Expression cassettes for producing microbial metabolites and uses thereof

Abstract

The invention belongs to the field of microorganisms, and relates to an expression cassette for producing microbial metabolites. Specifically, the invention relates to an expression cassette for expressing beta-carotene, which comprises the following elements: (1) a promoter; (2) a polynucleotide encoding a phytoene synthase; (3) a polynucleotide encoding a phytoene dehydrogenase; (4) a polynucleotide encoding a lycopene cyclase; (5) a polynucleotide encoding a geranyl and geranyl pyrophosphate synthase; and (6) a terminator. The invention also relates to a combination of expression cassettes comprising said expression cassette, a vector, a host cell, a recombinant microorganism, and the use of said expression cassette for producing a microbial metabolite.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms and relates to an expression cassette for producing microbial metabolites, a combination of expression cassettes, vectors, host cells, recombinant microorganisms, and uses thereof for producing microbial 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, 3R, 3'S-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] Lutein is present in large quantities in plant tissues such as green vegetables, red peppers, marigolds, corn seeds, wolfberries, and Physalis fruits, as well as in some non-photosynthetic bacteria. Studies have shown that the macular pigment of the human retina is composed of two types of xanthophylls present in the diet, namely lutein and zeaxanthin. These two pigments can absorb blue light waves that are damaging to the retina, thereby protecting the eyeball. At the same time, zeaxanthin can also prevent free radicals from invading biomembranes, quench singlet oxygen, and capture reactive oxygen free radicals, and has specific antioxidant functions. In the treatment of other chronic diseases, zeaxanthin has a special effect that is different from other carotenoids, and it also has unique physiological effects 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] After a lot of experiments, the inventors of the present application developed an expression cassette for expressing β-carotene, and based on the expression cassette, provided a combination of expression cassettes for expressing β-carotene and zeaxanthin, and a method for producing β-carotene and zeaxanthin, thereby providing the present invention.

[0008] Expression cassette

[0009] In a first aspect, the present invention provides an expression cassette for expressing β-carotene, comprising the following elements:

[0010] (1) Promoter;

[0011] (2) a polynucleotide encoding phytoene synthase;

[0012] (3) a polynucleotide encoding phytoene dehydrogenase;

[0013] (4) a polynucleotide encoding a lycopene cyclase;

[0014] (5) a polynucleotide encoding geranylgeranyl pyrophosphate synthase; and

[0015] (6)Terminator.

[0016] In some embodiments, the elements are directly linked to each other. In some embodiments, the elements are directly linked to each other in sequence from the 5' end to the 3' end.

[0017] In certain embodiments, the 5' end of the polynucleotide encoding phytoene synthase, the polynucleotide encoding phytoene dehydrogenase, the polynucleotide encoding lycopene cyclase, and the polynucleotide encoding geranylgeranyl pyrophosphate synthase are each independently linked to one or more ribosome binding sites (RBS).

[0018] In certain embodiments, the phytoene synthase, phytoene dehydrogenase, lycopene cyclase, and geranylgeranyl pyrophosphate synthase are independently derived from Enterobacter agglomerans, Pseudescherichia vulneris, Escherichia coli, Lactobacillus paracasei, Saccharomyces cerevisiae, Streptococcus pneumoniae, Pantoea ananas, or Enterobacteriaceae bacterium.

[0019] As used herein, the expression “the phytoene synthase, phytoene dehydrogenase, lycopene cyclase, and geranylgeranyl pyrophosphate synthase are independently derived from Enterobacter agglomerans, Pseudescherichia vulneris, Escherichia coli, Lactobacillus paracasei, Saccharomyces cerevisiae, Streptococcus pneumoniae, Pantoea ananas, or Enterobacteriaceae bacterium” means that the phytoene synthase, phytoene dehydrogenase, lycopene cyclase, and geranylgeranyl pyrophosphate synthase are independently derived from Enterobacter agglomerans, Pseudescherichia vulneris, Escherichia coli, Lactobacillus paracasei, Saccharomyces cerevisiae, Streptococcus pneumoniae, Pantoea ananas, or Enterobacteriaceae bacterium. vulneris, Escherichiacoli, Lactobacillus paracasei, Saccharomyces cerevisiae, Streptococcus pneumoniae, Pantoea ananas, or Enterobacteriaceae bacterium have the same sequence and function as the phytoene synthase, phytoene dehydrogenase, lycopene cyclase, and geranylgeranyl pyrophosphate synthase naturally contained in Bacillus vulneris, Escherichia coli, Lactobacillus paracasei, Saccharomyces cerevisiae, Streptococcus pneumoniae, Pantoea ananas, or Enterobacteriaceae bacterium. The phytoene synthase, phytoene dehydrogenase, lycopene cyclase, and geranylgeranyl pyrophosphate synthase can be isolated from Enterobacter agglomerans, Pseudescherichia vulneris, Escherichia coli, Lactobacillus paracasei, Saccharomyces cerevisiae, Streptococcus pneumoniae, Pantoea ananas, or Enterobacteriaceae bacterium, or can be artificially synthesized isolated proteins.At the same time, the phytoene synthase, phytoene dehydrogenase, lycopene cyclase, geranylgeranyl pyrophosphate synthase may contain naturally occurring mutations relative to their wild-type proteins, such as substitutions, deletions or additions of one or several amino acids (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions).

[0020] 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.

[0021] In certain embodiments, the expression cassette comprises the following elements in order from the 5' end to the 3' end:

[0022] (1) Promoter;

[0023] (2) RBS;

[0024] (3) a polynucleotide encoding phytoene synthase;

[0025] (4) RBS;

[0026] (5) a polynucleotide encoding phytoene dehydrogenase;

[0027] (6) RBS;

[0028] (7) a polynucleotide encoding a lycopene cyclase;

[0029] (8) RBS;

[0030] (9) a polynucleotide encoding geranylgeranyl pyrophosphate synthase; and

[0031] (10)Terminator.

[0032] In certain embodiments, the sequence of the promoter is shown in any one of SEQ ID NOs: 15, 19, 22, 24, and 34, the sequence of the terminator is shown in any one of SEQ ID NOs: 9, 20, 30, and 52, the sequence of the polynucleotide encoding phytoene synthase is shown in SEQ ID NO: 8, the sequence of the polynucleotide encoding phytoene dehydrogenase is shown in SEQ ID NO: 7, the sequence of the polynucleotide encoding lycopene cyclase is shown in SEQ ID NO: 6 or 28, and the sequence of the polynucleotide encoding geranylgeranyl pyrophosphate synthase is shown in SEQ ID NO: 3.

[0033] In certain embodiments, the sequences of the RBSs in (2), (4), (6) and (8) are shown in SEQ ID NOs: 25, 26, 27 and 29 respectively.

[0034] In certain embodiments, the sequence of the promoter is shown as SEQ ID NO:24, the sequence of the terminator is shown as SEQ ID NO:30, the sequence of the polynucleotide encoding phytoene synthase is shown as SEQ ID NO:8, the sequence of the polynucleotide encoding phytoene dehydrogenase is shown as SEQ ID NO:7, the sequence of the polynucleotide encoding lycopene cyclase is shown as SEQ ID NO:28, and the sequence of the polynucleotide encoding geranylgeranyl pyrophosphate synthase is shown as SEQ ID NO:3.

[0035] In certain embodiments, the expression cassette comprises the following elements in order from the 5' end to the 3' end:

[0036] (1) Promoter;

[0037] (2) RBS;

[0038] (3) a polynucleotide encoding phytoene synthase;

[0039] (4) RBS;

[0040] (5) a polynucleotide encoding phytoene dehydrogenase;

[0041] (6) RBS;

[0042] (7) a polynucleotide encoding a lycopene cyclase;

[0043] (8) RBS;

[0044] (9) a polynucleotide encoding geranylgeranyl pyrophosphate synthase; and

[0045] (10) terminator;

[0046] wherein the elements are directly connected, the sequence of the promoter is shown in SEQ ID NO:24, the sequence of the terminator is shown in SEQ ID NO:30, the sequence of the polynucleotide encoding phytoene synthase is shown in SEQ ID NO:8, the sequence of the polynucleotide encoding phytoene dehydrogenase is shown in SEQ ID NO:7, the sequence of the polynucleotide encoding lycopene cyclase is shown in SEQ ID NO:28, the sequence of the polynucleotide encoding geranylgeranyl pyrophosphate synthase is shown in SEQ ID NO:3, and the sequences of the RBS in (2), (4), (6) and (8) are shown in SEQ ID NOs:25, 26, 27 and 29, respectively.

[0047] In certain embodiments, the expression cassette further comprises a polynucleotide encoding isoprene pyrophosphate isomerase.

[0048] In certain embodiments, the 5' end of the polynucleotide encoding isoprene pyrophosphate isomerase is linked to one or more RBSs.

[0049] In certain embodiments, the isoprene pyrophosphate isomerase is derived from Enterobacter agglomerans, Pseudescherichia vulneris, Escherichia coli, Lactobacillus paracasei, Saccharomyces cerevisiae, Streptococcus pneumoniae, Pantoea ananas, or Enterobacteriaceae bacterium.

[0050] In certain embodiments, the expression cassette comprises the following elements in order from the 5' end to the 3' end:

[0051] (1) Promoter;

[0052] (2) RBS;

[0053] (3) a polynucleotide encoding geranylgeranyl pyrophosphate synthase;

[0054] (4) RBS;

[0055] (5) a polynucleotide encoding isoprene pyrophosphate isomerase;

[0056] (6) a polynucleotide encoding a lycopene cyclase;

[0057] (7) a polynucleotide encoding phytoene dehydrogenase;

[0058] (8) a polynucleotide encoding phytoene synthase; and

[0059] (9)Terminator.

[0060] In certain embodiments, the sequence of the promoter is shown in any one of SEQ ID NOs: 15, 19, 22, 24, and 34, the sequence of the terminator is shown in any one of SEQ ID NOs: 9, 20, 30, and 52, the sequence of the polynucleotide encoding phytoene synthase is shown in SEQ ID NO: 8, the sequence of the polynucleotide encoding phytoene dehydrogenase is shown in SEQ ID NO: 7, the sequence of the polynucleotide encoding lycopene cyclase is shown in SEQ ID NO: 6 or 28, the sequence of the polynucleotide encoding geranylgeranyl pyrophosphate synthase is shown in SEQ ID NO: 3, and the sequence of the polynucleotide encoding isoprene pyrophosphate isomerase is shown in SEQ ID NO: 5 or 47.

