CN106893726B - Promoter and recombinant yeast strain - Google Patents

Abstract

The invention relates to the technical field of genetic engineering, and discloses a promoter and a recombinant yeast strain. The promoter of the invention is processed by one or more of the following steps based on the full-length sequence of the yeast strain ALD6 promoter: (1) knocking out one or more conserved sequences on an ALD6 promoter; (2) knocking out one or more sequences containing conserved sequences on an ALD6 promoter; (3) mutating one or more conserved sequences on the ALD6 promoter; (4) replacing one or more conserved sequences in the ALD6 promoter; (5) replacing one or more sequences comprising a conserved sequence on the ALD6 promoter. According to the invention, through the intensive research on the yeast strain ALD6 promoter, the conditions of deletion, replacement, mutation and the like of a conserved sequence of the yeast strain ALD6 promoter are found to improve the yield of the yeast strain for producing the terpenoid, and the yeast strain can be used as an optimization means of a synthetic path of the yeast engineering bacteria to construct the recombinant engineering bacteria with more excellent yield.

Description

Promoter and recombinant yeast strain

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a promoter and a recombinant yeast strain.

Background

Terpenoids are a generic term for all isoprene polymers and derivatives thereof, and can be classified according to the number of isoprene units included in the molecule: monoterpenes, sesquiterpenes, diterpenes, triterpenes, tetraterpenes. Terpenes are ubiquitous in the plant world and have important medicinal and economic values, such as antitumor drug paclitaxel, antimalarial drug artemisinin, strong antioxidant carotenoid, lycopene, geraniol, artenadiene, etc. However, due to the factors of scarce plant resources, low active substance content, great difficulty in chemical synthesis and the like, the wide application of terpenes in the fields of medicines and the like is limited.

The synthetic biological cell factory is used for carrying out targeted modification aiming at host cells (microorganisms and the like) and a target biosynthesis path to generate direct benefits, the production cost can be reduced, and the production period can be shortened, so that the compound is efficiently produced by using the synthetic biological cell factory, and the application prospect is very wide. Saccharomyces cerevisiae is a well-known safe model microorganism, has a short growth cycle, is easy to culture at high density, can be used as edible and medicinal yeast, and is a very excellent host for synthesizing the substances.

A key scientific problem with current synthetic biological cell factories is the adaptation of host cells to heterologous biosynthetic pathways. On one hand, the metabolic flux of the heterologous biosynthesis pathway determines the yield of the target product, so that the heterologous pathway needs to be optimized, including the optimization of the expression of heterologous genes, the screening of gene sources, the supply of intracellular precursor substances and the like; on the other hand, the intrinsic metabolic and regulatory systems from the host cell also influence the productivity of the target biosynthetic pathway, which requires extensive systematic optimization of the underpan cells.

In s.cerevisiae, the gene ALD6 (under the control of the ALD6 promoter) encodes acetaldehyde dehydrogenase, catalyzing the production of acetate from acetaldehyde in the cytoplasm. The expression level of the gene ALD6 directly influences the accumulation of acetic acid in cells, but there is no report that the gene can influence the yield of terpenoids.

Disclosure of Invention

In view of the above, the present invention aims to provide a promoter, which can increase the yield of various terpenoids in a yeast strain;

another object of the present invention is to provide a recombinant yeast strain containing the above promoter sequence, which can increase the production of various terpenoids.

In order to achieve the above purpose, the invention provides the following technical scheme:

a promoter is treated on the basis of the full-length sequence of the ALD6 promoter of a yeast strain by one or more of the following steps:

(1) knocking out one or more conserved sequences on an ALD6 promoter;

(2) knocking out one or more sequences containing conserved sequences on an ALD6 promoter;

(3) mutating one or more conserved sequences on the ALD6 promoter;

(4) replacing one or more conserved sequences in the ALD6 promoter;

(5) replacing one or more sequences comprising a conserved sequence on the ALD6 promoter.

At present, the research on the functions of the ALD6 promoter of the yeast strain is only limited in the aspect of acetic acid accumulation, and the functions of other aspects are not reported, aiming at the current situation, the ALD6 promoter is researched from multiple aspects, and the deletion, the replacement and the mutation of the conserved sequence of the ALD6 promoter are found to improve the yield of the terpenoid production yeast strain.

Preferably, the promoter of the invention is a promoter processed by one of the following methods based on the full-length sequence of the promoter of the yeast strain ALD 6:

(1) knocking out one or more sequences containing conserved sequences on an ALD6 promoter;

(2) knocking out a conserved sequence on an ALD6 promoter;

(3) mutating a conserved sequence on an ALD6 promoter;

(4) knocking out one or more sequences containing conserved sequences on one ALD6 promoter, and mutating the conserved sequences on one ALD6 promoter;

(5) a sequence containing a conserved sequence on one or more segments of the ALD6 promoter is replaced with a resistance tag.

Preferably, the yeast strain of the invention is saccharomyces cerevisiae, can be selected from saccharomyces cerevisiae of CEN.PK series, such as saccharomyces cerevisiae CEN.PK2-1C or saccharomyces cerevisiae CEN.PK2-1D or saccharomyces cerevisiae CEN.PK2, and can also be selected from saccharomyces cerevisiae of BY series, such as BY4741 saccharomyces cerevisiae or BY4742 saccharomyces cerevisiae or BY4743 saccharomyces cerevisiae. All Saccharomyces cerevisiae ALD6 promoters that have been genomically sequenced have the same conserved sequence and also have substantially the same non-conserved sequence, and as a whole, all Saccharomyces cerevisiae ALD6 promoters are substantially identical. Wherein, the Saccharomyces cerevisiae CEN.PK2-1C and the Saccharomyces cerevisiae CEN.PK2-1D, BY4741 have the identical ALD6 promoter sequences.

