CN112852764B - Mutant protein of ketonizing enzyme and application - Google Patents

Abstract

Disclosed is a protein which is a mutant protein comprising a substitution of one or more amino acid residues selected from among the 6 th, 12 th, 105 th, 147 th, 152 th and 239 th positions of the amino acid sequence of a beta-carotene ketolase, and which has an improved ability to synthesize canthaxanthin and/or astaxanthin. The invention also provides related biological materials and derivatives of the protein, and application of the related biological materials and derivatives in preparation of canthaxanthin and/or astaxanthin. The astaxanthin yield and the ratio of the astaxanthin content of the protein in the invention in the carotenoid are both greatly improved, in particular to a CrtW mutant with 3 amino acid residue substitutions of A6T, T105A and L239M, and the astaxanthin yield and the ratio of the astaxanthin content in the carotenoid are respectively improved from 0.6 +/-0.10 mg/g cdw and 17 percent of beta-carotene ketolase to 3.21 +/-0.18 mg/g cdw and 92 percent.

Description

Mutant protein of ketonizing enzyme and application

Technical Field

The invention relates to mutant protein of ketolase and application thereof, belonging to the technical field of biology.

Background

Astaxanthin (3, 3 ' -dihydroxy-4, 4' -diketo-beta, beta ' -carotene) is a red carotenoid, which is ubiquitous in the marine environment. Astaxanthin is synthesized by microalgae and phytoplankton which are the primary producers of the marine food chain, and some zooplankton or crustacean such as salmon, shrimp, lobster, etc. cannot synthesize astaxanthin by themselves but accumulate astaxanthin in the body with a slight reddish color by ingestion of the microalgae and phytoplankton. The astaxanthin is added into the feed additive, so that not only can aquatic organisms present bright colors and attract consumers, but also nutrition can be provided for the growth and the propagation of the aquatic organisms, and therefore, the astaxanthin is widely used in the aquaculture industry. Astaxanthin is a natural compound with the strongest antioxidant capacity found in nature at present, and the antioxidant activity of the astaxanthin is 10 times of that of beta-carotene. Pharmacological and physiological studies have also found that astaxanthin has biological activities of antitumor, cardiovascular disease prevention, immunity enhancement, etc., and thus has been widely used in the healthcare industry (Tao, l.a., Wilczek, j., Odom, j.m., Cheng, q.o.,2006, Engineering a beta-carotene ketolase for an astaxanthine production, metabolic engineering.8, 523-531). The appearance of astaxanthin brings revolutionary changes to the antioxidant market, and the astaxanthin is the most potential novel antioxidant preparation.

Natural astaxanthin is found primarily in marine animals and microorganisms in the marine environment. According to the source of astaxanthin, the production modes of astaxanthin are mainly as follows: three methods of chemical synthesis, aquatic product processing and biosynthesis (Ausich, R.L.,1997.Commercial opportunities for fungal production by biotechnology. pure Appl chem.69, 2169-2173). The chemically synthesized astaxanthin has high yield and relatively low price, but the process is complex, the obtained product is a mixture of three stereoisomers, and the antioxidant activity is low. Meanwhile, the animal body has poor absorption capacity for chemically synthesized astaxanthin, and chemically synthesized astaxanthin is much lower than natural astaxanthin in terms of coloring, biological value and the like. The european union committee has only allowed natural astaxanthin as a food pigment and the us FDA has banned the addition of chemically synthesized astaxanthin to food, so that less effective chemically synthesized products are gradually eliminated and banned from use. The aquatic product processing method mainly extracts the shellfish aquatic product wastes such as shrimps, crabs and the like, and the method has the advantages of high cost, low product purity, high ash content and chitin content, low yield, and great environmental pollution caused by the extracted waste liquid, and is only used in a few countries.

Based on the biosynthesis pathway of astaxanthin, research has been carried out on genetic modification of model organisms such as escherichia coli, blue algae, yeast and the like which can not synthesize astaxanthin by using a synthetic biology method, and a series of exogenous astaxanthin synthesis genes are introduced to construct an astaxanthin-producing recombinant strain. Lemuth et al integrated crt series genes into E.coli chromosome, coordinated the ratio of beta-carotene ketolase (CrtW) and hydroxylase (CrtZ) by reducing the expression of crtZ, reduced zeaxanthin production, and constructed E.coli BW-ASTA high yield up to 1.4mg/g DCW (Lemuth, K., Steuer, K., Albermann, C.,2011.Engineering of a plasmid-free Escherichia coli strain for improved in vivo biosynthesis of microorganisms.10, 29).

Disclosure of Invention

The invention aims to solve the technical problem of improving the yield of astaxanthin/canthaxanthin.

In order to solve the above technical problems, the present invention provides a protein which is a mutant protein composed of β -carotene ketolase (CrtW) by substitution of one or more of the following amino acid residues 1) to 6):

1) the mutant protein has a substitution at amino acid residue 6 of the amino acid sequence of the β -carotene ketolase as compared to the β -carotene ketolase;

2) the mutant protein has a substitution at amino acid residue position 12 of the amino acid sequence of the β -carotene ketolase as compared to the β -carotene ketolase;

3) (ii) the mutant protein has a substitution at amino acid residue 105 of the amino acid sequence of the β -carotene ketolase as compared to the β -carotene ketolase;

4) (ii) the mutant protein has a substitution at amino acid residue 147 of the amino acid sequence of the β -carotene ketolase as compared to the β -carotene ketolase;

5) (ii) the mutant protein has a substitution at amino acid residue 152 of the amino acid sequence of the β -carotene ketolase as compared to the β -carotene ketolase;

6) the mutant protein has a substitution at amino acid residue 239 of the amino acid sequence of the β -carotene ketolase compared to the β -carotene ketolase;

the mutant proteins have higher canthaxanthin and/or astaxanthin production than the beta-carotene ketolase.

In the above protein, 1) the substitution at the 6 th amino acid residue in the amino acid sequence of the β -carotene ketolase is a threonine residue; 2) a substitution to a glutamine residue at the 12 th amino acid residue of the amino acid sequence of the β -carotene ketolase; 3) said substitution at amino acid residue 105 of the amino acid sequence of said β -carotene ketolase is a substitution to an alanine residue; 4) a substitution at amino acid residue 147 of the amino acid sequence of said β -carotene ketolase is a substitution to a threonine residue; 5) said substitution at amino acid residue 152 of the amino acid sequence of said β -carotene ketolase is a substitution to a threonine residue; 6) the substitution at the 239 th amino acid residue of the amino acid sequence of the β -carotene ketolase is a substitution of a leucine residue for a methionine residue.

In the above proteins, the beta-carotene ketolase may be derived from any species, e.g., from the genus Pseudomonas, e.g., Brevundimonas.

In the protein, the amino acid sequence of the beta-carotene ketolase is shown as a sequence 1 in a sequence table.

In the above protein, the mutant protein has a sequence in which only one or more amino acid residues selected from the group consisting of the 6 th, 12 th, 105 th, 147 th, 152 th and 239 th positions of the amino acid sequence of the β -carotene ketolase are substituted, and no other amino acid residue is substituted, as compared with the β -carotene ketolase.

In the above protein, the mutant protein is CrtW, MutW1, MutW2 or MutW 18:

the CrtW is a protein of which the amino acid sequence is shown in a sequence 3 in a sequence table;

the mutW1 is obtained by replacing the 105 th amino acid residue of the protein in the sequence 1 in the sequence table with alanine residue, replacing the 239 th amino acid residue with methionine residue and keeping other amino acid residues unchanged;

the MutW2 is obtained by replacing the 147 th amino acid residue of the protein in the sequence 1 in the sequence table with threonine residue and keeping other amino acid residues unchanged;

the MutW18 is obtained by replacing the 6 th amino acid residue of the protein in the sequence 1 in the sequence table with threonine residue, replacing the 12 th amino acid residue with glutamine residue, replacing the 152 th amino acid residue with threonine residue and keeping other amino acid residues unchanged.

The related biological materials of the protein are also in the protection scope of the invention, and the related biological materials are any one of the following B1) to B8):

B1) a nucleic acid molecule encoding the protein;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism comprising the recombinant vector of B4).

In the above-mentioned related biological materials, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA, and the like.

In the related biological material, the recombinant microorganism is escherichia coli, and specifically can be a beta-carotene producing strain.

The derivative of the protein is a fusion protein obtained by fusing tag proteins at the amino terminal or/and the carboxyl terminal of the protein or a fusion protein obtained by connecting a protein with a targeting function and the beta-carotene ketolase.