[0061] In certain embodiments, the sequences of the RBS in (2) and (4) are shown in SEQ ID NO: 12 and 4 respectively.

[0062] In certain embodiments, the sequence of the promoter is shown as SEQ ID NO:22, the sequence of the terminator is shown as SEQ ID NO:9, the sequence of the polynucleotide encoding phytoene synthase is shown as SEQ ID NO:8, the sequence of the polynucleotide encoding phytoene dehydrogenase is shown as SEQ ID NO:7, the sequence of the polynucleotide encoding lycopene cyclase is shown as SEQ ID NO:6, the sequence of the polynucleotide encoding geranylgeranyl pyrophosphate synthase is shown as SEQ ID NO:3, and the sequence of the polynucleotide encoding isoprene pyrophosphate isomerase is shown as SEQ ID NO:5.

[0063] In certain embodiments, the expression cassette comprises the following elements in order from the 5' end to the 3' end:

[0064] (1) Promoter;

[0065] (2) RBS;

[0066] (3) a polynucleotide encoding geranylgeranyl pyrophosphate synthase;

[0067] (4) RBS;

[0068] (5) a polynucleotide encoding isoprene pyrophosphate isomerase;

[0069] (6) a polynucleotide encoding a lycopene cyclase;

[0070] (7) a polynucleotide encoding phytoene dehydrogenase;

[0071] (8) a polynucleotide encoding phytoene synthase; and

[0072] (9) terminator;

[0073] wherein the elements are directly connected, the sequence of the promoter is shown in SEQ ID NO:22, the sequence of the terminator is shown in SEQ ID NO:9, the sequence of the polynucleotide encoding phytoene synthase is shown in SEQ ID NO:8, the sequence of the polynucleotide encoding phytoene dehydrogenase is shown in SEQ ID NO:7, the sequence of the polynucleotide encoding lycopene cyclase is shown in SEQ ID NO:6, the sequence of the polynucleotide encoding geranylgeranyl pyrophosphate synthase is shown in SEQ ID NO:3, the sequence of the polynucleotide encoding isoprene pyrophosphate isomerase is shown in SEQ ID NO:5, and the sequences of the RBS in (2) and (4) are shown in SEQ ID NOs:12 and 4, respectively.

[0074] Combination of expression cassettes

[0075] In a second aspect, the present invention provides a combination of expression cassettes for expressing β-carotene, comprising a first expression cassette, and one or more expression cassettes selected from a second expression cassette, a third expression cassette and a fourth expression cassette, wherein the first expression cassette is the expression cassette described in the first aspect of the present invention.

[0076] In certain embodiments, the second expression cassette comprises the following elements:

[0077] (1) Promoter;

[0078] (2) a polynucleotide encoding hydroxymethylglutaryl-CoA synthetase (HMGCS);

[0079] (3) a polynucleotide encoding 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR);

[0080] (4) a polynucleotide encoding acetyl-CoA acyltransferase (AACT); and

[0081] (5)Terminator.

[0082] In some embodiments, the elements are directly linked to each other. In some embodiments, the elements are directly linked to each other in sequence from the 5' end to the 3' end.

[0083] In certain embodiments, the 5' end of the polynucleotide encoding HMGCS, the polynucleotide encoding HMGR, and the polynucleotide encoding AACT are each independently linked to one or more RBSs.

[0084] In certain embodiments, the HMGCS, HMGR, and AACT are independently derived from Enterobacter agglomerans, Pseudescherichia vulneris, Escherichia coli, Lactobacillus paracasei, Saccharomyces cerevisiae, Streptococcus pneumoniae, Pantoea ananas, or Enterobacteriaceae bacterium.

[0085] In certain embodiments, the sequence of the promoter is shown in any one of SEQ ID NOs: 15, 19, 22, 24, and 34, the sequence of the terminator is shown in any one of SEQ ID NOs: 9, 20, 30, and 52, the sequence of the polynucleotide encoding HMGCS is shown in SEQ ID NO: 36, the sequence of the polynucleotide encoding HMGR is shown in SEQ ID NO: 38, and the sequence of the polynucleotide encoding AACT is shown in SEQ ID NO: 40.

[0086] In certain embodiments, the second expression cassette comprises the following elements in order from the 5' end to the 3' end:

[0087] (1) Promoter;

[0088] (2) RBS;

[0089] (3) a polynucleotide encoding hydroxymethylglutaryl-CoA synthetase (HMGCS);

[0090] (4) RBS;

[0091] (5) a polynucleotide encoding 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR);

[0092] (6) RBS;

[0093] (7) a polynucleotide encoding acetyl-CoA acyltransferase (AACT); and

[0094] (8)Terminator.

[0095] In certain embodiments, the sequences of the RBSs in (2), (4) and (6) are shown in SEQ ID NOs: 35, 37 and 39 respectively.

[0096] In certain embodiments, the sequence of the promoter is shown as SEQ ID NO:34, the sequence of the terminator is shown as SEQ ID NO:30, the sequence of the polynucleotide encoding HMGCS is shown as SEQ ID NO:36, the sequence of the polynucleotide encoding HMGR is shown as SEQ ID NO:38, and the sequence of the polynucleotide encoding AACT is shown as SEQ ID NO:40.

[0097] In certain embodiments, the second expression cassette comprises the following elements in order from the 5' end to the 3' end:

[0098] (1) Promoter;

[0099] (2) RBS;

[0100] (3) a polynucleotide encoding hydroxymethylglutaryl-CoA synthetase (HMGCS);

[0101] (4) RBS;

[0102] (5) a polynucleotide encoding 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR);

[0103] (6) RBS;

[0104] (7) a polynucleotide encoding acetyl-CoA acyltransferase (AACT); and

[0105] (8) terminator;

[0106] Among them, the elements are directly connected, the sequence of the promoter is shown in SEQ ID NO:34, the sequence of the terminator is shown in SEQ ID NO:30, the sequence of the polynucleotide encoding HMGCS is shown in SEQ ID NO:36, the sequence of the polynucleotide encoding HMGR is shown in SEQ ID NO:38, the sequence of the polynucleotide encoding AACT is shown in SEQ ID NO:40, and the sequences of the RBS in (2), (4) and (6) are shown in SEQ ID NOs:35, 37 and 39 respectively.

[0107] In certain embodiments, the third expression cassette comprises the following elements:

[0108] (1) Promoter;

[0109] (2) a polynucleotide encoding a gene related to mevalonate synthesis selected from mevalonate kinase (MVK);

[0110] (3) a polynucleotide encoding phosphomevalonate kinase (PMVK);

[0111] (4) a polynucleotide encoding mevalonate diphosphate decarboxylase (MVD); and

[0112] (5)Terminator.

[0113] In some embodiments, the elements are directly linked to each other. In some embodiments, the elements are directly linked to each other in sequence from the 5' end to the 3' end.

[0114] In certain embodiments, the 5' end of the polynucleotide encoding MVK, the polynucleotide encoding PMVK, and the polynucleotide encoding MVD are each independently linked to one or more RBSs.

[0115] In certain embodiments, the MVK, PMVK, and MVD genes are independently derived from Enterobacter agglomerans, Pseudescherichia vulneris, Escherichia coli, Lactobacillus paracasei, Saccharomyces cerevisiae, Streptococcus pneumoniae, Pantoea ananas, or Enterobacteriaceae bacterium.

[0116] In certain embodiments, the sequence of the promoter is as shown in any one of SEQ ID NOs: 15, 19, 22, 24, and 34, the sequence of the terminator is as shown in any one of SEQ ID NOs: 9, 20, 30, and 52, the sequence of the polynucleotide encoding MVK is as shown in SEQ ID NO: 44, the sequence of the polynucleotide encoding PMVK is as shown in SEQ ID NO: 45, and the sequence of the polynucleotide encoding MVD is as shown in SEQ ID NO: 46.

[0117] In certain embodiments, the third expression cassette comprises the following elements in order from the 5' end to the 3' end:

[0118] (1) Promoter;

[0119] (2) RBS;

[0120] (3) a polynucleotide encoding a gene related to mevalonate synthesis selected from mevalonate kinase (MVK);

[0121] (4) RBS;

[0122] (5) a polynucleotide encoding phosphomevalonate kinase (PMVK);

[0123] (6) RBS;

[0124] (7) a polynucleotide encoding mevalonate diphosphate decarboxylase (MVD); and

[0125] (8)Terminator.

[0126] In certain embodiments, the sequences of the RBS in (2), (4) and (6) are shown in SEQ ID NO: 35, 39 and 26 respectively.

[0127] In certain embodiments, the sequence of the promoter is shown in SEQ ID NO:34, the sequence of the terminator is shown in SEQ ID NO:30, the sequence of the polynucleotide encoding MVK is shown in SEQ ID NO:44, the sequence of the polynucleotide encoding PMVK is shown in SEQ ID NO:45, and the sequence of the polynucleotide encoding MVD is shown in SEQ ID NO:46.

[0128] In certain embodiments, the third expression cassette comprises the following elements in order from the 5' end to the 3' end:

[0129] (1) Promoter;

[0130] (2) RBS;

[0131] (3) a polynucleotide encoding a gene related to mevalonate synthesis selected from mevalonate kinase (MVK);

[0132] (4) RBS;

[0133] (5) a polynucleotide encoding phosphomevalonate kinase (PMVK);

[0134] (6) RBS;

[0135] (7) a polynucleotide encoding mevalonate diphosphate decarboxylase (MVD); and

[0136] (8) terminator;

[0137] Among them, the elements are directly connected, the sequence of the promoter is shown in SEQ ID NO:34, the sequence of the terminator is shown in SEQ ID NO:30, the sequence of the polynucleotide encoding MVK is shown in SEQ ID NO:44, the sequence of the polynucleotide encoding PMVK is shown in SEQ ID NO:45, the sequence of the polynucleotide encoding MVD is shown in SEQ ID NO:46, and the sequences of the RBS in (2), (4) and (6) are shown in SEQ ID NOs:35, 39 and 26 respectively.