The invention uses a promoter conserved sequence to predict a website UCSC Genome Browser (http:// Genome-asia. UCSC. edu/cgi-bin/hgBlat, the evolution conservation prediction of the BY4741 Saccharomyces cerevisiae ALD6 promoter (the sequence is shown as SEQ ID NO: 1) results in 9 sections of conserved sequences, the A promoter (ALD6 full-length promoter) shown in FIG. 1, the conserved sequence is nine conserved sequences of 1-377bp (numbered IX), 630-654bp (numbered VIII), 701-787bp (numbered VII), 846-900bp (numbered VI), 916-925bp (numbered V), 941-1116bp (numbered IV), 1184-1205bp (numbered III), 1227-1313bp (numbered II), and 1430-1469bp (numbered I) in the ALD6 full-length promoter.

Since the 9 conserved sequences are key factors for solving the problem, the sequence including the conserved sequence can be knocked out in the invention to achieve the intended purpose, and the sequence including the conserved sequence can be a sequence connected with adjacent sequences at the upstream, downstream or upstream and downstream positions of the conserved sequence, and the adjacent sequences can have non-conserved sequence, other conserved sequence, or both, can have the whole sequence of the non-conserved sequence or other conserved sequence, or can be a partial sequence.

As described above, the sequences comprising conserved sequences have various forms, and in the present invention, can be selected from 1-629bp sequence, 630-700bp sequence, 701-845bp sequence, 846-915bp sequence, 916-940bp sequence, 941-1183bp sequence, 1184-1226bp sequence, 1227-1429bp sequence, 1430-1479bp sequence, 378-654bp sequence, 655-787bp sequence, 787-900bp sequence, 788-900bp sequence, 901-925bp sequence, 926-1116bp sequence, 1117-1205bp sequence, 1117-1191bp sequence, 1206-1313bp sequence and 1314-1469bp sequence.

In the knockout treatment of related sequences, the present invention preferably performs gradient knockout according to the sequence from left to right of the a promoter in fig. 1, that is, performs multi-form knockout according to the gradually overlapping sequence of the knockout IX sequence, the knockout of the entire non-conserved sequence downstream of the IX sequence + IX, and the knockout of the entire non-conserved sequence downstream of the IX sequence + IX + VIII sequence, and for reasons of large number, the present invention does not list all the gradient knockout forms according to the above-described manner, but a person skilled in the art can realize other non-listed gradient knockout forms under the guidance of the above-described manner, which is also the protection scope of the present invention.

In the mutation treatment of the conserved sequence, the present invention preferably performs mutation on the entire conserved sequence, and the mutation is preferably a base substitution mutation, and in the specific implementation process, a base conversion mutation is exemplified, and the base conversion mutation is that pyrimidine base C is exchanged with T, and purine base A is exchanged with G.

In the substitution treatment of related sequences, sequences which do not influence the production function of the terpenoid compounds of the yeast strains can be selected for substitution, and in the specific implementation process of the invention, the invention combines the convenience of practical screening to select resistance tags for substitution, such as KanMX resistance tags.

For the convenience of understanding the technical idea of the present invention, the present invention is illustrated by way of example but not limitation, referring to the B-L promoter of fig. 1, each promoter is subjected to various treatments such as knocking out, mutation and replacement on the basis of the full-length a promoter, so as to obtain a promoter meeting the conditions of the present invention. Wherein, the A promoter is ALD6 full-length promoter, and is 1479bp in total; the promoter B is a promoter for knocking out an IX sequence; the C promoter is a promoter of which the IX-containing sequence (1-629bp) is knocked out; the D promoter is a promoter for knocking out the sequences (1-629bp and 630-700bp) containing IX and VIII; the E promoter is a promoter for carrying out base conversion mutation on the whole VI sequence; the F promoter is a promoter for knocking out the sequences (1-629bp, 630-700bp and 701-845bp) containing IX, VIII and VII; the G promoter is a promoter for performing base conversion mutation on the VI whole sequence on the basis of the F promoter; the H promoter is a promoter for knocking out the sequences (1-629bp, 630-700bp, 701-845bp and 846-915bp) containing IX, VIII, VII and VI; the I promoter is a promoter for knocking out sequences containing IX, VIII, VII, VI and V (1-629bp, 630-700bp, 701-845bp, 846-915bp and 916-940 bp); the J promoter is a promoter for knocking out sequences containing IX, VIII, VII, VI, V and IV (1-629bp, 630-700bp, 701-845bp, 846-915bp, 916-940bp and 941-1183 bp); the K promoter is a promoter for knocking out sequences containing IX, VIII, VII, VI, V, IV and III (1-629bp, 630-700bp, 701-845bp, 846-915bp, 916-940bp, 941-1183bp, 1184-1226 bp); the L promoter is a promoter in which the sequences comprising VI, V, IV, part III and part VII (787) -900bp, 901-925bp, 926-1116bp and 1117-1191bp) are replaced by a KanMX resistance tag.

According to the invention, the C, D, E, F, G, H, K, L promoter is adopted as a test object to respectively replace the original ADL6 promoter in the saccharomyces cerevisiae strains for production such as geraniol, arteannuadiene, geranylgeraniol, zymosterol and lycopene, and the like, and then the yield is compared with the original strains, and the result shows that after the promoter is adopted, the yield of geraniol, arteannuadiene, geranylgeraniol, zymosterol and lycopene of each strain is improved compared with that of the original strain.

Meanwhile, in order to verify whether the conserved sequence is a main influence factor, adjacent non-conserved sequences are reserved on the basis of the promoter disclosed by the invention for comparison, and the result shows that the two sequences are not obviously different in terpenoid yield, so that deletion, replacement, mutation and the like of the conserved sequence on the ALD6 promoter are shown as main influence factors.

