CN116024109A - Rhodosporidium toruloides engineering strain for high-yield nervonic acid and construction method and application thereof - Google Patents

Abstract

The invention discloses a rhodosporidium toruloides engineering strain for high-yield nervonic acid, a construction method and application thereof, wherein the engineering strain takes rhodosporidium toruloides as an original strain, and heterologously expresses KCS enzymes in fatty acid elongase complexes with different substrate preference; the KCR, HCD and ECR enzymes in the fatty acid elongase complex, and the malate enzyme ME1 are overexpressed. The strain for producing the nervonic acid with high yield constructed by the invention has the oil and the nervonic acid titer reaching 73.7g/L and 35.4g/L respectively on the level of a 50L fermentation tank, and has potential industrial production value.

Description

Rhodosporidium toruloides engineering strain for high-yield nervonic acid and construction method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a rhodosporidium toruloides engineering strain for producing nervonic acid, a construction method and application thereof.

Background

Nervonic Acid (NA), known as cis-15-tetracosadienoic acid (cis-15-tetracosenic acid), is an ultra-long chain fatty acid (Very long chain fatty acid, VLCFA), is an important nutrient substance in brain nerve tissue and cells, and has positive therapeutic and prophylactic effects on brain tissue development, alzheimer's disease, cardiovascular and cerebrovascular diseases, immune system diseases, and the like.

The special efficacy of nervonic acid makes it one of the research hotspots in the fields of medicine and health food. However, the source of the nervonic acid is still mainly from limited plant resources at present, however, the woody plants or the herbaceous plants cannot produce grease efficiently or realize large-scale and wide planting like crops such as rape due to the strict requirements on growth conditions, so that the nervonic acid is in short supply and has high market price. Realizing the industrialized high-efficiency production of the nervonic acid, effectively reducing the current high price and making the nervonic acid become a part of people's daily diet, thereby making a certain contribution to guaranteeing and improving the folk level. In recent years, with the development of metabolic engineering, system biology and synthetic biology, researchers can adopt a biotechnology method so as to realize efficient fermentation production of nervonic acid to meet the demands of future markets.

Although the plant genetic engineering method is adopted, the aim of accumulating a large amount of nervonic acid in oil crops can be fulfilled. However, oil crops have the disadvantages of slow growth rate, occupation of cultivated land, and the like compared with microbial cells. Therefore, the microorganism is more feasible to produce the nervonic acid, and the yeast with high oil yield is one of the best host choices. In the original FAE pathway in yeast, enzymes corresponding to KCS are Elo1, elo2 and Elo3, but specific substrates are all saturated fatty acids, so that the ultra-long chain fatty acid products in yeast are generally saturated fatty acids such as C20:0, C22:0, C24:0 and C26:0. Therefore, to achieve the synthesis of ultra-long chain unsaturated fatty acids in yeast, it is necessary to introduce a KCS enzyme gene capable of using C18:1 as a specific substrate. The KCS genes with different substrate preferences are integrated into R.toruloides, and finally the KCS genes of C.abyssinica and C.graeca are used for combined expression, so that the composition proportion of nervonic acid in grease reaches about 16% (the fermentation titer of nervonic acid is 7.9 g/L), but the proportion of erucic acid reaches about 10% (FILLET S, RONCHEL C, CALLEJO C, et al engineering Rhodosporidium toruloides for the production of very long-chain monounsaturated fatty acid-rich oil S.applied Microbiology and Biotechnology,2017,101 (19): 7271-7280), and the overall nervonic acid titer is lower, the proportion of erucic acid is higher, and the industrial production is far away. In recent years, researchers have also conducted metabolic engineering studies on the synthesis of nervonic acid in yarrowia lipolytica (Yarrowia lipolytica), the highest nervonic acid strain reported by Zhao Chen et al, which contains 6.4% of lipid, failed to further increase the yield of nervonic acid when KCS, which is preferred by three different substrates, atFAE1 (AtKCS), btFAE1 and CgKCS, was expressed in combination (Zhao Chen, meng Huimin, li Guxin, zhang Peiyu, li Fuli, wang Shian, jie Zhi, yarrowia lipolytica, the effect of synthesizing ultralong chain fatty acid and temperature, microbiological report, 2020,47 (01): 13-23). In summary, the modification of yarrowia lipolytica to produce nervonic acid still faces the defects of low titer, high erucic acid content and the like, and the aim of industrial production cannot be achieved, so that new technical means are needed to develop ideas in other host cells to solve the problems.

Disclosure of Invention

The invention aims to: aiming at the problems in the prior art, the invention provides a genetic engineering strain for producing the nervonic acid with high efficiency, and the genetic engineering strain can be used for stably and efficiently synthesizing the grease rich in the nervonic acid through fermentation.

The invention also provides a construction method and application of the genetic engineering strain for producing the nervonic acid.

The technical scheme is as follows: in order to achieve the above purpose, the engineering strain of rhodosporidium toruloides with high yield of nervonic acid disclosed by the invention takes rhodosporidium toruloides as an original strain, and heterologously expresses KCS enzymes in fatty acid elongase complexes with different substrate preference; the KCR, HCD and ECR enzymes in the fatty acid elongase complex, and the malate enzyme ME1 are overexpressed.

