CN117417934B

CN117417934B - Cross-strain promoter, multi-strain shuttle plasmid and application thereof

Info

Publication number: CN117417934B
Application number: CN202311415902.7A
Authority: CN
Inventors: 康振; 陈坚; 左文杰; 彭安祺; 堵国成; 王阳; 李江华
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2023-10-27
Filing date: 2023-10-27
Publication date: 2024-09-13
Anticipated expiration: 2043-10-27
Also published as: CN117417934A

Abstract

The invention discloses a strain-crossing promoter, a multi-strain shuttle plasmid and application thereof, in particular to a general promoter for escherichia coli, corynebacterium glutamicum, bacillus subtilis, saccharomyces cerevisiae and pichia pastoris, which can be active in the host and can start transcription of target genes. The invention effectively solves the problem that the construction of optimal chassis cells does not have enough universal promoter tools for carrying out different host expression verification on target genes in synthetic biology and metabolic engineering. By utilizing the strain-crossing promoter and the multi-strain shuttle plasmid constructed by the invention, the target gene expression condition can be rapidly screened in different host cells to obtain an optimal expression host, the synthetic biology tool box is effectively expanded, and a good basic element is provided for constructing a complex gene circuit.

Description

Cross-strain promoter, multi-strain shuttle plasmid and application thereof

Technical Field

The invention relates to the technical field of bioengineering, in particular to a cross-strain promoter, a multi-strain shuttle plasmid and application thereof.

Background

In fermentation engineering and cell metabolism engineering, it is necessary to construct metabolic pathways for the host microbial cells to synthesize exogenous related compounds. However, since exogenous genes are different, the microbial cells of different hosts have different host expression adaptability, so conventionally, for strange exogenous gene expression, research and characterization in a plurality of hosts including escherichia coli are often needed to screen out the most suitable exogenous gene expression host. Generally, after obtaining an optimal expression host, a series of genetic engineering operations such as gene amplification in E.coli cells are required, and then transferred to the expression host for expression.

For more convenient construction procedures, researchers have developed shuttle plasmid vectors that have the ability to transfer in different host cells, for example: bacillus subtilis-escherichia coli shuttle plasmid, escherichia coli-saccharomyces cerevisiae shuttle plasmid, and the like. However, at present, few researches on construction of multi-strain shuttle plasmids exist, and gene expression operation and metabolic pathway construction on more than two hosts are still complicated. Furthermore, there is incompatibility of natural promoters between different hosts, and when the same gene is required to be expressed between different hosts, each transfer of the gene expression cassette requires replacement of the promoter adapted to the host.

Therefore, it is a technical problem to be solved by those skilled in the art to provide a plasmid capable of shuttle among various host cells, stably existing and expressing a target gene carried thereon, and facilitating construction of metabolic pathways.

Disclosure of Invention

In order to solve the technical problems, the invention constructs a cross-strain promoter and multi-strain shuttle plasmid which can be simultaneously applied to genetic engineering operations of escherichia coli, bacillus subtilis, saccharomyces cerevisiae and pichia pastoris. The strain-crossing promoter carried by the plasmid can start gene expression of five microbial host cells at least comprising escherichia coli, bacillus subtilis, corynebacterium glutamicum, saccharomyces cerevisiae and pichia pastoris, and effectively improves gene expression and metabolic pathway construction efficiency.

It is a first object of the present invention to provide a trans-species promoter comprising a prokaryotic portion and a eukaryotic portion, which promoter is capable of promoting expression of a gene of interest in different prokaryotes and eukaryotes. The strain-crossing promoter comprises a transcription factor binding site, an UP sequence, a core skeleton, a ribosome binding site and a Kozak sequence, wherein:

(1) The UP sequence is located between the transcription factor binding site and the core backbone and the transcription factor binding site is located upstream of the core backbone, and the ribosome binding site and the Kozak sequence are located downstream of the core backbone; the transcription factor binding site is selected from one of sequences shown in SEQ ID NO.7-11, the UP sequence is selected from one of sequences shown in SEQ ID NO.3-6, and the sequence of the core skeleton is shown in SEQ ID NO. 2;

Or (2) the transcription binding site is located upstream of the core backbone and the UP sequence is located in the core backbone, the ribosome binding site and Kozak sequence are located downstream of the core backbone; the transcription factor binding site is selected from one of sequences shown in SEQ ID NO.7-11, the UP sequence is selected from one of sequences shown in SEQ ID NO.3-6, the sequence of the core skeleton is shown in SEQ ID NO.2, and the UP sequence insertion site is between the 18 th base and the 19 th base of the core skeleton sequence.

