Overproduction of a thermostable 4‐α‐glucanotransferase by codon optimization at N‐terminus region

Kim, Minsu; Jang, Jun-Hyuck; Kim, Young-Wan

doi:10.1002/jsfa.6084

Cited by 13 publications

(10 citation statements)

References 45 publications

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“…Studies have shown that in E . coli if rare codons occur near the 5’ end of an ORF, especially as clusters, the effect can be detrimental, resulting in very low to no expression of foreign genes [ 20 , 48 , 49 , 50 , 51 , 52 ]. By inserting the rarest codon AGA Arg at different positions near the 5’ end of a gene Kim and Lee [ 20 ] reported that positioning at the +2 and +3 positions had the most negative effect.…”

Section: Discussionmentioning

confidence: 99%

Elevation of the Yields of Very Long Chain Polyunsaturated Fatty Acids via Minimal Codon Optimization of Two Key Biosynthetic Enzymes

Xia

et al. 2016

PLoS ONE

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Eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17) and Docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19) are nutritionally beneficial to human health. Transgenic production of EPA and DHA in oilseed crops by transferring genes originating from lower eukaryotes, such as microalgae and fungi, has been attempted in recent years. However, the low yield of EPA and DHA produced in these transgenic crops is a major hurdle for the commercialization of these transgenics. Many factors can negatively affect transgene expression, leading to a low level of converted fatty acid products. Among these the codon bias between the transgene donor and the host crop is one of the major contributing factors. Therefore, we carried out codon optimization of a fatty acid delta-6 desaturase gene PinD6 from the fungus Phytophthora infestans, and a delta-9 elongase gene, IgASE1 from the microalga Isochrysis galbana for expression in Saccharomyces cerevisiae and Arabidopsis respectively. These are the two key genes encoding enzymes for driving the first catalytic steps in the Δ6 desaturation/Δ6 elongation and the Δ9 elongation/Δ8 desaturation pathways for EPA/DHA biosynthesis. Hence expression levels of these two genes are important in determining the final yield of EPA/DHA. Via PCR-based mutagenesis we optimized the least preferred codons within the first 16 codons at their N-termini, as well as the most biased CGC codons (coding for arginine) within the entire sequences of both genes. An expression study showed that transgenic Arabidopsis plants harbouring the codon-optimized IgASE1 contained 64% more elongated fatty acid products than plants expressing the native IgASE1 sequence, whilst Saccharomyces cerevisiae expressing the codon optimized PinD6 yielded 20 times more desaturated products than yeast expressing wild-type (WT) PinD6. Thus the codon optimization strategy we developed here offers a simple, effective and low-cost alternative to whole gene synthesis for high expression of foreign genes in yeast and Arabidopsis.

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Section: Discussionmentioning

confidence: 99%

Elevation of the Yields of Very Long Chain Polyunsaturated Fatty Acids via Minimal Codon Optimization of Two Key Biosynthetic Enzymes

Xia

et al. 2016

PLoS ONE

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“…The recombinant E. coli cells were cultured overnight at 37°C in Luria-Bertani broth [1% (w/v) Bacto-tryptone, 0.5% (w/v) yeast extract, and 0.5% (w/v) NaCl] supplemented with kanamycin (20 lg/mL). Production of the codon-optimized TTaGT (previously termed TTaGT-mut6) was carried out as described previously [16]. The protein concentration was determined according to the method of Bradford [19], using bovine serum albumin as a standard.…”

Section: Methodsmentioning

confidence: 99%

“…Using an Escherichia coli-B. subtilis shuttle vector equipped with a strong promoter, a thermostable aGTase from Thermus thermophilus (TTaGT) was successfully produced in B. subtilis [16]. Because aGTases are originally cytoplasmic proteins, the recombinant TTaGT in B. subtilis existed in the cytoplasm.…”

Section: Introductionmentioning

confidence: 99%

Complex formation of a 4-α-glucanotransferase using starch as a biocatalyst for starch modification

