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1.
Biochem Biophys Res Commun ; 704: 149710, 2024 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-38417345

RESUMO

IlvA1, a pyridoxal phosphate-dependent (PLP) enzyme, catalyzes the deamination of l-threonine and l-serine to yield 2-ketobutyric acid or pyruvate. To gain insights into the function of IlvA1, we determined its crystal structure from Pseudomonas aeruginosa to 2.3 Å. Density for a 2-ketobutyric acid product was identified in the active site and a putative allosteric site. Activity and substrate binding assays confirmed that IlvA1 utilizes l-threonine, l-serine, and L-allo-threonine as substrates. The enzymatic activity is regulated by the end products l-isoleucine and l-valine. Additionally, the efficiency of d-cycloserine and l-cycloserine inhibitors on IlvA1 enzymatic activity was examined. Notably, site-directed mutagenesis confirmed the active site residues and revealed that Gln165 enhances the enzyme activity, emphasizing its role in substrate access. This work provides crucial insights into the structure and mechanism of IlvA1 and serves as a starting point for further functional and mechanistic studies of the threonine deaminase in P. aeruginosa.


Assuntos
Butiratos , Pseudomonas aeruginosa , Treonina Desidratase , Pseudomonas aeruginosa/metabolismo , Treonina Desidratase/química , Treonina Desidratase/metabolismo , Fosfato de Piridoxal/metabolismo , Ciclosserina , Treonina/metabolismo , Fosfatos , Cristalografia por Raios X
2.
ACS Appl Mater Interfaces ; 15(14): 17705-17715, 2023 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-36995754

RESUMO

Virus-like particles (VLPs) derived from bacteriophage P22 have been explored as biomimetic catalytic compartments. In vivo colocalization of enzymes within P22 VLPs uses sequential fusion to the scaffold protein, resulting in equimolar concentrations of enzyme monomers. However, control over enzyme stoichiometry, which has been shown to influence pathway flux, is key to realizing the full potential of P22 VLPs as artificial metabolons. We present a tunable strategy for stoichiometric control over in vivo co-encapsulation of P22 cargo proteins, verified for fluorescent protein cargo by Förster resonance energy transfer. This was then applied to a two-enzyme reaction cascade. l-homoalanine, an unnatural amino acid and chiral precursor to several drugs, can be synthesized from the readily available l-threonine by the sequential activity of threonine dehydratase and glutamate dehydrogenase. We found that the loading density of both enzymes influences their activity, with higher activity found at lower loading density implying an impact of molecular crowding on enzyme activity. Conversely, increasing overall loading density by increasing the amount of threonine dehydratase can increase activity from the rate-limiting glutamate dehydrogenase. This work demonstrates the in vivo colocalization of multiple heterologous cargo proteins in a P22-based nanoreactor and shows that controlled stoichiometry of individual enzymes in an enzymatic cascade is required for the optimal design of nanoscale biocatalytic compartments.


Assuntos
Capsídeo , Treonina Desidratase , Capsídeo/química , Treonina Desidratase/análise , Glutamato Desidrogenase , Proteínas do Capsídeo/química , Nanotecnologia
3.
Plant Physiol ; 192(1): 527-545, 2023 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-36530164

RESUMO

The hormones salicylic acid (SA) and jasmonic acid (JA) often act antagonistically in controlling plant defense pathways in response to hemibiotrophs/biotrophs (hemi/biotroph) and herbivores/necrotrophs, respectively. Threonine deaminase (TD) converts threonine to α-ketobutyrate and ammonia as the committed step in isoleucine (Ile) biosynthesis and contributes to JA responses by producing the Ile needed to make the bioactive JA-Ile conjugate. Tomato (Solanum lycopersicum) plants have two TD genes: TD1 and TD2. A defensive role for TD2 against herbivores has been characterized in relation to JA-Ile production. However, it remains unknown whether TD2 is also involved in host defense against bacterial hemi/biotrophic and necrotrophic pathogens. Here, we show that in response to the bacterial pathogen-associated molecular pattern (PAMP) flagellin flg22 peptide, an activator of SA-based defense responses, TD2 activity is compromised, possibly through carboxy-terminal cleavage. TD2 knockdown (KD) plants showed increased resistance to the hemibiotrophic bacterial pathogen Pseudomonas syringae but were more susceptible to the necrotrophic fungal pathogen Botrytis cinerea, suggesting TD2 plays opposite roles in response to hemibiotrophic and necrotrophic pathogens. This TD2 KD plant differential response to different pathogens is consistent with SA- and JA-regulated defense gene expression. flg22-treated TD2 KD plants showed high expression levels of SA-responsive genes, whereas TD2 KD plants treated with the fungal PAMP chitin showed low expression levels of JA-responsive genes. This study indicates TD2 acts negatively in defense against hemibiotrophs and positively against necrotrophs and provides insight into a new TD2 function in the elaborate crosstalk between SA and JA signaling induced by pathogen infection.