Further, the sequence of the ribosome binding site is shown in SEQ ID NO. 12.

Further, the Kozak sequence is shown in SEQ ID NO. 13.

The strain-crossing promoter not only has a binding site sequence, an UP sequence and an RBS sequence for binding prokaryotes sigma 70, sigma N, sigma B and sigma D, but also has a binding sequence of transcription factors Cat8, tye7, hap4, abf1, gat1, gln3, gcr2, stp1, UPC2, gal4-like and Swi 5. The sequence arrangement and combination are rationally designed. The promoter has activity in cells such as escherichia coli, bacillus subtilis, corynebacterium glutamicum, saccharomyces cerevisiae, pichia pastoris and the like, and can start transcription of a target gene.

The second object of the present invention is to provide a multi-species shuttle plasmid carrying the above-mentioned cross-species promoter.

Further, the multi-species shuttle plasmid contains a replication site and/or an integration site of the host species.

Further, the multi-strain shuttle plasmid is a shuttle plasmid used between two or more of escherichia coli, bacillus subtilis, corynebacterium glutamicum, saccharomyces cerevisiae and pichia pastoris.

Further, one or more of an escherichia coli replicon, a bacillus subtilis replicon, a corynebacterium glutamicum replicon, a saccharomyces cerevisiae replicon or an integration site and a pichia pastoris integration site are connected to the multi-strain shuttle plasmid.

Further, the E.coli replication site was ColE1, the B.subtilis replication site was repB, and the Saccharomyces cerevisiae replication site was 2 μm ori.

Further, the saccharomyces cerevisiae genome integration site is a URA3 sequence, and the pichia genome integration site is a HIS sequence.

Further, the construction method of the multi-strain shuttle plasmid comprises the following steps:

S1, designing a strain-crossing promoter: specific primers are synthesized, wherein the primers have the binding site sequences of sigma 70, sigma N, sigma B and sigma D, and UP sequences, and homology arm sequences are arranged between the primers, so that the subsequent DNA assembly process is facilitated. The activity of endogenous constitutive promoters of Saccharomyces cerevisiae and Pichia pastoris and their core promoters are detected, and the transcription factor binding sites of the preferred promoters are analyzed, preferably, the DNA binding sequences of transcription factors Cat8, tye7, hap4, abf1, gat1, gln3, gcr2, stp1, UPC2, gal4-like, swi5 are synthesized.

S2, synthesizing an escherichia coli replication site ColE1 sequence, a bacillus subtilis replication site repB sequence, a saccharomyces cerevisiae replication site 2 mu m ori sequence and a pichia pastoris integration site HIS sequence.

S3, taking plasmids such as pY26 and the like as skeletons, and assembling a strain-crossing promoter and each strain replication site through a homologous arm to obtain the multi-strain shuttle plasmid.

It is a third object of the present invention to provide a host cell containing the cross-species promoter or multi-species shuttle plasmid.

The fourth object of the invention is to provide the application of the cross-strain promoter or multi-strain shuttle plasmid in genetic engineering. Such as application in the field of microbial cell metabolism engineering, including expression of exogenous genes and construction of cell metabolic pathways.

The fifth object of the invention is to provide the application of the cross-strain promoter or multi-strain shuttle plasmid in the field of synthetic biology, including construction of biosensor elements, genetic circuit design and chassis cell screening.

The sixth object of the present invention is to provide a method for screening genetically engineered host bacteria, comprising the steps of:

And connecting target genes to strain-crossing promoters in the multi-strain shuttle plasmid, then respectively introducing the target genes into host bacteria to be selected, and screening according to target signals.

