Method for producing bacterial metabolites

Abstract

The invention belongs to the field of microbial fermentation, particularly relates to a method for preparing bacterial metabolites, and further provides a method for extracting zeaxanthin from escherichia coli based on the method. The method has the advantages of simple process steps, high purification yield of the target product, avoiding of the use of high-toxicity solvents, suitableness for subsequent large-scale commercial production, and important value.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation and relates to a method for preparing bacterial metabolites. Background Art

[0002] Zeaxanthin, also known as zeaxanthin, CAS No. 144-68-3, molecular formula C 40 H 56 O2, molecular weight 566.88, is a carotenoid, a dihydroxy derivative of β-carotene. Zeaxanthin has three stereoisomers, namely 3S, 3'S-zeaxanthin, 3S, 3'R-meso-zeaxanthin, and 3R, 3'R-zeaxanthin. The structural formula of 3R, 3'R-zeaxanthin is shown below:

[0003]

[0004] According to the orientation of the linking group on the double bond, zeaxanthin is divided into cis-zeaxanthin and all-trans-zeaxanthin. Zeaxanthin in nature is mainly in the all-trans structure. Due to the presence of unsaturated double bonds in the molecule, it is easy to undergo cis isomerization under conditions such as light, heating and processing. The all-trans conformation of zeaxanthin has the strongest stability and physiological activity. The cis structure has greater changes in physical and chemical properties and physiological functions compared to the all-trans conformation, which limits the application of zeaxanthin in medicine and food.

[0005] Zeaxanthin is present in large quantities in plant tissues such as green vegetables, red peppers, marigolds, corn seeds, wolfberries and Physalis fruits, and in some non-photosynthetic bacteria. Studies have shown that the macular pigment of the human retina is composed of two xanthophylls present in the diet, namely lutein and zeaxanthin. The two pigments can absorb the function of blue light waves that damage the retina, thereby protecting the eyeball. At the same time, zeaxanthin can also prevent free radicals from invading biofilms, quench singlet oxygen and capture reactive oxygen free radicals, and has a specific antioxidant function. In the treatment of other chronic diseases, zeaxanthin has a special effect that is different from other carotenoids, and also has a unique physiological effect in preventing cardiovascular diseases, cataracts, preventing cancer, and enhancing immunity. In addition, zeaxanthin is also widely used in dietary pigments, agriculture, and cosmetics.

[0006] At present, there are four main ways to obtain zeaxanthin in industry: extraction from plants; chemical total synthesis; chemical transformation using other intermediates; and microbial fermentation. Plant extraction mainly obtains zeaxanthin from plants such as capsanthin, marigold, corn, wolfberry, and sour berries. If plant extraction is used, a large amount of organic solvent is often required, and supercritical extraction, ultrasound and other inefficient and expensive methods are used to obtain the final product. The process is complex, energy consumption is high, and the product yield is low. At the same time, the removal effect of other carotenoids is poor. As for the chemical synthesis strategy, as summarized in "Chang, An-Ni. Total synthesis of (3R, 3'R, 6'R)-lutein, (3R, 3'R)-zeaxanthin and their stereoisomers. University of Maryland, College Park, 2008", total synthesis often has complex process steps, serious process pollution, and high synthesis cost. At present, this method is still a long way from commercialization. The use of canthaxanthin, lutein, or astaxanthin can largely solve the problems caused by total synthesis, but the use of toxic reagents and harsh reaction parameters in the process have brought severe challenges to the industrial scale-up of zeaxanthin. Microbial synthesis has opened up a new way of thinking for the commercial production of zeaxanthin. Patents such as CN101203616A, CN103038358B, and CN105886518A have also conducted a lot of research on different genera of bacteria, and have made many attempts in strain construction, fermentation optimization, and downstream extraction. However, the low fermentation yield, low extraction yield, and complex process have seriously limited its industrial prospects. Summary of the invention

[0007] The inventors of the present application have developed a method for preparing bacterial metabolites through a large number of experiments, and based on this method, provided a method for extracting zeaxanthin from Escherichia coli, thereby providing the present invention.

[0008] In one aspect, the present invention provides a method for preparing a bacterial metabolite, comprising the following steps:

[0009] (1) collecting the bacteria;

[0010] (2) Add the following reagents to the bacteria:

[0011] Reagent I, wherein the reagent I is one or more selected from alcohols and esters, and the amount of the reagent I relative to the bacteria is 0.2 to 2.0 parts by weight;

[0012] Reagent II, wherein the reagent II is water, and the amount of the reagent II relative to the bacteria is 0.05 to 5.0 parts by weight; and

[0013] Reagent III, wherein the reagent III is one or more selected from inorganic acids and organic acids, and the amount of the reagent III relative to the bacteria is 0.01 to 0.02 parts by weight;

[0014] (3) separating the product of step (2) to obtain a solid phase product;

[0015] Wherein, the metabolites are one or more selected from zeaxanthin, β-carotene and lycopene.

[0016] As used herein, the expression "the amount of the reagent I relative to the bacteria is 0.2 to 2.0 parts by weight" means that the ratio of the weight of the reagent I to the weight of the bacteria is 0.2 to 2.0.

[0017] Unless otherwise specified or clearly contradicted by context, the meanings of the remaining similar expressions herein shall be defined in a similar manner to the above.

[0018] In certain embodiments, the alcohol is an alcohol containing 1, 2, 3, 4, or 5 carbon atoms.

[0019] In certain embodiments, the esters are esters comprising 1, 2, 3, 4, or 5 carbon atoms.

[0020] In certain embodiments, the alcohol is one or more selected from methanol, ethanol, ethylene glycol, propanol, isopropanol, butanol, chlorobutanol, benzyl alcohol, phenylethyl alcohol and phenoxyethanol.

[0021] In certain embodiments, the ester is ethyl acetate or acrylate.

[0022] In certain embodiments, the inorganic acid is one or more selected from hydrochloric acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, boric acid, silicic acid, hypochlorous acid and hydrofluoric acid.

[0023] In certain embodiments, the organic acid is one or more selected from formic acid, acetic acid, propionic acid, butyric acid, caprylic acid, adipic acid, oxalic acid, malonic acid, succinic acid, maleic acid, tartaric acid, benzoic acid, phenylacetic acid, phthalic acid, terephthalic acid, fumaric acid, valeric acid, caproic acid, capric acid, stearic acid, palmitic acid, acrylic acid, lactic acid, sorbic acid, citric acid and oxalic acid.

[0024] In certain embodiments, step (2) comprises the following steps:

[0025] Add the following reagents or their mixture to the bacteria:

[0026] Reagent I, wherein the reagent I is ethanol, and the amount of the ethanol relative to the bacteria is 0.2 to 2.0 parts by weight;

[0027] Reagent II, wherein the reagent II is water, and the amount of the water relative to the bacteria is 0.05 to 5.0 parts by weight; and

[0028] Reagent III, the reagent III is hydrochloric acid, and the amount of the hydrochloric acid used is 0.01 to 0.02 parts by weight relative to the bacteria.

[0029] In certain embodiments, the ethanol is used in an amount of 0.2-1.5 parts by weight relative to the bacteria; the water is used in an amount of 0.05-4.0 parts by weight relative to the bacteria; and the hydrochloric acid is used in an amount of 0.01-0.02 parts by weight relative to the bacteria.

[0030] In certain embodiments, the ethanol is used in an amount of 0.4-1.0 parts by weight relative to the bacteria; the water is used in an amount of 0.1-3.0 parts by weight relative to the bacteria; and the hydrochloric acid is used in an amount of 0.01-0.02 parts by weight relative to the bacteria.

[0031] In certain embodiments, the ethanol is used in an amount of 0.4-0.8 parts by weight relative to the bacteria; the water is used in an amount of 0.1-2.0 parts by weight relative to the bacteria; and the hydrochloric acid is used in an amount of 0.01-0.02 parts by weight relative to the bacteria.

[0032] In certain embodiments, the bacteria are one or more selected from Escherichia coli, lactic acid bacteria, streptococci, acetic acid bacteria, propionibacterium and bifidobacterium.

[0033] In certain embodiments, step (1) comprises the following steps:

[0034] The fermentation liquid of the bacteria is collected, and the fermentation liquid is centrifuged to collect the bacteria. The centrifugation conditions are: rotation speed 6000-12000 rcf, temperature 0-25° C., and time 10-40 min.