Assuntos
Infecções Bacterianas , Solanum lycopersicum , Solanum lycopersicum/genética , Treonina Desidratase/genética , Treonina Desidratase/metabolismo , Ciclopentanos/farmacologia , Ciclopentanos/metabolismo , Oxilipinas/farmacologia , Oxilipinas/metabolismo , Ácido Salicílico/farmacologia , Ácido Salicílico/metabolismo , Doenças das Plantas/microbiologia , Regulação da Expressão Gênica de Plantas , Botrytis/fisiologia
4.
Curr Microbiol ; 79(2): 42, 2022 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-34982257

RESUMO

Cysteine and homocysteine suppress the growth of Escherichia coli. It was explained by the inhibition of threonine deaminase (TD) (Harris in J Bacteriol 145(2):1031-1035, 1981). TD inhibition was detected by a decline in its product, 2-ketobutyrate (2-KB). We propose that cysteine or homocysteine may not inhibit TD activity. Instead, 2-KB binds to them forming stable cyclic adducts. This binding possibly leads to isoleucine limitation and growth inhibition.


Assuntos
Aminoácidos/farmacologia , Escherichia coli , Compostos de Sulfidrila/farmacologia , Treonina Desidratase , Escherichia coli/enzimologia , Treonina Desidratase/antagonistas & inibidores
5.
Appl Environ Microbiol ; 88(5): e0213021, 2022 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-35020456

RESUMO

A variety of the yeast Saccharomyces cerevisiae with intracellular accumulation of isoleucine (Ile) would be a promising strain for developing a distinct kind of sake, a traditional Japanese alcoholic beverage, because Ile-derived volatile compounds have a great impact on the flavor and taste of fermented foods. In this study, we isolated an Ile-accumulating mutant (strain K9-I48) derived from a diploid sake yeast of S. cerevisiae by conventional mutagenesis. Strain K9-I48 carries a novel mutation in the ILV1 gene encoding the His480Tyr variant of threonine deaminase (TD). Interestingly, the TD activity of the His480Tyr variant was markedly insensitive to feedback inhibition by Ile, but was not upregulated by valine, leading to intracellular accumulation of Ile and extracellular overproduction of 2-methyl-1-butanol, a fusel alcohol derived from Ile, in yeast cells. The present study demonstrated for the first time that the conserved histidine residue located in a linker region between two regulatory domains is involved in allosteric regulation of TD. Moreover, sake brewed with strain K9-I48 contained 2 to 3 times more 2-methyl-1-butanol and 2-methylbutyl acetate than sake brewed with the parent strain. These findings are valuable for the engineering of TD to increase the productivity of Ile and its derived fusel alcohols. IMPORTANCE Fruit-like flavors of isoleucine-derived volatile compounds, 2-methyl-1-butanol (2MB) and its acetate ester, contribute to a variety of the flavors and tastes of alcoholic beverages. Besides its value as aroma components in foods and cosmetics, 2MB has attracted significant attention as second-generation biofuels. Threonine deaminase (TD) catalyzes the first step in isoleucine biosynthesis and its activity is subject to feedback inhibition by isoleucine. Here, we isolated an isoleucine-accumulating sake yeast mutant and identified a mutant gene encoding a novel variant of TD. The variant TD exhibited much less sensitivity to isoleucine, leading to higher production of 2MB as well as isoleucine than the wild-type TD. Furthermore, sake brewed with a mutant yeast expressing the variant TD contained more 2MB and its acetate ester than that brewed with the parent strain. These findings will contribute to the development of superior industrial yeast strains for high-level production of isoleucine and its related fusel alcohols.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Bebidas Alcoólicas/análise , Etanol/metabolismo , Retroalimentação , Fermentação , Isoleucina , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Treonina Desidratase/genética , Treonina Desidratase/metabolismo
6.
Amino Acids ; 53(6): 903-915, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33938999

