CN109943493B - Mutant strain for realizing diversity of general enzyme catalytic functions and construction method thereof - Google Patents

Abstract

The invention belongs to the field of microorganisms, and discloses a method for constructing mutant strains of universal enzyme catalytic function diversity. The invention screened and obtained a recombinant Saccharomyces cerevisiae mutant strain that catalyzed two-step dehydrogenation reaction to produce zeta-carotene by BtCrtI, and a strain of lycopene that catalyzed four-step dehydrogenation to produce lycopene and three-step dehydrogenation reaction. A recombinant Saccharomyces cerevisiae mutant strain with a ratio of 5:1. The invention utilizes the recombinant Saccharomyces cerevisiae strain, obtains mutant strains of BtCrtI with different catalytic functions through the nature of random mutation, clarifies the key amino acid sites, realizes the controllability of the dehydrogenation step, enriches the functional information of CrtI, and clarifies the key amino acid sites. Lay the groundwork for the biosynthesis of important natural products.

Description

Mutant strain for realizing diversity of general enzyme catalytic functions and construction method thereof

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to a mutant strain for realizing the diversity of general enzyme catalytic functions and a construction method thereof, in particular to a recombinant saccharomyces cerevisiae mutant strain for generating zeta-carotene by catalyzing two-step dehydrogenation reaction with BtCrtI, a recombinant saccharomyces cerevisiae mutant strain for catalyzing the ratio of lycopene generated by four-step dehydrogenation to neurosporene generated by three-step dehydrogenation reaction to be 5:1, and a construction method thereof.

Background

In the biological metabolic process, a general enzyme which can catalyze multi-step continuous reaction exists. It is statistical that about 20% of enzymes in the biological metabolism can be classified as general enzymes, and play an important role in the life metabolic process of organisms, and especially the functional characteristics of the enzymes catalyzing multi-step reactions can effectively simplify the enzyme system in the organisms. The general enzymes show higher specific gravity in lower organisms, such as metabolic enzymes of Escherichia coli, and the proportion of the general enzymes reaches about 37%. Various types of general enzymes are contained in a biological system, wherein phytoene dehydrogenase PDS and zeta-carotene dehydrogenase ZDS of plant and algae sources can catalyze 2-step continuous dehydrogenation reactions, and phytoene can be catalyzed to generate 4-step continuous dehydrogenation reactions through synergistic action. The dehydrogenase CrtN derived from staphylococcus aureus catalyzes a polyene substrate of C30 in a biosynthesis pathway of the aureochrome to perform 4-step continuous dehydrogenation reaction at most. In the synthetic route of astaxanthin, hydroxylase CrtZ and ketolase CrtW take 2 steps of continuous hydroxylation reaction and 2 steps of continuous carbonylation reaction by taking beta-carotene as a substrate. In addition, there are many of these general enzymes in the hydrolases and synthetases that catalyze multiple sequential reactions.

Phytoene dehydrogenase (CrtI) is a typical representative of the general enzymes catalyzing multi-step sequential reactions, and is also the primary rate-limiting enzyme in the carotenoid synthesis pathway downstream of lycopene. CrtI can catalyze phytoene to generate multi-step continuous dehydrogenation reaction to sequentially generate a plurality of dehydrogenation products with different carbon-carbon double bond saturation degrees, such as neurosporene, lycopene and the like. In the research of catalytic reaction of dehydrogenase CrtI, it is found that CrtI of different biological sources can show different dominant reaction steps in catalytic multi-step dehydrogenation reaction, and dehydrogenation reactions of 3 steps, 4 steps or even 5 steps can occur. For example, Rrhodiobacter-derived CrtI mainly catalyzes 3-step continuous dehydrogenation reaction to generate neurosporene, Erwinia-derived CrtI mainly catalyzes 4-step continuous dehydrogenation reaction to generate lycopene, and Neurospora-derived CrtI catalyzes 5-step continuous dehydrogenation reaction to generate 3, 4-dehydrolycopene. For dehydrogenase CrtI of a specific biological source, the number of multi-step reaction steps has certain randomness, and the composition of the final product is mixed with intermediate products with different dehydrogenation steps.

In recent years, CrtI has not been studied for structural and functional diversity. Erwinia (p.ananatis) CrtI obtained by Dannert and Umeno et al in 2000 was able to catalyze the four-step dehydrogenation reaction to the six-step dehydrogenation reaction of phytoene. In 2001, a Wang and Liao research team utilizes an enzyme directed evolution strategy to mutate CrtI from three-step dehydrorhodobacter sphaeroides (Rheobacter sphaeroides), and the proportion of lycopene, a dehydrogenation product of four steps, is greatly improved through high-throughput screening. The group of Sandmann and Xiao in 2010 also obtained CrtI mutants derived from rhodobacter xylinum (rubivivax gelatinus) with a change in the product ratio from the production of the four-step dehydrogenation product lycopene to the production of the three-step dehydrogenation product neurosporene. In recent years, Schaub and Yu reported the crystal structure (PDB: 4DGK) of CrtI dehycocoenzyme derived from Erwinia (Pantoea ananatis), preliminarily elucidated the basic structural information of CrtI, and speculated the reaction mechanism of CrtI. However, the research is not combined with the activity experiment of the enzyme, and lacks of the structure and function correlation research and verification, which is necessary for the deep research and exploration of CrtI.

CrtI belongs to a flavoprotein family, the catalytic activity can be exerted only after Flavin Adenine Dinucleotide (FAD) is used as a prosthetic group and is assembled with a coenzyme-removed enzyme to form holoenzyme, and at present, the in-vitro assembly efficiency of phytoene dehydrogenase holoenzyme is low, the activity of the enzyme is not ideal, so that the possibility and the accuracy of in-vitro experiments of the holoenzyme are difficult, and the difficulty and the uncertainty of research are increased. Saccharomyces cerevisiae, as a well-known safe mode microorganism, has clear genetic background and simple gene operation, and has a very important position in the carotenoid industrialization aspect, but few reports on the research of CrtI functional diversity in eukaryotes are reported at present. In the research of lycopene biosynthesis, the applicant investigates the catalytic effect of dehydrogenase CrtI of various biological sources such as Agrobacterium aurantiacaum, Pantoea agglomerans, Blakeslea trispora and the like in Saccharomyces cerevisiae. Wherein the Blakeslea trispora-derived CrtI shows better substrate conversion efficiency, but still contains a small amount of the three-step dehydrogenation product, namely, the alternarin. Therefore, based on the CrtI from Blakeslea trispora as a research basis, if the CrtI catalytic step number and key site information influencing the functional diversity of the CrtI can be explored to understand the specific regulation function of the site, the structural and functional information of the CrtI can be enriched, important reference is provided for deep research of general enzymes catalyzing multi-step reactions such as CrtI, and the CrtI catalytic step number and the specific regulation function have important significance on the modification and application of the CrtI and biosynthesis of important natural products.

Disclosure of Invention

The invention aims to construct a CrtI mutant strain library in a saccharomyces cerevisiae strain, and the catalytic steps of the mutant strain are controlled by screening, namely the proportion of dehydrogenation products is changed, and the corresponding saccharomyces cerevisiae mutant strain is provided.

In order to realize the purpose of the invention, the invention adopts the following technical scheme:

the invention firstly provides a construction method of a mutant strain for realizing the diversity of catalytic functions of universal enzymes, which is characterized in that a mutant library of the universal enzymes is constructed, and the mutant strain with the diversity of the catalytic functions is screened by shaking flask fermentation.

Further, in some embodiments, the construction method performs sequencing analysis on the universal enzyme gene of the mutant strain with catalytic function diversity obtained by screening, and identifies key amino acid site information influencing the functional diversity of the universal enzyme.

The general enzymes in the construction method of the invention can be various types of general enzymes in the biological system, including but not limited to phytoene dehydrogenase CrtI, phytoene dehydrogenase PDS, zeta-carotene dehydrogenase ZDS from plants and algae, dehydrogenase CrtN from staphylococcus aureus, hydroxylase CrtZ and ketolase CrtW in the astaxanthin synthesis path.

