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1.
Microb Cell Fact ; 18(1): 128, 2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387595

RESUMO

BACKGROUND: Acetoin (AC) and 2,3-butanediol (2,3-BD) as highly promising bio-based platform chemicals have received more attentions due to their wide range of applications. However, the non-efficient substrate conversion and mutually transition between AC and 2,3-BD in their natural producing strains not only led to a low selectivity but also increase the difficulty of downstream purification. Therefore, synthetic engineering of more suitable strains should be a reliable strategy to selectively produce AC and 2,3-BD, respectively. RESULTS: In this study, the respective AC (alsS and alsD) and 2,3-BD biosynthesis pathway genes (alsS, alsD, and bdhA) derived from Bacillus subtilis 168 were successfully expressed in non-natural AC and 2,3-BD producing Corynebacterium crenatum, and generated recombinant strains, C. crenatum SD and C. crenatum SDA, were proved to produce 9.86 g L-1 of AC and 17.08 g L-1 of 2,3-BD, respectively. To further increase AC and 2,3-BD selectivity, the AC reducing gene (butA) and lactic acid dehydrogenase gene (ldh) in C. crenatum were then deleted. Finally, C. crenatumΔbutAΔldh SD produced 76.93 g L-1 AC in one-step biocatalysis with the yield of 0.67 mol mol-1. Meanwhile, after eliminating the lactic acid production and enhancing 2,3-butanediol dehydrogenase activity, C. crenatumΔldh SDA synthesized 88.83 g L-1 of 2,3-BD with the yield of 0.80 mol mol-1. CONCLUSIONS: The synthetically engineered C. crenatumΔbutAΔldh SD and C. crenatumΔldh SDA in this study were proved as an efficient microbial cell factory for selective AC and 2,3-BD production. Based on the insights from this study, further synthetic engineering of C. crenatum for AC and 2,3-BD production is suggested.

2.
Molecules ; 24(14)2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31336696

RESUMO

9α-Hydroxy-4-androstene-3,17-dione (9-OH-AD) is one of the significant intermediates for the preparation of ß-methasone, dexamethasone, and other steroids. In general, the key enzyme that enables the biotransformation of 4-androstene-3,17-dione (AD) to 9-OH-AD is 3-phytosterone-9α-hydroxylase (KSH), which consists of two components: a terminal oxygenase (KshA) and ferredoxin reductase (KshB). The reaction is carried out with the concomitant oxidation of NADH to NAD+. In this study, the more efficient 3-phytosterone-9α-hydroxylase oxygenase (KshC) from the Mycobacterium sp. strain VKM Ac-1817D was confirmed and compared with reported KshA. To evaluate the function of KshC on the bioconversion of AD to 9-OH-AD, the characterization of KshC and the compounded system of KshB, KshC, and NADH was constructed. The optimum ratio of KSH oxygenase to reductase content was 1.5:1. An NADH regeneration system was designed by introducing a formate dehydrogenase, further confirming that a more economical process for biological transformation from AD to 9-OH-AD was established. A total of 7.78 g of 9-OH-AD per liter was achieved through a fed-batch process with a 92.11% conversion rate (mol/mol). This enzyme-mediated hydroxylation method provides an environmentally friendly and economical strategy for the production of 9-OH-AD.

3.
Sheng Wu Gong Cheng Xue Bao ; 35(7): 1348-1358, 2019 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-31328491

RESUMO

The trehalose synthase (ScTreS) gene from Streptomyces coelicolor was successfully cloned and heterologously expressed in Escherichia coli BL21(DE3). The protein purified by Ni-NTA affinity column showed an apparent molecular weight (MW) of 62.3 kDa analyzed by SDS-PAGE. The optimum temperature of the enzyme was 35 °C and the optimum pH was 7.0; the enzyme was sensitive to acidic conditions. By homologous modeling and sequence alignment, the enzyme was modified by site-directed mutagenesis. The relative activities of the mutant enzymes K246A and A165T were 1.43 and 1.39 times that of the wild type, an increased conversion rate of 14% and 10% respectively. To optimize the synthesis conditions of trehalose, the mutant strain K246A was cultivated in a 5-L fermentor and used for whole-cell transformation. The results showed that with the substrate maltose concentration of 300 g/L at 35 °C and pH 7.0, the highest conversion rate reached 71.3%, and the yield of trehalose was 213.93 g/L. However, when maltose concentration was increased to 700 g/L, the yield of trehalose can reach 465.98 g/L with a conversion rate of 66%.


