CN-116445312-B - Yarrowia lipolytica genetically engineered bacterium for producing erythritol by utilizing glycerol

Abstract

The invention discloses a yarrowia lipolytica genetic engineering bacterium for producing erythritol by utilizing glycerol, which is obtained by screening strains with higher cell growth and erythritol yield through random insertion mutation mediated by non-homologous end connection. Furthermore, the genetically engineered bacterium overexpresses the glycerol dehydrogenase gene GCY3 or ARA1 of the glycerol metabolic dihydroxyacetone pathway and the dihydroxyacetone kinase gene DAK2. Furthermore, the genetically engineered bacteria overexpress a transketolase gene TKL1 and a transaldolase gene TAL1 of a non-oxidized pentose phosphate pathway. The invention also discloses a construction method and application of the genetically engineered bacterium. The dominant genetically engineered bacterium obtained by constructing the mutant library screening has the characteristic of high-concentration glycerol tolerance, and the substrate conversion rate and the glycerol utilization rate are both obviously higher than those of the original strain.

Inventors

• HUA QIANG
• LIU FENG
• TIAN JINGTAO
• WANG YATING
• ZHAO LINGXUAN
• CHEN JUN
• WEI LIUJING

Assignees

• 华东理工大学

Dates

Publication Date

20260512

Application Date

20230515

Claims (3)

1. 1. A yarrowia lipolytica genetic engineering bacterium for producing erythritol by using glycerol is characterized in that a glycerol dehydrogenase gene GCY3 and a dihydroxyacetone kinase gene DAK2 of a glycerol metabolic dihydroxyacetone pathway or a glycerol dehydrogenase gene ARA1 and a dihydroxyacetone kinase gene DAK2 are overexpressed on the basis of the yarrowia lipolytica genetic engineering bacterium with the preservation number of CCTCC NO: M20221813, wherein: The nucleotide sequence of the glycerol dehydrogenase gene GCY3 is shown as SEQ ID NO.2, the nucleotide sequence of the dihydroxyacetone kinase gene DAK2 is shown as SEQ ID NO.1, and the nucleotide sequence of the glycerol dehydrogenase gene ARA1 is shown as SEQ ID NO. 3.
2. 2. A yarrowia lipolytica genetically engineered strain for producing erythritol using glycerol, characterized in that the yarrowia lipolytica genetically engineered strain of claim 1 overexpresses the ketolase gene TKL1 and the transaldolase gene TAL1 of the non-oxidative pentose phosphate pathway, wherein: The nucleotide sequence of the transketolase gene TKL1 is shown as SEQ ID NO.4, and the nucleotide sequence of the transaldolase gene TAL1 is shown as SEQ ID NO. 5.
3. 3. Use of genetically engineered yarrowia lipolytica bacteria according to claim 1 or 2 for the production of erythritol.

Description

Yarrowia lipolytica genetically engineered bacterium for producing erythritol by utilizing glycerol Technical Field The invention relates to the technical field of genetic engineering, in particular to yarrowia lipolytica genetic engineering bacteria for producing erythritol by utilizing glycerol. Background Erythritol (R, S-1,2,3, 4-tetrol, CAS number 149-32-6) has a molecular formula of C 4H10O4, and has the advantages of 1, fresh taste, lower sweetness, 2, high stability, 3, obvious heat absorption effect when dissolved in water, 4, lower hygroscopicity, and 5, obvious solubility along with temperature change. Since it is not metabolized by the caries-causing bacteria of interest, it is non-caries and has been shown to reduce plaque. Erythritol has also been shown to be a free radical scavenger with antioxidant properties. Through available metabolism in animals and humans, toxicology and clinical studies have found that the ingestion of erythritol in large quantities over long periods of time is safe for humans. In addition, erythritol is an important four-carbon platform chemical, and can be subjected to chemical reactions such as hydrogen reduction (hydrodeoxygenation) through suitable catalyst components (active metals, additives and carriers) to generate chemicals such as butadiene, 1, 4-butanediol, 2, 5-dihydrofuran, tetrahydrofuran and the like. Currently, erythritol has been used in the food industry, the daily chemical industry, the pharmaceutical field, and the large-scale consumer grade chemical industry. The production method of erythritol mainly comprises a chemical synthesis method and a microbial fermentation method. However, the chemical synthesis method is accompanied with the problems of complex reaction, high cost, harsh reaction conditions, low product conversion rate, difficult subsequent purification and the like, and is mainly a microbial fermentation method in actual industrial production. Fermentation using industrial by-products as a carbon source is an important research content in the biotechnology field, and accords with the current policies of green manufacturing and recycling economy. The process of producing biodiesel generates a great amount of glycerol as a byproduct, and the effective utilization and conversion of glycerol by a microbiological technology greatly promotes the development of recycling economy. Yarrowia lipolytica (Yarrowia lipolytica) as a non-conventional yeast resistant to hyperosmotic conditions spontaneously produces erythritol as its own osmoprotectant and has broad substrate availability, a recognized safety (GARS) strain. Endogenous DNA double-strand breaks (DSBs) occur in many biological processes, such as DNA replication, three-dimensional genome folding, and gene transcription, and occur at high frequency. Most DSBs in yarrowia lipolytica are repaired by non-homologous end joining (NHEJ), and random integration of endogenous or exogenous genes in the yarrowia lipolytica genome can be achieved by using NHEJ, so that a mutant library of yarrowia lipolytica can be quickly constructed by the randomness of the insertion, providing a novel engineering strategy for the construction of efficient cell factories. Catabolism of glycerol depends on two pathways, one being the "G3P pathway" with glycerol-3-phosphate as an intermediary metabolite and the other being the "DHA pathway" with dihydroxyacetone as an intermediary metabolite. In the G3P pathway, glycerol is catalyzed by glycerol kinase to consume one molecule of ATP to produce glycerol-3-phosphate, which is then catalyzed by glycerol-3-phosphate dehydrogenase to produce dihydroxyacetone phosphate, whereas in the DHA pathway, glycerol is catalyzed by glycerol dehydrogenase to produce cytotoxic dihydroacetone, which is then catalyzed by dihydroxyacetone kinase to consume one molecule of ATP to produce dihydroacetone phosphate (DHAP). The glycerol metabolite monoacetone phosphate is converted into glyceraldehyde-3-phosphate by triose phosphate isomerase, and enters gluconeogenesis, glycolysis and tricarboxylic acid circulation paths. The current report of erythritol production using glycerol is not numerous, and studies to enhance glycerol metabolism in yarrowia lipolytica have focused on the G3P pathway, ignoring the impact of the potential DHA pathway on glycerol metabolism. Therefore, there is a need to develop genetically engineered yarrowia lipolytica bacteria that can produce erythritol by using glycerol, by deeply exploring the key gene of the DHA pathway of glycerol metabolism in yarrowia lipolytica. Disclosure of Invention The invention aims to optimize the synthetic route of producing erythritol by using glycerol by using yarrowia lipolytica through metabolic engineering means, so as to obtain the yarrowia lipolytica genetic engineering strain with optimized performance for producing erythritol by using glycerol. In order to achieve the above object, according to a first aspect of the present