CN106244615B

CN106244615B - Engineering bacterium, construction method thereof and application of engineering bacterium in preparation of geraniol

Info

Publication number: CN106244615B
Application number: CN201610738201.0A
Authority: CN
Inventors: 李霞; 姜国珍; 肖文海; 姚明东; 刘宏; 元英进
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2016-08-26
Filing date: 2016-08-26
Publication date: 2020-05-15
Anticipated expiration: 2036-08-26
Also published as: CN106244615A

Abstract

The invention relates to the field of genetic engineering, in particular to an engineering bacterium, a construction method thereof and application thereof in geraniol preparation. The IDI1 gene, the tHMG gene and the modified GES gene are expressed in the engineering bacteria provided by the invention. Compared with the strain in the prior art, the yield of the engineering bacteria for producing the geraniol is higher, the fermentation time is 120 hours, and the concentration of the geraniol can reach 360.06 mg/L.

Description

Engineering bacterium, construction method thereof and application of engineering bacterium in preparation of geraniol

Technical Field

The invention relates to the field of genetic engineering, in particular to an engineering bacterium, a construction method thereof and application thereof in geraniol preparation.

Background

Geraniol (Geraniol, 3, 7-dimethyl-2, 6-octadien-1-ol, C)₁₀H₁₈O, molecular weight 154.2493), yak-geraniol, is an acyclic monoterpene compound found in plant volatile oil. Because geraniol has a mild rose taste and is widely used in floral daily essences, the regulation of GB2760-1996 states that geraniol is an allowable edible spice and is mainly used for preparing essences such as apples, peaches, apricots, plums, strawberries, lemons, gingers, cinnamon, nutmeg and the like. It is used as the main agent of rose essence, is an indispensable flavoring material in various flower essences, and can be used in cosmetics, soap, etc. Geraniol is also a raw material for the production of citronellol, ionone, vitamin a, etc., and the ester formed by geraniol is also a good perfume. Besides being used as essence, geraniol has certain pharmacological activity, can be used for clinical treatment of antibiosis, anthelmintic and chronic bronchitis, and is also proved to be used for treatment of cancer. In addition, geraniol is considered to be a superior gasoline substitute to ethanol due to its high energy, low hygroscopicity, and relatively low volatility characteristics. But is naturally bornThe yield of the produced geraniol is limited, the chemical synthesis has safety problems, and a microbial cell factory is an excellent geraniol production platform in the face of increasing market demands. The microbial synthesis has the advantages of low cost, high yield, safe product, easy operation and the like. Currently, in the research of synthesizing geraniol by microorganisms, the adopted hosts mainly focus on escherichia coli, saccharomyces cerevisiae and methanococcus.

The method for producing geraniol by microorganisms appeared at the earliest in 2013, and the industrial biotechnology laboratory of the university of south of the Yangtze river achieved the production of geraniol in Saccharomyces cerevisiae by enhancing the supply of precursors. Subsequently, there are continuous cases of success in the microbial production of geraniol, but most of them are E.coli as host. The saccharomyces cerevisiae is used as a well-known safe mode microorganism, has clear genetic background and simple gene operation, and can be used for large-scale fermentation production. Compared with Escherichia coli, it has high content of vitamins and proteins, and can be used as edible medicine and feed, and no antibiotic substance is added during fermentation. Therefore, achieving high geraniol yields in saccharomyces cerevisiae would represent a great competitive power in the industrialization of the microbial production of geraniol. However, there have been few reports of geraniol synthesis using Saccharomyces cerevisiae and the yield has been low to date. Compared with the yield of the geraniol in the escherichia coli, the yield of the geraniol in the saccharomyces cerevisiae is still relatively low, and the industrial requirement can not be met far. Therefore, the development of recombinant saccharomyces cerevisiae with high geraniol yield is urgently needed.

Disclosure of Invention

In view of this, the technical problem to be solved by the present invention is to provide an engineering bacterium, a construction method thereof and an application thereof in geraniol preparation, wherein the strain provided by the present invention can ferment to produce geraniol, and the highest concentration of produced geraniol can reach 360 mg/L.

The invention provides a modified GES gene, the nucleotide sequence of which is shown in any one of SEQ ID NO 1-7; or a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotides in the nucleotide sequence shown in any one of SEQ ID NO. 1-7.

