcohols, e.g., isobutanol, using Escherichia coli have been developed with successful results. Here, we translated the isobutanol process from shake flasks to a 1-L bioreactor in order to characterize three E. coli strains. With in situ isobutanol removal from the bioreactor using gas stripping, the engineered E. coli strain (JCL260) produced more than 50 g/L in 72 h. In addition, the isobutanol production by the parental strain (JCL16) and the high isobutanol-tolerant mutant (SA481) were compared with JCL260. Interestingly, we found that the isobutanol-tolerant strain in fact produced worse than either JCL16 or JCL260. This result suggests that in situ product removal can properly overcome isobutanol toxicity in E. coli cultures. The isobutanol productivity was approximately twofold and the titer was 9% higher than n-butanol produced by Clostridium in a similar integrated system.
Assuntos
Reatores Biológicos/microbiologia , Butanóis/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Microbiologia Industrial/métodos , Engenharia GenéticaRESUMO
The production of isobutanol in microorganisms has recently been achieved by harnessing the highly active 2-keto acid pathways. Since these 2-keto acids are precursors of amino acids, we aimed to construct an isobutanol production platform in Corynebacterium glutamicum, a well-known amino-acid-producing microorganism. Analysis of this host's sensitivity to isobutanol toxicity revealed that C. glutamicum shows an increased tolerance to isobutanol relative to Escherichia coli. Overexpression of alsS of Bacillus subtilis, ilvC and ilvD of C. glutamicum, kivd of Lactococcus lactis, and a native alcohol dehydrogenase, adhA, led to the production of 2.6 g/L isobutanol and 0.4 g/L 3-methyl-1-butanol in 48 h. In addition, other higher chain alcohols such as 1-propanol, 2-methyl-1-butanol, 1-butanol, and 2-phenylethanol were also detected as byproducts. Using longer-term batch cultures, isobutanol titers reached 4.0 g/L after 96 h with wild-type C. glutamicum as a host. Upon the inactivation of several genes to direct more carbon through the isobutanol pathway, we increased production by approximately 25% to 4.9 g/L isobutanol in a pycldh background. These results show promise in engineering C. glutamicum for higher chain alcohol production using the 2-keto acid pathways.
Assuntos
Butanóis/metabolismo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Engenharia Genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
The proteomic response of a threonine-overproducing mutant of Escherichia coli was quantitatively analysed by two-dimensional electrophoresis. Evidently, 12 metabolic enzymes that are involved in threonine biosynthesis showed a significant difference in intracellular protein level between the mutant and native strain. The level of malate dehydrogenase was more than 30-fold higher in the mutant strain, whereas the synthesis of citrate synthase seemed to be severely inhibited in the mutant. Therefore, in the mutant, it is probable that the conversion of oxaloacetate into citrate was severely inhibited, but the oxidation of malate to oxaloacetate was significantly up-regulated. Accumulation of oxaloacetate may direct the metabolic flow towards the biosynthetic route of aspartate, a key metabolic precursor of threonine. Synthesis of aspartase (aspartate ammonia-lyase) was significantly inhibited in the mutant strain, which might lead to the enhanced synthesis of threonine by avoiding unfavourable degradation of aspartate to fumarate and ammonia. Synthesis of threonine dehydrogenase (catalysing the degradation of threonine finally back to pyruvate) was also significantly down-regulated in the mutant. The far lower level of cystathionine beta-lyase synthesis in the mutant seems to result in the accumulation of homoserine, another key precursor of threonine. In the present study, we report that the accumulation of important threonine precursors, such as oxaloacetate, aspartate and homoserine, and the inhibition of the threonine degradation pathway played a critical role in increasing the threonine biosynthesis in the E. coli mutant.
Assuntos
Escherichia coli/enzimologia , Mutação/fisiologia , Proteoma/fisiologia , Treonina/biossíntese , Ácido Aspártico/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glucose/metabolismoRESUMO
Genome engineering can be used to produce bacterial strains with a wide range of desired phenotypes. However, the incorporation of gene-sized DNA fragments is often challenging due to the intricacy of the procedure, off-target effects, and low insertion efficiency. Here we report a genome engineering method enabling the continuous incorporation of gene-sized double-stranded DNAs (dsDNAs) into the Escherichia coli genome. DNA substrates are inserted without introducing additional marker genes, by synchronously turning an endogenous counter-selectable marker gene ON and OFF. To accomplish this, we utilized λ Red protein-mediated recombination to insert dsDNAs within the promoter region of a counter-selectable marker gene, tolC. By repeatedly switching the marker gene ON and OFF, a number of desired gene-sized dsDNAs can be inserted consecutively. With this method, we successfully inserted approximately 13â kb gene clusters to generate engineered E. coli strains producing 1,4-butanediol (1,4-BDO).