Yoon

Kim

et al. 2017

Food Sci Biotechnol

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A 4-α-glucanotransferases from (TTαGT) possesses an extra substrate binding site, leading to facile purification of the intact enzyme using amylose as an insoluble binding matrix. Due to the cost of amylose and low recovery yield, starch was replaced for amylose as an alternative capturer in this study. Using gelatinized corn starch at pH 9 with 36-h incubation in the presence of 1 M ammonium sulfate increased the TTαGT-starch complex formation yield from 2 to 56%. In preparative-scale production, TTαGT produced in was recovered by 42.1% with the same specific activity as that of purified TTαGT. Structural and rheological analyses of the enzymatically modified starches revealed that the starch complex exhibited catalytic performance comparable to soluble TTαGT, suggesting that the starch complex can be used as a biocatalyst for modified starch production without elution of the enzyme from the complex.

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“…α-GTase transglycosylation activity is useful in carbohydrate chemistry. Starches that are modified by α-GTases show novel rheological and nutritional properties, such as thermoreversible gelation, fat-replacing properties, and hypocholesterolemic and hypoglycemic effects [9]. α-GTases from different bacteria have successfully modified the properties of various food materials, including increased water solubility, stability, functional effects, and taste [10].…”

Section: Introductionmentioning

confidence: 99%

“…α-GTases from different bacteria have successfully modified the properties of various food materials, including increased water solubility, stability, functional effects, and taste [10]. In addition, intramolecular glucan transferase produces cyclic glucans (cycloamyloses) with a higher degree of polymerization compared with cyclodextrins [9]. 1-Deoxynojirimycin (DNJ), an aza-sugar, has structural characteristics similar to those of cyclic monosaccharides; however, the oxygen is substituted with a nitrogen atom.…”

Section: Introductionmentioning

confidence: 99%

Transglycosylation Properties of a Novelα-1,4-Glucanotransferase fromBacteroides thetaiotaomicronand Its Application in Developing anα-Glucosidase-Specific Inhibitor

Choi

Kim

et al. 2018

Journal of Chemistry

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In this study, α-glucanotransferase from Bacteroides thetaiotaomicron was expressed in Escherichia coli and characterized. Conserved amino-acid sequence alignment showed that Bacteroides thetaiotaomicron α-glucanotransferase (BtαGTase) belongs to the glycoside hydrolase family 77. The enzyme exhibited optimal catalytic activity at 60°C and pH 3.0. BtαGTase catalyzed transglycosylation reactions that produced only glycosyl or maltosyl transfer products, which are preferable for the generation of transglycosylated products with high yield. The 1-deoxynojirimycin (DNJ) glycosylation product G1-DNJ was generated using BtαGTase, and the inhibitory effect of G1-DNJ was analyzed. A kinetic study of inhibition revealed that G1-DNJ inhibited α-glucosidase to a greater extent than did DNJ but did not show any inhibitory effects towards α-amylase, suggesting that G1-DNJ is a potential candidate for the prevention of diabetes.

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Overproduction of a thermostable 4‐α‐glucanotransferase by codon optimization at N‐terminus region

Abstract: The codon-optimized TTαGT that was produced in a GRAS microorganism, B. subtilis, without the selection antibiotics is potentially useful in the food industry as a food-grade enzyme.

Cited by 13 publications

References 45 publications

Elevation of the Yields of Very Long Chain Polyunsaturated Fatty Acids via Minimal Codon Optimization of Two Key Biosynthetic Enzymes

Elevation of the Yields of Very Long Chain Polyunsaturated Fatty Acids via Minimal Codon Optimization of Two Key Biosynthetic Enzymes

Complex formation of a 4-α-glucanotransferase using starch as a biocatalyst for starch modification

Transglycosylation Properties of a Novelα-1,4-Glucanotransferase fromBacteroides thetaiotaomicronand Its Application in Developing anα-Glucosidase-Specific Inhibitor

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