REFERENCE TO SEQUENCE LISTING

The instant application contains a Sequence Listing in ASCII format as a file named seq-new.txt, created on Aug. 24, 2021, of 48 kB in size, and which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to a genetically engineered strain, belonging to the technical field of bioengineering.

BACKGROUND

Phenylethanoids mainly have three structural analogs: 2-phenylethanol (β-phenylethanol), tyrosol (p-hydroxyphenylethanol, tyrosol, 4-hydroxyphenylethanol, 2-(4-hydroxyphenyl)ethanol), hydroxytyrosol (3,4-dihydroxyphenylethanol, hydroxytyrosol, 3,4-dihydroxyphenylethanol, 2-(3,4-dihydroxyphenyl)ethanol), which are all L-α-aromatic amino acid derivatives. 2-Phenylethanol is an aromatic alcohol with a delicate rose scent and is widely used in food, medicines, cosmetics, tobacco and daily chemicals. It is not only the basic component of all rose scent aromas, but also has synergetic and synergistic effects, and is a component required for a variety of fragrance formulae. Tyrosol is the core of the salidroside molecule, has good oxidation resistance, and is also an important pharmaceutical intermediate. It is mainly used in the synthesis of cardiovascular drugs, such as Metoprolol, Bxolol and the like. Hydroxytyrosol is regarded as one of the most powerful antioxidants and can eliminate free radicals in the body, restore the health of the internal organs of the human body, prevent brain failure and delay aging.

These compounds are currently produced by methods such as plant extraction, chemical synthesis, microbiological and the like. Chemical methods have been successful in the synthesis of 2-phenylethanol, tyrosol and hydroxytyrosol (Chinese patents 200880002426.3, 201310183569.1, 200910098983.6). Since these compounds are important food or health care raw materials, chemically synthesized products are not liked by people. These products currently on the market are mainly extracted from plants. For example, Chinese patent 201510625623.2 discloses a method for extracting high-purity 2-phenylethanol from rose petals; Chinese patent 201410743409.2 discloses a method for preparing high-purity tyrosol fromRhodiola crenulata; And Chinese patent 201710195462.7 discloses a method for extracting hydroxytyrosol from olive leaves. Due to the limitations of plant resources and contents thereof, these products are expensive, so the production by microbiological methods has received extensive attention.

L-phenylalanine, L-tyrosine, and L-dopa may be subjected to deamination, decarboxylation and aldehyde group reduction to form 2-phenylethanol, tyrosol and hydroxytyrosol. Based on this metabolic principle, several microbiological production methods have been developed.

Yeast has a metabolic pathway for the de novo synthesis of 2-phenylethanol, and can also directly transform L-phenylalanine to 2-phenylethanol through an amino acid catabolic pathway. As early as 1907, Ehrlich added L-phenylalanine in a yeast medium, so that the yield of 2-phenylethanol was greatly increased (Ber Dtsch Chem Ges, 1907.40:1027-1047). Chinese patents 02137575.5, 200710066884.0, 200810071807.9, 200910049170.8, 200910049565.8, 201310243384.5, 201410661174.2 and 201610256845.6 all disclose strains and methods for producing 2-phenylethanol by transforming L-phenylalanine with yeast. Romagnoli has developed a method for producing 2-phenylethanol by deleting yeast aromatic amino acid transaminase and using glucose as a substrate, the yield is about 3 mM, and a small amount of tyrosol is also produced (Deletion of theSaccharomyces cerevisiaeAro8 Gene, Encoding an Aromatic Amino Acid Transaminase, Enhances Phenylethanol Production from Glucose. Yeast, 2015,32(1): 29-45). Since the yeast grows slowly and consumes part of L-phenylalanine during the transformation process, the yield of the 2-phenylethanol is lower and the cost is higher.

There are still other methods for preparing 2-phenylethanol byEnterobactersp. (De-Novo Synthesis of 2-Phenylethanol byEnterobacterSp CGMCC 5087. BMC Biotechnology, 2014,14) or the submerged fermentation technique ofAntrodia camphorata(Chinese patent 201410041209.2), but they have lower efficiency than the yeast.