RESUMO

The peptidoglycan of the hyperthermophile Thermotoga maritima contains an unusual component, D-lysine (D-Lys), in addition to the typical D-alanine (D-Ala) and D-glutamate (D-Glu). In a previous study, we identified a Lys racemase that is presumably associated with D-Lys biosynthesis. However, our understanding of D-amino acid metabolism in T. maritima and other bacteria remains limited, although D-amino acids in the peptidoglycan are crucial for preserving bacterial cell structure and resistance to environmental threats. Herein, we characterized enzymatic and structural properties of TM0356 that shares a high amino acid sequence identity with serine (Ser) racemase. The results revealed that TM0356 forms a tetramer with each subunit containing a pyridoxal 5'-phosphate as a cofactor. The enzyme did not exhibit racemase activity toward various amino acids including Ser, and dehydratase activity was highest toward L-threonine (L-Thr). It also acted on L-Ser and L-allo-Thr, but not on the corresponding D-amino acids. The catalytic mechanism did not follow typical Michaelis-Menten kinetics; it displayed a sigmoidal dependence on substrate concentration, with highest catalytic efficiency (kcat/K0.5) toward L-Thr. Interestingly, dehydratase activity was insensitive to allosteric regulators L-valine and L-isoleucine (L-Ile) at low concentrations, while these L-amino acids are inhibitors at high concentrations. Thus, TM0356 is a biosynthetic Thr dehydratase responsible for the conversion of L-Thr to α-ketobutyrate and ammonia, which is presumably involved in the first step of the biosynthesis of L-Ile.


Assuntos
Proteínas de Bactérias/química , Thermotoga maritima/enzimologia , Treonina Desidratase/química , Proteínas de Bactérias/genética , Domínios Proteicos , Thermotoga maritima/genética , Treonina Desidratase/genética
7.
J Hazard Mater ; 417: 126102, 2021 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-34015711

RESUMO

Biomineralization is often used by microorganisms to sequester heavy metal ions and provides a potential means for remediating increasing levels of heavy metal pollution. Bacteria have been shown to utilize cysteine for the biomineralization of metal sulfide. Indeed, in the present study, the supplement of L-cysteine was found to significantly improve both cadmium resistance and removal abilities of a deep-sea bacterium Pseudomonas stutzeri 273 through cadmium sulfide (CdS) nanoparticle biomineralization. With a proteomic approach, threonine dehydratase of P. stutzeri 273 (psTD) was proposed to be a key factor enhancing bacterial cadmium resistance through catalyzing L-cysteine desulfuration, H2S generation and CdS nanoparticle biomineralization. Consistently, deletion of the gene encoding psTD in P. stutzeri 273 resulted in the decline of H2S generation, decrease of cadmium resistance, and reduction of cadmium removal ability, confirming the unique function of psTD directing the formation of CdS nanoparticles. Correspondingly, the single-enzyme biomineralization of CdS nanoparticle driven by psTD was further developed, and psTD was shown to act as a capping reagent for the mineralization reaction, which controlling the size and structure of nanocrystals. Our results provide important clues for the construction of engineered bacteria for cadmium bioremediation and widen the synthesis methods of nanomaterials.


