Improvement in γ-decalactone production by Yarrowia sp. after genome shuffling

2014 ◽  
Vol 68 (8) ◽  
Author(s):  
Yu-Ping Zhao ◽  
Xiao-Qing Mu ◽  
Yan Xu
Keyword(s):  
Cell Growth ◽  
Genome Shuffling ◽  
Fermentation Medium ◽  
Fatty Acyl ◽  
Culture Collection ◽  
Fermentation Time ◽  
Mutant Strains ◽  
Protoplast Preparation ◽  
Acyl Coa Oxidase ◽  
Initial Strains

AbstractIn this study, a modified genome shuffling method was used to improve γ-decalactone (GDL) production of Yarrowia sp. China General Microbiological Culture Collection Center (CGMCC 2.1405). Five UV mutant strains with higher GDL production or shorter fermentation time were selected as the initial strains for genome shuffling. Conditions of protoplast preparation, regeneration, inactivation, fusion, sporulation of recombinant fusants and ascospore isolation were optimized. Four hereditarily stable haploid recombinants with high GDL production were obtained by three rounds of genome shuffling. Among them, a high GDL-producing recombinant, G3-3.21, producing 3.75 g L−1 of GDL in the fermentation medium after 64 h was obtained. This value is 6.54-fold higher than that of the parent strains CGMCC 2.1405, at the peak production shortened by 8 h. Mathematical kinetic models of CGMCC 2.1405 and G3-3.21 were established to well predict the cell growth and GDL production. The cell growth of G3-3.21 was significantly faster than that of CGMCC 2.1405. The product synthesis constant associated with the strain growth of G3-3.21 was higher than that associated with CGMCC 2.1405. Long-chain fatty-acyl-CoA oxidase activities of G3-3.21 were 833 mU mg−1 and 6.83 times higher than that of CGMCC 2.1405.

scholarly journals Genome shuffling enhances stress tolerance of Zymomonas mobilis to two inhibitors

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Weiting Wang ◽  
Bo Wu ◽  
Han Qin ◽  
Panting Liu ◽  
Yao Qin ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Cell Growth ◽  
Stress Tolerance ◽  
Parental Strain ◽  
Zymomonas Mobilis ◽  
De Novo ◽  
Ethanol Yield ◽  
Genome Shuffling ◽  
De Novo Mutation ◽  
Fermentation Time

Abstract Background Furfural and acetic acid are the two major inhibitors generated during lignocellulose pretreatment and hydrolysis, would severely inhibit the cell growth, metabolism, and ethanol fermentation efficiency of Zymomonas mobilis. Effective genome shuffling mediated by protoplast electrofusion was developed and then applied to Z. mobilis. Results After two rounds of genome shuffling, 10 different mutants with improved cell growth and ethanol yield in the presence of 5.0 g/L acetic acid and 3.0 g/L furfural were obtained. The two most prominent genome-shuffled strains, 532 and 533, were further investigated along with parental strains in the presence of 7.0 g/L acetic acid and 3.0 g/L furfural. The results showed that mutants 532 and 533 were superior to the parental strain AQ8-1 in the presence of 7.0 g/L acetic acid, with a shorter fermentation time (30 h) and higher productivity than AQ8-1. Mutant 533 exhibited subtle differences from parental strain F34 in the presence of 3.0 g/L furfural. Mutations present in 10 genome-shuffled strains were identified via whole-genome resequencing, and the source of each mutation was identified as either de novo mutation or recombination of the parent genes. Conclusions These results indicate that genome shuffling is an efficient method for enhancing stress tolerance in Z. mobilis. The engineered strains generated in this study could be potential cellulosic ethanol producers in the future.

scholarly journals Identification and purification of a peroxisomal branched chain fatty acyl-CoA oxidase.

1991 ◽  
Vol 266 (36) ◽  
pp. 24676-24683
Author(s):  
P.P. Van Veldhoven ◽  
G. Vanhove ◽  
F. Vanhoutte ◽  
G. Dacremont ◽  
G. Parmentier ◽  
...  
Keyword(s):  
Fatty Acyl ◽  
Acyl Coa Oxidase ◽  
Branched Chain

scholarly journals Acyl-CoA synthetase and the peroxisomal enzymes of β-oxidation in human liver. Quantitative analysis of their subcellular localization

1984 ◽  
Vol 224 (3) ◽  
pp. 709-720 ◽  
Author(s):  
M Bronfman ◽  
N C Inestrosa ◽  
F O Nervi ◽  
F Leighton
Keyword(s):  
Human Liver ◽  
Fatty Acyl ◽  
Single Step ◽  
Synthetase Activity ◽  
Beta Oxidation ◽  
Analysis Of Results ◽  
Fractionation Method ◽  
Hydroxyacyl Coa Dehydrogenase ◽  
Acyl Coa Oxidase ◽  
Rate Limiting