Based on the beneficial effects, the invention provides the application of the promoter in constructing the yeast strain for producing the terpenoid and producing the terpenoid. Wherein the terpenoid is one or more of geraniol, artenadiene, geranylgeraniol, zymosterol and lycopene.

In addition, according to test results and application, the invention provides a recombinant terpenoid-producing yeast strain, and the promoter provided by the invention is used for replacing the original ALD6 promoter. In the actual construction process, the required promoter can be constructed in advance, and the promoter is integrated on a genome through self homologous recombination of yeast, or the promoter can be directly subjected to operations such as knockout, replacement, mutation and the like on the basis of the original ALD6 promoter. The yeast strain is a saccharomyces cerevisiae strain, can be selected from saccharomyces cerevisiae of CEN.PK series, such as saccharomyces cerevisiae CEN.PK2-1C or saccharomyces cerevisiae CEN.PK2-1D, and can also be selected from saccharomyces cerevisiae of BY series, such as BY4741 saccharomyces cerevisiae.

According to the technical scheme, through the deep research on the ALD6 promoter of the yeast strain, the conditions of deletion, replacement, mutation and the like of the conserved sequence of the yeast strain are found to improve the yield of the yeast strain for producing the terpenoid, and the yeast strain can be used as an optimization means of a synthesis path of the yeast engineering bacteria to construct the recombinant engineering bacteria with more excellent yield.

Drawings

FIG. 1 shows a schematic diagram of an ALD6 full-length promoter and a promoter of the present invention; wherein Roman numerals represent conserved sequences on the ALD6 promoter, English letters represent the number of each promoter, and VI conserved sequences in E and G promoters are subjected to base conversion mutation;

FIG. 2 is a schematic diagram showing the gene elements of gene fragment 1; wherein, two ends of TRP1LHA and TRP1RHA respectively represent the upstream and downstream homologous sequences of the TRP1 locus of the yeast;

FIG. 3 is a schematic diagram showing the gene elements of gene fragment 2; wherein, two ends LEU2LHA and LEU2RHA respectively represent the upstream and downstream homologous sequences of the LEU2 locus of the yeast;

FIG. 4 is a schematic diagram showing the gene elements of gene fragment 3; wherein, two ends of TRP1LHA and TRP1RHA respectively represent the upstream and downstream homologous sequences of the TRP1 locus of the yeast;

FIG. 5 is a schematic diagram showing the gene elements of gene fragment 4; wherein, two ends LEU2LHA and LEU2RHA respectively represent the upstream and downstream homologous sequences of the LEU2 locus of the yeast;

FIG. 6 is a schematic diagram showing the gene elements of gene fragment 5; wherein, two ends of TRP1LHA and TRP1RHA respectively represent the upstream and downstream homologous sequences of the TRP1 locus of the yeast;

FIG. 7 is a schematic diagram showing the gene elements of gene fragment 6; wherein, two ends LEU2LHA and LEU2RHA respectively represent the upstream and downstream homologous sequences of the LEU2 locus of the yeast;

FIG. 8 is a bar graph showing the geraniol production of different recombinant strains; wherein A represents a recombinant strain containing a full-length promoter A, C, D, E, F, G, H, K, L represents a recombinant strain containing a corresponding numbered promoter, and the abscissa represents the yield of geraniol in mg/L;

FIG. 9 is a bar graph showing the yields of artemisinine from different recombinant strains; wherein A represents a recombinant strain containing a full-length promoter A, C, D, E, F, G, H, K, L represents a recombinant strain containing a promoter with a corresponding number, and the abscissa represents the yield of artenadiene in mg/L;

FIG. 10 shows a bar graph of geranylgeraniol production by different recombinant strains; wherein A represents a recombinant strain containing a full-length promoter A, C, D, E, F, G, H, K, L represents a recombinant strain containing a corresponding numbered promoter, and the abscissa represents the geranylgeraniol yield in mg/L;

FIG. 11 is a bar graph showing the zymosterol production by different recombinant strains; wherein A represents a recombinant strain containing a full-length promoter A, C, D, E, F, G, H, K, L represents a recombinant strain containing a promoter with a corresponding number, and the abscissa represents the output of zymosterol in mg/g DCW;

FIG. 12 is a bar graph showing lycopene production by different recombinant strains; wherein A represents a recombinant strain containing a full-length promoter A, C, D, E, F, G, H, K, L represents a recombinant strain containing a correspondingly numbered promoter, and the abscissa represents the lycopene production in mg/g DCW.

Detailed Description

The invention discloses a promoter and a recombinant yeast strain, and a person skilled in the art can realize the promoter and the recombinant yeast strain by appropriately improving process parameters according to the content. 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. The promoters and strains of the present invention have been described in terms of preferred embodiments, and it will be apparent to those of ordinary skill in the art that modifications, variations, and combinations of the promoters and strains can be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.

In the specific embodiment of the invention, in order to facilitate the implementation of the shake flask fermentation test, the saccharomyces cerevisiae CEN.PK2-1C which is a quadruple auxotroph (leucine, tryptophan, histidine and uracil) saccharomyces cerevisiae is adopted, so that the correct strain can be screened conveniently, and meanwhile, in order to ensure that the saccharomyces cerevisiae does not consume an inducer, three genes of gal1, gal7 and gal10 in the saccharomyces cerevisiae are knocked out, and the change of the saccharomyces cerevisiae is only convenient for the implementation of the verification test and has no influence on the implementation of the final effect.