Assuntos
Streptomyces coelicolor , Biocatálise , Clonagem Molecular , Escherichia coli , Glucosiltransferases , Trealose
4.
Appl Microbiol Biotechnol ; 103(17): 7055-7070, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31273395

RESUMO

Thermostability plays an important role in the application of L-asparaginase in the pharmaceutical and food industries. Understanding the key residues and structures that influence thermostability in L-asparaginase is necessary to obtain suitable L-asparaginase candidates. In this study, special residues and structures that altered thermostability in thermophilic L-asparaginase and non-thermophilic L-asparaginase II were identified. Interchanging these special residues and structures of L-asparaginases from the four strains, that is, Pyrococcus yayanosii CH1 (PYA), Thermococcus gammatolerans (TGA), Bacillus subtilis (BSA II), and Escherichia coli (ECA II), revealed the 51st and 298th residues of PYA (corresponding to 57th, 305th residues of ECA II) as the key residues responsible for thermal stability of thermophilic L-asparaginase and non-thermophilic L-asparaginase II. Moreover, the C terminal tightness, loop rigidity, and low surface charge around activity sites were of great significance to the thermostability of L-asparaginase. This study therefore revealed the crucial amino acid residues and structures responsible for the difference in thermostability of the thermophilic and non-thermophilic L-asparaginase and provides a reference for engineering thermostability in L-asparaginase II.

5.
J Biotechnol ; 302: 85-91, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31254548

RESUMO

L-theanine, an amino acid known for its favourable taste and linked with health benefits, can be prepared by enzymatic synthesis using γ-glutamyltranspeptidase (GGT; E.C 2.3.2.2). In the present study, a novel GGT from Bacillus pumilus ML413 was expressed in Bacillus subtlis 168 and exhibited high stability at low temperature (40 °C) and alkaline pH 10, compared to the other GGTs. To enhance GGT production, firstly, PrsA lipoproteins was overexpressed in the host which resulted in the extracellular GGT activity doubled. Subsequently, a suitable poly (A/T) tail (TTTAAA) was selected and added to the 3'-terminal of ggt gene, which increased the mRNA stability of ggt gene by 58% and the activity of GGT by 60%. Finally, under optimized fed batch system, L-theanine yield was 53 g l-1 within 16 h. In this study, we demonstrate a convenient strategy of increasing theanine yield using GGT overexpressing in a safe host B. subtilis 168.

6.
J Ind Microbiol Biotechnol ; 46(8): 1155-1166, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31203489

RESUMO

L-Arginine is an important amino acid with extensive application in the food and pharmaceutical industries. The efficiency of nitrogen uptake and assimilation by organisms is extremely important for L-arginine production. In this study, a strain engineering strategy focusing on upregulate intracellular nitrogen metabolism in Corynebacterium crenatum for L-arginine production was conducted. Firstly, the nitrogen metabolism global transcriptional regulator AmtR was deleted, which has demonstrated the beneficial effect on L-arginine production. Subsequently, this strain was engineered by overexpressing the ammonium transporter AmtB to increase the uptake of NH4+ and L-arginine production. To overcome the drawbacks of using a plasmid to express amtB, Ptac, a strong promoter with amtB gene fragment, was integrated into the amtR region on the chromosome in the Corynebacterium crenatum/ΔamtR. The final strain results in L-arginine production at a titer of 60.9 g/L, which was 35.14% higher than that produced by C. crenatum SYPA5-5.

7.
Opt Express ; 27(12): 17199-17208, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31252933

RESUMO

Terahertz (THz) generation in a periodically poled lithium niobate crystal via cascaded difference-frequency generation based on Cherenkov-type quasi-phase matching (QPM) is proposed. Photon conversion efficiency is evaluated based on a promising structure that combines QPM and Cherenkov phase-matching with reduced wave-vector mismatch. Cascading processes contribute to photon conversion efficiency, and THz radiation with maximum photon conversion efficiency of 1154.2% in a 14-order cascaded Stokes process was obtained. Comparing the processes with and without Cherenkov-type radiation, with a 50-MW pump, power was boosted nearly 1.9 times for the former case. These results provide an experimental approach to high-energy THz-wave generation.