[0035] In certain embodiments, the centrifugation conditions are: rotation speed 8000-10000 rcf, temperature 20-25° C., time 25-40 min.

[0036] In certain embodiments, step (2) further comprises the following steps:

[0037] After adding the reagent to the bacteria, the mixture is stirred at a temperature of -20 to 0° C. and a rotation speed of 500 to 10,000 rpm for 8 to 16 hours. In some embodiments, the temperature is -10 to -5° C.

[0038] In certain embodiments, in step (3), a solid phase product is obtained by one or more methods selected from the following:

[0039] Precipitation, extraction, chromatography, distillation, evaporation, filtration, centrifugation, crystallization, drying and electrophoresis.

[0040] In certain embodiments, step (3) comprises the following steps:

[0041] The product obtained in step (2) is sequentially subjected to extraction, evaporation, filtration and drying to extract the target metabolite.

[0042] In certain embodiments, the extracting comprises the steps of:

[0043] At a temperature of 0 to 25° C., use 2 to 6 parts by weight of an extraction reagent relative to the product obtained in step (2) to perform 1 to 3 extractions for 6 to 8 hours, wherein the extraction reagent comprises one or more of alcohols and esters.

[0044] In certain embodiments, the alcohol is an alcohol containing 1, 2, 3, 4, or 5 carbon atoms.

[0045] In certain embodiments, the esters are esters comprising 1, 2, 3, 4, or 5 carbon atoms.

[0046] In certain embodiments, the alcohol is selected from methanol, ethanol, ethylene glycol, propanol, isopropanol, butanol, chlorobutanol, benzyl alcohol, phenylethyl alcohol, and phenoxyethanol.

[0047] In certain embodiments, the ester is selected from the group consisting of ethyl acetate, butyl acetate, butyl acetate, pentyl acetate, ethyl hexanoate, and ethyl butyrate.

[0048] In certain embodiments, the extracting comprises the steps of:

[0049] At a temperature of 0 to 10° C., 2 to 3 extractions are performed using ethyl acetate in an amount of 2 to 4 parts by weight relative to the product obtained in step (2) for a period of 6 to 8 hours.

[0050] In certain embodiments, the evaporating comprises the steps of:

[0051] At a temperature of 0-25°C and a vacuum degree of 10-1000 mbar, the rotary evaporation is carried out for 1-6 hours.

[0052] In certain embodiments, the temperature is 0-10° C., and the vacuum degree is 20-60 mbar.

[0053] In certain embodiments, step (3) further comprises the following steps:

[0054] The solid phase obtained by filtration is frozen at a temperature of -80 to -10°C for 1 to 8 hours.

[0055] In certain embodiments, it comprises the steps of:

[0056] (1) fermenting the bacteria, collecting the fermentation broth, and centrifuging the fermentation broth to collect the bacteria, wherein the centrifugation conditions are: a speed of 8000-10000 rcf, a temperature of 20-25° C., and a time of 25-40 min;

[0057] 2a) adding the following reagents or a mixture thereof to the bacteria:

[0058] Reagent I, wherein the reagent I is ethanol, and the amount of the ethanol relative to the bacteria is 0.4 to 0.8 parts by weight,

[0059] Reagent II, wherein the reagent II is water, and the amount of the water relative to the bacteria is 0.1 to 2.0 parts by weight, and

[0060] Reagent III, wherein the reagent III is hydrochloric acid, and the amount of the hydrochloric acid relative to the bacteria is 0.01 to 0.02 parts by weight;

[0061] 2b) stirring at a temperature of -10 to -5°C and a speed of 500 to 10,000 rpm for 8 to 16 hours;

[0062] 2c) centrifuging the bacterial solution obtained in 2b) to collect the solid phase of the solution, the centrifugation conditions being: rotation speed 8000-10000 rcf, temperature 20-25° C., time 25-40 min;

[0063] 3a) extracting the solid phase of the solution obtained in 2c) by using 2 to 4 parts by weight of ethyl acetate relative to the solid phase of the solution at a temperature of 0 to 10° C. for 2 to 3 times for 6 to 8 hours to obtain an extract phase;

[0064] 3b) evaporating the extract phase obtained in 3a) at a temperature of 0-25° C. and a vacuum degree of 20-200 mbar for 1-6 hours to obtain a concentrated slurry of the metabolite;

[0065] 3c) filtering the concentrated slurry obtained in 3b), freezing the solid phase obtained by filtration at a temperature of -80 to -10°C for 1 to 8 hours, and then freeze-drying the solid phase to obtain the metabolite.

[0066] In certain embodiments, the bacteria are one or more selected from Escherichia coli, lactic acid bacteria, streptococci, acetic acid bacteria, propionibacterium and bifidobacterium.

[0067] In certain embodiments, the bacterium is Escherichia coli, and the bacterium comprises a combination of exogenously introduced expression cassettes, the combination of expression cassettes comprising a first expression cassette, a second expression cassette, a third expression cassette, a fourth expression cassette, a fifth expression cassette, and a sixth expression cassette, and,

[0068] (a) The first expression cassette comprises the following elements in order from the 5' end to the 3' end:

[0069] (1) a promoter; (2) a ribosome binding site (RBS); (3) a polynucleotide encoding geranylgeranyl pyrophosphate synthase; (4) an RBS; (5) a polynucleotide encoding isoprene pyrophosphate isomerase; (6) a polynucleotide encoding lycopene cyclase; (7) a polynucleotide encoding phytoene dehydrogenase; (8) a polynucleotide encoding phytoene synthase; and (9) a terminator;

[0070] (b) The second expression cassette comprises the following elements in sequence from the 5' end to the 3' end:

[0071] (1) a promoter; (2) an RBS; (3) a polynucleotide encoding a phytoene synthase; (4) an RBS; (5) a polynucleotide encoding a phytoene dehydrogenase; (6) an RBS; (7) a polynucleotide encoding a lycopene cyclase; (8) an RBS; (9) a polynucleotide encoding a geranylgeranyl pyrophosphate synthase; and (10) a terminator;

[0072] (c) The third expression cassette comprises the following elements in sequence from the 5' end to the 3' end:

[0073] (1) Promoter; (2) RBS; (3) Polynucleotide encoding hydroxymethylglutaryl-CoA synthetase (HMGCS); (4) RBS; (5) Polynucleotide encoding 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR); (6) RBS; (7) Polynucleotide encoding acetyl-CoA acyltransferase (AACT); (8) Terminator;

[0074] (d) The fourth expression cassette comprises the following elements in sequence from the 5' end to the 3' end:

[0075] (1) a promoter; (2) an RBS; (3) a polynucleotide encoding a gene related to mevalonate synthesis selected from hydroxymevalonate kinase (MVK); (4) an RBS; (5) a polynucleotide encoding phosphomevalonate kinase (PMVK); (6) an RBS; (7) a polynucleotide encoding diphosphomevalonate decarboxylase (MVD); (8) a terminator;

[0076] (e) The fifth expression cassette comprises the following elements in sequence from the 5' end to the 3' end:

[0077] (1) Promoter; (2) RBS; (3) Polynucleotide encoding geranylgeranyl pyrophosphate synthase; (4) Terminator;

[0078] (f) The sixth expression cassette comprises the following elements in sequence from the 5' end to the 3' end:

[0079] (1) Promoter; (2) RBS; (3) Polynucleotide encoding β-carotene hydroxylase; (4) Terminator.

[0080] In certain embodiments, the phytoene synthase, phytoene dehydrogenase, lycopene cyclase, geranylgeranyl pyrophosphate synthase, isoprenyl pyrophosphate isomerase, HMGCS, HMGR, AACT, MVK, PMVK, MVD, β-carotene hydroxylase are independently derived from Enterobacter agglomerans, Pseudescherichia vulneris, Escherichia coli, Lactobacillus paracasei, Saccharomyces cerevisiae, Streptococcus pneumoniae, Pantoea ananas, or Enterobacteriaceae bacterium.

[0081] In certain embodiments, the sequence of the promoter is shown in any one sequence selected from SEQ ID NO:11, 12, 14, 18, and 27, and the sequence of the terminator is shown in any one sequence selected from SEQ ID NO:8, 15, 24, and 43.

