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Method for expressing vitreoscilla hemoglobin in phaeodactylum tricornutum for autotrophic and mixotrophic culture

A technology of Vibrio lucidum and hemoglobin, applied in the field of bioengineering, can solve the problems of affecting the accumulation of unsaturated fatty acids, the reduction of cell growth rate, and the damage of cell oxygen, so as to achieve the effects of improving expression efficiency, reducing photorespiration, and promoting accumulation.

Active Publication Date: 2020-11-06
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, during the growth process of algae cells, they are susceptible to high light stress, producing excessive reactive oxygen species (Reactive oxygen species, ROS), causing cellular oxygen damage, resulting in a decrease in cell growth rate and affecting the accumulation of unsaturated fatty acids.

Method used

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  • Method for expressing vitreoscilla hemoglobin in phaeodactylum tricornutum for autotrophic and mixotrophic culture
  • Method for expressing vitreoscilla hemoglobin in phaeodactylum tricornutum for autotrophic and mixotrophic culture
  • Method for expressing vitreoscilla hemoglobin in phaeodactylum tricornutum for autotrophic and mixotrophic culture

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Experimental program
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Effect test

Embodiment 1

[0073] The construction method of the VHb transformant, and the construction method of the expression VHb transformant of the growth of the VHb transformant and the high value-added product yield under autotrophic conditions, comprising the following steps:

[0074] (1) VHb gene codon optimization and construction of expression vector

[0075] In the present invention, the original VHb gene as shown in SEQ ID NO.1 is optimized for algae-biased codons. The optimized VHb gene nucleotide sequence is shown in SEQ ID NO.2, and the amino acid sequence is consistent with the original amino acid sequence, as shown in Shown in SEQ ID NO.3.

[0076] The pPha-T1-based expression vector was inserted into the VHb gene downstream of the fcp-A promoter to construct the VHb expression vector. The plasmid pPha-T1 was used as a template, and the primer pPha-BB-F / R was used to amplify the backbone fragment required for constructing the vector by PCR. The primer sequences are as follows, see Ta...

Embodiment 2

[0131] Cultivation of VHb expressing transformants under syntrophic conditions

[0132] The difference with Example 1 is that the autotrophic culture method adopted in step (5) in Example 1 is adjusted to a concurrent culture condition, and the f / 2 medium used for autotrophic culture is added until the final concentration of the medium is 2g / L-mannitol was used as an organic carbon source, and added to the medium with a final concentration of 0.2g / L tea polyphenols to promote the utilization of mannitol. The rest of the conditions were consistent with the autotrophic conditions, and the difference between the transformed strain and the wild-type growth state was compared. Its growth curve is as Figure 7 As shown, the contents of fucoxanthin and EPA are as follows Figure 8 shown.

[0133] The test results in Example 2 prove that the expression of the VHb gene can promote the efficiency of algae cells utilizing organic carbon for cell metabolism in the process of concurrent...

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Abstract

The invention provides a method for expressing vitreoscilla hemoglobin in phaeodactylum tricornutum for autotrophic and mixotrophic culture. codon optimization of a VHb gene and construction of an expression vector are mainly performed; the VHb expression vector is constructed, and is introduced into a cell in an electrotransformation manner by utilizing PCR amplification linearized fragments, andtransgenic algal strains are obtained by screening; the transgenic algal strains obtained by the invention can continuously improve the gene in the aspects of the growth efficiency of phaeodactylum tricornutum under autotrophic and mixotrophic culture conditions and the yield of high added-value products.

Description

technical field [0001] The invention relates to a method for expressing Vitella hyaline hemoglobin in Phaeodactylum tricornutum for autotrophic and concurrent culture, and belongs to the technical field of bioengineering. Background technique [0002] Marine microalgae play an important role in marine ecosystems, and as primary producers, they are an important food source for marine organisms. There are many kinds of marine microalgae, among which Phaeodactylum tricornutum has attracted people's attention because of its fast growth rate and high oil content. Its growth rate can reach 0.38g / L / d, the oil content after harvest can reach more than 48%, and the yield of EPA (eicosenoic acid) can reach 36mg / L / d. It has been used as a new source of EPA production in recent years. And as a typical diatom, its fucoxanthin content is between 15.42-16.51mg / g. However, during the growth process of algae cells, they are susceptible to high light stress, producing excessive reactive oxy...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/31C12N1/13C12P7/64C12P17/02C12R1/89
CPCC07K14/805C12N15/8247C12N15/825C12P7/6427C12P17/02
Inventor 范勇丁晓婷李福利姜尔颖师晓艺
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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