CN112920960B - Method for increasing palmitoleic acid content of saccharomyces cerevisiae - Google Patents

Abstract

The invention discloses a method for improving the palmitoleic acid content of saccharomyces cerevisiae. The invention provides a recombinant bacterium, which is obtained by carrying out the following 1) transformation in oil-producing saccharomyces cerevisiae: 1) improving the transcription activity of the OLE1 gene in the oil-producing saccharomyces cerevisiae to obtain the recombinant strain. In the invention, in a saccharomyces cerevisiae strain YS58 with more oil production, a transcription factor Mga2 or a truncated Mga2 gene fragment is overexpressed, and the expression level of an OLE1 gene for regulating and controlling delta 9 desaturase is improved, so that the content of palmitoleic acid in saccharomyces cerevisiae is improved, exogenous fatty acid elongase genes KCS and FAE are expressed at the same time, a saccharomyces cerevisiae genetic engineering strain is constructed, and the content of palmitoleic acid in saccharomyces cerevisiae is improved.

Description

Method for increasing content of palmitoleic acid in saccharomyces cerevisiae

Technical Field

The invention relates to the technical field of biology, in particular to a method for improving the palmitoleic acid content of saccharomyces cerevisiae.

Background

Palmitoleic acid (palmitoleic acid) is a 16-carbon monounsaturated fatty acid with a double bond at the 7 th carbon atom of the carbon end (C16:1 n-7). The compound has wide medical and cosmetic applications, has certain curative effect on chronic diseases such as obesity, diabetes, fatty liver and the like, and has application potential in the fields of blood sugar reduction, inflammation resistance, skin pigmentation improvement and food. At present, marine products such as fish oil are the main source of palmitoleic acid, but the source of palmitoleic acid is limited due to international prohibition of whale catching and shortage of fishery resources. Efforts have therefore been made to expand the possibilities of isolating such fatty acids from microbial sources.

Palmitoleic acid exists as a biological metabolite, mainly in the form of triglycerides, phospholipids or glycerol sugar esters. In s.cerevisiae, the formation of unsaturated fatty acids is due to the action of Δ 9 desaturase, the OLE1 gene is responsible for encoding Δ 9 desaturase, and transcription factor Mga2 regulates the metabolism of unsaturated fatty acids by controlling the transcription of OLE1 gene and the stability of its mRNA. Mga2 can sense unsaturated fatty acid level on cell membrane and bind ligand through Tryptophan residue of TM transmembrane domain, so as to regulate Mga2 transcription activity, while higher unsaturated fatty acid level on membrane can inhibit Mga2 transcription activation, so that fatty acid dehydrogenase expression cannot be activated. But the regulation and control effects of the IPT structural domain and the ankyrin repetitive sequence on the synthesis of unsaturated fatty acid are not clear yet.

Fatty acid de novo synthesis C2 to C4; c4 to C16; the 3-ketoacyl-ACP synthase (KCS) enzymes used in the three stages C16 to C18 are different. KCSIII is responsible for condensing acetyl-coa and formyl-coa into a four-carbon unit, then KCSI is responsible for extending the four-carbon unit to C16; the last step, KCSII, is responsible for the extension of C16 to C18, where the C16 and C18 series fatty acids are the major components of most animal plant cell fatty acid compositions.

Disclosure of Invention

An object of the present invention is to provide a recombinant bacterium.

The recombinant strain provided by the invention is obtained by carrying out the following 1) transformation in oil-producing saccharomyces cerevisiae:

1) improving the transcription activity of the OLE1 gene in the oil-producing saccharomyces cerevisiae to obtain the recombinant strain.

In the recombinant strain, the improvement of the transcriptional activity of the OLE1 gene in the grease-producing Saccharomyces cerevisiae is the improvement of the expression level (over-expression Mga2) of the transcription factor Mga2 of the OLE1 gene or the coding gene of a truncated body of the OLE1 gene in the grease-producing Saccharomyces cerevisiae;

the amino acid sequence of the transcription factor Mga2 is a sequence 4 in a sequence table;

the amino acid sequence of the truncated body of the transcription factor Mga2 is 1 st-975 th of a sequence 4 in a sequence table;

or the amino acid sequence of the truncated body of the transcription factor Mga2 is 1 st to 706 th in a sequence 4 in a sequence table.

Or the promoter of the coding gene of the transcription factor Mga2 or the truncation thereof is a saccharomyces cerevisiae strong promoter, such as PGK1 or TEF 1.

The Mga2 overexpression method is to clone the gene to YEp by adopting a T4 connection methodlac112-P _PGK -T _CYC On the vector, a saccharomyces cerevisiae strong promoter PGK1 is adopted to start expression.

In the recombinant bacteria, the recombinant bacteria are obtained by carrying out the transformation of 1) and 2) in oil-producing saccharomyces cerevisiae:

2) and (3) allowing the grease-producing saccharomyces cerevisiae to express fatty acid elongase.

The expression of KCS and FAE is realized by cloning a gene sequence to a pYES2.0 vector by adopting a T4 connection and seamless cloning method, and the expression is started by adopting a strong promoter TEF1 or PGK1, wherein the promoter of KCS is TEF1 or PGK1, and the promoter of FAE is PGK 1.

In the recombinant strain, 1 or 2 kinds of fatty acid elongases are used;

the fatty acid elongase is fatty acid elongase from crambe or cardamine hirsutum or protein of amino acid residues with homology of more than 95 percent with the amino acid sequence of the fatty acid elongase.

In the recombinant bacterium, the fatty acid elongase is a fatty acid elongase shown in a) and a fatty acid elongase shown in b) as follows:

a) the fatty acid elongase is KCS; the encoding gene is derived from beta-ketoacyl coenzyme A synthase gene of cardamine hirsuta (Cardaminehirsuta), and the nucleotide sequence is sequence 2 in the sequence table;

b) the fatty acid elongase is FAE; the coding gene is derived from beta-ketoacyl coenzyme A synthase gene of Crambe abyssinica, and the nucleotide sequence is the sequence 3 in the sequence table.

In the recombinant bacteria, the grease-producing saccharomyces cerevisiae takes saccharomyces cerevisiae YS58 as an example.

It is another object of the present invention to provide the following method.

The invention provides a method for preparing the recombinant bacterium of the first purpose, which is prepared according to the modified form of the recombinant bacterium of the first purpose.

The application of the recombinant bacterium in improving the palmitoleic acid content of the saccharomyces cerevisiae is also within the protection range of the invention;

or, the application of the recombinant bacteria in the preparation or production of palmitoleic acid is also within the protection scope of the invention.

Or, the invention provides a method for increasing the palmitoleic acid content of saccharomyces cerevisiae, which comprises the following steps: the improvement is carried out according to the improvement mode 1) in the recombinant strain, so that the palmitoleic acid content of the saccharomyces cerevisiae is increased;

or, the invention provides a method for increasing the palmitoleic acid content of saccharomyces cerevisiae, which comprises the following steps: the improvement is carried out according to the improvement modes 1) and 2) in the recombinant bacteria, so that the palmitoleic acid content of the saccharomyces cerevisiae is increased.

It is also an object of the present invention to provide a process for the preparation or production of palmitoleic acid.

The method provided by the invention comprises the following steps:

1) fermenting the recombinant bacteria in an YNB auxotrophic culture medium, and collecting fermentation products;

2) and extracting fatty acid in the fermentation product by a saponification method to obtain the palmitoleic acid.

The construction method of the saccharomyces cerevisiae gene engineering bacteria comprises the steps of transforming a vector into saccharomyces cerevisiae by using a saccharomyces cerevisiae gene transformation kit, coating the saccharomyces cerevisiae gene transformation kit on a corresponding auxotroph (Trp-deficient or Ura-deficient) solid culture medium, and screening the saccharomyces cerevisiae gene engineering bacteria successfully transformed into a recombinant vector.

The culture method of the saccharomyces cerevisiae gene engineering bacteria comprises the following steps: the engineering bacteria are inoculated into YNB culture medium and are continuously activated twice in a shaking flask at the temperature of 28 ℃ and the speed of 180 rpm. The seed solution was inoculated into YNB auxotrophic medium 28 ℃, shake-flask culture was carried out at 180rpm for 2 days at 18 ℃, and after further culture was carried out at 180rpm for 4 days, the cells were collected by centrifugation.

In the invention, in a saccharomyces cerevisiae strain YS58 with more oil production, a transcription factor Mga2 or a truncated Mga2 gene fragment is overexpressed, and the expression level of an OLE1 gene for regulating and controlling delta 9 desaturase is improved, so that the palmitoleic acid content of saccharomyces cerevisiae is improved, and meanwhile, exogenous fatty acid elongase genes KCS and FAE are expressed, so that a saccharomyces cerevisiae genetic engineering strain is constructed, and the palmitoleic acid content of saccharomyces cerevisiae is improved.