Escherichia coligrows faster and is a better option for genetically engineered bacteria. Chinese patent 201210276491.3 over-expresses phenylpyruvate decarboxylase and alcohol dehydrogenase in L-phenylalanine high-yieldEscherichia colito achieve a high yield of 2-phenylethanol, but the over-expressed 2 enzymes disturb the metabolism ofEscherichia coligrown by de novo synthesis of glucose, so the yield can only reach 130 mg/L. Chinese patent 201510650028.4 co-expresses 4 enzymes inEscherichia coli, namely aromatic amino acid aminotransferase, exogenous phenylpyruvate decarboxylase, phenyl alcohol dehydrogenase and glutamate dehydrogenase, for whole cell transformation of L-phenylalanine to produce 2-phenylethanol; due to the low concentration of α-ketoglutaric acid in the cells, it is difficult to provide sufficient ammonia receptors; when the α-ketoglutaric acid is added to the transformation system, the highest yield can be obtained, and glutamic acid can also be added to significantly increase the yield; and it is clear that the addition of other compounds will increase the cost of transformation and will also increase the complexity of the transformation and purification operations. Chinese patent 201610464256.7 discloses a non-cellular biosynthesis method, by which separately expressed transaminase, phenylpyruvate decarboxylase and alcohol dehydrogenase are co-fixed or L-phenylalanine is transformed in a solution volume to produce 2-phenylethanol; and it is found from the published documents that the system lacks sufficient ammonia receptors and NADH, so the process can not easily implement production. Chinese patent 201710256900.6 discloses: mixing three kinds ofEscherichia coliwet cells for respectively expressing phenylalanine dehydrogenase, 2-keto acid decarboxylase and alcohol dehydrogenase, adding coenzymes TPP and NAD, and controlling temperature and pH for transforming L-phenylalanine; and in this method, the NAD and TPP are expensive and will be decomposed and fail after reacting for certain time, the three enzymes need to be separately cultured, which greatly increases the reaction cost, and the independent cells also affect the NAD shuttle between cells to achieve coenzyme regeneration, so the transformation reaction is difficult to continue.

Chinese patent 201310133238.7 discloses a method for producing tyrosol from tyrosine or glucose by over-expressing 4-hydroxyphenyl decarboxylase derived fromSaccharomyces cerevisiaeinEscherichia coliand knocking out the phenylacetaldehyde dehydrogenase gene; however, when the tyrosine is used as a substrate, this strain lacks an effective deamination system; when the glucose is used for de novo synthesis, the tyrosol is toxic to the cells, affecting cell growth and product synthesis; and therefore, this strain is difficult to achieve efficient production of tyrosol. 201510242626.8 discloses a method of over-expressing a monooxygenase gene cluster HpaBC derived fromEscherichia coliinEscherichia coliand realizing de novo synthesis of hydroxytyrosol from glucose; the main disadvantage of this scheme is that the hydroxytyrosol is toxic to the cells and has the lower expression level of HpaBC; and therefore, the efficiency of producing hydroxytyrosol is more difficult to increase. Chinese patent 201710091999.9 discloses anEscherichia coliwhich over-expresses pyruvate decarboxylase and aromatic amino acid aminotransferase derived fromSaccharomyces cerevisiaeand application of the bacterium for producing tyrosol by whole cell transformation of L-tyrosine; And it is clear that the bacterium lacks sufficient ammonia receptors and a good aldehyde reduction system, so the yield is low. Chung et al., by over-expressing plant-derived aromatic aldehyde synthases inEscherichia coli, converting L-tyrosine into p-hydroxyphenylacetaldehyde in one step, and then producing tyrosol from the reduction system insideEscherichia coli; and in addition, over-expression of HpaBC achieved hydroxytyrosol, but the low expression efficiency of plant genes inEscherichia coliaffected the whole cell transformation effect (Production of Three Phenylethanoids, Tyrosol, Hydroxytyrosol, and Salidroside, Using Plant Genes Expressing inEscherichia coli. Scientific Reports, 2017).