Assuntos
Cisteína , Nanopartículas , Biomineralização , Cádmio , Compostos de Cádmio , Proteômica , Sulfetos , Treonina Desidratase
8.
Biochem Cell Biol ; 99(5): 636-644, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-33843274

RESUMO

Histone residues play an essential role in the regulation of various biological processes. In the present study, we utilized the H3/H4 histone mutant library to probe the functional aspects of histone residues in amino acid biosynthesis. We found that the histone residue H3R72 plays a crucial role in the regulation of isoleucine biosynthesis. Substitution of the arginine residue (H3R72) of histone H3 to alanine (H3R72A) renders yeast cells unable to grow in minimal medium. Histone mutant H3R72A requires external supplementation of either isoleucine, serine, or threonine for growth in minimal medium. We also observed that the H3R72 residue and leucine amino acid in synthetic complete medium might play a crucial role in determining the intake of isoleucine and threonine in yeast. Furthermore, gene deletion analysis of ILV1 and CHA1 in the H3R72A mutant confirmed that isoleucine is the sole requirement for growth in minimal medium. Altogether, we have identified that histone H3R72 residue may be crucial for yeast growth in minimal medium by regulating isoleucine biosynthesis through the Ilv1 enzyme in the budding yeast Saccharomyces cerevisiae.


Assuntos
Alanina/metabolismo , Histonas/metabolismo , Isoleucina/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Treonina Desidratase/metabolismo , Arginina/genética , Arginina/metabolismo , Histonas/genética , Mutação
9.
Sheng Wu Gong Cheng Xue Bao ; 37(12): 4231-4242, 2021 Dec 25.
Artigo em Chinês | MEDLINE | ID: mdl-34984870

RESUMO

2-Hydroxybutyric acid (2-HBA) is an important intermediate for synthesizing biodegradable materials and various medicines. Chemically synthesized racemized 2-HBA requires deracemization to obtain optically pure enantiomers for industrial application. In this study, we designed a cascade biosynthesis system in Escherichia coli BL21 by coexpressing L-threonine deaminase (TD), NAD-dependent L-lactate dehydrogenase (LDH) and formate dehydrogenase (FDH) for production of optically pure (S)-2-HBA from bulk chemical L-threonine (L-Thr). To coordinate the production rate and the consumption rate of the intermediate 2-oxobutyric acid in the multi-enzyme cascade catalytic reactions, we explored promoter engineering to regulate the expression levels of TD and FDH, and developed a recombinant strain P21285FDH-T7V7827 with a tunable system to achieve a coordinated multi-enzyme expression. The recombinant strain P21285FDH-T7V7827 was able to efficiently produce (S)-2-HBA with the highest titer of 143 g/L and a molar yield of 97% achieved within 16 hours. This titer was approximately 1.83 times than that of the highest yield reported to date, showing great potential for industrial application. Our results indicated that constructing a multi-enzyme-coordinated expression system in a single cell significantly contributed to the biosynthesis of hydroxyl acids.


Assuntos
Formiato Desidrogenases , Hidroxibutiratos , Escherichia coli/genética , Treonina Desidratase
10.
Sheng Wu Gong Cheng Xue Bao ; 37(12): 4254-4265, 2021 Dec 25.
Artigo em Chinês | MEDLINE | ID: mdl-34984872

RESUMO

Leucine dehydrogenase (LDH) is the key rate-limiting enzyme in the production of L-2-aminobutyric acid (L-2-ABA). In this study, we modified the C-terminal Loop region of this enzyme to improve the specific enzyme activity and stability for efficient synthesis of L-2-ABA. Using molecular dynamics simulation of LDH, we analyzed the change of root mean square fluctuation (RMSF), rationally designed the Loop region with greatly fluctuated RMSF, and obtained a mutant EsLDHD2 with a specific enzyme activity 23.2% higher than that of the wild type. Since the rate of the threonine deaminase-catalyzed reaction converting L-threonine into 2-ketobutyrate was so fast, the multi-enzyme cascade catalysis system became unbalanced. Therefore, the LDH and the formate dehydrogenase were double copied in a new construct E. coli BL21/pACYCDuet-RM. Compared with E. coli BL21/pACYCDuet-RO, the molar conversion rate of L-2-ABA increased by 74.6%. The whole cell biotransformation conditions were optimized and the optimal pH, temperature and substrate concentration were 7.5, 35 °C and 80 g/L, respectively. Under these conditions, the molar conversion rate was higher than 99%. Finally, 80 g and 40 g L-threonine were consecutively fed into a 1 L reaction mixture under the optimal conversion conditions, producing 97.9 g L-2-ABA. Thus, this strategy provides a green and efficient synthesis of L-2-ABA, and has great industrial application potential.