[0138] In certain embodiments, the fourth expression cassette comprises the following elements:

[0139] (1) Promoter;

[0140] (2) a polynucleotide encoding geranylgeranyl pyrophosphate synthase; and

[0141] (3)Terminator.

[0142] In some embodiments, the elements are directly linked to each other. In some embodiments, the elements are directly linked to each other in sequence from the 5' end to the 3' end.

[0143] In certain embodiments, the 5' end of the polynucleotide encoding geranylgeranyl pyrophosphate synthase is linked to one or more RBSs.

[0144] In certain embodiments, the geranylgeranyl pyrophosphate synthase is derived from Enterobacter agglomerans, Pseudescherichia vulneris, Escherichia coli, Lactobacillus paracasei, Saccharomyces cerevisiae, Streptococcus pneumoniae, Pantoea ananas, or Enterobacteriaceae bacterium.

[0145] In certain embodiments, the sequence of the promoter is as shown in any one of SEQ ID NOs: 15, 19, 22, 24, and 34, the sequence of the terminator is as shown in any one of SEQ ID NOs: 9, 20, 30, and 52, and the sequence of the polynucleotide encoding geranylgeranyl pyrophosphate synthase is as shown in SEQ ID NO: 3.

[0146] In certain embodiments, the fourth expression cassette comprises the following elements in order from the 5' end to the 3' end:

[0147] (1) Promoter;

[0148] (2) RBS;

[0149] (3) a polynucleotide encoding geranylgeranyl pyrophosphate synthase; and

[0150] (4)Terminator.

[0151] In certain embodiments, the sequence of the RBS is as shown in SEQ ID NO:12.

[0152] In certain embodiments, the sequence of the promoter is shown as SEQ ID NO:19, the sequence of the terminator is shown as SEQ ID NO:20, and the sequence of the polynucleotide encoding geranylgeranyl pyrophosphate synthase is shown as SEQ ID NO:3.

[0153] In certain embodiments, the fourth expression cassette comprises the following elements in order from the 5' end to the 3' end:

[0154] (1) Promoter;

[0155] (2) RBS;

[0156] (3) a polynucleotide encoding geranylgeranyl pyrophosphate synthase; and

[0157] (4) terminator;

[0158] Among them, the elements are directly connected, the sequence of the promoter is shown in SEQ ID NO:19, the sequence of the terminator is shown in SEQ ID NO:20, the sequence of the polynucleotide encoding geranylgeranyl pyrophosphate synthase is shown in SEQ ID NO:3, and the sequence of the RBS is shown in SEQ ID NO:12.

[0159] In certain embodiments, the combination of expression cassettes comprises at least two of the first expression cassettes, and comprises the second expression cassette, a third expression cassette, and a fourth expression cassette. In certain embodiments, the sequences of the at least two first expression cassettes are different from each other.

[0160] In certain embodiments, the combination of expression cassettes comprises two of the first expression cassettes, and comprises the second expression cassette, a third expression cassette, and a fourth expression cassette.

[0161] In a third aspect, the present invention provides a combination of expression cassettes for expressing β-carotene and zeaxanthin, comprising the expression cassette of the first aspect of the present invention, or the combination of expression cassettes of the second aspect of the present invention, and one or more fifth expression cassettes, wherein the fifth expression cassette comprises the following elements:

[0162] (1) Promoter;

[0163] (2) a polynucleotide encoding β-carotene hydroxylase; and

[0164] (3) terminator;

[0165] In some embodiments, the elements are directly linked to each other. In some embodiments, the elements are directly linked to each other in sequence from the 5' end to the 3' end.

[0166] In certain embodiments, the 5' end of the polynucleotide encoding the β-carotene hydroxylase is linked to one or more RBSs.

[0167] In certain embodiments, the β-carotene hydroxylase is derived from Enterobacter agglomerans, Pseudescherichia vulneris, Escherichia coli, Lactobacillus paracasei, Saccharomyces cerevisiae, Streptococcus pneumoniae, Pantoea ananas, or Enterobacteriaceae bacterium.

[0168] In certain embodiments, the sequence of the promoter is as shown in any one of SEQ ID NOs: 15, 19, 22, 24, and 34, the sequence of the terminator is as shown in any one of SEQ ID NOs: 9, 20, 30, and 52, and the sequence of the polynucleotide encoding β-carotene hydroxylase is as shown in any one of SEQ ID NOs: 51, 58, 61, 69, 75, and 78.

[0169] In certain embodiments, the fifth expression cassette comprises the following elements in order from the 5' end to the 3' end:

[0170] (1) Promoter;

[0171] (2) RBS;

[0172] (3) a polynucleotide encoding β-carotene hydroxylase; and

[0173] (4)Terminator.

[0174] In certain embodiments, the sequence of the RBS is as shown in SEQ ID NO:55.

[0175] In certain embodiments, the sequence of the promoter is shown as SEQ ID NO:15, the sequence of the terminator is shown as SEQ ID NO:52, and the sequence of the polynucleotide encoding β-carotene hydroxylase is shown as SEQ ID NO:58.

[0176] In certain embodiments, the fifth expression cassette comprises the following elements in order from the 5' end to the 3' end:

[0177] (1) Promoter;

[0178] (2) RBS;

[0179] (3) a polynucleotide encoding β-carotene hydroxylase; and

[0180] (4) terminator;

[0181] Wherein, the elements are directly connected, the sequence of the promoter is shown in SEQ ID NO: 15, the sequence of the terminator is shown in SEQ ID NO: 52, the sequence of the polynucleotide encoding β-carotene hydroxylase is shown in SEQ ID NO: 58, and the sequence of the RBS is shown in SEQ ID NO: 55.

[0182] In certain embodiments, the sequence of the promoter is shown as SEQ ID NO:15, the sequence of the terminator is shown as SEQ ID NO:52, and the sequence of the polynucleotide encoding β-carotene hydroxylase is shown as SEQ ID NO:61.

[0183] In certain embodiments, the fifth expression cassette comprises the following elements in order from the 5' end to the 3' end:

[0184] (1) Promoter;

[0185] (2) RBS;

[0186] (3) a polynucleotide encoding β-carotene hydroxylase; and

[0187] (4) terminator;

[0188] Wherein, the elements are directly connected, the sequence of the promoter is shown in SEQ ID NO: 15, the sequence of the terminator is shown in SEQ ID NO: 52, the sequence of the polynucleotide encoding β-carotene hydroxylase is shown in SEQ ID NO: 61, and the sequence of the RBS is shown in SEQ ID NO: 55.

[0189] In certain embodiments, the sequence of the promoter is shown as SEQ ID NO:15, the sequence of the terminator is shown as SEQ ID NO:52, and the sequence of the polynucleotide encoding β-carotene hydroxylase is shown as SEQ ID NO:69.

[0190] In certain embodiments, the fifth expression cassette comprises the following elements in order from the 5' end to the 3' end:

[0191] (1) Promoter;

[0192] (2) RBS;

[0193] (3) a polynucleotide encoding β-carotene hydroxylase; and

[0194] (4) terminator;

[0195] Wherein, the elements are directly connected, the sequence of the promoter is shown in SEQ ID NO: 15, the sequence of the terminator is shown in SEQ ID NO: 52, the sequence of the polynucleotide encoding β-carotene hydroxylase is shown in SEQ ID NO: 69, and the sequence of the RBS is shown in SEQ ID NO: 55.

[0196] In certain embodiments, the sequence of the RBS is shown in SEQ ID NO:68.

[0197] In certain embodiments, the sequence of the promoter is shown as SEQ ID NO:15, the sequence of the terminator is shown as SEQ ID NO:52, and the sequence of the polynucleotide encoding β-carotene hydroxylase is shown as SEQ ID NO:69.

[0198] In certain embodiments, the fifth expression cassette comprises the following elements in order from the 5' end to the 3' end:

[0199] (1) Promoter;

[0200] (2) RBS;

[0201] (3) a polynucleotide encoding β-carotene hydroxylase; and

[0202] (4) terminator;

[0203] Wherein, the elements are directly connected, the sequence of the promoter is shown in SEQ ID NO: 15, the sequence of the terminator is shown in SEQ ID NO: 52, the sequence of the polynucleotide encoding β-carotene hydroxylase is shown in SEQ ID NO: 69, and the sequence of the RBS is shown in SEQ ID NO: 68.

[0204] In certain embodiments, the fifth expression cassette further comprises a polynucleotide encoding a lycopene cyclase.

[0205] In certain embodiments, the 5' end of the polynucleotide encoding the lycopene cyclase is linked to one or more RBSs.

[0206] In certain embodiments, the lycopene cyclase is derived from Enterobacter agglomerans, Pseudescherichia vulneris, Escherichia coli, Lactobacillus paracasei, Saccharomyces cerevisiae, Streptococcus pneumoniae, Pantoea ananas, or Enterobacteriaceae bacterium.

[0207] In certain embodiments, the sequence of the polynucleotide encoding lycopene cyclase is shown as SEQ ID NO: 6 or 28.

[0208] In certain embodiments, the fifth expression cassette comprises the following elements in order from the 5' end to the 3' end:

[0209] (1) Promoter;

[0210] (2) RBS;

[0211] (3) a polynucleotide encoding β-carotene hydroxylase;

[0212] (4) RBS;

[0213] (5) a polynucleotide encoding a lycopene cyclase; and

[0214] (6)Terminator.

[0215] In certain embodiments, the sequences of the RBS in (2) and (4) are shown in SEQ ID NO: 64 and 65 respectively.

[0216] In certain embodiments, the sequence of the promoter is shown as SEQ ID NO:24, the sequence of the terminator is shown as SEQ ID NO:52, the sequence of the polynucleotide encoding β-carotene hydroxylase is shown as SEQ ID NO:51, and the sequence of the polynucleotide encoding lycopene cyclase is shown as SEQ ID NO:28.

[0217] In certain embodiments, the fifth expression cassette comprises the following elements in order from the 5' end to the 3' end:

[0218] (1) Promoter;

[0219] (2) RBS;

[0220] (3) a polynucleotide encoding β-carotene hydroxylase;

[0221] (4) RBS;

[0222] (5) a polynucleotide encoding a lycopene cyclase; and

[0223] (6) terminator;

[0224] Wherein, the elements are directly connected, the sequence of the promoter is shown in SEQ ID NO:24, the sequence of the terminator is shown in SEQ ID NO:52, the sequence of the polynucleotide encoding β-carotene hydroxylase is shown in SEQ ID NO:51, the sequence of the polynucleotide encoding lycopene cyclase is shown in SEQ ID NO:28, and the sequences of the RBS in (2) and (4) are shown in SEQ ID NOs:64 and 65 respectively.