In the derivatives of the above proteins, the tag protein (protein-tag) refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The tag protein can be Flag tag protein, His6 tag protein, MBP tag protein, HA tag protein, myc tag protein, GST tag protein or SUMO tag protein, etc.

The invention also provides the application of the protein, and/or the related biological material, and/or the derivative in preparing canthaxanthin and/or astaxanthin.

Experiments prove that compared with the original beta-carotene ketolase, the mutant beta-carotene ketolase obtained by replacing a plurality of or one of amino acid residues in the amino acid sequences of the beta-carotene ketolase A6T, P12Q, T105A, M147T, A152T and L239M has greatly improved astaxanthin yield and astaxanthin content in the ratio of carotenoid content, particularly the CrtW mutant with 3 amino acid residues of A6T, T105A and L239M has improved astaxanthin yield from 0.6 +/-0.10 mg/g cdw of the beta-carotene ketolase to 3.21 +/-0.18 mg/g cdw and improved astaxanthin content in the ratio of the carotenoid content from 17 percent to 92 percent of the beta-carotene ketolase. The ketolase mutant protein can greatly improve the yield of astaxanthin.

Drawings

FIG. 1 is a diagram showing screening and evaluation of a mutation in ketolase CrtW in example 1 of the present invention. FIG. 1A is a schematic diagram of astaxanthin synthesis, wherein CrtW is ketolase and CrtZ is hydroxylase; FIG. 1, panel B, is a histogram of canthaxanthin production using canthaxanthin production screening for a mutation in CrtW, where CrtW is an initiating β -carotene ketolase, MutW1, MutW2, MutW3, MutW4, MutW5, MutW6, MutW7, MutW8, MutW9, MutW10, MutW11, MutW12 are ketolase mutants; FIG. 1C is a bar graph showing the evaluation of astaxanthin production by high canthaxanthin-yielding CrtW mutants, wherein CrtW is an initiating β -carotene ketolase, MutW1, MutW2, MutW3, MutW4, MutW5 and MutW6 are ketolase mutants; FIG. 1, panel D, is a bar graph of screening for mutations in CrtW, which is the starting β -carotene ketolase, and MutW13, MutW14, MutW15, MutW16, MutW17, MutW18, which are ketolase mutants, using astaxanthin production.

FIG. 2 shows the effect of the mutation site of ketolase CrtW on astaxanthin synthesis in example 1 of the present invention. CrtW is a starting β -carotene ketolase; A6T, P12Q, T105A, M147T, A152T and L239M are mutants of single point mutation of ketolase; CrtW is a highly active mutant containing the A6T, T105A and L239M mutations.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

1. Bacterial strains

In the following examples, the strain M1-46 is described in the non-patent literature "Lu, J., Tang, J.L., Liu, Y., Zhu, X.N., Zhang, T.C., Zhang, X.L.,2012.combinatorial modification of galP and glk gene expression for improved expression. applied Microbiol Biotechnol.93,2455-2462", publicly available from the Tianjin Industrial Biotech institute for repeating the experiments of the present application, and is not useful for other applications.

In the examples described below, the Trans10 competent cells are available from Beijing Quanjin Biotechnology Ltd.

In the following examples, the strain CAR005 is a β -carotene Engineering strain described in non-patent literature "Zhao, j., Li, q.y., Sun, t., Zhu, x.n., Xu, h.t., Tang, j.l., Zhang, x.l., Ma, y.h.,2013.Engineering Central metabolism modules of Escherichia coli for improving β -carotenes production. metabolism engineering.17, 42-50", publicly available from the institute of biotechnology for repeating the experiments of the present application, and is not applicable for other uses.

2. Plasmids

In the following examples, plasmid pACYC184-M2-P12, publicly available from the institute of biotechnology and Industrial science of Tianjin, is described in non-patent document "Daguanping, Miao Liang Tian, Sutao, Li Qingyan, Xiaodong Guang, Zhang Li, 2015. Metabolic engineering of Escherichia coli to produce coenzyme Q₁₀Biology engineeringReport. 2015, 31 (2): 206- "219", publicly available from the institute for biotechnology in Tianjin industry for repeating the experiments of this application and not for other uses.

In the examples described below, plasmid pSC101 is publicly available from the institute for Biotechnology in the Tianjin industry, described in the non-patent document "Bernardi, A., Bernardi, F.,1984.Complete sequence of pSC101.nucleic acids research.12, 9415-26", and publicly available from the institute for Biotechnology in the Tianjin industry, for the purpose of repeating the experiments of the present application, and is not useful for other applications.

In the following examples, plasmid pXZ-CS is publicly available from the institute of Biotechnology in the Tianjin industry and is described in non-patent documents "Tan, Z., Zhu, X., Chen, J., Li, Q., Zhang, X.,2013.Activating phosphor enzyme in synthesis for improvement of secretion process.applied and environmental microbiology.79,4838-44", publicly available from the institute of Biotechnology in the Tianjin industry for repeating the experiments of the present application and is not available for other uses.

In the examples described below, plasmid pKD46 is a product of the CGSC E.coli Collection of Yale university, USA.

In the following examples, plasmid pACYC184-M-crt is publicly available from the institute for Biotechnology in the Tianjin industry and described in non-patent documents "ZHao, J., Li, Q.Y., Sun, T., Zhu, X.N., Xu, H.T., Tang, J.L., Zhang, X.L., Ma, Y.H.,2013.Engineering Central metabolism modules of Escherichia coli for improving β -carotene production.metabolism engineering.17, 42-50", which is publicly available from the institute for Biotechnology in the Tianjin industry to repeat the experiments of the present application and is not available for other uses.

3. Reagent

NewEngland Biolabs Phusion 5X buffer, 10XT4 ligation buffer, T4 polynucleotide kinase, T4 ligase (400,000 cohesive end units/ml), Phusion High-Fidelity DNA polymerase were all products of NEB.

PCR purification Kit Wash (Gel/PCR extraction Kit) is a product of BioMIGA Biotechnology Inc.

4. Culture medium

In the following examples, the LB solid medium is a medium made of sodium chloride, peptone, yeast extract, agar and water, and the contents of sodium chloride, peptone, yeast extract and agar are as follows: 10g/L sodium chloride, 10g/L peptone, 5g/L yeast extract and 15g/L agar.

In the following examples, the LB liquid medium is a medium made of sodium chloride, peptone, yeast extract and water, and the contents of sodium chloride, peptone and yeast extract are as follows: 10g/L sodium chloride, 10g/L peptone and 5g/L yeast extract.

LB solid medium containing chloramphenicol (final concentration of 34. mu.g/ml) was a medium made of sodium chloride, peptone, yeast extract, agar, chloramphenicol and water, and the contents of sodium chloride, peptone, yeast extract, agar, chloramphenicol were as follows: 10g/L sodium chloride, 10g/L peptone, 5g/L yeast extract, 15g/L agar, 34. mu.g/ml chloramphenicol.

LB liquid medium containing chloramphenicol (final concentration of 34. mu.g/ml) was a medium made of sodium chloride, peptone, yeast extract, chloramphenicol and water, and the contents of sodium chloride, peptone, yeast extract, chloramphenicol were as follows: 10g/L sodium chloride, 10g/L peptone, 5g/L yeast extract, 34. mu.g/ml chloramphenicol.

LB solid medium containing ampicillin (final concentration of 50. mu.g/ml) and chloramphenicol (final concentration of 34. mu.g/ml) was a medium made of sodium chloride, peptone, yeast extract, agar, ampicillin, chloramphenicol and water, and the contents of sodium chloride, peptone, yeast extract, agar, ampicillin and chloramphenicol were as follows: 10g/L sodium chloride, 10g/L peptone, 5g/L yeast extract, 15g/L agar, 50. mu.g/ml ampicillin, 34. mu.g/ml chloramphenicol.

The LB liquid medium (sodium chloride free) containing 10% sucrose was a medium made of sucrose, peptone, yeast extract and water, and the contents of sucrose, peptone and yeast extract were as follows: 10 percent of sucrose, 10g/L of peptone and 5g/L of yeast extract.

LB solid Medium (sodium chloride free) containing 6% sucrose A medium made of sucrose, peptone, yeast extract, agar and water, the contents of sucrose, peptone, yeast extract, agar being as follows: 6 percent of sucrose, 10g/L of peptone, 5g/L of yeast extract and 15g/L of agar by mass percentage.

In the following examples, unless otherwise specified, the first nucleotide of each nucleotide sequence is the 5 'terminal nucleotide of the corresponding DNA, and the last nucleotide is the 3' terminal nucleotide of the corresponding DNA.

