Recombinant cell and method for synthesizing methyl acetoin and derivative compounds thereof by using biological method

A technology of biosynthesis of methyl acetoin, applied in the field of production of methyl acetoin compound, biosynthesis of methyl acetoin compound and its derivative compounds

Active Publication Date: 2012-12-05
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the method for synthesizing methylacetoin by biological method has not been reported yet.

Method used

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  • Recombinant cell and method for synthesizing methyl acetoin and derivative compounds thereof by using biological method
  • Recombinant cell and method for synthesizing methyl acetoin and derivative compounds thereof by using biological method
  • Recombinant cell and method for synthesizing methyl acetoin and derivative compounds thereof by using biological method

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Experimental program
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Embodiment 1

[0065] The acoA and acoB genes (GenBank Accession No.936152; 939697) of Bacillus subtilis were cloned between the NcoI and SalI sites of the pACYduet1 (purchased from Novagen) plasmid, and the constructed pJXL32 plasmid (such as Figure 7 indicated) were electrotransformed into Escherichia coli BL21(DE3) (purchased from Invitrogen). The constructed cells were cultured in 100ml LB medium (LB medium, 10g sodium chloride dissolved in 1L water, 10g trypsin powder, and 5g yeast powder). The culture temperature was maintained at 30°C. When the cells grow to a certain stage, usually the exponential growth stage, IPTG (50mg / L final concentration) and acetone (1M final concentration) are added to the medium. Causes cells to produce methylacetoin.

[0066] The produced methyl acetoin is detected by gas chromatography-mass spectrometry and the results are as follows: Figure 12 , as shown in 13.

[0067] Methylacetoin or methylbutenone is separated from bacterial cultures by gas stri...

Embodiment 2

[0069] The bdhA (GenBank Accession No.939490) gene of Bacillus subtilis is cloned between the SalI and aflII sites on the pJXL32 described above, and the pJXL37 plasmid (such as Figure 8shown) were electrotransformed into E. coli BL21(DE3). The constructed cells were cultured in 100ml LB medium. The culture temperature was maintained at 30°C. When the cells grow to a certain stage, usually the exponential growth stage, IPTG (50mg / L final concentration) and acetone (1M final concentration) are added to the medium. Causes cells to produce 2-methyl-2,3-butanediol. The 2-methyl-2,3-butanediol that produces is detected by gas chromatography-mass spectrometry as Figure 10 , as shown in 11.

[0070] 2-Methyl-2,3-butanediol was separated from bacterial cultures by distillation. 2-Methyl-2,3-butanediol is catalyzed by lithium acetate to generate prenylation at 200 degrees Celsius, the specific operation reference (2).

Embodiment 3

[0072] The thl gene of Clostridium acetobutylicum (GenBank Accession No. 1119056 ), the atoAD gene of Escherichia coli (GenBank Accession No. 947525 ; 946719 ), and the abc gene of Clostridium acetobutylicum (GenBank Accession No. 1116170 ), connected together by overlap extension PCR, and then cloned on pET28a by homologous recombination (3). The constructed plasmid is called pJXL40 (such as Figure 9 shown). pJXL40 and pJXL32 were simultaneously electrotransformed into E. coli BL21(DE3). The constructed cells were cultured in 100ml LB medium. The culture temperature was maintained at 30°C. When the cells grow to a certain stage, usually the exponential growth stage, IPTG (50 mg / L final concentration) and glucose (20 g / L final concentration) are added to the medium. The cells produced methyl acetoin as analyzed by mass spectrometry.

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Abstract

The invention provides a method, a recombinant cell and an enzyme system relevant to the production of methyl acetoin and derivative compounds thereof, and particularly provides nucleic acid, polypeptide, a host cell, the method and material for producing the methyl acetoin and the derivative compounds thereof by using a biological method.

Description

technical field [0001] The invention relates to the field of producing acetoin compounds, in particular to the field of synthesizing methylacetoin compounds and derivatives thereof by biological methods. Background technique [0002] Methyl acetoin is an important chemical raw material for the synthesis of chemical products such as pigments, medicines, and polymers. [0003] Methylacetoin is traditionally produced by the petrochemical industry, which is highly polluting and unsustainable. [0004] A chemical synthesis route for the production of methylacetoin has been reported so far. However, the method for synthesizing methylacetoin by biological method has not been reported yet. Contents of the invention [0005] The present invention relates to the production of methylacetoin, and other organic compounds (such as 2-methyl-2,3-butanediol, methylbutenone, 2-methyl-3-hydroxyl-1- butene, isoprene) methods and materials. Specifically, the present invention provides meth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/26C12P7/18C12P7/04C12P5/02C12N1/19C12N1/21C12R1/645C12R1/225C12R1/46C12R1/07C12R1/185
Inventor 咸漠姜兴林杨建明
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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