CN103409332A - Construction of saccharomyces cerevisiae engineering bacteria generating beta-phenethyl alcohol, strain and application thereof - Google Patents

Abstract

The invention relates to the construction of Saccharomyces cerevisiae engineering bacteria producing β-phenylethanol, strains and applications thereof, and belongs to the technical field of bioengineering and food flavor manufacturing. The prepared β-phenylethanol flavor is a natural flavor and can be used to prepare various food flavors , Widely used in food, medicine, daily chemical and other industries. Using Saccharomycescerevisiae S288c as the starting strain, design and construct an engineered bacteria that overexpresses the coupling overexpression of aminotransferase gene (ARO8) and decarboxylase gene (ARO10) in the synthetic metabolic pathway of β-phenylethanol, and enhances the genes of two key enzymes in the main synthetic pathway Copying and expression, the selected engineered bacteria (transformants) are genetically stable after multiple passages, and the metabolic flux and yield of β-phenylethanol are significantly improved. Using L-phenylalanine as the main raw material, engineering bacteria are used to ferment and transform β-phenylethanol flavors, with single configuration and chirality and high aroma quality, which provides new ideas and technical support for the preparation of natural β-phenylethanol flavors.

Description

Construction, strain and application of Saccharomyces cerevisiae engineering bacteria producing β-phenylethanol

technical field

The invention relates to the construction, strain and application of Saccharomyces cerevisiae engineering bacteria producing β-phenylethanol, which belongs to the technical field of bioengineering and food flavor manufacturing. The prepared natural β-phenylethanol flavor can be used for the deployment of various food flavors and is widely used In food, medicine, daily chemical and other industries. the

Background technique

β-phenylethanol (2-phenylethanol, 2-PE), also known as 2-phenylethanol, is a kind of aromatic alcohol with elegant, delicate and long-lasting rose aroma. It has obvious aroma-enhancing effect when added in a small amount. The second largest spice of vanillin, it can be used to blend rose, clove, orange blossom, ylang-ylang, lily of the valley and other fruity flavors and wine flavors, widely used in food, medicine, daily chemical products, cosmetics and tobacco and other industries. 2-PE also has the effects of calming and refreshing, sterilizing, aphrodisiac and aphrodisiac. the

β-Phenylethyl alcohol naturally exists in a variety of plant and flower refined oils, such as rose, jasmine, narcissus, lily, etc., but in most cases the concentration is very low, such as the 2-PE content in rose flowers is relatively high, generally per ton of roses Flowers can only extract 1kg rose essential oil (2-PE content is about 60%). Therefore, natural 2-PE is difficult to produce on a large scale and cannot meet market demand. At present, β-phenylethanol is mainly produced by chemical synthesis methods such as benzene-ethylene oxide method and styrene oxide hydrogenation method. During the chemical synthesis process, the environmental pollution is serious, and the products often contain by-products such as biphenyl, monochloroethylbenzene, and chloroethanol that are difficult to remove, which affect the quality of their products, and it is difficult to meet the quality standards of edible and daily spices. With people's fashion pursuit of "green" and "natural", it is urgent to develop new technologies to produce natural β-phenylethanol on a large scale. the

Studies have shown that a variety of yeasts have the ability to synthesize β-phenylethanol, such as Saccharomyces cererisiae, Kluyveromyces marxianus, K.lactis, Pichia fermentans, Torulopsis utilis), Hansenula anomala, etc. However, there are large differences in the synthesis ability of 2-PE among different species and strains, and the breeding of high-yield 2-PE strains has become the primary condition for biotransformation production. the

There have been some studies on the synthetic pathway of β-phenylethanol in S.cerevisiae, its regulatory enzymes and its genes. Using L-phenylalanine (L-Phe) as a precursor, S. cererisiae is transformed into 2-PE through the Ehrlich pathway, which is the quickest and main way to biotransform 2-PE at present. The synthesis and metabolism of 2-PE is regulated by transaminase, The regulation of key enzymes such as phenylpyruvate decarboxylase; Ehrlich's first step transamination reaction is mainly participated by aromatic aminotransferase I (aro8p), encoded by the ARO8 gene, which encodes a polypeptide chain consisting of 950 residues, Its N-terminus is the region that binds to DNA, and the C-terminus is rich in aspartic acid. A large number of negative charges form multiple winding α-helices. This region mediates the interaction between proteins and endows the protein with the ability to activate transcription . Phenylpyruvate decarboxylase (Aro10p) is another key enzyme in the Ehrlich pathway. The phenylpyruvate decarboxylase of S.cererisiae is encoded by the ARO10 gene. The upstream regulatory sequence of the ARO10 gene gene has a 36bp upstream activation sequence and is regulated by aro8p Induced regulation. Therefore, it is believed that transamination reaction and decarboxylation reaction are two key steps for S. cerevisiae to utilize L-phenylalanine to synthesize and metabolize β-phenylethanol, which are respectively controlled by aromatic aminotransferase (aro8p) and phenylpyruvate decarboxylase (Aro10). adjust. Enhancing the copy and expression of S. cerevisiae aminotransferase gene (ARO8) and decarboxylase gene (ARO10) is conducive to enhancing the metabolic flux and yield of the target product β-phenylethanol. the