[0225] In certain embodiments, the zeaxanthin is all-trans-zeaxanthin.

[0226] In certain embodiments, the zeaxanthin is 3R,3'R-all-trans-zeaxanthin.

[0227] As used herein, the term "3R,3'R-all-trans-zeaxanthin" and "3R,3R'-all-trans-zeaxanthin" have the same meaning and can be used interchangeably.

[0228] In a fourth aspect, the present invention provides a combination of expression cassettes for expressing zeaxanthin, comprising the combination of expression cassettes described in the third aspect of the present invention, and a sixth expression cassette, wherein the sixth expression cassette comprises the following elements:

[0229] (1) Promoter;

[0230] (2) at least two polynucleotides encoding β-carotene hydroxylase;

[0231] (3)Terminator.

[0232] In some embodiments, the elements are directly linked to each other. In some embodiments, the elements are directly linked to each other in sequence from the 5' end to the 3' end.

[0233] In certain embodiments, the at least two polynucleotides encoding β-carotene hydroxylases are linked via a RBS.

[0234] In certain embodiments, the 5' ends of the at least two polynucleotides encoding β-carotene hydroxylases are each independently linked to one or more RBSs.

[0235] In certain embodiments, the 5' ends of the at least two polynucleotides encoding β-carotene hydroxylases are each independently linked to an RBS.

[0236] In certain embodiments, the at least two β-carotene hydroxylases are derived from different species selected from Enterobacter agglomerans, Pseudescherichia vulneris, Escherichia coli, Lactobacillus paracasei, Saccharomyces cerevisiae, Streptococcus pneumoniae, Pantoea ananas, or Enterobacteriaceae bacterium.

[0237] In certain embodiments, the sequence of the promoter is as shown in any one of SEQ ID NOs: 15, 19, 22, 24, and 34, the sequence of the terminator is as shown in any one of SEQ ID NOs: 9, 20, 30, and 52, and the sequence of the polynucleotide encoding β-carotene hydroxylase is as shown in any one of SEQ ID NOs: 51, 58, 61, 69, 75, and 78.

[0238] In certain embodiments, the sixth expression cassette comprises the following elements in order from the 5' end to the 3' end:

[0239] (1) Promoter;

[0240] (2) RBS;

[0241] (3) a polynucleotide encoding β-carotene hydroxylase;

[0242] (4) RBS;

[0243] (5) a polynucleotide encoding β-carotene hydroxylase;

[0244] (6) RBS;

[0245] (7) a polynucleotide encoding β-carotene hydroxylase;

[0246] (8) RBS;

[0247] (9) a polynucleotide encoding β-carotene hydroxylase; and

[0248] (10)Terminator.

[0249] In certain embodiments, the sequences of the RBSs in (2), (4), (6) and (8) are shown in SEQ ID NOs: 74, 76, 77 and 76 respectively.

[0250] In certain embodiments, the sequence of the promoter is as shown in SEQ ID NO: 15, the sequence of the terminator is as shown in SEQ ID NO: 30, and the sequences of the polynucleotides encoding β-carotene hydroxylase in (3), (5), (7) and (9) are as shown in SEQ ID NO: 75, 69, 58 and 78 respectively.

[0251] In certain embodiments, the sixth expression cassette comprises the following elements in order from the 5' end to the 3' end:

[0252] (1) Promoter;

[0253] (2) RBS;

[0254] (3) a polynucleotide encoding β-carotene hydroxylase;

[0255] (4) RBS;

[0256] (5) a polynucleotide encoding β-carotene hydroxylase;

[0257] (6) RBS;

[0258] (7) a polynucleotide encoding β-carotene hydroxylase;

[0259] (8) RBS;

[0260] (9) a polynucleotide encoding β-carotene hydroxylase; and

[0261] (10) terminator;

[0262] Wherein, the elements are directly connected, the sequence of the promoter is shown in SEQ ID NO: 15, the sequence of the terminator is shown in SEQ ID NO: 30, the sequences of the polynucleotides encoding β-carotene hydroxylase in (3), (5), (7) and (9) are shown in SEQ ID NOs: 75, 69, 58 and 78 respectively, and the sequences of the RBS in (2), (4), (6) and (8) are shown in SEQ ID NOs: 74, 76, 77 and 76 respectively.

[0263] In certain embodiments, the sequences of the RBSs in (2), (4), (6) and (8) are shown in SEQ ID NOs: 74, 55, 81 and 55 respectively.

[0264] In certain embodiments, the sequence of the promoter is as shown in SEQ ID NO: 15, the sequence of the terminator is as shown in SEQ ID NO: 30, and the sequences of the polynucleotides encoding β-carotene hydroxylase in (3), (5), (7) and (9) are as shown in SEQ ID NO: 75, 69, 58 and 78 respectively.

[0265] In certain embodiments, the sixth expression cassette comprises the following elements in order from the 5' end to the 3' end:

[0266] (1) Promoter;

[0267] (2) RBS;

[0268] (3) a polynucleotide encoding β-carotene hydroxylase;

[0269] (4) RBS;

[0270] (5) a polynucleotide encoding β-carotene hydroxylase;

[0271] (6) RBS;

[0272] (7) a polynucleotide encoding β-carotene hydroxylase;

[0273] (8) RBS;

[0274] (9) a polynucleotide encoding β-carotene hydroxylase; and

[0275] (10) terminator;

[0276] Wherein, the elements are directly connected, the sequence of the promoter is shown in SEQ ID NO: 15, the sequence of the terminator is shown in SEQ ID NO: 30, the sequences of the polynucleotides encoding β-carotene hydroxylase in (3), (5), (7) and (9) are shown in SEQ ID NOs: 75, 69, 58 and 78 respectively, and the sequences of the RBS in (2), (4), (6) and (8) are shown in SEQ ID NOs: 74, 55, 81 and 55 respectively.

[0277] In certain embodiments, the zeaxanthin is all-trans-zeaxanthin.

[0278] In certain embodiments, the zeaxanthin is 3R,3'R-all-trans-zeaxanthin.

[0279] Vectors, host cells, recombinant microorganisms

[0280] In another aspect, the present invention provides a vector comprising the expression cassette of the first aspect of the present invention, or a combination of the expression cassettes of the second aspect, the third aspect, or the fourth aspect of the present invention.

[0281] In certain embodiments, the vector is a cloning vector or an expression vector.

[0282] In certain embodiments, in the vector, the 5' end of the expression cassette is linked to an upstream homology arm, and the 3' end of the expression cassette is linked to a downstream homology arm.

[0283] In certain embodiments, the vector comprises a combination of expression cassettes according to the second aspect, the third aspect, or the fourth aspect of the invention, wherein the homology arms of the plurality of expression cassettes are different from each other.

[0284] In another aspect, the present invention provides a host cell comprising the expression cassette of the first aspect of the present invention, a combination of the expression cassettes of the second aspect, the third aspect, or the fourth aspect of the present invention, or a vector.

[0285] In another aspect, the present invention provides a recombinant microorganism comprising the expression cassette of the first aspect of the present invention, or a combination of the expression cassettes of the second aspect, the third aspect, or the fourth aspect of the present invention.

[0286] In certain embodiments, the expression cassette is integrated into the genome of the recombinant microorganism.

[0287] In certain embodiments, the recombinant microorganism comprises at least two expression cassettes of the first aspect of the invention, or a combination of expression cassettes of the second, third, or fourth aspects of the invention, wherein the different expression cassettes are each independently integrated into different sites of the genome of the recombinant microorganism.

[0288] In certain embodiments, the recombinant microorganism is capable of producing β-carotene and / or zeaxanthin.

[0289] In certain embodiments, the recombinant microorganism is selected from a fungus or a bacterium.

[0290] In certain embodiments, the fungus is a yeast (eg, Saccharomyces cerevisiae, Saccharomyces lipolytica, Kluyveromyces).

[0291] In certain embodiments, the bacteria are one or more selected from Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptococcus, Streptomyces, Acetobacter, Propionibacterium and Bifidobacterium.

[0292] use

[0293] On the other hand, the present invention provides the use of the expression cassette described in the first aspect of the present invention, the combination of the expression cassette described in the second aspect, the third aspect, or the fourth aspect of the present invention, the vector, the host cell, or the recombinant microorganism of the present invention in producing zeaxanthin and / or β-carotene.

[0294] In certain embodiments, the zeaxanthin is all-trans-zeaxanthin.

[0295] In certain embodiments, the zeaxanthin is 3R,3'R-all-trans-zeaxanthin.

[0296] In another aspect, the present invention provides a method for producing zeaxanthin and / or β-carotene, comprising the following steps:

[0297] The recombinant microorganism of the present invention is cultured under conditions that allow the growth and metabolism of the microorganism, and zeaxanthin and / or β-carotene are recovered from the culture of the cultured recombinant microorganism.

[0298] In certain embodiments, the recombinant microorganism is cultured by fermentation, and zeaxanthin and / or β-carotene is recovered from the fermentation product.

[0299] In another aspect, the present invention provides a method for producing β-carotene, comprising the following steps:

[0300] The recombinant microorganism is cultured under conditions that allow the growth and metabolism of the microorganism, and β-carotene is recovered from the culture of the cultured recombinant microorganism, wherein the recombinant microorganism comprises the expression cassette of the first aspect of the present invention, the combination of expression cassettes of the second aspect of the present invention, and / or the combination of expression cassettes of the third aspect of the present invention.

[0301] In certain embodiments, the recombinant microorganism is cultured by fermentation, and β-carotene is recovered from the fermentation product.

[0302] In another aspect, the present invention provides a method for producing β-carotene and zeaxanthin, comprising the following steps:

[0303] The recombinant microorganism is cultured under conditions that allow the growth and metabolism of the microorganism, and β-carotene and zeaxanthin are recovered from the culture of the cultured recombinant microorganism, wherein the recombinant microorganism comprises the expression cassette of the first aspect of the present invention, the combination of expression cassettes of the second aspect of the present invention, the combination of expression cassettes of the third aspect of the present invention, and / or the combination of expression cassettes of the fourth aspect of the present invention.

