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Actinobacillus succinogenes PZ improving yield of succinic acid, and construction method and application thereof

A technology of actinomycetes and engineering strains, applied in the field of bioengineering, can solve the problems of unsatisfactory industrial production, unpredictable results, heavy workload, etc., and achieve the effect of increasing the reduction level, weakening the rate-limiting steps, and increasing production

Active Publication Date: 2017-08-01
GUANGXI ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chinese patents ZL201210056568.6, ZL 201210122717.4, etc. have reported the method of breeding Actinobacillus succinate, but the fermentation level of succinic acid can only reach about 30g / L, which cannot meet the needs of industrial production; Chinese patent ZL 201180011827.7, ZL 200910080815.4 et al. conducted research on raw materials and fermentation methods, but the method used was random screening, with heavy workload and unpredictable results

Method used

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  • Actinobacillus succinogenes PZ improving yield of succinic acid, and construction method and application thereof
  • Actinobacillus succinogenes PZ improving yield of succinic acid, and construction method and application thereof
  • Actinobacillus succinogenes PZ improving yield of succinic acid, and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1 expression vector construction

[0048] Such as figure 1 As shown, in the original plasmid PGZRS-18 (gifted by Professor Wang Chunlai from Harbin Veterinary Institute, figure 1 A)的基础上添加pepck基因的启动子(克隆自产琥珀酸放线杆菌基因组,SEQ ID NO1所示序列,即tcgataaattgaaaatgcagcaatagaggaaacacggtttgtttgagtgaaaacagccgtgttttttcatttaccgccataaaaatttgaaacggatcacaaatcatgaaaaaaatacgttcaaattagaactaattatcgaaaatttgatctagttaacattttttaggtataaatagttttaaaatagatctagtttggatttttaattttaaattatcaatgaggtga)到载体PGZRS-18-pro( figure 1 B) and the ColE1 replicon gene of Escherichia coli (cloned from pUC18 vector GenBank: L08752.1), constructing the vector PGZRS-E ( figure 1 C).

[0049] Expression vector pGZRS-E and original vector pGZRS-18, the results of electrophoresis analysis using XbaI single enzyme digestion products are as follows: figure 2 As shown, where M: λ / HindIII DNA molecular weight marker; 1: expression vector pGZRS-E; 2: pGZRS-18 vector, the results show that the promoter of the pepck gene an...

Embodiment 2

[0050] Embodiment 2 expression gene acquisition and the construction of expression vector and genetically engineered bacteria

[0051] 1) The tandem expression genes pepck gene (GenBank: EU253567.1) and zwf gene (GenBank: BA000007.2) were synthesized by chemical synthesis (synthesized by Gene Synthesis Company);

[0052] 2) Genes expressed in tandem can also be obtained by overlapping PCR.

[0053] Primers are as follows:

[0054] P1: 5'-GCG TCTAGA ATGACTGACTTAAACAAACT-3' (SEQ ID NO 2)

[0055] P2: 5'-TACCGCCATTGCGTTTGTCGTCTAGATGCTTTTGGACCG

[0056] GCGCCAAC-3' (SEQ ID NO3)

[0057] P3: 5'-TTGGCGCCGGTCCAAAAGCAATGGCGGTAACGCAAAC

[0058] AGC-3' (SEQ ID NO 4)

[0059] P4: 5'-TGC GAGCTC TTACTCAAACTCATTCCAGG-3' (SEQ ID NO 5)

[0060] PCR reaction system

[0061]

[0062] PCR parameters

[0063]

[0064] The result is as follows:

[0065] (1) Amplify the pepck gene with primers P1 and P2; amplify the zwf gene with primers P3 and P4.

[0066] (2) Purify the pepck ...

Embodiment 3

[0077] Embodiment 3 engineering strain relevant enzyme activity assay

[0078] The engineering strain Actinobacillus succinogenesPZ obtained in Example 2 and the control strain Actinobacillus succinogenes GXAS137 were carried out in liquid fermentation culture:

[0079] Inoculate the single colony of Actinobacillus succinogenes on the plate into the seed medium, and cultivate it in an anaerobic incubator at 37°C for 16 hours, and the number of bacteria will reach more than 300 million, and then carry out the second inoculum according to 5% (V / V) inoculation amount. Level multiplication culture 8h, bacterial number reaches more than 300,000,000, promptly obtains liquid seed; By 5% (V / V) inoculum size, liquid seed is inoculated in the 250mL anaerobic bottle that fermentation medium is housed, and liquid capacity is 150mL, use the pH buffer to adjust the pH of the fermentation broth to maintain at 6.5-7.0, and fill it with N 2 In an environment with a rotating speed of 100r / min ...

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Abstract

The invention provides an actinobacillus succinogenes PZ improving yield of succinic acid, and a construction method thereof. The construction method comprises the steps of constructing an expression carrier, constructing recombinant plasmid and constructing the actinobacillus succinogenes PZ. The actinobacillus succinogenes starting strain is connected in series with and expressed excessively by gene pepck of a key rate-limiting enzyme, PEPCK, for generating succinic acid by using the actinobacillus succinogenes, and gene zwf of G6PDH improving the restoring force level in a body, so as to acquire the actinobacillus succinogenes PZ. The actinobacillus succinogenes PZ can improve the yield of succinic acid, and has the potential of industrial production application.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to an engineering strain of Actinobacillus succinate produced by improving the production of succinic acid, its construction method and application. Background technique [0002] Succinate is an important intermediate in the metabolism of many strict and facultative anaerobes. At present, it has been found that a variety of microorganisms can produce succinic acid through fermentation, and the research mainly focuses on Actinobacillus succinogenes and Escherichia coli (E.coli). In addition, Mannheimia succiniciproducens, Anaerobiospirillum succiniciproducens and some lactic acid bacteria, propionic acid producing bacteria and fungi can also produce a small amount of succinic acid. Among the numerous succinic acid-producing microorganisms, Actinobacillus succinogenes can use a variety of carbon sources (glucose, xylose, arabinose, lactose, etc.) 158g / L and 104g / L...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12P7/46C12R1/01
CPCC12N9/1205C12N9/88C12N15/74C12N2800/60C12N2820/55C12P7/46C12Y207/0101C12Y401/01049
Inventor 申乃坤张红岩王青艳朱婧李亿秦艳梁戈
Owner GUANGXI ACAD OF SCI
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