The GES genes provided by the invention are respectively from 7 different plants, wherein:

the nucleotide sequence shown in SEQ ID NO. 1 is modified from GES gene (CtGES) of Cinnamomum tenuipilum (Cinnamomum tenuipile);

the nucleotide sequence shown in SEQ ID NO. 2 is modified from GES gene (LdGES) of the sweet tongue grass (Lippia dulcis);

the nucleotide sequence shown in SEQ ID NO. 3 is modified from the GES gene (VvGES) of grape (vitas vinifera);

the nucleotide sequence shown in SEQ ID NO. 4 is modified from GES gene (PcGES) of Perilla citriodora (Perilla citriodora);

the nucleotide sequence shown in SEQ ID NO. 5 is modified from GES gene (CrGES) of Catharanthus roseus (Catharanthus roseus);

6 and 7 are modified from GES gene (VoGES) of valerian (Valeriana officinalis); wherein, the GES protein expressed by SEQ ID NO. 6(VoGES1) and SEQ ID NO. 7(VoGES2) has only one amino acid difference; .

Experiments show that the GES gene of nucleotide sequence shown in SEQ ID NO: 1-7 is further transferred into the yeast transformed with the IDIl gene and the tHMG gene, so that the yeast can generate geraniol. The DNA molecule which is obtained by substituting, deleting or adding one or more nucleotides in the nucleotide sequence shown in any one of SEQ ID NO. 1-7 and can encode protein with GES enzyme activity is also in the protection scope of the invention.

According to the analysis of the detection result, the GES is positioned in chloroplast, but the GES is not well folded in Saccharomyces cerevisiae, so that the activity of the GES is influenced, and the positioning sequence is truncated. The membrane localization sequence of the GES protein is located at the N-terminus, thus, the 5' end of the GES gene sequence is truncated. The membrane localization sequence of the GES protein is about 14 to 88 amino acids in length. The embodiment of the invention verifies the effect of the LdGES, CrGES, VoGES1 and VoGES2 after truncation, 28 amino acids are truncated at the N end of the LdGES, the CrGES is respectively truncated at the N end by 14 amino acids, 28 amino acids and 43 amino acids, and the VoGES1 and VoGES2 are respectively truncated at the N end by 58 amino acids and 88 amino acids, so that the gene expressing the truncated GES protein is transferred into yeast, and the yield of geraniol can be further improved.

The sequence of the modified GES gene provided by the invention is that one or more nucleotides are deleted from the 5' end of the nucleotide sequence shown in any one of SEQ ID NO. 1-7.

The plurality is 42 to 264, specifically 42, 69, 84, 129, 174, 264.

In the embodiment of the invention, the sequence of the modified GES gene is shown as any one of SEQ ID NO 8-15.

In some embodiments, 69 nucleotides are deleted from the 5' end of the nucleotide sequence set forth in SEQ ID NO. 2 (SEQ ID NO. 13);

42 nucleotides (SEQ ID NO:8), 84 nucleotides (SEQ ID NO:9) and 129 nucleotides (SEQ ID NO:10) are deleted at the 5' end of the nucleotide sequence shown in SEQ ID NO: 5;

174 nucleotides (SEQ ID NO:11) and 264 nucleotides (SEQ ID NO:12) are deleted at the 5' end of the nucleotide sequence shown in SEQ ID NO: 6;

174 nucleotides (SEQ ID NO:14) and 264 nucleotides (SEQ ID NO:15) are deleted at the 5' -end of the nucleotide sequence shown in SEQ ID NO: 7.

Experiments show that after 84 nucleotides are truncated at the 5' end of the LdGES gene, the N end of the LdGES enzyme is truncated by 28 amino acids, and after yeast is transformed, the yield of geraniol is improved from 4.53mg/L to 127.68mg/L, which is improved by about 28 times;

after the 5' end of the CrGES gene is truncated by 42 nucleotides, the N end of the CrGES enzyme is truncated by 14 amino acids, and after the CrGES enzyme is converted into yeast, the yield of geraniol is improved from 42.63mg/L to 283.36mg/L, which is improved by about 7 times;

after 84 nucleotides are truncated at the 5' end of the CrGES gene, 28 amino acids are truncated at the N end of the CrGES enzyme, and after the CrGES enzyme is converted into yeast, the yield of geraniol is improved from 42.63mg/L to 295.62mg/L, which is improved by about 7 times;

after the 5' end of the CrGES gene is truncated by 129 nucleotides, the N end of the CrGES enzyme is truncated by 43 amino acids, and after the CrGES enzyme is converted into yeast, the yield of geraniol is improved from 42.63mg/L to 360.06mg/L, which is improved by about 8 times;

the 5' end of the VoGES1 gene is truncated by 174 nucleotides, the N end of the VoGES1 enzyme is truncated by 58 amino acids, and after the gene is transformed into yeast, the yield of geraniol is improved from 2.73mg/L to 102.46mg/L and is improved by about 37.53 times;

after 264 nucleotides are truncated at the 5' end of the VoGES1 gene, 88 amino acids are truncated at the N end of the VoGES1 enzyme, and after the gene is converted into yeast, the yield of geraniol is improved to 12.41mg/L from 2.73mg/L and is improved by about 5 times;

after the 5' end of the VoGES2 gene is truncated by 174 nucleotides, the N end of the VoGES2 enzyme is truncated by 58 amino acids, and after the gene is converted into yeast, the yield of geraniol is improved from 9.51mg/L to 241.28mg/L, which is improved by about 25 times;

after 264 nucleotides are truncated at the 5' end of the VoGES2 gene, the N end of the VoGES2 enzyme is truncated by 88 amino acids, and after the gene is converted into yeast, the yield of geraniol is improved to 98.54mg/L from 9.51mg/L and is improved by about 10 times.

