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New oxidoreductases for enantioselective reactions

An active and functional technology, applied in the field of compositions for generating new variants-2-hydroxyacid dehydrogenase, can solve problems such as unpredictable correct alignment

Active Publication Date: 2018-01-05
DUKE UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among other things, these examples demonstrate that correct alignment of similar residues in IDH and HIDH sequences is unpredictable and requires experimental verification

Method used

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  • New oxidoreductases for enantioselective reactions
  • New oxidoreductases for enantioselective reactions
  • New oxidoreductases for enantioselective reactions

Examples

Experimental program
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Effect test

Embodiment 1

[0214] Preparation of HIDH constructs

[0215] A blunt-ended 1002 base pair (bp) fragment encoding Thermophila HIDH (TTC1012) lacking the 3'-stop codon was amplified from the Thermomicrobial HB27 gDNA library (ATCC). A blunt-ended 1110 bp fragment encoding LYS12 lacking the 3'-stop codon was amplified from a S. cerevisiae gDNA library using KAPA Taq DNA polymerase (KAPA Biosystems, Woford, MA). These fragments were cloned into pTrcHis2-TOPO (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. Mutations were performed using the QuikChange Site-Directed Mutagenesis Kit (Agilent). All constructs were verified by sequencing with pTrcHis2-for and pTrcHis2-rev primers. The constructs are listed in Table 1, SEQ ID NO: 1-22.

Embodiment 2

[0217] Purification of HIDH mutants

[0218] The expression and purification procedure for homoisocitrate dehydrogenase was adapted from published methods. Lin et al., Biochemistry 46:890-898 ​​(2007). Expression constructs were transformed into BL21-DE3 E. coli (Stratagene). Single colonies were inoculated into 5 mL LB-Amp medium starter cultures and grown with shaking (37° C. 225 rpm) for 6 h. Starter culture (5 mL) was added with 45 mL LB-Amp, shaken for 2 h, and induced with 1 mM IPTG and shaken for another 2 h. Centrifuge at 4,500×g at 4°C to collect the precipitate and dissolve in 2 mL of buffer B (500 mM NaCl, 10 mM MgCl 2 , 20 mM imidazole, 2 mM β-mercaptoethanol, 10 mM Tris, pH 7.5, supplemented with 1× EDTA-free cOmplete Mini from Roche TM protease inhibitors) and sonicated on ice for 6 cycles of 15 sec each on a Sonifer 250 (Branson). The lysate was clarified by centrifugation at 13,000 xg at 4°C. Lysates were loaded onto Ni-NTA spin columns (Qiagen) that had ...

Embodiment 3

[0220] Preparation of crude lysates of HIDH mutants

[0221] To obtain a crude lysate, 0.5 mL starter culture as described above was added to 4.5 mL LB-Amp and shaken for 2 h followed by an additional 2 h of incubation with 1 mM IPTG. The pellets from these cultures were collected by centrifugation at 4,500 xg at 4°C. These pellets were resuspended on ice in 500 μL buffer A (0.2% Triton-X 100, 1 mM PMSF, 0.5 mM EDTA, 10 mM Tris, pH 7.5) and sonicated on ice for 6 cycles of 15 s each on a Sonifer 250 (Branson) . Then, it was centrifuged down at 13,000×g 4°C for 15 minutes, and the clarified lysate was normalized to 700 ng / μL with lysis buffer and aliquoted for storage at -80°C. Protein concentrations were determined using BioRad protein assay reagents according to the manufacturer's instructions.

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Abstract

The present application describes compositions and methods for producing oxidoreductases for enantioselective reactions. The present application describes compositions and methods for producing novel variants of (R)-2-hydroxyacid dehydrogenases capable of enzymatically converting 1-carboxy-2-ketoacids into 1-carboxy-( R)‑2‑hydroxy acid, or its reverse reaction. Illustrative examples include (a) (R)-2-hydroxyadipate dehydrogenase and its use for converting 2-oxoadipate to (R)-2-hydroxyadipate or its reverse reaction and (b) (R)-2-hydroxyglutarate dehydrogenase and its use for converting 2-oxoglutarate into (R)-2-hydroxyglutarate or its reverse reaction. The present application also describes methods for producing unnatural microbial organisms to enzymatically convert 2-oxoadipate to (E)-2-hexenedioic acid or adipate, or to enzymatically convert 2-oxopentanedioate Compositions and methods for the conversion of diacids to (E)-2-glutaconate or glutaric acid or the reverse reaction of each.

Description

[0001] related application [0002] This application is a non-provisional application claiming the benefit of priority of US Provisional Patent Application No. 61 / 604,630, filed February 29, 2012, which is hereby incorporated by reference in its entirety. This application is related to US Patent Application No. 13 / ___,___ filed February 27, 2013. technical field [0003] Described herein are compositions and methods for producing oxidoreductases for enantioselective reactions. Described herein are compositions and methods for producing novel variant (R)-2-hydroxyacid dehydrogenases capable of enzymatically converting 1-carboxy-2-keto acids (i.e., 1-carboxy- 2-oxoacids, α-ketocarboxylic acids, α-oxoacids) into 1-carboxy-(R)-2-hydroxyacids (i.e., 1-carboxy-D-2-hydroxyacids, (R)- α-hydroxycarboxylic acid), or its reverse reaction. Illustrative examples include (a) (R)-2-hydroxyadipate dehydrogenase and its use for converting 2-oxoadipate to (R)-2-hydroxyadipate or its reverse ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/40C12P7/00C12N1/20C12N1/00
CPCC12N9/0006C07K2319/21C12N9/0008C12N15/52
Inventor Z·J·雷特曼H·阎B·D·宙一J·H·萨普森
Owner DUKE UNIV
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