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Bacterial strain for producing farnesene and application of bacterial strain

A technology of farnesene and strains, applied in the field of synthetic biology, can solve problems such as failure to implement industrial production and unsatisfactory protein expression, and achieve good industrial application prospects, increase production, and experimental rationality

Active Publication Date: 2013-08-14
SHENZHEN ACTION TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But now only a few farnesene synthases have been cloned, and their protein expression in microorganisms is not ideal. The use of microorganisms to produce farnesene has been moving forward slowly, and industrial production has not been implemented.
Existing reports (Wang, C., et al., 2011. Metabolic engineering of Escherichia coli for α-farnesene production. Metabolic Engineering. 13, 648-655) the highest yield of farnesene is only 0.38g / L

Method used

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  • Bacterial strain for producing farnesene and application of bacterial strain
  • Bacterial strain for producing farnesene and application of bacterial strain
  • Bacterial strain for producing farnesene and application of bacterial strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 In Vitro Reconstruction System Optimization of Mevalonate Pathway

[0040] The experimental method of the in vitro reconstitution system is the same as that established by Yu et al. in 2011 (Yu, X., et al., 2011. .108,18643-18648.), through the study of each protein in the mevalonate pathway, it was shown that increasing ERG13, ERG8, IspA and Idi can promote the production of terpenoids, especially the effect of increasing Idi is more obvious ( image 3 ), the optimized ratio of each protein in the synthesis of farnesene in the MVA pathway obtained from the in vitro reconstruction experiment is AtoB:ERG13:tHMG1:ERG12:ERG8:MVD1:Idi:IspA:AFS=1:10:2:5:5:2:5 : 2: 2, compared with the equimolar amounts of each protein, the reaction rate is significantly improved ( Figure 4 ).

Embodiment 2

[0041] Embodiment 2 Mevalonate pathway plasmid construction

[0042] Escherichia coli BL21(DE3) genomic DNA and Saccharomyces cerevisiae INVSC1 genomic DNA were purified with Qiagen's Blood and Cell Culture DNA Mini Kit.

[0043] Plasmid pMH1 contains the first three genes of the mevalonate pathway: atoB gene (acetoacetyl-CoA thioesterase) from Escherichia coli BL21 (DE3), erg13 (HMG-CoA synthase, 3- hydroxymethyl-glutaryl-CoA synthase) and thmg1 (HMG-CoA reductase, 3-hydroxymethyl-glutaryl-CoA reductase, deletes the transmembrane region of HMG1). Plasmid pFZ81 contains the last four genes of the mevalonate pathway: erg12 (mevalonate kinase), erg8 (mevalonate-5-phosphate kinase) and mvd1 (mevalonate-5-phosphate kinase) from Saccharomyces cerevisiae INVSC1 pyrophosphate kinase), derived from the idi (isopentenyl pyrophosphate isomerase) gene of Escherichia coli BL21 (DE3). All genes were amplified by PCR, and the primers used are listed in Table 1.

[0044] The specific cons...

Embodiment 3

[0050] Example 3 Farnesene produces metabolic pathway plasmid construction

[0051] The afs gene from Malus x domestica was codon-optimized by Nanjing KingScript Biotechnology Co., Ltd. for gene synthesis (sequence shown in SEQ ID NO.1), and then inserted into the plasmid through NdeI and XhoI restriction sites In pET21a(+), the plasmid pFZ35 ( Figure 7 ).

[0052] IspA gene was amplified from E. coli genomic DNA with primers IspA-BamHI and IspA-EcoRI-SpeI, digested with BamHI and EcoRI and inserted into plasmid pET28a(+) to obtain pFZ32. Then insert the XbaI-XhoI fragment containing ispA derived from pFZ32 into the SpeI-XhoI fragment of pFZ35 to obtain plasmid pFZ38, the plasmid schematic diagram is as follows Figure 8 As shown, the sequence (excluding the backbone vector sequence) is shown in SEQ ID NO.4.

[0053] The Idi gene was amplified by PCR using BL21(DE3) genomic DNA as a template (see Table 1 for primers Idi-NdeI and Idi-XhoI), and then digested with NdeI and X...

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Abstract

The invention discloses a bacterial strain for producing farnesene and an application of the bacterial strain, belonging to the field of synthetic biology. The bacterial strain for producing the farnesene contains related genes for synthesizing the farnesene through a mevalonic acid pathway and codon optimization; and the sequence of a farnesene synthetic gene afs optimized by codons is as shown by SEQ ID NO.1. The bacterial strain can be used for producing the farnesene, and a seed solution of the bacterial strain is inoculated into a culture medium containing a carbon source to carry out prokaryotic expression to obtain the farnesene. The gene for synthesizing the farnesene is subjected to codon optimization or the farnesene is synthesized by using the mevalonic acid pathway to ensure that each protein is closer to a ratio of AtoB:ERG13:tHMG1:ERG12:ERG8:MVD1:Idi:IspA:AFS=1:10:2:5:5:2:5:2:2, so that the production of the farnesene can be further promoted. By adopting the bacterial strain, the output of the farnesene is greatly improved to be more than 1g / L.

Description

technical field [0001] The invention belongs to the field of synthetic biology and relates to a strain for producing farnesene and an application thereof. Background technique [0002] Farnesene was first isolated from apple trees and plays an important role in plant defense. Because it is an important chemical raw material, for example, it can be used for synthetic rubber, and more importantly, farnesene is a precursor of excellent bioenergy farnesane, which has a broad market value and has been widely recognized by people in recent years. focus on. [0003] The content of farnesene in plants is very low, and it cannot be used in the chemical market by extracting natural products. Therefore, the production of farnesene by microorganisms seems to be the best way for people to utilize this compound. The biggest bottleneck of microbial fermentation lies in how to achieve industrialization, and the necessary condition for industrialization is that its output needs to meet the...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P5/02C12R1/19
Inventor 刘天罡朱发银邓子新
Owner SHENZHEN ACTION TECH CO LTD
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