Wherein the KCS enzyme in the fatty acid elongase compound with different substrate preference comprises one or more of exogenous KCS enzyme ELOVL6 or MACLE1 with substrate preference for C16:0 fatty acyl-CoA, exogenous KCS enzyme TnKCS or AtKCS with relative substrate preference for C18:1 fatty acyl-CoA, exogenous KCS enzyme CraKCS or MoKCS or BnKCS with relative substrate preference for C20:1 fatty acyl-CoA, and exogenous KCS enzyme LaKCS or CgKCS with relative substrate preference for C22:1 fatty acyl-CoA, and the sequence of the coding genes of the above enzymes is respectively shown in SEQ ID NO 1-9.

Preferably, the KCS enzymes in the fatty acid elongase complex that heterologously express different substrate preferences include AtKCS, bnKCS, ELOVL and LaKCS.

Wherein, the exogenous KCS enzyme with substrate preference to C16:0 fatty acyl-CoA is: ELOVL6 from mammal, and its coding gene sequence is shown in SEQ ID NO. 1; MACLE1 from Mortierella alpina S-4, the sequence of the coding gene is shown in SEQ ID NO. 2.

Wherein, the exogenous KCS gene with relative substrate preference to C18:1 acyl-CoA is: the nucleotide sequence of the TnKCS of the source Teesdalia nudicaulis is shown as SEQ ID NO. 3; arabidopsis thaliana, the nucleotide sequence of which is shown in SEQ ID NO. 4.

Wherein, the exogenous KCS gene with relative substrate preference to C20:1 acyl-CoA is: the nucleotide sequence of the CraKCS of the source Crambe abyssinica is shown as SEQ ID NO. 5; moKCS derived from Malania oleifera has a nucleotide sequence shown in SEQ ID NO. 6; bnKCS derived from Brassica napus has a nucleotide sequence shown in SEQ ID NO. 7.

Wherein, the exogenous KCS gene with relative substrate preference to C22:1 acyl-CoA is: the LaKCS of Lunaria annua has a nucleotide sequence shown in SEQ ID NO. 8; the CgKCS from Cardamine graeca has the nucleotide sequence shown in SEQ ID NO. 9.

Preferably, KCR, HCD and ECR enzymes in the over-expressed fatty acid elongase complex are genes which are derived from rhodosporidium toruloides host and have the functions and are respectively shown in SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12 or have homology with the amino acid sequences coded by the genes to be 30% or higher.

Preferably, KCR, HCD and ECR enzymes in the over-expressed fatty acid elongase complex are AtKCR, atHCD and AtECR from Arabidopsis thaliana, and the gene sequences for encoding the above three enzymes are SEQ ID NO. 13, SEQ ID NO. 14 and SEQ ID NO. 15 respectively; or a gene having this function, which has an amino acid sequence homology of 30% or higher with the amino acid sequence encoded by the above gene.

Preferably, the over-expression endogenous malic enzyme ME1 is derived from rhodosporidium toruloides host, and the gene sequence for encoding the above enzyme is shown as SEQ ID NO. 16; or a malic enzyme gene of other biological origin having a homology of 30% or higher with the amino acid sequence encoded by the above gene.

Further, the KCR, HCD and ECR enzyme genes and the malic enzyme ME1 gene in the over-expression FAE pathway are endogenous genes of rhodosporidium toruloides.

Wherein the promoter used for expressing or over-expressing the coding genes of the enzymes is a constitutive promoter P _TEF 、P _GPD And P _LDP The nucleotide sequences are respectively shown as SEQ ID NO.17, SEQ ID NO. 18 and SEQ ID NO. 19.

Preferably, the overexpression of the endogenous gene employs P _LDP Expressing a promoter; the exogenous genes AtKCS and BnKCS adopt P _GPD Expressing a promoter; the exogenous genes ELOVL6 and LaKCS adopt P _TEF And (3) expressing a promoter.

Further, constitutive promoter P was used _TEF 、P _GPD And P _LDP The expression of each gene means a combination of these three promoters with any gene that is expressed or overexpressed.

Preferably, the overexpression or expression of the gene is mediated by agrobacterium, and the expression cassette is integrated into the host genome for stable expression.

The invention relates to a construction method of rhodosporidium toruloides engineering strain for high-yield nervonic acid, which comprises the following steps:

(1) Synthesizing heterologous genes of KCS enzymes in fatty acid elongase complexes with different substrate preference, extracting plasmids after transformation, and mediating the plasmids with the heterologous genes into rhodosporidium toruloides wild fungi;

(2) Synthesizing KCR, HCD and ECR enzymes and malic enzyme ME1 genes in the fatty acid elongase complex, extracting plasmids after transformation, and mediating the plasmids with KCR, HCD, ECR, ME genes into rhodosporidium toruloides constructed in the step (1).

The rhodosporidium toruloides engineering strain for producing the high-yield nervonic acid is applied to the fermentation production of oil containing the nervonic acid or the preparation of the nervonic acid.

The expressed or overexpressed enzymes of the invention include: exogenous 3-ketoacyl-CoA synthetases (KCS) having relative substrate preference for C16:0, C18:1, C20:1 and C22:1; 3-ketoacyl-CoA reductase (KCR), 3-hydroxyacyl-CoA dehydratase (HCD), and trans-2, 3-enol-CoA reductase (ECR) in the endogenous or exogenous fatty acid elongase complex; endogenous or exogenous Malic enzyme (ME 1).