It is a seventh object of the present invention to provide an expression system for host selection comprising the above-described cross-species promoter or multi-species shuttle plasmid.

An eighth object of the present invention is to provide a recombinant Pichia pastoris containing at least an MT-OX enzyme encoding gene and a EEVS enzyme encoding gene which are promoted by the trans-species promoter.

Further, pichia pastoris GS115 is used as an original strain.

A ninth object of the present invention is to provide a method for producing Gadusol, which uses the recombinant Pichia pastoris for fermentation production.

The invention has the beneficial effects that:

The invention realizes the simultaneous construction of molecular operation plasmids of escherichia coli, bacillus subtilis, saccharomyces cerevisiae and pichia pastoris and promoters for exogenous gene expression, solves the problems of lack of multi-host genetic operation plasmid vector tools and incapability of identifying natural promoters among different hosts, and effectively improves the construction efficiency of gene expression and metabolic pathways. The invention provides a good foundation for research and application of metabolic pathway construction, synthesis biological gene expression element development and the like of cell factories.

Drawings

FIG. 1 is a schematic representation of the rational design of the insertion site of a prokaryotic sigma element across bacterial promoters.

FIG. 2 shows the results of 6 UP sequence activity assays and mutation analyses.

FIG. 3 is a preliminary screening of promoter UP mutation library sequences by flow cytometry and detection of the relative intensities of green fluorescent proteins of transformants.

Fig. 4 is a graph of the stronger UP sequence frequency LOGO obtained by the flow screening.

FIG. 5 is a test for the activity of an endogenous promoter in a yeast cell.

FIG. 6 is the fluorescence intensity of different transcription binding sequences in Saccharomyces cerevisiae and Pichia pastoris cells.

FIG. 7 is a schematic diagram of the construction process of the cross-species promoter pshuttle-21.

FIG. 8 is a schematic diagram of the construction process of a multi-species shuttle plasmid.

FIG. 9 is a microscopic photograph of the structure of the main elements of the cross-species promoter and its expression of green fluorescent protein in different microbial cells.

FIG. 10 shows the results of activity assays for combinations of different promoter elements.

FIG. 11 is a liquid chromatogram using cross-species promoter expression gadusol.

FIG. 12 is a mass spectrum of the application of cross species promoter expression gadusol.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.

The primers used in the following examples are as follows:

Construction of PCR primer across strain promoters

Note that: wherein N represents any one of A\T\C\G nucleotides.

Shuttle plasmid PCR sequencing primer

The media used in the examples below were as follows:

LB medium: 10g/L of sodium chloride, 5g/L of yeast powder and 10g/L of peptone.

YPD medium: yeast powder 10g/L, peptone 20g/L, glucose 20g/L.

BMGY medium: peptone: 20.0g/L, yeast extract powder: 10.0g/L, 100mmol of phosphate buffer, pH6.0, 100ml of filter sterilized 13.4% YNB solution, 2ml of filter sterilized 0.02% biotin solution, and 40ml of glycerol were added in a sterile operation. The total volume was 1L.

The solid culture medium is added with 2% of agar powder on the basis of liquid.

Example 1: cross-strain promoter and multi-strain shuttle plasmid vector construction method

(1) Design of strain-crossing promoters

1. Prokaryotic sigma factor fused promoter core skeleton

The design sequence and the structure of the sigma 70, sigmaN, sigmaB, sigmaD binding sites are shown in figure 1, and primer SigmaBD-F, sigmaBD-R is used by taking promoter Pbs as a framework (GGCGCGCCCCTCCTTGACACTGAATTTAGCATGTGATATAATTAACTTAATATTCTACCCAAGCTTATAAAAGAGCACTGTTGGGCGTGAGTGGAGGCGCCGGAAAAAAGCATCGAAAAAA,SEQ ID NO.1),; sigmaN-F, sigmaN-R introduces a sigma factor conserved sequence at a specific site, integrates the sigma factor conserved sequence into a promoter Pbs step by step through a step-by-step PCR reaction, designs a cross-strain promoter core structure, and has the sequence of

CTGGCACACCTTTTGCATCCTCCTTGACACTGAATTTAGCATGTGATATAATTAACTTAATATTCTACCCAAGCTTATAAAAGAGTTTAATTGGGCGCCGATATGGGTATCAGAAAAAAGCATCGAAAAAA(SEQ ID NO.2).

