Method for large-scale production of CMP-sialic acid through coupled fermentation of genetically engineered bacteria and yeast

A technology of genetically engineered bacteria and genetically engineered strains, applied in the field of fermentation engineering, can solve the problems of high CTP production cost, reduced production cost, high price and the like, and achieves the effects of low price, broad market prospect and high yield

Pending Publication Date: 2022-01-04
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In order to solve the problem that the current sialyllactose synthesis precursor CMP-Neu5Ac is expensive and difficult to obtain, which leads to high production costs of CTP, the present invention constructs a genetically engineered bacterium that heterologously expresses CMP-sialidase Neu5Ac in Escherichia coli, Add genetically engineered bacteria and yeast into the reaction system containing CMP and Neu5Ac, and construct a method for coupling fermentation to produce CMP-Neu5Ac, which realizes the efficient production of CMP-Neu5Ac and reduces production costs

Method used

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  • Method for large-scale production of CMP-sialic acid through coupled fermentation of genetically engineered bacteria and yeast
  • Method for large-scale production of CMP-sialic acid through coupled fermentation of genetically engineered bacteria and yeast
  • Method for large-scale production of CMP-sialic acid through coupled fermentation of genetically engineered bacteria and yeast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1: Construction of engineering bacteria expressing CMP-sialidase gene heterologously

[0048] 1. Acquisition of CMP-sialidase gene (neuA)

[0049] 分别以来源于Neisseria meningitidis M0579、Neisseria meningitidisstrain M22819、Pasteurella multocida ATCC43137、Haemophillus ducreyi 35000HP的neuA基因序列为模板(GenBank号分别为CP007668.1(1012519…1013205)、CP016646.1(1413007…1414122)、CP008918. 1 (1975338…1976009), AE017143.1 (540594…541283)), and primers were designed according to the nucleotide sequence (respectively, the enzyme cutting sites are Nde I and Sal I). The corresponding forward and reverse primers were used to amplify the neuA gene, and the resulting amplified products were detected by agarose gel electrophoresis. The size of the amplified product was about 0.7kb (neuA), and its size was completely consistent with the size of the target gene. .

[0050] 2. Construction of recombinant protein expression plasmids

[0051] After the PCR products obtained in step 1 were purif...

Embodiment 2

[0060] Embodiment 2: Transformation and synthesis of CMP-Neu5Ac by single-gene engineering bacteria

[0061] Transformation conditions: 20mM MgCl 2 , 1mM DTT, 150mM Tris, 60mM CTP, 60mM Neu5Ac and 50g / L JM109(DE3) / pET28a-neuA cells (wet weight), react at 30°C and 200r / min for 2h.

[0062] (1) Optimization of IPTG induction concentration

[0063] JM109(DE3) / pET28a-nst were respectively inoculated into 10 mL LB liquid medium containing 20 μg / mL Kan, cultured in shake flasks at 37 °C and 200 r / min for 12 h, and then transferred at an inoculum volume of 2% (v / v). Into 100mL LB liquid medium containing 20μg / mL Kan, shake the flask at 37°C and 200r / min and culture until OD600 After about 0.6, add IPTG with final concentrations of 0.1, 0.2, 0.5, 0.8, 1.0 and 1.5mmol / L to induce, respectively. The induction temperature is 16°C, and the bacteria are collected after 200r / min induction for 20h. The obtained bacterial cells catalyzed the reaction in the above transformation solution, an...

Embodiment 3

[0068] Example 3: Synthesis of CMP-Neu5Ac through coupled fermentation of brewer's yeast and engineering strains

[0069] 1. Large-scale acquisition of genetically engineered bacterial cells

[0070] There are two sources of engineering bacteria cells: (1) high-density fermentation medium contains lactose, and the expression of lactose-induced enzymes; (2) IPTG induction.

[0071] Method 1, high-density fermentation: first pick a single colony of engineering bacteria and inoculate it in 50ml LB liquid medium with 20μg / ml Kan, at 37°C, 200r / min, and culture overnight (12h) to the logarithmic phase of growth. Then inoculate 500ml high-density fermentation medium containing 25μg / ml Kan according to 5% (5mL / 100mL) inoculum amount, culture in shake flask at 37°C and 200r / min for 2h. Then culture at 20°C and 200r / min for 20h. Centrifuge at 6000rmp to collect the cells, wash the cell sludge once with 0.5% normal saline, and then collect by centrifugation, the cells are used for fur...

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Abstract

The invention discloses a method for large-scale production of CMP-sialic acid through coupled fermentation of genetically engineered bacteria and yeast, and belongs to a fermentation engineering technology. According to the method, on the basis of industrial escherichia coli, CMP-sialidase Neu5Ac is heterologously expressed in the escherichia coli, a genetic engineering strain is constructed, any one of the constructed engineering strain and yeast are subjected to mixed fermentation, CMP and sialic acid are taken as substrates to synthesize the CMP-sialic acid, a feasible way is provided for industrial production of the sialylation step of acid breast milk oligosaccharide-sialylated oligosaccharide, the yield is high, the price is low, and the bottleneck problem that a large amount of sialylated oligosaccharide is difficult to synthesize due to the fact that the CMP-sialic acid is high in price is solved. The yield of the CMP-sialic acid can reach 24.5 g / L after the reaction is carried out for 4 hours under the condition of a fermentation tank, so that the method has obvious social benefits and wide market prospects.

Description

technical field [0001] The invention relates to a method for large-scale production of CMP-sialic acid by coupled fermentation of genetically engineered bacteria and yeast, which belongs to fermentation engineering technology. Background technique [0002] HMO (Human milk oligosaccharides) is a general term for all oligosaccharides contained in human milk, also known as human milk oligosaccharides. It is the second largest carbohydrate component and the third largest nutrient component in breast milk after lactose, accounting for 10% of dry matter. At present, more than 200 kinds of HMO have been identified, and HMO can be divided into three main types: (1) A. Fucosylated neutral HMO accounts for 35-50% of the total, representing substance 2 '-Fucosylgalactose (2'-FL) is the highest content of all HMOs, accounting for nearly 30%; (2) non-fucosyl neutral HMOs, accounting for 42-55% of the total, representing Substance Lactose-N-neotetraose (LNnT); (3). Sialylated acidic HMO...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/26C12P39/00C12N1/19C12R1/865C12R1/19C12R1/125C12R1/645
CPCC12P19/26C12P39/00
Inventor 张洪涛游星周文黎玉詹晓北
Owner JIANGNAN UNIV
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