Example 1

The major problem limiting astaxanthin production is the low activity of beta-carotene ketolase (CrtW) on the substrate beta-carotene or zeaxanthin (see panel a in fig. 1). Therefore, the invention mainly constructs and screens the mutation of CrtW to obtain the mutation with the best catalytic efficiency, so that the metabolic flow can maximally flow to the astaxanthin metabolic pathway, and the astaxanthin yield can be improved to meet the requirement of industrial production. The specific process is as follows:

1. construction of pSC102-crtW plasmid

1.1, construction of pACYC184-M2-Pm46 plasmid

PCR amplification was performed with the genome of M1-46 strain as DNA template, using the primer pair FRT-PacI/M46-down-PmeI:

FRT-PacI：5’-CAATAATTAAGTGTAGGCTGGAGCTGCTTCGAAG-3’；

M46-down-PmeI：5’-CAAGTTTAAACCGAATTGGTGGGGCGAGAG-3’。

the amplification system is as follows: new England Biolabs Phusion 5 Xbuffer 10U l, dNTP 1U l (each dNTP 10mM), DNA template 20ng, primers (10U M) 2U l, Phusion High-Fidelity DNA polymerase (2.5U/. mu.l) 0.5U l, distilled water 33.5U l, total volume 50U l.

Amplification conditions were 98 ℃ pre-denaturation for 2 min (1 cycle); denaturation at 98 ℃ for 10 seconds, annealing at 56 ℃ for 10 seconds, and extension at 72 ℃ for 30 seconds (30 cycles); extension at 72 ℃ for 5min (1 cycle).

The PCR amplification product was digested with restriction enzymes PacI and PmeI, ligated with plasmid pACYC184-M2-P12 digested with the same enzymes, and the ligation product was transformed into Trans10 competent cells by calcium chloride transformation: add 50. mu.l Trans10 competent cells, ice-wash for 30min, heat-shock at 42 ℃ for 30 sec, immediately place on ice for 2 min, add 250. mu.l LB liquid medium, incubate at 200rpm, 37 ℃ for 1 h. The transformed cells were plated on LB solid medium plates containing chloramphenicol (final concentration: 34. mu.g/ml), cultured overnight at 37 ℃, and single colonies were picked to extract plasmid DNA, verified by restriction with restriction enzymes PacI and PmeI, and analyzed by sequencing. Sequencing results will contain M1-46 sequence between PacI and PmeI sites, and other sequences of pACYC184-M2-P12 vector are kept unchanged, and the obtained recombinant expression vector is named pACYC184-M2-Pm 46.

1.2 construction of constitutive Low copy plasmid pSC102

The pSC102 plasmid comprises the coding sequence of replication initiation site ori and replication protein RepA of low-copy plasmid pSC101 and constitutive artificial regulatory promoter element M1-46, and the specific process is as follows:

1.2.1 PCR amplification with primer pair 101ori-up/101ori-down using the DNA of the low copy plasmid pSC101 as template:

101ori-up：5’-GCGCCTGTAGTGCCATTT-3’；

101ori-down：5’-GCGCTATTTCTTCCAGAATTG-3’。

the amplification system is as follows: new England Biolabs Phusion 5 Xbuffer 10U l, dNTP 1U l (each dNTP 10mM), DNA template 20ng, primers (10U M) 2U l, Phusion High-Fidelity DNA polymerase (2.5U/. mu.l) 0.5U l, distilled water 33.5U l, total volume 50U l.

Amplification conditions were 98 ℃ pre-denaturation for 2 min (1 cycle); denaturation at 98 ℃ for 10 seconds, annealing at 56 ℃ for 10 seconds, and extension at 72 ℃ for 30 seconds (30 cycles); extension at 72 ℃ for 5min (1 cycle).

PCR amplification yielded a DNA fragment I containing the replication initiation site ori of pSC101 and the coding sequence of the replication protein RepA.

1.2.2, using plasmid pACYC184-M2-Pm46 obtained in step 1.1 as a template, and performing PCR amplification by using a primer pair ori-Cm-up/ori-AP 1-down.

ori-Cm-up：

5’-TGGCAATTCTGGAAGAAATAGCGCTGTGACGGAAGATCACTTCG-3’；

ori-AP1-down：

5’-TGGGGGTAAATGGCACTACAGGCGCTTATCTCTGGCGGTGTTGAC-3’。

The amplification system is as follows: new England Biolabs Phusion 5 Xbuffer 10U l, dNTP 1U l (each dNTP 10mM), DNA template 20ng, primers (10U M) 2U l, Phusion High-Fidelity DNA polymerase (2.5U/. mu.l) 0.5U l, distilled water 33.5U l, total volume 50U l.

Amplification conditions were 98 ℃ pre-denaturation for 2 min (1 cycle); denaturation at 98 ℃ for 10 seconds, annealing at 56 ℃ for 10 seconds, and extension at 72 ℃ for 30 seconds (30 cycles); extension at 72 ℃ for 5min (1 cycle).

And obtaining a DNA fragment II by PCR amplification, wherein the DNA fragment II contains a resistance gene cat and a constitutive artificial regulatory element M1-46.

1.2.3, cleaning the DNA fragment I obtained in step 1.2.1 and the DNA fragment II obtained in step 1.2.2, and then performing PCR ligation by using CPEC technology (Quan, J., Tian, J.,2009.Circular polymerase extension of complex gene libraries and pathwalls. plos one.4, e 6441). Taking 1 μ l of the product after CPEC ligation, transforming Tans10 competent cells by adopting a calcium chloride transformation method: DNA fragment I and DNA fragment II were added to 50. mu.l of Trans10 competent cells, ice-washed for 30 minutes, heat-shocked at 42 ℃ for 30 seconds, immediately placed on ice for 2 minutes, added to 250. mu.l of LB liquid medium, and incubated at 200rpm and 37 ℃ for 1 hour. The transformed cells were plated on LB solid medium plates containing chloramphenicol (final concentration: 34. mu.g/ml), cultured overnight at 37 ℃, and 5 clones were selected for strain PCR with primers 101ori-up/101ori-down, extracted for plasmid sequencing analysis. The plasmid with the correct sequence was designated pSC102 (standard for correct sequence: pSC102 plasmid contains the replication origin ori of the low copy plasmid pSC101 and the coding sequence for the replication protein RepA and the constitutive artificial regulatory promoter elements M1-46).

1.3 construction of plasmid pSC102-CrtW

The crtW gene (nucleotide sequence shown as sequence 2 in the sequence listing) containing the F222L mutation based on the crtW gene (GeneBank No: AB181388, 17-JUN-2020) of Brevmdimonas sp.SD212 was synthesized by King-Shamir organism as the starting crtW gene in this example. The amino acid sequence of the protein beta-carotene ketolase (CrtW) which is the starting beta-carotene ketolase of the embodiment, beta-carotene ketolase (CrtW) is shown as a sequence 1 in a sequence table. PCR amplification is carried out by using a primer pair 46-RBS-up/crtW-SacI-down by using a crtW gene with an amino acid sequence of a sequence 2 in a sequence table as a template.

46-RBS-up：5’-AGCTTTGTTTAAACCAGGAAACAGCTATGAC-3’；

crtW-SacI-down：5’-GCATGAGCTCTTACGATTCACCACGCCAC-3’。

The amplification system is as follows: new England Biolabs Phusion 5 Xbuffer 10U l, dNTP 1U l (each dNTP 10mM), DNA template 20ng, primers (10U M) 2U l, Phusion High-Fidelity DNA polymerase (2.5U/. mu.l) 0.5U l, distilled water 33.5U l, total volume 50U l.

Amplification conditions were 98 ℃ pre-denaturation for 2 min (1 cycle); denaturation at 98 ℃ for 10 seconds, annealing at 56 ℃ for 10 seconds, and extension at 72 ℃ for 30 seconds (30 cycles); extension at 72 ℃ for 5min (1 cycle).

The PCR amplification product is cut by restriction enzymes PmeI and SacI to obtain a crtW gene fragment, and simultaneously the plasmid pSC102 obtained in the step 1.2.3 is cut by restriction enzymes PmeI and SacI to obtain a plasmid pSC102 fragment. The crtW gene fragment and the pSC102 plasmid fragment were ligated with T4 DNA ligase for 2h at 25 ℃. Then, the Trans10 competent cells were transformed by calcium chloride transformation: the ligation product was added to 50. mu.l of Trans10 competent cells, ice-washed for 30 minutes, heat-shocked at 42 ℃ for 30 seconds, immediately placed on ice for 2 minutes, added to 250. mu.l of LB liquid medium, incubated at 200rpm, and at 37 ℃ for 1 hour. The transformed cells were plated on LB solid medium plate containing chloramphenicol (final concentration: 34. mu.g/ml) and cultured overnight at 37 ℃.5 clones were picked, subjected to strain PCR with primer AP1-up/crtW-down200, extracted for plasmid sequencing analysis:

AP1-up：5’-TTATCTCTGGCGGTGTTGAC-3’；

crtW-down200：5’-GGTGCACGTCCGGCAAATCT-3’。

the recombinant expression vector with the nucleotide sequence shown as sequence 2 in the sequence table is named pSC102-CrtW by replacing the fragment between the recognition sites of restriction endonucleases PmeI and SacI (a small fragment including the recognition site of PmeI and the recognition site of SacI) of the pSC102 vector with the starting crtW gene, and keeping other sequences of the pSC102 vector unchanged.

