Strain and method for biosynthesis of isopentyldiol

A technology for biosynthesis and isoprene glycol, applied in the direction of microorganism-based methods, biochemical equipment and methods, botany equipment and methods, etc.

Active Publication Date: 2020-06-30
HUAANTANG BIOTECH GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, no natural biological pathway for IPDO synthesis has been found, which prompted us to develop a new IPDO biosynthetic pathway

Method used

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  • Strain and method for biosynthesis of isopentyldiol
  • Strain and method for biosynthesis of isopentyldiol
  • Strain and method for biosynthesis of isopentyldiol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1 bacterial strain, plasmid and substratum

[0027] The Escherichia coli strains and plasmids used in the present invention are listed in Table 1. Escherichia coli strains DH5a and BL21 were used for gene cloning and expression. Methylobacillus flagellatus KT and lactobacillus lactis were purchased from Leibniz Institute DSMZ (https: / / www.dsmz.de / ) and used to amplify the pathway enzymes for the synthesis of IPDO.

[0028] The pET28a(+) plasmid was used for protein expression in E. coli BL21 strain, and the primers for constructing the plasmid are shown in Table 2.

[0029] Shake flask fermentation: a single colony was cultured overnight in 5mL LB medium, and then inoculated in 10mL Fe II medium (30g / L glucose, 0.5g / L MgSO 4 ·7H 2 O,3g / L KH 2 PO 4 ,12g / L K 2 HPO 4 ,4g / L(NH 4 ) 2 SO 4 , 1g / L yeast extract, 2g / L monosodium citrate and 0.1g / L FeSO 4 ·7H 2 Cultivate at 37° C. in (0), the rotating speed is 200 rpm, and the fermentation time is 48 hour...

Embodiment 2

[0034] Embodiment 2 is used for the plasmid construction of IPDO production

[0035] Taking the construction of pZA-ky as an example, the steps of plasmid pZA-ky construction are as follows: use primers pky-kdc-LF / LR and pky-yqhd-LF / LR to amplify (2-keto acid decarboxylase) kdc and (aldehyde ketone reductase) yqhD genes. Using pZA as a template, a linearized vector with terminal extension of 15-20bp was obtained by PCR using primers pky-vec-LF / LR. After the three fragments were processed for gel recovery, the fusion HD cloning kit (Clontech, TaKara Bio USA Inc., USA) was used to ligate the PCR fragment and the linearized vector in a fusion cloning reaction.

Embodiment 3

[0036] Embodiment 3 gas chromatography analyzes IPDO (GC)

[0037] The concentration of isopentyl glycol-phenylboronic acid was determined by GC (Agilent 7890B, USA). The non-polar capillary column used here is HP5ms UI, 30m x 250μm x 0.25μm, and the carrier gas helium is constant at 80kpa. The FID temperature was set at 350 °C, the injector temperature was 270 °C, the injection volume was 0.2 μL, and the separation was 1:5. The temperature was maintained at 100 °C for 2 min and increased to 270 °C with a gradient of 20 °C / min. Add 1,3-propanediol (1,3-PDO) as an internal reference while testing the IPDO standard.

[0038] The effect example is constructed by the synthetic pathway from α-KIC to IPDO

[0039] The mfl gene was amplified from Methylflagellate KT and connected to pET28a vector to obtain pET-mfl. The plasmid map of pET-mfl is as follows figure 2 shown. The kdc and yqhD genes were amplified from the genomes of Lactobacillus and Escherichia coli, respectively, a...

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Abstract

The invention relates to the field of biosynthesis, in particular to a strain and method for biosynthesis of isopentyldiol. According to the strain, by taking alpha-ketoisocaproate (alpha-KIC) as a reaction substrate, the isopentyldiol (IPDO) can be produced by over-expression of isoleucine dioxygenase (IDO), 2-keto acid decarboxylase (Kdc) and aldo-keto reductase. After escherichia coli BL21 (DE3) is introduced into the path, the isopentyldiol (IPDO) of 21.57 mg / L is produced after a finally constructed recombinant escherichia coli engineering strain IP01 is added into a shake flask with the50mM substrate alpha-KIC and is cultured for 48h.

Description

technical field [0001] The invention relates to the field of biosynthesis, in particular to strains and methods for biosynthesizing isopentyl glycol. Background technique [0002] Isopentyl glycol (IPDO) is an important ingredient widely used in the cosmetic industry. IPDO is odorless and non-irritating, and is a safe and environmentally friendly material. Due to these properties, it is used as a humectant, solubilizer and preservative booster. Compared with other polyols such as propylene glycol and butylene glycol, IPDO shows better skin affinity and makeup removal effect. Therefore, the demand for IPDO in the cosmetic industry is growing rapidly. So far, IPDO is mainly obtained by chemical synthesis. Chemical reactions usually require harsh reaction conditions, such as high pressure or high temperature, and unnecessary by-products may be harmful to the human body, which hinders its application in the cosmetic industry. Compared with chemical synthesis, biocatalysis c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/60C12N15/53C12N1/21C12P7/18C12R1/19
CPCC12N15/70C12N15/52C12N9/88C12N9/0006C12N9/0071C12P7/18C12Y401/01072C12Y101/01021C12Y114/11
Inventor 张炽坚何廷刚艾勇刘永飞马成伟曾安平
Owner HUAANTANG BIOTECH GRP CO LTD
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