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Method for producing isoprene by utilizing blue algae

A technology of isoprene and isoprenyl pyrophosphate, applied in the field of genetic engineering, can solve the problems of low yield and high cost, and achieve the effects of increasing yield, expanding application scope and widening industrial application prospects

Active Publication Date: 2013-07-24
SHANGHAI RES & DEV CENT OF INDAL BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In order to solve the defects of low yield and high cost in the existing production methods of isoprene, the inventors of the present application compared isoprene synthases from different sources such as Eucalyptus blueus, poplar, kudzu vine, etc., and studied It was expressed in Synechomonas after codon optimization, and further optimized the metabolic flow of the isoprene synthesis pathway, resulting in a significant increase in the production of isoprene synthesized by Synechomonas genetically engineered bacteria

Method used

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  • Method for producing isoprene by utilizing blue algae
  • Method for producing isoprene by utilizing blue algae
  • Method for producing isoprene by utilizing blue algae

Examples

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Embodiment 1

[0076] Example 1 1. Construction of expression vector integrating Synechocystis genome

[0077] 1.1. Construction of plasmid pKS2

[0078] Plasmid pKS2 is a homologous recombination vector inserted at glgX (slr0237) site in Synechocystis sp. PCC6803 genome. Using the Synechocystis sp. PCC6803 genome as a template, the primer pair P5 and P6 amplified the upstream homology arm glgX-up sequence (glgXup), and the primer pair P7 and P8 amplified the downstream homology arm psbA2-down sequence (glgXdn), and loaded in sequence into the pBluescript KS+ plasmid, and then introduce the spectinomycin marker (Spe), and at the same time introduce the endogenous promoter of Synechocystis PCC6803 encoding psbA2 (P psbA2 ) and the trrnB terminator sequence (trrnB) of Escherichia coli to obtain the homologous recombination vector pKS2 (SEQ ID NO: 47) expressing the target gene, the plasmid schematic diagram is as follows figure 2 shown.

[0079] 1.2. Construction of plasmid pKS3

[0080...

Embodiment 2

[0083] Example 2 , codon optimization

[0084] The isoprene synthase encoding gene e1ispS derived from Eucalyptus globulus, the original DNA sequence is from the GenBank database (GenBank: AB266390.1), and the sequence is shown in SEQ ID NO:50. The isoprene synthase coding gene p1ispS derived from poplar (Populus alba), the original DNA sequence is from GenBank database (GeneBank: AB198180), and the sequence is shown in SEQ ID NO: 51. The isoprene synthase p2ispS derived from Populus alba x Populus tremula, the original sequence is from the GenBank database (GenBank: AJ294819.1), and the sequence is shown in SEQ ID NO: 52. Among them, none of the e1ispS, p1ispS and p2ispS sequences contain a signal peptide, and p2fulllength (SEQ ID NO: 53) is a full-length sequence containing a signal peptide. The above DNA sequence was synthesized by Nanjing GenScript Company.

[0085] By using the GeneDesigner software of DNA2.0 Company, according to the codon preference of Synechocystis...

Embodiment 3

[0091] Example 3 , promoter screening

[0092] Using the Synechocystis sp. PCC6803 genome as a template, the following promoters were cloned: the primer pair P37 and P38 cloned the promoter P of psbA psbA 2 (SEQ ID NO: 57), primer pair P41 and P42 clone rbc operon promoter P rbc (SEQ ID NO: 58), primer pair P43 and P44 clone groESL operon promoter P groESL (SEQ ID NO:59), primer pair P45 and P46 clone Ni 2+ inducible promoter P nrsB (SEQ ID NO: 60), primer pair P47 and P48 clone ftsQ operon promoter P ftsQ (SEQ ID NO: 61). Using the plasmid pTrcHis (purchased from Invitrogen) as a template, the primer pair P49 and P50 were used to clone the promoter P trc (SEQ ID NO: 62).

[0093] The above-mentioned promoters are used to replace the pP in Example 2 respectively cpc The P amplified by the primer pair P39 and P40 in the k1*ispS plasmid cpc Promoter (SEQ ID NO: 63), resulting in plasmids expressing k1*ispS using different promoters. The above plasmids were respectivel...

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Abstract

The invention discloses a method for producing isoprene by utilizing blue algae, and the method comprises the following steps of: (1) expressing an isoprene synthetase coding gene derived from blue gum and an isoprene synthetase coding gene with an optimized codon in the blue algae; (2) applying the promotor of a cpc operon to express a mediate terpenoid synthetase coding gene contained in the blue algae; (3) enhancing the expression of an prenyl pyrophosphate isomerase coding gene; (4) expressing a fusion protein which contains a prenyl pyrophosphate isomerase and an isoprene synthetase; and (5) applying an SMUO tag to express a heterologous protein in the blue algae. The method disclosed by the invention can be used for carrying out genetic modification on the blue algae and expressing the isoprene synthetase (IspS) of a plant source in the blue algae, outstandingly increases the output of isoprene produced through the gene engineering blue algae through multiple methods, greatly widens the application range of the gene engineering blue algae by applying research results to express other heterologous proteins in the gene engineering blue algae and has wide industrial application prospect.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a method for producing isoprene by using cyanobacteria. Background technique [0002] Isoprene is an important bulk basic raw material, mainly used in the synthesis of isoprene rubber for the manufacture of tires. At the same time, it is used to produce various fine chemical products such as methyl heptenone and lavender alcohol. It is also used in the production of lithographic rubber for integrated circuits and synthetic lubricating oil additives. In addition, recent research has shown that isoprene can also be converted into biofuels, such as jet fuel. [0003] At present, isoprene is mainly produced from petroleum-based raw materials in industry, that is, the C5 fraction in oil refining by-products is separated by solvent extraction distillation. The preparation method is expensive and the purity of isoprene is low. With the increasing shortage of fossil reso...

Claims

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Application Information

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IPC IPC(8): C12P5/02C12N15/60C12N15/74C12N9/90C12N9/88C12R1/01
CPCC12Y402/03027C12N9/88C12N9/90C12P5/026
Inventor 杨琛高翔刘邓高方
Owner SHANGHAI RES & DEV CENT OF INDAL BIOTECH
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