8.
Food Chem ; 286: 146-153, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-30827588

RESUMO

Vaccinium bracteatum Thunb. leaves (VBTL) are used to juicing and dye rice to produce 'Wu mi', which displays a deep blue color. However, little information is known about the formation mechanism of the pigments. In this research, non-targeted metabolic profiling of VBTL and VBTL juice samples at different growth stages was studied for searching the pigment precursors through UPLC-QToF-MS and multivariate data analysis. The results showed the L* and b* values of 'Wu mi' produced by spring leaves (stages of 2WAB, 4WAB and 6WAB) were significantly lower than other growth stages. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) of the VBTL and VBTL juice samples showed distinct classifications. Further variable importance in projection (VIP) plot in PLS-DA model demonstrated the discriminatory potential biomarkers between VBTL and VBTL juice samples. Some of the identified biomarkers were tentatively identified as the precursor compounds of the iridoid-derived pigments.


Assuntos
Espectrometria de Massas/métodos , Metabolômica/métodos , Folhas de Planta/metabolismo , Vaccinium myrtillus/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Análise Discriminante , Sucos de Frutas e Vegetais/análise , Análise dos Mínimos Quadrados , Espectrometria de Massas/estatística & dados numéricos , Metabolômica/estatística & dados numéricos , Análise Multivariada , Oryza
9.
ACS Synth Biol ; 8(4): 734-743, 2019 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-30840437

RESUMO

Optically pure 1,2-amino alcohols are highly valuable products as intermediates for chiral pharmaceutical products. Here we designed an environmentally friendly non-natural biocatalytic cascade for efficient synthesis of 1,2-amino alcohols from cheaper epoxides. A redesignated ω-transaminase PAKω-TA was tested and showed good bioactivity at a lower pH than other reported transaminases. The cascade was efficiently constructed as a single one-pot E. coli recombinant, by coupling SpEH (epoxide hydrolase), MnADH (alcohol dehydrogenase), and PAKω-TA. Furthermore, RBS regulation strategy was used to overcome the rate limiting step by increasing expression of MnADH. For cofactor regeneration and amino donor source, an interesting point was involved as that a cofactor self-sufficient system was designed by expression of GluDH. It established a "bridge" between the cofactor and the cosubstrate, such that the cofactor self-sufficient system could release cofactor (NADP+) and cosubstrate (l-Glutamine) regenerated simultaneously. The recombinant E. coli BL21 (SGMP) with cofactor self-sufficient whole-cell cascade biocatalysis showed high ee value (>99%) and high yield, with 99.6% conversion of epoxide ( S)-1a to 1,2-amino alcohol ( S)-1d in 10 h. It further converted ( S)-2a-5a to ( S)-2d-5d with varying conversion rates ranging between 65-96.4%. This study first provides one-step synthesis of optically pure 1,2-amino alcohols from ( S)-epoxides employing a synthetic redox-self-sufficient cascade.

10.
Molecules ; 24(3)2019 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-30736411

RESUMO

In this study, the Micrococcus luteus K-3 glutaminase was successfully over-expressed in the GRAS (Generally Recognized as Safe) Bacillus subtilis strain 168 by integration of the Mglu gene in the 16S rDNA locus. This was done in order to screen a strain producing high levels of recombinant glutaminase from the selected candidates. The transcription of the glutaminase genes in the B. subtilis 168 chromosome and the expression of glutaminase protein was further assessed by qPCR, SDS-PAGE analysis and an enzyme activity assay. To further increase the production of glutaminase, the nprB and nprE genes, which encode specific proteases, were disrupted by integration of the Mglu gene. After continuous cell culturing without the addition of antibiotics, the integrated recombinant strains showed excellent genetic stability, demonstrating favorable industrialization potential. After the fermentation temperature was optimized, a 5-L bioreactor was used for fed-batch fermentation of the recombinant glutaminase producing strain at 24 °C, and the highest enzyme activity achieved was approximately 357.6 U/mL.