The invention has the following beneficial effects: the method improves the palmitoleic acid content in the saccharomyces cerevisiae, and provides a reference for breaking through the limitation of palmitoleic acid sources and expanding microbial sources. The invention takes the regulation and control of the transcription factor of the delta 9 desaturase gene as the starting point to construct the saccharomyces cerevisiae gene engineering bacteria, can effectively improve the transcription activity of the OLE1 gene, and is beneficial to the synthesis of unsaturated fatty acid; meanwhile, the transcription factor and the fatty acid elongase are jointly regulated and controlled, so that the palmitoleic acid content of the saccharomyces cerevisiae is effectively improved, and the palmitoleic acid content of the saccharomyces cerevisiae reaches 60%. The exogenous KCS gene is responsible for the extension of long-chain monounsaturated fatty acid, when the unsaturated fatty acid in the cell membrane is excessive, the synthesis of palmitoleic acid can be inhibited, and partial long-chain fatty acid is consumed by using fatty acid elongase genes KCS and FAE, so that the production of saccharomyces cerevisiae palmitoleic acid is driven.

The invention has the following advantages:

1) overexpression of the transcription factor Mga2 can effectively improve the expression of OLE 1. And with the continuous truncation of Mga2 gene, the content of monounsaturated fatty acid in the saccharomyces cerevisiae is increased, wherein after truncation of IPT structural domain and ankyrin repetitive sequence of Mga2, the accumulation of palmitoleic acid in saccharomyces cerevisiae genetic engineering bacteria is facilitated.

2) The fatty acid elongase is commonly used in the synthesis of the ultra-long chain fatty acid, but is not applied in the synthesis of the palmitoleic acid, and the improvement of the palmitoleic acid content by the synergistic action of the palmitoleic acid elongase and the transcription factor Mga2 is not reported in documents. When Mga2 is over-expressed, the fatty acid elongase genes KCS and FAE are expressed at the same time, which is more beneficial to improving the palmitoleic acid content of the saccharomyces cerevisiae.

3) According to the invention, two fatty acid elongases are used and are respectively from crambe and cardamine hirsute, C18:1 is extended to C22:1 by using FAE genes of the fatty acid elongases, C22:1 is extended to C24:1 by using KCS genes of the fatty acid elongases, and related researches show that the content of palmitoleic acid can be improved by transferring two exogenous genes.

Drawings

FIG. 1 is a schematic representation of the location of the various domains of transcription factor Mga2 and the truncation location of the three truncated fragments.

FIG. 2 is a schematic diagram of the design of a seamless ligation primer involved in the construction of a heterologous expression of a fatty acid elongase gene.

FIG. 3 is a graph showing the effect of overexpressing transcription factor Mga2 and truncated fragments of overexpressing transcription factor Mga2 on palmitoleic acid content in Saccharomyces cerevisiae.

FIG. 4 shows the effect of heterologous expression of fatty acid elongase genes KCS and FAE on the palmitoleic acid content of Saccharomyces cerevisiae while overexpressing transcription factors.

FIG. 5 is a gas chromatography analysis chart of the standard and each sample.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

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

The present invention is further illustrated by the following specific examples, but the scope of the invention is not limited thereto.

The culture media used in the following examples are as follows, wherein the percentages are by mass:

final concentration composition of LB medium: 0.5 percent of yeast powder, 1 percent of peptone, 0.5 percent of sodium chloride, 1.7 percent of agar (used for a solid culture medium), a solvent is deionized water, and the pH value is natural.

Final concentration composition of YPD medium: 1% yeast powder, 2% peptone, 2% glucose, 1.7% agar (for solid medium), deionized water as solvent, and natural pH.

Final concentration composition of YNB auxotrophic medium: 0.17% nitrogen source without amino yeast (Shanghai-derived leaf Biotechnology Co., Ltd., S24483), 0.5% ammonium sulfate, 0.13% Dropout Supplement-Trp (Beijing Ku Lai Pake Co., Ltd., PM2250) or 0.13% Dropout Supplement-Ura (Beijing Ku Lai Pake Co., Ltd., PM2270), 2% glucose, deionized water as solvent, pH5.0, addition of D-S amino acid determined by the selection marker of the expression vector, and addition of 1.7% agar to the solid medium based on the above components.

The amino acid sequence of the transcription factor Mga2 in the following embodiment is sequence 4 in the sequence table, and the nucleotide sequence of the gene coded by the transcription factor is sequence 1 in the sequence table;

the nucleotide sequence of the gene coded by the fatty acid elongase KCS is sequence 2;

the nucleotide sequence of the gene coded by the fatty acid elongase FAE is sequence 3.

Example 1 Effect of overexpression of transcription factor Mga2 on the content of palmitoleic acid in Saccharomyces cerevisiae

Construction of over-expressed transcription factor Mga2 gene engineering bacteria

1. Construction of Mga2 overexpression vector

The yeast genome is extracted by using a standard yeast extraction kit of Bomeide company to clone Mga2 genes, BamHI and SmaI enzyme cleavage sites are selected and are connected to a vector YEplac112-P with a saccharomyces cerevisiae strong promoter PGK promoter and a CYC terminator _PGK -T _CYC (Shanghai ze leaf Biotech Co., Ltd., ZY 1369).

The successfully ligated plasmids were transferred to Trans10 competent cells for validation screening and plasmid amplification. Selecting a single colony grown from a plate for colony PCR verification, selecting the single colony successfully verified by the colony PCR, inoculating the single colony into an LB test tube with Amp resistance for culturing for about 12 hours, extracting plasmids, performing enzyme digestion verification to further determine whether the single colony is a required carrier, selecting a plasmid with a correct enzyme digestion verification result for sequencing, and determining that Mga2 gene fragments are successfully connected into YEplac112-P through a sequence comparison result _PGK -T _CYC The vector, the plasmid with the correct sequencing was named YEplac112-P _PGK -MGA2-T _CYC And Mga2 is an overexpression vector.

YEplac112-P _PGK -MGA2-T _CYC The recombinant vector is obtained by replacing Mga2 gene shown in sequence 1 in a sequence table with YEplac112-P _PGK -T _CYC The vector obtained between BamHI and SmaI enzyme cutting sites of the vector expresses Mga2 gene, and the promoter of Mga2 gene is P _PGK The terminator is T _CYC。

2. Construction of truncated Mga2 Gene overexpression vectors

Similar to the Mga2 gene overexpression vector construction, the gene for the Mga2 truncation was cloned using the primers listed in table 1.

Table 1 shows the sequences of the primers used

The wMga2 gene is obtained by using a DNA molecule shown in a sequence 1 as a template and using MGA2-F and MGA2-R for amplification;

MGA2 of a truncated tMga2(975aa) _975aa The gene uses MGA2-F and MGA2 as templates _975aa The nucleotide sequence of the gene is obtained by amplification of-R, and the nucleotide sequence is 1 st to 2925 th of the sequence 1;

MGA2 of a truncated tMga2(706aa) _706aa The gene uses MGA2-F and MGA2 as templates _706aa The nucleotide sequence of the gene is obtained by amplification of-R, and the nucleotide sequence is 1 st to 2118 th of the sequence 1;

MGA2 of a truncated tMga2(529aa) _529aa The gene uses a DNA molecule shown in sequence 1 as a template, and MGA2-F and MGA2 _529aa The nucleotide sequence of the gene is 1 st to 1587 th positions of the sequence 1.

The above described truncated body is shown in figure 1.

According to YEplac112-P _PGK -T _CYC Plasmid restriction site and MGA2 gene self sequence characteristics BamHI and SmaI restriction sites are selected to be connected with MGA2 truncated fragment to respectively construct YEplac112-P _PGK -MGA2 _975aa -T _CYC 、YEplac112-P _PGK -MGA2 _706aa -T _CYC 、YEplac112-P _PGK -MGA2 _529aa -T _CYC An expression vector.