[0082] In certain embodiments, the sequence of the polynucleotide encoding phytoene synthase is shown in SEQ ID NO:7, the sequence of the polynucleotide encoding phytoene dehydrogenase is shown in SEQ ID NO:6, the sequence of the polynucleotide encoding lycopene cyclase is shown in any one of SEQ ID NOs:5 and 22, the sequence of the polynucleotide encoding geranylgeranyl pyrophosphate synthase is shown in SEQ ID NO:2, the sequence of the polynucleotide encoding isoprene pyrophosphate isomerase is shown in SEQ ID NO:4 or 39, the sequence of the polynucleotide encoding HMGCS is shown in SEQ ID NO:29, the sequence of the polynucleotide encoding HMGR is shown in SEQ ID NO:31, the sequence of the polynucleotide encoding AACT is shown in SEQ ID NO:33, the sequence of the polynucleotide encoding MVK is shown in SEQ ID NO:36, the sequence of the polynucleotide encoding PMVK is shown in SEQ ID NO:37, the sequence of the polynucleotide encoding MVD is shown in SEQ ID NO:38, the sequence of the polynucleotide encoding β-carotene hydroxylase is shown in SEQ ID NO: Any sequence among NO:42, 48, 49, 53, 57, 60.

[0083] In certain embodiments, the combination of expression cassettes comprises two or more second expression cassettes.

[0084] In certain embodiments, the combination of expression cassettes:

[0085] (a) The first expression cassette comprises the following elements in order from the 5' end to the 3' end:

[0086] (1) a promoter; (2) a ribosome binding site (RBS); (3) a polynucleotide encoding geranylgeranyl pyrophosphate synthase; (4) an RBS; (5) a polynucleotide encoding isoprene pyrophosphate isomerase; (6) a polynucleotide encoding lycopene cyclase; (7) a polynucleotide encoding phytoene dehydrogenase; (8) a polynucleotide encoding phytoene synthase; and (9) a terminator;

[0087] wherein the elements are directly connected, the sequence of the promoter is shown in SEQ ID NO:11, the sequence of the terminator is shown in SEQ ID NO:8, the sequence of the polynucleotide encoding phytoene synthase is shown in SEQ ID NO:7, the sequence of the polynucleotide encoding phytoene dehydrogenase is shown in SEQ ID NO:6, the sequence of the polynucleotide encoding lycopene cyclase is shown in SEQ ID NO:5, the sequence of the polynucleotide encoding geranylgeranyl pyrophosphate synthase is shown in SEQ ID NO:2, the sequence of the polynucleotide encoding isoprene pyrophosphate isomerase is shown in SEQ ID NO:4, and the sequences of the RBS in (2) and (4) are shown in SEQ ID NOs:10 and 3, respectively;

[0088] (b) The second expression cassette comprises the following elements in sequence from the 5' end to the 3' end:

[0089] (1) a promoter; (2) an RBS; (3) a polynucleotide encoding a phytoene synthase; (4) an RBS; (5) a polynucleotide encoding a phytoene dehydrogenase; (6) an RBS; (7) a polynucleotide encoding a lycopene cyclase; (8) an RBS; (9) a polynucleotide encoding a geranylgeranyl pyrophosphate synthase; and (10) a terminator;

[0090] wherein the elements are directly connected, the sequence of the promoter is shown in SEQ ID NO:18, the sequence of the terminator is shown in SEQ ID NO:24, the sequence of the polynucleotide encoding phytoene synthase is shown in SEQ ID NO:7, the sequence of the polynucleotide encoding phytoene dehydrogenase is shown in SEQ ID NO:6, the sequence of the polynucleotide encoding lycopene cyclase is shown in SEQ ID NO:22, the sequence of the polynucleotide encoding geranylgeranyl pyrophosphate synthase is shown in SEQ ID NO:2, and the sequences of the RBS in (2), (4), (6) and (8) are shown in SEQ ID NOs:19, 20, 21 and 23 respectively;

[0091] (c) The third expression cassette comprises the following elements in sequence from the 5' end to the 3' end:

[0092] (1) Promoter; (2) RBS; (3) Polynucleotide encoding hydroxymethylglutaryl-CoA synthetase (HMGCS); (4) RBS; (5) Polynucleotide encoding 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR); (6) RBS; (7) Polynucleotide encoding acetyl-CoA acyltransferase (AACT); (8) Terminator;

[0093] wherein the elements are directly connected, the sequence of the promoter is shown in SEQ ID NO:27, the sequence of the terminator is shown in SEQ ID NO:24, the sequence of the polynucleotide encoding HMGCS is shown in SEQ ID NO:29, the sequence of the polynucleotide encoding HMGR is shown in SEQ ID NO:31, the sequence of the polynucleotide encoding AACT is shown in SEQ ID NO:33, and the sequences of the RBS in (2), (4) and (6) are shown in SEQ ID NOs:28, 30 and 32 respectively;

[0094] (d) The fourth expression cassette comprises the following elements in sequence from the 5' end to the 3' end:

[0095] (1) a promoter; (2) an RBS; (3) a polynucleotide encoding a gene related to mevalonate synthesis selected from hydroxymevalonate kinase (MVK); (4) an RBS; (5) a polynucleotide encoding phosphomevalonate kinase (PMVK); (6) an RBS; (7) a polynucleotide encoding diphosphomevalonate decarboxylase (MVD); (8) a terminator;

[0096] wherein the elements are directly connected, the sequence of the promoter is shown in SEQ ID NO:27, the sequence of the terminator is shown in SEQ ID NO:24, the sequence of the polynucleotide encoding MVK is shown in SEQ ID NO:36, the sequence of the polynucleotide encoding PMVK is shown in SEQ ID NO:37, the sequence of the polynucleotide encoding MVD is shown in SEQ ID NO:38, and the sequences of the RBS in (2), (4) and (6) are shown in SEQ ID NOs:28, 32 and 20, respectively;

[0097] (e) The fifth expression cassette comprises the following elements in sequence from the 5' end to the 3' end:

[0098] (1) Promoter; (2) RBS; (3) Polynucleotide encoding geranylgeranyl pyrophosphate synthase; (4) Terminator;

[0099] wherein the elements are directly connected, the sequence of the promoter is shown in SEQ ID NO: 14, the sequence of the terminator is shown in SEQ ID NO: 15, the sequence of the polynucleotide encoding geranylgeranyl pyrophosphate synthase is shown in SEQ ID NO: 2, and the sequence of the RBS is shown in SEQ ID NO: 10; and / or,

[0100] (f) The sixth expression cassette comprises the following elements in sequence from the 5' end to the 3' end:

[0101] (1) Promoter; (2) RBS; (3) Polynucleotide encoding β-carotene hydroxylase; (4) Terminator;

[0102] Wherein, the elements are directly connected, the sequence of the promoter is shown in SEQ ID NO: 12, the sequence of the terminator is shown in SEQ ID NO: 43, the sequence of the polynucleotide encoding β-carotene hydroxylase is shown in SEQ ID NO: 48, and the sequence of the RBS is shown in SEQ ID NO: 46.

[0103] In certain embodiments, the combination of expression cassettes further comprises a seventh expression cassette, wherein the seventh expression cassette comprises the following elements in sequence from the 5' end to the 3' end:

[0104] (1) Promoter; (2) RBS; (3) Polynucleotide encoding β-carotene hydroxylase; (4) RBS; (5) Polynucleotide encoding lycopene cyclase; (6) Terminator.

[0105] In certain embodiments, the sequence of the promoter is shown in any one of SEQ ID NOs: 11, 12, 14, 18, and 27, the sequence of the terminator is shown in any one of SEQ ID NOs: 8, 15, 24, and 43, and the sequence of the polynucleotide encoding β-carotene hydroxylase is shown in any one of SEQ ID NOs: 42, 48, 49, 53, 57, and 60.