The strain in the construction method can be yeast, algae, mould and bacteria.

In some embodiments, the construction method wherein the universal enzyme is Blakeslea trispora-derived CrtI and the strain is saccharomyces cerevisiae.

In some embodiments, the construction method specifically comprises the steps of taking recombinant saccharomyces cerevisiae for producing phytoene as a chassis strain, constructing a mutant library of a coded BtCrtI gene by using error-prone PCR, assembling the mutant libraries of a promoter Gal7, a terminator Cyc1t and the BtCrtI gene on a plasmid PRS416 by homologous recombination in a yeast body, directly constructing the BtCrtI mutant strain in the chassis, and screening the saccharomyces cerevisiae strain with catalytic function diversity by shake flask fermentation;

further, in some embodiments, the construction method further comprises the step of performing sequencing analysis on the BtCrtI gene of the BtCrtI mutant strain to identify key amino acid site information that affects BtCrtI functional diversification.

The construction method of the invention can screen and obtain the mutant strains with the diversity of the general enzyme catalysis functions. For example, a CrtI mutant strain library derived from Blakeslea trispora is constructed in a saccharomyces cerevisiae strain, and the catalytic steps of the mutant strain are controlled by screening to obtain the corresponding saccharomyces cerevisiae mutant strain.

The invention provides a recombinant saccharomyces cerevisiae mutant strain which is used for producing zeta-carotene by BtCrtI catalysis two-step dehydrogenation reaction and a recombinant saccharomyces cerevisiae mutant strain which is used for producing lycopene by catalysis four-step dehydrogenation and neurosporene by three-step dehydrogenation reaction in a ratio of 5: 1.

Wherein, the 453 th histidine of BtCrtI gene of the recombinant Saccharomyces cerevisiae mutant strain which generates zeta-carotene by BtCrtI catalytic two-step dehydrogenation reaction is mutated into arginine, and the recombinant Saccharomyces cerevisiae mutant strain is named as SyBE _ Sc04020004 (H453R).

The 136 th histidine of BtCrtI gene from Blakeslea trispora of the recombinant saccharomyces cerevisiae mutant strain with the ratio of lycopene generated by catalyzing four-step dehydrogenation to neurosporene generated by three-step dehydrogenation reaction being 5:1 is mutated into arginine, and the recombinant saccharomyces cerevisiae mutant strain is named as SyBE _ Sc04020003 (H136R).

Wild type BtCrtI is dehydrogenated in four steps, the ratio of lycopene to neurosporene is as high as 19: the dehydrogenation ratios of the two strains 1SyBE _ Sc04020004(H453R) and SyBE _ Sc04020003(H136R) are changed compared with the wild type, and a foundation is provided for researching the multi-step dehydrogenation function of dehydrogenase CrtI and even multi-step reaction general enzymes. Research on the dehydrogenation function of CrtI provides guidance for the modification and application of dehydrogenase CrtI, and is further beneficial to the production of carotenoid. The mutant sites of BtCrtI can be discovered according to the two mutant strains, thereby being beneficial to deep exploration on the mechanism of the multi-step dehydrogenation function. Carotenoids of different dehydrogenation saturations can also be obtained, for example, recombinant Saccharomyces cerevisiae mutant strain SyBE _ Sc04020004(H453R) which produces zeta-carotene by BtCrtI catalyzed two-step dehydrogenation reaction can be used to produce zeta-carotene.

The invention also provides a construction method of the SyBE _ Sc04020004(H453R) and the SyBE _ Sc04020003 (H136R).

Wherein, the construction method of the SyBE _ Sc04020004(H453R) recombinant saccharomyces cerevisiae mutant strain comprises the following steps:

step A, constructing a recombinant plasmid PRS416-BtCrtI with the 453 th histidine mutated into arginine of the BtCrtI gene⁴⁵³；

And step B, transforming the recombinant plasmid into saccharomyces cerevisiae SyBE _ Sc04020001, screening a transformant, and culturing the screened transformant by using a YPD culture medium to obtain a recombinant saccharomyces cerevisiae mutant strain.

Further, the recombinant plasmid PRS416-BtCrtI in the step A⁴⁵³The construction method specifically comprises the steps of taking PRS416 plasmid containing BtCrtI gene as a template, amplifying fragments GAL7-H453R and H453R-cyc1T, splicing the two fragments by an OE-PCR method to obtain a linear fragment with two ends containing XbaI and NotI enzyme cutting sites, treating the linear fragment and the PRS416 plasmid by XbaI and NotI endonucleases, and connecting the linear fragment and the PRS416 plasmid by using T4 ligase.

The construction method of the SyBE _ Sc04020003(H136R) recombinant saccharomyces cerevisiae mutant strain comprises the following steps:

step A, constructing a recombinant plasmid PRS416-BtCrtI with the 136 th histidine mutated into arginine of the BtCrtI gene¹³⁶；

And step B, transforming the recombinant plasmid into saccharomyces cerevisiae SyBE _ Sc04020001, screening a transformant, and culturing the screened transformant by using a YPD culture medium to obtain a recombinant saccharomyces cerevisiae mutant strain.

Further, the recombinant plasmid PRS416-BtCrtI in the step A¹³⁶The construction method specifically comprises the steps of taking PRS416 plasmid containing BtCrtI gene as a template, amplifying fragments GAL7-H136R and H136R-cyc1T, splicing the two fragments by an OE-PCR method to obtain a linear fragment with two ends containing XbaI and NotI enzyme cutting sites, treating the linear fragment and the PRS416 plasmid by XbaI and NotI endonucleases, and connecting the linear fragment and the PRS416 plasmid by using T4 ligase.

In the above construction method, the process of screening transformants is divided into two rounds. The first round is color screening, the color shades of dehydrogenation products of different dehydrogenation steps are different, and the color of the dehydrogenation product lycopene obtained in four steps from colorless substrate phytoene to dark red is darker and darker, so that the strain with lighter color is selected on a plate containing D galactose in the first round. The second round of screening is to screen out the target strains by fermentation and HPLC detection to determine the distribution and proportion of products.

According to the technical scheme, the invention provides a method for constructing the mutant strain with the catalytic function diversity of the universal enzyme, a mutant library of the universal enzyme is constructed, and the mutant strain with the catalytic function diversity is screened by shake flask fermentation. Further constructing a CrtI mutant strain library derived from Blakeslea trispora in the saccharomyces cerevisiae strain, and screening to control the catalytic steps of the mutant strain to obtain the corresponding saccharomyces cerevisiae mutant strain. The invention obtains a recombinant saccharomyces cerevisiae mutant strain which generates zeta-carotene by BtCrtI catalysis two-step dehydrogenation reaction and a recombinant saccharomyces cerevisiae mutant strain which generates lycopene by catalysis four-step dehydrogenation and neurosporene by three-step dehydrogenation reaction in a ratio of 5: 1. According to the invention, the recombinant saccharomyces cerevisiae strain is utilized, BtCrtI mutant strains with different catalytic functions are obtained through the random mutation property, the key amino acid sites are defined, the controllability of the dehydrogenation step is realized, the functional information of CrtI is enriched, and a foundation is laid for the biosynthesis of important natural products.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows a diagram of a CrtI catalyzed multi-step dehydrogenation reaction pathway;

FIG. 2 is a diagram showing a process of constructing a Saccharomyces cerevisiae chassis according to example 1;

FIG. 3 shows the construction of the wild-type CrtI strain of example 2;

FIG. 4 shows a diagram of the construction of a library of the example 3CrtI mutant strains;

FIG. 5 is a graph comparing the ratio of dehydrogenation products of the wild type and different mutant strains of example 4, wherein L is Lycopene (Lycopene) and N is Neurosporene (Neurosporene).

Detailed Description

The invention discloses a mutant strain for realizing the diversity of general enzyme catalytic functions and a construction method thereof. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and products of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.