YEplac112-P _PGK -MGA2 _975aa -T _CYC The recombinant vector is MGA2 shown in 1 st-2925 th site of a sequence 1 in a sequence table _975aa Gene replacement YEplac112-P _PGK -T _CYC The vector obtained by cutting the fragment between BamHI and SmaI enzymes of the vector expresses MGA2 _975aa Gene, MGA2 _975aa The promoter of the gene is P _PGK The terminator is T _CYC。

YEplac112-P _PGK -MGA2 _706aa -T _CYC The recombinant vector is MGA2 shown in 1 st-2118 th site of a sequence 1 in a sequence table _706aa Gene replacement YEplac112-P _PGK -T _CYC The vector obtained by cutting the fragment between BamHI and SmaI enzymes of the vector expresses MGA2 _706aa Gene, MGA2 _706aa The promoter of the gene is P _PGK The terminator is T _CYC。

YEplac112-P _PGK -MGA2 _529aa -T _CYC The recombinant vector is MGA2 shown in 1 st to 1587 th sites of a sequence 1 in a sequence table _529aa Gene replacement YEplac112-P _PGK -T _CYC The vector obtained by cutting the fragment between BamHI and SmaI enzymes of the vector expresses MGA2 _529aa Gene, MGA2 _529aa The promoter of the gene is P _PGK The terminator is T _CYC。

3. Construction of Saccharomyces cerevisiae Gene engineering bacteria

The 4 expression vectors successfully constructed in the steps 1 and 2 are respectively introduced into the YS58 strain by using a lithium acetate transformation method, screened and expressed, and the specific steps are as follows:

the host bacteria is Saccharomyces cerevisiae YS58 (Shanghai Beino Biotechnology Co., Ltd.), the classical yeast transformation kit is used for yeast transformation, and the source is Beijing Kulai bock technology Co., Ltd., SK 2400. YPD medium was activated twice, cultured to logarithmic growth phase to prepare competent cells, harvested by refrigerated centrifugation, washed twice with water, and suspended in 1/10-concentration LiAc. A premix of 240. mu.l of PEG Solution (SL 9510, Ku-Laplace technology Co., Ltd. in Beijing), 36. mu.l of LiAc Solution (YT 0002, Ku-Laplace technology Co., Ltd. in Beijing), 10. mu.l of Carrier DNA (YT 0003, Ku-Laplace technology Co., Ltd. in Beijing), and 5. mu.l of plasmid (any of the above 4 expression vectors) was prepared and supplemented with sterile water to a final volume of 360. mu.l. The premix was injected into YS58 competent cells, sequentially washed with water at 30 ℃ and 42 ℃ for 30 minutes, centrifuged, and then resuspended in sterile water and applied to Trp-deficient selection medium (PM 2251, Beijing blue Boster Bio Inc.), followed by culturing at 30 ℃ for 2-4 days to select positive transformants.

Empty plasmid YEplac112-P _PGK -T _CYC And recombinant plasmid YEplac112-P _PGK -MGA2-T _CYC 、YEplac112-P _PGK -MGA2 _975aa -T _CYC 、YEplac112-P _PGK -MGA2 _706aa -T _CYC 、YEplac112-P _PGK -MGA2 _529aa -T _CYC The recombinant bacteria YS01, YS02, YS03, YS04 and YS05 are obtained by transferring the strain into a host bacterium YS58 in sequence.

Second, influence of over-expression transcription factor on palmitoleic acid content of saccharomyces cerevisiae

1. Engineering bacteria culture

Activating the genetically engineered bacteria and the host bacteria: preparing YNB auxotrophic culture medium, preparing nitrogen source and glucose solution separately to prevent ammonia carbonyl reaction, autoclaving at 115 deg.C under 0.12MPa for 25 min.

And (3) obtaining a seed solution, namely inoculating the prepared genetically engineered bacteria YS01, YS02, YS03, YS04 and YS05 into an YNB auxotrophic culture medium, culturing for 48h in a constant-temperature shaking table at 180rpm and 28 ℃, repeatedly activating twice to obtain the seed solution, and standing for use.

Fermentation culture: and inoculating the seed solution obtained by repeated activation twice into an YNB auxotrophic culture medium, wherein the bacterial strain YS01 is a control bacterial strain, shaking the flask for 2 days at 28 ℃, and then transferring to 18 ℃ for continuous culture for four days after carrying out shake flask fermentation culture at 180rpm, so as to obtain YS01 fermentation product, YS02 fermentation product, YS03 fermentation product, YS04 fermentation product and YS05 fermentation product.

2. Extraction of the product

The wall breaking efficiency of the saccharomyces cerevisiae is not high, intracellular products cannot be completely released to the outside of cells, the accuracy of a detection result is directly influenced, and a saponification method is selected to extract the yeast grease according to laboratory conditions and the efficiency of a grease extraction method pointed out in literature, and the method specifically comprises the following steps:

6000g of fermentation products of the strains are centrifuged for 5min, thallus is collected and washed twice, 15mL of 10% (volume percentage content) KOH-methanol solution (composed of 10g/100mLKOH, 95% methanol and 5% deionized water) is added, and the mixture is transferred to a ground conical flask. Then placing the mixture in a water bath kettle at the temperature of 80 ℃ for condensing and refluxing for 2 hours to cause strong base to carry out saponification reaction with intracellular esters, collecting condensed and refluxed liquid to obtain fatty acid salt, and acidifying the fatty acid salt by hydrochloric acid (adding 4mL of hydrochloric acid with the concentration of 6M) to obtain fatty acid.

3. Product handling and detection

Preparation of fatty acid methyl ester: adding 4mL of 1% (volume percentage) sulfuric acid-methanol solution into the fatty acid obtained by the 2 saponification method, uniformly mixing, transferring into a clean test tube with a plug, and reacting for 1h in a water bath kettle at 60 ℃; cooling at room temperature, and adding 5mL of saturated NaCl solution to precipitate impurities such as protein; then adding 1mL of normal hexane, and collecting an organic phase, namely the extracted fatty acid methyl ester; the sample was dehydrated with anhydrous sodium sulfate.

GC detection of fatty acid methyl esters: and (3) carrying out gas phase detection and analysis on the obtained fatty acid methyl ester by using a DB-WAX chromatographic column of an Shimadzu gas analyzer, using high-purity nitrogen as a detection carrier, and separating the fatty acid methyl ester by adopting a two-stage heating program, wherein the initial column temperature is 62 ℃, the time lasts for 5min, the heating increasing rate is 3 ℃/min, the column temperature is gradually increased from 60 ℃ to 215 ℃, and the retention time is 32 min.

The standard substance is a mixed standard of 37 fatty acid methyl esters (Shanghai-sourced folk biotechnology, B25881).

The results are shown in FIG. 5, wherein A is a fatty acid methyl ester mixed standard gas chromatography analysis chart, and B is a YS01 fatty acid gas chromatography analysis chart; c is YS02 fatty acid gas chromatography diagram, D is YS03 fatty acid gas chromatography diagram; e is a YS04 fatty acid gas chromatography analysis chart; f is YS05 fatty acid gas chromatography analysis chart; g is YS06 fatty acid gas chromatography diagram; h is YS07 fatty acid gas chromatography diagram, and I is YS08 fatty acid gas chromatography diagram. The target product obtained in the sample is the methyl palmitoleate, and the retention time of the methyl palmitoleate in the sample is consistent with that of the methyl palmitoleate in the standard and is 10.200-10.450 min.

Palmitoleic acid C16: 1-area of methyl palmitoleate peak/area of total peak X100%

Palmitoleic acid C16:1 content results are shown in fig. 3, and it can be seen that the strain YS02 overexpressing the Mga2 transcription factor compared to the empty plasmid strain YS01, C16:1, the content of the strain YS04 palmitoleic acid reaches 46 percent, and is improved by about 9 percent compared with a control strain YS 01.

This is consistent with the expected results, and it can be seen that the IPT domain and ankyrin repeat are not essential domains for P120 activation, and even the domain is truncated to facilitate the activation and expression of Mga2, thereby increasing the palmitoleic acid content in Saccharomyces cerevisiae.

Example 2 Effect of overexpression of Mga2 transcription factor and simultaneous heterologous expression of fatty acid elongase genes KCS and FAE on the content of palmitoleic acid in Saccharomyces cerevisiae

Construction of gene engineering bacteria for synchronously heterogeneously expressing fatty acid elongase genes KCS and FAE by overexpression of Mga2 transcription factors

The fatty acid elongase genes KCS and FAE are from foreign plants cardamine hirsute and crambe and are not easy to obtain, so the sequences are directly synthesized by Jinwei Zhi company in Suzhou and are connected with YEplac112-P _PGK -T _CYC On a carrier.

The nucleotide sequence of the fatty acid elongase gene KCS is a sequence 2 in the sequence table;

the nucleotide sequence of the fatty acid elongase gene FAE is a sequence 3 in the sequence table.