The invention relates to a construction method of genetic engineering for producing nervonic acid in high yield, which comprises the following steps: overexpression of KCR, HCD and ECR potentiates endogenous FAE pathways; overexpression of ME1 enhances NADPH supply; four exogenous 3-ketoacyl-CoA synthetases (KCS) having relative substrate preference for acyl-CoA are expressed C16:0, C18:1, C20:1 and C22:1 to achieve a push-pull effect between each product and substrate. The invention realizes the great improvement of the nervonic acid by over-expressing KCR, HCD and ECR enzyme genes in FAE in rhodosporidium toruloides for the first time, especially the enhancement of the FAE pathway, and the enhancement of the nervonic acid is improved by more than 2 times.

The beneficial effects are that: compared with the prior art, the invention has the following advantages:

the invention takes rhodosporidium toruloides with high oil yield as chassis cells, and the KCR, HCD and ECR in FAE are over-expressed in oleaginous microorganisms for the first time to strengthen the FAE path; overexpression of the ME1 gene increases intracellular NADPH supply; and the purpose of high-yield nervonic acid of the recombinant strain is realized by combining the combined expression of all KCS.

The fermentation titer of the nervonic acid reaches 18.2g/L by adopting cheap and easily available raw materials such as glucose, glycerol, corn steep liquor and the like as a culture medium and carrying out fed-batch fermentation on the level of a 50L fermentation tank. When raw materials such as glucose, yeast powder and ammonium sulfate are used as a culture medium, the fermentation titer of the nervonic acid can reach 35.4g/L, and the level is the highest fermentation level in all the current literature reports.

Drawings

FIG. 1 shows fatty acid composition after shake flask fermentation of NA0315 strain;

FIG. 2 is a graph showing the time course of each parameter in a 5L tank fed-batch fermentation of NA0315 strain;

FIG. 3 is a graph showing the time course of each parameter in a 50L tank fed-batch fermentation (one) of NA0315 strain;

FIG. 4 is a plot of time course for each parameter in a 50L tank fed-batch fermentation (II) with NA0315 strain.

Detailed Description

The invention is further illustrated by the following examples. The following examples are illustrative, not limiting, and are not intended to limit the scope of the invention.

The methods of detection, mediation, etc. in the subsequent examples and the primers used are referred to below.

The titres, compositions and total fatty acid titres of each fatty acid in the examples were all measured by a gas chromatograph equipped with FID detector (agilent 7890A). Sample pretreatment: 1mL of the fermentation culture was centrifuged at 12000g/min for 1 minute, the supernatant was removed, and after washing the cells with deionized water three times, 2mL of methanol, 1mL of 1mg/mL of nonadecanoic acid (dissolved in toluene) and 0.2mL of acetyl chloride were added, after 60 minutes in a boiling water bath, cooled to room temperature, 3mL of 6% potassium carbonate solution was added, centrifuged at 5000g/min for 5 minutes, the upper organic phase was sucked up, and the cells were filtered through a 0.25 μm organic filter head, and 1. Mu.L of the cells were analyzed by gas chromatography. Chromatographic conditions: the chromatographic column is Agilent HP88 capillary column (100 m×0.25mm×0.20 μm), the sample inlet temperature is 250deg.C, the detector temperature is 270 deg.C, and the temperature program is: the initial temperature was maintained at 120℃for 1 minute, then increased to 175℃at a rate of 10℃per minute, maintained for 10 minutes, increased to 210℃at a rate of 5℃per minute, maintained for 5 minutes, increased to 230℃at a rate of 5℃per minute, and maintained for 7 minutes.

Biomass measurement: taking 1mL of bacterial liquid into a 1.5mL centrifuge tube which is dried to constant weight in advance, centrifuging for 1 minute under the condition of 12000g/min, removing supernatant, washing thalli with deionized water for three times, drying to constant weight in an oven at 80 ℃, weighing, and calculating biomass.

Glucose determination: after diluting the fermentation broth 100 times, 25. Mu.L was taken for detection by a biosensing analyzer (SBA 40-E, institute of biological sciences, shandong province).

Glycerol assay: high performance liquid chromatography is used, with specific reference to SN/T2544-2010.

Construction of plasmids: by primer design, all PCR fragments carry a 15-20bp arm homologous to the linearized plasmid, which are then ligated by means of a recombinase. And (3) transforming the recombinant plasmid into escherichia coli DH5 alpha, and carrying out bacterial liquid PCR and sequencing verification. The recombinant enzyme is purchased from Vazyme one-step cloning kit (C112/C115), and specific operation steps are shown in the specification of the product.

The culture medium used comprises:

LB, tryptone 10g/L, yeast powder 5g/L and NaCl 10g/L;

YPD, glucose 20g/L, peptone 20g/L, yeast powder 10g/L;

induction medium: k (K) ₂ HPO ₄ 1.74g/L，KH ₂ PO ₄ 1.36g/L，NaCl 1.74g/L，MgSO ₄ ·7H ₂ O 0.49g/L，CaCl2 0.08g/L，FeSO ₄ ·7H ₂ O 2.5mg/L，(NH ₄ ) ₂ SO ₄ 0.53g/L, 1.80g/L glucose, 7.81 g/L4-maleinate, 5.0g/L glycerol, 0.04g/L acetosyringone and 15g/L agar;

shake flask screening medium: glucose 100g/L; 10g/L corn steep liquor; 1.0g/L of ammonium sulfate; potassium dihydrogen phosphate 0.75g/L; 0.4g/L of magnesium sulfate heptahydrate; 0.3g/L of calcium chloride.