In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.

Example 1 obtaining of Saccharomyces cerevisiae with high yield of phytoene

1. Construction of a Modularly Integrated plasmid

The saccharomyces cerevisiae strain with high GGPP yield has the strain number of SyBE _ Sc14C10 (Chenyan; design construction and fermentation process optimization of saccharomyces cerevisiae with high lycopene yield [ D ]; Tianjin university; 2017).

In order to realize the production of phytoene in saccharomyces cerevisiae, a yeast promoter Gal1-10, a gene CrtB, a terminator Pgk1t and an upstream integration homologous gene are obtained by PCR amplificationElements such as an arm and a downstream integration homology arm are integrated, a used gene CrtB is optimized according to a saccharomyces cerevisiae codon, is synthesized by Jinwei Zhi Co, Suzhou after a common restriction enzyme cutting site is properly avoided, and is modularly assembled by applying overlap extension PCR (OE-PCR) to obtain L-arm-P_GAL1-CrtB-Tpgk 1-R-arm is named Module1(SEQ ID No. 1); the assembled DNA fragment is connected with a blunt-end vector pJET1.2/blunt (purchased from thermo company), transformed into Escherichia coli competent DH5 alpha, subjected to colony PCR screening, and subjected to single-enzyme digestion, double-enzyme digestion verification and sequencing verification on the quality-improved plasmid so as to ensure that the target fragment is connected correctly and the base sequence is not mutated, thereby constructing a correct recombinant plasmid (figure 2).

2. Modular integration construction of recombinant saccharomyces cerevisiae strain for producing phytoene

Firstly, cutting the Module1 integration plasmid by using a PmeI cutting site to obtain a Module1 integration fragment L-arm-P_GAL1And (3) -CrtB-Tpgk 1-R-arm, independently transforming the fragments into a yeast strain with high GGPP yield by a lithium acetate method, and integrating the yeast strain with the genome through recombination of left and right homologous sequences of homologous arms and sequences on the yeast genome. After transformation, an SC-TRP solid plate (6.7 g/L of synthetic yeast nitrogen source YNB, 20g/L of glucose, 2g/L of mixed amino acid powder lacking tryptophan, leucine, histidine and uracil, 2% agar powder) is adopted for screening, obtained transformants are subjected to streak purification culture, yeast genomes are extracted for PCR verification, correct positive clones are screened if sequencing results are completely correct, and the recombinant strains with correct verification are preserved with glycerol and named as SyBE _ Sc 04020001.

Example 2 construction of wild type BtCrtI in Saccharomyces cerevisiae

1. Construction of Saccharomyces cerevisiae strains catalyzing phytoene to produce lycopene by CrtI (FIG. 3)

GAL7 promoter, CYC1t terminator and BtCrtI gene synthesized by Suzhou Jinwei company according to the codon of saccharomyces cerevisiae after optimizing and properly avoiding the restriction sites commonly used are spliced together by an OE-PCR method to obtain a linear fragment GAL7 promoter-BtCrtI-CYC 1t terminator with both ends containing XbaI and NotI restriction sites, namely PGAL7-BtCrtI-TCYC1(SEQ ID NO. 2); and then, treating the linear fragment and a PRS416 plasmid by XbaI and NotI endonucleases, connecting the linear fragment and the PRS416 plasmid by using T4 ligase, transforming the linear fragment and the PRS416 plasmid into escherichia coli competent DH5 alpha, carrying out colony PCR screening, and carrying out single-enzyme digestion, double-enzyme digestion verification and sequencing verification on the upgraded plasmid to ensure that the target fragment is connected correctly and the base sequence is not mutated, thereby constructing a correct recombinant plasmid. And finally, introducing the constructed PRS416 plasmid containing BtCrtI (plasmid PRS416-BtCrtI) into Saccharomyces cerevisiae SyBE _ Sc04020001 by a yeast lithium acetate conversion method, preserving the glycerol strain and naming the glycerol strain as SyBE _ Sc 04020002.

2. Fermentation measurement of yield and proportion of dehydrogenation products of wild type strain

Test materials: strain SyBE _ Sc04020002

Seed culture medium: 40g/L glucose, 20g/L peptone and 10g/L yeast extract powder;

fermentation medium: 40g/L glucose, 20g/L peptone, 10g/L yeast extract powder and 10g/L D-galactose.

Picking single colony, inoculating into 5ml YPD seed culture medium, culturing at 30 deg.C and 250rpm for about 18 hr; at the initial OD₆₀₀Transferring to 25ml seed culture medium at 0.2 deg.C, culturing at 30 deg.C and 250rpm to OD₆₀₀5-8 percent; the secondary seeds were inoculated into 50ml YPDG fermentation medium to make the initial OD₆₀₀The culture was carried out at 30 ℃ and 250rpm for 48h at 0.1.

Taking two 500-microliter fermentation liquid, centrifuging at 4000rpm for 1min, collecting thalli, and washing once; drying one part of the thallus at 80 ℃ to constant weight, and weighing to calculate the dry weight of the cells; the other part of the thallus is used for product extraction, and the specific steps are as follows: cells were resuspended in 1ml 3M HCl, boiled in a boiling water bath for 2min, and then immediately ice-cooled for 3min, and relatively fine particles were observed. Cells were centrifuged at 12000rpm for 1min and the supernatant was discarded, washed with sterile water for 2 times, added with a small amount of quartz sand and 1ml of acetone containing 1% (w/v) BHT, vortexed for 5min, and finally centrifuged at 12000rpm for 5min to observe the cells becoming white, and then the acetone phase was collected, filtered through a 0.22 μm organic filter and subjected to supernatant detection. It is worth noting that the fat-soluble carotenoid is unstable in quality and is easily decomposed by light, and the extraction process should be performed in the shade as much as possible.

And (4) HPLC detection: the carotenoids were measured by high performance liquid chromatography HPLC. A BDSHypersil C18 column is used as a chromatographic column, and the model is as follows: 150mm × 4.6mm,5 μm; an ultraviolet detector Waters e2489UV/Vis is adopted; the pump is Waters e 2695; the prepared mobile phase is acetonitrile: methanol: dichloromethane ═ 21:21:8(v/v/v), flow rate 1.0ml/min, column temperature 30 ℃. The detection wavelengths of the lycopene, the neurosporene, the zeta-carotene, the phytofluene and the phytoene under an ultraviolet detector are 471nm, 440nm, 401nm, 349nm and 287nm respectively.

And (3) test results: d-galactose in the fermentation medium is an inducer and is responsible for turning on the transcription of GAL7 promoter. Transcription from the GAL promoter is inhibited by glucose in the presence of glucose in the initial medium; as fermentation proceeds, glucose is rapidly consumed, and when glucose is consumed, the glucose repression effect is released and D-galactose turns on transcription of GAL7 promoter, thereby gradually accumulating lycopene. After 72h fermentation, the yield of the respective dehydrogenation product was determined as: lycopene reaches 264.0mg/L, neurosporene reaches 13.89mg/L, and a two-step dehydrogenation product zeta-carotene and a one-step dehydrogenation product hexahydrolycopene are not detected. It follows that the dehydrogenation reaction catalyzed by wild-type BtCrtI in saccharomyces cerevisiae results in a ratio of the four-step dehydrogenation product to the three-step dehydrogenation product of 19: 1.

