Bacterial strain for producing lycopene and application of bacterial strain

A lycopene and strain technology, applied in the field of synthetic biology, can solve the problems of instability, low natural extraction yield, low plant fruit content, etc., and achieve the effects of increasing yield, shortening fermentation cycle, and improving industrial application prospects.

Active Publication Date: 2015-06-10
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This natural product of plant origin is well accepted by consumers, however this method of production has a number of disadvantages
First of all, industrial production based on plant source materials is heavily dependent on its output, which is unstable due to uncontrollable factors such as weather; the cost of large-scale planting remains high; it has obvious seasonality; the content in plant fruits is very low, and natural low extraction yield

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 The in vitro reconstitution system optimizes the mevalonate pathway

[0041] In vitro reconstitution and steady-state analysis of the fatty acid synthase from Escherichia coli. Proceedings of the National Academy of Sciences (Yu, X., et al., 2011. 108,18643-18648.), through the study of the various proteins of the mevalonate pathway, it has been shown that increasing ERG13, ERG8 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 of the MVA pathway obtained in the in vitro reconstruction experiment is AtoB:ERG13:tHMG1:ERG12:ERG8:MVD1:Idi=1:10:2:5:5:2:5, which is equivalent to the equimolar of each protein Ratio, the reaction rate is significantly improved ( Figure 4 )

Embodiment 2

[0042] Example 2 Construction of Mevalonate Pathway Plasmid

[0043] Purified by Qiagen's Blood and Cell Culture DNA Mini Kit, E. coli BL21 (DE3) genomic DNA and Saccharomyces cerevisiae INVSC1 genomic DNA were obtained.

[0044] The plasmid pMH1 contains the first three genes of the mevalonate pathway: the atoB gene (acetoacetyl-CoA thioesterase) from E. coli BL21 (DE3), and the erg13 (HMG-CoA synthase from S. cerevisiae INVSC1, 3- Hydroxymethyl-glutaryl-CoA synthase) and thmg1 (HMG-CoA reductase, 3-hydroxymethyl-glutaryl-CoA reductase, deletes the transmembrane region of HMG1). The plasmid pFZ81 contains the last four genes of the mevalonate pathway: erg12 (mevalonate kinase), erg8 (mevalonate-5-phosphokinase) and mvd1 (mevalonate-5- Pyrophosphate kinase), derived from the idi (isopentenyl pyrophosphate isomerase) gene of E. coli BL21 (DE3). All genes were amplified by PCR, and the primers used are shown in Table 1.

[0045] The specific construction method is as follows:

[0046...

Embodiment 3

[0051] Example 3 Construction of Plasmid of Lycopene Production Metabolic Pathway

[0052] The crtE, crtB and crtI genes derived from Pantoea ananatis were codon-optimized by Nanjing GenScript Biotechnology Co., Ltd. for gene synthesis (sequence shown in SEQ ID NO.1-3), and then passed through NdeI and XhoI enzymes The cutting site was inserted into the plasmid pET28a(+) to obtain plasmids pFZ21, pFZ22 and pFZ23. Then the XbaI-XhoI fragments derived from pFZ22 and pFZ23 containing crtB and crtI were inserted into the SpeI-XhoI fragment of pFZ21 to obtain plasmid pFZ27, and finally the XbaI-XhoI fragment of pFZ27 was inserted into plasmid pET21a(+) for production The plasmid pFZ110 of lycopene Figure 7 As shown, the sequence (without backbone vector sequence) is shown in SEQ ID NO.6.

[0053] Idi gene was amplified by PCR with BL21(DE3) genomic DNA as template (primers Idi-NdeI, Idi-XhoI are shown in Table 1), and then digested with NdeI and XhoI and inserted into the pET28a(+) pl...

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Abstract

The invention discloses a bacterial strain for producing lycopene and an application of the bacterial strain, and belongs to the field of synthetic biology. The bacterial strain for producing lycopene contains a mevalonate pathway and a related gene which is synthetized by the lycopene optimized by a codon; and the synthetic gene sequence of the lycopene optimized by the codon is shown in SEQ ID NO.1-3. The bacterial strain can be used for producing the lycopene; and a seed solution of the bacterial strain is inoculated into a culture medium containing a carbon source to carry out prokaryotic expression, so as to obtain the lycopene. The gene synthetized by the lycopene is optimized by the codon or each protein of the mevalonate pathway is closer to A to B:ERG13:tHMG1:ERG12:ERG8:MVD1:Idi=1:10:2:5:5:2:5; and production of the lycopene can be further promoted. By adopting the bacterial strain, the yield of the lycopene can be greatly improved to over 1g / L; and the fermentation cycle is shortened by over 50%.

Description

Technical field [0001] The invention belongs to the field of synthetic biology and relates to a strain for producing lycopene and its application. Background technique [0002] Lycopene is an important antioxidant, which has anti-aging, anti-tumor and lowering the risk of stroke, cardiovascular and cerebrovascular diseases, so it can be used as a natural health food or medicine. Lycopene has important uses in the fields of food, cosmetics and medicine, and has broad market value. [0003] At present, the development and production of lycopene in the world mainly include natural extraction, chemical synthesis, and microbial fermentation. Natural extraction is mainly obtained by planting tomatoes in large quantities and extracting and purifying the fruit after ripening. This plant-derived natural product has been widely accepted by consumers, but this production method has many disadvantages. First of all, industrial production based on plant-derived materials is heavily dependent...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P5/02C12R1/19
Inventor 刘天罡朱发银邓子新
Owner WUHAN UNIV
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