High-yield succinic acid strain and its application

A succinic acid and strain technology, applied in the directions of bacteria, electricity/wave energy treatment of microorganisms, biochemical equipment and methods, etc., can solve the problem of reducing the effective conversion efficiency of carbon, the high cost of succinic acid production, and hindering the biological process of succinic acid. industrialization process, etc.

Active Publication Date: 2011-12-21
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, there are a large number of by-products in the existing engineering bacteria. The existence of by-products not only reduces the effective conversion efficiency of carbon, but also greatly increases the compl

Method used

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  • High-yield succinic acid strain and its application
  • High-yield succinic acid strain and its application
  • High-yield succinic acid strain and its application

Examples

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Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Rational design to obtain engineering strains for succinic acid production

[0021] The following 9 genes in Escherichia coli MG1655 were sequentially knocked out by pDS132 traceless knockout system: adhE, ackA, pta, focA, pflB, iclR, ldhA, poxB, pepc.

[0022] Refer to the literature of the pDS132 traceless knockout system, the knockout and screening methods and steps of each gene are the same. The specific operation is as follows:

[0023]For the knockout of the adhE gene, first use the sequence as the primer 1 (adhE-1) of SEQ ID NO: 1 and the sequence as the primer 2 of SEQ ID NO: 2 (adhE-2); the sequence is the primer 2 of SEQ ID NO: 3 Primer 3 (adhE-3) and primer 4 (adhE-4) whose sequence is SEQ ID NO: 4 amplify two homologous arms of about 1 kb respectively from the genomic DNA of Escherichia coli MG1655, and then pass primer 1 and primer 4. Using the left and right homology arms as a template, obtain a fusion fragment of about 2 kb through fusion PCR...

Embodiment 2

[0032] Example 2: High-yielding succinic acid mutant strain obtained through irrational mutagenesis screening

[0033] Through genome analysis and metabolic pathway analysis, the SLEcS14 mutant strain has the potential to re-mutate and adjust itself to improve cell growth and succinic acid synthesis ability. The present invention uses ultraviolet mutagenesis combined with culture conditions to screen succinic acid high-yielding bacteria.

[0034] The SLEcS14 strain was used as the initial strain for mutagenesis, cultured in LB liquid medium (10g / L tryptone, 5g / L yeast extract, 5g / L NaCl) at 37°C to obtain a mid-log phase bacterial liquid, centrifuged the bacterial liquid, and precipitated After washing twice with normal saline, adjust the OD550 to 0.3 with LB liquid medium, select the irradiation time with a lethal rate of 70-80% as the irradiation time for mutagenesis, and then transfer the mutated bacterial solution into a 100ml anaerobic bottle containing 20ml of NBS basic ...

Embodiment 3

[0039] Embodiment 3: SLEcS16 mutant strain anaerobic fermentation

[0040] Because the pH of fermentation acid production in the anaerobic bottle is not well controlled, the growth of bacteria and the production of succinic acid are affected. Fill 3L of liquid in a 5L fermenter for sealed fermentation, the medium is NBS, the temperature is 37°C, 200rpm, feed 1.2MKOH and 2.6MK 2 CO 3 Lye controls pH7.0. The fermentation results are shown in the table below. At the end of 188 hours, the succinic acid fermentation process produced 19.62 g / L of succinic acid, almost no by-products, only a very small amount of acetic acid and lactic acid, and no ethanol and formic acid. The ratio of succinic acid to by-products was 15.45: 1.

[0041] Fermentation time (h) OD 550 Succinic acid (g / L) Lactic acid (g / L) Acetic acid(g / L) 0 0.15 0.12 0.00 0.00 24 0.52 0.56 0.00 0.04 48 0.82 1.66 0.06 0.09 72 1.27 3.80...

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Abstract

The invention relates to a bacterial strain for the high yield of succinic acid and application thereof. Escherichia coli is modified in order to remove or decrease the activities of adhE, ackA, pta, focA, pflB, iclR, ldhA, poxB and pepc genes; and the gene knockout bacterial strain then receives ultraviolet mutagenesis, and is screened by culture medium with glucose as the only carbon source, so that an engineering bacterial strain which can produce the succinic acid on basal medium and with the glucose as the only carbon source by anaerobic fermentation can be obtained. The invention adopts the succinic acid-producing escherichia coli as the starting strain, and by means of a rational design, the inveniton obtains the engineering bacterial strain with the glucose as the only carbon source theoretically, which carries out anaerobic fermentation in the basal medium and only produces the succinic acid and no other byproducts. Then by means of irrational metabolic evolution integrating ultraviolet mutagenesis with growth screening, a mutant strain for producing the succinic acid is obtained. The bacterial strain disclosed by the invention nearly produces no other heteroacids in the fermentation of the succinic acid, so the production cost of the succinic acid can be remarkably reduced.

Description

technical field [0001] The invention belongs to the technical field of screening and application of industrial bacteria, and specifically relates to the screening and application of high-yield strains of succinic acid, that is, the high-yield strains are obtained by using genetic engineering methods combined with metabolic engineering transformation and traditional mutagenesis techniques, and the screened Escherichia coli For the fermentative production of succinic acid. Background technique [0002] Succinic acid, also known as succinic acid, is widely used in medicine, pesticides, dyes, spices, paints, food, plastics and other industries. It can also be used as a C4 platform compound to synthesize some important chemical products such as butanediol, tetrahydrofuran, and γ-butylene Lactone, n-methylpyrrolidone (NMD), 2-pyrrolidone, etc., can also be used to synthesize degradable biopolymers, such as polybutene succinic acid (PBS) and polyamide (Journal of Process Engineerin...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N15/09C12N13/00C12N15/01C12P7/46C12R1/19
Inventor 孙际宾刘娇郑平孙村民
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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