2. Screening of ketolase CrtW dominant mutations by production of canthaxanthin

2.1 construction of beta-Carotene producing Strain YL-CAR002

Firstly, a beta-carotene production strain needs to be constructed to be used for constructing zeaxanthin production strains and platform bacteria of a CrtW ketolase mutant plasmid library. The construction of the beta-carotene production strain starts from a beta-carotene engineering strain CAR005, a two-step homologous recombination method is adopted to knock out a crtX gene (GenBank No: JX876608, 17-DEC-2012) to obtain recombinant Escherichia coli YL-CAR002, and the method specifically comprises the following 4 steps:

2.1.1, PCR amplification with primers crtX-cat-sacB-up/crtX-cat-sacB-down using pXZ-CS plasmid as template:

crtX-cat-sacB-up：

5’-ACCATTTTGTTCAGGCCTGGTTTGAGAAAAAACTCGCTGCCGTCAGTTAATGTGACGGAAGATCACTTCGCA-3’；

crtX-sacB-down：

5’-AGCCCGTTGGCCAGTCCCGCGCCCACCAGAATCAGATCATACCGCGGCATTTATTTGTTAACTGTTAATT-3’。

the amplification system is as follows: new England Biolabs Phusion 5 Xbuffer 10U l, dNTP 1U l (each dNTP 10mM), DNA template 20ng, primers (10U M) 2U l, Phusion High-Fidelity DNA polymerase (2.5U/. mu.l) 0.5U l, distilled water 33.5U l, total volume 50U l.

Amplification conditions were 98 ℃ pre-denaturation for 2 min (1 cycle); denaturation at 98 ℃ for 10 seconds, annealing at 56 ℃ for 10 seconds, and extension at 72 ℃ for 30 seconds (30 cycles); extension at 72 ℃ for 5min (1 cycle).

The DNA fragment 1 of 2718bp was obtained by PCR amplification. The DNA fragment 1 comprises a cat-sacB DNA fragment and homologous recombination fragments of about 50bp which knock out the crtX gene.

DNA fragment 1 was used for the first homologous recombination: plasmid pKD46 was first transformed into the engineered beta-carotene strain CAR005 by calcium chloride transformation, and then DNA fragment 1 was electrically transformed into the engineered beta-carotene strain CAR005 harboring plasmid pKD 46.

The electrotransformation and screening process comprises the following steps: first, an electrotransformation competent cell of the β -carotene engineering strain CAR005 carrying the plasmid pKD46 was prepared by the method referred to "Dower, w.j., Miller, j.f., Ragsdale, c.w.,1988.High efficiency transformation of e.coli by High voltage electrolysis. nucleic Acids res.16, 6127-45"; mu.l of electrotransformation competent cells of the β -carotene engineering strain CAR005 carrying the plasmid pKD46 were placed on ice, 50ng of the DNA fragment 1 were added, placed on ice for 2 minutes and transferred to a 0.2cm Bio-Rad cuvette. A MicroPulser (Bio-Rad) electroporator was used with a shock parameter of 2.5 kv. After the electric shock, 1ml of LB liquid medium was quickly transferred to an electric shock cup, and after 5 blows, transferred to a test tube, and incubated at 75rpm and 30 ℃ for 2 hours. 200. mu.l of the incubated bacterial solution was applied to a plate containing LB solid medium containing ampicillin (final concentration: 50. mu.g/ml) and chloramphenicol (final concentration: 34. mu.g/ml), and after overnight culture at 30 ℃,5 single colonies were selected for PCR verification using primers cat-up/crtW-YZ-down:

cat-up：5’-ATGAAACCGCTGATTGCATCTA-3’；

crtW-YZ-down：5’-TTACGATTCACCACGCCACAG-3’。

the single colony that was verified to contain DNA fragment 1 by sequencing was designated YL-CAR 002C.

2.1.2 reverse PCR amplification with plasmid pACYC184-M-crt DNA as template DNA and primers ant-crtX-F/ant-crtX-R:

ant-crtX-F：5’-AAGGAGATATACC ATGCCGCGGTATGATCTG-3’；

ant-crtX-R：5’-TTAACTGACGGCAGCGAG-3’。

the amplification system is as follows: new England Biolabs Phusion 5 Xbuffer 10U l, dNTP 1U l (each dNTP 10mM), DNA template 20ng, primers (10U M) 2U l, Phusion High-Fidelity DNA polymerase (2.5U/. mu.l) 0.5U l, distilled water 33.5U l, total volume 50U l.

Amplification conditions were 98 ℃ pre-denaturation for 2 min (1 cycle); denaturation at 98 ℃ for 10 seconds, annealing at 56 ℃ for 10 seconds, and extension at 72 ℃ for 30 seconds (30 cycles); extension at 72 ℃ for 5min (1 cycle).

A8657 bp PCR product was obtained, which did not contain the crtX gene.

2.1.3, phosphorylating the 8657bp PCR product obtained in the step 2.1.2, wherein the 8657bp PCR product can be self-ligated without enzyme digestion, and the plasmid obtained by self-ligation is used for the second homologous recombination, and the specific steps are as follows: the 8657bp PCR product is firstly cleaned by a PCR purification kit, 30ng of the PCR amplification product after cleaning and purification is taken, 2 mu l of 10XT4 connecting buffer solution and 1 mu l T4 polynucleotide kinase are added, distilled water is supplemented to 20 mu l, and the reaction is carried out for 30 minutes at 37 ℃; mu. l T4 ligase (400,000 cohesive end units/ml) was added and the reaction was carried out at room temperature for 2 hours to obtain a ligated product. The ligation products were transferred to Trans10 competent cells by calcium chloride transformation: add 10. mu.l ligation product to 50. mu.l Trans10 competent cells and ice-wash for 30 min; heat shock was performed at 42 ℃ for 30 seconds, immediately placed on ice for 2 minutes, and 250. mu.l of LB liquid medium was added thereto, and incubated at 37 ℃ for 1 hour. And (3) coating 200 mu l of bacterial liquid on an LB solid medium plate containing chloramphenicol (the final concentration is 34 mu g/ml), after overnight culture, selecting 5 positive single colonies, carrying out liquid culture on positive clones, and extracting positive clone plasmids for sequencing verification. The sequencing result proved that the PCR amplification product of step 2.1.2 was self-ligated and the plasmid was correctly constructed, and the correct plasmid (not containing the crtX gene) was named pACYC 184-M-crtEYIB.

2.1.4, using DNA of plasmid pACYC184-M-crtEYIB as template, and crt-cluster-F/crt-cluster-R as primer to make amplification:

crt-cluster-F：

5’-AATTCAAGGAGATATACCATGATGACGGTCTGTGCAGAA-3’；

crt-cluster-R：

5’-GCAGTCGACGCTGCGAGAACGTCA-3’。

the 4510bp DNA fragment 2 was obtained by amplification.

DNA fragment 2 was used for the second homologous recombination and was electrotransformed into strain YL-CAR002C obtained in step 2.1.1. Electrotransformation and screening processes: firstly, preparing an electrotransformation competent cell of YL-CAR002C carrying pKD46 plasmid; mu.l of electroporation competent cells of YL-CAR002C harboring pKD46 plasmid were placed on ice, 50ng of DNA fragment 2 were added, placed on ice for 2 min and transferred to a 0.2cm Bio-Rad cuvette. The electroporation apparatus was used in accordance with the MicroPluser (Bio-Rad) method, and the electric shock parameter was 2.5 kv. After electric shock, 1ml of LB liquid medium was quickly transferred to a cuvette, and after 5 blows, transferred to a test tube, 75 rotations, and incubated at 30 ℃ for 4 hours. The culture broth was transferred to LB liquid medium (sodium chloride-free) containing 10% sucrose, 50ml of the medium was placed in a 250ml flask, and after 24 hours of culture, streaked on LB solid medium (sodium chloride-free) containing 6% sucrose. The single clone was verified by PCR with the primer crt-cluster-F/crt-cluster-R, and the single colony in which the DNA fragment 2 was verified by sequencing was named YL-CAR 002.