The presence of acyl-CoA synthetase (EC 6.2.1.3) in peroxisomes and the subcellular distribution of beta-oxidation enzymes in human liver were investigated by using a single-step fractionation method of whole liver homogenates in metrizamide continuous density gradients and a novel procedure of computer analysis of results. Peroxisomes were found to contain 16% of the liver palmitoyl-CoA synthetase activity, and 21% and 60% of the enzyme activity was localized in mitochondria and microsomal fractions respectively. Fatty acyl-CoA oxidase was localized exclusively in peroxisomes, confirming previous results. Human liver peroxisomes were found to contribute 13%, 17% and 11% of the liver activities of crotonase, beta-hydroxyacyl-CoA dehydrogenase and thiolase respectively. The absolute activities found in peroxisomes for the enzymes investigated suggest that in human liver fatty acyl-CoA oxidase is the rate-limiting enzyme of the peroxisomal beta-oxidation pathway, when palmitic acid is the substrate.

scholarly journals Application of DNA Shuffling as a Tool for Hydrolase Activity Improvement of Pseudomonas Strain

2020 ◽  
Vol 11 (1) ◽  
pp. 7735-7745
Keyword(s):  
Protein Engineering ◽  
Protoplast Fusion ◽  
Concentration Gradient ◽  
First Generation ◽  
Human Life ◽  
Genome Shuffling ◽  
Dna Shuffling ◽  
High Concentration ◽  
Mutant Strains ◽  
High Concentrations

Biotechnology is considered one of the most influential technologies in various areas of human life, including health, economics, and the environment. Protein engineering is one of the major biotechnology tools in the field of modification and advancement of biocatalysts capabilities. Among the most effective protein engineering methods, in particular, to improve the industrial strain capabilities, is the shuffling genome method. This study aimed to follow knowledge and biocatalysts engineering techniques based on DNA shuffling methods. In the first step, two procedures were followed (DES method and compatibility according to the concentration gradient of Diazinon) to obtain mutant strains. Acquired mutant strains from both methods were resistant to high concentrations of poison up to 3000 mg/L. The activity of these strains also demonstrated their elevated activity compared to parent samples. The highest activity was related to four strains IR1.G1, IR1.D8, IR1.D4, and IR1.D5, which were 0.234 U/ml, 0.1 U/ml, 0.098 U/ml, and 0.066 U/ml, respectively. The improved strain was obtained via the concentration gradient of the diazinon method (IRL1.G1 strain) in comparison with IRL1.D8 strain (owning highest activity through DES method) possesses excessive activity in 3000 mg/L concentration of Diazinon. The evaluated results of first-generation genome shuffling of strains (the first round of protoplast fusion) also indicated that those shuffled strains with the ability to grow in the vicinity of the toxin (3000 mg/L concentration of Diazinon) showed better activity than obtained mutated strains by both methods (concentration gradient of the toxin and the DES method). In the final stage, the best results were related to IRL1.F2, IRL1.F3, and IRL1.F1 shuffled strains with 0.541 mg/L, 0.523 mg/L, and 0.509 mg/L, respectively. The highest activity belonged to the IRL1.F2 genome shuffled strain (first round of protoplast fusion). This strain could grow in a high concentration of toxin, and also, the activity was increased 30, 3.6, and 2.3 times in comparison with the parent strain (IRL1), IRL.D8 mutant, and IRL1.G1, respectively.

scholarly journals Mutagenesis development of actinoplanes sp. KCTC 9161 by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and screening for acarbose production

2018 ◽  
Vol 14 (4) ◽  
pp. 753-760
Author(s):  
Do Thi Tuyen ◽  
Nguyen The Duong ◽  
Le Thanh Hoang
Keyword(s):  
Type Ii Diabetes ◽  
Wild Type Strain ◽  
Fermentation Medium ◽  
Original Strain ◽  
Wild Type ◽  
Chromatography Method ◽  
Mutant Strains ◽  
Insulin Dependent ◽  
Mutant Lines ◽  
Layer Chromatography