Example 3 construction of wild type BtCrtI in Saccharomyces cerevisiae

1. Error-prone PCR construction of BtCrtI mutant library

Test materials:

strain SyBE _ Sc04020001 and plasmid PRS416-BtCrtI

10 × error-prone PCR buffer: 70mm magnesium chloride hexahydrate, 500mm potassium chloride, 100mm tris (hydroxymethyl) aminomethane, 0.1w/v gelatin

10 × error-prone PCR dNTPs: 10mm dCTPs and dTTPs, 2mm dGTPs and dATPs

10mm manganese chloride tetrahydrate

GAL7 promoter and CYC1t terminator are amplified by PCR, plasmid containing BtCrtI is used as a template, and BtCrtI gene is amplified by random mutation of error-prone PCR, wherein the configuration method of an error-prone PCR system (100 mu l) is as follows: 10. mu.l of 10 Xerror-prone PCR buffer, 10. mu.l of 10 Xerror-prone PCR dNTPs, 4. mu.l of each of the upper and lower primers, 2. mu.l of the template, 5. mu.l of manganese chloride solution, 1. mu.l of fast taq enzyme, and 64. mu.l of supplemented water were added.

It should be noted that there is 40bp of homologous sequence among promoter, gene and terminator, which is convenient for assembling the three components in the following. Wherein, the homologous sequence (68bp) between the PRS416 vector and the promoter GAL7 (SEQ ID NO. 3): TCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCACTAGTTCTAGA, respectively; homologous sequence (40bp) between promoter GAL7 and BtCrtI (SEQ ID No. 4): ATTCCCTCAAAAATGTCTGATCAGAAGAAGCACATTGTCG, respectively; homologous sequence (40bp) between BtCrtI sequence and terminator CYC1(SEQ ID No. 5): CTCTAACGATATTAGGATATAAGGCCGCATCATGTAATTA, respectively; homologous sequence (73bp) between terminator CYC1 and PRS416 vector (SEQ ID NO. 6): GCGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCA are provided.

The error-prone PCR cycle settings were: the pre-denaturation step is 94 ℃ for 3min, the denaturation step is 94 ℃ for 5s, the annealing step is 55 ℃ for 15s, and the elongation step is 72 ℃ for 50s, which are carried out for 30 cycles without complete elongation stage. After the gel is recovered, three linear sheet ends of a promoter, a BtCrtI gene mutation library and a terminator are obtained. Meanwhile, the plasmid PRS416 is cut by XbaI and NotI endonucleases to obtain a linear vector; then, introducing three linear fragments and the PRS416 linear vector after enzyme digestion treatment into saccharomyces cerevisiae by a yeast lithium acetate conversion method, assembling and realizing the formation of circular plasmids through homologous sequences contained among the fragments, wherein BtCrtI genes with different mutation points are connected in the plasmids, thereby successfully constructing a BtCrtI mutation library, screening by an SC-URA + galactose solid plate (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L, mixed amino acid powder of tryptophan, leucine, histidine and uracil 2g/L, agar powder of 2 percent) after conversion, because the colors of dehydrogenation products are different, such as zeta-carotene is yellow, streptococerucin is pink, lycopene is dark red, the colors of all mutants are also different, and because the experiment aim is to find out mutants which are prone to two-step dehydrogenation and three-step dehydrogenation, therefore, the lighter strains were picked for subsequent validation analysis.

2. Fermentation measurement of yield and proportion of each dehydrogenation product of mutant strain

Test materials: individual mutant strains picked on plates

The test method comprises the following steps: exactly the same as in example 2.

And (3) test results: d-galactose in the fermentation medium is an inducer and is responsible for turning on the transcription of GAL7 promoter. Transcription from the GAL promoter is inhibited by glucose in the presence of glucose in the initial medium; as the fermentation proceeds, glucose is rapidly consumed, and when glucose is consumed, the glucose repression effect is released, and D-galactose turns on the transcription of GAL promoter, thereby gradually accumulating each dehydrogenation product. After fermentation for 72h, only two-step dehydrogenation products of zeta-carotene and one-step dehydrogenation product of phytofluene are detected by one mutant, a small amount of zeta-carotene and phytofluene is detected by the other mutant, and the ratio of the four-step dehydrogenation product of lycopene to the three-step dehydrogenation product of neurosporene is as follows: 5:1.

Example 4 BtCrtI Gene sequencing analysis and validation of mutant strains

1. BtCrtI gene sequencing of mutant strains

Selecting single colonies of two mutant strains to an SC-URA culture medium for culture, extracting yeast plasmids after culturing for 20H at 30 ℃, then converting the yeast plasmids into escherichia coli competence DH5 alpha, carrying out colony PCR screening, carrying out single and double enzyme digestion verification and sequencing verification on the quality-improved grains to ensure that a target fragment is correctly connected, detecting the position of a base sequence with mutation, and finding out that the strain generating a two-step dehydrogenation product has the mutation from histidine H to arginine R at 453 amino acid positions and the strain tending to the three-step dehydrogenation product has the mutation from histidine H to arginine R at 136 positions.

2. Phenotypic validation of mutant sites

Introduction of mutation points: primers were designed at positions 136 and 453, respectively, and mutation points were introduced to mutate histidine H to arginine R. Using prs416 plasmid containing BtCrtI gene as a template, amplifying fragments GAL7-H136R (SEQ ID NO.7), H136R-cyc1t (SEQ ID NO.8), GAL7-H453R (SEQ ID NO.9) and H453R-cyc1t (SEQ ID NO.10), and splicing the two fragments at the 136 th site and the 453 th site by an OE-PCR method to obtain enzyme cutting sites with the two ends containing XbaI and NotI. And then, treating the linear fragment and a PRS416 plasmid by XbaI and NotI endonucleases, connecting the linear fragment and the PRS416 plasmid by using T4 ligase, transforming the linear fragment and the PRS416 plasmid into escherichia coli competent DH5 alpha, carrying out colony PCR screening, and carrying out single-enzyme digestion, double-enzyme digestion verification and sequencing verification on the upgraded plasmid to ensure that the target fragment is connected correctly and the base sequence is not mutated, thereby constructing a correct recombinant plasmid. Finally, the constructed PRS416 plasmid containing the mutant BtCrtI is introduced into Saccharomyces cerevisiae SyBE _ Sc04020001 by a yeast lithium acetate transformation method, and the glycerol strain is preserved and named as SyBE _ Sc04020003(H136R) and SyBE _ Sc04020004 (H453R).

Fermentation verification of the mutant: the strain was fermented in exactly the same manner as in example 2.

As a result: d-galactose in the fermentation medium is an inducer and is responsible for turning on the transcription of GAL7 promoter. Transcription from the GAL promoter is inhibited by glucose in the presence of glucose in the initial medium; as the fermentation proceeds, glucose is rapidly consumed, and when glucose is consumed, the glucose repression effect is released, and D-galactose turns on the transcription of GAL promoter, thereby gradually accumulating each dehydrogenation product. After fermentation for 72H, the ratio of dehydrogenation products of the two mutant strains is the same as that of the previous fermentation, the H453R mutant only detects two-step dehydrogenation products of zeta-carotene and one-step dehydrogenation product of phytofluene, the H136R mutant only detects a small amount of zeta-carotene and phytofluene, and the ratio of the four-step dehydrogenation product of lycopene to the three-step dehydrogenation product of neurosporene is as follows: 5:1 (FIG. 5).