Based on the above analysis, the patent application starts from the adjustment of the key rate-limiting enzymes in the above two key steps, by cloning the ARO8 and ARO10 genes and integrating overexpression in S. The metabolic flux and yield of the product β-phenylethanol revealed the molecular regulation mechanism of L-phenylalanine catabolism. The above S.cerevisiae engineered bacteria ferment L-phenylalanine to transform and prepare β-phenylethanol fragrance, which has a single configuration and chirality and high aroma quality, which provides new ideas and support for the preparation of natural β-phenylethanol fragrance. the

Contents of the invention

The invention provides a construction of Saccharomyces cerevisiae engineering bacteria producing β-phenylethanol, strains and applications thereof. Using Saccharomyces cerevisiae S288c as the starting strain, design and construct engineering bacteria coupled with overexpression of key enzyme genes (AR08, AR10) in the synthetic metabolic pathway of β-phenylethanol to enhance the expression of key enzyme genes in the main synthetic pathway; The engineering bacterium (transformant) Paro8-10 has been passed down for many times, heredity is stable, and the metabolic flux and yield of β-phenylethanol have been significantly improved; Under the base and optimized fermentation conditions, L-phenylalanine can be efficiently converted to produce natural β-phenylethanol flavor. the

Instructions on the Preservation of Biological Material Samples

Biological material sample preservation unit: General Microbiology Center of China Committee for the Collection of Microbial Cultures. the

Address of depository unit: Institute of Microbiology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing. the

Deposit date: December 11, 2012. the

Deposit number: CGMCC No.6950. the

Classification name: Saccharomyces cerevisiae (Saccharomyces cerevisiae) Paro8-10. the

Detailed ways

The present invention provides a construction of Saccharomyces cerevisiae engineering bacteria producing β-phenylethanol, bacterial strains and applications thereof, specific implementation methods (technical solutions):

(1) ARO8 and ARO10 genes were amplified from Saccharomyces cerevisiae DNA by PCR technology, connected to shuttle plasmids respectively, and recombinant plasmid expression vectors were constructed, and introduced into S. cerevisiae S288c to overexpress ARO8 and ARO10 genes respectively, and detect their Gene transcription activity, analysis of transaminase, decarboxylase expression level, metabolic flux. the

(2) Connect the ARO8 and ARO10 genes to the shuttle plasmid expression vector at the same time to construct the recombinant plasmid coupling expression vector, and introduce it into S. cerevisiae S288c to screen the positive transformants to overexpress the ARO8 and ARO10 gene coupling to construct a high-yield β- S.cerevisiae engineering bacteria of phenylethyl alcohol. Analyze transaminase, decarboxylase gene transcription and expression levels, metabolic flux of engineering bacteria, clarify key metabolic nodes, and analyze molecular regulatory mechanisms. the

(3) Prepare fermentation medium with L-phenylalanine as the main raw material, optimize the medium and fermentation conditions of engineering bacteria, and use fermenter (bioreactor) to efficiently transform and produce natural β-phenylethanol fragrance. the

technical effect

By regulating the single overexpression and coupled overexpression of key rate-limiting enzyme functional genes such as transamination reaction and decarboxylation reaction in the synthetic metabolic pathway of β-phenylethanol, the metabolic pathway and metabolic flux of β-phenylethanol biosynthesis were optimized. Under the medium and fermentation conditions, compared with the wild-type S.cerevisiae S288c strain, the conversion rate and yield of the target product β-phenylethanol were greatly improved by the selected engineered bacteria, and the optimization strategy and technical effect of engineering bacteria construction were obvious. the

Claims (3)

1. A Saccharomyces cerevisiae engineering bacterium producing β-phenylethanol is characterized in that the aminotransferase gene (ARO8 gene) and the decarboxylase gene (ARO10 gene) of Saccharomyces cerevisiae (Saccharomycescerevisiae) are coupled and overexpressed to construct a high-yield β-phenylethanol The engineering bacteria of ethanol Saccharomyces cerevisiae, the selected engineering bacteria have been subcultured for many times, the genetics are stable, and the metabolic flux and yield of β-phenylethanol are significantly improved. 2. a method for constructing a brewing yeast engineering bacterium producing β-phenylethanol, characterized in that suitable primers are designed to clone the aminotransferase gene (ARO9 gene) and decarboxylase gene (ARO10 gene) of Saccharomyces cerevisiae, and ARO9 The gene and ARO10 gene were coupled to the shuttle plasmid to obtain the recombinant plasmid, and the recombinant plasmid was introduced into Saccharomyces cerevisiae, and the engineering bacteria were screened. 3. An application of Saccharomyces cerevisiae engineering bacteria producing β-phenylethanol, which is characterized in that L-phenylalanine is used as the main raw material, and the engineering bacteria efficiently utilizes L-phenylalanine in the fermenter to transform into β-phenylethanol Fragrance, preparation of natural β-phenylethanol fragrance.