As can be seen, the conversion of the nucleotide sequence shown in SEQ ID NO. 10 can increase the yield of geraniol to 360.06 mg/L.

The yield of geraniol can be increased to more than 200mg/L by converting the nucleotide sequence shown by any one of SEQ ID NO 8-10 and SEQ ID NO 14.

The output of geraniol can be increased to more than 100mg/L by converting the nucleotide sequence shown by any one of SEQ ID NO 8-11 and SEQ ID NO 13-14.

The yield of geraniol can be increased to more than 98.5mg/L by converting the nucleotide sequence shown by any one of SEQ ID NO 8-11 and SEQ ID NO 13-15.

The invention provides an application of a modified GES gene in construction of an engineering bacterium for producing geraniol.

The invention also provides an engineering bacterium, which expresses IDI1 gene, tHMG gene and modified GES gene; the sequence of the IDI1 gene is shown as SEQ ID NO. 16; the tHMG gene sequence is shown in SEQ ID NO: 17.

According to the biosynthetic pathway of geraniol (FIG. 1), HMG CoA is reacted with HMGR, IDIl, ERG20, GES to form geraniol. According to the invention, the Chassis bacteria express IDI1 gene, tHMG gene and modified GES gene, so that high-yield of geraniol in the engineering bacteria is realized. The engineering bacteria of the invention are selected from algae, yeast, mold or bacteria. Wherein the yeast is preferably yeast of lipolytic genus, yeast of Kluyveromyces genus or Saccharomyces cerevisiae; the saccharomyces cerevisiae is selected from CEN.PK series and BY series; the mould is preferably streptomycete; the bacterium is preferably Escherichia coli or Bacillus subtilis.

In the present example, the starting strain was the yeast cen. pk2-1C.

The IDIl gene and the tHMG gene are from the CEN.PK2-1 genome of yeast.

The IDIl gene and the tHMG gene are integrated at the GAL80 gene position of the CEN.PK2-1C of the yeast, and the GAL80 gene of the CEN.PK2-1C of the yeast is knocked out.

The modified GES gene is introduced by adopting centromere plasmid.

The genotype of the engineering bacteria provided by the invention is CEN. PK2-1C, delta gal80: (tHMG-P)_GAL1,10-IDI1-His3，pRS415-GPM1t-P_GAL7-GES-GPDt。

Wherein the gene sequence of the GES is shown as any one of SEQ ID NO 1-7, or is a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotides in the nucleotide sequence shown as any one of SEQ ID NO 1-7. In the embodiment of the invention, the sequence of the modified GES gene is shown as any one of SEQ ID NO 8-15.

The engineering bacteria provided by the invention are applied to the production of geraniol.

The engineering bacteria provided by the invention can improve the output of geraniol to 360.1 mg/L.

In order to construct the engineering bacteria provided by the invention, the invention also provides a knockout box, which comprises a GAL80 left homologous arm, an ADH1t terminator, a GAL1/10 promoter, a TDH2t terminator, a His nutritional tag and a GAL80 right homologous arm which are connected in sequence.

The length of the left homology arm of gal80 is 400bp, and the length of the right homology arm of gal80 is 400 bp.

Preferably, the backbone of the knockout cassette is pRS 425K.

Wherein, the left homology arm of GAL80, the right homology arm of GAL80, the terminator of ADH1t, the promoter of GAL1/10 and the terminator of TDH2t are from the yeast CEN. PK2-1. The His nutritional tag is from yeast S288C.

The primer pair for amplifying the left homologous arm of gal80 is shown as SEQ ID NO: 20-21.

The primer pair for amplifying the ADH1t terminator is shown as SEQ ID NO. 22-23.

The primer pair for amplifying GAL1,10 promoter is shown in SEQ ID NO. 24-25.

The primer pair for amplifying the TDH2t terminator is shown as SEQ ID NO 26-27.

The primer pair for amplifying the right homologous arm of gal80 is shown as SEQ ID NO: 28-29.

The primer pair for amplifying the His nutritional tag is shown as SEQ ID NO: 30-31.