The PCR reaction conditions were: 98 ℃ for 3min; cycling for 32 times at 98 ℃ for 10s,55 ℃ for 15s and 72 ℃ for 2min for 30 s; and at 72℃for 10min. After each reaction, the plasmid is transformed into competent cells of escherichia coli, sequencing verification is carried out by picking up transformants, the sequencing primer is seqUP, and plasmids are extracted from correctly sequenced transformants.

2. Detection of prokaryotic promoter upstream UP sequence Activity and library establishment

Firstly, six promoter sequences are synthesized and integrated into the upstream of a core promoter sequence for activity detection, as shown in figure 2, strong 1, 4 and 6 UP sequences are selected for sequence analysis, and the degenerate sequences are taken as follows: TTGCACATATGAAGATCAAAAAAAAATCCGTGAGA (SEQ ID NO. 3).

Secondly, according to blast analysis of the UP sequence No. 1-6, the mutation sequence is defined as follows: TTCAGTGTCAAGGAGG-NNNNN-G-NN-T-N-T-N-TTTTTGATC-N-TCAT-N-T-NN-GGCGCGCCGGATCC, N represents random four nucleotides. Random sequence library is constructed by synthesizing random UP sequence primers UPN-F and UPN-R, introducing the primers into the upstream of Pbs promoter, constructing random sequence, and constructing random sequence library by PCR and homologous assembly. The PCR reaction conditions were: 98 ℃ for 3min; cycling for 32 times at 98 ℃ for 10s,55 ℃ for 15s and 72 ℃ for 2min for 30 s; and at 72℃for 10min. 11 total parallel PCR reactions were performed to increase library coverage. The competent cells of the escherichia coli TOP10 high-efficiency are taken, the competent cells are transformed by adopting a chemical transformation method, the transformed cells are cultured for 40min at 37 ℃ by using 1ml of LB culture medium, the cells are collected by centrifugation, and the cells are fully suspended by using PBS solution, so that the OD (optical density) of the cells is about 0.3. High throughput screening was performed using a flow cytometer, and cells with a fluorescence intensity of 0.1% before were collected. The collected cells were suspended in 2ml of LB medium, cultured at 37℃for 20min, and plated. Culturing the plates overnight, picking about 1000 transformants on a plurality of LB resistance plates by using an automatic bacterial picking workstation Q-pix system, inoculating into a 96-well plate, culturing for 12 hours at 37 ℃, measuring GFP relative fluorescence by using a TECAN microplate reader by taking an original promoter Pbs as a control, and further screening the fluorescence intensity to obtain a series of mutant promoters with the intensity about 225% -125% that of the original Pbs promoters, as shown in figure 3.

The first ten transformants with promoter activity stronger than the positive control were screened for fluorescence intensity, plasmid extraction was performed and sequenced. And (3) comparing the sequences of the sequencing results to obtain a plurality of better UP sequences shown in the table 1.

TABLE 1 UP sequence screening results

And carrying out flow screening on the S1-S10, and obtaining a strong UP sequence frequency LOGO chart shown in figure 4. These UP sequences obtained by mutation and screening are used to construct the prokaryotic element portion of the trans-species promoter. The fusion sequence (SEQ ID NO. 3) of the strong UP sequences S1, S2 and S3 (SEQ ID NO. 4-6) obtained by screening and the UP _1\4\6 sequence is used for the next step of cross-strain promoter design.