Acarbose has been widely used in the therapy of type II diabetes (non-insulin dependent) because it controls blood sugar contents of patients after meals. Acarbose, a pseudo-oligosaccharide, acts as a competitive -glucosidase inhibitor. Acarbose is produced by the strains of Bacillus, Streptomyces and Actinoplanes sp. The aim of this study was to develop mutagenesis for an Actinoplanes sp. strain and screening for acarbose production. The spores of Actinoplanes sp. KCTC 9161 strain were subjected to be mutated by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) for screening and finding mutant strains that were capable of production of higher acarbose (an inhibitor of α-glucosidase) higher than wild type strain. Firstly, the original NTG solution was prepared in phosphate buffer 0.05 M, pH 6.9 and the safety concentration of NTG was determined at 5 mg/ml. Then, the spores were incubated with different NTG amounts and duration. The living colonies were transferred to fermentation medium. The results obtained showed that 15 mutant strains were produced higher acarbose than wild type when used thin layer chromatography method for analysis and comparing with standard acarbose (Sigma). Three cell lines among total tested 15 mutant lines of Actinoplanes sp. KCTC 9161 produced acarbose at a higher level or indicated a higher inhibitory activity toward α-glucosidase than the original strain. Enzymatic inhibitory ativity of α-glucosidase of three mutant strains (Actinoplanes sp. KCTC- L4, L11, L14) was increased 1.3 fold higher than wild type and Actinoplanes sp. KCTC spores were very sensitive to NTG toxic, 98% spores could not survive at the treatment condition of 50 µg NTG for 30 minutes. In addition, an applicable protocol for mutating Actinoplanes sp. using NTG was suggested for further research.

scholarly journals Experimental Design to Improve Cell Growth and Ethanol Production in Syngas Fermentation by Clostridium carboxidivorans

Catalysts ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 59 ◽  
Author(s):  
Carolina Benevenuti ◽  
Alanna Botelho ◽  
Roberta Ribeiro ◽  
Marcelle Branco ◽  
Adejanildo Pereira ◽  
...  
Keyword(s):  
Cell Growth ◽  
Ethanol Production ◽  
Yeast Extract ◽  
Biomass Gasification ◽  
Culture Collection ◽  
Medium Composition ◽  
Syngas Fermentation ◽  
The Cost ◽  
Clostridium Carboxidivorans ◽  
Peptone Yeast Extract Glucose

Fermentation of gases from biomass gasification, named syngas, is an important alternative process to obtain biofuels. Sequential experimental designs were used to increase cell growth and ethanol production during syngas fermentation by Clostridium carboxidivorans. Based on ATCC (American Type Culture Collection) 2713 medium composition, it was possible to propose a best medium composition for cell growth, herein called TYA (Tryptone-Yeast extract-Arginine) medium and another one for ethanol production herein called TPYGarg (Tryptone-Peptone-Yeast extract-Glucose-Arginine) medium. In comparison to ATCC® 2713 medium, TYA increased cell growth by 77%, reducing 47% in cost and TPYGarg increased ethanol production more than four-times, and the cost was reduced by 31%. In 72 h of syngas fermentation in TPYGarg medium, 1.75-g/L of cells, 2.28 g/L of ethanol, and 0.74 g/L of butanol were achieved, increasing productivity for syngas fermentation.

Differential induction of peroxisomal β-oxidation enzymes by clofibric acid and aspirin in piglet tissues

2001 ◽  
Vol 281 (5) ◽  
pp. R1553-R1561 ◽  
Author(s):  
Xing Xian Yu ◽  
Jack Odle ◽  
James K. Drackley
Keyword(s):  
Body Weight ◽  
Liver Weight ◽  
Fatty Acyl ◽  
Clofibric Acid ◽  
Heart Weight ◽  
Final Body Weight ◽  
Daily Food ◽  
Newborn Pigs ◽  
Acyl Coa Oxidase ◽  
Total Carnitine

Peroxisomal β-oxidation (POX) of fatty acids is important in lipid catabolism and thermogenesis. To investigate the effects of peroxisome proliferators on peroxisomal and mitochondrial β-oxidation in piglet tissues, newborn pigs (1–2 days old) were allowed ad libitum access to milk replacer supplemented with 0.5% clofibric acid (CA) or 1% aspirin for 14 days. CA increased ratios of liver weight to body weight ( P < 0.07), kidney weight to body weight ( P< 0.05), and heart weight to body weight ( P < 0.001). Aspirin decreased daily food intake and final body weight but increased the ratio of heart weight to body weight ( P < 0.01). In liver, activities of POX, fatty acyl-CoA oxidase (FAO), total carnitine palmitoyltransferase (CPT), and catalase were 2.7-, 2.2-, 1.5-fold, and 33% greater, respectively, for pigs given CA than for control pigs. In heart, these variables were 2.2-, 4.1-, 1.9-, and 1.8-fold greater, respectively, for pigs given CA than for control pigs. CA did not change these variables in either kidney or muscle, except that CPT activity was increased ∼110% ( P < 0.01) in kidney. Aspirin increased only hepatic FAO and CPT activities. Northern blot analysis revealed that CA increased the abundance of catalase mRNA in heart by ∼2.2-fold. We conclude that 1) POX and CPT in newborn pigs can be induced by peroxisomal proliferators with tissue specificity and 2) the relatively smaller induction of POX in piglets (compared with that in young or adult rodents) may be related to either age or species differences.

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