[0106] In certain embodiments, the seventh expression cassette comprises the following elements in order from the 5' end to the 3' end:

[0107] (1) Promoter; (2) RBS; (3) Polynucleotide encoding β-carotene hydroxylase; (4) RBS; (5) Polynucleotide encoding lycopene cyclase; (6) Terminator;

[0108] Wherein, the elements are directly connected, the sequence of the promoter is shown in SEQ ID NO: 18, the sequence of the terminator is shown in SEQ ID NO: 43, the sequence of the polynucleotide encoding β-carotene hydroxylase is shown in SEQ ID NO: 48, the sequence of the polynucleotide encoding lycopene cyclase is shown in SEQ ID NO: 22, and the sequences of the RBS in (2) and (4) are shown in SEQ ID NO: 50 and 51, respectively. In certain embodiments, the combination of expression cassettes further comprises an eighth expression cassette, and the eighth expression cassette comprises the following elements from the 5' end to the 3' end:

[0109] (1) a promoter; (2) an RBS; (3) at least two polynucleotides encoding β-carotene hydroxylase; (4) a terminator;

[0110] Wherein, the at least two polynucleotides encoding β-carotene hydroxylase are connected via RBS.

[0111] In certain embodiments, the sequence of the promoter is shown in any one of SEQ ID NOs: 11, 12, 14, 18, and 27, the sequence of the terminator is shown in any one of SEQ ID NOs: 8, 15, 24, and 43, and the sequence of the polynucleotide encoding β-carotene hydroxylase is shown in any one of SEQ ID NOs: 42, 48, 49, 53, 57, and 60.

[0112] In certain embodiments, the eighth expression cassette comprises the following elements in order from the 5' end to the 3' end:

[0113] (1) a promoter; (2) an RBS; (3) a polynucleotide encoding a β-carotene hydroxylase; (4) an RBS; (5) a polynucleotide encoding a β-carotene hydroxylase; (6) an RBS; (7) a polynucleotide encoding a β-carotene hydroxylase; (8) an RBS; (9) a polynucleotide encoding a β-carotene hydroxylase; and (10) a terminator;

[0114] Wherein, the elements are directly connected, the sequence of the promoter is shown in SEQ ID NO: 12, the sequence of the terminator is shown in SEQ ID NO: 24, the sequences of the polynucleotides encoding β-carotene hydroxylase in (3), (5), (7) and (9) are shown in SEQ ID NOs: 57, 53, 42 and 60, respectively, and the sequences of the RBS in (2), (4), (6) and (8) are shown in SEQ ID NOs: 56, 58, 59 and 58, respectively. In certain embodiments, each expression cassette contained in the combination of expression cassettes is independently integrated into the genome of the bacterium.

[0115] In certain embodiments, each of the expression cassettes is independently integrated into the genome of the bacterium at a different site.

[0116] Definition of terms

[0117] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Meanwhile, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0118] When the terms "for example," "such as," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0119] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0120] As used herein, the term "gene" refers to a polymeric form of nucleotides of any length, which are ribonucleotides or deoxyribonucleotides. The term includes double-stranded and single-stranded DNA and RNA. Also included are known types of modifications, such as methylation, "capping", and substitution of one or more naturally occurring nucleotides with analogs. Preferably, a gene comprises a coding sequence encoding a polypeptide. A "coding sequence" is a nucleotide sequence that is transcribed into mRNA and / or translated into a polypeptide when it is placed or under the control of an appropriate regulatory sequence. The boundaries of the coding sequence are determined by the translation start codon at the 5'-end and the translation stop codon at the 3'-end. The coding sequence may include, but is not limited to, mRNA, cDNA, recombinant nucleic acid sequence or genomic DNA, and in some cases introns may also be present.

[0121] As used herein, the term "transformation" is the process of introducing a heterologous nucleic acid (e.g., a nucleic acid construct, a vector, an expression cassette, etc.) into a host cell or organism. Specifically, "transformation" refers to a process in which a DNA molecule is transiently transformed into an organism, or stably transformed or integrated into the genome of an organism or capable of autonomous replication.

[0122] As used herein, the term "converted / recombinant" refers to a host organism, such as a bacterium, fungus or plant, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the host's genome or the nucleic acid molecule can exist as an extrachromosomal molecule. Such extrachromosomal molecules can replicate automatically. The transformed cell, tissue or plant is understood to include not only the final product of the transformation process, but also its genetically modified progeny. A "non-transformed" or "non-recombinant" host refers to a wild-type organism or a naturally occurring organism, such as a bacterium, fungus or plant.

[0123] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0124] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells, etc. Host cells may include a single cell or a cell population.

[0125] In the present invention, unless otherwise specified, the “first” (e.g., the first expression cassette), “second” (e.g., the second expression cassette), “third” (e.g., the third expression cassette), “fourth” (e.g., the fourth expression cassette), “fifth” (e.g., the fifth expression cassette), “sixth” (e.g., the sixth expression cassette), “seventh” (e.g., the seventh expression cassette), and “eighth” (e.g., the eighth expression cassette) are mainly for reference distinction and do not have a typical order meaning.

[0126] Advantageous Effects of the Invention

[0127] The present invention provides a method for preparing bacterial metabolites, and based on the method, further provides a method for extracting zeaxanthin from Escherichia coli. The method has simple process steps, high purification yield of the target product, and avoids the use of highly toxic solvents, is suitable for subsequent large-scale commercial production, and has important value. BRIEF DESCRIPTION OF THE DRAWINGS

[0128] Figure 1: Liquid chromatogram of fermentation broth. This chromatogram is derived from the detection of the sample at the end of fermentation. The component at the retention time of 1.320 min is zeaxanthin, the component at the retention time of 3.252 min is β-carotene component, the component at the retention time of 3.876 min is lycopene component, and the components at other retention times are other fermentation by-products.

[0129] Figure 2 :Liquid chromatogram of the cell lysate. The chromatogram is derived from the detection of the cell lysate sample. The retention time of 1.319 min is zeaxanthin, the retention time of 3.253 min is the β-carotene component, the retention time of 3.875 min is the lycopene component, and the components at other retention times are other fermentation by-products.

[0130] Figure 3 : Liquid chromatogram of the extraction phase. The chromatogram is derived from the detection of the extraction liquid sample. The retention time of 1.319min is zeaxanthin, the retention time of 3.253min is the β-carotene component, the retention time of 3.875min is the lycopene component, and the other retention time components are other fermentation by-products.

[0131] Figure 4 : Liquid chromatogram of the product. This chromatogram is derived from the detection of the dried sample. The retention time of 1.245 min is zeaxanthin, the retention time of 3.233 min is β-carotene component, the retention time of 3.718 min is lycopene component, and the other retention time components are other fermentation by-products. DETAILED DESCRIPTION

[0132] The invention is now described in the following non-limiting examples.

[0133] Those skilled in the art will appreciate that the embodiments describe the present invention by way of example and are not intended to limit the scope of protection claimed in the present application. The experimental methods in the embodiments are conventional methods unless otherwise specified. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0134] Preparation Example 1: Preparation of modified Escherichia coli for producing zeaxanthin

[0135] (1) Transformed strain BOTA01009 producing β-carotene

[0136] The pEcCas9 plasmid was transferred into C43 (DE3) (purchased from Weidi Biotechnology, CAT#: EC1040) by conventional E. coli chemical transformation methods. The strain contained Kan resistance and grew in LB+Kan resistance medium. The resulting strain was numbered BOTA01003. The expression cassettes 1-5 for β-carotene synthesis were amplified using pAC-BETAipi (purchased from Miaoling, CAT# P9847) as a template. The element composition and specific sequence of the expression cassettes 1-5 are shown in Tables 1-5 below. The expression cassettes 1-5 were transferred into BOTA01003, and the fragments from the promoter to the terminator in the expression cassettes 1-5 were inserted into the adhE, gdhA, IS6, IS10, and ZapB sites of the BOTA01003 cell genome by CRIPSP / Cas9, respectively, to obtain a modified strain for producing β-carotene, which was numbered BOTA01009.