Sequence listing

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taacgacatt actatatata taatatagga agcatttaat agaacagcat cgtagaagta 360

cggattagaa gccgccgagc gggcgacagc cctccgacgg aagactctcc tccgtgcgtc 420

ctcgtcttca ccggtcgcgt tcctgaaacg cagatgtgcc tcgcgccgca ctgctccgaa 480

caataaagat tctacaatac tagcttttat ggttatgaag aggaaaaatt ggcagtaacc 540

tggccccaca aaccttcaaa ttaacgaatc aaattaacaa ccataggatg ataatgcgat 600

tagtttttta gccttatttc tggggtaatt aatcagcgaa gcgatgattt ttgatctatt 660

aacagatata taaatggaaa agctgcataa ccactttaac taatactttc aacattttca 720

gtttgtatta cttcttattc aaatgtcata aaagtatcaa caaaaaattg ttaatatacc 780

tctatacttt aacgtcaagg agaaaaaact ataatgtcac aaccaccatt attggaccac 840

gctacacaaa ctatggcaaa cggttctaaa tctttcgcta ctgctgctaa attattcgac 900

ccagcaacaa gaagatctgt attgatgttg tacacctggt gtagacattg cgatgacgtt 960

atagatgacc aaactcacgg ttttgcttca gaagctgcag ccgaagaaga agctacacaa 1020

agattggcaa gattaagaac tttgacatta gctgcattcg aaggtgccga aatgcaagat 1080

ccagcttttg ccgctttcca agaagttgca ttaacccatg gtattactcc tagaatggct 1140

ttggatcact tagacggttt tgcaatggat gtcgcccaaa caagatacgt aaccttcgaa 1200

gacactttaa gatattgtta ccatgtcgcc ggtgttgtcg gtttgatgat ggctagagta 1260

atgggtgtta gagatgaaag agttttagat agagcatgtg acttgggttt agccttccaa 1320

ttgacaaaca tagctagaga tataatagat gacgcagcca tagacagatg ctatttgcca 1380

gctgaatggt tacaagatgc aggtttgact cctgaaaatt acgctgcaag agaaaacaga 1440

gccgctttag ccagagttgc tgaaagattg atagatgcag ccgaaccata ttacatctct 1500

tcacaagctg gtttgcatga tttgccacct agatgcgcat gggccattgc taccgcaaga 1560

tctgtttaca gagaaatcgg tattaaagtc aaggctgcag gtggttccgc atgggataga 1620

agacaacaca cttctaaagg tgaaaagatc gctatgttga tggccgctcc tggtcaagtt 1680

attagagcaa agaccaccag agtcacccca agaccagccg gtttatggca aagacctgtt 1740

taaattgaat tgaattgaaa tcgatagatc aatttttttc ttttctcttt ccccatcctt 1800

tacgctaaaa taatagttta ttttattttt tgaatatttt ttatttatat acgtatatat 1860

agactattat ttatctttta atgattatta agatttttat taaaaaaaaa ttcgctcctc 1920

ttttaatgcc tttatgcagt ttttttttct cgatatttct atgttcgggt tcagcgtatt 1980

ttaagtttaa taactcgaaa attctgcgtt cgttatatat gtgtactttg cagttatgac 2040

gccagatggc agtagtggaa gatattcttt attgaaaaat agcttgtcac cttacgtaca 2100

atcttgatcc ggagcttttc tttttttgcc gattaagaat tcggtcgaaa aaagaaaagg 2160

agagggccaa gagggagggc attggtgact attgagcacg tgagtatacg tgattaagca 2220

cacaaaggca gcttggagta tgtctgttat taatttcaca ggtagttctg gtccattggt 2280

gaaagtttgc ggcttgcaga gcacagaggc cgcagaatgt gctctagatt ccgatgctga 2340

cttgctgggt attatatgtg tgcccaatag aaagagaaca attgacccgg ttattgcaag 2400

gaaaatttca agtcttgtaa aagcatataa aaatagttca ggcactccga aatacttggt 2460

tggcgt 2466

<210> 2

<211> 2729

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tttgccagct tactatcctt cttgaaaata tgcactctat atcttttagt tcttaattgc 60

aacacataga tttgctgtat aacgaatttt atgctatttt ttaaatttgg agttcagtga 120

taaaagtgtc acagcgaatt tcctcacatg tagggaccga attgtttaca agttctctgt 180

accaccatgg agacatcaaa gattgaaaat ctatggaaag atatggacgg tagcaacaag 240

aatatagcac gagccgcgaa gttcatttcg ttacttttga tatcgctcac aactattgcg 300

aagcgcttca gtgaaaaaat cataaggaaa agttgtaaat attattggta gtattcgttt 360

ggtaaagtag agggggtaat ttttcccctt tattttgttc atacattctt aaattgcttt 420

gcctctcctt ttggaaagct atacttcgga gcactgttga gcgaaggctc attagatata 480

ttttctgtca ttttccttaa cccaaaaata agggaaaggg tccaaaaagc gctcggacaa 540

ctgttgaccg tgatccgaag gactggctat acagtgttca caaaatagcc aagctgaaaa 600

taatgtgtag ctatgttcag ttagtttggc tagcaaagat ataaaagcag gtcggaaata 660

tttatgggca ttattatgca gagcatcaac atgataaaaa aaaacagttg aatattccct 720

caaaaatgtc tgatcagaag aagcacattg tcgtcatagg tgctggaata ggaggtactg 780

caacagcagc aaggttagca agggagggtt tcagagtcac tgtcgtcgag aagaacgact 840

tctctggagg aaggtgctct ttcattcacc acgacggtca caggttcgac cagggacctt 900

cattgtactt gatgcctaag ttgtttgagg acgctttcgc tgacttagac gagaggatag 960

gagaccactt ggacttatta agatgtgaca acaattacaa agtccatttc gacgacggtg 1020

acgctgtcca attgtcatca gacttaacaa agatgaaggg tgagttggac aggattgagg 1080

gacctttagg attcggtagg ttcttagatt tcatgaaaga gacacacgtc cactacgagc 1140

agggtacatt cattgctata aagagaaact tcgaaactat atgggactta ataaggttac 1200

agtacgtccc agagattttt aggttgcact tattcggtaa gatatacgac agagcatcaa 1260

aatacttcca aacaaaaaag atgaggatgg cttttacttt tcaaacaatg tacatgggta 1320

tgtcacctta cgacgcacct gcagtctact cattgttgca atatacagag ttcgcagagg 1380

gaatttggta cccaaggggt ggtttcaaca tggtcgtcca aaagttggag tctatagctt 1440

ctaagaagta cggagctgag ttcaggtacc aatctcctgt cgctaagatt aacactgtcg 1500

ataaagacaa gagggtcact ggtgtcactt tggagtctgg agaagtcatt gaggcagacg 1560

ctgtcgtctg caacgctgac ttggtctacg cttaccacca cttgttgcca ccttgcaact 1620

ggacaaagaa gactttggca tctaagaaat taacatcttc atcaatttct ttttactggt 1680

caatgtctac taaggtccct caattggacg tccacaacat tttcttggct gaggcttaca 1740

aggagtcatt cgacgagatt tttaacgatt tcggtttgcc ttctgaagca tctttctacg 1800

tcaacgttcc ttcaaggata gacgagtctg cagcacctcc aaataaggac tcaattatag 1860

ttttagttcc aattggtcac atgaagtcta agacaggtaa ctcagcagag gagaactacc 1920

cagagttggt caacagggct agaaagatgg tcttggaggt catagagagg aggttgggag 1980

tcaacaactt cgctaacttg atagaacacg aggaggtcaa cgacccatca gtctggcaat 2040

ctaagttcaa cttgtggagg ggatcaatat taggtttatc acatgatgtc tttcaggttt 2100

tgtggttcag accttcaaca aaggactcta ctaacagata tgacaattta tttttcgtcg 2160

gtgcatcaac tcaccctggt acaggagtcc caatagtctt ggcaggatct aaattaactt 2220

ctgaccaggt ctgtaagtca ttcggacaaa accctttgcc taggaagtta caggactctc 2280

agaagaaata tgcacctgag caaacaagga agactgagtc acactggatt tattactgct 2340

tagcatgcta ctttgtcact ttcttgttct tctatttctt tcctagggac gacactacta 2400

ctccagcatc ttttattaat cagttgttgc caaacgtctt ccaaggacag aactctaacg 2460

atattaggat ataaggccgc atcatgtaat tagttatgtc acgcttacat tcacgccctc 2520

cccccacatc cgctctaacc gaaaaggaag gagttagaca acctgaagtc taggtcccta 2580

tttatttttt tatagttatg ttagtattaa gaacgttatt tatatttcaa atttttcttt 2640

tttttctgta caaacgcgtg tacgcatgta acattatact gaaaaccttg cttgagaagg 2700

ttttgggacg ctcgaaggct ttaatttgc 2729

<210> 3

<211> 68

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tcgaggtcga cggtatcgat aagcttgata tcgaattcct gcagcccggg ggatccacta 60

gttctaga 68

<210> 4

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

attccctcaa aaatgtctga tcagaagaag cacattgtcg 40

<210> 5

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ctctaacgat attaggatat aaggccgcat catgtaatta 40

