Bacterial strain for producing lycopene and application of bacterial strain

A technology of lycopene and bacterial strains, which is applied in the field of synthetic biology, can solve the problems of instability, low natural extraction yield, and low plant fruit content, and achieve the effects of increasing yield, shortening fermentation cycle, and good industrial application prospects

Active Publication Date: 2013-08-14
WUHAN UNIV
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  • 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 In Vitro Reconstruction System Optimization of Mevalonate Pathway

[0041] 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 and Idi can promote the production of terpenoids, especially the effect of increasing Idi is more obvious ( image 3 ), the optimal ratio of each protein in the MVA pathway obtained from the in vitro reconstruction experiment is AtoB:ERG13:tHMG1:ERG12:ERG8:MVD1:Idi=1:10:2:5:5:2:5, which is equivalent to each protein in equimolar ratio, the reaction rate is significantly increased ( Figure 4 )

Embodiment 2

[0042] Embodiment 2 Mevalonate pathway plasmid construction

[0043] Escherichia coli BL21(DE3) genomic DNA and Saccharomyces cerevisiae INVSC1 genomic DNA were purified with Qiagen's Blood and Cell Culture DNA Mini Kit.

[0044] 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.

[0045] The specific cons...

Embodiment 3

[0051] Example 3 Lycopene Production Metabolic Pathway Plasmid Construction

[0052] The crtE, crtB and crtI genes from Pantoea ananatis were codon-optimized by Nanjing KingScript Biotechnology Co., Ltd. for gene synthesis (sequence shown in SEQ ID NO.1-3), followed by NdeI and XhoI enzymes The cleavage site was inserted into plasmid pET28a(+) to obtain plasmids pFZ21, pFZ22 and pFZ23. Then insert the XbaI-XhoI fragment containing crtB and crtI derived from pFZ22 and pFZ23 into the SpeI-XhoI fragment of pFZ21 to obtain plasmid pFZ27, and finally insert the XbaI-XhoI fragment of pFZ27 into the plasmid pET21a (+) to obtain for production Lycopene plasmid pFZ110 The schematic diagram of the plasmid is as follows Figure 7 As shown, the sequence (excluding the backbone vector sequence) is shown in SEQ ID NO.6.

[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 XhoI ...

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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 an application thereof. Background technique [0002] Lycopene is an important antioxidant, which has the effects of anti-aging, anti-tumor and reducing 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, microbial fermentation and other methods. Natural extraction is mainly obtained by planting tomatoes in large quantities, and extracting and purifying the fruits after they mature. Natural products of plant origin are well accepted by consumers, however this method of production has a number of disadvantages. First of all, industrial production...

Claims

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Application Information

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Patent Type & Authority Applications(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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