[0137] Table 1: Element composition and specific sequence of expression cassette 1

[0138] name Sequence source SEQ ID NO: Element 1 adhE upstream homology arm Escherichia coli 1 Element 2 PRO330 promoter synthetic sequence 11 Element 3 RBS synthetic sequence 10 Element 4 crtE_PSEVU Pseudescherichia vulneris 2 Element 5 RBS Enterobacter agglomerans 3 Element 6 idi_PSEVU Pseudescherichia vulneris 4 Element 7 crtY_PSEVU Pseudescherichia vulneris 5 Element 8 crtI_PSEVU Pseudescherichia vulneris 6 Element 9 crtB_PSEVU Pseudescherichia vulneris 7 Element 10 Terminator Enterobacter agglomerans 8 Element 11 adhE downstream homology arm Escherichia coli 9

[0139] Table 2: Element composition and specific sequence of expression cassette 2

[0140]

[0141]

[0142] Table 3: Element composition and specific sequence of expression cassette 3

[0143] name Sequence source SEQ ID NO: Element 1 IS6 upstream homology arm Escherichia coli 17 Element 2 pTrc promoter synthetic sequence 18 Element 3 RBS synthetic sequence 19 Element 4 crtB_PSEVU Pseudescherichia vulneris 7 Element 5 RBS(DNA1764) synthetic sequence 20 Element 6 crtI_PSEVU Pseudescherichia vulneris 6 Element 7 RBS synthetic sequence 21 Element 8 crtY_PSEVU Pseudescherichia vulneris 22 Element 9 RBS synthetic sequence 23 Element 10 crtE_PSEVU Pseudescherichia vulneris 2 Element 11 BBa_B1008 terminator synthetic sequence 24 Element 12 IS6 downstream homology arm Escherichia coli 25

[0144] Table 4: Element composition and specific sequence of expression cassette 4

[0145]

[0146]

[0147] Table 5: Element composition and specific sequence of expression cassette 5

[0148] name Sequence source SEQ ID NO: Element 1 ZapB upstream homology arm Escherichia coli 35 Element 2 M1-93 promoter synthetic sequence 27 Element 3 RBS synthetic sequence 28 Element 4 ERG12_YEAST Saccharomyces cerevisiae 36 Element 5 RBS synthetic sequence 32 Element 6 mvaK2_STRR6 Streptococcus pneumoniae 37 Element 7 RBS synthetic sequence 20 Element 8 mvaD_STRP2 Streptococcus pneumoniae 38 Element 9 RBS synthetic sequence 30 Element 10 idi_YEAST Saccharomyces cerevisiae 39 Element 11 BBa_B1008 terminator synthetic sequence 24 Element 12 ZapB downstream homology arm Escherichia coli 40

[0149] (2) Transformed strain BOTA01010 producing zeaxanthin

[0150] The expression cassette 6 for zeaxanthin synthesis was amplified using pAC-BETAipi as a template, and its element composition and specific sequence are shown in Table 6. The expression cassette 6 was transferred into BOTA01009, and the fragment from the promoter to the terminator in the expression cassette 6 was inserted into the flgA site of the BOTA01009 cell genome by CRIPSP / Cas9 to obtain a modified strain producing zeaxanthin, which was numbered BOTA01010.

[0151] Table 6: Element composition and specific sequence of expression cassette 6

[0152]

[0153]

[0154] (3) Transformed strains BOTA01016 and BOTA01017 producing zeaxanthin

[0155] The expression cassette 7-9 for zeaxanthin synthesis was amplified using pAC-BETAipi as a template, and its element composition and specific sequence are shown in Tables 7-9 below. The expression cassette 7 was transferred into BOTA01009, and the fragment from the promoter to the terminator in the expression cassette 7 was inserted into the SS9 site of the BOTA01009 cell genome by CRIPSP / Cas9 to obtain a modified strain producing zeaxanthin, which was numbered BOTA01013.

[0156] Table 7: Element composition and specific sequence of expression cassette 7

[0157] name Sequence source SEQ ID NO: Element 1 SS9 upstream homology arm Escherichia coli 41 Element 2 pTrc promoter synthetic sequence 18 Element 3 RBS synthetic sequence 50 Element 4 crtZ_PANAN-2 Pantoea ananas 48 Element 5 RBS synthetic sequence 51 Element 6 crtY_PSEVU Pseudescherichia vulneris 22 Element 7 BBa_B1002 terminator synthetic sequence 43 Element 8 SS9 downstream homology arm Escherichia coli 44

[0158] The expression cassette 8 was transferred into BOTA01013, and the fragment from the promoter to the terminator in the expression cassette 8 was inserted into the IdhA site of the BOTA01013 cell genome by CRIPSP / Cas9 to obtain a modified strain producing zeaxanthin, which was numbered BOTA01016.

[0159] Table 8: Element composition and specific sequence of expression cassette 8

[0160]

[0161]

[0162] The expression cassette 9 was transferred into BOTA01013, and the fragment from the promoter to the terminator in the expression cassette 9 was inserted into the IGRD33 site of the BOTA01013 cell genome by CRIPSP / Cas9 to obtain a modified strain producing zeaxanthin, which was numbered BOTA01017.

[0163] Table 9: Element composition and specific sequence of expression cassette 9

[0164]

[0165] (4) Transformed strain BOTA01019 producing zeaxanthin

[0166] The expression cassette 10 for zeaxanthin synthesis was amplified using pAC-BETAipi as a template, and the element composition and specific sequence of the promoter to terminator part were the same as those of the expression cassette 3. The two ends of the expression cassette 10 were connected to the upstream and downstream homology arm sequences of the insertion site (different from the aforementioned insertion site, i.e., other sites except adhE, gdhA, IS6, IS10, ZapB, flgA, SS9, IdhA, and IGRD33 sites), and transferred into BOTA01010, and the fragment from the promoter to the terminator in the expression cassette 10 was inserted into the BOTA01010 cell genome by CRIPSP / Cas9, to obtain a modified strain producing zeaxanthin, which was numbered BOTA01019.

[0167] The modified E. coli bacteria BOTA01010, BOTA01016, BOTA01017, and BOTA01019 obtained in Preparation Example 1 were used in subsequent E. coli fermentation broth experiments and zeaxanthin extraction experiments.

[0168] Preparation Example 2: Preparation of Escherichia coli fermentation broth

[0169] (1) Primary fermentation

[0170] Experimental reagents and consumables: 250 ml shake flask, TB culture medium.

[0171] Experimental steps: The volume of the culture medium was 20%, and the modified E. coli strains BOTA01010, BOTA01016, BOTA01017, and BOTA01019 obtained in Preparation Example 1 were inoculated respectively. The inoculation amount of the strain was 0.6% v / v, the shaking flask speed was 200 rpm, and the culture was carried out at 30° C. for 8-10 hours. The harvested primary fermentation liquid was used for subsequent fermentation.

[0172] (2) Secondary fermentation

[0173] Experimental reagents and consumables: 3L fermenter, initial fermentation medium (formula shown in Table 10), feed (formula shown in Table 13).

[0174] Table 10: Fermentation initial medium formulation

[0175] Element concentration glycerin 30g / L Disodium hydrogen phosphate 25.6g / L Potassium dihydrogen phosphate 8g / L Sodium chloride 1.0g / L Ammonium chloride 2.2g / L Magnesium sulfate heptahydrate 0.49g / L TMS solution (formula as shown in Table 11) 10mL / L Vitamin solution (formula as shown in Table 12) 10mL / L

[0176] Table 11: TMS solution formula

[0177] Element concentration Ferrous Sulfate Heptahydrate 10g / L Zinc sulfate heptahydrate 5.25g / L Copper sulfate pentahydrate 3.0g / L Manganese sulfate 0.5g / L Boric acid 0.2g / L Calcium chloride 2.0g / L Sodium molybdate dihydrate 0.2g / L

[0178] Table 12: Vitamin solution formula

[0179] Element concentration Biotin 0.1g / L Choline chloride 0.1g / L Folic acid 0.1g / L Inositol 0.2g / L Niacinamide 0.1g / L Calcium Pantothenate 0.1g / L Pyridoxal hydrochloride 0.1g / L Thiamine Hydrochloride 0.1g / L Riboflavin 0.01g / L Sodium chloride 8.5g / L

[0180] Table 13: Feed formula

[0181]

[0182]

[0183] Experimental steps: The inoculation amount of the primary fermentation broth was 10% v / v, the stirring speed was 300-1200rpm, the ventilation ratio was 1.0VVM, and the dissolved oxygen control was set to 30%. The pH of the fermentation process was controlled at 6.8 with 5M ammonia water. After culturing for 28-32 hours at 30°C, the carbon source in the initial culture medium was exhausted and feeding began. During the fed-batch culture, a constant dissolved oxygen feeding control strategy was adopted, and the dissolved oxygen control value was 30%. The fermentation was terminated after 60 hours of fed-batch culture. The obtained fermentation broth was used for subsequent experiments.