Assuntos
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , DNA Ribossômico/genética , Endopeptidases/genética , Fermentação , Glutaminase/biossíntese , Regulação Bacteriana da Expressão Gênica , Glutaminase/metabolismo , Temperatura Ambiente
11.
J Ind Microbiol Biotechnol ; 46(5): 635-647, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30790119

RESUMO

Cholesterol oxidase, steroid C27 monooxygenase and 3-ketosteroid-Δ1-dehydrogenase are key enzymes involved in microbial catabolism of sterols. Here, three isoenzymes of steroid C27 monooxygenase were firstly characterized from Mycobacterium neoaurum as the key enzyme in sterol C27-hydroxylation. Among these three isoenzymes, steroid C27 monooxygenase 2 exhibits the strongest function in sterol catabolism. To improve androst-1,4-diene-3,17-dione production, cholesterol oxidase, steroid C27 monooxygenase 2 and 3-ketosteroid-Δ1-dehydrogenase were coexpressed to strengthen the metabolic flux to androst-1,4-diene-3,17-dione, and 3-ketosteroid 9α-hydroxylase, which catalyzes the androst-1,4-diene-3,17-dione catabolism, was disrupted to block the androst-1,4-diene-3,17-dione degradation pathway in M. neoaurum JC-12. Finally, the recombinant strain JC-12S2-choM-ksdd/ΔkshA produced 20.1 g/L androst-1,4-diene-3,17-dione, which is the highest reported production with sterols as substrate. Therefore, this work is hopes to pave the way for efficient androst-1,4-diene-3,17-dione production through metabolic engineering.


Assuntos
Androstadienos/química , Isoenzimas/metabolismo , Micobactérias não Tuberculosas/metabolismo , Fitosteróis/metabolismo , Esteróis/química , Hidrocarboneto de Aril Hidroxilases/química , Microbiologia Industrial , Engenharia Metabólica , Metabolismo , Oxigenases de Função Mista/metabolismo , Oxirredutases/química , Plasmídeos/metabolismo , Polienos/metabolismo , Esteroide Hidroxilases/química
12.
Microb Cell Fact ; 18(1): 12, 2019 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-30678678

RESUMO

BACKGROUND: Styrene monooxygenase (SMO) catalyzes the first step of aromatic alkene degradation yielding the corresponding epoxides. Because of its broad spectrum of substrates, the enzyme harbors a great potential for an application in medicine and chemical industries. RESULTS: In this study, we achieved higher enzymatic activity and better stability towards styrene by enlarging the ligand entrance tunnel and improving the hydrophobicity through error-prone PCR and site-saturation mutagenesis. It was found that Asp305 (D305) hindered the entrance of the FAD cofactor according to the model analysis. Therefore, substitution with amino acids possessing shorter side chains, like glycine, opened the entrance tunnel and resulted in up to 2.7 times higher activity compared to the wild-type enzyme. The half-lives of thermal inactivation for the variant D305G at 60 °C was 28.9 h compared to only 3.2 h of the wild type SMO. Moreover, overexpression of SMO in Pseudomonas putida KT2440 with NADH regeneration was carried out in order to improve biotransformation efficiency for epoxide production. A hexadecane/buffer (v/v) biphasic system was applied in order to minimize the inactivation effect of high substrate concentrations on the SMO enzyme. Finally, SMO activities of 190 U/g CDW were measured and a total amount of 20.5 mM (S)-styrene oxide were obtained after 8 h. CONCLUSIONS: This study offers an alternative strategy for improved SMO expression and provides an efficient biocatalytic system for epoxide production via engineering the entrance tunnel of the enzyme's active site.


Assuntos
Compostos de Epóxi/metabolismo , Oxigenases/metabolismo , Pseudomonas putida/enzimologia , Sítios de Ligação , Biocatálise , Escherichia coli/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Meia-Vida , Cinética , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , NAD/metabolismo , Oxigenases/genética , Estabilidade Proteica , Pseudomonas putida/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Temperatura Ambiente
13.
Food Chem ; 276: 547-553, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30409631

RESUMO

The use of selected Saccharomyces cerevisiae PS7314, Lactobacillus rossiae NOS7307, Lactobacillus brevis NOS7311, and Lactobacillus plantarum NOS7315 as mono-culture or co-culture for production of sourdoughs, their breads showed different physical and organoleptic properties. The pH of breads fermented with sourdoughs incubated with mono-culture or co-culture all decreased. An opposite trend was found for TTA. The use of single lactobacillus for the dough fermentation decreased the specific volume of bread, which was 4.15-19.10% lower than that of control bread (CB). However, the synergetic fermentation helped the improvement of bread quality. Compared to CB, the mixed culture 4 sourdough remarkably decreased the hardness by 52.08%, increased the specific volume by 5.29%, improved porosity of final product by 24.90%, and gave a preferable sensory characteristic to bread. Thus, the MC4 could be recommended for replacing spontaneous sourdough for improving the quality of bread.


