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Pyruvate carboxylase mutant N315F with improved enzymatic activity and application of pyruvate carboxylase mutant N315F

A technology of pyruvate carboxylase and mutants, which is applied in the fields of genetic engineering and fermentation engineering, can solve problems such as carbon flow loss, achieve the effects of reducing liquid loading, increasing dissolved oxygen, and increasing production

Active Publication Date: 2015-11-18
JIANGNAN UNIV
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  • Abstract
  • Description
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Problems solved by technology

However, the batch fermentation of Saccharomyces cerevisiae under the conditions of high concentration of sugar and aeration will produce a large amount of ethanol. For the fermentation with carboxylic acid as the target product, the large accumulation of ethanol will cause a large loss of carbon flow

Method used

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  • Pyruvate carboxylase mutant N315F with improved enzymatic activity and application of pyruvate carboxylase mutant N315F
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  • Pyruvate carboxylase mutant N315F with improved enzymatic activity and application of pyruvate carboxylase mutant N315F

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Embodiment 1

[0029] The localization of embodiment 1 RoPYC (pyruvate carboxylase from Rhizopus oryzae) in Saccharomyces cerevisiae

[0030] 1. TEF1promoter and RoPYC gene ORFs were cloned into pGFP33 vector

[0031] According to the principle of the homologous recombination kit, design primers with more than 15 bases homologous to the vector at both ends, as shown in Table 1, the lowercase letters are homologous arms, and the uppercase letters are PCR primers.

[0032] Table 1 Primers for amplifying the RoPYC gene

[0033]

[0034] Using pY15TEF1-RoPYC as a template (Xuetal.FumaricacidproductioninSaccharomycescerevisiaebbyinsilicoaidedmetabolicengineering, 2012), RoPYC-F, RoPYC-R (sequences are shown in SEQIDNO.3, SEQIDNO.4 respectively) as primers to amplify the ORF frame of TEF1p and RoPYC (obtained PCR The product contains the nucleotide sequence encoding the amino acid sequence SEQIDNO.1), the amplified product is connected to the pGFP33 vector, transformed into large intestine com...

Embodiment 2

[0039] Example 2 RoPYC site-directed mutation and expression

[0040] Site-directed mutagenesis was performed by PCR method, saturation mutation was performed on the N315 site of RoPYC, and the asparagine at the 315th site was mutated into other 19 amino acids. The entire plasmid was amplified by PCR using the pY15TEF1-RoPYC plasmid as a template, F and R containing mutation sites as primers, and Takara's high-fidelity enzyme PrimeSTARGXL. The enzyme digestion system contains 1 μL of PCR product and 1 μL of DpnI enzyme, with a total volume of 20 μL, digested overnight at 37°C. Fragment purification of digested products. Take 5 μL of the purified product and transform it into 30 μL of competent cells Trans1-T1, smear the LA plate, inoculate the grown transformant in the LA medium, extract the plasmid and send it to Shanghai Sangon for sequencing.

[0041] Among them, the primers F(Phe) and R(Phe) (sequences shown in SEQ ID NO.5 and SEQ ID NO.6 respectively) used for N315F mut...

Embodiment 3

[0046] Example 3 Expression of Mutant N315F and Production of Fumaric Acid

[0047] Compared the specific enzyme activity of pyruvate carboxylase mutant N315F and the parent enzyme, and the impact of different loading liquid volumes on the accumulation of fumaric acid in the genetically engineered bacteria expressing N315F, the results are as follows: image 3 and Figure 4 shown.

[0048] Culture conditions: the genetically engineered bacteria seeds cultivated at 30°C and 220rpm for 24h were transferred to fermentation culture at an inoculum size of 5%, and then cultivated at 30°C and 220rpm for 96h.

[0049] Depend on image 3 It can be seen that the enzyme activity of the mutant N315F increased by 18.6% compared with the parents.

[0050] Depend on Figure 4 It can be seen that reducing the amount of liquid and increasing dissolved oxygen is beneficial to the accumulation of fumaric acid. When the amount of liquid was 40mL / 250mL, the output of fumaric acid reached 318 ±...

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Abstract

The invention discloses a pyruvate carboxylase mutant N315F with improved enzymatic activity and application of the pyruvate carboxylase mutant N315F, and belongs to the field of genetic engineering and fermentation engineering. A N315 locus of the pyruvate carboxylase of rhizopus oryzae is mutated to phenylalanine, so that the enzymatic activity of the obtained mutant is increased by 18.6 percent. A gene FUM1 is knocked out on the basis of knocking out PDC1 and ADH1; meanwhile, when the pyruvate carboxylase mutant N315F is excessively expressed, the fumaric acid yield is increased by 22 percent. Meanwhile, by reducing loaded liquid and increasing the dissolved oxygen, the fumaric acid yield can reach 318+ / -11.2 mg / L, which is increased by 25.7 percent compared with the fumaric acid yield (253+ / -9.0 mg / L) when the loaded liquid is 100mL / 250mL. By adopting the pyruvate carboxylase mutant N315F and the application thereof, a synthetic path for a carbon metabolic flow to enter the fumaric acid from pyruvic acid is effectively reinforced, a condition is created for efficiently producing the fumaric acid and other dicarbonxylic acid for establishing the engineering yeast, and the industrial application value and prospect are good.

Description

technical field [0001] The invention relates to a pyruvate carboxylase mutant N315F with improved enzyme activity and application thereof, belonging to the fields of genetic engineering and fermentation engineering. Background technique [0002] As a eukaryotic model microorganism, Saccharomyces cerevisiae has the following advantages: rich genetic information, convenient operation of metabolic transformation; simple nutritional requirements, low cost of separation and extraction process; good growth under low pH conditions (even pH<3.0); Tolerant to high concentrations of substrates; certified by the FDA as GRAS (General Regarded As Safe) microorganisms, fermented products have the advantages of safety and become fermented to produce carboxylic acids (lactic acid, pyruvic acid, malic acid, fumaric acid, succinic acid, α-ketoglutadiene Potentially optimal microorganisms for acid, etc.). However, batch fermentation of Saccharomyces cerevisiae under conditions of high suga...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N1/19C12P7/46C12P7/44C12N15/52
CPCC12N9/93C12P7/44C12P7/46C12Y604/01001
Inventor 徐国强蒋伶活吴满珍李鹏越
Owner JIANGNAN UNIV
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