The construction method of the knockout box provided by the invention comprises the following steps:

step 1: sequentially splicing the left homologous arm of GAL80, ADH1t terminator, GAL1/10 promoter, TDH2t terminator, His nutritional tag and the right homologous arm of GAL80 by an OE-PCR method to obtain a fragment of GAL80up-ADH1t-P_GAL1/10-T_TDH2t-His-gal80down；

Step 2: fragment gal80up-ADH1t-P_GAL1/10-T_TDH2tThe His-gal80down was ligated to the multicopy plasmid pRS425K via the NotI cleavage site to give Δ gal80, ADH1t-P_GAL1,10TDH2t-His3 knock-out cassette.

The OE-PCR takes a primer shown as SEQ ID NO. 20 as an upstream primer and takes a primer shown as SEQ ID NO. 31 as a downstream primer.

Further, IDIl and tHMG are ligated into the knockout cassette to obtain the knockout cassette Δ gal 80:ADH1 t-IDI1-P_GAL1/10-tHMG-T_TDH2tHis3, the specific method is as follows:

step 1: the tHMG fragment passes through BsmBI enzyme cutting site and knockout box delta gal 80:ADH1 t-P_GAL1,10Connecting TDH2t-His3, performing NotI enzyme digestion, recovering the knockout box, and transferring the knockout box to a pLD2 vector to obtain a knockout box containing a tHMG gene;

step 2: the IDI1 fragment was ligated to the knockout cassette containing IDIl gene via BsaI cleavage site to obtain pLD2-gal80up-ADH1t-IDI1-P_GAL1/10-tHMG-T_TDH2t-His-gal80down。

The invention providesKnockout box delta gal80 ADH1t-IDI1-P_GAL1/10-tHMG-T_TDH2tApplication of His3 in construction of engineering bacteria for producing geraniol.

In order to construct the engineering bacteria for producing the geraniol, the invention also provides a centromere plasmid containing the modified GES gene, and the construction method and the plasmid map of the centromere plasmid are shown in figure 3. The centromere plasmid takes pRS415 plasmid as a framework and comprises GPM1t, GAL7 promoter, GES gene and GPDt which are connected in sequence.

The centromere plasmid is constructed by expression box pLD2-GPM1t-P_GAL7Starting with GPDt, the modified GES gene is digested and ligated into an expression cassette by BsaI enzyme to obtain pLD2-GPM1t-P_GAL7-GES-GPDt. The expression cassette was transferred to the single copy plasmid pRS415 using the NotI cleavage site.

The invention does not limit the preparation method of the GES gene fragment, and in order to connect the GES gene into an expression cassette, a sequence gcggccgcggtctcca (SEQ ID NO:18) is added at the 5' end of the GES gene; 3' addition sequence taaaggagaccgcggccgc (SEQ ID NO: 19).

The construction method of the engineering bacteria provided by the invention transforms the IDI1 gene, the tHMG gene and the modified GES gene into the initial bacteria.

Specifically, the construction method of the engineering bacteria provided by the invention comprises the following steps:

step 1: using a knockout box delta gal80 to knock out ADH1t-IDI1-P_GAL1/10-tHMG-T_TDH2tHis3 transformation of IDI1 gene and tHMG gene into starter bacteria to obtain the strain of the chassis;

step 2: transforming the centromere plasmid containing the modified GES gene into the chassis bacteria to obtain the engineering bacteria provided by the invention.

In the present invention, the starting bacterium is a yeast, and the preferred yeast is CEN. PK2-1C.

The transformation in the step 1 and the step 2 adopts a lithium acetate high-efficiency transformation method.

The invention also provides a preparation method of geraniol, and the engineering bacteria provided by the invention are fermented.

The fermentation is two-phase fermentation; the organic phase of the two-phase fermentation adopts isopropyl myristate.

The invention adopts a two-phase fermentation method to reduce the damage of geraniol to the saccharomyces cerevisiae cells as much as possible.

The culture medium for the fermentation comprises: 6.7g/L of yeast nitrogen source YNB is synthesized; 20g/L of glucose; the mixed amino acid powder lacking leucine is 2 g/L.

The volume of the isopropyl myristate is 20% of the fermentation medium.

The temperature of the fermentation is 30 ℃, and the fermentation is oscillated at 200 rpm.

The fermentation specifically comprises the following steps: at the initial OD₆₀₀Inoculating 0.2 ═ to fresh 5ml fermentation medium, culturing at 30 deg.C and 250rpm for about 12h until the thallus grows to middle logarithmic phase, and using initial OD₆₀₀Inoculating 0.2 of the strain into 50ml of fermentation medium, simultaneously adding 20 percent by volume of isopropyl myristate to perform two-phase fermentation at the temperature of 30 ℃ and the rpm of 200, supplementing 10g/L of absolute ethyl alcohol when the fermentation time is 20h, and finishing the fermentation after the fermentation time is 120 h.