Shake flask screening culture conditions: and placing the mixture under the conditions of a shaking table at 30 ℃ and 200rpm, and measuring residual sugar by using a biological sensing analyzer (SBA-40E) in the culture process, so as to culture until the sugar consumption is complete.

Agrobacterium-mediated rhodosporidium toruloides: the correct plasmid will be verified and transformed by electroporation to Agrobacterium tumefaciens AGL1. Subsequently, agrobacterium harboring the desired plasmid and rhodosporidium toruloides were cultured in LB and YPD media, respectively, for 16-18 hours, and their OD600 was measured, followed by dilution with sterile water to 0.6 and 0.8, respectively. Adding 100 mu L of each diluted bacterial solution to an induction plate of an induction culture medium containing acetosyringone, culturing for 48-72 hours at 24 ℃, coating the co-culture on a YPD plate containing corresponding antibiotics (rhodosporidium toruloides can grow on YPD), culturing for 48 hours at 30 ℃, randomly picking 20-30 bacterial strains, inoculating the bacterial strains to a shake flask screening culture medium, placing the shake flask screening culture medium under the conditions of 30 ℃ and a shaking table at 200rpm, measuring residual sugar by adopting a biological sensing analyzer (SBA-40E) in the culturing process, culturing until sugar consumption is complete, and selecting the bacterial strain with the highest nervonic acid titer.

Agrobacterium AGL1 was purchased from Shanghai Di Biol Co, pZPK-P _PGK -HYG ^R -T _NOS -P _GPD -MCS-T _HSP Plasmid and rhodosporidium toruloides NP11 were presented as a group of subjects Zhao Zongbao by the institute of great company of China academy of sciences (Sun Wenyi. Research based on Agrobacterium-mediated genetic recombination System of rhodosporidium toruloides [ D)]2017.), the examples of the present invention used NP11 strain as the starting strain. pZPK-P _PGK -G418 ^R -T _NOS -P _TEF -MCS-T _HSP And pZPK-P _PGK -BLE ^R -T _NOS -P _LDP -MCS-T _HSP The plasmid is pZPK-P _PGK -HYG ^R -T _NOS -P _GPD -MCS-T _HSP Is a plasmid skeleton, and is modified. The nucleotide sequences of the Ble and G418 genes are respectively shown as SEQ ID NO. 20 and SEQ ID NO. 21, and are respectively synthesized on pUC57-KAN plasmid by biological company to respectively obtain the Ble containing synthetic genepUC57-KAN and pUC57-KAN containing synthetic gene G418. Specifically, ecoRI and EcoRV were used for the plasmid pZPK-P _PGK -HYG ^R -T _NOS -P _GPD -MCS-T _HSP Performing double enzyme digestion to linearize the enzyme; with pZPK-P _PGK -HYG ^R -T _NOS -P _GPD -MCS-T _HSP The plasmid is used as a template, and primers PGK-F and PGK-R1, and primers NOS-F1 and NOS-R1 are adopted to amplify PGK and NOS fragments respectively; amplifying the G418 fragment by using pUC57-KAN plasmid containing synthetic gene G418 as a template and adopting primers G418-F and G418-R; the TEF fragment (PTEF) was amplified using the Rhodotorula genome as a template and the primers TEF-F and TEF-R (SEQ ID NO. 17). The PGK, NOS, G and TEF fragments were then inserted into linearized plasmid pZPK-P using the above plasmid construction method using a recombinase kit _PGK -HYG ^R -T _NOS -P _GPD -MCS-T _HSP Construction of plasmid pZPK-P _PGK -G418 ^R -T _NOS -P _TEF -MCS-T _HSP 。

In the same way as above, a plasmid pZPK-P was constructed _PGK -BLE ^R -T _NOS -P _LDP -MCS-T _HSP . Specifically, ecoRI and EcoRV were used for the plasmid pZPK-P _PGK -HYG ^R -T _NOS -P _GPD -MCS-T _HSP Performing double enzyme digestion to linearize the enzyme; with pZPK-P _PGK -HYG ^R -T _NOS -P _GPD -MCS-T _HSP The plasmid is used as a template, and primers PGK-F and PGK-R2, and primers NOS-F2 and NOS-R2 are adopted to amplify PGK and NOS fragments respectively; using pUC57-KAN plasmid containing synthetic gene Ble as template, and adopting primers BLE-F and BLE-R to amplify Ble fragment; using rhodotorula genome as template, using primer LDP-F and LDP-R to amplify LDP fragment (PLDP) (SEQ ID NO. 19); then adopting the plasmid construction method to construct plasmid pZPK-P _PGK -BLE ^R -T _NOS -P _LDP -MCS-T _HSP 。

The primers used in the examples are shown in the following table:

example 1

Establishment of nervonic acid synthesis pathway

Heterologous genes ELOVL6 (SEQ ID NO: 1), MACLE1 (SEQ ID NO: 2), tnKCS (SEQ ID NO: 3), atKCS (SEQ ID NO: 4), craKCS (SEQ ID NO: 5), moKCS (SEQ ID NO: 6), bnKCS (SEQ ID NO: 7), laKCS (SEQ ID NO: 8) and CgKCS (SEQ ID NO: 9) were synthesized by the biological company onto pUC57-KAN plasmids, respectively, which were synthesized by the biological company to obtain pUC57-KAN containing the synthetic gene ELOVL6, pUC57-KAN containing the synthetic gene MACLE1, pUC57-KAN containing the synthetic gene AtKCS, pUC57-KAN containing the synthetic gene TnKCS, pUC57-KAN containing the synthetic gene CraKCS, pUC57-KAN containing the synthetic gene BnKCS, pUC57-KAN containing the synthetic gene LaKCS, pUC57-KAN containing the synthetic gene CgKAS, pUC57-KAN containing the synthetic gene.