Assuntos
Aminobutiratos , Escherichia coli , Escherichia coli/genética , Leucina Desidrogenase/genética , Treonina Desidratase
11.
Bioengineered ; 11(1): 1124-1136, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33084479

RESUMO

Corynebacterium glutamicum is a safe and popular industrial microorganism that it is gram-positive bacteria with thick cell walls, which hinder the extracellular secretion of products. Surfactant has good surface or interface activity and can destroy the cell membrane of microorganisms. In this study, the surfactant SDS was used to artificially destroy the cell membrane of Corynebacterium glutamicum, increase the permeability of the cell membrane, and increase the ability of the strain to secrete L-isoleucine. This is the first time that surfactants have been applied to the fermentation of Corynebacterium glutamicum. Results indicated that after optimization, the output of L-isoleucine reached 43.67 g/L, which was 13.01% higher than that without sodium dodecyl sulfate. The yield of the by-products, such as valine, leucine, and alanine, was reduced by 72.30%, 64.30%, 71.70%, respectively. This method can promote the production of L-isoleucine while minimizing the damage of SDS to the strain.


Assuntos
Corynebacterium glutamicum/metabolismo , Isoleucina/metabolismo , Dodecilsulfato de Sódio/farmacologia , Acetolactato Sintase/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Corynebacterium glutamicum/efeitos dos fármacos , Fermentação , Treonina Desidratase/metabolismo
12.
Org Biomol Chem ; 18(32): 6236-6240, 2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32729605

RESUMO

4-Fluorothreonine (4-FT) is the only naturally occurring fluorinated amino acid antibiotic. Although two conserved proteins in the 4-FT pathway have been found to be involved in self-detoxification mechanisms, the 4-FT-producing strains may also require an alternative pathway to degrade the intracellular 4-FT. In this study, we examined the possible degradation role of three enzymes involved in threonine metabolite pathways toward 4-FT as a possible degradation route to avoid in vivo 4-FT accumulation. Among these three enzymes, threonine deaminase was found to catalyse a defluorination reaction to generate 4-hydroxy-α-ketobutyrate, which is supposed to be further metabolised by an aldolase that likely is a unique occurrence in the 4-FT-producing strains. Our finding may constitute a 4-FT degradation pathway as a complementary resistance mechanism.


Assuntos
Treonina Desidratase/metabolismo , Treonina/análogos & derivados , Biocatálise , Halogenação , Estrutura Molecular , Treonina/química , Treonina/metabolismo
13.
Sheng Wu Gong Cheng Xue Bao ; 36(4): 782-791, 2020 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-32347072

RESUMO

L-2-aminobutyric acid (L-ABA) is an important chemical raw material and chiral pharmaceutical intermediate. The aim of this study was to develop an efficient method for L-ABA production from L-threonine using a trienzyme cascade route with Threonine deaminase (TD) from Escherichia. coli, Leucine dehydrogenase (LDH) from Bacillus thuringiensis and Formate dehydrogenase (FDH) from Candida boidinii. In order to simplify the production process, the activity ratio of TD, LDH and FDH was 1:1:0.2 after combining different activity ratios in the system in vitro. The above ratio was achieved in the recombinant strain E. coli 3FT+L. Moreover, the transformation conditions were optimized. Finally, we achieved L-ABA production of 68.5 g/L with a conversion rate of 99.0% for 12 h in a 30-L bioreactor by whole-cell catalyst. The environmentally safe and efficient process route represents a promising strategy for large-scale L-ABA production in the future.