Assuntos
Pão/análise , Pão/microbiologia , Fenômenos Físicos , Sensação , Leveduras/metabolismo , Fermentação , Especificidade da Espécie
14.
Enzyme Microb Technol ; 119: 10-16, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30243381

RESUMO

Catalase catalyzes the decomposition of hydrogen peroxide to water and oxygen. The main role of this enzyme is to prevent cell damage caused by reactive oxygen species (ROS). However, endogenous catalase is sensitive to high temperature and possesses limited activity. To satisfy requirements for this critical bottleneck, in this work, we improved the thermo-stability of a heme-catalase (KatX2) from a high oxidative stress resistance Bacillus pumilus ML413 through the construction of a disulfide bond between S286C and D289C. After the site-directed mutagenesis targeting the disulfide bond between S286C and D289C into the wild-type catalase, a potential improvement of thermo-stability half-life at 60 °C was increased by 48 min compared to the wild-type half-life. Unexpectedly, a catalytic efficiency of KatX2 S286C/D289C mutant was increased by 40% when compared to the wild-type KatX2. More importantly, this unprecedented highly stable KatX2 recombinant mutant S286C/D289C exhibits higher catalytic efficiency and thermo-stability with no change on the catalase secondary structure. Thus, this rational design based KatX2 could be adopted as a potential biocatalyst in industry.

15.
Biotechnol J ; 2018 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-30052323

RESUMO

Unnatural amino acids (UAAs) play a key role in modern medicinal chemistry such as small molecules and peptide-based drugs with fast-growing markets. Low efficiency for natural enzymes including leucine dehydrogenase (LeuDH, EC1.4.1.9) are one major challenge for UAA production. Here, rational engineering of LeuDH from Bacillus cereus with a structure-based design approach is studied. The results achieve higher enzymatic activity and stability toward α-keto acid reduction by improving the hydrophobic and rigidity of enzymatic substrate entrance tunnel. High catalytic efficiency for variant E116V is associated with the presence of more hydrophobic tunnels that allows easy substrate diffusion, which is confirmed in absorbance spectroscopy study. For variant T45M/E116V, melting temperature and half-lives of thermal inactivation at 60 °C is 62.8 °C and 29.2 h, respectively, much higher than 48.4 °C and 3.4 h of wild type. Structural analysis indicates that an additional hydrogen bond in ß5 fold is formed in variant T45M, which results in a more rigid ß5 fold leading to better stability. Furthermore, asymmetric synthesis of α-amino acids with coenzyme regeneration reveals higher productivities for variant T45M/E116V. This study indicates the importance of substrate entrance tunnel for enzymatic activities and stability, the engineered LeuDH would better serve UAA production.

16.
Front Psychol ; 9: 772, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29875721

RESUMO

Previous studies have shown that smiling, fairness, intention, and the results being openness to the proposer can influence the responses in ultimatum games, respectively. But it is not clear that how the four factors might interact with each other in twos or in threes or in fours. This study examined the way that how the four factors work in resource distribution games by testing the differences between average rejection rates in different treatments. Two hundred and twenty healthy volunteers participated in an intentional version of the ultimatum game (UG). The experiment used a 2 × 2 × 2 × 2 mixed design with "openness" as a between subjects factor and the other three as within subjects factors, and the participants were assigned as recipients. The results revealed that fairness or perceived good intention reduced the subject's average rejection rates. There was a significant interaction between facial expressions and openness. With fair offers, the average rejection rate for informed was lower than that of uninformed; but when unfair, no difference between the corresponding average rejection rates was found. The interaction effect of smiling and openness was also significant, the average rejection rate for informed offers was lower when the proposer was smiling and no rejection rate difference between uninformed offers and informed offers when no smiling. No other interaction effect was found.

