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Recombinant saccharomyces cerevisiae engineering bacteria and application thereof in synthesis of nano-selenium

A technology for recombining Saccharomyces cerevisiae and Saccharomyces cerevisiae, which is applied in application, genetic engineering, fermentation and other directions, can solve the problems of difficulty in separation and purification of synthetic nano-selenium, and achieve the effects of reducing costs, increasing yield, and simplifying separation and purification steps.

Pending Publication Date: 2022-03-29
ZHEJIANG UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The invention provides a recombinant Saccharomyces cerevisiae engineering bacterium and its application in the synthesis of nano-selenium. The method screens out Saccharomyces cerevisiae strains with a high conversion rate of sodium selenite and carries out vesicle transport and metabolic transformation to obtain a saccharomyces cerevisiae strain that can tolerate higher concentrations of selenium The recombinant Saccharomyces cerevisiae strain of sodium selenate is a fermentation bacterium, which is inoculated into a fermentation medium containing a high concentration of selenite for fermentation, and the selenite is reduced to biological elemental nano-selenium particles with a particle size of about 100nm, and the generated nano-selenium particles Selenium can be more easily separated from bacterial strains by gradient centrifugation, which solves the problem of difficult separation and purification of nano-selenium synthesized by existing methods, and improves the output of nano-selenium

Method used

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  • Recombinant saccharomyces cerevisiae engineering bacteria and application thereof in synthesis of nano-selenium
  • Recombinant saccharomyces cerevisiae engineering bacteria and application thereof in synthesis of nano-selenium
  • Recombinant saccharomyces cerevisiae engineering bacteria and application thereof in synthesis of nano-selenium

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1, the screening of sodium selenite high transformation efficiency bacterial strain

[0046] 1. Preparation of fermentation broth

[0047] Eleven strains of Saccharomyces cerevisiae purchased from China Industrial Microbiology Culture Collection Center (CICC) preserved in the laboratory at -80°C were inoculated into YPD solid medium, and activated at 30°C for 24 hours to obtain activated strains. Pick a ring of activated strains in YPD liquid medium, shake culture at 30°C for 15 hours, and use it as seed solution. The seed solution was inoculated into the YPD liquid medium of 30mL with the inoculation amount of volume concentration 10%, after cultivating for 24h at 30°C, adding sodium selenite (Na 2 SeO 3 , Sodium selenite was first dissolved in water to make 10000mg / L aqueous solution, and then added after filtering with a filter membrane with a pore size of 0.22 μm), and oscillated on a reciprocating shaker at 30°C for 48h to obtain fermentation broths of...

Embodiment 2

[0060] Embodiment 2, utilize TEM electron microscope, SEM electron microscope and XPS bionano elemental selenium to carry out characterization

[0061] 1. Fermentation of strains

[0062] The 1406 strain of Saccharomyces cerevisiae screened in Example 1 was inoculated into YPD solid medium, and cultured at 30° C. for 24 hours for activation to obtain activated strains. Pick a ring of activated strains in YPD liquid medium, shake culture at 30°C for 15 hours, and use it as seed solution. The seed solution was inoculated into the YPD liquid medium of 30mL with the inoculation amount of volume concentration 10%, after cultivating for 24h at 30°C, adding sodium selenite (Na 2 SeO 3 , sodium selenite was first dissolved in water to make a 10000mg / L aqueous solution, and then added after filtering through a filter membrane with a pore size of 0.22μm), and oscillated on a reciprocating shaker at 30°C for 48h to obtain 39mL of fermentation broth.

[0063] 2. Scanning electron microsc...

Embodiment 3

[0074] Example 3. Modification of vesicle transport pathway

[0075] 1. Construction and transformation of gRNA plasmid for CRISPR-Cas9 system

[0076] (1) Strain p426_Cas9_gRNA-Hyg

[0077] Using the plasmid p426_Cas9_gRNA-HIS3b (Addgene, No. 87402) as a template, primers P1 / P2 were used to amplify the p426_Cas9_gRNA-HIS3b vector fragment (denoted as fragment 1) without the URA3 fragment.

[0078] Using the plasmid pRS41H (EUROSCARF, Germany) as a template, the hygromycin hyg resistance gene was amplified using primers P3 / P4 (the nucleotide sequence is shown in SEQ ID NO: 6, which is denoted as fragment 2). Fragment 1 and Fragment 2 were incubated using a one-step cloning kit, and then transformed into Escherichia coli DH5α competent. Positive transformants were selected and verified by colony PCR using primers P5 / P6, and the obtained strain was designated as p426_Cas9_gRNA-Hyg.

[0079] (2) Strain p426_Cas9_gRNA-Hyg-YPRC

[0080] Utilize chopchop website, design target s...

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Abstract

The invention discloses a recombinant saccharomyces cerevisiae engineering bacterium and application thereof in synthesis of nano-selenium. The recombinant saccharomyces cerevisiae engineering bacterium is constructed by inserting a Sec13p gene and a Sso1p gene into a single copy site YPRC delta 15 of saccharomyces cerevisiae. The two genes Sec13p and Sso1p for transportation of the brewer's yeast vesicles are improved, the vesicle transportation mechanism of nano-selenium is enhanced, the excretion effect of nano-selenium particles is improved, the separation and purification steps of the nano-selenium particles and the brewer's yeast are simplified, the cost is reduced for industrial production of the nano-selenium particles, and the yield is increased.

Description

(1) Technical field [0001] The invention relates to the synthesis of nano-selenium, in particular to a method for improving the secretion of yeast strains to synthesize nano-selenium. The biological nano-selenium particles produced by transformation are uniform in size, and in addition to being used as a trace element supplement, it also has the functions of scavenging free radicals and inhibiting bacteria. , can be used in feed additives, health products and cosmetics. (2) Background technology [0002] Selenium is an essential trace element for the life activities of mammals. Studies have shown that the dose of dietary selenium reaching the supra-nutritional level has a good effect on the prevention and treatment of cancer, but these doses are close to the chronic toxicity dose of selenium, and the toxicity of selenium is not only dependent on the ingested dose, but also affected by its chemical form influence. In nature, the existence forms of selenium include organic s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/31C12P3/00C12R1/865
CPCC07K14/395C12P3/00Y02E50/10
Inventor 孙杰袁梦杰王宇光章银军王梦玲林一诺史玉娜黄润东李圳炀过海清占伟杰
Owner ZHEJIANG UNIV OF TECH
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