By adopting the engineering bacteria provided by the invention for fermentation, the transcription of GES is inhibited by glucose due to the existence of glucose in the initial culture medium; as the fermentation proceeded, glucose was gradually consumed, glucose was consumed for about 19h of fermentation, glucose suppression was released, and P was deleted in the case of GAL80 knockout_GALThe promoter is automatically turned on and the GES begins transcription and translation, thereby gradually accumulating geraniol. By P_GALThe growth of thalli and the production of products are separated, thereby reducing the adverse effect of product toxicity on cell growth and further making the further improvement of the output of the geraniol possible.

After the fermentation is finished, the method also comprises the step of extraction.

The extraction is as follows: and (3) taking the isopropyl myristate layer, and removing water to obtain a solution containing geraniol.

The invention provides an engineering bacterium, a construction method thereof and application thereof in geraniol production, wherein the IDI1 gene, tHMG gene and a modified GES gene are expressed in the engineering bacterium. Compared with the strain in the prior art, the yield of the engineering bacteria for producing the geraniol is higher and the fermentation time is 120h, and the concentration of the geraniol can reach 360.06 mg/L.

Drawings

FIG. 1 shows a scheme for the synthesis of geraniol using recombinant s.cerevisiae;

FIG. 2 shows a Chassis strain knockout cassette construction diagram;

FIG. 3 shows a GES gene expression cassette construction diagram;

FIG. 4 shows a graph of cell growth in different fermentation environments.

Detailed Description

The invention provides an engineering bacterium, a construction method thereof and application thereof in geraniol preparation, and a person skilled in the art can realize the engineering bacterium by appropriately improving process parameters by referring 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. While the methods and applications of this 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 in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

In the present invention, the precursor for synthesizing geraniol is GPP, which synthesizes geraniol under the action of geraniol synthase (GES). The GES gene sources and sequences adopted by the invention are shown in Table 1.

In the present invention, the meanings indicated by the abbreviations in English are as follows:

GES geraniol synthase

tHMG 3-hydroxy-3-methylglutaryl coenzyme A reductase coding gene

IDI1 Isopentenyl Pyrophosphate isomerase

GAL80up GAL left homology arm

GAL80down GAL right homology arm

P_GAL1GAL1 promoter

T_TDH2tTDH2t terminator

P_GAL1/10GAL1/10 promoter

△ ga80l: Gal80 is knocked out and replaced by the following sequence

Glucose

Acetyl-CoA Acetyl-CoA

HMG-CoA 3-hydroxy-3-methylglutaryl coenzyme

mevalonate mevalonic acid

IPP isoprene Pyrophosphate

DMAPP dimethyl propenyl diphosphate-triammonium salt

GPP Geranyl pyrophosphate

Geraniol Geraniol

ALD Gene aldehyde dehydrogenase Gene

OE-PCR overlap extension PCR

The saccharomyces cerevisiae engineering strain, the construction method thereof, the plasmid used in the application and other biological materials can be purchased from the market, and the primer sequences can be synthesized by biological companies.

The invention is further illustrated by the following examples:

EXAMPLE 1 construction of Chassis strains

With CEN. PK2-1C as an initial strain, a Saccharomyces cerevisiae strain CEN. PK2-1C, delta GAL80, in which GAL80 is knocked out and idi1(SEQ ID NO:16) and tHMGR (SEQ ID NO:17) genes are integrated at the GAL80 position is constructed_GAL1,10IDI1-His3, resulting in the final chassis strain SyBE _ Sc 020201. The specific construction process is as follows:

the primer sequences used in the construction are shown in table 1:

table 1: primer sequences

Amplification of the left homology arm of gal80	SEQ ID NO:20～21
		Amplification of the ADH1t terminator	SEQ ID NO:22～23
Amplification ofGAL1,10 promoter	SEQ ID NO:24～25
		Amplification of TDH2t terminator	SEQ ID NO:26～27
Amplification of the Right homology arm of gal80	SEQ ID NO:28～29
		Amplified His nutritional tag	SEQ ID NO:30～31
Amplification of OE-PCR	SEQ ID NO:20、31
		Amplification of Gene IDI1	SEQ ID NO:35～36
Amplification of Gene tHMG	SEQ ID NO:37～38
		Colony PCR	SEQ ID NO:35-38