Currently, multi-enzyme tandem whole-cell catalyzed simple precursor generation of high-added-value products has been widely used (Constructing Biocatalytic Cascades: In Vitro and in Vivo Approaches to de Novo Multi-Enzyme Pathways, ACS Catal., 2017, 7(1), 710-724), and efficient transformation of inexpensive substrates by whole cells is sometimes more cost effective than de novo synthesis with glucose.

Based on the defects in various current methods, the present disclosure constructs a multi-enzyme co-expressedEscherichia coli, which realizes the whole cell catalytic transformation of aromatic L-α-amino acids and can respectively catalyze L-phenylalanine, L-tyrosine and L-dopa to produce 2-phenylethanol, tyrosol and hydroxytyrosol.

SUMMARY

Based on the defects of various current methods, the present disclosure provides a recombinantEscherichia colistrain capable of producing Phenylethanoids at low cost. At the same time, the present disclosure aims to solve the technical problems of the construction and application of the strain.

A first object of the present disclosure is to provide a recombinantEscherichia colistrain capable of producing Phenylethanoids at low cost; and the recombinantEscherichia colistrain simultaneously expresses 4 enzymes, namely L-amino acid oxidase, α-keto acid decarboxylase, alcohol dehydrogenase, and an enzyme capable of reducing NAD(P) to NAD(P)H.

In an implementation, the L-amino acid oxidase is fromProteus mirabilisATCC 29906 orCosenzaea myxofaciensATCC 19692.

In an implementation, the amino acid sequence of the L-amino acid oxidase is a sequence of which the accession NO. in NCBI is WP_004244224.1 (SEQ ID NO:1) or OAT30925.1 (SEQ ID NO:2) and which is hereby incorporated by reference in its entirety.

In an implementation, the nucleotide sequence of the L-amino acid oxidase is a sequence of which the accession NO. in NCBI is NZ_GG668576 REGION: 1350390 . . . 1351805 (SEQ ID NO:3) and which is hereby incorporated by reference in its entirety, or LXEN01000066 REGION: 20563 . . . 21963 (SEQ ID NO:4) and which is hereby incorporated by reference in its entirety.

In an implementation, the α-keto acid decarboxylase is fromProteus mirabilisATCC 29906 orLactococcus lactisATCC 19435.

In an implementation, the amino acid sequence of the α-keto acid decarboxylase is a sequence of which the accession NO. in NCBI is BC_004247067.1 (SEQ ID NO:5) and which is hereby incorporated by reference in its entirety, or WP_025016816.1 (SEQ ID NO:6) and which is hereby incorporated by reference in its entirety.

In an implementation, the nucleotide sequence of the α-keto acid decarboxylase is a sequence of which the accession NO. in NCBI is NZ_GG668593 REGION: 50463 . . . 52112 (SEQ ID NO:7) and which is hereby incorporated by reference in its entirety, or NZ_LKLC01000008 REGION: 208327 . . . 209973 (SEQ ID NO:8) and which is hereby incorporated by reference in its entirety.

In an implementation, the alcohol dehydrogenase is fromEscherichia coliBL21(DE3).

In an implementation, the amino acid sequence of the alcohol dehydrogenase is a sequence of which the accession NO. in NCBI is WP_001318460.1 (SEQ ID NO:9) and which is hereby incorporated by reference in its entirety, or WP_000692754.1 (SEQ ID NO:10) and which is hereby incorporated by reference in its entirety.

In an implementation, the nucleotide sequence of the alcohol dehydrogenase is a sequence of which the accession NO. in NCBI is NC_012892 REGION: 4406777 . . . 4407796 (SEQ ID NO:11) and which is hereby incorporated by reference in its entirety, or NC_012892 REGION: 312506 . . . 313555 (SEQ ID NO:12) and which is hereby incorporated by reference in its entirety.