The pUC57-KAN containing the synthetic gene AtKCS is used as a template, the AtKCS-F and the AtKCS-R are used as primers to amplify the AtKCS fragment (SEQ ID NO: 4), run nucleic acid gel, and gel and recover. Plasmid pZPK-P using EcoRV and NcoI endonuclease pairs _PGK -HYG ^R -T _NOS -P _GPD -MCS-T _HSP Double enzyme cutting and nucleic acid gel running are carried out, and gel recovery is carried out. Then, the fragments are inserted into linearized plasmids by adopting a recombinase kit, then the linearized plasmids are transformed into DH5 alpha competent cells, transformants are selected after the culture, the plasmids are extracted after bacterial liquid PCR verifies that the bands are correct, and the plasmids are sent to a sequencing company for sequencing. Thereby successfully constructing pZPK-P _PGK -HYG ^R -T _NOS -P _GPD -AtKCS-T _HSP . In the same way, plasmid pZPK-P was constructed _PGK -HYG ^R -T _NOS -P _GPD -BnKCS-T _HSP 。

pZPK-P amplification Using primers ConExp-F1 and ConExp-R1 _PGK -HYG ^R -T _NOS -P _GPD -AtKCS-T _HSP P on plasmid _GPD -AtKCS-T _HSP Expression cassette, and the expression cassette is inserted into the XbaI single enzyme digestion linearization pZPK-P by adopting a recombinase kit _PGK -HYG ^R -T _NOS -P _GPD -BnKCS-T _HSP Thereby constructing the coexpression plasmid pZPK-P _PGK -HYG ^R -T _NOS -P _GPD -AtKCS-T _HSP -P _GPD -BnKCS-T _HSP . Then the same primers ConExp-F1 and ConExp-R1 are adopted to amplify the corresponding expression cassette, and the expression cassette is inserted into the corresponding plasmid which is linearized by single digestion of XbaI, thereby constructing the coexpression plasmid pZPK-P _PGK -HYG ^R -T _NOS -P _GPD -TnKCS-T _HSP -P _GPD -CraKCS-T _HSP And pZPK-P _PGK -HYG ^R -T _NOS -P _GPD -AtKCS-T _HSP -P _GPD -MoKCS-T _HSP 。

By adopting the same method, plasmid pZPK-P is constructed _PGK -G418 ^R -T _NOS -P _TEF -ELOVL6-T _HSP And pZPK-P _PGK -G418 ^R -T _NOS -P _TEF -LaKCS-T _HSP . pZPK-P amplification Using primers ConExp-F2 and ConExp-R2 _PGK -G418 ^R -T _NOS -P _TEF -ELOVL6-T _HSP Amplification of P on plasmid _TEF -ELOVL6-T _HSP Expression cassette inserted into KpnI single enzyme digestion linearization pZPK-P _PGK -G418 ^R -T _NOS -P _TEF -LaKCS-T _HSP Thereby constructing the coexpression plasmid pZPK-P _PGK -G418 ^R -T _NOS -P _TEF -ELOVL6-T _HSP -P _TEF -LaKCS-T _HSP . Then the same primers ConExp-F2 and ConExp-R2 are adopted to amplify the corresponding expression cassette, and the expression cassette is inserted into the corresponding plasmid which is linearized by KpnI single enzyme digestion, thereby constructing a coexpression plasmid, pZPK-P _PGK -G418 ^R -T _NOS -P _TEF -MACLE1-T _HSP -P _TEF -CgKCS-T _HSP 。

Will carry the plasmid pZPK-P _PGK -HYG ^R -T _NOS -P _GPD -AtKCS-T _HSP -P _GPD -BnKCS-T _HSP 、pZPK-P _PGK -HYG ^R -T _NOS -P _GPD -TnKCS-T _HSP -P _GPD -CraKCS-T _HSP And pZPK-P _PGK -HYG ^R -T _NOS -P _GPD -AtKCS-T _HSP -P _GPD -MoKCS-T _HSP After the agrobacterium mediating rhodosporidium yeast wild strain, strains NA0101, NA0129 and NA0142 are obtained respectively.

Shake flask fermentation was performed under the above shake flask selection medium and culture conditions, and the fatty acid composition and the nervonic acid titer of each strain were determined by the above gas chromatography fatty acid detection method. Wherein, the titer of the nervonic acid of the NA0101 strain is the highest and is 0.35g/L; and the ratio of nervonic acid to erucic acid in the fatty acid composition is 2.7% and 6.2%, respectively. Subsequently by carrying the plasmid pZPK-P _PGK -G418 ^R -T _NOS -P _TEF -ELOVL6-T _HSP -P _TEF -LaKCS-T _HSP And pZPK-P _PGK -G418 ^R -T _NOS -P _TEF -MACLE1-T _HSP -P _TEF -CgKCS-T _HSP The agrobacterium-mediated strain NA0101, respectively obtained NA0210 and NA0228 strains after the measurement. Wherein, the titer of the nervonic acid of the NA0210 strain is the highest and is 2.4g/L; the fatty acid composition was 19.2% and 3.5% for nervonic acid and erucic acid, respectively.