Meanwhile, the saccharomyces cerevisiae CEN. PK2-1C does not have the capability of producing terpenoids per se, lacks part of enzymes, and needs to construct an exogenous gene element to be introduced into the strain to become a production strain. Wherein, the yeast strain for producing the geraniol by fermentation needs to contain the following gene segments integrated on the genome of the yeast strain by the homologous recombination of the yeast strain per se:

a gene segment 1 formed by sequentially splicing an upstream homologous sequence at a yeast trp1 site, a GAL1 promoter, a geraniol synthase encoding gene GES, a PGK1 terminator and a downstream homologous sequence at a yeast trp1 site, a schematic diagram is shown in figure 2, and the geraniol synthase encoding gene GES is derived from catharanthus roseus (Catharanthus roseus);

a gene segment 2 formed by sequentially splicing an upstream homologous sequence of a yeast LEU2 locus, an LEU2 marker, an ACT1 terminator, a truncated HMG-CoA reductase gene tHMGR1, a GAL10 promoter and a downstream homologous sequence of a yeast LEU2 locus, and a schematic diagram is shown in FIG. 3.

The yeast strain for producing the arteannuin by fermentation needs to contain the following gene segments integrated on the genome of the yeast strain by the homologous recombination of the yeast strain per se:

a gene segment 2 formed by sequentially splicing an upstream homologous sequence of a yeast LEU2 locus, an LEU2 marker, an ACT1 terminator, a truncated HMG-CoA reductase gene tHMGR1, a GAL10 promoter and a downstream homologous sequence of a yeast LEU2 locus;

the gene fragment 3 is formed by sequentially splicing an upstream homologous sequence of a yeast trp1 site, a GAL1 promoter, an artesundiene synthetase encoding gene ADS, a PGK1 terminator and a downstream homologous sequence of a yeast trp1 site, and is schematically shown in figure 4, wherein the artesundiene synthetase encoding gene ADS is derived from Artemisia annua (Artemisia annua).

The yeast strain for the fermentative production of geranylgeraniol needs to contain the following gene segments integrated into its genome by yeast self-homologous recombination:

an upstream homologous sequence of the LEU2 locus of the yeast, an LEU2 marker, an ACT1 terminator, a truncated HMG-CoA reductase gene tHMGR1, a GAL10 promoter, a GAL1 promoter, a geranylgeraniol synthase encoding gene GGPPS, a GPM1 terminator and a downstream homologous sequence of the LEU2 locus of the yeast are sequentially spliced to form a gene segment 4, the schematic diagram is shown in figure 5, and the geranylgeraniol synthase encoding gene GGPPS is derived from Taxus media (Taxus x media).

The yeast strain for fermentative production of zymosterol needs to contain the following gene segments integrated into its genome by yeast self-homologous recombination:

a homologous sequence at the upstream of the LEU2 locus of the yeast, an LEU2 marker, an ACT1 terminator, a truncated HMG-CoA reductase gene tHMGR1, a GAL10 promoter and a homologous sequence at the downstream of the LEU2 locus of the yeast are sequentially spliced to form a gene segment 2.

The yeast strain for producing lycopene by fermentation needs to contain the following gene segments integrated on the genome of the yeast by yeast self-homologous recombination:

the gene fragment 1 in CN105087406A, named as gene fragment 5 in the present invention, is shown in fig. 6, wherein the source of gene crtB is Pantoea agglomerans (Pantoea agglomerans) and is marked as PacrtB, the source of gene crtI is Blakeslea trispora (Blakeslea trispora) and is marked as Bt crtI:

the gene fragment 2 in CN105087406A is named as gene fragment 6 in the invention, the schematic diagram is shown in figure 7, the source of the gene crtE is Pantoea agglomerans (Pantoea agglomerans) which is marked as PacrtE, and the gene crtE is shown in SEQ ID NO: 5:

if the above-mentioned several target products are required to be simultaneously fermented and produced, the gene fragments introduced according to the requirements can be introduced one by one. The gene elements, homologous sequences and the like are obtained BY designing and synthesizing appropriate primers BY using the genome of the saccharomyces cerevisiae strain BY4741 as a template and performing PCR amplification, and specifically refer to the description in patent CN 105087406A; the heterologous genes are obtained by artificial synthesis after codon optimization and appropriate avoidance of common restriction enzyme cutting sites, wherein the GES sequence of a geraniol synthetase encoding gene derived from catharanthus roseus (Catharanthus roseus) is shown as SEQ ID NO. 2, the ADS encoding gene derived from Artemisia annua (Artemisia annua) is shown as SEQ ID NO. 3, and the GGPPS encoding gene derived from geranylgeraniol synthetase derived from Taxus media (Taxus x media) is shown as SEQ ID NO. 4.

The invention is further illustrated by the following examples.

Example 1: the promoter of the invention

The corresponding promoter is obtained on the basis of the ALD6 full-length promoter in the following manner:

the promoter B is a promoter for knocking out an IX sequence;

the C promoter is a promoter of which the IX-containing sequence (1-629bp) is knocked out;

the D promoter is a promoter for knocking out the sequences (1-629bp and 630-700bp) containing IX and VIII;

the E promoter is a promoter for carrying out base conversion mutation on the whole VI sequence;

the F promoter is a promoter for knocking out the sequences (1-629bp, 630-700bp and 701-845bp) containing IX, VIII and VII;

the G promoter is a promoter for performing base conversion mutation on the VI whole sequence on the basis of the F promoter;

the H promoter is a promoter for knocking out the sequences (1-629bp, 630-700bp, 701-845bp and 846-915bp) containing IX, VIII, VII and VI;

the I promoter is a promoter for knocking out sequences containing IX, VIII, VII, VI and V (1-629bp, 630-700bp, 701-845bp, 846-915bp and 916-940 bp);

the J promoter is a promoter for knocking out sequences containing IX, VIII, VII, VI, V and IV (1-629bp, 630-700bp, 701-845bp, 846-915bp, 916-940bp and 941-1183 bp);

the K promoter is a promoter for knocking out sequences containing IX, VIII, VII, VI, V, IV and III (1-629bp, 630-700bp, 701-845bp, 846-915bp, 916-940bp, 941-1183bp, 1184-1226 bp);

the L promoter is a promoter in which the sequences comprising VI, V, IV, part III and part VII (787) -900bp, 901-925bp, 926-1116bp and 1117-1191bp) are replaced by a KanMX resistance tag.

