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Three-dimensional structure of isoprene synthase and its use thereof for generating variants

An isoprene synthase, three-dimensional structure technology, applied in the directions of enzymes, lyases, carbon-oxygen lyases, etc., can solve the problems of difficulty in obtaining polypeptide crystals and easy crystallization of polypeptides.

Inactive Publication Date: 2012-09-05
DANISCO US INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In some cases peptides are easily crystallized, while in other cases peptide crystals have proven to be very difficult to obtain
There is no detailed theory to guide attempts to crystallize macromolecules, therefore most attempts at crystal growth of macromolecules have been empirical in nature (MacPherson, 2004)

Method used

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  • Three-dimensional structure of isoprene synthase and its use thereof for generating variants
  • Three-dimensional structure of isoprene synthase and its use thereof for generating variants
  • Three-dimensional structure of isoprene synthase and its use thereof for generating variants

Examples

Experimental program
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Effect test

Embodiment 1

[0312] Embodiment 1. Construction of MBD kudzu IspS

[0313] This example describes the construction of a vector expressing a maltose binding protein-kudzu isoprene synthase fusion molecule.

[0314] I. Construction of pTrcKudzu

[0315] A synthetic gene encoding the isoprene synthase (IspS) of Pueraria lobata and codon-optimized for Escherichia coli was purchased from DNA2.0 (Menlo Park, CA) as plasmid p9795 ( figure 2 and 3) provide. The insert was removed by digestion with BspLU11I / PstI, gel purified and religated into NcoI / PstI-digested pTrcHis2B (Invitrogen, Carlsbad, California). The resulting plasmid was called pTrcKudzu ( Figure 4 and 5). The stop codon in the insert precedes the PstI site, resulting in a construct in which the His-tag is not attached to the IspS protein.

[0316] II. Construction of plasmid pMAL-C4X Kudzu

[0317] According to the manufacturer's instructions, using plasmid pTrcKudzu as DNA template, primers EL-959 and EL-960 of Table 1-1, 10 m...

Embodiment 2

[0324] Example 2. IspS variants used for crystal structure experiments

[0325] This example describes the generation of affinity-tagged isoprene synthase (IspS) enzymes for expression, purification and crystallization.

[0326] I. Strain Construction

[0327] For constructs in the pET200D-TOPO vector (Invitrogen), gel extraction and purification (Qiagen) from Populus alba, P. PCR product of Yang's IspS enzyme. The PCR reaction of the pET200 construct was as follows: The reaction mixture was: 1 μl (template)-pET24a-P.alba, 5 μl 10X PfuUltraII Fusion buffer, 1 μl dNTP (10 mM), 1 μl primer (50uM), primer F-(MCM219 or 218), 1 μl Primer (50uM) Primer R-(MCM182), 41μl diH 2 O and 1 μl of PfuUltra II Fusion DNA polymerase from Stratagene; cycling parameters were: 95°C for 1 min, 29 cycles of "95°C for 1 min, 55°C for 20 sec, 72°C for 27 sec", followed by 72°C for 3 min, Then at 4°C until cooling, using an Eppendorf Mastercycler. Similar responses were performed for aspen, populus...

Embodiment 3

[0372] Three-dimensional structure of embodiment 3.IspS

[0373] Seven constructs of plant isoprene synthase (IspS) were prepared, and it was hoped that one or more would yield crystals suitable for x-ray diffraction. These constructs are: a construct containing N-terminal histidine-tagged maltose-binding protein and kudzu IspS (MBP-kudzu); a construct containing N-terminal histidine-tagged full-length P. alba IspS (MD08-99 ); remove the first 19 residues of P. alba IspS (MD08-100) at the N-terminus, and the construct will also remove the histidine tag after purification; full-length untagged P. alba IspS ( Strain RM11608-2); a truncated P. alba IspS construct characterized by two additional residues (MD09-167) preceding the generation of the double arginine motif; a P. americana IspS was produced comprising N-terminal histidine tag and N-terminal truncation (MD08-104); another construct consisting of IspS from P. japonicus with N-terminal histidine tag and N-terminal truncat...

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Abstract

The present invention provides a three-dimensional structures of P. tremuloides isoprene synthase and P. alba isoprene synthase. The invention also provides methods of using the three dimensional structure to design isoprene synthases with improved activity for increased isoprene production in microbial host cells. Biosynthetically produced isoprene of the present invention finds use in the manufacture of rubber and elastomers.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Provisional Patent Application 61 / 172,199, filed April 23, 2009, and US Provisional Patent Application 61 / 255,831, filed October 28, 2009, the contents of which are hereby incorporated by reference in their entirety. technical field [0003] This application relates to the crystallization and three-dimensional structure determination of Populus tremuloides isoprene synthase and Populus alba isoprene synthase. The present application also provides methods of producing variants of isoprene synthase to increase isoprene production in microbial host cells. Background technique [0004] Isoprenoids are polymers of isoprene that find use in pharmaceuticals, nutraceuticals, fragrances, perfumes, and rubber products. However, natural isoprenoid supplies are limited due to ecological considerations. For this reason, and to provide an isoprenoid composition with fewer impurities and gr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88
CPCC12P5/007C07K2299/00C12N9/88C12Y402/03027
Inventor Z·Q·贝克R·R·博特C·L·莱福D·H·韦尔斯J·V·米勒
Owner DANISCO US INC
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