1. Construction of MGA2 and KCS double-gene expression engineering bacteria YS07

pYES2-P _TEF1 -MGA2 _706aa -T _CYC -P _PGK -KCS-T _CYC The construction method of the expression vector comprises the following steps:

1) the KCS gene (SEQ ID NO: 2) was ligated into pYES2-P using Not I and Xho I _TEF1 Vector (the vector is P _TEF1 (SEQ ID NO: 5) the pYES2-P vector (vector obtained by substituting the Gal1 promoter in the pYES2.0 vector (Saimer Feishell science and technology (China) Co., Ltd.)) was constructed _TEF1 -KCS-T _CYC (T _CYC Self-carrying vector pYES 2);

2) the CYC and PGK fragments are seamlessly connected by using Overlap PCR to form T _CYC -P _PGK (SEQ ID NO: 6), schematic primer design as shown in FIG. 2 a; then, the T is put into _CYC -P _PGK Double digestion with BamHI and EcoRI into pYES2-P obtained in 1) above _TEF1 -KCS-T _CYC In (1), obtaining pYES2-P _TEF1 -T _CYC -P _PGK -KCS-T _CYC ；

3) Mixing MGA2 _706aa The gene (SEQ ID NO: 1, 1-2118) was amplified by a PCR instrument and ligated to pYES2-P described above _TEF1 -T _CYC -P _PGK -KCS-T _CYC The Kpn I and BamH I enzyme cutting sites are verified by colony PCR and sequencing to obtain pYES2-P _TEF1 -MGA2 _706aa -T _CYC -P _PGK -KCS-T _CYC Two-gene vector expressing MGA2 _706aa Genes and KCS genes, wherein the MGA2 corresponding promoter is TEF1, and the KCS corresponding promoter is PGK 1.

The primers used in the construction are shown in Table 2.

Will pYES2-P _TEF1 -MGA2 _706aa -T _CYC -P _PGK -KCS-T _CYC The double-gene vector is transferred into a host bacterium YS58 to obtain a saccharomyces cerevisiae gene engineering bacterium YS 07.

Secondly, constructing MGA2, KCS and FAE three-gene expression engineering bacteria YS08

1. Construction of pYES2-P _TEF1 -KCS-T _CYC -P _PGK -FAE-T _CYC Double-gene expression vector

1) From a company-synthesized support _PGK -FAE and KCS-T _CYC Two fragments are amplified by using the method that the Overlap PCR utilizes homologous sequences to seamlessly connect the two fragments to form KCS-T _CYC -P _PGK -FAE (seq id No. 7), schematic of fig. 2 b;

2) reacting KCS-T _CYC -P _PGK the-FAE fragment was digested simultaneously with EcoRI and Not I to join pYES2-P _TEF1 Vector construction pYES2-P _TEF1 -KCS-T _CYC -P _PGK -FAE-T _CYC A double gene vector.

2. Three-gene vector pYES2-P _TEF1 -KCS-T _CYC -P _PGK -FAE-T _CYC -P _PGK -MGA2 _706aa -T _CYC

In pYES2-P _TEF1 -KCS-T _CYC -P _PGK -FAE-T _CYC The construction is carried out on the basis of the double-gene vector, and specifically comprises the following steps:

1) the CYC and PGK fragments are seamlessly connected, and then T is connected _CYC -P _PGK And MGA2 _706aa Seamless join, get T _CYC -P _PGK -MGA2 _706aa (SEQ ID NO: 8), which is schematically shown in FIG. 2 c.

2) Then, the product is processedWill T _CYC -P _PGK -MGA2 _706aa Ligation to pYES2-P _TEF1 -KCS-T _CYC -P _PGK -FAE-T _CYC NotI and XhoI double restriction sites of the double-gene vector to obtain pYES2-P _TEF1 -KCS-T _CYC -P _PGK -FAE-T _CYC -P _PGK -MGA2 _706aa -T _CYC Three gene vectors, the primers used are shown in Table 2.

The recombinant vector pYES2-P _TEF1 -KCS-T _CYC -P _PGK -FAE-T _CYC -P _PGK -MGA2 _706aa -T _CYC Transferring the strain into a host strain YS58 to obtain a saccharomyces cerevisiae gene engineering strain YS 08.

Table 2 shows the primers used

Influence of heterologous expression of fatty acid elongase genes KCS and FAE on palmitoleic acid content of saccharomyces cerevisiae while over-expressing transcription factors

1. Engineering bacteria culture

The same culture method as that of example 1 was followed, except that YS07 and YS08 were cultured by fermentation and pYES2-P was introduced into the host strain YS58 _TEF1 Vector strain YS06 was obtained as a control strain.

2. Extraction of the product

The same procedure as in example 1.

3. Product handling and detection

The same procedure as in example 1.

As shown in FIG. 4, when the transcription factor Mga2 was overexpressed and the KCS gene was overexpressed, the palmitoleic acid content was significantly increased, and the palmitoleic acid content of the strain YS07 was about 51%. On the basis, the effect of simultaneous expression of the KCS gene and the FAE gene is more obvious, and the palmitoleic acid content of the final strain YS08 can reach 60%, so that the palmitoleic acid content is obviously improved compared with that of a control strain.

The above embodiments are only described to help understanding the method of the present invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, it is possible to make various improvements and modifications to the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.