All the promoters can be obtained by total synthesis on the basis of knowing the sequences of the promoters, and the ALD6 promoter can also be obtained by primer amplification, and the corresponding promoters are obtained by processing according to a conventional knockout method, a replacement method and a mutation method.

Example 2: shake flask fermentation test (comparison between full-Length promoter and promoter of the invention)

1. Construction of test strains

Basic strains:

according to the description in patent CN105087406A, three genes of gal1, gal7 and gal10 in Saccharomyces cerevisiae CEN.PK2-1C are knocked out to construct a recombinant Saccharomyces cerevisiae strain A (a control strain, corresponding to a full-length promoter A);

with reference to the description in patent CN105087406A, three genes of gal1, gal7 and gal10 in Saccharomyces cerevisiae CEN.PK2-1C are knocked out, the C, D, E, F, G, H, K, L promoter in example 1 is used for replacing the original ALD6 promoter through yeast homologous recombination, and recombinant Saccharomyces cerevisiae strains C, D, E, F, G, H, K and L are constructed, and correspond to respective promoter self-numbering;

geraniol producing strain:

integrating a gene segment 1 and a gene segment 2 on the basis of the recombinant saccharomyces cerevisiae strain A to obtain a geraniol production strain A;

integrating a gene segment 1 and a gene segment 2 on the basis of the recombined saccharomyces cerevisiae strains C, D, E, F, G, H, K and L to obtain geraniol production strains C, D, E, F, G, H, K and L;

artemisia apiacea diene production strain:

integrating a gene fragment 3 and a gene fragment 2 on the basis of the recombinant saccharomyces cerevisiae strain A to obtain an artenadiene production strain A;

integrating a gene fragment 3 and a gene fragment 2 on the basis of the recombined saccharomyces cerevisiae strains C, D, E, F, G, H, K and L to obtain artenadiene production strains C, D, E, F, G, H, K and L;

geranylgeraniol producing strain:

integrating a gene segment 4 on the basis of the recombinant saccharomyces cerevisiae strain A to obtain a geranylgeraniol production strain A;

integrating a gene segment 4 on the basis of the recombined saccharomyces cerevisiae strain C, D, E, F, G, H, K and L to obtain geranylgeraniol production strains C, D, E, F, G, H, K and L;

zymosterol-producing strain:

integrating a gene segment 2 on the basis of the recombinant saccharomyces cerevisiae strain A to obtain a zymosterol production strain A;

integrating the gene fragment 2 on the basis of the recombined saccharomyces cerevisiae strains C, D, E, F, G, H, K and L to obtain a zymosterol production strain C, D, E, F, G, H, K and L;

lycopene producing strain:

integrating gene segments 5 and 6 on the basis of the recombinant saccharomyces cerevisiae strain A to obtain a lycopene production strain A;

integrating gene segments 5 and 6 on the basis of the recombined saccharomyces cerevisiae strains C, D, E, F, G, H, K and L to obtain lycopene production strains C, D, E, F, G, H, K and L;

the corresponding gene fragments are respectively transformed into each strain by a lithium acetate method, and are recombined with trp1 or leu2 sites on a yeast genome through upstream and downstream homologous sequences of trp1 or leu2 to be integrated on the genome. After transformation, an SD-TRP or SD-LEU solid plate (6.7 g/L of synthetic yeast nitrogen source YNB, 20g/L of glucose, 2g/L of mixed amino acid powder of single-lacking tryptophan or leucine, 2% agar powder) is adopted for screening, the obtained transformant is subjected to streak purification culture, a yeast genome is extracted for PCR verification, and the recombinant strains which are verified to be correct are preserved with glycerol and named respectively.

2. Construction of Gene fragments

Gene fragment 1, gene fragment 3 and gene fragment 5 were prepared by using the corresponding gene elements according to the method of patent CN105087406A example 3;

gene fragment 2, gene fragment 4 and gene fragment 6 were prepared by using the corresponding gene elements according to the method of patent CN105087406A example 4;

3. fermentation process

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

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

(Note: for geraniol, artenadiene, geranylgeraniol producing strains, 20% of n-dodecane was added to the fermentation medium)

Inoculating the above strain into 5mL seed culture medium, culturing at 30 deg.C and 250rpm for 14-16h, and determining initial thallus concentration OD₆₀₀Transferred to fresh 25mL seed medium at 0.2, cultured at 30 ℃ and 250rpm to the middle of logarithmic growth, and the initial cell concentration OD₆₀₀The cells were inoculated into 50mL of each fermentation medium at 0.5, cultured at 30 ℃ and 250rpm, and the cell density (OD) was monitored during the fermentation₆₀₀) And the yield.

4. Yield detection

Geraniol, artenadiene, geranylgeraniol: after fermentation for 48 hours, 12000g of fermentation liquor is centrifuged for 5min, and then an upper organic phase is taken out, diluted by normal hexane and subjected to GC-MS detection.

Zymosterol: after fermenting for 48 hours, taking two equal parts of fermentation liquor, centrifuging for 2min at 4000g, collecting thalli, and washing twice with water. Placing one part of the thalli at 80 ℃ to dry 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 method comprises the following steps: resuspending the cells with 1mL of 2N NaOH, placing in a boiling water bath to boil for 10min, and then immediately carrying out ice-bath for 3 min; centrifuging the crushed cells at 12000rpm at 4 deg.C for 4min, discarding supernatant, adding 300uL methanol solution containing 1.5M NaOH, saponifying at 60 deg.C for 4h, adding 300uL n-hexane, vortex oscillating for 10min, centrifuging, and collecting organic phase; the aqueous phase was vortexed with 300uL of n-hexane and then shaken for 10min, and finally centrifuged to collect the organic phase. And (3) carrying out vacuum freeze drying on the collected organic phase, adding 100ul of a derivatization reagent MSTFA, incubating at 30 ℃ for 2h, and finally diluting with n-hexane and carrying out GC-MS detection.