Assuntos
DNA/genética , Genoma Bacteriano/genética , Engenharia Metabólica/métodos , Regiões Promotoras Genéticas/genética , Proteínas da Membrana Bacteriana Externa/genética , Vias Biossintéticas/genética , Butileno Glicóis/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Marcadores Genéticos , Proteínas de Membrana Transportadoras/genética , Modelos Genéticos , Família Multigênica/genética , Recombinação Genética , Reprodutibilidade dos TestesRESUMO
Hexanoic acid can be used for diverse industrial applications and is a precursor for fine chemistry. Although some natural microorganisms have been screened and evolved to produce hexanoic acid, the construction of an engineered biosynthetic pathway for producing hexanoic acid in yeast has not been reported. Here we constructed hexanoic acid pathways in Kluyveromyces marxianus by integrating 5 combinations of seven genes (AtoB, BktB, Crt, Hbd, MCT1, Ter, and TES1), by which random chromosomal sites of the strain are overwritten by the new genes from bacteria and yeast. One recombinant strain, H4A, which contained AtoB, BktB, Crt, Hbd, and Ter, produced 154mg/L of hexanoic acid from galactose as the sole substrate. However, the hexanoic acid produced by the H4A strain was re-assimilated during the fermentation due to the reverse activity of AtoB, which condenses two acetyl-CoAs into a single acetoacetyl-CoA. This product instability could be overcome by the replacement of AtoB with a malonyl CoA-acyl carrier protein transacylase (MCT1) from Saccharomyces cerevisiae. Our results suggest that Mct1 provides a slow but stable acetyl-CoA chain elongation pathway, whereas the AtoB-mediated route is fast but unstable. In conclusion, hexanoic acid was produced for the first time in yeast by the construction of chain elongation pathways comprising 5-7 genes in K. marxianus.
Assuntos
Caproatos/metabolismo , Kluyveromyces/genética , Kluyveromyces/metabolismo , Engenharia Metabólica/métodos , Biotecnologia , Caproatos/análise , Etanol/metabolismo , Fermentação , Galactose/metabolismo , Glucose/metabolismo , Redes e Vias MetabólicasRESUMO
While Kluyveromyces marxianus is a promising yeast strain for biotechnological applications, genetic engineering of this strain is still challenging, especially when multiple genes are to be transformed. Sequential gene integration, which takes advantage of repetitive insertion/excision of the URA3 gene as a marker, has been the best option until now, because the URA3-deletion mutant is the only precondition for this method. However, we found that the introduced gene is co-excised during the URA3 excision step for next gene introduction, resulting in a very low cumulative probability (<1.57×10â»6 % for 4 genes) of integrating all genes of interest. To overcome this extremely low probability, and to reduce labor and time, all 4 genes were simultaneously transformed. Surprisingly, the infamously high 'non-homologous end joining' activity of K. marxianus enabled simultaneous integration of all 4 genes in a single step, with a probability of 7.9%. Various K. marxianus strains could also be similarly transformed. Our finding not only reduces the labor and time required for such procedures, but also removes a number of preconditions, such as pre-made vectors, selection markers and knockout mutants, which are needed to introduce many genes into K. marxianus.
Assuntos
Cromossomos Fúngicos , Genes Fúngicos , Engenharia Genética/métodos , Kluyveromyces/genética , Proteínas Fúngicas/genética , Deleção de Sequência , Transformação GenéticaRESUMO
One of the major challenges in using electrical energy is the efficiency in its storage. Current methods, such as chemical batteries, hydraulic pumping, and water splitting, suffer from low energy density or incompatibility with current transportation infrastructure. Here, we report a method to store electrical energy as chemical energy in higher alcohols, which can be used as liquid transportation fuels. We genetically engineered a lithoautotrophic microorganism, Ralstonia eutropha H16, to produce isobutanol and 3-methyl-1-butanol in an electro-bioreactor using CO(2) as the sole carbon source and electricity as the sole energy input. The process integrates electrochemical formate production and biological CO(2) fixation and higher alcohol synthesis, opening the possibility of electricity-driven bioconversion of CO(2) to commercial chemicals.
Assuntos
Biocombustíveis , Butanóis/metabolismo , Dióxido de Carbono/metabolismo , Cupriavidus necator/genética , Cupriavidus necator/metabolismo , Pentanóis/metabolismo , Reatores Biológicos , Cupriavidus necator/crescimento & desenvolvimento , Eletricidade , Técnicas Eletroquímicas , Eletrodos , Formiatos/metabolismo , Genes Bacterianos , Engenharia GenéticaRESUMO
Biofuels are currently produced from carbohydrates and lipids in feedstock. Proteins, in contrast, have not been used to synthesize fuels because of the difficulties of deaminating protein hydrolysates. Here we apply metabolic engineering to generate Escherichia coli that can deaminate protein hydrolysates, enabling the cells to convert proteins to C4 and C5 alcohols at 56% of the theoretical yield. We accomplish this by introducing three exogenous transamination and deamination cycles, which provide an irreversible metabolic force that drives deamination reactions to completion. We show that Saccharomyces cerevisiae, E. coli, Bacillus subtilis and microalgae can be used as protein sources, producing up to 4,035 mg/l of alcohols from biomass containing â¼22 g/l of amino acids. These results show the feasibility of using proteins for biorefineries, for which high-protein microalgae could be used as a feedstock with a possibility of maximizing algal growth and total CO(2) fixation.