2.2 construction of CrtW mutant pools

The method comprises the following specific steps:

2.2.1, error-prone PCR was performed using the pSC102-CrtW plasmid constructed in step 1 as a template and a primer set 46-RBS-up/crtW-SacI-down (the primer sequence was the same as in step 1.3).

Error-prone PCR reaction system: taq PCR Buffer 10 Xbuffer 5. mu.l, dNTP (10mM) 1. mu.l, dATP (10mM) 2. mu.l, dTTP (10mM) 2. mu.l, MnCl₂Mu.l (25mM), 1. mu.l each of primers (10. mu.M), 1. mu.l of DNA template (20 ng/. mu.l), 1. mu.l of Taq DNA polymerase (2.5U/. mu.l), and the balance water, in a total volume of 50. mu.l.

Error-prone PCR amplification conditions: pre-denaturation at 94 ℃ for 3min (1 cycle); denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, and extension at 72 ℃ for 1min (30 cycles); extension at 72 ℃ for 10 min (1 cycle).

And performing error-prone PCR amplification to obtain a CrtW mutant library.

2.2.2, cleaning the CrtW mutant library obtained in the step 2.2.1, then cutting the CrtW mutant library by restriction enzymes PmeI and SacI to obtain a CrtW mutant library fragment, and cutting the plasmid pSC102 constructed in the step 1.2 by the restriction enzymes PmeI and SacI to obtain a plasmid pSC102 fragment. And (3) connecting the CrtW mutant library fragment and the plasmid pSC102 fragment for 2h at 25 ℃ by using T4 DNA ligase, wherein the connected product is the CrtW mutant library.

2.3 screening of CrtW mutations by production of canthaxanthin

1ul of the pool of CrtW mutants obtained in step 2.2 was transformed into YL-CAR002 (constructed in step 2.1) electroporation competent cells. The transformed cells were plated on LB solid medium plate containing chloramphenicol (final concentration: 34. mu.g/ml) and grown overnight at 37 ℃. The pool volume was 140,000 CFU/. mu.g DNA, the colony color of the strain was orange-red, while the starting strain containing CrtW (YL-CAR 002 containing pSC 102-CrtW) was yellow.

The colony color was observed, and the redder the colony color, the higher the canthaxanthin, 105 clones were initially screened with the naked eye from 7000 single clones. The 105 strains are subjected to canthaxanthin yield analysis by adopting a carotenoid content analysis method (comprising astaxanthin, canthaxanthin, zeaxanthin and the like), and the starting bacterium CrtW (pSC 102-CrtW-containing YL-CAR002) is used as a control, wherein the carotenoid content analysis method comprises the following specific steps:

(1) seed culture: the monoclonal to be tested was picked up, inoculated into a small test tube (15 mm. times.100 mm) containing 3ml of LB liquid medium containing chloramphenicol (final concentration: 34. mu.g/ml), and cultured overnight at 30 ℃ and 250rpm to obtain a seed solution for inoculation into the fermentation medium.

(2) And (3) amplification culture: the seed culture solution was inoculated in 50ml of LB liquid medium (250ml triangular flask) at an inoculum size of 1% (V/V) and cultured at 37 ℃ for 48 hours at 250 rpm.

(3) The analysis method comprises the following steps: centrifuging 2ml of the bacterial liquid cultured in the step (2) at 13,000rpm for 3min to collect thalli, cleaning the thalli with sterilized water, adding 750 mu l of an extracting agent (liquid formed by mixing acetonitrile, methanol and dichloromethane according to a volume ratio of 21: 21: 8), and carrying out ultrasonic extraction on ice bath for 30 min; centrifuging at 13,000rpm for 3min, collecting the supernatant in a new centrifuge tube, adding 750 μ l of the extract into the thallus precipitate, and repeating ultrasonic extraction; combining the corresponding test tube extracts, and centrifuging at 13,000rpm for 3 min; the supernatant was filtered through a 0.22 μm organic filter and analyzed for carotenoid content using High Performance Liquid Chromatography (HPLC).

HPLC detection conditions: a DAD detector, Symmetry C18 column (250 mm. times.4.6 mm, 5 μm) was used, the column incubator was controlled at 30 ℃ and the gradient mobilities (mobile phase A and mobile phase B) were mixed at a flow rate of 0.8 ml/min. The mobile phase A is a mixed organic solvent (liquid formed by mixing acetonitrile, methanol and dichloromethane according to the volume ratio of 21: 21: 8), and the mobile phase B is a methanol water solution with the volume percentage of 10%. Taking the liquid obtained by mixing the mobile phase A and the mobile phase B as a mobile phase to carry out gradient elution, wherein the conditions are as follows:

mobile phase A: 80 to 100 percent (0 to 18min) by volume, 100 to 80 percent (more than 18 to 25min) by volume and 80 percent (more than 25 to 30min) by volume; mobile phase B: the volume percentage content is 20 to 0 percent (0 to 18min), 0 to 20 percent (more than 18 to 25min) and 20 percent (more than 25 to 30 min). The amount of each sample was 20. mu.l, the detection time was 30 minutes, and the detection wavelength was 476 nm.

12 strains with improved canthaxanthin yield are obtained by co-screening 105 strains, and corresponding mutations are MutW1, MutW2, MutW3, MutW4, MutW5, MutW6, MutW7, MutW8, MutW9, MutW10, MutW11 and MutW12, wherein the results of re-screening the 12 strains by using CrtW as a control (the strains marked as CrtW in B, C and D in figure 1) are shown in figure 1B, wherein the 6 mutations of MutW1, MutW2, MutW3, MutW4, MutW5 and MutW6 have higher canthaxanthin yield which is 1.2-1.6 times that of the control CrtW.

3. Screening of ketolase CrtW dominant mutations by production of astaxanthin

3.1 construction of zeaxanthin producer YL-Z004

From the beta-carotene producing strain YL-CAR002 constructed in the step 2, the crtZ gene (GenBank No: ADU76136, 09-JAN-2011) from Pantoea agglomerans is integrated on the mgsA locus on the chromosome by a two-step homologous recombination method to obtain the zeaxanthin producing strain, which specifically comprises the following steps:

3.1.1, using the DNA of YL-CAR002C strain constructed in step 2.1.1 as template DNA, PCR amplification was performed with primers mgsA-cat-up/mgsA-sacB-down:

mgsA-cat-up：

5’-TTTCGGTCTTTATCTTGCAGCGATAAGTGCTTACAGTAATCTGTAGGAAATGTGACGGAAGATCACTTCGCA-3’；

mgsA-sacB-down：

5’-AAAACCGTAAGAAACAGGTGGCGTTTGCCACCTGTGCAATATTACTTCAGACGGTCCGCGTTATTTGTTAACTGTTAATTGTCCT-3’。

the amplification system is as follows: new England Biolabs Phusion 5 Xbuffer 10U l, dNTP 1U l (each dNTP 10mM), DNA template 20ng, primers (10U M) 2U l, Phusion High-Fidelity DNA polymerase (2.5U/. mu.l) 0.5U l, distilled water 33.5U l, total volume 50U l.

Amplification conditions were 98 ℃ pre-denaturation for 2 min (1 cycle); denaturation at 98 ℃ for 10 seconds, annealing at 56 ℃ for 10 seconds, and extension at 72 ℃ for 30 seconds (30 cycles); extension at 72 ℃ for 5min (1 cycle).

The amplified DNA fragment (i) has a size of about 3Kb and comprises a 50bp sequence homologous to the upstream of ATG of mgsA gene, a cat-sacB DNA fragment and a 50bp sequence homologous to the downstream of TAA of mgsA gene.

The DNA fragment (r) was used for the first homologous recombination and was electroporated into E.coli YL-CAR002 (constructed in step 2.1) harboring pKD 46. The electrotransformation and screening process comprises the following steps: first, an electrotransformation competent cell of E.coli YL-CAR002 carrying plasmid pKD46 was prepared by the method described in "Dower, W.J., Miller, J.F., Ragsdale, C.W.,1988.High efficiency transformation of E.coli by High voltage electrophoresis. nucleic Acids Res.16, 6127-45"; mu.l of electrotransformation competent cells of E.coli YL-CAR002 carrying plasmid pKD46 were placed on ice, 50ng of DNA fragment (r) were added, placed on ice for 2 minutes and transferred to a 0.2cm Bio-Rad cuvette. A MicroPulser (Bio-Rad) electroporator was used with a shock parameter of 2.5 kv. After the electric shock, 1ml of LB liquid medium was quickly transferred to an electric shock cup, and after 5 blows, transferred to a test tube, and incubated at 75rpm and 30 ℃ for 2 hours. 200. mu.l of the incubated bacterial suspension was applied to a plate containing LB solid medium containing ampicillin (final concentration: 50. mu.g/ml) and chloramphenicol (final concentration: 34. mu.g/ml), and after overnight culture at 30 ℃,5 single colonies were selected for PCR verification. PCR validation was performed using primers cat-up/mgsA-down:

cat-up：5’-ATGAAACCGCTGATTGCATCTA-3’；

mgsA-down：5’-AAAAGCCGTCACGTTATTG-3’

the PCR contained a 700bp band, which was the resulting strain YL-Z004C.