Assuntos
Aminobutiratos , Formiato Desidrogenases , Leucina Desidrogenase , Treonina Desidratase , Treonina , Aminobutiratos/síntese química , Bacillus thuringiensis/enzimologia , Candida/enzimologia , Escherichia coli/enzimologia , Formiato Desidrogenases/metabolismo , Leucina Desidrogenase/metabolismo , Treonina/metabolismo , Treonina Desidratase/metabolismo
14.
Mutagenesis ; 35(2): 189-195, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-31769492

RESUMO

Evaluation of the functional impact of germline BRCA1 variants that are likely to be associated to breast and ovarian cancer could help to investigate the mechanism of BRCA1 tumorigenesis. Expression of pathogenic BRCA1 missense variants increased homologous recombination (HR) and gene reversion (GR) in yeast. We thought to exploit yeast genetics to shed light on BRCA1-induced genome instability and tumorigenesis. We determined the effect on GR of several neutral and pathogenic BRCA1 variants in the yeast strain RSY6wt and its isogenic DSB repair mutants, such as mre11∆, rad50∆ and rad51∆. In the RSY6wt, four out of five pathogenic and two out of six neutral variants significantly increased GR; rad51∆ strain, the pathogenic variants C61G and A1708E induced a weak but significant increase in GR. On the other hand, in rad50∆ mutant expressing the pathogenic variants localised at the BRCT domain, a further GR increase was seen. The neutral variant N132K and the VUS A1789T induced a weak GR increase in mre11∆ mutant. Thus, BRCA1 missense variants require specific genetic functions and presumably induced GR by different mechanisms. As DNA repair is regulated by cell cycle, we determined the effect on GR of BRCA1 variants in cell cycle-arrested RSYwt cells. GR is highly BRCA1-inducible in S-phase-arrested cells as compared to G1 or G2. Sequence analysis of genomic DNA from ILV1 revertant clones showed that BRCA1-induced ilv1-92 reversion by base substitution when GR is at least 6-fold over the control. Our study demonstrated that BRCA1 may interfere with yeast DNA repair functions that are active in S-phase causing high level of GR. In addition, we confirmed here that yeast could be a reliable model to investigate the mechanism and genetic requirements of BRCA1-induced genome instability. Finally, developing yeast-based assays to characterise BRCA1 missense variants could be useful to design more precise therapies.


Assuntos
Proteína BRCA1/genética , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Rad51 Recombinase/genética , Proteínas de Saccharomyces cerevisiae/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinogênese , Pontos de Checagem do Ciclo Celular/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/genética , Feminino , Instabilidade Genômica/genética , Humanos , Mutação de Sentido Incorreto/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Saccharomyces cerevisiae/genética , Treonina Desidratase/genética
15.
Science ; 365(6453): 595-598, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31395784

RESUMO

In synthetic biology, methods for stabilizing genetically engineered functions and confining recombinant DNA to intended hosts are necessary to cope with natural mutation accumulation and pervasive lateral gene flow. We present a generalizable strategy to preserve and constrain genetic information through the computational design of overlapping genes. Overlapping a sequence with an essential gene altered its fitness landscape and produced a constrained evolutionary path, even for synonymous mutations. Embedding a toxin gene in a gene of interest restricted its horizontal propagation. We further demonstrated a multiplex and scalable approach to build and test >7500 overlapping sequence designs, yielding functional yet highly divergent variants from natural homologs. This work enables deeper exploration of natural and engineered overlapping genes and facilitates enhanced genetic stability and biocontainment in emerging applications.


Assuntos
Genes Essenciais , Homologia de Genes , Engenharia Genética/métodos , Aptidão Genética , Instabilidade Genômica , Proteínas de Bactérias/genética , DNA Recombinante , Mutagênese , Mutação Silenciosa , Biologia Sintética , Treonina Desidratase/genética
16.
Nat Commun ; 9(1): 3818, 2018 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-30232330

RESUMO

The preparation of α-functionalized organic acids can be greatly simplified by adopting a protocol involving the catalytic assembly of achiral building blocks. However, the enzymatic assembly of small amino acids and aldehydes to form numerous α-functionalized organic acids is highly desired and remains a significant challenge. Herein, we report an artificially designed chiral-group-resetting biocatalytic process, which uses simple achiral glycine and aldehydes to synthesize stereodefined α-functionalized organic acids. This cascade biocatalysis comprises a basic module and three different extender modules and operates in a modular assembly manner. The engineered Escherichia coli catalysts, which contained different module(s), provide access to α-keto acids, α-hydroxy acids, and α-amino acids with excellent conversion and enantioselectivities. Therefore, this biocatalytic process provides an attractive strategy for the conversion of low-cost achiral starting materials to high-value α-functionalized organic acids.