Example 2

Overexpression of malic enzyme

Extracting rhodosporidium toruloides genome by using a yeast genome extraction kit (Shanghai) and (B518227), using the genome as a template, amplifying an ME1 fragment (SEQ ID NO: 16) by using primers ME-F and ME-R, and recovering the gel after nucleic acid gel running. Plasmid pZPK-P using EcoRV and NcoI endonucleases _PGK -BLE ^R -T _NOS -P _LDP -MCS-T _HSP Double enzyme cutting and nucleic acid gel running are carried out, and gel recovery is carried out. Then, the fragments are inserted into linearized plasmids by using a recombinase kit, then the linearized plasmids are transformed into DH5 alpha competent cells, transformants are selected, bacterial liquid PCR is performed to verify that the bands are correct, the plasmids are extracted, and the plasmids are sent to a sequencing company for sequencing. Thus, the pZPK-P was successfully constructed _PGK -BLE ^R -T _NOS -P _LDP -RtME1-T _HSP A plasmid. After electrotransformation of this plasmid into Agrobacterium AGL1, rhodosporidium toruloides NA0210 of example 1 was mediated in co-induction medium, resulting in NA0337 strain. And control bacteriaCompared with the NA0210 strain, the total grease of the NA0347 strain is increased from 11.0g/L to 14.5g/L, and the total grease is improved by 31.8%. And the fatty acid composition has no obvious change, and the final nervonic acid titer of NA0347 is 3.3g/L.

Example 3

Enhancement of endogenous FAE pathway

Extracting rhodosporidium toruloides genome by using a yeast genome extraction kit (Shanghai) and (B518227), using the genome as a template, amplifying a KCR fragment (RtKCR) (SEQ ID NO: 10) by using primers KCR-F and KCR-R, and performing gel recovery after nucleic acid gel running. Plasmid pZPK-P using EcoRV and NcoI endonucleases _PGK -BLE ^R -T _NOS -P _LDP -MCS-T _HSP Double enzyme cutting and nucleic acid gel running are carried out, and gel recovery is carried out. Then, the fragments are inserted into linearized plasmids by using a recombinase kit, then the linearized plasmids are transformed into DH5 alpha competent cells, transformants are selected, bacterial liquid PCR is performed to verify that the bands are correct, the plasmids are extracted, and the plasmids are sent to a sequencing company for sequencing. Thus, the pZPK-P was successfully constructed _PGK -BLE ^R -T _NOS -P _LDP -RtKCR-T _HSP A plasmid. Subsequently, ECR fragment (RtECR) (SEQ ID NO: 12) was amplified using primers ECR-F and ECR-R, HCD fragment (RtHCD) (SEQ ID NO: 11) was amplified using primers HCD-F and HCD-R, and pZPK-P was constructed separately in the same manner _PGK -BLE ^R -T _NOS -P _LDP -RtECR-T _HSP 、pZPK-P _PGK -BLE ^R -T _NOS -P _LDP -RtHCD-T _HSP . Subsequently pZPK-P was amplified using the primers ConExp-F2 and ConExp-R3 _PGK -BLE ^R -T _NOS -P _LDP -RtECR-T _HSP P on plasmid _LDP -RtECR-T _HSp The expression cassette is inserted into KpnI single enzyme digestion linearization pZPK-P by adopting a recombinase kit _PGK -BLE ^R -T _NOS -P _LDP -RtKCR-T _HSP Thereby constructing the coexpression plasmid pZPK-P _PGK -BLE ^R -T _NOS -P _LDP -RtECR-T _HSP -P _LDP -RtKCR-T _HSP . Subsequently pZPK-P was amplified using the same primers ConExp-F2 and ConExp-R3 _PGK -BLE ^R -T _NOS -P _LDP -RtHCD-T _HSP Corresponding P on plasmid _LDP -RtHCD-T _HSP Expression cassette inserted into KpnI single enzyme digestion linearization pZPK-P _PGK -BLE ^R -T _NOS -P _LDP -RtECR-T _HSP -P _LDP -RtKCR-T _HSPP On the plasmid, thereby constructing the coexpression plasmid pZPK-P _PGK -BLE ^R -T _NOS -P _LDP -RtHCD-T _HSP -P _LDP -RtECR-T _HSP -P _LDP -RtKCR-T _HSP . Further, the pZPK-P was amplified using the primers ConExp-F2 and ConExp-R3 _PGK -BLE ^R -T _NOS -P _LDP -RtME1-T _HSP P on plasmid _LDP -RtME1-T _HSP Expression cassette inserted into KpnI single enzyme digestion linearization pZPK-P _PGK -BLE ^R -T _NOS -P _LDP -RtHCD-T _HSP -P _LDP -RtECR-T _HSP -P _LDP -RtKCR-T _HSP Construction of Co-expression plasmid pZPK-P _PGK -BLE ^R -T _NOS -P _LDP -RtME-T _HSP -P _LDP -RtHCD-T _HSP -P _LDP -RtECR-T _HSP -P _LDP -RtKCR-T _HSP 。