3.1.2, PCR amplification was performed using the genome of Pantoea agglomerans as a template and the primers mgsA-crtZ-up/mgsA-crtZ-down:

mgsA-crtZ-up：

5’-TTTCGGTCTTTATCTTGCAGCGATAAGTGCTTACAGTAATCTGTAGGAAAATGTTGTGGATTTGGAATG-3’；

mgsA-crtZ-down：

5’-AAAACCGTAAGAAACAGGTGGCGTTTGCCACCTGTGCAATATTACTTCAGACGGTCCGCGTTACTTCCCGGGTGGCG-3’。

the amplification system is as follows: new England Biolabs Phusion 5 Xbuffer 10U l, dNTP 1U l (each dNTP 10mM), DNA template 20ng, primers (10U M) 2U l, Phusion High-Fidelity DNA polymerase (2.5U/. mu.l) 0.5U l, distilled water 33.5U l, total volume 50U l.

Amplification conditions were 98 ℃ pre-denaturation for 2 min (1 cycle); denaturation at 98 ℃ for 10 seconds, annealing at 56 ℃ for 10 seconds, and extension at 72 ℃ for 30 seconds (30 cycles); extension at 72 ℃ for 5min (1 cycle).

Amplifying to obtain DNA segment. DNA fragment (c) contains upstream homologous 50bp sequence of mgsA gene, crtZ gene and downstream homologous 50bp sequence of mgsA gene.

The DNA fragment (c) was used for the second homologous recombination and was electrotransformed into YL-CAR002C (constructed in step 2.1.1) containing pKD 46. The electrotransformation and screening process comprises the following steps: first, an electrotransformation competent cell of E.coli YL-CAR002C carrying plasmid pKD46 was prepared, as described in "Dower, W.J., Miller, J.F., Ragsdale, C.W.,1988.High efficiency transformation of E.coli by High voltage electrophoresis. nucleic Acids Res.16, 6127-45"; mu.l of electrotransformation competent cells of E.coli YL-CAR002C harboring plasmid pKD46 were placed on ice, 50ng of DNA fragment 2 min added, and transferred to a 0.2cm Bio-Rad cuvette. A MicroPulser (Bio-Rad) electroporator was used with a shock parameter of 2.5 kv. After the electric shock, 1ml of LB liquid medium was quickly transferred to an electric shock cup, and after 5 blows, transferred to a test tube, and incubated at 75rpm and 30 ℃ for 2 hours. 200. mu.l of the incubated bacterial suspension was applied to a plate containing LB solid medium containing ampicillin (final concentration: 50. mu.g/ml) and chloramphenicol (final concentration: 34. mu.g/ml), and after overnight culture at 30 ℃,5 single colonies were selected for PCR verification. PCR verification was performed using primers mgsA-up/crtZ-KpnI-down:

mgsA-up：5’-CAGCTCATCAACCAGGTCAA-3’；

crtZ-KpnI-down：5’-CGGGGTACCTTACTTCCCGGATGCGGGCTC-3’。

the strain which was verified to contain the DNA fragment (c) was named YL-Z004.

3.2 evaluation of astaxanthin production by the MutW1-6 mutation

The mutant MutW1, MutW2, MutW3, MutW4, MutW5, MutW6 and the original CrtW plasmid with high canthaxanthin yield screened in the step 2 are respectively transformed into an electric transformation competent cell of Escherichia coli YL-Z004 (constructed in the step 3.1) to obtain 6 recombinant strains and an original strain. The astaxanthin production was analyzed for these strains in the same manner as for the carotenoid content in step 2.3.

Astaxanthin production is shown in FIG. 1, panel C, and astaxanthin production was improved in all of the 6 recombinant strains. Wherein the astaxanthin yield of MutW1 is 2.76mg/g dcw, which accounts for 79% of the total carotenoids; the content of MutW2 astaxanthin was 78% of the total carotenoids (see table 2).

3.3 screening of the CrtW mutation by production of astaxanthin

Mu.l of the pool of CrtW mutants obtained in step 2.2 was electroporated into the electroporation competent cells of YL-Z004 (constructed in step 3.1.2). The transformed cells were plated on LB solid medium plates containing chloramphenicol (final concentration: 34. mu.g/ml), grown overnight at 37 ℃ and had a pool volume of 2,000,000 CFU/. mu.g DNA.

The colony color was observed, and astaxanthin production was higher in terms of redder colony color, and 59 clones were visually primary-screened from 10 ten thousand single clones. The 59 strains were analyzed for astaxanthin production using YL-Z004 containing plasmid pSC102-CrtW as a control, in step 2.3 for carotenoid content, to obtain 6 mutants of MutW13, MutW14, MutW15, MutW16, MutW17 and MutW18 with improved astaxanthin production. MutW18 produced the highest amount of astaxanthin, reaching 2.59mg/g DCW, with an astaxanthin content of 74% of the total carotenoids (see FIG. 1, panel D).

4. Mutation site analysis of CrtW ketolase mutant

For gene sequencing analysis of the mutations MutW1, MutW2 and MutW18, the information on nucleotide changes and amino acids at the mutation sites of the CrtW mutant (MutW1) contained in MutW1, the CrtW mutant (MutW2) contained in MutW2-YL-Z004 and the CrtW mutant (MutW18) contained in MutW18-YL-Z004 are summarized in Table 1, relative to the starting crtW gene of sequence 2 in the sequence Listing, and the encoded CrtW (sequence 1 in the sequence Listing, starting beta-carotene ketolase) thereof:

the recombinant plasmid pSC102-MutW1 contains mutant MutW1, is a recombinant vector obtained by replacing the CrtW gene of pSC102-CrtW with the MutW1 gene and keeping other nucleotides of pSC102-CrtW unchanged; the MutW1 gene is a mutant gene obtained by replacing the nucleotide at position 165 with C, the nucleotide at position 313 with G, the nucleotide at position 507 with A, the nucleotide at position 522 with G, and the nucleotide at position 717 with A in the CDS (SEQ ID NO: 2) of the CrtW gene, while keeping the other nucleotides of CrtW unchanged.

The recombinant plasmid pSC102-MutW2 contains mutant MutW2, is a recombinant vector obtained by replacing the CrtW gene of pSC102-CrtW with the MutW2 gene and keeping other nucleotides of pSC102-CrtW unchanged; the mutW2 gene is a mutant gene obtained by replacing the 24 th nucleotide of the CDS (SEQ ID NO: 2) of the CrtW gene with C, the 61 st nucleotide with T, the 75 th nucleotide with A, the 285 th nucleotide with C, the 440 th nucleotide with C, and the 502 th nucleotide with T, while keeping the other nucleotides of CrtW unchanged.

The recombinant plasmid pSC102-MutW18 contains mutant MutW18, is a recombinant vector obtained by replacing the CrtW gene of pSC102-CrtW with the MutW18 gene and keeping other nucleotides of pSC102-CrtW unchanged; the mutW18 gene is a mutant gene obtained by replacing the 16 th nucleotide of CDS (SEQ ID NO: 2) of the CrtW gene with A, the 35 th nucleotide with A, the 234 th nucleotide with A, and the 454 th nucleotide with A, while keeping the other nucleotides of CrtW unchanged.

According to table 1, MutW1 has 2 non-synonymous mutations and 3 synonymous mutations; MutW2 has 1 nonsynonymous mutation and 3 synonymous mutations; MutW18 has 3 non-synonymous mutations and 1 synonymous mutation.

TABLE 1 sequence analysis of CrtW mutants

5. Ketolase CrtW single mutation site analysis

According to the mutation information of table 1, 6 non-synonymous mutations A6T, P12Q, T105A, M147T, a152T and L239M were introduced into the crtW gene, respectively. The specific process is as follows:

5.1 construction of plasmids containing A6T Single-Point mutation

Taking the DNA (starting crtW gene with a sequence 2 in a sequence table, and coded CrtW as starting beta-carotene ketolase with a sequence 1 in the sequence table) of the pSC102-CrtW plasmid constructed in the step 1 as a template, and carrying out reverse PCR amplification on A6T-up/A6T-down by using a primer pair:

A6T-up：5’-ATGACCGCCGCAGTCACAGAACCGC-3’；

A6T-down：5’-TGACTGCGGCGGTCATAGCTGTTTCCTG-3’。

amplification systems and amplification conditions reference was made to the amplification systems and amplification conditions of step 1.1. The amplified product is treated with DpnI, and after enzyme linking, a plasmid containing A6T single point mutation is obtained.