Lycopene: after fermenting for 48 hours, taking two equal parts of fermentation liquor, centrifuging for 2min at 4000g, collecting thalli, and washing twice with water. Placing one part of the thalli at 80 ℃ to dry 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 method comprises the following steps: resuspending the cells with 3N HCl, boiling in a boiling water bath for 2min, and immediately ice-cooling for 3 min; centrifuging the crushed cells at 12000rpm at 4 ℃ for 4min, discarding the supernatant, washing with water for 2 times, adding acetone, and vortexing for 5 min; and finally, centrifugally collecting an acetone phase, filtering by using a filter membrane of 2 mu m, and detecting by using an ultraviolet liquid phase, wherein the detection wavelength of the lycopene is 471 nm.

5. Results

The results are shown in fig. 8-fig. 12, and it is obvious that the original strain contains ALD6 full-length promoter, i.e. a promoter, which is lower in the yields of terpenoids such as geraniol, artesundiene, geranylgeraniol, zymosterol and lycopene, whereas the yields of terpenoids are improved after the promoters of the invention are replaced.

Example 3: shake flask fermentation test (removal of conserved sequence and removal of alignment between sequence promoters containing the conserved sequence)

In order to verify that conserved sequences are the main influencing factors, the present invention provides the following groups of promoters which are different only in the presence of non-conserved sequences, and recombinant strains were constructed in the manner of example 2 to compare the yields of terpenoids, and the results are shown in Table 1.

Group 1: promoter B and promoter C, the difference is that the promoter B has a 378-629bp non-conserved sequence more than the promoter C;

group 2: promoter I and promoter I + 926-;

group 3: promoter J and promoter J +1117-1183bp non-conserved sequences;

TABLE 1

As can be seen from Table 1, the yield difference between the groups is not obvious, which indicates that the treatment of the conserved sequence can realize the improvement of the yield of terpenoids, and the non-conserved sequence has no significant influence on the yield.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

