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Method for manufacturing 3-hydroxypropionic acid from microbial fermentation glycerol

A technology of microbial fermentation and hydroxypropionic acid, applied in the field of microbial fermentation and bioengineering, can solve the problems of no glycerol dehydratase enzyme activity, increase the difficulty of operation, and it is difficult to completely cut off the production path, so as to avoid energy consumption and rate limitation. , The effect of eliminating the steps of gene manipulation and good industrial application prospects

Inactive Publication Date: 2015-08-12
EAST CHINA UNIV OF SCI & TECH
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Problems solved by technology

[0008] So far, some progress has been made in the research on the preparation of 3-hydroxypropionic acid by constructing genetically engineered bacteria, but there are still many problems: 1) The pathway modification itself requires cumbersome molecular biology operations, and the later cultivation often requires the addition of antibiotics and induction Agents, etc., increase the difficulty of operation and increase the production cost
With K.pneumoniae as the host, a large amount of 1,3-propanediol is produced during the production of 3-HP. The latter is the natural terminal product of K.pneumoniae. Difficult to improve
Using E.coli as the host, the yield of 3-HP is relatively high, but because it does not have glycerol dehydratase activity, the construction process is more complicated
Moreover, E.coli itself cannot produce the coenzyme VB of glycerol dehydratase 12 , needs to be added in the fermentation system, because it is expensive, which greatly increases the fermentation cost
3) The balance of glycerol dehydratase and aldehyde dehydrogenase in recombinant bacteria also restricts the improvement of 3-HP production
Glycerol dehydratase found so far is only active under anaerobic and microaerobic conditions, and under these conditions NAD(P)H cannot generate NAD(P)+, the cofactor of aldehyde dehydrogenase in time, which not only makes 3 -Reduced rate of HP production also leads to accumulation of highly cytotoxic 3-hydroxypropanal, which leads to cell death and aborts fermentation

Method used

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  • Method for manufacturing 3-hydroxypropionic acid from microbial fermentation glycerol
  • Method for manufacturing 3-hydroxypropionic acid from microbial fermentation glycerol
  • Method for manufacturing 3-hydroxypropionic acid from microbial fermentation glycerol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 , Construction of Klebsiella pneumoniae (K.pneumoniae) glycerol dehydratase enhanced strain

[0033] The gene dhaB (GenBank accession No.U30903) encoding glycerol dehydratase was amplified from Klebsiella pneumoniae by PCR technology, and it was connected to the expression vector pKM13 (Ping Zheng, Kirsten Wereath, etc., Process Biochemistry 41 (2006) 2160–2169), the recombinant plasmid pKM13-dhaB was obtained, and the recombinant plasmid was transformed into Klebsiella pneumoniae ATCC25955 to obtain the recombinant strain ATCC25955 / pKM13-dhaB.

[0034] The specific construction process is:

[0035] (1) Construction of expression plasmids:

[0036] According to Klebsiella pneumoniae dhaT gene sequence design two primers, its sequence is as follows:

[0037] Primer 1: 5'-ccg gaattc atgaaaagatcaaaacgatttgc-3'

[0038] Primer 2: 5'-tgc tctaga ttaattcgcctgaccggccag-3'

[0039] Klebsiella pneumoniae genomic DNA was used as a template to amplify the target ...

Embodiment 2

[0045] Example 2 , Construction of Gluconobacter oxydans (G.oxydans) Alcohol Dehydrogenase Enhanced Strain

[0046] The gene adhAB encoding membrane-bound alcohol dehydratase was amplified from Gluconobacter oxydans DSM2003 by PCR technology, and was connected to the expression vector pBBR1MCS-5 with DNA ligase to obtain the recombinant plasmid pBBR1MCS-5-adhAB, which was transformed into Gluconobacter DSM2003 was oxidized to obtain the recombinant strain DSM2003 / pBBR1MCS-5-adhAB.