5.2 construction of plasmid containing P12Q Single-Point mutation

And (2) performing reverse PCR amplification by using the DNA of the pSC102-CrtW plasmid constructed in the step 1 as a template DNA and using a primer pair P12Q-up/P12Q-down:

P12Q-up：5’-AACCGCGTATTGTCCAGCGTCAAACCT-3’；

P12Q-down：5’-TGGACAATACGCGGTTCTGCGACTGC-3’。

amplification systems and amplification conditions reference was made to the amplification systems and amplification conditions of step 1.1. The amplified product is treated with DpnI, and after enzyme linking, a plasmid containing a P12Q single point mutation is obtained.

5.3 construction of plasmid containing T105A Single-Point mutation

And (2) performing reverse PCR amplification by using the DNA of the pSC102-CrtW plasmid constructed in the step 1 as a template DNA and using a primer pair T105A-up/T105A-down:

T105A-up：5’-TTCGATCGCCTGAAAGCCGCACATC-3’；

T105A-down：5’-CTTTCAGGCGATCGAAACGAAAACCG-3’。

amplification systems and amplification conditions reference was made to the amplification systems and amplification conditions of step 1.1. The amplified product was treated with DpnI and enzymatically ligated to obtain a plasmid containing the single point mutation of T105A.

5.4 construction of plasmid containing M147T Single Point mutation

And (2) performing reverse PCR amplification by using the DNA of the pSC102-CrtW plasmid constructed in the step 1 as a template DNA and using a primer pair M147T-up/M147T-down:

M147T-up：5’-TCGGCTGGCGCGAAACGGCAGTCC-3’；

M147T-down：5’-GTTTCGCGCCAGCCGAAGTAGGTACGG-3’。

amplification systems and amplification conditions reference was made to the amplification systems and amplification conditions of step 1.1. The amplified product was treated with DpnI and enzymatically ligated to obtain a plasmid containing the M147T single point mutation.

5.5 construction of plasmid containing A152T Single-Point mutation

Taking the DNA of the pSC102-CrtW plasmid constructed in the step 1 as a template DNA, and performing inverse PCR amplification by using a primer pair A152T-up/A152T-down:

A152T-up：5’-ATGGCAGTCCTGACGACTCTGGTTCTG-3’；

A152T-down：5’-TCGTCAGGACTGCCATTTCGCGCC-3’。

amplification systems and amplification conditions reference was made to the amplification systems and amplification conditions of step 1.1. The amplified product was treated with DpnI and enzymatically ligated to obtain a plasmid containing the A152T single point mutation.

5.6 construction of plasmid containing L239M Single-Point mutation

And (2) performing reverse PCR amplification by using the DNA of the pSC102-CrtW plasmid constructed in the step 1 as a template DNA and using a primer pair L239M-up/L239M-down:

L239M-up：5’-CGTCCGTGGTGGCGTATGTGGCGTG-3’；

L239M-down：5’-TACGCCACCACGGACGCCACGGGG-3’。

amplification systems and amplification conditions reference was made to the amplification systems and amplification conditions of step 1.1. The amplified product is treated by DpnI, and after enzyme connection, a plasmid containing the L239M single-point mutation is obtained.

6. High activity ketolase CrtW mutation

Combining mutation sites by using a site-directed mutagenesis technology, introducing the A6T mutation for improving the yield of astaxanthin into a mutant gene of MutW1(T105A and L239M), and the specific method comprises the following steps: PCR amplification was performed using pSC102-MutW1 DNA containing the MutW1 mutation as a template and primers A6T-up/A6T-down, and the amplification system and amplification conditions were referred to the amplification system and amplification conditions of step 1.1. The amplified product was treated with DpnI and transformed to give a plasmid containing the A6T, T105A and L239M mutations, designated pSC 102-crtW. pSC102-crtW contains crtW gene with nucleotide sequence of sequence 4, and the amino acid sequence of the encoded mutant CrtW is sequence 3 of the sequence table.

Plasmids containing controls and mutations (CrtW, MutW1, Mut2, MutW18, CrtW) were transformed into YL-Z004 and the effect of the mutations on astaxanthin production was evaluated. Astaxanthin content analysis was performed according to the carotenoid content analysis method of step 2.3, and compared with the astaxanthin content of the starting control, and the results are shown in table 2 and fig. 2.

TABLE 2 evaluation of astaxanthin production by ketolase CrtW mutant

The mutant CrtW had an astaxanthin production of 3.21mg/g cdw, the astaxanthin content representing 92% of the total carotenoid production. The CrtW combined mutations A6T, T105A and L239M are high-activity ketonizing enzymes, and can obviously improve the yield of astaxanthin.

The main strains and plasmids constructed in this example are shown in tables 3 and 4, respectively.

Table 3 Main strains constructed in this example

Name of Strain	Related features
		YL-CAR002C	CAR005，ΔcrtX::cat-sacB
YL-CAR002	CAR005，ΔcrtX
		YL-Z004C	YLCAR002，ΔmgsA:::cat-sacB
YL-Z004	YLCAR002，mgsA:37-crtZ

TABLE 4 major plasmids constructed in this example

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

<120> ketonizing enzyme mutant protein and use thereof

<130> GNCSY202054

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<170> SIPOSequenceListing 1.0

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Met Thr Ala Ala Val Ala Glu Pro Arg Ile Val Pro Arg Gln Thr Trp

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Ile Gly Leu Thr Leu Ala Gly Met Ile Val Ala Gly Trp Gly Ser Leu

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His Val Tyr Gly Val Tyr Phe His Arg Trp Gly Thr Ser Ser Leu Val

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Ile Val Pro Ala Ile Val Ala Val Gln Thr Trp Leu Ser Val Gly Leu

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Phe Ile Val Ala His Asp Ala Met His Gly Ser Leu Ala Pro Gly Arg

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Pro Arg Leu Asn Ala Ala Val Gly Arg Leu Thr Leu Gly Leu Tyr Ala

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Gly Phe Arg Phe Asp Arg Leu Lys Thr Ala His His Ala His His Ala

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Ala Pro Gly Thr Ala Asp Asp Pro Asp Phe Tyr Ala Pro Ala Pro Arg

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Ala Phe Leu Pro Trp Phe Leu Asn Phe Phe Arg Thr Tyr Phe Gly Trp

130 135 140

Arg Glu Met Ala Val Leu Thr Ala Leu Val Leu Ile Ala Leu Phe Gly

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Leu Gly Ala Arg Pro Ala Asn Leu Leu Thr Phe Trp Ala Ala Pro Ala

165 170 175

Leu Leu Ser Ala Leu Gln Leu Phe Thr Phe Gly Thr Trp Leu Pro His

180 185 190

Arg His Thr Asp Gln Pro Phe Ala Asp Ala His His Ala Arg Ser Ser

195 200 205

Gly Tyr Gly Pro Val Leu Ser Leu Leu Thr Cys Phe His Leu Gly Arg

210 215 220

His His Glu His His Leu Thr Pro Trp Arg Pro Trp Trp Arg Leu Trp

225 230 235 240

Arg Gly Glu Ser

<210> 2

<211> 735

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgaccgccg cagtcgcaga accgcgtatt gtcccgcgtc aaacctggat tggcctgacc 60

ctggctggta tgattgttgc tggctggggc tccctgcatg tttatggtgt ctactttcat 120

cgttggggca ccagctctct ggtgattgtt ccggcaatcg tcgctgtgca gacgtggctg 180

tcagtgggtc tgtttattgt tgcgcatgat gccatgcacg gttcgctggc gccgggtcgt 240

ccgcgtctga acgcggccgt gggccgcctg accctgggcc tgtatgccgg ttttcgtttc 300

gatcgcctga aaaccgcaca tcacgctcat cacgcagctc cgggtacggc agatgacccg 360

gacttttatg caccggctcc gcgtgctttt ctgccgtggt tcctgaactt tttccgtacc 420

tacttcggct ggcgcgaaat ggcagtcctg acggctctgg ttctgatcgc actgtttggt 480

ctgggtgcac gtccggcaaa tctgctgacc ttctgggcag caccggcact gctgagcgca 540

ctgcagttgt ttaccttcgg cacgtggctg ccgcatcgtc acaccgatca accgtttgca 600

gacgcacatc acgcacgtag ttccggttac ggtccggttc tgtctctgct gacgtgcttc 660

catctgggtc gtcaccatga acatcatctg accccgtggc gtccgtggtg gcgtctgtgg 720

cgtggtgaat cgtaa 735

<210> 3

<211> 244

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Thr Ala Ala Val Thr Glu Pro Arg Ile Val Pro Arg Gln Thr Trp