[0184] Taking the modified bacteria BOTA01010 as an example, the zeaxanthin extraction experiments in the following Examples 1 to 5 were carried out. Unless otherwise specified, the detection method of the zeaxanthin content in the following examples is described below:

[0185] 1) Chromatographic column: CHIRALPAK AD-H LC column 4.6mm*250 5μm;

[0186] 2) Mobile phase: A1: isopropanol B1: n-hexane;

[0187] 3) Injection volume: 2.5ul;

[0188] 4) Seal wash: 10% isopropyl alcohol;

[0189] 5) Needle wash: methanol: ethyl acetate = 1:1;

[0190] 6) Column temperature: 30°C;

[0191] 7) Flow rate: 0.7 ml / min;

[0192] 8) Gradient elution, the conditions are shown in Table 14;

[0193] Table 14: Gradient elution conditions

[0194] Time / min A / ％ B / ％ Flow rate / (ml / min) 0 5.5 94.5 0.7 10 5.5 94.5 0.7 40 15 85 0.7 41 5.5 94.5 0.7 45 5.5 94.5 0.7

[0195] 9) Running time: 45min;

[0196] 10) Detection wavelength: 452nm.

[0197] Example 1: Zeaxanthin Extraction Example 1

[0198] (1) Preliminary isolation of bacteria

[0199] According to the preparation example, 1812 g of fermentation liquid (containing 1008 mg of zeaxanthin, the spectrum is as shown in Figure 1 As shown), centrifuged at 10000rcf for 30min at room temperature, and 453g of wet cells were harvested;

[0200] (2) Bacterial cell disruption

[0201] To the harvested wet cells, 317 g of ethanol, 91 g of purified water, and 9.0 g of 1 mol / L hydrochloric acid were added, and the mixture was stirred at -10 °C and 3000 rpm for 12 hours to obtain a broken cell solution (containing 907 mg of zeaxanthin, as shown in the spectrum). Figure 2 The bacterial solution was centrifuged at 10000 rcf for 30 min at room temperature to harvest 281 g of wet bacteria;

[0202] (3) Extraction

[0203] Use twice the mass of ethyl acetate (562g), extract at 6℃ for 6 hours twice, and harvest 620g of extract in total (containing 506mg of zeaxanthin, as shown in the spectrum Figure 3 As shown), 215g of raffinate residue;

[0204] (4) Concentration

[0205] The raffinate was concentrated at 0°C and 60 mbar until no liquid was distilled out to obtain 66 g of concentrated slurry; the concentrated slurry was filtered and the filter cake was washed with purified water; the wet filter cake was pre-frozen in a -80°C refrigerator for 2 hours, and then placed in a freeze dryer for 15 hours to obtain 573 mg of the product, with a product content of up to 76% and a downstream purification yield of 43% (containing 435 mg of zeaxanthin, as shown in the spectrum Figure 4 shown).

[0206] Example 2: Zeaxanthin Extraction Example 2

[0207] (1) Preliminary isolation of bacteria

[0208] According to the preparation example, 1588 g of fermentation liquid (containing 476 mg of zeaxanthin) was harvested, and centrifuged at 10000 rcf for 30 min at room temperature to harvest 480 g of wet cells;

[0209] (2) Bacterial cell disruption

[0210] Add 333 g of ethanol, 95 g of purified water, and 9.5 g of 1 mol / L hydrochloric acid to the harvested wet cells, and stir thoroughly at -10°C and 3000 rpm for 12 hours to obtain a broken cell solution (containing 446 mg of zeaxanthin); centrifuge the broken cell solution at 10000 rcf for 30 min at room temperature to harvest 302 g of wet cells;

[0211] (3) Extraction

[0212] Use 4 times the mass of ethyl acetate (1208g), extract twice at 6°C for 6 hours, harvest a total of 1261g (containing 389mg zeaxanthin), and extract residue 242g;

[0213] (4) Concentration

[0214] The raffinate was concentrated at 0°C and 60 mbar until no liquid was distilled out to obtain 45 g of concentrated slurry; the concentrated slurry was filtered and the filter cake was washed with purified water; the wet filter cake was pre-frozen in a -80°C refrigerator for 2 hours, and then placed in a freeze dryer for 15 hours to obtain 828 mg of product (containing 366 mg of zeaxanthin), with a product content of 44% and a downstream purification yield of up to 77%.

[0215] Compared with Example 1, Example 2 adjusts the amount of ethyl acetate used during extraction, increasing the volume from 2 times to 4 times. The broken bacterial liquid before extraction in Example 1 contains 907 mg of zeaxanthin, and the solution after extraction with 2 times the volume of ethyl acetate (before concentration) contains 506 mg of zeaxanthin, and the yield of zeaxanthin after extraction is about 55.79%. The broken bacterial liquid before extraction in Example 2 contains 446 mg of zeaxanthin, and the solution after extraction with 2 times the volume of ethyl acetate (before concentration) contains 389 mg of zeaxanthin, and the yield of zeaxanthin after extraction is about 87.22%. Therefore, the yield of zeaxanthin extracted with 4 times the volume of ethyl acetate is significantly higher than that with 2 times the volume of ethyl acetate, and the subsequent examples all use 4 times the volume of ethyl acetate for extraction.

[0216] Example 3: Zeaxanthin Extraction Example 3

[0217] (1) Preliminary isolation of bacteria

[0218] According to the preparation example, 6444 g of fermentation liquid (containing 6894 mg of zeaxanthin) was harvested, and centrifuged at 10000 rcf for 30 min at room temperature to harvest 2020 g of wet cells;

[0219] (2) Bacterial cell disruption

[0220] To the harvested wet cells, 1414 g of ethanol, 3030 g of purified water, and 40.0 g of 1 mol / L hydrochloric acid were added, and the mixture was stirred at -10°C and 3000 rpm for 12 hours to obtain a broken cell solution (containing 6393 mg of zeaxanthin); the broken cell solution was centrifuged at 10000 rcf for 30 min at room temperature to harvest 1756 g of wet cells;

[0221] (3) Extraction

[0222] Use 4 times the mass of ethyl acetate (7024g), extract twice at 6°C for 6 hours, harvest a total of 7108g (containing 3779mg zeaxanthin), and extract residue 1663g;

[0223] (4) Concentration

[0224] The raffinate was concentrated at 0°C and 60 mbar until no liquid was distilled out to obtain 121 g of concentrated slurry; the concentrated slurry was filtered and the filter cake was washed with purified water; the wet filter cake was pre-frozen in a -80°C refrigerator for 2 hours, and then placed in a freeze dryer for 15 hours to obtain 4597 mg of product (containing 3629 mg of zeaxanthin), with a product content of 79% and a downstream purification yield of up to 53%.

[0225] Compared with Examples 1 and 2, Example 3 adjusts the mass ratio of each reagent during the bacteriostasis and increases the amount of purified water.

[0226] Example 4: Zeaxanthin Extraction Example 4

[0227] (1) Preliminary isolation of bacteria

[0228] According to the preparation example, 1533 g of fermentation liquid (containing 1211 mg of zeaxanthin) was harvested, and centrifuged at 10000 rcf for 30 min at room temperature to harvest 502 g of wet cells;

[0229] (2) Bacterial cell disruption

[0230] Add 351 g of ethanol, 753 g of purified water, and 10.0 g of 1 mol / L hydrochloric acid to the harvested wet cells, and stir thoroughly at 0°C and 3000 rpm for 12 hours to obtain a broken cell solution (containing 1002 mg of zeaxanthin); centrifuge the broken cell solution at 10000 rcf for 30 min at room temperature to harvest 252 g of wet cells;

[0231] (3) Extraction

[0232] Use 4 times the mass of ethyl acetate (1008g), extract twice at 6°C for 6 hours, harvest a total of 1035g (containing 902mg of zeaxanthin), and extract residue 221g;

[0233] (4) Concentration

[0234] The raffinate was concentrated at 0°C and 60 mbar until no liquid was distilled out to obtain 42 g of concentrated slurry; the concentrated slurry was filtered and the filter cake was washed with purified water; the wet filter cake was pre-frozen in a -80°C refrigerator for 2 hours, and then placed in a freeze dryer for 15 hours to obtain 1129 mg of product (containing 756 mg of zeaxanthin), with a product content of 67% and a downstream purification yield of up to 62%.