Assuntos
Biocatálise , Ácidos Carboxílicos/química , Ácidos Carboxílicos/metabolismo , Aldeídos/metabolismo , Biotransformação , Corynebacterium glutamicum/enzimologia , Glicina/metabolismo , Especificidade por Substrato , Treonina Desidratase
17.
Biochemistry ; 57(41): 6003-6012, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30226377

RESUMO

The biosynthesis of branched-chain amino acids or BCAAs (l-isoleucine, l-leucine, and l-valine) is essential in eubacteria, but mammals are branched-chain amino acid auxotrophs, making the enzymes in the pathway excellent targets for antibacterial drug development. The biosynthesis of l-isoleucine, l-leucine, and l-valine is very efficient, requiring only eight enzymes. Threonine dehydratase (TD), a pyridoxal 5'-phosphate (PLP)-dependent enzyme encoded by the ilvA gene, is the enzyme responsible for the conversion of l-threonine (l-Thr) to α-ketobutyrate, ammonia, and water, which is the first step in the biosynthesis of l-isoleucine. We have cloned, expressed, and biochemically characterized the reaction catalyzed by Mycobacterium smegmatis TD (abbreviated as MsIlvA) using steady-state kinetics and kinetic isotope effects. We show here that in addition to l-threonine, l-allo-threonine and l-serine are also used as substrates by TD, and all exhibit sigmoidal, non-Michaelis-Menten kinetics. Curiously, ß-chloro-l-alanine was also a substrate rather than an inhibitor as expected. The enzymatic activity of TD is sensitive to the presence of allosteric regulators, including the activator l-valine or the end product feedback inhibitor of the BCAA pathway in which TD is involved, l-isoleucine. Primary deuterium kinetic isotopes are small, suggesting Cα proton abstraction is only partially rate-limiting. Solvent kinetic isotopes were significantly larger, indicating that a proton transfer occurring during the reaction is also partially rate-limiting. Finally, we demonstrate that l-cycloserine, a general inhibitor of PLP-dependent enzymes, is an excellent inhibitor of threonine deaminase.


Assuntos
Proteínas de Bactérias/química , Mycobacterium smegmatis/enzimologia , Treonina Desidratase/química , Regulação Alostérica/fisiologia , Proteínas de Bactérias/metabolismo , Catálise , Cinética , Especificidade por Substrato/fisiologia , Treonina Desidratase/metabolismo
18.
Biotechnol Lett ; 40(11-12): 1551-1559, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30259222

RESUMO

OBJECTIVE: To strengthen NADH regeneration in the biosynthesis of L-2-aminobutyric acid (L-ABA). RESULTS: L-Threonine deaminase (L-TD) from Escherichia coli K12 was modified by directed evolution and rational design to improve its endurance to heat treatment. The half-life of mutant G323D/F510L/T344A at 42 °C increased from 10 to 210 min, a 20-fold increase compared to the wild-type L-TD, and the temperature at which the activity of the enzyme decreased by 50% in 15 min increased from 39 to 53 °C. The mutant together with thermostable L-leucine dehydrogenase from Bacillus sphaericus DSM730 and formate dehydrogenase from Candida boidinii constituted a one-pot system for L-ABA biosynthesis. Employing preheat treatment in the one-pot system, the biosynthesis of L-ABA and total turnover number of NAD+/NADH were 0.993 M and 16,469, in contrast to 0.635 M and 10,531 with wild-type L-TD, respectively. CONCLUSIONS: By using the engineered L-TD during endured preheat treatment, the one-pot system has achieved a higher productivity of L-ABA and total turnover number of coenzyme.