SEQUENCE LISTING

<110> Beijing university of chemical industry

<120> method for improving palmitoleic acid content of saccharomyces cerevisiae

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 3342

<212> DNA

<213> Artificial sequence

<400> 1

atgcagcaga acagtgagtt cttaacggaa acacctggaa gcgaccctca tatatctcaa 60

ttgcacgcga atagcgtaat ggaatcacag ctcttggacg atttcctcct gaacgggtct 120

cccatgtacc aggatgatag catggcgcat attaatattg atgagggtgc taatttccaa 180

aattttatca agacagatga gggtgattcg cccaacctgt tgtctttcga aggtatcggt 240

aacaatactc atgtcaacca aaacgtgtcc actccactgg aggaggaaat ggaaagtaac 300

agagccttga aggaggaaga agaggacgag catgaaaata aggtttttaa tgaaaaaaat 360

ataggcaacc ctgctcatga cgagattgta tttggaagaa aggagacgat tcaatctgtt 420

tacataaatc ctttagatta ccttaaagtg aacgcagcgc agctaccttt ggatgtagag 480

gtctcaggtt tgccacaagt atctagagtg gaaaatcaac tgaaactgaa agtgaaaatt 540

acgtctgaaa caccactaaa ccaaagcatg ctttacttgc ctagcgattc catttcaaga 600

gaaaagtttt atttaaaaaa aaatatcgag gatttttcag aagacttcaa gaaaaatctt 660

ctgtacatca atgcgtttgt tctatgtgcg gtcagcaaca gaacgacaaa tgtttgtacc 720

aagtgtgtta agcgagaaca aagaagagcc gctagaagga aatcaggtat tgcagacaat 780

ttactctggt gtaataatat taatagaagg ttagtcgtgt tcaataacaa acaggttttc 840

cccataatga aaactttcga taatgttaag gagtttgaat taactaccag gctagtttgt 900

tattgcaggc accataaggc aaataatggc tttgtcatat tattcactat aacagattgg 960

caaaatagac tgttgggtaa gtttacgaca acacctatta tgatcacgga tagaaaacca 1020

gcaaatatgg ataccaccaa gtttaataac actactacct cgtccagaag gcagctaacg 1080

gaagaagaat ctaccacaga atattattca acggataaca accaattgag caaagacgaa 1140

aatatgccat ttcaatatac ttatcaacac aacccatatg ataatgacag tcaaatgaat 1200

aatattccac tgaaagacaa aaacgtacca ttcccatatt ccatctctca acagacagat 1260

ttgcttcaga acaataactt atcactgaac ctttctctgc ccaatcagca tattccatca 1320

ccaacatcta tgagcgaaga aggctcagaa tcatttaact atcatcatcg cgataatgac 1380

aatcccgtcc gtactatctc tttgacaaat attgaacaac agagtcaatt gaaccaacgg 1440

aaaagagcac gcaataattt ggaaaatgac attggtaaac ccctattcaa gcattccttt 1500

tcaaattcaa tcagtgcaac aaatacgatg aatccagctt tacattcaat gcaagatttc 1560

tcaatgaaaa acaacaacaa taatttgcca tcaattaatc gcgttatacc ttcacaaggc 1620

ccaatcaatg gtggtatcga agttacatta ctgggttgta acttcaaaga tggtctttct 1680

gtaaagttcg gctctaatct tgccctttct acgcaatgct ggagtgagac cacgatcgtc 1740

acttatctcc ctcccgctgc ctacgcgggt caagttttcg tctctattac tgatacgaat 1800

aatgaaaata ataacgatga ccttccccaa gaaattgaga tcaatgacaa taaaaaggcc 1860

atatttacct atgttgatga tactgatagg caactgattg aattggcttt gcaaattgtg 1920

ggattaaaaa tgaatggtaa gttagaagat gcaagaaata tcgcgaagag gattgttggc 1980

aatgattctc ctgatagcgg tacaaatggc aacagctgtt caaaaagcac aggtccctct 2040

ccaaaccaac acagtatgaa tctgaacaca agtgttcttt actccgatga agtcttgata 2100

caaaaagtta taaaatcatt gaacataaat tccaatattt ccatatgtga ttcattaggg 2160

agaactttat tacatcttgc ctgtttgaaa aattactcaa gccttgtgta tacattgatt 2220

aaaaagggtg ctcgtgttaa cgatattgat tcctttgggc taactccatt acattttgct 2280

tgcataagtg gtgaccctaa aattattaag atgcttttaa attgtaaagt aaattattca 2340

ctgaggtcac acaacggatt aactgcaaga gaagtattca tagcaaacca cattcattca 2400

aaggaaatag acaaaaaaca agataacaga gacaaccata agtttgttca taatgacact 2460

tatatcagcg aagtattgtc attgtttgaa gaattccaaa acggtacgaa gtttaccgat 2520

agtgtagaaa cagacagtaa ttattctatt agcaggaaat attcacaatc cagtttcaat 2580

tcaagcctgc tagacaatga atctttgaat gagaacttat tcgaaagcca aagcatgata 2640

aatcccactt ctatggagat tcagcatcca accttgcaac tatttgagaa ttcaagttac 2700

tctgagtacg accaaagtga tttcgaagaa gacggggatg aagatctgtt cgtcactgac 2760

gaagtagaaa aaccaggtgt tgcatgcagg gaggaacaaa gcgaactcct tgatattgga 2820

tctagcgcca acgaacccga agaggataat ggtagtacat ctctctggaa tagagtttta 2880

catcgaatta atgatgactt accaaaatat gaggatctgt tcccgttgtc ttggggtaaa 2940

gatgataaat tgaaaaccac aaatcaagac agtattgtgg agcagtcagc atctaatatt 3000

gaaaactctg aaaattcgga ggaagaggat tatgaggaag aggaagaatt tttgaaaaaa 3060

cagtttaaca gattcttcca aaacaaacaa aacttccgaa atgataaaat gttaatattt 3120

ttctggatac ccttaacact actacttttg acatggttca tcatgtacaa atttggcaac 3180

caagatagtt ccatcaatca tataagcgaa ttaatctcag agtacttgag aattgcatta 3240

gcaaagttct tgctgggaaa tgaaaggatg aaaactgcat tcaggtcaaa attatcaaac 3300

ctgcaaacaa caagaatgtt gaacgattta attgtcagtt ag 3342

<210> 2

<211> 1521

<212> DNA

<213> Artificial sequence

<400> 2

atgacttcta ttaatgttaa attgttgtac cattacgttt tgactaactt ttttaatttg 60

tgtttgttcc cattgactgc tttccctgct ggtaaggctt ctcaattgac tactaatgat 120

ttacatcatt tgtattctta cttgcatcat aatttaatta cagttacttt gttgtttgct 180

tttactgttt ttggttctat tttgtatatt gttacaagac caaaaccagt ttatttggtt 240

gattattctt gttatttgcc accaagacat ttgtcttgtg gtatttctag agttatggaa 300

attttttatg aaataagaaa atctgatcca tctagagagg tccctttcga tgacccatct 360

tctttggaat ttttgagaaa gattcaggag aggtctggtt tgggtgatga gacttacggt 420

ccacaaggtt tggtccacga catgccattg aggatgaact ttgctgctgc tagggaggag 480

actgagcagg tcattaacgg tgctttggaa aaattgtttg aaaatacaaa agttaatcca 540

agagaaattg gtattttggt tgttaattct tctatgttta atccaactcc atctttgtct 600

gctatggttg ttaatacttt taaattgaga tctaatatta aatctttttc attgggtggt 660

atgggttgct ctgctggtat tattgctatt gatttggcta aagatttgtt gcatgttcat 720

aaaaatactt atgcattggt tgtttctact gaaaatatta ctcattctac ttatactggt 780

gataacagat ctatgatggt ttctaattgt ttgtttagaa tgggtggtgc tgctattttg 840

ttgtctaata aagctggtga tagaagaaga tctaaatata agttggctca cactgttaga 900

actcacactg gtgctgacga ccagtctttt aggtgcgtta ggcaagagga cgacgacaga 960

ggtaagattg gtgtttgttt gtctaaagat attactgctg ttgctggtaa aactgttact 1020

aaaaatattg ctactttggg tccattggtt ttgccattgt cagaaaaatt tttgtatgtt 1080

gtttctttga tggctaaaaa attgtttaaa aataaaatta aacatactta tgttccagat 1140

tttaagttgg ctattgatca tttttgtatt cacgctggtg gtagggctgt cattgatgtt 1200

ttggagaaaa atttggcttt gtctcctgtt gatgttgaag cttctagatc tactttgcat 1260

agatttggta atacttcttc ttcttctatt tggtatgaat tggcttatat tgaagctaaa 1320