3. Sequence analysis and rational combination of eukaryotic promoter transcription factors

Preferably, the endogenous constitutive promoters of Pichia and Saccharomyces cerevisiae and the core structural region (core) thereof are used for activity detection, and green fluorescent protein is used as an indicator gene, as shown in FIG. 5. The transcription factor binding sites of GAP promoter, GCW14 promoter and TEF1 promoter with strong activity are analyzed to obtain transcription factor collection. The related transcription factor conserved binding sequence is inquired through a transcription factor database yeastract-plus and a transcription factor prediction website https:// www.genomatix.de, and the transcription factors with transcription negative regulation and control function are removed, so that 12 forward activation transcription factors are obtained, and the forward activation transcription factors are shown in table 2:

TABLE 2 partial transcription factor conserved binding sequences

The transcription factor sequences were aligned and combined by using a simple set of degenerate sequence codes as shown below. This code implements a function minimum superstring that accepts any number and length of DNA sequences as parameters and returns the minimum parallel strings of these DNA.

The code is as follows:

From the above codes, transcription factor binding sequences comprising 12 to 1 from short to long were selected, resulting in the following combinations as shown in the following table:

TABLE 3 transcription factor binding sequences

(Taking 12TF as an example), these 12 sequences were ligated by PCR upstream of the promoter sequence SEQ ID NO.1 using primers TF-R1/TF-R2/TF-F1/TF-F2, and fluorescence intensity verification was performed in Saccharomyces cerevisiae and Pichia pastoris cells, denoted 1TF to 12TF, as shown in FIG. 6.

Comparing and selecting the following preferred sequences for alternative combination: 2TF, 5TF, 6TF, 11TF, 12TF (SEQ ID NO. 7-11).

4. The prokaryotic cell fraction was combined with sequences selected from eukaryotic cell promoters and designed to span the species promoter pshuttle. The composition structure is shown in Table 4, the specific combination is shown in example 3, the composition mainly comprises a prokaryotic element and a eukaryotic element, and the structure schematic diagram is shown in FIG. 7. Wherein the nucleotide sequence of the RBS sequence is: AAGGAGGAAAAAA (SEQ ID NO. 12), the nucleotide sequence of the Kozak sequence is TCTGTAACA (SEQ ID NO. 13).

(2) Multi-strain shuttle plasmid PSH design

As shown in FIG. 8, the resistance element, part of the replication element and the backbone of plasmid PEBS、pY26、PEBS2(Yang S,Liu Q,Zhang Y,Du G,Chen J,Kang Z.Construction and Characterization of Broad-Spectrum Promoters for Synthetic Biology.ACS Synth Biol.2018 Jan 19;7(1):287-291.)、PAO815 were selected, respectively, and E.coli, B.subtilis, saccharomyces cerevisiae replication site or genome integration site, and Pichia genome integration site were synthesized by PCR reaction. After assembly construction, the replication or integration region of the shuttle plasmid PSH is formed. The GFP gene DNA sequence is synthesized by a company, an escherichia coli replication site (E coli ori) is ColE1, a bacillus subtilis replication site (B.s ori) is repB, a saccharomyces cerevisiae replication site is 2 mu m ori (on a PEBS2 plasmid), a saccharomyces cerevisiae genome integration site is a URA3 sequence, and a pichia pastoris genome integration site is an HIS sequence. ColE1, repB, 2 μm ori, URA3 or HIS was assembled by Gibbs. Transformants were picked and sent to DNA sequencing to verify if the construction was successful. The sequencing primer is as follows: seq-1 to seq-9.

Example 2: detection of activity of strain-crossing promoters in different host cells

The designed multi-strain universal promoter library is verified in different microbial cells by expressing green fluorescent protein.

The transformation method of each strain is as follows:

Coli: add 1 μl of pre-chilled plasmid solution with cross strain promoters to competent cells and mix gently. Then the mixture of cells and plasmids was placed on ice for 30 minutes, the mixture of competent cells and plasmids was placed in a 42℃water bath for heat shock for 90 seconds, the mixture was taken out of the 42℃water bath, immediately placed on ice, and after adding 450. Mu.l of LB medium, placed on a 37℃shaker, vibrated at 225rpm, post-cultured for 1 hour, centrifuged to aspirate 100. Mu.l of bacterial liquid onto an LB-band resistant agar plate, and spread evenly on a plate. LB plates were incubated upside down at 37℃overnight.