[0304] In certain embodiments, the recombinant microorganism is cultured by fermentation, and β-carotene and zeaxanthin are recovered from the fermentation product.

[0305] In another aspect, the present invention provides a method for producing zeaxanthin, comprising the following steps:

[0306] The recombinant microorganism is cultured under conditions that allow the growth and metabolism of the microorganism, and zeaxanthin is recovered from the culture of the cultured recombinant microorganism, wherein the recombinant microorganism comprises the combination of expression cassettes described in the third aspect of the present invention and / or the combination of expression cassettes described in the fourth aspect of the present invention.

[0307] In certain embodiments, the recombinant microorganism is cultured by fermentation, and zeaxanthin is recovered from the fermentation product.

[0308] In certain embodiments, the zeaxanthin is all-trans-zeaxanthin.

[0309] In certain embodiments, the zeaxanthin is 3R,3'R-all-trans-zeaxanthin.

[0310] In certain embodiments, the microorganism is selected from fungi or bacteria.

[0311] In certain embodiments, the fungus is a yeast (eg, Saccharomyces cerevisiae, Saccharomyces lipolytica, Kluyveromyces).

[0312] In certain embodiments, the bacteria are one or more selected from Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptococcus, Streptomyces, Acetobacter, Propionibacterium and Bifidobacterium.

[0313] Definition of terms

[0314] 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.

[0315] 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."

[0316] 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.

[0317] 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.

[0318] 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.

[0319] 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.

[0320] 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.

[0321] 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.

[0322] 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), and "sixth" (e.g., the sixth expression cassette) are mainly for reference distinction and do not have a typical order meaning.

[0323] Advantageous Effects of the Invention

[0324] The present invention provides an expression cassette for producing microbial metabolites (such as zeaxanthin and β-carotene), and further provides the use thereof for producing microbial metabolites (such as zeaxanthin and β-carotene) based on the method. The construction method of the expression cassette is mature and stable, and can be transferred into different strains for the production of related metabolites. The strain transferred into the expression cassette has a significantly improved metabolite (such as zeaxanthin and β-carotene) yield, which is suitable for subsequent large-scale commercial production. Moreover, the zeaxanthin produced by the combination of the expression cassettes of the present invention is a relatively rare 3R, 3'R-all-trans zeaxanthin, which has important value. BRIEF DESCRIPTION OF THE DRAWINGS

[0325] Figure 1 : Chromatogram of zeaxanthin structure identification. The horizontal axis represents the time when the peak is detected, and the vertical axis represents the peak signal intensity. "3R,3S-meso-zeaxanthin" is 3R,3'S-meso-zeaxanthin, and "3R,3R'-all-trans-zeaxanthin" is 3R,3'R-all-trans-zeaxanthin. DETAILED DESCRIPTION

[0326] The invention is now described in the following non-limiting examples.

[0327] 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.

[0328] Unless otherwise specified, the culture method of the transformants in the following examples is described below:

[0329] Pick a single clone into a 96-well deep-well plate containing 400 μl of TB medium containing 5 g / L glucose, add 50 mg / L Kan into the medium, and culture at 30°C, 800 rpm for 1 day. Transfer 10 μl of the culture solution to 400 μl of TB medium and culture at 30°C, 800 rpm for 3 days.

[0330] If not otherwise specified, the extraction and detection methods of β-carotene and zeaxanthin in the following embodiments refer to the following description:

[0331] Take 50ul of bacterial solution, add 750μl of extraction solution (methanol: ethyl acetate = 1:1), shake at 2000rpm for 20 minutes, ultrasonic at 25℃ for 10 minutes, shake at 30 degrees and 1500rpm for 60 minutes, centrifuge at 25℃, 4000rpm for 20 minutes. Take the supernatant for detection. The detection conditions are as follows:

[0332] 1) Chromatographic column: CHIRALPAK AD-H LC column 4.6mm*250 5μm;

[0333] 2) Mobile phase: A1: isopropanol B1: n-hexane;

[0334] 3) Injection volume: 2.5ul;

[0335] 4) Seal wash: 10% isopropyl alcohol;

[0336] 5) Needle wash: methanol: ethyl acetate = 1:1;

[0337] 6) Column temperature: 30°C;

[0338] 7) Flow rate: 0.7 ml / min;

[0339] 8) Gradient elution, the conditions are shown in Table 1;

[0340] Table 1: Gradient elution conditions

[0341] 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

[0342] 9) Running time: 45min;

[0343] 10) Wavelength: 452nm.

[0344] Example 1: Construction of Escherichia coli producing β-carotene

[0345] (1) Construction of Escherichia coli strains

[0346] The pEcCas9 plasmid was transferred into C43 (DE3) (purchased from Weidi Biotechnology, catalog number CAT#: EC1040) by conventional Escherichia coli chemical transformation method. The strain has Kan resistance and grows in LB+Kan resistance medium. The resulting strain is numbered BOTA01003. pAC-BETAipi (purchased from Miaoling, item number P9847) was used as a template to amplify the expression cassette for β-carotene synthesis, which includes the pa promoter from Enterobacter agglomerans; the gene crtE encoding geranylgeranyl pyrophosphate synthase, the gene crtB encoding phytoene synthase, the gene crtI encoding phytoene dehydrogenase, the gene crtY encoding lycopene cyclase, and the gene idi encoding isoprene pyrophosphate isomerase from Pseudescherichia vulneris; the 5' end of the gene also includes a ribosome binding site RBS from Enterobacter agglomerans. Using the SOE method, homology arm sequences of the adhE site with a length of about 500 bp were introduced on both sides of the expression cassette. The resulting expression cassette DNA sequence is ADD-1977 (its element composition and specific sequence are shown in Table 2 below). By using the electric shock method, the pTargetF plasmid IPCC511 with adhE cleavage site and ADD-1977 were co-transformed into C43 (DE3), and the transformant BOTA01004 was screened by LB+Kan+Spe. The pTargetF plasmid was discarded from the transformant, and the obtained host bacteria numbered BOTA01005, and the pTargetF plasmid IPCC581 with a cleavage site on the pa promoter and the DNA sequence ADD-2359 with the pTrc promoter and the upstream homology arm of adhE, and the crtE gene as the downstream homology arm were co-transformed into BOTA01005, and the transformants were screened by LB+Kan+Spe, and the obtained strain was BOTA01002, in which the promoter of the β-carotene expression cassette was replaced by the pa promoter with the pTrc promoter, and a new RBS (DNA1918) was added in front of the crtE gene. The element composition and specific sequence of the expression cassette are shown in Table 3 below. Based on BOTA01002, the pTargetF plasmid was discarded to obtain strain BOTA01006.

[0347] Then, in BOTA01006, the pTargetF plasmid with the ispA cleavage site; the DNA sequence ADD-2553 (whose element composition and specific sequence are shown in Table 4 below) with the ispA upstream homology arm, promoter (PRO397), RBS (DNA5336), and ispA downstream homology arm (i.e., the first 500 bp of nucleotides of the ispA gene coding frame); the pTargetF plasmid with the gdhA cleavage site; the DNA sequence ADD-2553 (whose element composition and specific sequence are shown in Table 4 below) with the gdhA upstream homology arm, promoter J23100, RBS (DNA1918), and a Pseudescherichia vulneris), the terminator rrnB_T2, and the DNA sequence ADD-2236 of the downstream homologous arm of gdhA (its element composition and specific sequence are shown in Table 5 below) were co-transformed into BOTA01002, the promoter of the ispA gene was replaced by the pTrc promoter, RBS was added in front of the ispA gene, and a gene crtE encoding geranylgeranyl pyrophosphate synthase from Escherichia vulneris was added at the gdhA site to obtain strain BOTA01007.

[0348] Table 2: ADD-1977 element composition and specific sequence

[0349] ADD-1977 name SEQ ID NO: Element 1 adhE upstream homology arm 1 Element 2 pa promoter 2 Element 3 crtE_PSEVU 3 Element 4 RBS(DNA11666) 4 Element 5 idi_PSEVU 5 Element 6 crtY_PSEVU 6 Element 7 crtI_PSEVU 7 Element 8 crtB_PSEVU 8 Element 9 Pa terminator 9 Element 10 adhE downstream homology arm 10

[0350] Table 3: Element composition and specific sequence of expression cassette in BOTA01002

[0351]

[0352]

[0353] Table 4: ADD-2553 component composition and specific sequence

[0354] ADD-2553 name SEQ ID NO: Element 1 ispA upstream homology arm 14 Element 2 Promoter (PRO397) 15 Element 3 RBS(DNA5336) 16 Element 4 ispA downstream homology arm 17

[0355] Table 5: ADD-2236 component composition and specific sequence

[0356] ADD-2236 name SEQ ID NO: Element 1 gdhA upstream homology arm 18 Element 2 J23100 promoter 19 Element 3 RBS(DNA1918) 12 Element 4 crtE_PSEVU 3 Element 5 rrnB_T2 terminator 20 Element 6 gdhA downstream homology arm 21

[0357] (2) Experimental results

[0358] The transformants obtained in (1) were cultured and the β-carotene production was detected. The transformation information of the strains and the β-carotene production are shown in Table 6. The β-carotene expression cassette driven by the pa promoter was inserted into the C43 (DE3) genome, and the resulting strain BOTA01004 did not detect the production of β-carotene. After replacing the pa promoter with the strong promoter pTrc promoter, the obtained strain BOTA01005 and its IPCC-lost strain BOTA01002 were able to produce 10 mg / L β-carotene. On this basis, the strain BOTA01007 was obtained in which the promoter of ispA was replaced with a strong promoter and the gene crtE encoding geranylgeranyl pyrophosphate synthase from Escherichia wounding was overexpressed in the genome, which was able to produce 20 mg / L β-carotene.

[0359] The transformation information of the strains in Example 1 and the β-carotene production of each transformed strain are summarized in Table 6 below.

[0360] Table 6: Transformation information of strains and β-carotene production

[0361]

[0362] Example 2: Optimizing the expression of genes related to β-carotene synthesis by promoter-ribosome binding site library

[0363] The genes related to β-carotene synthesis involve the expression of at least four genes, namely, the gene crtE encoding geranylgeranyl pyrophosphate synthase, the gene crtB encoding phytoene synthase, the gene crtI encoding phytoene dehydrogenase, and the gene crtY encoding lycopene cyclase.