ggtagaatga aaaaaggtaa taaagtttgg caaattgcta ttggttctgg tttcaagtgc 1380

aactctgctg tttgggtcgc tttgtgcaac gtcaagccat ctgtcaactc tccatgggag 1440

cactgcattg atcgttatcc tgttgaaatt aattatggtt cttctaaatc tgaaactaga 1500

gctcaaaatg gtagatctta a 1521

<210> 3

<211> 1521

<212> DNA

<213> Artificial sequence

<400> 3

atgacttcta ttaatgttaa attgttgtac cattacgttt tgactaactt ttttaatttg 60

tgtttgttcc cattgactgc tttccctgct ggtaaggctt ctcaattgac tactaatgat 120

ttacatcatt tgtattctta cttgcatcat aatttaatta cagttacttt gttgtttgct 180

tttactgttt ttggttctat tttgtatatt gttacaagac caaaaccagt ttatttggtt 240

gattattctt gttatttgcc accaagacat ttgtcttgtg gtatttctag agttatggaa 300

attttttatg aaataagaaa atctgatcca tctagagagg tccctttcga tgacccatct 360

tctttggaat ttttgagaaa gattcaggag aggtctggtt tgggtgatga gacttacggt 420

ccacaaggtt tggtccacga catgccattg aggatgaact ttgctgctgc tagggaggag 480

actgagcagg tcattaacgg tgctttggaa aaattgtttg aaaatacaaa agttaatcca 540

agagaaattg gtattttggt tgttaattct tctatgttta atccaactcc atctttgtct 600

gctatggttg ttaatacttt taaattgaga tctaatatta aatctttttc attgggtggt 660

atgggttgct ctgctggtat tattgctatt gatttggcta aagatttgtt gcatgttcat 720

aaaaatactt atgcattggt tgtttctact gaaaatatta ctcattctac ttatactggt 780

gataacagat ctatgatggt ttctaattgt ttgtttagaa tgggtggtgc tgctattttg 840

ttgtctaata aagctggtga tagaagaaga tctaaatata agttggctca cactgttaga 900

actcacactg gtgctgacga ccagtctttt aggtgcgtta ggcaagagga cgacgacaga 960

ggtaagattg gtgtttgttt gtctaaagat attactgctg ttgctggtaa aactgttact 1020

aaaaatattg ctactttggg tccattggtt ttgccattgt cagaaaaatt tttgtatgtt 1080

gtttctttga tggctaaaaa attgtttaaa aataaaatta aacatactta tgttccagat 1140

tttaagttgg ctattgatca tttttgtatt cacgctggtg gtagggctgt cattgatgtt 1200

ttggagaaaa atttggcttt gtctcctgtt gatgttgaag cttctagatc tactttgcat 1260

agatttggta atacttcttc ttcttctatt tggtatgaat tggcttatat tgaagctaaa 1320

ggtagaatga aaaaaggtaa taaagtttgg caaattgcta ttggttctgg tttcaagtgc 1380

aactctgctg tttgggtcgc tttgtgcaac gtcaagccat ctgtcaactc tccatgggag 1440

cactgcattg atcgttatcc tgttgaaatt aattatggtt cttctaaatc tgaaactaga 1500

gctcaaaatg gtagatctta a 1521

<210> 4

<211> 1113

<212> PRT

<213> Artificial sequence

<400> 4

Met Gln Gln Asn Ser Glu Phe Leu Thr Glu Thr Pro Gly Ser Asp Pro

1 5 10 15

His Ile Ser Gln Leu His Ala Asn Ser Val Met Glu Ser Gln Leu Leu

20 25 30

Asp Asp Phe Leu Leu Asn Gly Ser Pro Met Tyr Gln Asp Asp Ser Met

35 40 45

Ala His Ile Asn Ile Asp Glu Gly Ala Asn Phe Gln Asn Phe Ile Lys

50 55 60

Thr Asp Glu Gly Asp Ser Pro Asn Leu Leu Ser Phe Glu Gly Ile Gly

65 70 75 80

Asn Asn Thr His Val Asn Gln Asn Val Ser Thr Pro Leu Glu Glu Glu

85 90 95

Met Glu Ser Asn Arg Ala Leu Lys Glu Glu Glu Glu Asp Glu His Glu

100 105 110

Asn Lys Val Phe Asn Glu Lys Asn Ile Gly Asn Pro Ala His Asp Glu

115 120 125

Ile Val Phe Gly Arg Lys Glu Thr Ile Gln Ser Val Tyr Ile Asn Pro

130 135 140

Leu Asp Tyr Leu Lys Val Asn Ala Ala Gln Leu Pro Leu Asp Val Glu

145 150 155 160

Val Ser Gly Leu Pro Gln Val Ser Arg Val Glu Asn Gln Leu Lys Leu

165 170 175

Lys Val Lys Ile Thr Ser Glu Thr Pro Leu Asn Gln Ser Met Val Tyr

180 185 190

Leu Pro Ser Asp Ser Ile Ser Arg Glu Lys Phe Tyr Leu Lys Lys Asn

195 200 205

Ile Glu Asp Phe Ser Glu Asp Phe Lys Lys Asn Leu Leu Tyr Ile Asn

210 215 220

Ala Phe Val Leu Cys Ala Val Ser Asn Arg Thr Thr Asn Val Cys Thr

225 230 235 240

Lys Cys Val Lys Arg Glu Gln Arg Arg Ala Ala Arg Arg Lys Ser Gly

245 250 255

Ile Ala Asp Asn Leu Leu Trp Cys Asn Asn Ile Asn Arg Arg Leu Val

260 265 270

Val Phe Asn Asn Lys Gln Val Phe Pro Ile Met Lys Thr Phe Asp Asn

275 280 285

Val Lys Glu Phe Glu Leu Thr Thr Arg Leu Val Cys Tyr Cys Arg His

290 295 300

His Lys Ala Asn Asn Gly Phe Val Ile Leu Phe Thr Ile Thr Asp Trp

305 310 315 320

Gln Asn Arg Leu Leu Gly Lys Phe Thr Thr Thr Pro Ile Met Ile Thr

325 330 335

Asp Arg Lys Pro Ala Asn Met Asp Thr Thr Lys Phe Asn Asn Thr Thr

340 345 350

Thr Ser Ser Arg Arg Gln Leu Thr Glu Glu Glu Ser Thr Thr Glu Tyr

355 360 365

Tyr Ser Thr Asp Asn Asn Gln Leu Ser Lys Asp Glu Asn Met Pro Phe

370 375 380

Gln Tyr Thr Tyr Gln His Asn Pro Tyr Asp Asn Asp Ser Gln Met Asn

385 390 395 400

Asn Ile Pro Leu Lys Asp Lys Asn Val Pro Phe Pro Tyr Ser Ile Ser

405 410 415

Gln Gln Thr Asp Leu Leu Gln Asn Asn Asn Leu Ser Leu Asn Leu Ser

420 425 430

Leu Pro Asn Gln His Ile Pro Ser Pro Thr Ser Met Ser Glu Glu Gly

435 440 445

Ser Glu Ser Phe Asn Tyr His His Arg Asp Asn Asp Asn Pro Val Arg

450 455 460

Thr Ile Ser Leu Thr Asn Ile Glu Gln Gln Ser Gln Leu Asn Gln Arg

465 470 475 480

Lys Arg Ala Arg Asn Asn Leu Glu Asn Asp Ile Gly Lys Pro Leu Phe

485 490 495

Lys His Ser Phe Ser Asn Ser Ile Ser Ala Thr Asn Thr Met Asn Pro

500 505 510

Ala Leu His Ser Met Gln Asp Phe Ser Met Lys Asn Asn Asn Asn Asn

515 520 525

Leu Pro Ser Ile Asn Arg Val Ile Pro Ser Gln Gly Pro Ile Asn Gly

530 535 540

Gly Ile Glu Val Thr Leu Leu Gly Cys Asn Phe Lys Asp Gly Leu Ser

545 550 555 560

Val Lys Phe Gly Ser Asn Leu Ala Leu Ser Thr Gln Cys Trp Ser Glu

565 570 575

Thr Thr Ile Val Thr Tyr Leu Pro Pro Ala Ala Tyr Ala Gly Gln Val

580 585 590

Phe Val Ser Ile Thr Asp Thr Asn Asn Glu Asn Asn Asn Asp Asp Leu

595 600 605

Pro Gln Glu Ile Glu Ile Asn Asp Asn Lys Lys Ala Ile Phe Thr Tyr

610 615 620

Val Asp Asp Thr Asp Arg Gln Leu Ile Glu Leu Ala Leu Gln Ile Val

625 630 635 640

Gly Leu Lys Met Asn Gly Lys Leu Glu Asp Ala Arg Asn Ile Ala Lys

645 650 655

Arg Ile Val Gly Asn Asp Ser Pro Asp Ser Gly Thr Asn Gly Asn Ser

660 665 670

Cys Ser Lys Ser Thr Gly Pro Ser Pro Asn Gln His Ser Met Asn Leu

675 680 685

Asn Thr Ser Val Leu Tyr Ser Asp Glu Val Leu Ile Gln Lys Val Ile

690 695 700

Lys Ser Leu Asn Ile Asn Ser Asn Ile Ser Ile Cys Asp Ser Leu Gly

705 710 715 720

Arg Thr Leu Leu His Leu Ala Cys Leu Lys Asn Tyr Ser Ser Leu Val

725 730 735

Tyr Thr Leu Ile Lys Lys Gly Ala Arg Val Asn Asp Ile Asp Ser Phe