<210> 6

<211> 73

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gcggccgcca ccgcggtgga gctccagctt ttgttccctt tagtgagggt taattgcgcg 60

cttggcgtaa tca 73

<210> 7

<211> 1162

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ctagtctaga tttgccagct tactatcctt cttgaaaata tgcactctat atcttttagt 60

tcttaattgc aacacataga tttgctgtat aacgaatttt atgctatttt ttaaatttgg 120

agttcagtga taaaagtgtc acagcgaatt tcctcacatg tagggaccga attgtttaca 180

agttctctgt accaccatgg agacatcaaa gattgaaaat ctatggaaag atatggacgg 240

tagcaacaag aatatagcac gagccgcgaa gttcatttcg ttacttttga tatcgctcac 300

aactattgcg aagcgcttca gtgaaaaaat cataaggaaa agttgtaaat attattggta 360

gtattcgttt ggtaaagtag agggggtaat ttttcccctt tattttgttc atacattctt 420

aaattgcttt gcctctcctt ttggaaagct atacttcgga gcactgttga gcgaaggctc 480

attagatata ttttctgtca ttttccttaa cccaaaaata agggaaaggg tccaaaaagc 540

gctcggacaa ctgttgaccg tgatccgaag gactggctat acagtgttca caaaatagcc 600

aagctgaaaa taatgtgtag ctatgttcag ttagtttggc tagcaaagat ataaaagcag 660

gtcggaaata tttatgggca ttattatgca gagcatcaac atgataaaaa aaaacagttg 720

aatattccct caaaaatgtc tgatcagaag aagcacattg tcgtcatagg tgctggaata 780

ggaggtactg caacagcagc aaggttagca agggagggtt tcagagtcac tgtcgtcgag 840

aagaacgact tctctggagg aaggtgctct ttcattcacc acgacggtca caggttcgac 900

cagggacctt cattgtactt gatgcctaag ttgtttgagg acgctttcgc tgacttagac 960

gagaggatag gagaccactt ggacttatta agatgtgaca acaattacaa agtccatttc 1020

gacgacggtg acgctgtcca attgtcatca gacttaacaa agatgaaggg tgagttggac 1080

aggattgagg gacctttagg attcggtagg ttcttagatt tcatgaaaga gacacacgtc 1140

aggtacgagc agggtacatt ca 1162

<210> 8

<211> 1614

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tacgagcagg gtacattcat tgctataaag agaaacttcg aaactatatg ggacttaata 60

aggttacagt acgtcccaga gatttttagg ttgcacttat tcggtaagat atacgacaga 120

gcatcaaaat acttccaaac aaaaaagatg aggatggctt ttacttttca aacaatgtac 180

atgggtatgt caccttacga cgcacctgca gtctactcat tgttgcaata tacagagttc 240

gcagagggaa tttggtaccc aaggggtggt ttcaacatgg tcgtccaaaa gttggagtct 300

atagcttcta agaagtacgg agctgagttc aggtaccaat ctcctgtcgc taagattaac 360

actgtcgata aagacaagag ggtcactggt gtcactttgg agtctggaga agtcattgag 420

gcagacgctg tcgtctgcaa cgctgacttg gtctacgctt accaccactt gttgccacct 480

tgcaactgga caaagaagac tttggcatct aagaaattaa catcttcatc aatttctttt 540

tactggtcaa tgtctactaa ggtccctcaa ttggacgtcc acaacatttt cttggctgag 600

gcttacaagg agtcattcga cgagattttt aacgatttcg gtttgccttc tgaagcatct 660

ttctacgtca acgttccttc aaggatagac gagtctgcag cacctccaaa taaggactca 720

attatagttt tagttccaat tggtcacatg aagtctaaga caggtaactc agcagaggag 780

aactacccag agttggtcaa cagggctaga aagatggtct tggaggtcat agagaggagg 840

ttgggagtca acaacttcgc taacttgata gaacacgagg aggtcaacga cccatcagtc 900

tggcaatcta agttcaactt gtggagggga tcaatattag gtttatcaca tgatgtcttt 960

caggttttgt ggttcagacc ttcaacaaag gactctacta acagatatga caatttattt 1020

ttcgtcggtg catcaactca ccctggtaca ggagtcccaa tagtcttggc aggatctaaa 1080

ttaacttctg accaggtctg taagtcattc ggacaaaacc ctttgcctag gaagttacag 1140

gactctcaga agaaatatgc acctgagcaa acaaggaaga ctgagtcaca ctggatttat 1200

tactgcttag catgctactt tgtcactttc ttgttcttct atttctttcc tagggacgac 1260

actactactc cagcatcttt tattaatcag ttgttgccaa acgtcttcca aggacagaac 1320

tctaacgata ttaggatata aggccgcatc atgtaattag ttatgtcacg cttacattca 1380

cgccctcccc ccacatccgc tctaaccgaa aaggaaggag ttagacaacc tgaagtctag 1440

gtccctattt atttttttat agttatgtta gtattaagaa cgttatttat atttcaaatt 1500

tttctttttt ttctgtacaa acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt 1560

gagaaggttt tgggacgctc aaggaaaaaa gcggccgcgc aaattaaagc cttc 1614

<210> 9

<211> 2112

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ctagtctaga tttgccagct tactatcctt cttgaaaata tgcactctat atcttttagt 60