[0235] Compared with Example 3, Example 4 adjusted the stirring temperature of the bacteria breaking from -10°C to 0°C. The broken bacteria liquid before extraction in Example 3 contained 6894 mg of zeaxanthin, and the broken bacteria liquid obtained after the bacteria breaking at -10°C contained 6393 mg of zeaxanthin, and the zeaxanthin yield after the bacteria breaking was about 92.73%. The broken bacteria liquid before extraction in Example 4 contained 1211 mg of zeaxanthin, and the broken bacteria liquid obtained after the bacteria breaking at 0°C contained 1002 mg of zeaxanthin, and the zeaxanthin yield after the bacteria breaking was about 82.74%. Therefore, the zeaxanthin yield of bacteria breaking at -10°C is higher than that of bacteria breaking at 0°C.

[0236] Example 5: Zeaxanthin Extraction Example 5

[0237] (1) Preliminary isolation of bacteria

[0238] According to the preparation example, 1858 g of fermentation liquid (containing 1792 mg of zeaxanthin) was harvested, and centrifuged at 10000 rcf for 30 min at room temperature to harvest 580 g of wet cells;

[0239] (2) Bacterial cell disruption

[0240] Add 406 g of ethanol, 870 g of purified water, and 11.6 g of 1 mol / L hydrochloric acid to the harvested wet cells, and stir thoroughly at -10°C and 3000 rpm for 12 hours to obtain a broken cell solution (containing 1689 mg of zeaxanthin); centrifuge the broken cell solution at 10000 rcf for 30 min at room temperature to harvest 335 g of wet cells;

[0241] (3) Extraction

[0242] Use 4 times the mass of ethyl acetate (1340g), extract twice at 6°C for 6 hours, harvest a total of 1402g (containing 1532mg of zeaxanthin), and extract residue 258g;

[0243] (4) Concentration

[0244] The raffinate was concentrated at 20° C. and 120 mbar until no liquid was distilled out to obtain 48 g of concentrated slurry; the concentrated slurry was filtered and the filter cake was washed with purified water; the wet filter cake was pre-frozen in a -80° C. refrigerator for 2 hours, and then placed in a freeze dryer for 15 hours to obtain 1823 mg of product (containing 1156 mg of zeaxanthin), with a product content of 63% and a downstream purification yield of up to 64%.

[0245] In Example 5, compared with Examples 3 and 4, the temperature and pressure during concentration are adjusted.

[0246] Example 6: Zeaxanthin Extraction Example 6

[0247] (1) Preliminary isolation of bacteria

[0248] According to the preparation example, 1722 g of fermentation liquid (containing 982 mg of zeaxanthin) was harvested, and centrifuged at 10000 rcf for 30 min at room temperature to harvest 436 g of wet cells;

[0249] (2) Bacterial cell disruption

[0250] To the harvested wet cells, 87 g of purified water and 8.7 g of 1 mol / L hydrochloric acid were added, and the mixture was stirred at -10°C and 3000 rpm for 12 hours to obtain a broken cell solution (containing 933 mg of zeaxanthin); the broken cell solution was centrifuged at 10000 rcf for 30 min at room temperature to harvest 268 g of wet cells;

[0251] (3) Extraction

[0252] Use 4 times the mass of ethyl acetate (1052g), extract twice at 6°C for 6 hours, and harvest a total of 1089g of extract (containing 304mg of zeaxanthin) and 203g of residue;

[0253] (4) Concentration

[0254] The raffinate was concentrated at 0°C and 60 mbar until no liquid was distilled out to obtain 57 g of concentrated slurry; the concentrated slurry was filtered and the filter cake was washed with purified water; the wet filter cake was pre-frozen in a -80°C refrigerator for 2 hours, and then placed in a freeze dryer for 15 hours to obtain 273 mg of the product, with a downstream purification yield of 20% (containing 196 mg of zeaxanthin).

[0255] Compared with Example 2, in Example 6, no ethanol is added to the (2) crushing system.

[0256] Example 7: Zeaxanthin Extraction Example 7

[0257] (1) Preliminary isolation of bacteria

[0258] According to the preparation example, 1822 g of fermentation liquid (containing 998 mg of zeaxanthin) was harvested, and centrifuged at 10000 rcf for 30 min at room temperature to harvest 458 g of wet cells;

[0259] (2) Bacterial cell disruption

[0260] Add 321 g of ethanol and 91 g of purified water to the harvested wet cells, stir thoroughly at -10°C and 3000 rpm for 12 hours to obtain a broken cell solution (containing 949 mg of zeaxanthin); centrifuge the broken cell solution at 10000 rcf for 30 min at room temperature to harvest 278 g of wet cells;

[0261] (3) Extraction

[0262] Use 4 times the mass of ethyl acetate (1112g), extract twice at 6°C for 6 hours, and harvest a total of 1150g of extract (containing 285mg of zeaxanthin) and 224g of residue;

[0263] (4) Concentration

[0264] The raffinate was concentrated at 0°C and 60 mbar until no liquid was distilled out to obtain 66 g of concentrated slurry; the concentrated slurry was filtered and the filter cake was washed with purified water; the wet filter cake was pre-frozen in a -80°C refrigerator for 2 hours, and then placed in a freeze dryer for 15 hours to obtain 389 mg of the product, with a downstream purification yield of 26% (containing 261 mg of zeaxanthin).

[0265] Compared with Example 2, in Example 7, no hydrochloric acid is added to the (2) crushing system.

[0266] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. A method for preparing bacterial metabolites, comprising the following steps: (1) Collecting bacteria; (2) Add the following reagents to the bacteria: Reagent I, wherein the reagent I is one or more selected from alcohols and esters, and the amount of the reagent I relative to the bacteria is 0.2 to 2.0 parts by weight; Reagent II, wherein the reagent II is water, and the amount of the reagent II relative to the bacteria is 0.05 to 5.0 parts by weight; and Reagent III, wherein the reagent III is one or more selected from inorganic acids and organic acids, and the amount of the reagent III relative to the bacteria is 0.01 to 0.02 parts by weight; (3) separating the product of step (2) to obtain a solid phase product; Wherein, the metabolites are one or more selected from zeaxanthin, β-carotene and lycopene; Preferably, the alcohol is one or more selected from methanol, ethanol, ethylene glycol, propanol, isopropanol, butanol, chlorobutanol, benzyl alcohol, phenylethyl alcohol and phenoxyethanol; Preferably, the ester is ethyl acetate or acrylate; Preferably, the inorganic acid is one or more selected from hydrochloric acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, boric acid, silicic acid, hypochlorous acid and hydrofluoric acid; Preferably, the organic acid is one or more selected from formic acid, acetic acid, propionic acid, butyric acid, caprylic acid, adipic acid, oxalic acid, malonic acid, succinic acid, maleic acid, tartaric acid, benzoic acid, phenylacetic acid, phthalic acid, terephthalic acid, fumaric acid, valeric acid, caproic acid, capric acid, stearic acid, palmitic acid, acrylic acid, lactic acid, sorbic acid, citric acid and oxalic acid.

2. The preparation method according to claim 1, wherein Step (2) comprises the following steps: The following reagents were added to the bacteria: Reagent I, wherein the reagent I is ethanol, and the amount of the ethanol relative to the bacteria is 0.2 to 2.0 parts by weight; Reagent II, wherein the reagent II is water, and the amount of the water relative to the bacteria is 0.05 to 5.0 parts by weight; and Reagent III, wherein the reagent III is hydrochloric acid, and the amount of the hydrochloric acid relative to the bacteria is 0.01 to 0.02 parts by weight; Preferably, the ethanol is used in an amount of 0.4-0.8 parts by weight relative to the bacteria; the water is used in an amount of 0.1-2.0 parts by weight relative to the bacteria; and the hydrochloric acid is used in an amount of 0.01-0.02 parts by weight relative to the bacteria.