Using Saccharomyces cerevisiae CEN. PK2-1 genome as template, designing upstream and downstream primers, PCR amplification gal80 upstream and downstream 400bp homology arms, ADH1t terminator and P_GAL1,10Promoter, TDH2t terminator, His nutritional tag, and then gal80 upstream homology arm, ADH1t terminator, P_GAL1,10The promoter, the TDH2t terminator, the His nutritional tag and the downstream homologous arm of gal80 are spliced by an OE-PCR method to obtain a product with NotI enzyme cutting sites at two ends and P_GAL1Two BsaI cutting sites are contained between the promoter and the TDH2t terminator, and the BsaI cutting sites are arranged at P_GAL10Two Bsm are included between the promoter and the ADH1t terminatorFragment gal80up of BI-ADH 1t-P_GAL1/10-T_TDH2tHis-gal80down, linked to the multicopy plasmid pRS425K via the NotI cleavage site to give Δ gal80, ADH1t-P_GAL1,10TDH2t-His3 knock-out cassette. Using a saccharomyces cerevisiae CEN.PK2-1 genome as a template, designing upstream and downstream primers to respectively amplify genes IDI1 and tHMG, firstly connecting the amplified tHMG with a knockout box through a BsmBI enzyme digestion site, transferring the tHMG into escherichia coli competence TransT1, performing colony PCR screening, performing NotI enzyme digestion verification and sequencing verification on an upgraded plasmid to obtain a correct plasmid pRS425K-gal80up-ADH1t-P_GAL1/10-tHMG-T_TDH2t-His-gal80down, digesting the plasmid with NotI enzyme, recovering the knockout box, transferring the knockout box to pLD2 vector, screening with blue and white spots, colony PCR verification and NotI enzyme digestion verification to obtain correct clone, wherein the plasmid is pLD2-gal80up-ADH1t-P_GAL1/10-tHMG-T_TDH2tHis-gal80 down. Then the IDI1 obtained by amplification is connected to the plasmid obtained in the last step through BsaI restriction enzyme cutting sites to obtain pLD2-gal80up-ADH1t-IDI1-P_GAL1/10-tHMG-T_TDH2tHis-gal80down, also after sequencing and enzyme digestion verification, to obtain the correct clone.

After the construction of the knockout cassette is completed, the cassette is digested with NotI endonuclease to obtain fragment gal80up-ADH1t-IDI1-P_GAL1/10-tHMG-T_TDH2tHis-GAL80down, transforming the obtained fragment into Saccharomyces cerevisiae strain CEN. PK2-1C by lithium acetate high efficiency transformation method, and integrating into Saccharomyces cerevisiae genome by homologous recombination of about GAL80 homologous sequence and GAL80 site on yeast genome. After transformation, an SC-drop solid medium (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, the obtained transformant is subjected to streak purification and enrichment culture, yeast genome is extracted for PCR verification, and a correctly verified recombinant strain is preserved with glycerol and named as SyBE _ Sc020201(CEN. PK2-1C, delta gal80:: tHMG-P)_GAL1,10-IDI1-His3)。

EXAMPLE 2 construction of Saccharomyces cerevisiae strains for the production of geraniol

1. Obtaining exogenous functional gene elements

The exogenous genes include geraniol synthase gene GES for synthesizing geraniol: selecting 9 functional genes from different sources to screen the optimal gene source for synthesizing the geraniol (as shown in Table 2), and the invention totally relates to 10 exogenous genes for synthesizing the geraniol. The genes are obtained by artificial synthesis through adding gcggccgcggtctcca (SEQ ID NO:18) and 3 'taaaggagaccgcggccgc (SEQ ID NO:19) at the 5' end of the gene after codon optimization of saccharomyces cerevisiae and appropriate avoidance of common restriction sites.

TABLE 2 sources and sequences of engineered GES genes

Wherein the VoGES1 and VoGES2 encode proteins that differ by only one amino acid. The 5' end deletion fragment is obtained by the following mode: shtml predicts the membrane localization sequences of LdGES, CrGES and VoGES through the website http:// www.cbs.dtu.dk/index, and performs gradient truncation according to the prediction result. The truncation method is as follows: the synthesized genes LdGES, CrGES and VoGES are used as templates, upstream and downstream primers (table 3) are designed and amplified to obtain truncated tGES with BsaI enzyme cutting sites at two ends. LdGES is truncated by 23 amino acids at the N-terminus, CrGES is truncated by 14, 28 and 43 amino acids at the N-terminus, respectively, and VoGES1 and VoGES2 are truncated by 58 and 88 amino acids at the N-terminus, respectively.

TABLE 3 upstream and downstream primers for amplification of truncated sequences

Segment of interest	Primer sequences
		SEQ ID NO:8	SEQ ID NO:46、SEQ ID NO:47
SEQ ID NO:9	SEQ ID NO:39、SEQ ID NO:42
		SEQ ID NO:10	SEQ ID NO:40、SEQ ID NO:42
SEQ ID NO:11	SEQ ID NO:41、SEQ ID NO:42
		SEQ ID NO:12	SEQ ID NO:43、SEQ ID NO:45
SEQ ID NO:13	SEQ ID NO:44、SEQ ID NO:45
		SEQ ID NO:14	SEQ ID NO:43、SEQ ID NO:45
SEQ ID NO:15	SEQ ID NO:44、SEQ ID NO:45

