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A tunable multienzyme cascade for the synthesis of optically pure allylic epoxy ketones or alcohols

An epoxy ketone and optical technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, and the use of vectors to introduce foreign genetic materials, etc., to achieve high ee and de values, efficient biosynthesis routes, and easy operation

Active Publication Date: 2020-02-21
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, there is no biological method reported to synthesize optically pure epoxy ketones by asymmetric epoxidation of α,β-unsaturated ketones

Method used

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  • A tunable multienzyme cascade for the synthesis of optically pure allylic epoxy ketones or alcohols
  • A tunable multienzyme cascade for the synthesis of optically pure allylic epoxy ketones or alcohols
  • A tunable multienzyme cascade for the synthesis of optically pure allylic epoxy ketones or alcohols

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Construction of biocatalyst E.coli BL21ΔnemA (pRSFD-REAB)

[0028] (1) Construction method of Escherichia coli expression strain E.coli BL21ΔnemA

[0029] Since the N-acetylmaleimide reductase expressed by the nemA gene in the E.coli BL21 genome can efficiently reduce the C=C bond of the substrate ɑ,β-unsaturated ketone, we introduced λRed recombination and knocked out E.coli The nemA gene in the BL21 genome.

[0030] Using P1 / P3 and P4 / P2 as primers (Table 1) and E. coli BL21 genomic DNA as a template, homologous sequences on both sides of the nemA gene were amplified respectively. PCR amplification conditions: pre-denaturation at 94°C for 5min, denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 0.5min, 30 cycles, and final extension at 72°C for 10min.

[0031] Using FRTf and FRTr as primers (Table 1) and plasmid pKD4 as a template, the kanamycin resistance gene fragment was amplified. Due to the different lengths of the fragme...

Embodiment 2

[0043] Example 2: Synthesis of (3R,4S)-4-phenyl-3,4-epoxy-2-butanone

[0044] Take 1.5 g of the bacteria from Example 1 and resuspend in 10 ml potassium dihydrogen phosphate / dipotassium hydrogen phosphate buffer (0.1M, pH=7.0), add 20 mM model substrate 4-phenyl-3-butene-2 - Ketone (1a in Table 2), 30°C, 230rpm, react for 2h. Take 1ml of the reaction solution, add 800μl of ethyl acetate to extract, centrifuge at 12,000 for 2min, take the organic phase, add appropriate amount of anhydrous sodium sulfate to dry, distill under reduced pressure, dissolve the product in 1ml of isopropanol, and filter by organic membrane for HPLC detection. Shimadzu Prominence LC-20AD system Daicel chiral column AD-H was used, and the detector was PDA. Detection conditions, isopropanol:n-hexane=10:90, 0.5ml / min, column temperature 35°C. The product is (3R,4S)-4-phenyl-3,4-epoxy-2-butanone, the conversion rate is >99%, and the enantioselectivity ee value is >99%. The product was determined by NMR-...

Embodiment 3

[0046] Embodiment 3: Synthesis of other chiral allyl epoxy ketones

[0047] Take 2 g of the bacteria from Example 1 and resuspend in 10 ml potassium dihydrogen phosphate / dipotassium hydrogen phosphate buffer (0.1 M, pH=7.0), add 10 mM substrate, react at 30° C., 230 rpm for 2 h. The reaction solution treatment method is the same as in Example 2.

[0048] Substrates include 4-phenyl-3-buten-2-one and derivatives obtained by halogen and methyl substitution at o- / m- / p-position respectively (Table 2). Conversion 70-99% and ee 85-99%. It has good conversion efficiency and excellent enantioselectivity ee value (>99%) for most substrates.

[0049]

[0050] Table 2

[0051]

[0052]

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Abstract

The invention discloses a new method for synthesizing two types of optically pure epoxy compounds which can regulate multi-enzyme cascade reaction. In this method, recombinant bacteria co-expressing carbonyl reductase and styrene monooxygenase in Escherichia coli are used as biocatalysts, ɑ, β-unsaturated ketones are used as substrates, and isopropanol is used as a switch to regulate and synthesize chiral alkenes. Propylene oxide ketone / alcohol.

Description

[0001] manual technical field [0002] The invention belongs to the technical field of microorganism and enzyme engineering, and specifically relates to a recombinant Escherichia coli that co-expresses carbonyl reductase and styrene monooxygenase as a biocatalyst to catalyze the synthesis of optically pure allylic rings from ɑ, β-unsaturated ketones. Oxyketone or alcohol approach. Background technique [0003] Optically pure epoxy compounds are one of the most important building blocks in organic synthesis, and numerous chiral functional groups can be derived by ring opening with nucleophiles. Optically pure allyl epoxy ketone / alcohol is a special multifunctional epoxy compound with carbonyl / hydroxyl in the ortho position. It not only has the common properties of epoxy compounds, but also has the special reactivity of hydroxyl / carbonyl. Therefore, they have a wider range of derivatization. A large number of chiral functional groups can be derived through functional group tr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P17/02C12N1/21C12N15/70C12R1/19
Inventor 吴中柳刘育昌
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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