3. The preparation method according to claim 1 or 2, wherein: The bacteria are one or more selected from Escherichia coli, lactic acid bacteria, streptococci, acetic acid bacteria, propionic acid bacteria and bifidobacteria.

4. The preparation method according to claim 1, wherein Step (1) comprises the following steps: Collecting the fermentation broth of the bacteria, centrifuging the fermentation broth to collect the bacteria, the centrifugation conditions are: speed 6000-12000 rcf, temperature 0-25° C., time 10-40 min; Preferably, the centrifugal conditions are: rotation speed 8000-10000 rcf, temperature 20-25° C., time 25-40 min.

5. The preparation method according to claim 1, wherein Step (2) further comprises the following steps: After adding the reagent to the bacteria, stirring at a temperature of -20 to 0°C and a speed of 500 to 10,000 rpm for 8 to 16 hours; Preferably, the temperature is -10 to -5°C.

6. The preparation method according to claim 1, wherein In step (3), a solid phase product is obtained by one or more methods selected from the following: precipitation, extraction, chromatography, distillation, evaporation, filtration, centrifugation, crystallization, drying, and electrophoresis; Preferably, step (3) comprises the following steps: The product obtained in step (2) is sequentially subjected to extraction, evaporation, filtration and drying to extract the target metabolite.

7. The method of claim 6, wherein: The extraction comprises the following steps: At a temperature of 0 to 25° C., using 2 to 6 parts by weight of an extraction reagent relative to the product obtained in step (2), performing 1 to 3 extractions for 6 to 8 hours, wherein the extraction reagent comprises: one or more of alcohols and esters; Preferably, the extraction comprises the following steps: At a temperature of 0 to 10° C., 2 to 3 extractions are performed using ethyl acetate in an amount of 2 to 4 parts by weight relative to the product obtained in step (2) for a period of 6 to 8 hours.

8. The method of claim 6, wherein: The evaporation comprises the following steps: At a temperature of 0-25°C, a vacuum degree of 10-1000 mbar, and rotary evaporation for 1-6 hours; Preferably, the temperature is 0-10° C., and the vacuum degree is 20-60 mbar.

9. The method of claim 6, wherein: Step (3) further comprises the following steps: The solid phase obtained by filtration is frozen at a temperature of -80 to -10°C for 1 to 8 hours.

10. The method of claim 1, comprising the steps of: (1) fermenting the bacteria, collecting the fermentation broth, and centrifuging the fermentation broth to collect the bacteria, wherein the centrifugation conditions are: a speed of 8000-10000 rcf, a temperature of 20-25° C., and a time of 25-40 min; 2a) adding the following reagents to the bacteria: Reagent I, wherein the reagent I is ethanol, and the amount of the ethanol relative to the bacteria is 0.4 to 0.8 parts by weight, Reagent II, wherein the reagent II is water, and the amount of the water relative to the bacteria is 0.1 to 2.0 parts by weight, and Reagent III, wherein the reagent III is hydrochloric acid, and the amount of the hydrochloric acid relative to the bacteria is 0.01 to 0.02 parts by weight; 2b) stirring at a temperature of -10 to -5°C and a speed of 500 to 10,000 rpm for 8 to 16 hours; 2c) centrifuging the bacterial solution obtained in 2b) to collect the solid phase of the solution, the centrifugation conditions being: rotation speed 8000-10000 rcf, temperature 20-25° C., time 25-40 min; 3a) extracting the solid phase of the solution obtained in 2c) by using 2 to 4 parts by weight of ethyl acetate relative to the solid phase of the solution at a temperature of 0 to 10° C. for 2 to 3 times for 6 to 8 hours to obtain an extract phase; 3b) evaporating the extract phase obtained in 3a) at a temperature of 0-25° C. and a vacuum degree of 20-200 mbar for 1-6 hours to obtain a concentrated slurry of the metabolite; 3c) filtering the concentrated slurry obtained in 3b), freezing the solid phase obtained by filtration at a temperature of -80 to -10°C for 1 to 8 hours, and then freeze-drying the solid phase to obtain the metabolite; Preferably, the bacteria are one or more selected from Escherichia coli, lactic acid bacteria, streptococci, acetic acid bacteria, propionic acid bacteria and bifidobacteria.

11. The method according to any one of claims 1 to 10, wherein The bacterium is Escherichia coli, and the bacterium contains a combination of exogenously transferred expression cassettes, the combination of expression cassettes comprising a first expression cassette, a second expression cassette, a third expression cassette, a fourth expression cassette, a fifth expression cassette, and a sixth expression cassette, and, (a) The first expression cassette comprises the following elements in order from the 5' end to the 3' end: (1) a promoter; (2) a ribosome binding site (RBS); (3) a polynucleotide encoding geranylgeranyl pyrophosphate synthase; (4) an RBS; (5) a polynucleotide encoding isoprene pyrophosphate isomerase; (6) a polynucleotide encoding lycopene cyclase; (7) a polynucleotide encoding phytoene dehydrogenase; (8) a polynucleotide encoding phytoene synthase; and (9) a terminator; (b) The second expression cassette comprises the following elements in sequence from the 5' end to the 3' end: (1) a promoter; (2) an RBS; (3) a polynucleotide encoding a phytoene synthase; (4) an RBS; (5) a polynucleotide encoding a phytoene dehydrogenase; (6) an RBS; (7) a polynucleotide encoding a lycopene cyclase; (8) an RBS; (9) a polynucleotide encoding a geranylgeranyl pyrophosphate synthase; and (10) a terminator; (c) The third expression cassette comprises the following elements in sequence from the 5' end to the 3' end: (1) Promoter; (2) RBS; (3) Polynucleotide encoding hydroxymethylglutaryl-CoA synthetase (HMGCS); (4) RBS; (5) Polynucleotide encoding 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR); (6) RBS; (7) Polynucleotide encoding acetyl-CoA acyltransferase (AACT); (8) Terminator; (d) The fourth expression cassette comprises the following elements in sequence from the 5' end to the 3' end: (1) a promoter; (2) an RBS; (3) a polynucleotide encoding a gene related to mevalonate synthesis selected from hydroxymevalonate kinase (MVK); (4) an RBS; (5) a polynucleotide encoding phosphomevalonate kinase (PMVK); (6) an RBS; (7) a polynucleotide encoding diphosphomevalonate decarboxylase (MVD); (8) a terminator; (e) The fifth expression cassette comprises the following elements in sequence from the 5' end to the 3' end: (1) Promoter; (2) RBS; (3) Polynucleotide encoding geranylgeranyl pyrophosphate synthase; (4) Terminator; (f) The sixth expression cassette comprises the following elements in sequence from the 5' end to the 3' end: (1) Promoter; (2) RBS; (3) Polynucleotide encoding β-carotene hydroxylase; (4) Terminator; Preferably, the phytoene synthase, phytoene dehydrogenase, lycopene cyclase, geranylgeranyl pyrophosphate synthase, isoprenyl pyrophosphate isomerase, HMGCS, HMGR, AACT, MVK, PMVK, MVD, β-carotene hydroxylase are independently derived from Enterobacter agglomerans, Pseudescherichia vulneris, Escherichia coli, Lactobacillus paracasei, Saccharomyces cerevisiae, Streptococcus pneumoniae, Pantoea ananas, or Enterobacteriaceae bacterium.

12. The method of claim 11, wherein: The combination of expression cassettes further comprises a seventh expression cassette, wherein the seventh expression cassette comprises the following elements in sequence from the 5' end to the 3' end: (1) Promoter; (2) RBS; (3) Polynucleotide encoding β-carotene hydroxylase; (4) RBS; (5) Polynucleotide encoding lycopene cyclase; (6) Terminator.

13. The method of claim 12, wherein: The combination of expression cassettes further comprises an eighth expression cassette, wherein the eighth expression cassette comprises the following elements in sequence from the 5' end to the 3' end: (1) a promoter; (2) an RBS; (3) at least two polynucleotides encoding β-carotene hydroxylase; (4) a terminator; Wherein, the at least two polynucleotides encoding β-carotene hydroxylase are connected via RBS.

14. The method according to any one of claims 10 to 13, wherein Each expression cassette contained in the combination of expression cassettes is independently integrated into the genome of the bacterium; Preferably, each of the expression cassettes is independently integrated into the genome of the bacterium at a different site.

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