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Rhamnolipid producing plasmid, construction method thereof, engineered escherichia coli and application

A technology of Escherichia coli and rhamnolipid, which is applied in the field of Escherichia coli engineering bacteria and application, rhamnolipid production plasmid and its construction, and can solve the problems of low yield of rhamnolipid and the like

Active Publication Date: 2017-05-31
自然资源部天津海水淡化与综合利用研究所
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These works have not carried out codon optimization and gene cluster structure optimization for the rhamnolipid synthesis gene for the E. coli host, and the rhamnolipid production is low

Method used

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  • Rhamnolipid producing plasmid, construction method thereof, engineered escherichia coli and application
  • Rhamnolipid producing plasmid, construction method thereof, engineered escherichia coli and application
  • Rhamnolipid producing plasmid, construction method thereof, engineered escherichia coli and application

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Embodiment 1

[0062] Embodiment 1 produces the construction of rhamnolipid engineering strain

[0063] 1. Construction of rhamnolipid synthesis gene vector

[0064] (1) Codon optimization and synthesis of SyrhlA gene, SyrhlB gene and SyrhlC gene

[0065] In this example, the original sequences of the rhamnosyltransferase genes rhlA, rhlB, and rhlC were all derived from Pseudomonas aeruginosa PAO1, and the rhlA was paired with reference to the Escherichia coli preferred codon table (see GenBank GI for the original gene sequence: 9949623), and properly avoid commonly used restriction sites. The optimized gene was named SyrhlA (sequence shown in Sequence Table 3), with NdeI and BamHI restriction enzyme sites added at both ends, synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and connected to pET21-21a ( +) Between the NdeI and BamHI sites of the vector, the plasmid DJ201 was obtained. The same method is used to optimize the codon of rhlB (the original sequence is GenBank GI:9949622)...

Embodiment 2

[0077] Embodiment 2 rhamnolipid-producing Escherichia coli engineering bacteria shake flask fermentation verification

[0078] 1. Fermentation process

[0079] Pick a single colony of Escherichia coli engineering bacteria BL202 in a 5mL liquid LB test tube containing 100μg / mL ampicillin, culture overnight at 37°C with shaking at 180r / min, and inoculate 1% of the inoculum into a 50mL test tube containing 100μg / mL ampicillin In the fermentation medium, 37°C, 180r / min shaking culture to OD 600 When the temperature is 0.6-0.8, 1 mM IPTG is added to induce the expression of the gene, and cultured for 46 hours to obtain a fermented liquid.

[0080] Ferment the engineering bacteria BL208, BL208 / 402, and BL208 / 301 with the same culture method, wherein 50 μg / mL chloramphenicol is added to the medium of BL208, and 50 μg / mL chloramphenicol and 50 μg of chloramphenicol are added to the medium of BL208 / 402 / mL kanamycin sulfate, 50 μg / mL chloramphenicol and 100 μg / mL ampicillin were adde...

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Abstract

The invention discloses a rhamnolipid producing plasmid, a construction method thereof, engineered escherichia coli and an application. Known rhamnosyltransferase genes rhlA, rhlB and rhlC are optimized and synthesized according to preference of an escherichia coli codon and named SyrhlA, SyrhlB and SyrhlC respectively; synthetic escherichia coli RBS (ribosome bind site) is connected to upstream SyrhlB, SyrhlA is connected to upstream RBS, and a gene cluster SyrhlA-RBS-SyrhlB is constructed; SyrhlA-RBS-SyrhlB and SyrhlC are constructed in plasmid pACYCDuet-1 containing double promoters, the plasmid is introduced into escherichia coli BL21(DE3) for expression, so that rhamnolipid is synthesized from escherichia coli with glucose as a carbon source, and the yield of rhamnolipid synthesized from escherichia coli is increased greatly.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a rhamnolipid production plasmid and its construction method, Escherichia coli engineering bacteria and application. Background technique [0002] Rhamnolipid is an important species of biosurfactant, which can significantly reduce surface tension, has excellent detergency, emulsification and flocculation capabilities, and has non-toxic and biodegradable characteristics. It is widely used in chemical industry, medicine, agriculture and environmental restoration, etc. The field has broad application prospects. [0003] Rhamnolipids were first discovered in 1949 by Jarvis and Johnson in Pseudomonas aeruginosa. Pseudomonas aeruginosa can synthesize rhamnolipids with various structures, mainly including disaccharide and double lipid structure, monosaccharide and double lipid structure, disaccharide and monolipid structure and monosaccharide and monolipid structure. Among them, the length...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/66C12N1/21C12P19/44C12R1/19
CPCC12N9/0006C12N9/1029C12N9/1051C12N15/66C12N15/70C12N2800/22C12P19/44C12Y101/01133C12Y203/01039
Inventor 杜瑾张爱君姜天翔郝建安王静
Owner 自然资源部天津海水淡化与综合利用研究所
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