FAE pathway-enhancing plasmid pZPK-P _PGK -BLE ^R -T _NOS -P _LDP -RtHCD-T _HSP -P _LDP -RtECR-T _HSP -P _LDP -RtKCR-T _HSP After electrotransformation to agrobacterium AGL1, rhodosporidium toruloides NA0210 is mediated in a co-induction culture medium, and finally the NA0354 strain is obtained, wherein in the fatty acid composition, the nervonic acid accounts for 42%, the erucic acid accounts for only about 4%, and the nervonic acid titer reaches 4.3g/L. Co-expression of the plasmid pZPK-P by FAE and ME1 _PGK -BLE ^R -T _NOS -P _LDP -RtME-T _HSP -P _LDP -RtHCD-T _HSP -P _LDP -RtECR-T _HSP -P _LDP -RtKCR-T _HSP After electrotransformation to Agrobacterium AGL1, rhodosporidium toruloides NA0210 was transduced in co-induction medium, and NA0315 strain was finally obtained. As shown in FIG. 1, after shaking flask fermentation, the NA0315 strain has a fatty acid composition with a nervonic acid content of 41% and an erucic acid content of about 4%, and is usefulThe acid titer reaches 5.3g/L.

Example 4

Example 4 the same construction procedure was used as in example 3, except that: KCR, HCD and ECR enzymes in the over-expressed fatty acid elongase complex are AtKCR, atHCD and AtECR from Arabidopsis thaliana, and the gene sequences for encoding the above three enzymes are SEQ ID NO. 13, SEQ ID NO. 14 and SEQ ID NO. 15 respectively.

Example 5

NA0315 strain 5L fermentation tank batch fed-batch fermentation

Seed liquid culture medium: glucose 60g/L; 10g/L corn steep liquor; 1.0g/L of ammonium sulfate; potassium dihydrogen phosphate 0.75g/L; 0.4g/L of magnesium sulfate heptahydrate; 0.3g/L of calcium chloride.

Fermentation initial medium: glucose 100g/L; corn steep liquor 40g/L; ammonium sulfate 4.0g/L; potassium dihydrogen phosphate 0.75g/L; 0.4g/L of magnesium sulfate heptahydrate; 0.3g/L of calcium chloride.

Seed liquid preparation: NA0315 strain was activated on YPD plates, cultured at 30℃for 48 hours, inoculated into 500mL Erlenmeyer flasks containing 100mL of seed solution, and cultured at 30℃for 60 hours, and used for fermenter inoculation.

The fermentation process comprises the following steps: the initial liquid loading amount of the 5L fermentation tank is 2.25L of fermentation initial culture medium; the inoculation amount is 0.25L; ventilation is 1VVM; the temperature is 30 ℃; the dissolved oxygen DO is coupled with the rotating speed, sectional control is adopted, the dissolved oxygen is controlled at 45% level when the fermentation starts to the glucose is exhausted, and then the dissolved oxygen is controlled at 5% level when the fermentation ends; sampling about 10mL every 8 hours, and measuring glucose (or glycerol) concentration and fermentation titer of biomass, grease and nervonic acid; when the glucose in the initial culture medium is exhausted, 200g of glycerol is added, when the glycerol concentration is lower than 20g/L, 200g of glycerol is added again, and the glycerol feeding is performed three times until the glycerol concentration is close to 0g/L, and the fermentation is finished.

The fermentation time course curve is shown in FIG. 2, and after 96 hours of fermentation, the biomass reaches 84g/L, and the total lipid and nervonic acid titers reach 46.1g/L and 16.3g/L, respectively. And the fatty acid composition of the grease is consistent with the shake flask fermentation result, and the proportions of the nervonic acid and the erucic acid are about 36 percent and 3.5 percent respectively.

Example 6

NA0315 strain 50L fermentation tank pilot scale-up (I)

1. The seed medium and the fermentation initiation medium were the same as in example 5.

The fermentation process comprises the following steps: the initial liquid loading amount of the 50L fermentation tank is 30L, and the inoculation amount is 0.9L seed liquid; ventilation is 1VVM; the temperature is 30 ℃; the initial stirring speed is 300rpm, and when the glucose is exhausted, the dissolved oxygen is coupled with the speed to control DO at about 5%; sampling about 10mL every 8 hours, and measuring glucose (or glycerol) concentration and fermentation titer of biomass, grease and nervonic acid; when the glucose in the initial culture medium is exhausted, 2Kg of glycerol is added, when the glycerol concentration is lower than 20g/L, 2Kg of glycerol is added again, and the glycerol feeding is performed three times until the glycerol concentration is close to 0g/L, and the fermentation is finished. The fermentation time course curve is shown in FIG. 3, the final biomass reached 87g/L, and the nervonic acid titer reached 45.2g/L and 18.2g/L, respectively. And the fatty acid composition of the grease is consistent with the shake flask fermentation result, and the proportions of the nervonic acid and the erucic acid are about 40% and 3.7%, respectively.

Example 7

NA0315 strain 50L fermentation tank pilot scale-up (II)

Seed liquid culture medium: glucose 60g/L; yeast powder 10g/L; ammonium sulfate 10g/L; potassium dihydrogen phosphate 0.75g/L; 0.4g/L of magnesium sulfate heptahydrate; 0.3g/L of calcium chloride.