Assuntos
Aminobutiratos/metabolismo , Proteínas de Escherichia coli/química , NAD/metabolismo , Treonina Desidratase/química , Aminobutiratos/análise , Evolução Molecular Direcionada/métodos , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Temperatura Alta , Mutação , Treonina Desidratase/genética , Treonina Desidratase/metabolismo
19.
Microb Cell Fact ; 17(1): 38, 2018 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-29523149

RESUMO

BACKGROUND: To produce 1-propanol as a potential biofuel, metabolic engineering of microorganisms, such as E. coli, has been studied. However, 1-propanol production using metabolically engineered Saccharomyces cerevisiae, which has an amazing ability to produce ethanol and is thus alcohol-tolerant, has infrequently been reported. Therefore, in this study, we aimed to engineer S. cerevisiae strains capable of producing 1-propanol at high levels. RESULTS: We found that the activity of endogenous 2-keto acid decarboxylase and alcohol/aldehyde dehydrogenase is sufficient to convert 2-ketobutyrate (2 KB) to 500 mg/L 1-propanol in yeast. Production of 1-propanol could be increased by: (i) the construction of an artificial 2 KB biosynthetic pathway from pyruvate via citramalate (cimA); (ii) overexpression of threonine dehydratase (tdcB); (iii) enhancement of threonine biosynthesis from aspartate (thrA, thrB and thrC); and (iv) deletion of the GLY1 gene that regulates a competing pathway converting threonine to glycine. With high-density anaerobic fermentation of the engineered S. cerevisiae strain YG5C4231, we succeeded in producing 180 mg/L 1-propanol from glucose. CONCLUSION: These results indicate that the engineering of a citramalate-mediated pathway as a production method for 1-propanol in S. cerevisiae is effective. Although optimization of the carbon flux in S. cerevisiae is necessary to harness this pathway, it is a promising candidate for the large-scale production of 1-propanol.


Assuntos
1-Propanol/metabolismo , Butiratos/metabolismo , Engenharia Metabólica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Vias Biossintéticas , Etanol/metabolismo , Fermentação , Glucose/metabolismo , Ácido Pirúvico/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Treonina Desidratase/metabolismo
20.
Appl Microbiol Biotechnol ; 102(4): 1783-1795, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29305698

RESUMO

Higher alcohols significantly influence the quality and flavor profiles of Chinese Baijiu. ILV1-encoded threonine deaminase, LEU1-encoded α-isopropylmalate dehydrogenase, and LEU2-encoded ß-isopropylmalate dehydrogenase are involved in the production of higher alcohols. In this work, ILV1, LEU1, and LEU2 deletions in α-type haploid, a-type haploid, and diploid Saccharomyces cerevisiae strains and ILV1, LEU1, and LEU2 single-allele deletions in diploid strains were constructed to examine the effects of these alterations on the metabolism of higher alcohols. Results showed that different genetic engineering strategies influence carbon flux and higher alcohol metabolism in different manners. Compared with the parental diploid strain, the ILV1 double-allele-deletion diploid mutant produced lower concentrations of n-propanol, active amyl alcohol, and 2-phenylethanol by 30.33, 35.58, and 11.71%, respectively. Moreover, the production of isobutanol and isoamyl alcohol increased by 326.39 and 57.6%, respectively. The LEU1 double-allele-deletion diploid mutant exhibited 14.09% increased n-propanol, 33.74% decreased isoamyl alcohol, and 13.21% decreased 2-phenylethanol production, which were similar to those of the LEU2 mutant. Furthermore, the LEU1 and LEU2 double-allele-deletion diploid mutants exhibited 41.72 and 52.18% increased isobutanol production, respectively. The effects of ILV1, LEU1, and LEU2 deletions on the production of higher alcohols by α-type and a-type haploid strains were similar to those of double-allele deletion in diploid strains. Moreover, the isobutanol production of the ILV1 single-allele-deletion diploid strain increased by 27.76%. Variations in higher alcohol production by the mutants are due to the carbon flux changes in yeast metabolism. This study could provide a valuable reference for further research on higher alcohol metabolism and future optimization of yeast strains for alcoholic beverages.


Assuntos
Bebidas Alcoólicas/microbiologia , Ciclo do Carbono/genética , Etanol/metabolismo , Microbiologia de Alimentos/métodos , Hidroliases/genética , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Treonina Desidratase/genética , 3-Isopropilmalato Desidrogenase/genética , 3-Isopropilmalato Desidrogenase/metabolismo , China , Fermentação , Deleção de Genes , Humanos , Hidroliases/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Treonina Desidratase/metabolismo
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