Bacillus subtilis: the colonies of the hay 168comk cells were streaked overnight and diluted to OD _600nm of about 0.3 with LB and 30% xylose for 1.5h. About 2. Mu.g of plasmid was mixed with it, incubated for 2 hours, centrifuged and the corresponding plates were plated.

Corynebacterium glutamicum and yeast: the corresponding electroporation transformation method was used.

As shown in fig. 9 (wherein a is a transcription factor binding sequence, B is an UP sequence, C is a core backbone fused with sigma factor, D is an RBS sequence and a Kozak sequence), to successfully express green fluorescent protein transcribed across a seed promoter in escherichia coli, bacillus subtilis, corynebacterium glutamicum, saccharomyces cerevisiae, pichia pastoris, and the highlight part is green fluorescent.

Example 3: screening of promoters across species

The PSH plasmid of example 1 was used as an expression vector, and E.coli, bacillus subtilis, corynebacterium glutamicum, saccharomyces cerevisiae, and Pichia pastoris were used as host bacteria, respectively, to determine the activity of the strain-crossing promoter. A cross species promoter library was constructed according to the elements selected in example 1 above, wherein the UP sequence was selected from SEQ ID NO.3-6 and the transcription factor binding site was selected from SEQ ID NO.7-11. The specific composition is shown in the following table: group A (selected transcription factor binding sequence), group B (selected UP sequence), group C (selected core backbone of fused sigma factor: SEQ ID NO. 2), group D (ribosome binding site, kozak sequence). The cross-species promoter combination sequence Pshuttle-1 is A1B1CD, pshuttle-2 is A1B2CD, and the combination of these derivatives Pshuttle-20 is A5B4CD.

TABLE 4 selection across strain promoter elements

As a result, as shown in FIG. 10 (measurement result of E.coli, control was wild-type cell), pshuttle-15 and Pshuttle-21 (see FIG. 7) were found to be optimal, and the combinations were A4B3CD and A5B4CD, respectively. Note that the 21 st sequence is A5B4CD (consistent with the Pshuttle-20 packet sequence numbers), but differs from Pshuttle-20 in that the sequence B4 is inserted after the group C italicized sequence, the other sequences are all normally aligned.

Example 4: construction gadusol of synthetic gene expression cassette based on strain-crossing promoter

Gadusol is a natural compound with anti-uv effect, which is structurally related to and has similar physiological functions as the retinoid amino acids (MAAs). Gadusol are commonly found in fungi, algae and marine invertebrates and are present in relatively high amounts in certain fish eggs in the ocean, which function to protect somatic or embryonic cells from ultraviolet radiation and provide antioxidant effects. At present gadusol is mainly extracted from algae, marine fish eggs and the like, and has high cost and low extraction quantity. In the aspect of microbial fermentation, gadusol only performs de-novo synthesis in the saccharomyces cerevisiae-based chassis cells, the synthesis yield of gadusol is low, and no chassis cells capable of efficiently expressing gadusol exist at present.

The shuttle plasmid PSH designed in the above example and the preferred promoters Pshuttle-21 in the cross strain promoter library are used to construct a gene expression cassette for synthesizing the ultraviolet resistant compound gadusol: MT-OX enzyme (NCBI gene number LOC 100003999) and EEVS enzyme (NCBI gene number zgc: 113054) were transformed into Saccharomyces cerevisiae and Pichia pastoris, respectively, and subjected to detection of expression of gadusol in the chassis host. Wherein the shuttle plasmid sequence used is shown in SEQ ID NO. 14.

The constructed plasmid expressing gadusol is transformed into the target host. The conversion process is described in example 2.

The Saccharomyces cerevisiae and Pichia pastoris transformants are picked up respectively, inoculated into YPD medium for 16-18h, inoculated into 500mL shake flasks containing fresh 100mL BMGY medium at 1%, cultured at 220rpm for 7d at 30 ℃ and centrifuged, and cells are collected.