2. Construction of Saccharomyces cerevisiae Strain for production of Geraniol

The expression cassette pLD2-GPM1t-P which is available in the laboratory is utilized_GAL7GPDt (SEQ ID NO: 48) and 10 synthetic GES are cut by BsaI enzyme and connected into an expression box to obtain pLD2-GPM1t-P_GAL7-GES-GPDt. Transferring the expression cassette to a single-copy plasmid pRS415 by using NotI restriction enzyme sites, transforming Escherichia coli TransT1, and obtaining correct clone after blue-white spot screening, colony PCR verification and sequencing verification, wherein the plasmid is pRS415-GPM1t-P_GAL7-GES-GPDt. High efficiency through lithium acetateThe transformation method comprises the steps of respectively transferring the obtained plasmids into an optimized saccharomyces cerevisiae strain SyBE _ Sc020201, screening by using an SC-drop solid plate (6.7 g/L of synthetic yeast nitrogen source YNB, 20g/L of glucose, 2g/L of mixed amino acid powder of tryptophan, leucine, histidine and uracil, and 2% of agar powder) after transformation, carrying out streak purification and enrichment culture on obtained transformants, carrying out colony PCR verification, storing glycerol strains on recombinant strains which are verified to be correct, and respectively naming: the correspondence between the species designation and the transferred sequence is shown in Table 4:

TABLE 4 bacterial species naming and GES Gene sequences transfer

SyBE_Sc020202	SEQ ID NO:1
		SyBE_Sc020204	SEQ ID NO:32
SyBE_Sc020205	SEQ ID NO:33
		SyBE_Sc020206	SEQ ID NO:2
SyBE_Sc020207	SEQ ID NO:3
		SyBE_Sc020208	SEQ ID NO:4
SyBE_Sc020209	SEQ ID NO:5
		SyBE_Sc020210	SEQ ID NO:6
SyBE_Sc020211	SEQ ID NO:7
		SyBE_Sc020212	SEQ ID NO:34
SyBE_Sc020215	SEQ ID NO:13
		SyBE_Sc020216	SEQ ID NO:8
SyBE_Sc020217	SEQ ID NO:9
		SyBE_Sc020218	SEQ ID NO:10
SyBE_Sc020219	SEQ ID NO:11
		SyBE_Sc020220	SEQ ID NO:12
SyBE_Sc020221	SEQ ID NO:14
		SyBE_Sc020222	SEQ ID NO:15

EXAMPLE 3 preparation of Geraniol

Test materials: EXAMPLE 2 Saccharomyces cerevisiae strain for the production of geraniol constructed

The test method comprises the following steps:

seed culture medium: 6.7g/L of synthetic yeast nitrogen source YNB, 20g/L of glucose and 2g/L of leucine-deficient mixed amino acid powder

Fermentation medium: 6.7g/L of synthetic yeast nitrogen source YNB, 20g/L of glucose and 2g/L of leucine-deficient mixed amino acid powder

Selecting single colony from solid streak plate, inoculating in 5ml SC-Leu liquid culture medium, culturing at 30 deg.C and 250rpm for about 20 hr until thallus growth reaches logarithmic phase, and taking initial OD₆₀₀Inoculating 0.2 to fresh 5ml SC-Leu liquid culture medium, culturing at 30 deg.C and 250rpm for about 12 hr until the thallus grows to middle logarithmic phase, and using initial OD₆₀₀Inoculating the mixture into 50ml of fermentation medium Sc-Leu, simultaneously adding 20% isopropyl myristate, performing two-phase fermentation at 30 ℃ and 200rpm, supplementing 10g/L of absolute ethyl alcohol when 20h is needed, and finishing the fermentation after 120 h.

The geraniol extraction method comprises the following steps: pouring the fermentation liquor into a 50ml centrifuge tube, centrifuging at 12000rpm for 5min, transferring the upper isopropyl myristate into a 2ml centrifuge tube by using a pipette, adding a proper amount of dried anhydrous sodium sulfate, turning over for several times, standing for about 10min for dewatering, filtering by using a 0.22 mu m organic filter membrane, diluting by using n-hexane, and detecting and analyzing by using GC-MS. The results are shown in Table 5:

TABLE 5 geraniol yields for each strain

The experimental results are as follows: transcription of GES is inhibited by glucose in the presence of glucose in the initial medium; as the fermentation proceeded, glucose was gradually consumed, glucose was consumed for about 19h of fermentation, glucose suppression was released, and P was deleted in the case of GAL80 knockout_GALThe promoter is automatically turned on and the GES begins to turnRecording and translation, thereby gradually accumulating geraniol. By P_GALThe growth of thalli and the production of products are separated, thereby reducing the adverse effect of product toxicity on cell growth and further making the further improvement of the output of the geraniol possible.

As can be seen from Table 4, after yeast transformed with a sequence that is not truncated, the yield of geraniol in SyBE _ Sc020209 is the highest, and is significantly better than that of other yeast transformed with a sequence that is not truncated (p)<0.01). SyBE _ Sc0202010 and SyBE _ Sc020211 differ by approximately 4-fold in yield, though only by one amino acid. From this, it was found that P was used as the optimal gene source_GALThe promoter has positive significance for efficiently producing geraniol.

