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Microorganisms and their uses

A product, Saccharomyces cerevisiae technology, applied in the direction of peptides, fungi, fermentation, etc., can solve the problems of low content, unstable content, chemical reagent residue, etc., and achieve the effect of high yield, high purity and high efficiency

Active Publication Date: 2020-05-15
HUBEI GUANGJI PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The extraction of natural products is mainly to obtain lycopene through the extraction and purification of mature fruits. However, this production method is affected by many uncontrollable factors such as climate, variety, geographical location, maturity, etc., and has obvious seasonality, unstable content, and The cost of large-scale planting and breeding is relatively high, and the content is usually relatively low
In addition, it is very difficult to extract and purify high-purity lycopene, which together makes the finished product of lycopene very expensive
The chemical synthesis method has the advantages of easy-to-obtain and cheap raw materials, mild reaction conditions, fast reaction rate, and easy separation of the product from the reaction system. performance and scope of use are limited

Method used

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  • Microorganisms and their uses
  • Microorganisms and their uses
  • Microorganisms and their uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Construction of the first generation engineering strain J1011-3

[0039] In this example, the inventor introduced the construction process of the J1011-3 strain in detail.

[0040] First construct ΔLEU2: pGAL1-BtCarG (Blakeslea trispora) knock-out box, namely knock-out box fragment 1, design upstream and downstream primers to PCR amplify each fragment so that there are 60-80bp overlapping fragments between each other, and then homologous All fragments are recombined together by means of recombination, and ΔLEU2: pGAL1-BtCarG (Blakesleatrispora) knockout box is obtained by restriction digestion linearization, such as knockout box fragment 1 such as figure 1 As shown, knockout box fragment 1 has the nucleotide sequence shown in SEQ ID NO:1. Using the yeast’s own homologous recombination mechanism, the fragments were respectively integrated into the yeast 30000B genome by the lithium acetate method. The integration site was LEU2. Since the LEU2 in the original yeast i...

Embodiment 2

[0048] Example 2 Selection of promoter and terminator

[0049] In order to be induced with galactose, the genes GAL1, GAL7, and GAL10 that metabolize galactose need to be knocked out. Therefore, the background strain of the characteristic promoter is the knockout gene GAL1,7,10. The price of galactose is relatively high and it is not suitable for industrial production. Therefore, GAL80 was knocked out on the basis of knocking out genes GAL1, 7, and 10, in order to achieve regulation of gene expression by controlling the amount of glucose. The obtained promoter information is used to construct and control the expression of genes related to carotenoid synthesis.

[0050] Such as Figure 4 As shown, both the inducible promoter and the constitutive promoter in the ΔGAL1 / 7 / 10 / 80 strain and the ΔGAL1 / 7 / 10 strain are basically the same in the stationary phase. It can be seen that knocking out GAL80 can induce gene expression without galactose, and the induction intensity is equivalent t...

Embodiment 3

[0059] Example 3 Construction of the second generation engineering strain

[0060] In this example, the inventors adjusted the number of copies of the three genes BtCarG (Blakeslea trispora), PaCrtB (Pantoea Agglomerans) and McCrtI (Mucorcircinelloides) on the J1011-3 strain based on the J1011-3 strain to obtain the second Generation strains.

[0061] The specific characteristics of the second-generation engineered strains are shown in Table 1. Select the appropriate promoter and terminator according to the design, use the upstream and downstream primers to PCR amplify each fragment so that there are 60-80bp overlapping fragments between each other, and then recombine all fragments together by homologous recombination. The corresponding fragments were obtained by enzyme digestion and linearization, and the fragments were integrated into the genome of yeast J1011-3 through yeast transformation using the yeast's own homologous recombination mechanism. After culturing the seeds, the...

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Abstract

The invention provides a microorganism, application thereof and a method for producing lycopene. Overexpression of the microorganism is selected from at least one of genes including tHMG1, BtCarG, PaCrtB, McCrtI, INO2, yap1, spt15-5, taf25-3, GapN, PYC2, SMAE1, MDH2, POS5, pntAB, ADH2, ACS6, ALD6, EUTE, ERG12, IDI1, ERG10, MVD1, ERG13 and ERG8, and silence is selected from at least one of genes including GAL1, GAL7, GAL10, GAL80, ROX1, VBA5, DOS2, Ypl062W, Yjl064W, Yer130C, Yer134C, Ynr063W, Exg1, Yor292C, Sfk1 and Mef1. The method for producing the lycopene from the microorganism is high in yield and efficiency and short in production period.

Description

Technical field [0001] The present invention relates to the field of bioengineering. Specifically, the present invention relates to microorganisms and their uses. More specifically, the present invention relates to microorganisms, methods for obtaining lycopene, and uses of microorganisms in preparing lycopene. Background technique [0002] The development and production of lycopene mainly include natural product extraction, chemical synthesis, and microbial fermentation. Natural product extraction is mainly through the extraction and purification of mature fruits to obtain lycopene. However, this production method is affected by many uncontrollable factors such as climate, variety, geographic location, and maturity. It has obvious seasonality, content instability, and The cost of large-scale planting and breeding is relatively high, and the content is usually relatively low. In addition, high-purity lycopene is very difficult in extraction and purification technology, which tog...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12P5/02
CPCC07K14/39C12P5/007
Inventor 刘天罡沈佳石彬叶紫玲马田刘然
Owner HUBEI GUANGJI PHARMA
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