740 745 750

Gly Leu Thr Pro Leu His Phe Ala Cys Ile Ser Gly Asp Pro Lys Ile

755 760 765

Ile Lys Met Leu Leu Asn Cys Lys Val Asn Tyr Ser Leu Arg Ser His

770 775 780

Asn Gly Leu Thr Ala Arg Glu Val Phe Ile Ala Asn His Ile His Ser

785 790 795 800

Glu Glu Val Asp Lys Lys Gln Glu Asn Arg Asp Asn His Lys Phe Val

805 810 815

His Asn Asp Thr Tyr Ile Ser Glu Val Leu Ser Leu Phe Glu Glu Phe

820 825 830

Gln Asn Gly Thr Lys Phe Thr Asp Ser Val Glu Thr Asp Ser Asn Tyr

835 840 845

Ser Ile Ser Arg Lys Tyr Ser Gln Ser Ser Phe Asn Ser Ser Leu Leu

850 855 860

Asp Asn Glu Ser Leu Asn Glu Asn Leu Phe Glu Ser Gln Ser Met Ile

865 870 875 880

Asn Pro Thr Ser Met Glu Ile Gln His Pro Thr Leu Gln Leu Phe Glu

885 890 895

Asn Ser Ser Tyr Ser Glu Tyr Asp Gln Ser Asp Phe Glu Glu Asp Gly

900 905 910

Asp Glu Asp Leu Phe Val Thr Asp Glu Val Glu Lys Pro Gly Val Ala

915 920 925

Cys Arg Glu Glu Gln Ser Glu Leu Leu Asp Ile Gly Ser Ser Ala Asn

930 935 940

Glu Pro Glu Glu Asp Asn Gly Ser Thr Ser Leu Trp Asn Arg Val Leu

945 950 955 960

His Arg Ile Asn Asp Asp Leu Pro Lys Tyr Glu Asp Leu Phe Pro Leu

965 970 975

Ser Trp Gly Lys Asp Asp Lys Leu Lys Thr Thr Asn Gln Asp Ser Ile

980 985 990

Val Glu Gln Ser Ala Ser Asn Ile Glu Asn Ser Glu Asn Ser Glu Glu

995 1000 1005

Glu Asp Tyr Glu Glu Glu Glu Glu Phe Leu Lys Lys Gln Phe Asn

1010 1015 1020

Arg Phe Phe Gln Asn Lys Gln Asn Phe Arg Asn Asp Lys Met Leu

1025 1030 1035

Ile Phe Phe Trp Ile Pro Leu Thr Leu Leu Leu Leu Thr Trp Phe

1040 1045 1050

Ile Met Tyr Lys Phe Gly Asn Gln Asp Ser Ser Ile Asn His Ile

1055 1060 1065

Ser Glu Leu Ile Ser Glu Tyr Leu Arg Ile Ala Leu Ala Lys Phe

1070 1075 1080

Leu Leu Gly Asn Glu Arg Met Lys Thr Ala Phe Arg Ser Lys Leu

1085 1090 1095

Ser Asn Leu Gln Thr Thr Arg Met Leu Asn Asp Leu Ile Val Ser

1100 1105 1110

<210> 5

<211> 412

<212> DNA

<213> Artificial sequence

<400> 5

atagcttcaa aatgtttcta ctcctttttt actcttccag attttctcgg actccgcgca 60

tcgccgtacc acttcaaaac acccaagcac agcatactaa atttcccctc tttcttcctc 120

tagggtgtcg ttaattaccc gtactaaagg tttggaaaag aaaaaagaga ccgcctcgtt 180

tctttttctt cgtcgaaaaa ggcaataaaa atttttatca cgtttctttt tcttgaaaat 240

tttttttttt gatttttttc tctttcgatg acctcccatt gatatttaag ttaataaacg 300

gtcttcaatt tctcaagttt cagtttcatt tttcttgttc tattacaact ttttttactt 360

cttgctcatt agaaagaaag catagcaatc taatctaagt tttaattaca aa 412

<210> 6

<211> 998

<212> DNA

<213> Artificial sequence

<400> 6

tcatgtaatt agttatgtca cgcttacatt cacgccctcc ccccacatcc gctctaaccg 60

aaaaggaagg agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt 120

tagtattaag aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt 180

acgcatgtaa cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt 240

taatttgcac gcacagatat tataacatct gcacaatagg catttgcaag aattactcgt 300

gagtaaggaa agagtgagga actatcgcat acctgcattt aaagatgccg atttgggcgc 360

gaatccttta ttttggcttc accctcatac tattatcagg gccagaaaaa ggaagtgttt 420

ccctccttct tgaattgatg ttaccctcat aaagcacgtg gcctcttatc gagaaagaaa 480

ttaccgtcgc tcgtgatttg tttgcaaaaa gaacaaaact gaaaaaaccc agacacgctc 540

gacttcctgt cttcctattg attgcagctt ccaatttcgt cacacaacaa ggtcctagcg 600

acggctcaca ggttttgtaa caagcaatcg aaggttctgg aatggcggga aagggtttag 660

taccacatgc tatgatgccc actgtgatct ccagagcaaa gttcgttcga tcgtactgtt 720

actctctctc tttcaaacag aattgtccga atcgtgtgac aacaacagcc tgttctcaca 780

cactcttttc ttctaaccaa gggggtggtt tagtttagta gaacctcgtg aaacttacat 840

ttacatatat ataaacttgc ataaattggt caatgcaaga aatacatatt tggtcttttc 900

taattcgtag tttttcaagt tcttagatgc tttctttttc tcttttttac agatcatcaa 960

ggaagtaatt atctactttt tacaacaaat ataaaaca 998

<210> 7

<211> 4065

<212> DNA

<213> Artificial sequence

<400> 7

atgacttcta ttaatgttaa attgttgtac cattacgttt tgactaactt ttttaatttg 60

tgtttgttcc cattgactgc tttccctgct ggtaaggctt ctcaattgac tactaatgat 120

ttacatcatt tgtattctta cttgcatcat aatttaatta cagttacttt gttgtttgct 180

tttactgttt ttggttctat tttgtatatt gttacaagac caaaaccagt ttatttggtt 240

gattattctt gttatttgcc accaagacat ttgtcttgtg gtatttctag agttatggaa 300

attttttatg aaataagaaa atctgatcca tctagagagg tccctttcga tgacccatct 360

tctttggaat ttttgagaaa gattcaggag aggtctggtt tgggtgatga gacttacggt 420

ccacaaggtt tggtccacga catgccattg aggatgaact ttgctgctgc tagggaggag 480

actgagcagg tcattaacgg tgctttggaa aaattgtttg aaaatacaaa agttaatcca 540

agagaaattg gtattttggt tgttaattct tctatgttta atccaactcc atctttgtct 600

gctatggttg ttaatacttt taaattgaga tctaatatta aatctttttc attgggtggt 660

atgggttgct ctgctggtat tattgctatt gatttggcta aagatttgtt gcatgttcat 720

aaaaatactt atgcattggt tgtttctact gaaaatatta ctcattctac ttatactggt 780

gataacagat ctatgatggt ttctaattgt ttgtttagaa tgggtggtgc tgctattttg 840

ttgtctaata aagctggtga tagaagaaga tctaaatata agttggctca cactgttaga 900

actcacactg gtgctgacga ccagtctttt aggtgcgtta ggcaagagga cgacgacaga 960

ggtaagattg gtgtttgttt gtctaaagat attactgctg ttgctggtaa aactgttact 1020

aaaaatattg ctactttggg tccattggtt ttgccattgt cagaaaaatt tttgtatgtt 1080

gtttctttga tggctaaaaa attgtttaaa aataaaatta aacatactta tgttccagat 1140

tttaagttgg ctattgatca tttttgtatt cacgctggtg gtagggctgt cattgatgtt 1200

ttggagaaaa atttggcttt gtctcctgtt gatgttgaag cttctagatc tactttgcat 1260

agatttggta atacttcttc ttcttctatt tggtatgaat tggcttatat tgaagctaaa 1320

ggtagaatga aaaaaggtaa taaagtttgg caaattgcta ttggttctgg tttcaagtgc 1380

aactctgctg tttgggtcgc tttgtgcaac gtcaagccat ctgtcaactc tccatgggag 1440

cactgcattg atcgttatcc tgttgaaatt aattatggtt cttctaaatc tgaaactaga 1500

gctcaaaatg gtagatctta aggatcctct agagtcgaca tcatgtaatt agttatgtca 1560

cgcttacatt cacgccctcc ccccacatcc gctctaaccg aaaaggaagg agttagacaa 1620

cctgaagtct aggtccctat ttattttttt atagttatgt tagtattaag aacgttattt 1680

atatttcaaa tttttctttt ttttctgtac agacgcgtgt acgcatgtaa cattatactg 1740

aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt taatttgcac gcacagatat 1800