[0364] In this embodiment, promoter-ribosome binding site library strategy is used to express co-regulation of 4 genes related to beta-carotene synthesis: crtB derived from Pseudescherichia vulneris, crtI derived from Pseudescherichia vulneris, crtY derived from Pseudescherichia vulneris and crtE derived from Pseudescherichia vulneris. The whole expression cassette is driven by pTrc promoter, and each gene has 6 possible RBS ribosome binding site sequences, and 6 possible RBS have different translation strengths. SOE PCR is used to amplify the upstream homology arm of IS6 site, the downstream homology arm and the promoter, 4 genes and terminator into a fragment with a fragment size of about 5000bp.

[0365] First, the strength of each RBS on protein translation efficiency was calculated using the RBS calculator. The calculation results are shown in Table 7 below.

[0366] Table 7: The strength of RBS in β-carotene synthesis-related genes on protein translation efficiency

[0367]

[0368] Design primers for each gene amplification, and the primer sequences are shown in Table 8 below.

[0369] Table 8: Primers for amplification of genes related to β-carotene synthesis

[0370]

[0371]

[0372] First, the upstream primers for amplifying each gene were mixed and PCR amplified together with the downstream primers. PCR was performed using 2xphanta max master mix. The method was as follows: start: 95°C for 30 seconds; 30 cycles: 95°C for 15 seconds, 60°C for 15 seconds, 72°C for 30 seconds / kb; final extension: 72°C for 5 minutes. The amplified PCR products were recovered by gel excision, mixed according to a molar ratio of 1:1:1:1, and added to the PCR reaction system, with the upstream primer pCT043-pCT048 mixed primer as the upstream primer and pCT079 as the downstream primer, to obtain a library fragment with a length of 5504bp. Gel excision was performed and the concentration was determined by Nanodrop.

[0373] The library fragment and IPCC555 with IS6 site were co-transformed into BOTA01007, and the transformants were selected for culture detection. The β-carotene content produced by the transformants was significantly improved, and the highest clone β-carotene production increased from 20 mg / L to 50 mg / L, an increase of 2.5 times. The registration information of the strain with the highest β-carotene production is BOTA01008, and its genotype insertion information is ADD-3479. The element composition and specific sequence of ADD-3479 are shown in Table 9 below.

[0374] Table 9: ADD-3479 component composition and specific sequence

[0375] ADD-3479 name SEQ ID NO: Element 1 IS6 upstream homology arm 23 Element 2 pTrc promoter 24 Element 3 RBS(DNA5378) 25 Element 4 crtB_PSEVU 8 Element 5 RBS(DNA1764) 26 Element 6 crtI_PSEVU 7 Element 7 RBS(DNA5358) 27 Element 8 crtY_PSEVU-2 28 Element 9 RBS(DNA1040) 29 Element 10 crtE_PSEVU 3 Element 11 BBa_B1008 terminator 30 Element 12 IS6 downstream homology arm 31

[0376] Example 3: Increasing the MVA pathway to increase the β-carotene content of Escherichia coli

[0377] Using BOTA01008 as the base bacteria, the expression cassette ADD-3585 of the MVA pathway related gene was integrated into the IS10 site by means of CRISPR / Cas9, and the expression cassette included from the 5' end to the 3' end: (1) M1-93 promoter, (2) RBS (DNA1774), (3) a gene sequence encoding hydroxymethylglutaryl-CoA synthetase, wherein the hydroxymethylglutaryl-CoA synthetase was hydroxymethylglutaryl-CoA synthetase BWK52_2116c derived from Lactobacillus paracasei, and the sequence was obtained after codon optimization in Escherichia coli, (4) RBS (DNA1035), (5) a gene sequence encoding 3-hydroxy-3-methylglutaryl-CoA reductase, wherein the 3-hydroxy-3-methylglutaryl-CoA reductase was derived from Lactobacillus paracasei. paracasei), the sequence was obtained after codon optimization of Escherichia coli, (6) RBS (DNA1038), (7) a gene sequence encoding acetyl-CoA acyltransferase, the acetyl-CoA acyltransferase being acetyl-CoA acyltransferase atoB from Escherichia coli, and (8) BBa_B1008 terminator. The element composition and specific sequence of ADD-3585 are shown in Table 10 below.

[0378] Table 10: ADD-3585 component composition and specific sequence

[0379] ADD-3585 name SEQ ID NO: Element 1 IS10 upstream homology arm 33 Element 2 M1-93 promoter 34 Element 3 RBS(DNA1774) 35 Element 4 BWK52_2116c_LACPA 36 Element 5 RBS(DNA1035) 37 Element 6 BWK52_2117c_LACPA 38 Element 7 RBS(DNA1038) 39 Element 8 atoB_ECOLI 40 Element 9 BBa_B1008 terminator 30 Element 10 IS10 downstream homology arm 41

[0380] On the basis of the above transformation, the expression cassette ADD-2872 of mevalonate synthesis-related genes was integrated into the ZapB site by CRISPR / Cas9. The expression cassette included from the 5' end to the 3' end: (1) M1-93 promoter, (2) RBS (DNA1774), (3) a gene sequence encoding mevalonate kinase, wherein the mevalonate kinase was mevalonate kinase ERG12 from Saccharomyces cerevisiae, and the sequence was obtained after codon optimization in Escherichia coli, (4) RBS (DNA1038), (5) a gene sequence encoding phosphomevalonate kinase, wherein the phosphomevalonate kinase was from Streptococcus pneumoniae. pneumoniae), the sequence was obtained after codon optimization in E. coli, (6) RBS (DNA1764), (7) a gene sequence encoding mevalonate diphosphate decarboxylase, the mevalonate diphosphate decarboxylase was mevalonate diphosphate decarboxylase mvaD from Streptococcus pneumoniae, the sequence was obtained after codon optimization in E. coli, (8) RBS (DNA1035), (9) a gene sequence encoding isoprene pyrophosphate isomerase, the isoprene pyrophosphate isomerase was isoprene pyrophosphate isomerase idi from Saccharomyces cerevisiae, (10) BBa_B1008 terminator. The element composition and specific sequence of ADD-2872 are shown in Table 11 below. The resulting strain is BOTA01009. The β-carotene production in BOTA01009 was detected to be increased to 100-114 mg / L.

[0381] Table 11: ADD-2872 component composition and specific sequence

[0382] ADD-2872 name SEQ ID NO: Element 1 ZapB upstream homology arm 43 Element 2 M1-93 promoter 34 Element 3 RBS(DNA1774) 35 Element 4 ERG12_YEAST 44 Element 5 RBS(DNA1038) 39 Element 6 mvaK2_STRR6 45 Element 7 RBS(DNA1764) 26 Element 8 mvaD_STRP2 46 Element 9 RBS(DNA1035) 37 Element 10 idi_YEAST 47 Element 11 BBa_B1008 terminator 30 Element 12 ZapB downstream homology arm 48

[0383] Example 4: Construction of Escherichia coli producing zeaxanthin

[0384] Using BOTA01009 as the base strain, crtZ (the expression cassette is ADD-3706, the element composition and specific sequence are shown in Table 12 below) driven by pTrc from Pantoea ananas (Erwinia uredovora) was inserted into the flgA site of the genome by CRIPSP / Cas9, and the resulting strain was BOTA01010, which can produce 33 mg / L zeaxanthin and 40 mg / L β-carotene.

[0385] Table 12: ADD-3706 component composition and specific sequence

[0386]

[0387]

[0388] Using BOTA01009 as the base strain, crtZ (expression cassette is ADD-4578, element composition and specific sequence are shown in Table 13 below) derived from Pseudescherichia vulneris driven by pTrc was inserted into the SS3 site of the genome by CRIPSP / Cas9, and the resulting strain was BOTA01011, which can produce 30 mg / L zeaxanthin and 50 mg / L β-carotene.

[0389] Table 13: ADD-4578 component composition and specific sequence

[0390] ADD-4587 name SEQ ID NO: Element 1 SS3 upstream homology arm 60 Element 2 Promoter (PRO397) 15 Element 3 RBS(DNA2260) 55 Element 4 crtZ_PSEVU-1 61 Element 5 BBa_B1002 terminator 52 Element 6 SS3 downstream homology arm 62

[0391] Using BOTA01009 as the base strain, crtZ (expression cassette ADD-3710, element composition and specific sequence shown in Table 14 below) from Escherichia coli was inserted into the flgA site to obtain the strain BOTA01012, which can produce 30 mg / L zeaxanthin and 40 mg / L β-carotene. The crtZ sequence was optimized according to the Escherichia coli codon.

[0392] Table 14: ADD-3710 component composition and specific sequence

[0393]

[0394]

[0395] Using BOTA01009 as the base bacteria, the promoter-ribosome binding site library of co-regulated expression of crtY from Pantoea ananas and crtZ from Pseudescherichia vulneris was inserted at the SS9 site, and about 80 transformants were selected to detect the yield of zeaxanthin. Among them, the clone BOTA01013 with the highest yield (its expression cassette is ADD-5748, and the element composition and specific sequence are shown in Table 15 below) produced 40 mg / L zeaxanthin and 50 mg / L β-carotene.

[0396] Table 15: ADD-5748 component composition and specific sequence

[0397] ADD-5748 name SEQ ID NO: Element 1 SS9 upstream homology arm 50 Element 2 pTrc promoter 24 Element 3 RBS(DNA11670) 64 Element 4 crtZ_PANAN-1 51 Element 5 RBS(DNA11671) 65 Element 6 crtY_PSEVU 28 Element 7 BBa_B1002 terminator 52 Element 8 SS9 downstream homology arm 53

[0398] The results showed that adding a copy of crtY while expressing crtZ could increase the production of zeaxanthin and β-carotene at the same time.

[0399] Example 5: Multi-copy crtZ insertion increases zeaxanthin production

[0400] Using BOTA01013 as the host bacteria, the crtZ gene derived from Enterobacteriaceae bacterium DC416 driven by pTrc was inserted into the ldhA site, the ribosome binding site strengths were calculated, and 71k and 3.4k were selected, respectively, to obtain strains BOTA01014 (whose expression cassette was ADD-5224, and the element composition and specific sequence were shown in Table 16 below) and BOTA01016 (whose expression cassette was ADD-5221, and the element composition and specific sequence were shown in Table 17 below), which were able to produce 60 mg / L β-carotene, 60 mg / L zeaxanthin; and 40 mg / L β-carotene, 40 mg / L zeaxanthin, respectively.