In an implementation, the enzyme capable of reducing NAD(P) to NAD(P)H is formate dehydrogenase, glucose dehydrogenase or phosphite dehydrogenase.

In an implementation, the enzyme capable of reducing NAD(P) to NAD(P)H is formate dehydrogenase fromKomagataella phaffiiATCC 76273, glucose dehydrogenase fromBacillus subtilisATCC 13952, or phosphite dehydrogenase fromPseudomonas abietaniphilaATCC 700689.

In an implementation, the amino acid sequence of the enzyme capable of reducing NAD(P) is a sequence of which the accession NO. in NCBI is AOA63544.1 (SEQ ID NO:13) and which is hereby incorporated by reference in its entirety, WP_013351020.1 (SEQ ID NO:14) and which is hereby incorporated by reference in its entirety or WP_003118429.1 (SEQ ID NO:15) and which is hereby incorporated by reference in its entirety.

In an implementation, the nucleotide sequence of the enzyme capable of reducing NAD(P) is a sequence of which the accession NO. in NCBI is CP014710 REGION: 1836993 . . . 1838090 (SEQ ID NO:16) and which is hereby incorporated by reference in its entirety, NZ_CP009748 REGION: 386154 . . . 38693 (SEQ ID NO:17) and which is hereby incorporated by reference in its entirety, or NZ_FNCO01000027 REGION: 29475 . . . 30485 (SEQ ID NO:18) and which is hereby incorporated by reference in its entirety.

In an implementation, the recombinantEscherichia colistrain co-expresses genes encoding the 4 enzymes using double plasmids pRSFDuet-1 and pETDuet-1; and the pRSFDuet-1 is loaded with L-amino acid oxidase genes and α-keto acid decarboxylase genes, and the pETDuet-1 is loaded with alcohol dehydrogenase genes and enzyme genes capable of reducing NAD(P).

In an implementation, the recombinantEscherichia colistrain is obtained by transforming 2 co-expressed recombinant plasmids into a hostEscherichia coliB21.

A second object of the present disclosure is to provide a method for producing phenylethanoids, which utilizes the recombinantEscherichia colistrain of the present disclosure.

In an implementation, the phenylethanoids is any of 2-phenylethanol, tyrosol and hydroxytyrosol.

In an implementation, the substrate for producing the phenylethanoids is any one or more of L-phenylalanine, L-tyrosine and L-dopa.

In an implementation, the production of the phenylethanoids is carried out by whole cell transformation.

In an implementation, a whole cell transformation production system comprises a fresh cell wet weight of 10-200 g/L, a substrate concentration of 0.5-20 g/L, a hydrogen donor concentration of 1-20 g/L, and a pH of 4.0-8.0; and the reaction is carried out at 15-40° C. for 0.5-48 hours.

In an implementation, the method for obtaining cells comprises: inoculating the recombinantEscherichia colistrain into an LB fermentation medium, and after the OD600of the cells reaches 0.6-0.8, adding IPTG to induce expression; after the induction of expression is completed, collecting the cells by centrifugation.

In an implementation, when the constructed three enzyme co-expressed plasmid contains glucose dehydrogenase, the hydrogen donor is glucose; when the constructed three enzyme co-expressed plasmid contains formate dehydrogenase, the hydrogen donor is sodium formate; and when the constructed three enzyme co-expressed plasmid contains phosphite dehydrogenase, the hydrogen donor is phosphorous acid.

Beneficial Effects of the Disclosure:

The present disclosure constructs a novel four-enzyme co-expressedEscherichia coliengineered strain, which can be applied to the production of phenylethanoids. The L-amino acid oxidase, α-keto acid decarboxylase and alcohol dehydrogenase selected by the disclosure have the characteristics of poor substrate specificity and high activity, so the same engineered strain can produce a variety of phenylethanoids under the condition of using aromatic-α-amino acids, and can also be used for producing derivatized alcohols of other amino acids. The production process is simple and the raw materials are easily available, so the strain has good industrial application prospects.

