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Escherichia coli engineering bacterium for producing acetoin as well as construction method and application of escherichia coli engineering bacterium in whole-cell catalytic production of acetoin

A technology of Escherichia coli and construction method, applied in microorganism-based methods, applications, genetic engineering and other directions, can solve the problems of low yield, expensive substrate acetaldehyde, difficult to store, etc., and achieves lower production costs, mild reaction conditions, The effect of high product yield

Active Publication Date: 2021-11-05
TIANJIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the yield of acetoin is high, the substrate 2,3-butanediol is expensive
Zhang et al. [3] Using the whole cell of Corynebacterium crenatum to catalyze the production of acetoin from glucose, the bacteria were reused three times, and the yield of acetoin reached 76.93g / L, but the yield was low at 0.67mol / mol
Among the current whole-cell catalytic methods, in the catalytic system with acetaldehyde as the substrate, the yield of acetoin is the highest, reaching 222g / L [4] , but the catalytic system requires high cell density (30gCDW / L), and its substrate acetaldehyde is expensive, dangerous and difficult to store, which is not conducive to the industrial production of acetoin. Therefore, it is urgent to develop a cheap and safe substrate whole-cell catalysis

Method used

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  • Escherichia coli engineering bacterium for producing acetoin as well as construction method and application of escherichia coli engineering bacterium in whole-cell catalytic production of acetoin
  • Escherichia coli engineering bacterium for producing acetoin as well as construction method and application of escherichia coli engineering bacterium in whole-cell catalytic production of acetoin
  • Escherichia coli engineering bacterium for producing acetoin as well as construction method and application of escherichia coli engineering bacterium in whole-cell catalytic production of acetoin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] The NADH oxidase is derived from Lactococcus lactis subsp.lactisIO-1 Example 1: Knocking out the genes pta-ackA and poxB on the Escherichia coli genome using the λ-Red recombination method

[0068] With pta-ackA-U-F (SEQ ID NO.22) and pta-ackA-U-R (SEQ ID NO.23) as primers (Table 1),

[0069] Using the Escherichia coli genome as a template, pta-ackA-U (SEQ ID No.12) was obtained by PCR reaction;

[0070] With pta-ackA-tet-1 (SEQ ID NO.24) and pta-ackA-tet-2 (SEQ ID NO.25) as primers,

[0071] Using pTKS / CS as a template, pta-ackA-tet-U (SEQ ID No.13) was obtained by PCR reaction;

[0072] Using pta-ackA-tet-3 (SEQ ID NO.26) and pta-ackA-tet-4 (SEQ ID NO.27) as primers and pTKS / CS as template, pta-ackA-tet was obtained by PCR reaction -D (SEQ ID No. 14);

[0073] With pta-ackA-D-F (SEQ ID NO.28) and pta-ackA-D-R (SEQ ID NO.29) as primers, with Escherichia coli genome as template, by PCR reaction, obtain pta-ackA-D (SEQ ID No. .15).

[0074] Then using pta-ackA-U-F a...

Embodiment 2

[0084] Embodiment 2: Construction of expression vector pET28a-aldC-S2-alsS-ldh-nox and acetoin-producing Escherichia coli engineering bacteria

[0085] The amino acid sequence of the α-acetolactate synthase used in the present invention is shown in SEQ ID No.1, derived from Bacillus subtilis 168; the amino acid sequence of the α-acetolactate decarboxylase used is shown in SEQ ID No.2 Shown, derived from lactate dehydrogenase Bacillus subtilis 168; the amino acid sequence of lactate dehydrogenase used is SEQ ID No.3; derived from Bacillus coagulans 2-6, the amino acid sequence of NADH oxidase used is SEQ ID No.3 ID No.4, derived from Lactococcus lactissubsp.lactis IO-1.

[0086]Using the primer LDH-F (SEQ ID NO.38) as the upstream primer, the primer LDH-R (SEQ ID NO.39) as the downstream primer, and the vector pET28a-ldh (SEQ ID NO.10) as the template, by PCR, A fragment ldh-1 containing the gene ldh (SEQ ID NO. 8) encoding lactate dehydrogenase was prepared. Using NOX-F (SEQ...

Embodiment 3

[0091] Embodiment 3: the application of acetoin-producing Escherichia coli engineered bacteria in whole-cell catalytic production of acetoin comprises the following steps:

[0092] 1) With the acetoin-producing Escherichia coli engineering bacteria BL-3 in Example 2, a single colony was picked from the plate, inoculated into 5mL LB medium, cultivated for 10h, and transferred to 200mL LB with 1% inoculum size culture medium at 37°C, 220rpm to OD 600 0.6-0.8, add IPTG (isopropyl β-D-1-thiogalactopyranoside) with a final concentration of 0.5mM, induce at 25°C for 12h, collect the bacteria at 4°C, 4500rpm, and use phosphate buffer solution (pH=6.0) to resuspend and wash the bacteria once, then centrifuge the bacteria at 4500 rpm at 4°C, and finally resuspend the bacteria with a small amount of phosphate buffer (pH=6.0);

[0093] 2) Suspend the thalline obtained in step 1) in the transformation solution containing lactic acid, so that the OD 600 =15, the concentration of lactic a...

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Abstract

The invention discloses an escherichia coli engineering bacterium for producing acetoin as well as a construction method and application of the escherichia coli engineering bacterium in whole-cell catalytic production of acetoin. The construction method of the escherichia coli engineering bacterium for producing acetoin comprises the following steps: co-expressing alpha-acetolactate synthetase, alpha-acetolactate decarboxylase, lactic dehydrogenase and NADH oxidase to obtain the escherichia coli engineering bacterium for producing acetoin; and constructing a host bacterium used in the escherichia coli engineering bacterium as mutant escherichia coli BL-2. The acetoin is produced by taking lactic acid as a substrate, the product yield is high, the substrate cost is low, extra NAD+ does not need to be added in a reaction system, and the production cost is further reduced. According to the method, the acetoin yield reaches 87.5 mM or above, the yield reaches 3.65 mM / h or above, and the yield reaches 0.444 mol / mol or above, namely the theoretical yield is 88.8% or above. The method is mild in reaction condition and simple in process.

Description

technical field [0001] The invention belongs to the field of bioengineering technology and application, and specifically relates to acetoin-producing Escherichia coli engineering bacteria, a construction method, and an application in whole-cell catalytic production of acetoin. Background technique [0002] Acetoin, also known as 3-hydroxy-2-butanone, monomer is colorless or light yellow liquid, dimer is white crystalline powder, spontaneous combustion, soluble in water, soluble in ethanol, propylene glycol, slightly soluble in ether. Acetoin exists in many foods and has a milky smell. It is often used as a food additive to enhance the milky smell in food. Acetoin is one of the 4-C platform compounds, listed by the US Department of Energy as one of the 30 platform compounds worthy of priority development, and is widely used in the fields of materials, pharmaceutical production and chemical synthesis. Acetoin is also a precursor of the important chemical 2,3-butanediol, liqu...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N9/10C12N9/88C12N9/04C12N9/02C12N15/53C12N15/54C12N15/60C12N15/11C12P7/26C12R1/19
CPCC12N15/70C12N9/1022C12N9/88C12N9/0006C12N9/0036C12P7/26C12Y202/01006C12Y401/01005C12Y101/01027C12Y106/99003Y02E50/10
Inventor 陈涛崔真真郑美玉王智文
Owner TIANJIN UNIV
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