SEQUENCE LISTING

<110> Tianjin university

<120> a promoter and a recombinant yeast strain

<130>MP1701567

<160>5

<170>PatentIn version 3.3

<210>1

<211>1479

<212>DNA

<213> Artificial sequence

<400>1

accaagttcc cgctcgattt gattcatgaa gaaggacaaa agaactattt aatgttcatg 60

aagatgattg aggaagaaaa ggaaaaaatt agaatacagc aggagcaaat gggcgggcaa 120

acatttacgc tgaaagacta tgttgaaggt aacttgcctt cgccagaaga acaaatgaaa 180

atacaattgg agaagcagaa ggaggtagac gccttatttg aagaggaaaa gaaaaagaag 240

aagattgctg aatccaaata attttcatgt aaaccctctt ctcatgtatc tacgtatcta 300

tgtgtgtatg taaatgtacc tgtacactcc ccacaccctc attttgttac tgtcatgtga 360

ataaaactta tgtatattgc taacttacta ccactgcacc tcctaacatc accatactac 420

gtacaaacac gcctatttat tttttctatg ttaaatttta acgatgtaga cacacctaat 480

gatctgatgc gctttgcata tctcatattc cttcactagc ataaaaatcc aaaaaaaaag 540

aatatttagg ccgaatggaa ttattcgtaa cgtcatacga aaaaagtttc aattcgtaca 600

atgcctggca tgttcattcg aatataaggc cgccgccttc cagtcagggt agccaaaagt 660

ataatcccgg gtggaaacta aactaaaaac cgtactcaca actttccgcg gacgctaaca 720

gacaaataga cacactatca ggtcaggaac tgccgtcaca tacgacactg cccctcacgt 780

aagggcatga tagaattgga ttatgtaaaa ggtgaagata ccattgtaga agcaaccagc 840

acgtcgccgt ggctgatgag gtctcctctt gcccgggccg cagaaaagag gggcagtggc 900

ctgtttttcg acataaatga ggggcatggc cagcaccgag acgtcattgt tgcatatggc 960

gtatccaagc cgaaacggcg ctcgcctcat ccccacggga ataaggcagc cgacaaaaga 1020

aaaacgaccg aaaaggaacc agaaagaaaa aagagggtgg gcgcgccgcg gacgtgtaaa 1080

aagatatgca tccagcttct atatcgcttt aactttaccg ttttgggcat cgggaacgta 1140

tgtaacattg atctcctctt gggaacggtg agtgcaacga atgcgatata gcaccgacca 1200

tgtgggcaaa ttcgtaataa attcggggtg agggggattc aagacaagca accttgttag 1260

tcagctcaaa cagcgattta acggttgagt aacacatcaa aacaccgttc gaggtcaagc 1320

ctggcgtgtt taacaagttc ttgatatcat atataaatgt aataagaagt ttggtaatat 1380

tcaattcgaa gtgttcagtc ttttacttct cttgttttat agaagaaaaa acatcaagaa 1440

acatctttaa catacacaaa cacatactat cagaataca 1479

<210>2

<211>1146

<212>DNA

<213> Artificial sequence

<400>2

atggcttact ctgctatggc aactatgggt tataatggta tggctgcatc ttgtcatact 60

ttgcatccaa catcaccatt aaaaccattt catggtgcat ctacatcatt ggaagctttt 120

aatggtgaac atatgggttt gttgagaggt tactctaaga gaaagttgtc ttcatacaag 180

aacccagctt caagatcttc aaatgctact gttgcacaat tgttgaatcc accacaaaag 240

ggtaaaaagg cagttgaatt cgatttcaat aagtacatgg attctaaagc tatgacagtt 300

aatgaagcat tgaataaggc tattccatta agatatccac aaaagatata tgaatctatg 360

agatactcat tgttagctgg tggtaaaaga gttagaccag ttttgtgtat tgctgcatgt 420

gaattagttg gtggtactga agaattggct attccaacag cttgtgcaat cgaaatgatc 480

catactatgt ctttgatgca tgatgatttg ccatgtatcg ataacgatga tttgagaaga 540

ggtaaaccaa caaaccataa gatcttcggt gaagatactg ctgttacagc tggtaatgct 600

ttgcattcat acgcattcga acatatcgct gtttctactt caaaaacagt tggtgcagat 660

agaatcttga gaatggtttc tgaattaggt agagctactg gttcagaagg tgttatgggt 720

ggtcaaatgg ttgatattgc atctgaaggt gacccatcaa tcgatttgca aactttggaa 780

tggatccata tccataagac agctatgttg ttggaatgtt ctgttgtttg tggtgcaatt 840

attggtggtg cttcagaaat cgttatcgaa agagcaagaa gatacgctag atgtgttggt 900

ttgttgttcc aagttgttga tgatatctta gatgttacta agtcttcaga tgaattgggt 960

aaaacagctg gtaaagattt gatctctgat aaggctacat acccaaagtt gatgggttta 1020

gaaaaggcaa aggaattttc tgatgaattg ttgaacagag ctaagggtga attgtcatgt 1080

tttgatccag ttaaagctgc accattgtta ggtttggcag attacgttgc ttttagacaa 1140

aattaa 1146

<210>3

<211>1641

<212>DNA

<213> Artificial sequence

<400>3

atgtctttga ctgaagaaaa gccaatcaga ccaatcgcta acttcccacc atctatctgg 60

ggtgaccaat tcttgatcta cgaaaagcaa gttgaacaag gtgttgaaca aatcgttaac 120

gacttgaaga aggaagttag acaattgttg aaggaagctt tggacatccc aatgaagcac 180

gctaacttgt tgaagttgat cgacgaaatc caaagattgg gtatcccata ccacttcgaa 240

agagaaatcg accacgcttt gcaatgtatc tacgaaactt acggtgacaa ctggaacggt 300

gacagatctt ctttgtggtt cagattgatg agaaagcaag gttactacgt tacttgtgac 360

gttttcaaca actacaagga caagaacggt gctttcaagc aatctttggc taacgacgtt 420

gaaggtttgt tggaattgta cgaagctact tctatgagag ttccaggtga aatcatcttg 480

gaagacgctt tgggtttcac tagatctaga ttgtctatca tgactaagga cgctttctct 540

actaacccag ctttgttcac tgaaatccaa agagctttga agcaaccatt gtggaagaga 600

ttgccaagaa tcgaagctgc tcaatacatc ccattctacc aacaacaaga ctctcacaac 660

aagactttgt tgaagttggc taagttggaa ttcaacttgt tgcaatcttt gcacaaggaa 720

gaattgtctc acgtttgtaa gtggtggaag gctttcgaca tcaagaagaa cgctccatgt 780

ttgagagaca gaatcgttga atgttacttc tggggtttgg gttctggtta cgaaccacaa 840

tactctagag ctagagtttt cttcactaag gctgttgctg ttatcacttt gatcgacgac 900

acttacgacg cttacggtac ttacgaagaa ttgaagatct tcactgaagc tgttgaaaga 960

tggtctatca cttgtttgga cactttgcca gaatacatga agccaatcta caagttgttc 1020

atggacactt acactgaaat ggaagaattc ttggctaagg aaggtagaac tgacttgttc 1080

aactgtggta aggaattcgt taaggaattc gttagaaact tgatggttga agctaagtgg 1140

gctaacgaag gtcacatccc aactactgaa gaacacgacc cagttgttat catcactggt 1200

ggtgctaact tgttgactac tacttgttac ttgggtatgt ctgacatctt cactaaggaa 1260

tctgttgaat gggctgtttc tgctccacca ttgttcagat actctggtat cttgggtaga 1320

agattgaacg acttgatgac tcacaaggct gaacaagaaa gaaagcactc ttcttcttct 1380

ttggaatctt acatgaagga atacaacgtt aacgaagaat acgctcaaac tttgatctac 1440

aaggaagttg aagacgtttg gaaggacatc aacagagaat acttgactac taagaacatc 1500

ccaagaccat tgttgatggc tgttatctac ttgtgtcaat tcttggaagt tcaatacgct 1560

ggtaaggaca acttcactag aatgggtgac gaatacaagc acttgatcaa gtctttgttg 1620

gtttacccaa tgtctatcta a 1641

<210>4

<211>1182

<212>DNA

<213> Artificial sequence

<400>4

atggcttata ccgcaatggc agcaggaact cagtcattgc agttgaggac agtcgcctct 60

taccaggagt gcaactcaat gaggtcttgc ttcaagttga ccccattcaa gtcattccac 120