tcttaattgc aacacataga tttgctgtat aacgaatttt atgctatttt ttaaatttgg 120

agttcagtga taaaagtgtc acagcgaatt tcctcacatg tagggaccga attgtttaca 180

agttctctgt accaccatgg agacatcaaa gattgaaaat ctatggaaag atatggacgg 240

tagcaacaag aatatagcac gagccgcgaa gttcatttcg ttacttttga tatcgctcac 300

aactattgcg aagcgcttca gtgaaaaaat cataaggaaa agttgtaaat attattggta 360

gtattcgttt ggtaaagtag agggggtaat ttttcccctt tattttgttc atacattctt 420

aaattgcttt gcctctcctt ttggaaagct atacttcgga gcactgttga gcgaaggctc 480

attagatata ttttctgtca ttttccttaa cccaaaaata agggaaaggg tccaaaaagc 540

gctcggacaa ctgttgaccg tgatccgaag gactggctat acagtgttca caaaatagcc 600

aagctgaaaa taatgtgtag ctatgttcag ttagtttggc tagcaaagat ataaaagcag 660

gtcggaaata tttatgggca ttattatgca gagcatcaac atgataaaaa aaaacagttg 720

aatattccct caaaaatgtc tgatcagaag aagcacattg tcgtcatagg tgctggaata 780

ggaggtactg caacagcagc aaggttagca agggagggtt tcagagtcac tgtcgtcgag 840

aagaacgact tctctggagg aaggtgctct ttcattcacc acgacggtca caggttcgac 900

cagggacctt cattgtactt gatgcctaag ttgtttgagg acgctttcgc tgacttagac 960

gagaggatag gagaccactt ggacttatta agatgtgaca acaattacaa agtccatttc 1020

gacgacggtg acgctgtcca attgtcatca gacttaacaa agatgaaggg tgagttggac 1080

aggattgagg gacctttagg attcggtagg ttcttagatt tcatgaaaga gacacacgtc 1140

aggtacgagc agggtacatt cattgctata aagagaaact tcgaaactat atgggactta 1200

ataaggttac agtacgtccc agagattttt aggttgcact tattcggtaa gatatacgac 1260

agagcatcaa aatacttcca aacaaaaaag atgaggatgg cttttacttt tcaaacaatg 1320

tacatgggta tgtcacctta cgacgcacct gcagtctact cattgttgca atatacagag 1380

ttcgcagagg gaatttggta cccaaggggt ggtttcaaca tggtcgtcca aaagttggag 1440

tctatagctt ctaagaagta cggagctgag ttcaggtacc aatctcctgt cgctaagatt 1500

aacactgtcg ataaagacaa gagggtcact ggtgtcactt tggagtctgg agaagtcatt 1560

gaggcagacg ctgtcgtctg caacgctgac ttggtctacg cttaccacca cttgttgcca 1620

ccttgcaact ggacaaagaa gactttggca tctaagaaat taacatcttc atcaatttct 1680

ttttactggt caatgtctac taaggtccct caattggacg tccacaacat tttcttggct 1740

gaggcttaca aggagtcatt cgacgagatt tttaacgatt tcggtttgcc ttctgaagca 1800

tctttctacg tcaacgttcc ttcaaggata gacgagtctg cagcacctcc aaataaggac 1860

tcaattatag ttttagttcc aattggtcac atgaagtcta agacaggtaa ctcagcagag 1920

gagaactacc cagagttggt caacagggct agaaagatgg tcttggaggt catagagagg 1980

aggttgggag tcaacaactt cgctaacttg atagaacacg aggaggtcaa cgacccatca 2040

gtctggcaat ctaagttcaa cttgtggagg ggatcaatat taggtttatc aagggatgtc 2100

tttcaggttt tg 2112

<210> 10

<211> 663

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gatgtctttc aggttttgtg gttcagacct tcaacaaagg actctactaa cagatatgac 60

aatttatttt tcgtcggtgc atcaactcac cctggtacag gagtcccaat agtcttggca 120

ggatctaaat taacttctga ccaggtctgt aagtcattcg gacaaaaccc tttgcctagg 180

aagttacagg actctcagaa gaaatatgca cctgagcaaa caaggaagac tgagtcacac 240

tggatttatt actgcttagc atgctacttt gtcactttct tgttcttcta tttctttcct 300

agggacgaca ctactactcc agcatctttt attaatcagt tgttgccaaa cgtcttccaa 360

ggacagaact ctaacgatat taggatataa ggccgcatca tgtaattagt tatgtcacgc 420

ttacattcac gccctccccc cacatccgct ctaaccgaaa aggaaggagt tagacaacct 480

gaagtctagg tccctattta tttttttata gttatgttag tattaagaac gttatttata 540

tttcaaattt ttcttttttt tctgtacaaa cgcgtgtacg catgtaacat tatactgaaa 600

accttgcttg agaaggtttt gggacgctca aggaaaaaag cggccgcgca aattaaagcc 660

ttc 663

Claims (4)

1. A recombinant Saccharomyces cerevisiae mutant strain for producing zeta-carotene by BtCrtI catalytic two-step dehydrogenation reaction is characterized in that the construction method of the recombinant Saccharomyces cerevisiae mutant strain comprises the following steps:

step A, constructing a recombinant plasmid PRS416-BtCrtI with the 453 th histidine mutated into arginine of the BtCrtI gene⁴⁵³；

The BtCrtI is a CrtI from Blakeslea trispora;

splicing GAL7 promoter, CYC1t terminator and gene BtCrtI synthesized by optimizing Saccharomyces cerevisiae codon and appropriately avoiding common restriction enzyme cutting sites by an OE-PCR method to obtain a linear fragment GAL7 promoter-BtCrtI-CYC 1t terminator with two ends containing XbaI and NotI restriction enzyme cutting sites, namely PGAL7-BtCrtI-TCYC1, wherein the sequence of the PGAL7-BtCrtI-TCYC1 is shown as SEQ ID number 2; then, the linear fragment and the PRS416 plasmid are treated by XbaI and NotI endonucleases and then are connected by using T4 ligase to obtain the PRS416 plasmid containing BtCrtI gene, namely the plasmid PRS 416-BtCrtI;

introducing a mutation point, designing a primer at the 453 position of BtCrtI, and introducing the mutation point to mutate histidine H into arginine R; amplifying fragments GAL7-H453R and H453R-cycle by using plasmid PRS416-BtCrtI as a template, splicing the two fragments at the 453 bit by an OE-PCR method to obtain a linear fragment of which two ends contain XbaI and NotI enzyme cutting sites, treating the linear fragment and the PRS416 plasmid by XbaI and NotI endonucleases, and connecting by using T4 ligase; the GAL7-H453R is shown as SEQ ID number 9, and the H453R-cycle is shown as SEQ ID number 10;

step B, transforming the recombinant plasmid into saccharomyces cerevisiae SyBE _ Sc04020001, screening transformants, and culturing the screened transformants by using a YPD culture medium to obtain a recombinant saccharomyces cerevisiae mutant strain; the SyBE _ Sc04020001 is a high-yield GGPP brewing yeast strain SyBE _ Sc14C10 adopting L-arm-P_GAL1-CrtB-Tpgk 1-R-arm is obtained by homologous recombination integrating the CrtB gene at a specific position in the genome;

obtaining a yeast promoter Ga11-10, a gene CrtB, a terminator Pgk1t, an upstream integration homologous arm and a downstream integration homologous arm through PCR amplification, optimizing the used gene CrtB according to a saccharomyces cerevisiae codon, artificially synthesizing the gene CrtB after appropriately avoiding common restriction enzyme cutting sites, and modularly assembling the yeast promoter Ga11-10, the gene CrtB, the terminator Pgk1t, the upstream integration homologous arm and the downstream integration homologous arm by using overlap extension PCR to obtain the L-arm-P_GAL1-CrtB-Tpgk 1-R-arm, named Modulel, said L-arm-P_GAL1The sequence of-CrtB-Tpgk 1-R-arm is shown as SEQ ID number 1;

the Saccharomyces cerevisiae strain SyBE _ Sc14C10 for high yield of GGPP is obtained by firstly obtaining delta gal1 delta gal7 delta gal 10:: HIS3 and then obtaining delta yp1062w: KanMX on the basis of CEN.PK2-1C.

2. The method for constructing a mutant strain of recombinant Saccharomyces cerevisiae as claimed in claim 1, which comprises:

step A, constructing a recombinant plasmid PRS416-BtCrtI with the 453 th histidine mutated into arginine of the BtCrtI gene⁴⁵³(ii) a The BtCrtI is a CrtI from Blakeslea trispora;

splicing GAL7 promoter, CYC1t terminator and gene BtCrtI synthesized by optimizing Saccharomyces cerevisiae codon and appropriately avoiding common restriction enzyme cutting sites by an OE-PCR method to obtain a linear fragment GAL7 promoter-BtCrtI-CYC 1t terminator with two ends containing XbaI and NotI restriction enzyme cutting sites, namely PGAL7-BtCrtI-TCYC1, wherein the sequence of the PGAL7-BtCrtI-TCYC1 is shown as SEQ ID number 2; then, the linear fragment and the PRS416 plasmid are treated by XbaI and NotI endonucleases and then are connected by using T4 ligase to obtain the PRS416 plasmid containing BtCrtI gene, namely the plasmid PRS 416-BtCrtI;

introducing a mutation point, designing a primer at the 453 position of BtCrtI, and introducing the mutation point to mutate histidine H into arginine R; amplifying fragments GAL7-H453R and H453R-cycle by using plasmid PRS416-BtCrtI as a template, splicing the two fragments at the 453 bit by an OE-PCR method to obtain a linear fragment of which two ends contain XbaI and NotI enzyme cutting sites, treating the linear fragment and the PRS416 plasmid by XbaI and NotI endonucleases, and connecting by using T4 ligase; the GAL7-H453R is shown as SEQ ID number 9, and the H453R-cycle is shown as SEQ ID number 10;

step B, transforming the recombinant plasmid into saccharomyces cerevisiae SyBE _ Sc04020001, screening transformants, and culturing the screened transformants by using a YPD culture medium to obtain a recombinant saccharomyces cerevisiae mutant strain; the SyBE _ Sc04020001 is a high-yield GGPP brewing yeast strain SyBE _ Sc14C10 adopting L-arm-P_GAL1-CrtB-Tpgk 1-R-arm is obtained by homologous recombination integrating the CrtB gene at a specific position in the genome;

obtaining a yeast promoter Ga11-10, a gene CrtB, a terminator Pgk1t, an upstream integration homologous arm and a downstream integration homologous arm through PCR amplification, optimizing the used gene CrtB according to a saccharomyces cerevisiae codon, artificially synthesizing the gene CrtB after appropriately avoiding common restriction enzyme cutting sites, and modularly assembling the yeast promoter Ga11-10, the gene CrtB, the terminator Pgk1t, the upstream integration homologous arm and the downstream integration homologous arm by using overlap extension PCR to obtain the L-arm-P_GAL1-CrtB-Tpgk 1-R-arm, named Modulel, said L-arm-P_GAL1The sequence of-CrtB-Tpgk 1-R-arm is shown as SEQ ID number 1;

the Saccharomyces cerevisiae strain SyBE _ Sc14C10 for high yield of GGPP is obtained by firstly obtaining delta gal1 delta gal7 delta gal 10:: HIS3 and then obtaining delta yp1062w: KanMX on the basis of CEN.PK2-1C.