17.
J Agric Food Chem ; 66(23): 5802-5811, 2018 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-29771121

RESUMO

Geniposide is the main bioactive constituent of gardenia fruit. Skeletal-muscle fibrosis is a common and irreversibly damaging process. Numerous studies have shown that geniposide could improve many chronic diseases, including metabolic syndrome and tumors. However, the effects of geniposide on skeletal-muscle fibrosis are still poorly understood. Here, we found that crude extracts of gardenia fruit pomace could significantly decrease the expression of profibrotic genes in vitro. Moreover, geniposide could also reverse profibrotic-gene expression induced by TGF-ß and Smad4, a regulator of skeletal-muscle fibrosis. In addition, geniposide treatment could significantly downregulate profibrotic-gene expression and improve skeletal-muscle injuries in a mouse model of contusion. These results together suggest that geniposide has an antifibrotic effect on skeletal muscle through the suppression of the TGF-ß-Smad4 signaling pathway.


Assuntos
Frutas/química , Gardenia , Iridoides/uso terapêutico , Músculo Esquelético/patologia , Extratos Vegetais/uso terapêutico , Animais , Fibrose/genética , Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Transdução de Sinais/efeitos dos fármacos , Proteína Smad4/metabolismo , Proteína Smad4/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia
18.
Nat Commun ; 9(1): 1846, 2018 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-29748556

RESUMO

Our understanding of the molecular mechanisms behind bacteria-phage interactions remains limited. Here we report that a small protein, SrpA, controls core cellular processes in response to phage infection and environmental signals in Pseudomonas aeruginosa. We show that SrpA is essential for efficient genome replication of phage K5, and controls transcription by binding to a palindromic sequence upstream of the phage RNA polymerase gene. We identify potential SrpA-binding sites in 66 promoter regions across the P. aeruginosa genome, and experimentally validate direct binding of SrpA to some of these sites. Using transcriptomics and further experiments, we show that SrpA, directly or indirectly, regulates many cellular processes including cell motility, chemotaxis, biofilm formation, pyocyanin synthesis and protein secretion, as well as virulence in a Caenorhabditis elegans model of infection. Further research on SrpA and similar proteins, which are widely present in many other bacteria, is warranted.

19.
J Ind Microbiol Biotechnol ; 45(6): 393-404, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29728854

RESUMO

L-Ornithine is a non-protein amino acid with extensive applications in the food and pharmaceutical industries. In this study, we performed metabolic pathway engineering of an L-arginine hyper-producing strain of Corynebacterium crenatum for L-ornithine production. First, we amplified the L-ornithine biosynthetic pathway flux by blocking the competing branch of the pathway. To enhance L-ornithine synthesis, we performed site-directed mutagenesis of the ornithine-binding sites to solve the problem of L-ornithine feedback inhibition for ornithine acetyltransferase. Alternatively, the genes argA from Escherichia coli and argE from Serratia marcescens, encoding the enzymes N-acetyl glutamate synthase and N-acetyl-L-ornithine deacetylase, respectively, were introduced into Corynebacterium crenatum to mimic the linear pathway of L-ornithine biosynthesis. Fermentation of the resulting strain in a 5-L bioreactor allowed a dramatically increased production of L-ornithine, 40.4 g/L, with an overall productivity of 0.673 g/L/h over 60 h. This demonstrates that an increased level of transacetylation is beneficial for L-ornithine biosynthesis.

20.
Sci Rep ; 8(1): 7915, 2018 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-29784948

RESUMO

L-asparaginase, which catalyses the hydrolysis of L-asparagine to L-aspartate, has attracted the attention of researchers due to its expanded applications in medicine and the food industry. In this study, a novel thermostable L-asparaginase from Pyrococcus yayanosii CH1 was cloned and over-expressed in Bacillus subtilis 168. To obtain thermostable L-asparaginase mutants with higher activity, a robust high-throughput screening process was developed specifically for thermophilic enzymes. In this process, cell disruption and enzyme activity assays are simultaneously performed in 96-deep well plates. By combining error-prone PCR and screening, six brilliant positive variants and four key amino acid residue mutations were identified. Combined mutation of the four residues showed relatively high specific activity (3108 U/mg) that was 2.1 times greater than that of the wild-type enzyme. Fermentation with the mutant strain in a 5-L fermenter yielded L-asparaginase activity of 2168 U/mL.

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