[0401] Table 16: ADD-5224 component composition and specific sequence

[0402] ADD-5224 name SEQ ID NO: Element 1 ldhA upstream homology arm 67 Element 2 Promoter (PRO397) 15 Element 3 RBS(DNA1775) 68 Element 4 crtZ_ENTBA 69 Element 5 BBa_B1002 terminator 52 Element 6 ldhA downstream homology arm 70

[0403] Table 17: ADD-5221 component composition and specific sequence

[0404] ADD-5221 name SEQ ID NO: Element 1 ldhA upstream homology arm 67 Element 2 Promoter (PRO397) 15 Element 3 RBS(DNA2260) 55 Element 4 crtZ_ENTBA 69 Element 5 BBa_B1002 terminator 52 Element 6 ldhA downstream homology arm 70

[0405] Using BOTA01013 as the host bacteria, a promoter-ribosome binding site expression library containing multiple crtZ from different bacterial species was inserted into the IGRD33 site, and 80 transformants were selected for detection. The detection found that clone BOTA01017 (whose expression cassette was ADD-7342, and the element composition and specific sequence were shown in Table 18 below) could produce 65 mg / L zeaxanthin, and there was no accumulation of β-carotene, and clone BOTA01018 (whose expression cassette was ADD-7343, and the element composition and specific sequence were shown in Table 19 below) could produce 71 mg / L zeaxanthin, and there was accumulation of 17 mg / L β-carotene.

[0406] Table 18: ADD-7342 component composition and specific sequence

[0407]

[0408]

[0409] Table 19: ADD-7343 component composition and specific sequence

[0410]

[0411]

[0412] The above results indicate that the co-expression of crtZ sequences from multiple species can improve the conversion efficiency of β-carotene to zeaxanthin compared with a single crtZ gene.

[0413] Example 6: Co-regulation of crtY, crtE, crtI, crtB promoter-ribosome binding site to increase zeaxanthin production

[0414] Using BOTA01010 as the host, a promoter-ribosome binding site library was constructed for crtY, crtE, crtI, and crtB according to the method of Example 2, and integrated into other sites in the genome, that is, one copy of each of the crtY, crtE, crtI, and crtB genes was added. 80 transformants were selected for testing, and the clone with the highest yield produced 60 mg / L zeaxanthin and 60 mg / L β-carotene, and the strain was numbered BOTA01019.

[0415] Example 7: Development of zeaxanthin fermentation process

[0416] Preparation of fermentation broth:

[0417] First level 250ml shake bottle fermentation

[0418] Culture medium: TB culture medium. Filling volume is 20%, inoculation volume is 0.6% v / v, shake flask speed is 200 rpm, culture at 30°C for 8-10 hours.

[0419] 2L fermentation tank fermentation conversion to produce zeaxanthin

[0420] The initial fermentation medium formula is: 30g / L glycerol, 25.6g / L disodium hydrogen phosphate, 8g / L potassium dihydrogen phosphate, 1.0g / L sodium chloride, 2.2g / L ammonium chloride, 0.49g / L magnesium sulfate heptahydrate, 10mL / L TMS solution, 10mL / L vitamin solution. The inoculation amount is 10% v / v, the stirring speed is 300-1200rpm, the ventilation ratio is 1.0VVM, and the dissolved oxygen control is set to 30%. The pH of the fermentation process is controlled at 6.8 with 5M ammonia water. At 30°C, the carbon source in the initial culture medium is exhausted after 28-32 hours of cultivation, and feeding begins. The feed medium formula is: 800g / L glycerol, 9.4g / L ammonium sulfate, 5g / L magnesium chloride hexahydrate, and 10mL / L TMS solution. During the fed-batch culture, a constant dissolved oxygen feeding control strategy is adopted, and the dissolved oxygen control value is 30%. The fermentation is terminated after 60 hours of fed-batch culture. TMS solution formula: 10g / L ferrous sulfate heptahydrate, 5.25g / L zinc sulfate heptahydrate, 3.0g / L copper sulfate pentahydrate, 0.5g / L manganese sulfate, 0.2g / L boric acid, 2.0g / L calcium chloride, 0.2g / L sodium molybdate dihydrate. Vitamin solution formula: 0.1g / L biotin, 0.1g / L choline chloride, 0.1g / L folic acid, 0.1g / L inositol, 0.2g / L nicotinamide, 0.1g / L calcium pantothenate, 0.1g / L pyridoxal hydrochloride, 0.1g / L thiamine hydrochloride, 0.01g / L riboflavin, 8.5g / L sodium chloride. The test found that BOTA01019 can produce 1.15g / L zeaxanthin, and its zeaxanthin content is 9.4mg / g. Strain BOTA01016 can produce 1.1-1.2g / L zeaxanthin and 120mg / L β-carotene. Strain BOTA01017 can produce 1.3g / L zeaxanthin and 88.94mg / L β-carotene, and its zeaxanthin content is 13.02mg / g.

[0421] Example 8: Zeaxanthin structure identification

[0422] The zeaxanthin produced by the modified strain in the above-mentioned embodiment (for example, BOTA01019 obtained in Example 6) was subjected to chromatogram detection with a zeaxanthin standard sample (purchased from Aike Reagent Co., Ltd.). The detection method was the same as the detection method for β-carotene and zeaxanthin in the above-mentioned embodiment. The detection results were as follows: Figure 1 As shown, the same peak time indicates that zeaxanthin with the same structure is detected. Compared with the standard sample, the zeaxanthin produced by the modified strain of the present application is all 3R, 3'R-all-trans zeaxanthin, and does not contain 3R, 3'S-meso zeaxanthin.

[0423] 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. An expression cassette for expressing β-carotene, comprising the following elements: (1) Promoter; (2) a polynucleotide encoding phytoene synthase; (3) a polynucleotide encoding phytoene dehydrogenase; (4) a polynucleotide encoding a lycopene cyclase; (5) a polynucleotide encoding geranylgeranyl pyrophosphate synthase; and (6) terminator; in, The elements are directly connected to each other; preferably, the elements are directly connected to each other in sequence from the 5' end to the 3' end; Preferably, the 5' ends of the polynucleotide encoding phytoene synthase, the polynucleotide encoding phytoene dehydrogenase, the polynucleotide encoding lycopene cyclase, and the polynucleotide encoding geranylgeranyl pyrophosphate synthase are each independently connected to one or more ribosome binding sites (RBS); Preferably, the phytoene synthase, phytoene dehydrogenase, lycopene cyclase and geranylgeranyl pyrophosphate synthase are independently derived from Enterobacter agglomerans, Pseudescherichia vulneris, Escherichia coli, Lactobacillus paracasei, Saccharomyces cerevisiae, Streptococcus pneumoniae, Pantoea ananas, or Enterobacteriaceae bacterium.

2. The expression cassette of claim 1, comprising the following elements in order from the 5' end to the 3' end: (1) Promoter; (2) RBS; (3) a polynucleotide encoding phytoene synthase; (4) RBS; (5) a polynucleotide encoding phytoene dehydrogenase; (6) RBS; (7) a polynucleotide encoding a lycopene cyclase; (8) RBS; (9) a polynucleotide encoding geranylgeranyl pyrophosphate synthase; and (10) terminator; Preferably, the sequence of the promoter is shown in any one of SEQ ID NOs: 15, 19, 22, 24, and 34, the sequence of the terminator is shown in any one of SEQ ID NOs: 9, 20, 30, and 52, the sequence of the polynucleotide encoding phytoene synthase is shown in SEQ ID NO: 8, the sequence of the polynucleotide encoding phytoene dehydrogenase is shown in SEQ ID NO: 7, the sequence of the polynucleotide encoding lycopene cyclase is shown in SEQ ID NO: 6 or 28, and the sequence of the polynucleotide encoding geranylgeranyl pyrophosphate synthase is shown in SEQ ID NO: 3; Preferably, the sequences of the RBS in (2), (4), (6) and (8) are shown in SEQ ID NO: 25, 26, 27 and 29 respectively.

3. The expression cassette of claim 1, further comprising a polynucleotide encoding isoprene pyrophosphate isomerase; Preferably, the 5' end of the polynucleotide encoding isoprene pyrophosphate isomerase is linked to one or more RBSs; Preferably, the isoprene pyrophosphate isomerase is derived from Enterobacter agglomerans, Pseudescherichia vulneris, Escherichia coli, Lactobacillus paracasei, Saccharomyces cerevisiae, Streptococcus pneumoniae, Pantoea ananas, or Enterobacteriaceae bacterium; Preferably, the expression cassette comprises, in order from the 5' end to the 3' end: (1) Promoter; (2) RBS; (3) a polynucleotide encoding geranylgeranyl pyrophosphate synthase; (4) 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) terminator; Preferably, the sequence of the promoter is shown in any one of SEQ ID NOs: 15, 19, 22, 24, and 34, the sequence of the terminator is shown in any one of SEQ ID NOs: 9, 20, 30, and 52, the sequence of the polynucleotide encoding phytoene synthase is shown in SEQ ID NO: 8, the sequence of the polynucleotide encoding phytoene dehydrogenase is shown in SEQ ID NO: 7, the sequence of the polynucleotide encoding lycopene cyclase is shown in SEQ ID NO: 6 or 28, the sequence of the polynucleotide encoding geranylgeranyl pyrophosphate synthase is shown in SEQ ID NO: 3, and the sequence of the polynucleotide encoding isoprene pyrophosphate isomerase is shown in SEQ ID NO: 5 or 47; Preferably, the sequences of the RBS in (2) and (4) are shown in SEQ ID NO: 12 and 4 respectively.

4. A combination of expression cassettes for expressing β-carotene, comprising a first expression cassette, and one or more expression cassettes selected from a second expression cassette, a third expression cassette, and a fourth expression cassette, wherein: The first expression cassette is the expression cassette according to any one of claims 1 to 3.