ggtgtcaact tcaacgttcc ttctttaggt gccgccaact gcgaaatcat gggtcacttg 180

aaattgggtt ctttgccata caaacagtgt tcagtatcat ctaagtcaac taagactatg 240

gcccagttgg tagatttggc agagaccgag aaagccgagg gaaaggatat cgagttcgat 300

tttaacgagt atatgaagtc taaggctgtc gctgttgatg cagccttgga taaggccatc 360

cctttggagt atccagagaa gatccatgag tctatgaggt actcattgtt ggccggagga 420

aaaagggtca gacctgcatt atgcatcgct gcttgcgagt tagtaggtgg ttctcaggac 480

ttggccatgc caaccgcatg tgccatggaa atgattcata ccatgtcatt gattcacgat 540

gatttgcctt gcatggacaa cgacgacttc agaaggggaa agcctaccaa tcacaaggtt 600

ttcggagagg acactgctgt tttagccggt gacgcattgt tatctttcgc ttttgaacac 660

atcgccgttg ccacatcaaa aactgtccca tctgacagga ccttgagagt catttctgag 720

ttgggtaaaa ccatcggttc acagggattg gtcggaggtc aggtagtcga catcacttct 780

gagggagacg ccaacgtcga cttaaagaca ttggagtgga ttcacattca caagactgcc 840

gtcttgttgg aatgctctgt tgtttctgga ggaatcttgg gtggagctac cgaggatgag 900

attgctagaa taagaagata cgccaggtgc gtcggtttgt tgttccaggt tgtcgacgac 960

attttggatg tcaccaagtc ttcagaggaa ttgggaaaga ccgccggtaa agacttattg 1020

accgacaagg ctacctaccc taagttgatg ggtttggaga aggccaaaga gtttgcagca 1080

gaattagcta ccagggcaaa ggaagagttg tcatcattcg accagatcaa ggcagcccct 1140

ttgttaggat tggccgatta catcgctttc aggcaaaact aa 1182

<210>5

<211>924

<212>DNA

<213> Artificial sequence

<400>5

atggtttctg gttctaaggc tggtgtctca ccacacaggg agattgaggt catgaggcag 60

tctattgacg atcacttggc tggtttgttg cctgagactg actctcagga cattgtctca 120

ttggcaatga gggagggtgt catggctcca ggtaagagga taaggccttt gttgatgttg 180

ttggcagcta gggacttgag gtaccagggt tctatgccta ctttgttgga cttggcttgc 240

gctgtcgaat tgactcacac tgcatcattg atgttggacg acatgccttg catggacaac 300

gcagaattaa ggaggggtca gccaacaaca cacaagaagt tcggtgagtc agtcgcaatt 360

ttggcatcag ttggtttgtt atcaaaggct ttcggattga ttgctgcaac tggtgactta 420

ccaggtgaga ggagggcaca ggctgtcaac gagttgtcta ctgctgtcgg agtccaggga 480

ttggtcttgg gtcagttcag ggacttgaac gacgcagctt tggacaggac tccagacgct 540

atattgtcta caaaccactt aaagacagga attttgttct ctgctatgtt gcagatagtc 600

gctattgcat ctgcttcttc tccatctaca agggagactt tgcacgcttt cgcattggac 660

ttcggtcagg ctttccagtt gttggacgac ttgagggacg atcacccaga gacaggaaag 720

gacaggaaca aggatgcagg taaatcaact ttggtcaaca ggttaggtgc agacgctgct 780

aggcagaagt taagggagca cattgactct gctgacaagc acttgacttt cgcttgccca 840

cagggaggtg ctattaggca gttcatgcac ttgtggttcg gtcaccactt agctgactgg 900

tcacctgtca tgaagatagc ttaa 924

Claims (7)

1. A promoter is characterized in that on the basis of the full-length sequence of the promoter of an ALD6 yeast strain, the sequence is shown as SEQ ID NO:1, and one or more of the following treatments are carried out:

(1) knocking out one or more conserved sequences on an ALD6 promoter;

(2) knocking out one or more sequences containing conserved sequences on an ALD6 promoter;

(3) carrying out base conversion mutation on the 846-900bp conserved sequence on the ALD6 promoter;

(4) knocking out one or more sequences containing conserved sequences on the ALD6 promoter, and carrying out base substitution mutation on the 846-900bp conserved sequences on the ALD6 promoter;

(5) replacing one or more conserved sequences in the ALD6 promoter;

(6) replacing a sequence comprising a conserved sequence on one or more ALD6 promoters;

the conserved sequence is selected from nine segments of conserved sequences of 1-377bp, 630-654bp, 701-787bp, 846-900bp, 916-925bp, 941-1116bp, 1184-1205bp, 1227-1313bp and 1430-1469bp in the full-length sequence of the ALD6 promoter;

the sequence containing the conserved sequence is a sequence connected with adjacent sequences at the upstream, downstream or upstream and downstream positions of the conserved sequence and is selected from a 1-629bp sequence, a 630-19 bp sequence, a 701-containing 845bp sequence, a 846-915bp sequence, a 916-940bp sequence, a 941-1183bp sequence, a 1184-containing 1226bp sequence, a 1227-containing 1429bp sequence, a 1430-containing 1479bp sequence, a 378-654bp sequence, a 655-containing 787bp sequence, a 787-containing 900bp sequence, a 788-containing 900bp sequence, a 901-containing 925bp sequence, a 926-containing 1116bp sequence, a 1117-containing 1205bp sequence, a 1117-containing 1191bp sequence, a 1206-containing 1313bp sequence and a 1314-containing 1469bp sequence;

in the substitution treatment of the related sequences, sequences that do not affect the terpenoid production function of the yeast strain can be selected for substitution.

2. The promoter according to claim 1, wherein the promoter is obtained by processing one of the following steps based on the full-length sequence of the promoter of the yeast strain ALD 6:

(1) knocking out one or more sequences containing conserved sequences on an ALD6 promoter;

(2) knocking out a conserved sequence on an ALD6 promoter;

(3) a sequence containing a conserved sequence on one or more segments of the ALD6 promoter is replaced with a resistance tag.

3. The promoter according to claim 2, wherein the resistance tag is a KanMX resistance tag.

4. Use of the promoter according to any one of claims 1 to 3 for the construction of yeast strains for terpenoid production and for the production of terpenoids.

5. The use according to claim 4, wherein the terpenoids are one or more of geraniol, artenadiene, geranylgeraniol, zymosterol, lycopene.

6. A recombinant terpenoid producing yeast strain characterized in that the ALD6 promoter is replaced by a promoter according to any one of claims 1 to 3.

7. The yeast strain of claim 6, wherein the yeast strain is a Saccharomyces cerevisiae strain.

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