[0047] The specific construction process is:

[0048] (1) Construction of expression plasmids:

[0049] Two primers were designed according to the gene sequence of Gluconobacter oxydans alcohol dehydrogenase (G.oxydans 621H GGOX1068), the sequences of which are as follows:

[0050] Primer 1: 5'-AAA GTC GAC ATGAACGACACGGTGCCCGAGC-3'

[0051] Primer 2: 5'-TC TCTAGA CAGGGGTGGGGACGCTTATTGTGCG-3'

[0052] Genomic DNA of Gluconobacter oxydans DSM2003 was used as a template to amplify the target...

Embodiment 3

[0058] Example 3 , A seed culture of microorganisms

[0059] Seed medium formula (g / L): peptone 10, yeast powder 5, NaCl 10, glycerol 2, adjust the pH to 7.0 with 2mol / L KOH, and sterilize at 121°C for 20 minutes.

[0060] Take a 250ml triangular flask, and fill the seed medium with 50ml of liquid. According to Table 1, use an inoculation loop to inoculate one ring of strains preserved on a slant. The culture temperature is 28-37°C. Oxygen cultivation was carried out for 10-15 hours, and the rotation speed of the shaker was 150-200 r / min.

[0061] Described strain is: Klebsiella pneumoniae (Klebsiella pneumoniae) ATCC25955, freund's Citrobacter freundii (Citrobacter freundii) DSM30040, butyric acid Clostridium (Clostridia butyricum) DSM5431, Pasteurian nitrogen-fixing Clostridium (Clostridia pastruianu) DSM525.

[0062] Table 1. Microbial seed culture conditions

[0063]

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Abstract

The invention relates to a method for manufacturing 3-hydroxypropionic acid from microbial fermentation glycerol. The method is implemented by the aid of first microorganisms and second microorganisms. The first microorganisms have glycerol dehydration activity, and the glycerol can be converted into 3-hydroxy-propionaldehyde or 1, 3-propylene glycol by the first microorganisms. The second microorganisms have glycol dehydration activity, and the 3-hydroxy-propionaldehyde or the 1, 3-propylene glycol glycerol can be converted into the 3-hydroxypropionic acid by the second microorganisms. The first microorganisms can be selected from Klebsiella pneumoniae and the like and genetically engineered bacteria of the Klebsiella pneumoniae and the like; the second microorganisms are Gluconobacter oxydans and genetically engineered bacteria of the Gluconobacter oxydans. The method has the advantages that the glycerol is used as a substrate, the two types of microorganisms are subjected to sectioned mixed fermentation to manufacture the 3-hydroxypropionic acid, the concentration of final products can be improved as compared with an existing single-bacterium fermentation method mainly on the basis of genetically engineered bacteria, the rate of converting the glycerol into the 3-hydroxypropionic acid can be increased as compared with the existing single-bacterium fermentation method, lactic acid in fermentation liquor can be converted into acetic acid, accordingly, the concentration of the lactic acid of byproducts can be reduced, and separation and purification stress can be relieved; the concentration of the 3-hydroxypropionic acid can reach 75gL<-1> at least, and accordingly the method has an excellent industrial application prospect.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to the field of microbial fermentation, in particular to a method for producing 3-hydroxypropionic acid by fermenting glycerol with microorganisms. Background technique [0002] 3-hydroxypropionic acid (3-HP) is an important platform compound, which can generate acrylic acid, malonic acid, 1,3-propanediol, etc. through dehydration, redox, esterification, etc. an important chemical substance. It can also be used as a monomer to synthesize an environmentally friendly polymer material - polyhydroxyalkanoic acid. In addition, it can also be used as a chemical raw material for the production of coatings, adhesives, water treatment chemicals and personal care products. In 2004, the US Department of Energy listed it as one of the 12 most valuable chemical products. [0003] At present, the preparation of 3-hydroxypropionic acid is mainly chemical method, such as acrylic acid hydra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/42C12P39/00C12P7/18
CPCC12P7/42C12P7/18C12P39/00
Inventor 赵莉魏东芝林金萍
Owner EAST CHINA UNIV OF SCI & TECH
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