5. The combination of expression cassettes according to claim 4, wherein: The second expression cassette comprises the following elements: (1) Promoter; (2) a polynucleotide encoding hydroxymethylglutaryl-CoA synthetase (HMGCS); (3) a polynucleotide encoding 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR); (4) a polynucleotide encoding acetyl-CoA acyltransferase (AACT); and (5) terminator; Preferably, the elements are directly connected to each other; preferably, the elements are directly connected to each other in sequence from the 5' end to the 3' end; Preferably, the 5' ends of the polynucleotide encoding HMGCS, the polynucleotide encoding HMGR, and the polynucleotide encoding AACT are each independently connected to one or more RBSs; Preferably, the HMGCS, HMGR, and AACT are independently derived from Enterobacter agglomerans, Pseudescherichia vulneris, Escherichia coli, Lactobacillus paracasei, Saccharomyces cerevisiae, Streptococcus pneumoniae, Pantoea ananas, or Enterobacteriaceae bacterium; Preferably, the sequence of the promoter is shown in any one of SEQ ID NOs: 15, 19, 22, 24, and 34, the sequence of the terminator is shown in any one of SEQ ID NOs: 9, 20, 30, and 52, the sequence of the polynucleotide encoding HMGCS is shown in SEQ ID NO: 36, the sequence of the polynucleotide encoding HMGR is shown in SEQ ID NO: 38, and the sequence of the polynucleotide encoding AACT is shown in SEQ ID NO:

40.

6. The combination of expression cassettes according to claim 4, wherein: The third expression cassette comprises the following elements: (1) Promoter; (2) a polynucleotide encoding a gene related to mevalonate synthesis selected from mevalonate kinase (MVK); (3) a polynucleotide encoding phosphomevalonate kinase (PMVK); (4) a polynucleotide encoding mevalonate diphosphate decarboxylase (MVD); and (5) terminator; Preferably, the elements are directly connected to each other; preferably, the elements are directly connected to each other in sequence from the 5' end to the 3' end; Preferably, the 5' ends of the polynucleotide encoding MVK, the polynucleotide encoding PMVK, and the polynucleotide encoding MVD are each independently connected to one or more RBSs; Preferably, the MVK, PMVK, and MVD genes are independently derived from Enterobacter agglomerans, Pseudescherichia vulneris, Escherichia coli, Lactobacillus paracasei, Saccharomyces cerevisiae, Streptococcus pneumoniae, Pantoea ananas, or Enterobacteriaceae bacterium; Preferably, the sequence of the promoter is as shown in any one of SEQ ID NO:15, 19, 22, 24, 34, the sequence of the terminator is as shown in any one of SEQ ID NO:9, 20, 30, 52, the sequence of the polynucleotide encoding MVK is as shown in SEQ ID NO:44, the sequence of the polynucleotide encoding PMVK is as shown in SEQ ID NO:45, and the sequence of the polynucleotide encoding MVD is as shown in SEQ ID NO:

46.

7. The combination of expression cassettes according to claim 4, wherein: The fourth expression cassette comprises the following elements: (1) Promoter; (2) a polynucleotide encoding geranylgeranyl pyrophosphate synthase; and (3) terminator; Preferably, the elements are directly connected to each other; preferably, the elements are directly connected to each other in sequence from the 5' end to the 3' end; Preferably, the 5' end of the polynucleotide encoding geranylgeranyl pyrophosphate synthase is linked to one or more RBSs; Preferably, the geranylgeranyl pyrophosphate synthase is derived from Enterobacter agglomerans, Pseudescherichia vulneris, Escherichia coli, Lactobacillus paracasei, Saccharomyces cerevisiae, Streptococcus pneumoniae, Pantoea ananas, or Enterobacteriaceae bacterium; Preferably, the sequence of the promoter is as shown in any one of SEQ ID NO:15, 19, 22, 24, 34, the sequence of the terminator is as shown in any one of SEQ ID NO:9, 20, 30, 52, and the sequence of the polynucleotide encoding geranylgeranyl pyrophosphate synthase is as shown in SEQ ID NO:

3.

8. The combination of expression cassettes of claim 4, comprising at least two of the first expression cassettes, and comprising the second expression cassette, a third expression cassette and a fourth expression cassette; preferably, the sequences of the at least two first expression cassettes are different from each other.

9. A combination of expression cassettes for expressing β-carotene and zeaxanthin, comprising the expression cassette of any one of claims 1 to 3, or the combination of the expression cassettes of any one of claims 4 to 8, and one or more fifth expression cassettes, wherein the fifth expression cassette comprises the following elements: (1) Promoter; (2) a polynucleotide encoding β-carotene hydroxylase; and (3) terminator; Preferably, the elements are directly connected to each other; preferably, the elements are directly connected to each other in sequence from the 5' end to the 3' end; Preferably, the 5' end of the polynucleotide encoding β-carotene hydroxylase is connected to one or more RBSs; Preferably, the β-carotene hydroxylase is derived from Enterobacter agglomerans, Pseudescherichia vulneris, Escherichia coli, Lactobacillus paracasei, Saccharomyces cerevisiae, Streptococcus pneumoniae, Pantoea ananas, or Enterobacteriaceae bacterium; Preferably, the sequence of the promoter is as shown in any one of SEQ ID NOs: 15, 19, 22, 24, and 34, the sequence of the terminator is as shown in any one of SEQ ID NOs: 9, 20, 30, and 52, and the sequence of the polynucleotide encoding β-carotene hydroxylase is as shown in any one of SEQ ID NOs: 51, 58, 61, 69, 75, and 78; Preferably, the fifth expression cassette further comprises a polynucleotide encoding a lycopene cyclase; Preferably, the 5' end of the polynucleotide encoding lycopene cyclase is linked to one or more RBSs; Preferably, the lycopene cyclase is derived from Enterobacter agglomerans, Pseudescherichia vulneris, Escherichia coli, Lactobacillus paracasei, Saccharomyces cerevisiae, Streptococcus pneumoniae, Pantoea ananas, or Enterobacteriaceae bacterium; Preferably, the sequence of the polynucleotide encoding lycopene cyclase is as shown in SEQ ID NO: 6 or 28; Preferably, the zeaxanthin is all-trans zeaxanthin; Preferably, the zeaxanthin is 3R,3'R-all-trans zeaxanthin.

10. A combination of expression cassettes for expressing zeaxanthin, comprising the combination of expression cassettes according to claim 9, and a sixth expression cassette, wherein the sixth expression cassette comprises the following elements: (1) Promoter; (2) at least two polynucleotides encoding β-carotene hydroxylase; (3) terminator; Preferably, the elements are directly connected to each other; preferably, the elements are directly connected to each other in sequence from the 5' end to the 3' end; Preferably, the at least two polynucleotides encoding β-carotene hydroxylase are connected via an RBS; preferably, the 5' ends of the at least two polynucleotides encoding β-carotene hydroxylase are independently connected to an RBS; Preferably, the at least two β-carotene hydroxylases are derived from different species, the species being selected from Enterobacter agglomerans, Pseudescherichia vulneris, Escherichia coli, Lactobacillus paracasei, Saccharomyces cerevisiae, Streptococcus pneumoniae, Pantoea ananas, or Enterobacteriaceae bacterium; Preferably, the sequence of the promoter is as shown in any one of SEQ ID NOs: 15, 19, 22, 24, and 34, the sequence of the terminator is as shown in any one of SEQ ID NOs: 9, 20, 30, and 52, and the sequence of the polynucleotide encoding β-carotene hydroxylase is as shown in any one of SEQ ID NOs: 51, 58, 61, 69, 75, and 78; Preferably, the zeaxanthin is all-trans zeaxanthin; Preferably, the zeaxanthin is 3R,3'R-all-trans zeaxanthin.

11. A vector comprising the expression cassette of any one of claims 1 to 3, or a combination of the expression cassettes of any one of claims 4 to 10; Preferably, the vector is a cloning vector or an expression vector; Preferably, in the vector, the 5' end of the expression cassette is connected to the upstream homology arm, and the 3' end of the expression cassette is connected to the downstream homology arm; Preferably, the vector comprises a combination of expression cassettes according to any one of claims 4 to 10, wherein the homology arms of the plurality of expression cassettes are different from each other.

12. A host cell comprising the expression cassette of any one of claims 1 to 3, a combination of expression cassettes of any one of claims 4 to 10, or the vector of claim 11.

13. A recombinant microorganism comprising an expression cassette according to any one of claims 1 to 3, or a combination of expression cassettes according to any one of claims 4 to 10; Preferably, the expression cassette is integrated into the genome of the recombinant microorganism; Preferably, the recombinant microorganism comprises at least two expression cassettes according to any one of claims 1 to 3, or a combination of expression cassettes according to any one of claims 4 to 10, wherein: The different expression cassettes are each independently integrated into different sites of the genome of the recombinant microorganism; Preferably, the recombinant microorganism is capable of producing β-carotene and / or zeaxanthin; Preferably, the recombinant microorganism is selected from fungi or bacteria; Preferably, the fungus is a yeast (e.g., Saccharomyces cerevisiae, Saccharomyces lipolytica, Kluyveromyces); Preferably, the bacteria are one or more selected from Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptococcus, Streptomyces, acetic acid bacteria, propionibacterium and Bifidobacterium.

14. Use of the expression cassette of any one of claims 1 to 3, the combination of the expression cassettes of any one of claims 4 to 10, the vector of claim 11, the host cell of claim 12, or the recombinant microorganism of claim 13 in producing zeaxanthin and / or β-carotene; Preferably, the zeaxanthin is all-trans zeaxanthin; Preferably, the zeaxanthin is 3R,3'R-all-trans zeaxanthin.

15. A method for producing zeaxanthin and / or β-carotene, comprising the following steps: Cultivating the recombinant microorganism according to claim 13 under conditions that allow the growth and metabolism of the microorganism, and recovering zeaxanthin and / or β-carotene from the culture of the cultivated recombinant microorganism; Preferably, the recombinant microorganism is cultured by fermentation, and zeaxanthin and / or β-carotene is recovered from the fermentation product; Preferably, the microorganism is selected from fungi or bacteria; Preferably, the fungus is a yeast (e.g., Saccharomyces cerevisiae, Saccharomyces lipolytica, Kluyveromyces); Preferably, the bacteria are one or more selected from Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptococcus, Streptomyces, acetic acid bacteria, propionibacterium and Bifidobacterium; Preferably, the zeaxanthin is all-trans zeaxanthin; Preferably, the zeaxanthin is 3R,3'R-all-trans zeaxanthin.

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