1 5 10 15

Ile Gly Leu Thr Leu Ala Gly Met Ile Val Ala Gly Trp Gly Ser Leu

20 25 30

His Val Tyr Gly Val Tyr Phe His Arg Trp Gly Thr Ser Ser Leu Val

35 40 45

Ile Val Pro Ala Ile Val Ala Val Gln Thr Trp Leu Ser Val Gly Leu

50 55 60

Phe Ile Val Ala His Asp Ala Met His Gly Ser Leu Ala Pro Gly Arg

65 70 75 80

Pro Arg Leu Asn Ala Ala Val Gly Arg Leu Thr Leu Gly Leu Tyr Ala

85 90 95

Gly Phe Arg Phe Asp Arg Leu Lys Ala Ala His His Ala His His Ala

100 105 110

Ala Pro Gly Thr Ala Asp Asp Pro Asp Phe Tyr Ala Pro Ala Pro Arg

115 120 125

Ala Phe Leu Pro Trp Phe Leu Asn Phe Phe Arg Thr Tyr Phe Gly Trp

130 135 140

Arg Glu Met Ala Val Leu Thr Ala Leu Val Leu Ile Ala Leu Phe Gly

145 150 155 160

Leu Gly Ala Arg Pro Ala Asn Leu Leu Thr Phe Trp Ala Ala Pro Ala

165 170 175

Leu Leu Ser Ala Leu Gln Leu Phe Thr Phe Gly Thr Trp Leu Pro His

180 185 190

Arg His Thr Asp Gln Pro Phe Ala Asp Ala His His Ala Arg Ser Ser

195 200 205

Gly Tyr Gly Pro Val Leu Ser Leu Leu Thr Cys Phe His Leu Gly Arg

210 215 220

His His Glu His His Leu Thr Pro Trp Arg Pro Trp Trp Arg Met Trp

225 230 235 240

Arg Gly Glu Ser

<210> 4

<211> 735

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgaccgccg cagtcacaga accgcgtatt gtcccgcgtc aaacctggat tggcctgacc 60

ctggctggta tgattgttgc tggctggggc tccctgcatg tttatggtgt ctactttcat 120

cgttggggca ccagctctct ggtgattgtt ccggcaatcg tcgccgtgca gacgtggctg 180

tcagtgggtc tgtttattgt tgcgcatgat gccatgcacg gttcgctggc gccgggtcgt 240

ccgcgtctga acgcggccgt gggccgcctg accctgggcc tgtatgccgg ttttcgtttc 300

gatcgcctga aagccgcaca tcacgctcat cacgcagctc cgggtacggc agatgacccg 360

gacttttatg caccggctcc gcgtgctttt ctgccgtggt tcctgaactt tttccgtacc 420

tacttcggct ggcgcgaaat ggcagtcctg acggctctgg ttctgatcgc actgtttggt 480

ctgggtgcac gtccggcaaa tctgctaacc ttctgggcag cgccggcact gctgagcgca 540

ctgcagttgt ttaccttcgg cacgtggctg ccgcatcgtc acaccgatca accgtttgca 600

gacgcacatc acgcacgtag ttccggttac ggtccggttc tgtctctgct gacgtgcttc 660

catctgggtc gtcaccatga acatcatctg accccgtggc gtccgtggtg gcgtatgtgg 720

cgtggtgaat cgtaa 735

Claims (15)

1. A protein characterized by: the protein is a mutant protein formed by mutating beta-carotene ketolase shown as a sequence 1 in a sequence table in the following 1) -9):

1) the mutant protein is obtained by replacing the 6 th amino acid residue of the amino acid sequence of the beta-carotene ketolase with threonine residue and keeping other amino acid residues unchanged;

2) the mutant protein is obtained by replacing the 12 th amino acid residue of the amino acid sequence of the beta-carotene ketolase with glutamine residue and keeping other amino acid residues unchanged;

3) the mutant protein is obtained by replacing the 105 th amino acid residue of the amino acid sequence of the beta-carotene ketolase with alanine residue and keeping other amino acid residues unchanged;

4) the mutant protein is obtained by replacing the 147 th amino acid residue of the amino acid sequence of the beta-carotene ketolase with threonine residue and keeping other amino acid residues unchanged;

5) the mutant protein is obtained by replacing the 152 th amino acid residue of the amino acid sequence of the beta-carotene ketolase with threonine residue and keeping other amino acid residues unchanged;

6) the mutant protein is obtained by replacing the 239 th amino acid residue of the amino acid sequence of the beta-carotene ketolase with methionine residue and keeping other amino acid residues unchanged;

7) the mutant protein is obtained by replacing amino acid residue at position 105 of the amino acid sequence of the beta-carotene ketolase with alanine residue, replacing amino acid residue at position 239 with methionine residue and keeping other amino acid residues unchanged;

8) the mutant protein is obtained by replacing the 6 th amino acid residue of the amino acid sequence of the beta-carotene ketolase with a threonine residue, replacing the 12 th amino acid residue with a glutamine residue, and replacing the 152 th amino acid residue with a threonine residue, and keeping other amino acid residues unchanged;

9) the mutant protein is obtained by replacing the 6 th amino acid residue of the amino acid sequence of the beta-carotene ketolase with threonine residue, replacing the 105 th amino acid residue with alanine residue, replacing the 239 th amino acid residue with methionine residue and keeping other amino acid residues unchanged.

2. The protein of claim 1, wherein: the mutant protein is CrtW, MutW1, MutW2 or MutW 18:

the CrtW is a protein of which the amino acid sequence is shown in a sequence 3 in a sequence table;

the mutW1 is obtained by replacing the 105 th amino acid residue of the protein in the sequence 1 in the sequence table with alanine residue, replacing the 239 th amino acid residue with methionine residue and keeping other amino acid residues unchanged;

the MutW2 is obtained by replacing the 147 th amino acid residue of the protein in the sequence 1 in the sequence table with threonine residue and keeping other amino acid residues unchanged;

the MutW18 is obtained by replacing the 6 th amino acid residue of the protein in the sequence 1 in the sequence table with threonine residue, replacing the 12 th amino acid residue with glutamine residue, replacing the 152 th amino acid residue with threonine residue and keeping other amino acid residues unchanged.

3.A protein-related biomaterial according to any one of claims 1or 2, characterized in that: the relevant biological material is a nucleic acid molecule encoding the protein.

4. A protein-related biomaterial according to any one of claims 1or 2, characterized in that: the relevant biological material is an expression cassette containing a nucleic acid molecule encoding the protein.

5. A protein-related biomaterial according to any one of claims 1or 2, characterized in that: the relevant biological material is a recombinant vector containing a nucleic acid molecule encoding the protein.

6. A protein-related biomaterial according to any one of claims 1or 2, characterized in that: the relevant biological material is a recombinant vector containing an expression cassette of a nucleic acid molecule encoding the protein.

7. A protein-related biomaterial according to any one of claims 1or 2, characterized in that: the relevant biological material is a recombinant microorganism containing a nucleic acid molecule encoding the protein.

8. A protein-related biomaterial according to any one of claims 1or 2, characterized in that: the relevant biological material is a recombinant microorganism containing an expression cassette for a nucleic acid molecule encoding the protein.

9. A protein-related biomaterial according to any one of claims 1or 2, characterized in that: the relevant biological material is a recombinant microorganism containing a recombinant vector containing a nucleic acid molecule encoding the protein.

10. A protein-related biomaterial according to any one of claims 1or 2, characterized in that: the relevant biological material is a recombinant microorganism containing a recombinant vector containing an expression cassette of a nucleic acid molecule encoding the protein.

11. The related biological material according to any one of claims 7 to 10, wherein: the recombinant microorganism is Escherichia coli.

12. A derivative of the protein of any one of claims 1or 2, characterized in that: is a fusion protein obtained by fusing tag protein at the amino terminal or/and the carboxyl terminal of the protein.

13. Use of a protein according to any one of claims 1or 2 for the preparation of canthaxanthin and/or astaxanthin.

14. Use of a related biomaterial according to any one of claims 7 to 11 in the preparation of canthaxanthin and/or astaxanthin.

15. Use of the derivative according to claim 12 for the preparation of canthaxanthin and/or astaxanthin.

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