3. A recombinant saccharomyces cerevisiae mutant strain with the ratio of lycopene produced by catalytic four-step dehydrogenation to neurosporene produced by three-step dehydrogenation being 5:1 is characterized in that the construction method of the recombinant saccharomyces cerevisiae mutant strain comprises the following steps:

step A, constructing a recombinant plasmid PRS416-BtCrtI with the 136 th histidine mutated into arginine of the BtCrtI gene¹³⁶(ii) a The BtCrtI is a CrtI from Blakeslea trispora;

splicing GAL7 promoter, CYC1t terminator and gene BtCrtI synthesized by optimizing Saccharomyces cerevisiae codon and appropriately avoiding common restriction enzyme cutting sites by an OE-PCR method to obtain a linear fragment GAL7 promoter-BtCrtI-CYC 1t terminator with two ends containing XbaI and NotI restriction enzyme cutting sites, namely PGAL7-BtCrtI-TCYC1, wherein the sequence of the PGAL7-BtCrtI-TCYC1 is shown as SEQ ID number 2; then, the linear fragment and the PRS416 plasmid are treated by XbaI and NotI endonucleases and then are connected by using T4 ligase to obtain the PRS416 plasmid containing BtCrtI gene, namely the plasmid PRS 416-BtCrtI;

introducing mutation points, namely designing a primer at the 136 th site of BtCrtI, and introducing the mutation points to mutate histidine H into arginine R; using plasmid PRS416-BtCrtI as a template, amplifying fragments GAL7-H136R and H136R-cyc1T, splicing the two fragments at the 136 th position by an OE-PCR method to obtain a linear fragment of which two ends contain XbaI and NotI enzyme cutting sites, treating the linear fragment and the PRS416 plasmid by XbaI and NotI endonucleases, and connecting the linear fragment and the PRS416 plasmid by using T4 ligase; the GAL7-H136R is shown as SEQ ID NO.7, and the H136R-cycle is shown as SEQ ID number 8;

step B, transforming the recombinant plasmid into saccharomyces cerevisiae SyBE _ Sc04020001, screening transformants, and culturing the screened transformants by using a YPD culture medium to obtain a recombinant saccharomyces cerevisiae mutant strain; the SyBE _ Sc04020001 is a high-yield GGPP brewing yeast strain SyBE _ Sc14C10 adopting L-arm-P_GAL1-CrtB-Tpgk 1-R-arm is obtained by homologous recombination integrating the CrtB gene at a specific position in the genome;

obtaining a yeast promoter Ga11-10, a gene CrtB, a terminator Pgk1t, an upstream integration homologous arm and a downstream integration homologous arm through PCR amplification, wherein the gene CrtB is used according to a saccharomyces cerevisiae codonPerforming optimization and appropriately avoiding common restriction enzyme cutting sites, performing artificial synthesis, and performing modular assembly on a yeast promoter Ga11-10, a gene CrtB, a terminator Pgk1t, an upstream integration homologous arm and a downstream integration homologous arm by using overlap extension PCR to obtain the L-arm-P_GAL1-CrtB-Tpgk 1-R-arm, named Modulel, said L-arm-P_GAL1The sequence of-CrtB-Tpgk 1-R-arm is shown as SEQ ID number 1;

the Saccharomyces cerevisiae strain SyBE _ Sc14C10 for high yield of GGPP is obtained by firstly obtaining delta gal1 delta gal7 delta gal 10:: HIS3 and then obtaining delta yp1062w: KanMX on the basis of CEN.PK2-1C.

4. The method for constructing a mutant strain of recombinant Saccharomyces cerevisiae as claimed in claim 3, which comprises:

step A, constructing a recombinant plasmid PRS416-BtCrtI with the 136 th histidine mutated into arginine of the BtCrtI gene¹³⁶(ii) a The BtCrtI is a CrtI from Blakeslea trispora;

splicing GAL7 promoter, CYC1t terminator and gene BtCrtI synthesized by optimizing Saccharomyces cerevisiae codon and appropriately avoiding common restriction enzyme cutting sites by an OE-PCR method to obtain a linear fragment GAL7 promoter-BtCrtI-CYC 1t terminator with two ends containing XbaI and NotI restriction enzyme cutting sites, namely PGAL7-BtCrtI-TCYC1, wherein the sequence of the PGAL7-BtCrtI-TCYC1 is shown as SEQ ID number 2; then, the linear fragment and the PRS416 plasmid are treated by XbaI and NotI endonucleases and then are connected by using T4 ligase to obtain the PRS416 plasmid containing BtCrtI gene, namely the plasmid PRS 416-BtCrtI;

introducing mutation points, namely designing a primer at the 136 th site of BtCrtI, and introducing the mutation points to mutate histidine H into arginine R; using plasmid PRS416-BtCrtI as a template, amplifying fragments GAL7-H136R and H136R-cyc1T, splicing the two fragments at the 136 th position by an OE-PCR method to obtain a linear fragment of which two ends contain XbaI and NotI enzyme cutting sites, treating the linear fragment and the PRS416 plasmid by XbaI and NotI endonucleases, and connecting the linear fragment and the PRS416 plasmid by using T4 ligase; the GAL7-H136R is shown as SEQ ID NO.7, and the H136R-cycle is shown as SEQ ID number 8;

step B, weighingThe plasmid group is transformed into saccharomyces cerevisiae SyBE _ Sc04020001, transformants are screened, and the screened transformants are cultured by YPD culture medium to obtain recombinant saccharomyces cerevisiae mutant strains; the SyBE _ Sc04020001 is a high-yield GGPP brewing yeast strain SyBE _ Sc14C10 adopting L-arm-P_GAL1-CrtB-Tpgk 1-R-arm is obtained by homologous recombination integrating the CrtB gene at a specific position in the genome;

obtaining a yeast promoter Ga11-10, a gene CrtB, a terminator Pgk1t, an upstream integration homologous arm and a downstream integration homologous arm through PCR amplification, optimizing the used gene CrtB according to a saccharomyces cerevisiae codon, artificially synthesizing the gene CrtB after appropriately avoiding common restriction enzyme cutting sites, and modularly assembling the yeast promoter Ga11-10, the gene CrtB, the terminator Pgk1t, the upstream integration homologous arm and the downstream integration homologous arm by using overlap extension PCR to obtain the L-arm-P_GAL1-CrtB-Tpgk 1-R-arm, named Modulel, said L-arm-P_GAL1The sequence of-CrtB-Tpgk 1-R-arm is shown as SEQ ID number 1;

the Saccharomyces cerevisiae strain SyBE _ Sc14C10 for high yield of GGPP is obtained by firstly obtaining delta gal1 delta gal7 delta gal 10:: HIS3 and then obtaining delta yp1062w: KanMX on the basis of CEN.PK2-1C.

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