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Construction and application of saccharomyces cerevisiae strain for efficiently synthesizing hemoglobin or myoglobin from different sources

A technology of hemoglobin and myoglobin, applied in the field of genetic engineering, can solve food safety hazards and other problems, and achieve the effects of solving potential safety hazards, efficient expression, and efficient production

Pending Publication Date: 2021-10-26
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existence of these miscellaneous proteins will bring great food safety hazards

Method used

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  • Construction and application of saccharomyces cerevisiae strain for efficiently synthesizing hemoglobin or myoglobin from different sources
  • Construction and application of saccharomyces cerevisiae strain for efficiently synthesizing hemoglobin or myoglobin from different sources
  • Construction and application of saccharomyces cerevisiae strain for efficiently synthesizing hemoglobin or myoglobin from different sources

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Construction of recombinant plasmids and recombinant bacteria for heterologous expression of hemoglobin / myoglobin from different animal and plant sources with different expression strategies

[0041] Select hemoglobin from soybean and clover, pig hemoglobin α subunit, porcine hemoglobin β subunit, bovine hemoglobin α subunit, bovine hemoglobin β subunit, and myoglobin from pig and bovine respectively, through codon optimization , the gene c2 (shown in SEQ ID NO.1) of synthetic soybean hemoglobin, the gene clover (shown in SEQ ID NO.2) of clover hemoglobin, the gene (shown in SEQ ID NO.3) of coding pig myoglobin, coding The gene of bovine myoglobin (shown in SEQ ID NO.4), the gene HBA1 of porcine hemoglobin α subunit (shown in SEQ ID NO.5), the gene HBB of porcine hemoglobin β subunit (shown in SEQ ID NO.6) , the gene HBA of bovine hemoglobin α subunit (shown in SEQ ID NO.7), and the gene HBB encoding bovine hemoglobin β subunit (shown in SEQ ID NO.8).

[004...

Embodiment 2

[0045] Example 2: Knocking out the gal80 gene and utilizing glucose for fermentation

[0046] Using Cas9-CRISPER technology to knock out the switch gene gal80 (GenBank No. 854954) that controls the expression of hemoglobin / myoglobin induced by the galactose-inducible promoter GAL in the genome of Saccharomyces cerevisiae CEN.PK2-1D, the principle is as follows Figure 5 As shown, the gal80 mutant Saccharomyces cerevisiae in which gal80 was knocked out was constructed, and the recombinant plasmid constructed in Example 1 was transferred into the gal80 mutant Saccharomyces cerevisiae, and the recombinant strain was obtained by screening and verification, and the fermentation mode directly induced by glucose was realized. And then realize the expression of hemoglobin / myoglobin.

[0047] Shake Flask Fermentation: Hemoglobin or myoglobin is produced by fermentation at the level of shake flasks, and an additional 10ug / ml heme is added to YNB medium, YPD medium or other Saccharomyces...

Embodiment 3

[0049] Example 3: Construction of recombinant bacteria that freely co-express porcine / bovine hemoglobin α subunit and its stable protein AHSP

[0050] The role of the hemoglobin α-subunit stabilizing protein AHSP is as follows: Figure 5 As shown, the gene AHSP2 (shown in SEQ ID NO.9) encoding porcine hemoglobin and the gene HBA1 (shown in SEQ ID NO.5) connected to the α subunit of porcine hemoglobin were connected to MCS1, MCS1, MCS2 was constructed to obtain a recombinant plasmid, and the recombinant plasmid was transformed into Saccharomyces cerevisiae CEN.PK2-1D to construct a recombinant bacterium.

[0051] The gene AHSP3 (shown in SEQ ID NO.10) encoding bovine hemoglobin and the gene HBA (shown in SEQ ID NO.7) encoding bovine hemoglobin α subunit were respectively connected to the MCS1 and MCS2 sites of pESC, and the co-expression was constructed The recombinant plasmid, and the co-expression recombinant plasmid was transformed into Saccharomyces cerevisiae CEN.PK2-1D, ...

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Abstract

The invention discloses construction and application of a saccharomyces cerevisiae strain for efficiently synthesizing hemoglobin or myoglobin from different sources, and belongs to the technical field of genetic engineering. Proper expression vectors, chaperonin and promoters are selected, a host is modified, heterologous expression of the hemoglobin or myoglobin from different sources is achieved in saccharomyces cerevisiae, safety of the hemoglobin and myoglobin is guaranteed, efficient production of the hemoglobin or myoglobin is achieved, and the potential safety hazard that an expression host is not a food-grade host in the previous report is solved. At a shake flask level, the yield of soybean hemoglobin can at least reach 120 mg L<-1>, the yield of clover hemoglobin can at least reach 20 mg L<-1>, the yield of porcine myoglobin can at least reach 99 mg L<-1>, the yield of bovine myoglobin can at least reach 89 mg L<-1>, the yield of porcine hemoglobin can at least reach 20 mg L<-1>, and the yield of bovine hemoglobin can at least reach 23 mg L<-1>. The result lays a foundation for application of the hemoglobin / myoglobin in the field of processing of foods such as artificial meat.

Description

technical field [0001] The invention relates to the construction and application of brewer's yeast strains for efficiently synthesizing hemoglobin or myoglobin from different sources, and belongs to the technical field of genetic engineering. Background technique [0002] With the rapid development of stem cell culture, it has become possible to produce small amounts of artificial meat in the laboratory. Due to high cost and insufficient market acceptance, artificial meat is still in the early stages of development. In addition, current artificial meat cannot well simulate the color and texture of real meat. Therefore, it is essential to satisfy the true color and nutritional flavor of muscle tissue, and these characteristics are mainly provided by heme protein and myoglobin. [0003] Hemoglobin is an iron-binding protein ubiquitous in nature. It has important physiological functions such as iron supplementation, oxygen transport, and respiration in organisms, and it also ...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/12C12N15/29C12P21/02C12R1/865
CPCC07K14/805C12N15/81C12N2800/22
Inventor 赵鑫锐薛技科毛炳杰孙小焱王淼周景文堵国成陈坚李江华
Owner JIANGNAN UNIV
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