After the fermentation culture, the cells were collected by centrifugation. The cells were washed twice with PBS, suspended in 80% methanol, disrupted by a high pressure homogenized cell disrupter, the disrupted cell solution was collected, the supernatant was collected after centrifugation, and after shaking for 30min, the supernatant was collected after-20℃overnight, and the supernatant was collected after centrifugation again. Concentrating gadusol the primary extract by centrifugation under vacuum, re-dissolving with PBS, centrifuging to obtain supernatant, and preserving at-20deg.C.

Gadusol was dissolved in phosphate buffer at pH 7.0 and loaded on a Hypersil GOLD aQ 5 μm liquid chromatography column (specification 4.6 mm. Times.250 mm) with the following buffers: methanol-5 mM phosphate buffer (1% MeOH for 20 min, then gradient from 1-95% MeOH over 20 min), flow rate 0.3mL/min, detection wavelength 296nm. The results are shown in FIGS. 11-12, and the isolated gadusol was analyzed by ultra high performance liquid chromatography tandem quadrupole time of flight mass spectrometer (MALDI SYNAPT MS) to rapidly screen the best gadusol expression host and successfully detect the highest gadusol production in Pichia pastoris cells, as shown in Table 5, with a yield of about 100mg/l after 7d fermentation, which is the highest yield for all primary chassis cells at present.

TABLE 5 expression gadusol yield across seed promoters

The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.

Claims

1. A strain-crossing promoter is characterized in that,

(1) The strain-crossing promoter comprises a transcription factor binding site, an UP sequence, a core skeleton, a ribosome binding site and a Kozak sequence, wherein: the UP sequence is located between the transcription factor binding site and the core backbone and the transcription factor binding site is located upstream of the core backbone, and the ribosome binding site and the Kozak sequence are located downstream of the core backbone; the transcription factor binding site is selected from one of sequences shown in SEQ ID NO.7-11, the UP sequence is selected from one of sequences shown in SEQ ID NO.3-6, and the sequence of the core skeleton is shown in SEQ ID NO. 2;

Or (2) the nucleotide sequence of the trans-strain promoter is ：CATATTCCGTTCGTCCGAATCAGCTGCTTGGTTGTCATGACCAGATAAGATTAGCGGCTCGTATAAAACGGAGGTCGTGTACCCGACCAGCCTGGCACACCTTTTGCATCAATATGACGATCAAAAATAGATCCCCCTTGGCGCGCCCCTCCTTGACACTGAATTTAGCATGTGATATAATTAACTTAATATTCTACCCAAGCTTATAAAAGAGTTTAATTGGGCGCCGATATGGGTATCAGAAAAAAGCATCGAAAAAAAAGGAGGAAAAAATCTGTAACA.

2. A multi-species shuttle plasmid carrying the cross-species promoter of claim 1.

3. The multi-species shuttle plasmid of claim 2 wherein the multi-species shuttle plasmid comprises a replication site and/or an integration site for a host species.

4. The multi-species shuttle plasmid of claim 2, wherein the multi-species shuttle plasmid is used for shuttle between two or more hosts among escherichia coli, bacillus subtilis, corynebacterium glutamicum, saccharomyces cerevisiae, pichia pastoris.

5. A host cell comprising the cross species promoter of claim 1 or the multi-species shuttle plasmid of any one of claims 2-4.

6. Use of the cross-species promoter of claim 1 or the multi-species shuttle plasmid of any one of claims 2-4 in genetic engineering or biosynthesis.

7. A method for screening genetically engineered host bacteria, comprising the steps of: after the target genes are linked to the strain-crossing promoters in the multi-strain shuttle plasmid according to any one of claims 2 to 4, the target genes are then introduced into host strains to be selected, respectively, and screened according to the target signals.

8. An expression system capable of being shuttled in different hosts, wherein the expression system comprises the cross-species promoter of claim 1 or the multi-species shuttle plasmid of any one of claims 2-4.

9. A recombinant pichia pastoris, wherein the recombinant pichia pastoris comprises at least the following modifications: overexpression of MT-OX enzyme-encoding gene and EEVS enzyme-encoding gene, which are promoted by the trans-species promoter of claim 1.

10. A method of producing Gadusol, wherein the recombinant pichia pastoris of claim 9 is used for fermentation production.