After transformation of the truncated sequences, the geraniol yields of most yeast strains were greatly improved, with the geraniol yields of SyBE _ Sc020218 up to 360mg/L being significantly better than other strains (p < 0.01).

For three different truncated CrGES, the yield of the geraniol is obviously improved, and the yield of the geraniol obtained by completely truncating the positioning sequence according to a prediction result is the highest and is obviously superior to that of a partially truncated strain (p < 0.01); the geraniol yields after the truncation of LdGES, VoGES1 and VoGES2 are improved to a certain extent, but the effect of completely truncating the positioning sequences according to the prediction results of VoGES1 and VoGES2 is not as good as that of partial truncations.

Example 4 Effect of two-phase fermentation on cell growth

1. Construction of control strains

The plasmids pRS415 are respectively transferred into an optimized saccharomyces cerevisiae strain SyBE _ Sc020201 through a lithium acetate high-efficiency conversion method, an SC-Leu solid plate (6.7 g/L of synthetic yeast nitrogen source YNB, 20g/L of glucose, 2g/L of leucine-deficient mixed amino acid powder and 2% of agar powder) is adopted for screening after conversion, colony PCR verification is carried out on the obtained transformants after streak purification culture, and the correctly verified recombinant strains are preserved with glycerol and are respectively named as SyBE _ Sc 020214.

2. Comparison of the Effect of two-phase and aqueous fermentation on cell growth and Geraniol production

Test materials: SyBE _ Sc020209 and SyBE _ Sc020214

The test method comprises the following steps:

Selecting single colony from solid streak plate, inoculating in 5ml SD-Leu liquid culture medium, culturing at 30 deg.C and 250rpm for about 20 hr until thallus growth reaches logarithmic phase, and taking initial OD₆₀₀Inoculating 0.2 to fresh 5ml SC-Leu liquid culture medium, culturing at 30 deg.C and 250rpm for about 12 hr until the thallus grows to middle logarithmic phase, and using initial OD₆₀₀Inoculated into 50ml of fermentation medium Sc-Leu, and fermented at 30 ℃ and 200 rpm. For the inoculation of SyBE _ Sc020209, 20% of isopropyl myristate or n-decane was added for two-phase fermentation, and for the inoculation of SyBE _ Sc020214, 20% of isopropyl myristate was added for two-phase fermentation. Furthermore, single phase fermentation was also carried out with the aqueous phase for SyBE _ Sc020209 at the same time. Adding 10g/L absolute ethyl alcohol when 19h, and finishing fermentation after 120 h.

The experimental results are as follows: as shown in fig. 4, the growth of the control group SyBE _ Sc020209 without adding isopropyl myristate after 19h is significantly lower than that of the experimental group, which indicates that the glucose is gradually consumed after about 19h, geraniol starts to accumulate and generates toxicity to cells, and isopropyl myristate can well relieve the toxicity of geraniol to cells; the growth of the cells of the experimental group added with n-decane is obviously inferior to that of the cells of other groups, which shows that n-decane has greater toxicity to the cells; the growth of the SyBE _ Sc020214 control group added with isopropyl myristate is basically consistent with that of the SyBE _ Sc020209 experimental group added with isopropyl myristate, and further shows that isopropyl myristate can well relieve cytotoxicity generated by geraniol. Therefore, the two-phase fermentation by using the isopropyl myristate as the organic phase has positive significance for the efficient production of the geraniol by the saccharomyces cerevisiae.

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

Claims

1. A modified GES gene which is a CrGES gene truncated by 129 nucleotides at the 5' end; the nucleotide sequence of the CrGES gene is shown as SEQ ID NO. 5.

2. Use of the modified GES gene of claim 1 in the construction of a geraniol-producing engineered bacterium.

3. An engineered bacterium which expresses IDI1 gene, tHMG gene and the modified GES gene of claim 1; the sequence of the IDI1 gene is shown as SEQ ID NO. 16; the tHMG gene sequence is shown in SEQ ID NO: 17.

4. The engineered bacterium of claim 3, wherein the starting strain is the yeast cen. pk2-1C.

5. Use of the engineered bacterium of any one of claims 3 to 4 in geraniol production.

6. The method of constructing an engineered bacterium according to any one of claims 3 to 4, wherein the IDI1 gene, tHMG gene and the modified GES gene according to claim 1 are transformed into a starting bacterium.

7. A method for producing geraniol, characterized by fermenting the engineered bacterium according to any one of claims 3 to 4.

8. The method of claim 7, wherein the fermentation is a two-phase fermentation; the organic phase of the two-phase fermentation adopts isopropyl myristate.