CN-122012548-A - Sucrose non-fermentation 1 related protein kinase gene RkSnf and application thereof

Abstract

The invention discloses a sucrose non-fermentation 1 related protein kinase gene RkSnf, the coding nucleotide sequence of which is shown as SEQ ID NO. 2, a knock-out gene RkSnf plasmid is constructed according to the gene and is transferred into rhodosporidium YM25235, experimental results show that the total carotenoid synthesis amount of a RkSnf gene knock-out strain is obviously improved compared with that of a wild rhodosporidium YM25235, and the result shows that the gene knock-out engineering bacterium constructed based on the gene provided by the invention can be used for carotenoid synthesis, and the invention has important theoretical significance and potential economic value for research on regulating and controlling carotenoid synthesis.

Inventors

• ZHANG QI
• YANG MINGJING
• HUANG XIAOLAN
• CHEN YUAN
• QIU JINGWEN

Assignees

• 昆明理工大学

Dates

Publication Date

20260512

Application Date

20260304

Priority Date

20250421

Claims (2)

1. 1. A sucrose non-fermentation 1 related protein kinase gene RkSnf has the coding nucleotide sequence shown in SEQ ID NO. 2.
2. 2. Use of the sucrose non-fermented protein kinase 1 gene RkSnf of claim 1 knocked out to promote production of carotenoids by rhodosporidium (Rhodosporidium kratochvilovae).

Description

Sucrose non-fermentation 1 related protein kinase gene RkSnf and application thereof Technical Field The invention belongs to the technical field of genetic engineering, and particularly relates to a sucrose non-fermented protein kinase 1 gene RkSnf and application thereof in promoting rhodosporidium (Rhodosporidium kratochvilovae) to produce carotenoid. Background Carotenoids (Carotenoid) are a general term for important natural pigments, which belong to the class of terpenes, have multiple conjugated double bonds and are generally yellow, orange or red in color. The structure of carotenoids is based on isoprenoids, and most typical carotenoid chemical structures contain 40 carbon atoms and are polymerized from 8 isoprenoids. Carotenoids have a wide variety of biological functions. Carotene is a precursor of vitamin A in human body, but human and animals cannot synthesize the Carotene by themselves, and the intake of carotenoid can improve the immunity of human body. In addition, carotenoids also have coloring, antioxidant, anti-apoptotic and anticancer effects. Microbial cells sense and transduce some external stimuli through signal transduction pathways, synthesizing metabolites such as unsaturated fatty acids, carotenoids, etc., that can help the microorganism resist the hostile environment. In yeast, sucrose non-fermentation 1 (SNF 1) related protein kinase (SnRK) is homologous to mammalian AMPK protein complex, which is a trimer composed of one of SNF1 catalytic subunit, SNF4 regulatory subunit and Gal83/Sip1/Sip2 protein family. In the absence of glucose, respiratory metabolism, glycogen accumulation and gluconeogenesis of microorganisms are promoted by inhibiting the transcription factor Mig1 or by stimulating the transcription activators Cat8 and Sip 4. In addition to being involved in the derepression of glucose control genes, SNF 1-related protein kinases are involved in a variety of physiological processes including meiosis and sporulation, senescence, autophagy and biofilm formation. SNF 1-related protein kinase is also involved in lipid metabolism in yeast, and it can activate transcription factors, enhance expression of fatty acid oxidation-related genes, thereby enhancing beta-oxidation of fatty acids, and play an important role in regulation of intracellular acetyl CoA homeostasis and global histone acetylation. Studies have shown that SNF 1-related protein kinases are also involved in yeast responses to environmental stresses, such as heat shock, alkaline pH, and salt stress. The Snf1 subunit is a catalytic core of SNF1 related protein kinase, has protein kinase activity and plays a key role in energy metabolism, stress response and carbon source utilization of yeast cells. It comprises a typical serine/threonine protein kinase domain that helps cells maintain homeostasis and viability by regulating carbon metabolism, gene expression, and activating stress response pathways. The Snf1 subunit plays an important role in carbon metabolism, and the Snf1 subunit inhibits the glycolytic pathway and reduces the consumption of glucose by phosphorylating various metabolic enzymes and regulating the expression of transcription factors and carbon metabolic pathways under the condition of low glucose and phosphorylating glycolytic related enzymes. It can also activate key enzymes in the gluconeogenic pathway, promoting the conversion of non-sugar substances to glucose. And the Snf1 subunit can regulate and control the metabolism of fatty acid, inhibit the synthesis of fatty acid, promote the oxidation of fatty acid and provide energy for cells. Disclosure of Invention The invention provides a sucrose non-fermentation 1 related protein kinase gene RkSnf1, which is separated from rhodosporidium (Rhodosporidium kratochvilovae) YM25235, wherein the nucleotide sequence of the genome of the sucrose non-fermentation 1 related protein kinase is shown as SEQ ID NO. 1 (2877 bp), the nucleotide sequence of the encoding nucleotide sequence of the genome is shown as SEQ ID NO. 2 (2331 bp), the amino acid sequence of the encoding gene is shown as SEQ ID NO. 3, the gene RkSnf1 in rhodosporidium (Rhodosporidium kratochvilovae) is knocked out, and the knocking out of the gene promotes the synthesis of carotenoid in rhodosporidium. In order to achieve the above object of the present invention, the technical scheme of the present invention is as follows: 1. Extracting rhodosporidium YM25235 strain genome by adopting a CTAB method, taking the genome as a template, adopting primers RkSnf1-F, rkSnf1-R, obtaining a sucrose non-fermented protein kinase 1 gene RkSnf sequence by PCR amplification, designing gRNA by using a CRISPR online website, and comprehensively evaluating the gRNA by using the online website to obtain gRNA1 and gRNA2 sequences; 2. According to the sequences of gRNA1 and gRNA2, synthesizing primers RkSnf-gRNA 1-F, rkSnf 1-gRNA 1-R, rkSnf1-sgRNA2-F, rkSnf1-sgRNA2-R by a biological engineering company, using