Fermentation initial medium: glucose 100g/L; 30g/L yeast powder; 30.0g/L of ammonium sulfate; 3.0g/L of monopotassium phosphate; 1.5g/L of magnesium sulfate heptahydrate; calcium chloride 1.2g/L.

Seed liquid preparation: NA0315 strain was activated on YPD plates, cultured at 30℃for 48 hours, inoculated into 500mL Erlenmeyer flasks containing 100mL of seed solution, and cultured at 30℃for 60 hours, and used for fermenter inoculation.

The fermentation process comprises the following steps: the initial liquid loading amount of the 50L fermentation tank is 30L, and the inoculation amount is 0.9L seed liquid; ventilation is 1VVM; the temperature is 30 ℃; the initial stirring speed is 300rpm, and when the glucose is exhausted, the dissolved oxygen is coupled with the speed to control DO at about 5%; sampling about 10mL every 8 hours, and measuring the glucose concentration and the fermentation titer of biomass, grease and nervonic acid; when the glucose in the initial culture medium is exhausted, 800g/L glucose is added for 3L, when the glucose concentration is lower than 10g/L, 3L glucose is added again, the glucose feeding is performed three times, and when the glucose concentration is close to 0g/L, the fermentation is finished. The fermentation time course curve is shown in FIG. 4, the final biomass reaches 110g/L, the titers of the grease and the nervonic acid reach 73.7g/L and 35.4g/L respectively, and the percentages of the nervonic acid and the erucic acid in the grease are about 48% and 2% respectively.

Claims (9)

1. The rhodosporidium toruloides engineering strain is characterized in that the engineering strain takes rhodosporidium toruloides as an original strain and heterologously expresses KCS enzymes in fatty acid elongase complexes with different substrate preference; the KCR, HCD and ECR enzymes in the fatty acid elongase complex are overexpressed, and the malate enzyme ME1.

2. The engineered strain of rhodosporidium toruloides of claim 1, wherein the KCS enzyme in the heterologous expression of different substrate preference fatty acid elongase complex comprises one or more of exogenous KCS enzyme ELOVL6 or MALCE1 having substrate preference for c16:0 acyl-coa, exogenous KCS enzyme TnKCS or atccs having relative substrate preference for c18:1 acyl-coa, exogenous KCS enzyme CraKCS or MoKCS or BnKCS having relative substrate preference for c20:1 acyl-coa, exogenous KCS enzyme LaKCS or CgKCS having relative substrate preference for c22:1 acyl-coa, the sequence of the encoding gene of each of which is shown in SEQ ID NOs 1-9, respectively.

3. The rhodosporidium toruloides engineered strain of claim 2 wherein KCS enzymes in the heterologous expression of fatty acid elongase complexes of different substrate preference comprise AtKCS, bnKCS, ELOVL and LaKCS.

4. The rhodosporidium toruloides engineering strain with high yield of nervonic acid according to claim 1, wherein KCR, HCD and ECR enzymes in the over-expressed fatty acid elongase complex are preferably derived from rhodosporidium toruloides host, and the sequences of genes for encoding the above three enzymes are respectively shown as SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12, or genes with the same or higher than 30% of amino acid sequence homology with other organisms encoded by the above genes.

5. The rhodosporidium toruloides engineering strain with high yield of nervonic acid according to claim 1, wherein KCR, HCD and ECR enzymes in the over-expressed fatty acid elongase complex are AtKCR, atHCD and AtECR from arabidopsis thaliana, and the gene sequences for encoding the above three enzymes are SEQ ID NO 13, SEQ ID NO 14 and SEQ ID NO 15 respectively; or a gene having this function, which has an amino acid sequence homology of 30% or higher with the amino acid sequence encoded by the above gene.

6. The rhodosporidium toruloides engineering strain with high yield of nervonic acid according to claim 1, wherein the overexpression endogenous malic enzyme ME1 is derived from rhodosporidium toruloides host, and the gene sequence for encoding the enzyme is shown as SEQ ID NO. 16; or a malic enzyme gene of other biological origin having a homology of 30% or higher with the amino acid sequence encoded by the above gene.

7. The rhodosporidium toruloides engineering strain with high yield of nervonic acid according to claim 1, wherein the promoters adopted for expressing or over-expressing the coding genes of the enzymes are constitutive promoters PTEF, PGPD and PLDP, and the nucleotide sequences of the promoters are shown as SEQ ID NO.17, SEQ ID NO. 18 and SEQ ID NO.19 respectively.

8. A method for constructing a rhodosporidium toruloides engineering strain with high yield of nervonic acid as claimed in claim 1, which is characterized by comprising the following steps:

(1) Synthesizing heterologous genes of KCS enzymes in fatty acid elongase complexes with different substrate preference, extracting plasmids after transformation, and mediating the plasmids with the heterologous genes into rhodosporidium toruloides wild fungi;

(2) Synthesizing KCR, HCD and ECR enzymes and malic enzyme ME1 genes in the fatty acid elongase complex, extracting plasmids after transformation, and mediating the plasmids with KCR, HCD, ECR, ME genes into rhodosporidium toruloides constructed in the step (1).

9. An application of the rhodosporidium toruloides engineering strain for producing the nervonic acid with high yield in the fermentation production of oil containing the nervonic acid or the preparation of the nervonic acid.

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