tataacatct gcacaatagg catttgcaag aattactcgt gagtaaggaa agagtgagga 1860

actatcgcat acctgcattt aaagatgccg atttgggcgc gaatccttta ttttggcttc 1920

accctcatac tattatcagg gccagaaaaa ggaagtgttt ccctccttct tgaattgatg 1980

ttaccctcat aaagcacgtg gcctcttatc gagaaagaaa ttaccgtcgc tcgtgatttg 2040

tttgcaaaaa gaacaaaact gaaaaaaccc agacacgctc gacttcctgt cttcctattg 2100

attgcagctt ccaatttcgt cacacaacaa ggtcctagcg acggctcaca ggttttgtaa 2160

caagcaatcg aaggttctgg aatggcggga aagggtttag taccacatgc tatgatgccc 2220

actgtgatct ccagagcaaa gttcgttcga tcgtactgtt actctctctc tttcaaacag 2280

aattgtccga atcgtgtgac aacaacagcc tgttctcaca cactcttttc ttctaaccaa 2340

gggggtggtt tagtttagta gaacctcgtg aaacttacat ttacatatat ataaacttgc 2400

ataaattggt caatgcaaga aatacatatt tggtcttttc taattcgtag tttttcaagt 2460

tcttagatgc tttctttttc tcttttttac agatcatcaa ggaagtaatt atctactttt 2520

tacaacaaat ataaaacagg taccatgact tctattaatg ttaaattatt gtatcattac 2580

gttattacta atttgttcaa tttatgcttt tttccattaa ctgctattgt tgctggtaaa 2640

gcttcaagat tgactattga tgatttgcat catttgtact attcttattt gcaacataat 2700

gttattacta ttgctccatt gtttgctttt acagtttttg gttctatttt gtatattgtt 2760

actagaccaa aacctgttta tttagttgaa tattcttgtt atttgccacc aactcaatgt 2820

agatcttcaa tttctaaagt tatggatatt ttttaccaag tcagaaaggc tgacccattc 2880

aggaacggta cttgcgacga ttcttcatgg ttggattttt tgagaaaaat tcaggaaaga 2940

tctggtttgg gtgacgagac acacggtcct gaaggtttgt tgcaggttcc accaaggagg 3000

actttcgctg ctgctagaga agaaactgaa caagttattg ttggtgcttt gaaaaacttg 3060

tttgaaaata ctaaagttaa tccaaaagat attggtattt tggttgttaa ttcttctatg 3120

tttaatccaa ctccatcttt gtctgctatg gttgttaata cttttaaatt gagatctaat 3180

gttagatctt ttaatttggg tggtatgggt tgctctgctg gtgttattgc tattgatttg 3240

gctaaagatt tgttgcatgt tcataaaaat acttatgctt tggttgtttc aactgaaaac 3300

attacataca atatttatgc tggtgataat agatctatga tggtttctaa ttgtttgttt 3360

agagttggtg gtgctgctat tttgttgtca aacaaaccta gagatagaag aagatctaaa 3420

tatgaattgg ttcacactgt cagaactcac actggtgctg acgacagatc tttcaggtgc 3480

gtccaacaag gtgacgacga gaacggtaag actggtgtct ctttgtctaa agatattaca 3540

gaagttgctg gtagaactgt taaaaagaat attgctactt tgggtccatt gattttacca 3600

ttgtctgaaa aattgttgtt ttttgttact tttatggcta aaaaattgtt taaagataaa 3660

gttaaacatt attatgttcc agattttaaa ttggctattg atcatttttg cattcacgct 3720

ggtggtaggg ctgttattga cgttttggag aaaaatttgg gtttagctcc aattgatgtt 3780

gaagcttcta gatctacttt gcatagattt ggtaatactt cttcttcttc tatttggtat 3840

gaattggctt atattgaagc taaaggtaga atgaaaaaag gtaataaagt ttggcaaatt 3900

gctttgggtt ctggtttcaa gtgcaactct gctgtctggg tcgctttgtc taacgtcaag 3960

gcttctacta actctccatg ggagcactgc attgacagat acccagttaa aattgattct 4020

gattctgcta aatctgaaac tagagctcaa aatggtagat cttaa 4065

<210> 8

<211> 3116

<212> DNA

<213> Artificial sequence

<400> 8

tcatgtaatt agttatgtca cgcttacatt cacgccctcc ccccacatcc gctctaaccg 60

aaaaggaagg agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt 120

tagtattaag aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt 180

acgcatgtaa cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt 240

taatttgcac gcacagatat tataacatct gcacaatagg catttgcaag aattactcgt 300

gagtaaggaa agagtgagga actatcgcat acctgcattt aaagatgccg atttgggcgc 360

gaatccttta ttttggcttc accctcatac tattatcagg gccagaaaaa ggaagtgttt 420

ccctccttct tgaattgatg ttaccctcat aaagcacgtg gcctcttatc gagaaagaaa 480

ttaccgtcgc tcgtgatttg tttgcaaaaa gaacaaaact gaaaaaaccc agacacgctc 540

gacttcctgt cttcctattg attgcagctt ccaatttcgt cacacaacaa ggtcctagcg 600

acggctcaca ggttttgtaa caagcaatcg aaggttctgg aatggcggga aagggtttag 660

taccacatgc tatgatgccc actgtgatct ccagagcaaa gttcgttcga tcgtactgtt 720

actctctctc tttcaaacag aattgtccga atcgtgtgac aacaacagcc tgttctcaca 780

cactcttttc ttctaaccaa gggggtggtt tagtttagta gaacctcgtg aaacttacat 840

ttacatatat ataaacttgc ataaattggt caatgcaaga aatacatatt tggtcttttc 900

taattcgtag tttttcaagt tcttagatgc tttctttttc tcttttttac agatcatcaa 960

ggaagtaatt atctactttt tacaacaaat ataaaacaat gcagcagaac agtgagttct 1020

taacggaaac acctggaagc gaccctcata tatctcaatt gcacgcgaat agcgtaatgg 1080

aatcacagct cttggacgat ttcctcctga acgggtctcc catgtaccag gatgatagca 1140

tggcgcatat taatattgat gagggtgcta atttccaaaa ttttatcaag acagatgagg 1200

gtgattcgcc caacctgttg tctttcgaag gtatcggtaa caatactcat gtcaaccaaa 1260

acgtgtccac tccactggag gaggaaatgg aaagtaacag agccttgaag gaggaagaag 1320

aggacgagca tgaaaataag gtttttaatg aaaaaaatat aggcaaccct gctcatgacg 1380

agattgtatt tggaagaaag gagacgattc aatctgttta cataaatcct ttagattacc 1440

ttaaagtgaa cgcagcgcag ctacctttgg atgtagaggt ctcaggtttg ccacaagtat 1500

ctagagtgga aaatcaactg aaactgaaag tgaaaattac gtctgaaaca ccactaaacc 1560

aaagcatgct ttacttgcct agcgattcca tttcaagaga aaagttttat ttaaaaaaaa 1620

atatcgagga tttttcagaa gacttcaaga aaaatcttct gtacatcaat gcgtttgttc 1680

tatgtgcggt cagcaacaga acgacaaatg tttgtaccaa gtgtgttaag cgagaacaaa 1740

gaagagccgc tagaaggaaa tcaggtattg cagacaattt actctggtgt aataatatta 1800

atagaaggtt agtcgtgttc aataacaaac aggttttccc cataatgaaa actttcgata 1860

atgttaagga gtttgaatta actaccaggc tagtttgtta ttgcaggcac cataaggcaa 1920

ataatggctt tgtcatatta ttcactataa cagattggca aaatagactg ttgggtaagt 1980

ttacgacaac acctattatg atcacggata gaaaaccagc aaatatggat accaccaagt 2040

ttaataacac tactacctcg tccagaaggc agctaacgga agaagaatct accacagaat 2100

attattcaac ggataacaac caattgagca aagacgaaaa tatgccattt caatatactt 2160

atcaacacaa cccatatgat aatgacagtc aaatgaataa tattccactg aaagacaaaa 2220

acgtaccatt cccatattcc atctctcaac agacagattt gcttcagaac aataacttat 2280

cactgaacct ttctctgccc aatcagcata ttccatcacc aacatctatg agcgaagaag 2340

gctcagaatc atttaactat catcatcgcg ataatgacaa tcccgtccgt actatctctt 2400

tgacaaatat tgaacaacag agtcaattga accaacggaa aagagcacgc aataatttgg 2460

aaaatgacat tggtaaaccc ctattcaagc attccttttc aaattcaatc agtgcaacaa 2520

atacgatgaa tccagcttta cattcaatgc aagatttctc aatgaaaaac aacaacaata 2580

atttgccatc aattaatcgc gttatacctt cacaaggccc aatcaatggt ggtatcgaag 2640

ttacattact gggttgtaac ttcaaagatg gtctttctgt aaagttcggc tctaatcttg 2700

ccctttctac gcaatgctgg agtgagacca cgatcgtcac ttatctccct cccgctgcct 2760

acgcgggtca agttttcgtc tctattactg atacgaataa tgaaaataat aacgatgacc 2820

ttccccaaga aattgagatc aatgacaata aaaaggccat atttacctat gttgatgata 2880

ctgataggca actgattgaa ttggctttgc aaattgtggg attaaaaatg aatggtaagt 2940

tagaagatgc aagaaatatc gcgaagagga ttgttggcaa tgattctcct gatagcggta 3000

caaatggcaa cagctgttca aaaagcacag gtccctctcc aaaccaacac agtatgaatc 3060

tgaacacaag tgttctttac tccgatgaag tcttgataca aaaagttata aaatca 3116

Claims (6)

1. A recombinant bacterium is obtained by carrying out the following transformation 1) and 2) in oil-producing saccharomyces cerevisiae:

1) improving the transcription activity of the OLE1 gene in the oil-producing saccharomyces cerevisiae to obtain a recombinant strain;

2) allowing the lipid-producing saccharomyces cerevisiae to express a fatty acid elongase;

the transcriptional activity of the OLE1 gene in the grease-producing Saccharomyces cerevisiae is improved by the expression level of the transcription factor Mga2 of the OLE1 gene or the coding gene of the truncated body of the OLE1 gene in the grease-producing Saccharomyces cerevisiae;

the amino acid sequence of the transcription factor Mga2 is a sequence 4 in a sequence table;

the amino acid sequence of the truncated body of the transcription factor Mga2 is 1 st-975 th of a sequence 4 in a sequence table;

or the amino acid sequence of the truncated body of the transcription factor Mga2 is the 1 st to 706 th position of the sequence 4 in the sequence table;

the fatty acid elongase is the fatty acid elongase shown in the following a);

or the fatty acid elongase is a fatty acid elongase shown in a) and a fatty acid elongase shown in b) as follows:

a) the fatty acid elongase is a protein coded by the nucleotide shown in a sequence 2;

b) the fatty acid elongase is protein coded by nucleotide shown in sequence 3.

2. The recombinant bacterium according to claim 1, wherein: the grease-producing saccharomyces cerevisiae is saccharomyces cerevisiae YS 58.

3. A method for preparing a recombinant bacterium by the modification of 1) and 2) according to any one of claims 1 to 2.

4. Use of the recombinant bacterium of any one of claims 1-2 to increase palmitoleic acid content of saccharomyces cerevisiae;

or, the use of the recombinant bacterium of any one of claims 1-2 for the production of palmitoleic acid.

5. A method for increasing the palmitoleic acid content of saccharomyces cerevisiae comprises the following steps: the improvement of the palmitoleic acid content of the saccharomyces cerevisiae is realized according to the improvement of 1) and 2) in the recombinant bacteria of any one of claims 1-2.

6. A process for preparing palmitoleic acid, comprising the steps of:

1) fermenting the recombinant strain of any one of claims 1-2 in a YNB auxotrophic medium and collecting the fermentation product;

2) and extracting fatty acid in the fermentation product by a saponification method to obtain the palmitoleic acid.

Images