Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
LB medium, yeast powder 5 g/L, tryptone 10 g/L and NaCl 5 g/L.
YPD medium, yeast powder 10 g/L, peptone 20 g/L, glucose 20 g/L.
SC medium, yeast nitrogen source base (Yeast Nitrogen base, YNB) 1.7: 1.7 g/L, ammonium sulfate (NH4)2SO4, 5: 5 g/L, amino acid mixture 1.8: 1.8 g/L, glucose 20: 20 g/L.
The composition of the amino acid mixture was 0.5g adenine (Adenine), 2.0g alanine (Alanine), 2.0g arginine (Argnine), 2.0g asparagine (ASPARAGINE), 2.0g aspartic acid (ASPARTIC ACID), 2.0g Cysteine (Cysteine), 2.0g glutamine (Glutamine), 2.0g glutamic acid (Glutamic acid), 2.0g Glycine (Glycine), 2.0g Histidine (HISTIDINE), 2.0g inositol (Inositol), 2.0g isoleucine (Isoleucine), 4.0g leucine (Leucine), 2.0g Lysine (Lysine), 2.0g methionine (Methionine), 2.0g aminobenzoic acid (para-Aminobenzoic acid), 0.2g Phenylalanine (PHENYLALANINE), 2.0g Proline (Proline), 2.0g serine (Serine), 2.0g threonine (Threonine), 2.0g tryptophan (Tryptophan), 2.0g Tyrosine (Valine) 2.0g Valine (Valine) 2.0g, and Valine (Valine) 2.0.67.
SC-URA- culture medium, based on SC culture medium, without uracil.
SC-URA--LEU- culture medium, based on SC culture medium, without adding uracil and leucine.
The corresponding solid medium can be obtained by adding 18g/L agarose into the culture medium.
SC culture medium is selected as shake flask fermentation medium, and the concentration of glucose is adjusted to 40g/L.
YPD-5FOA solid Medium 0.5 g/L of 5-FOA (5-fluoroorotic acid) was added to the YPD solid medium.
The method for shake flask fermentation culture comprises the following steps:
1) Streaking was performed on the SC solid medium plates, and culturing was performed at 30℃for 36 hours.
2) Culturing seed solution, adding 5ml SC liquid culture medium into 50ml sterile test tube, scraping lawn from the plate with 200 μl yellow gun head, blowing and mixing into liquid culture medium, and culturing at 30deg.C for 12 hr.
3) 30Ml of SC liquid medium was added to a 100ml baffle-less shake flask, the OD600 of the seed solution was measured, and the inoculation volume in the shake flask was calculated, with an initial OD600 of 0.1 in the shake flask.
4) After 48h of incubation 10% by volume (i.e. 3 ml) of the organic phase dodecane was added.
5) Culturing for 144h.
The testing method comprises the following steps:
1) And (3) detecting OD600, namely diluting the fermentation liquor with deionized water, measuring an absorbance value (600 nm) by using an ultraviolet spectrophotometer, and ensuring that the range of the diluted OD600 is within 0.2-0.8, wherein the measured value is multiplied by the dilution multiple to obtain the OD600.
2) Detection of Homogentisate (HGA) to determine HGA concentration, the yeast strain was cultured in SC medium and the supernatant of the fermentation broth was sampled. After centrifugation of the fermentation broth, 1mL supernatant was collected, mixed with 40. Mu.L of glacial acetic acid, and the impurities were removed by passing through a membrane (aqueous filter membrane with a pore size of 0.22 μm) by means of a syringe for HPLC analysis. HGAs were detected by HPLC (Sieimer-Fei, USA) equipped with a C18-H column (4.6X1250 mm, 5 μm, kromasil 100-5-C18 (W), sweden) using a UV/VIS detector at 290 nm. The sample was eluted with a solution (A) of 0.01M KH2PO4 and methanol (B) at a flow rate of 0.8 mL/min in a ratio of 90%/10% at 40 ℃.
3) Detection of delta-tocotrienol to determine delta-tocotrienol, the organic phase in the fermentation broth was collected by centrifugation and 1ml of the organic phase was collected and purified by filtration through a syringe (filter membrane of the organic phase having a pore size of 0.22 μm).
Detection was performed using GC-MS with the device GCMS-QP2010SE (shimadzu, japan). The column was 5% phenyl-95% polydimethylilexane, 30m X0.25 mm, 0.25 μm thick. High purity helium (99.999%) was used as carrier gas at a constant flow rate of 1.0 mL/min. The gas chromatograph oven temperature was initially set to 60 ℃ and then programmed to 300 ℃ at a rate of 40 ℃/min for 11 minutes. The total run time was 17 minutes (including a 6 minute solvent delay time). 1 mu L of sample solution is injected by adopting a non-split sample injection mode, and the temperature of a sample injection port is constantly maintained at 300 ℃.
Yarrowia lipolytica (Yarrowia lipolytica) Po1f, available from American type culture Collection under the accession number ATCC MYA-2613.
Example 1
1) The strain ATCC MYA-2613 delta Ku70-URA is named YL01 by taking yarrowia lipolytica ATCC MYA-2613 as a starting strain and knocking out the endogenous Ku70 gene in order to improve the homologous recombination efficiency of the yarrowia lipolytica.
The Ku70 gene is knocked out by using a homologous recombination method, and the specific method is as follows:
(1) The method comprises the steps of taking yarrowia lipolytica ATCC MYA-2613 genome DNA as a template, amplifying Ku70-up (shown as SEQ ID NO. 1) by using a primer Ku 70-up-F/Ku 70-up-R to obtain a Ku70-up fragment, and amplifying Ku70-down (shown as SEQ ID NO. 2) by using a primer Ku 70-down-F/Ku 70-down-R to obtain a Ku70-down fragment, namely obtaining upstream and downstream homology arms of the knocked-out Ku 70.
The specific primer sequences are as follows:
Ku70-up-F:CGTTGCGCTTGGGCTTGGGGCACTTCTGC;SEQ ID NO.3。
Ku70-up-R:cgttttacaacGTTCGTGGTTCGTGTTTCGTGTTCGT;SEQ ID NO.4。
Ku70-down-F:CTACGGCTACCTGCTGCTTCCAAACGATATGAGGATGAGT;SEQ ID NO.5。
Ku70-down-R:CAAATGCCTGATGGTGTGCCAGGAGGTGGAC;SEQ ID NO.6。
The gene amplification system was PrimestarMax (Takara). Mu.L, 2. Mu.L of each of the upstream and downstream primers, 1. Mu.L of the template, and 20. Mu.L of ddH2 O.
The gene amplification procedure was 98℃for 3 minutes, 98℃for 10 seconds, 57℃for 30 seconds, 72℃for 10 s/kb,35 cycles, and 72℃for 10 minutes.
And (3) carrying out agarose gel electrophoresis and product recovery on the PCR product to obtain gene fragments Ku70-up and Ku70-down.
(2) The screening mark used for homologous recombination is 3HA-URA-3HA, the 3HA-URA-3HA gene sequence synthesized by Huada gene (shown as SEQ ID NO. 7) is used as a template, and a primer Ku 70-URA-F/Ku 70-URA-R is used for amplifying the 3HA-URA-3HA to obtain a screening mark 3HA-URA-3HA fragment 1 of the knocked-out/integrated plasmid.
The primer sequences were as follows:
Ku70-URA-F:CGAACCACGAACgttgtaaaacgacggccagtcgaacc;SEQ ID NO.8。
Ku70-URA-R:GAAGCAGCAGGTAGCCGTAGGTCTCGTACTGCTTGAC;SEQ ID NO.9。
(3) The plasmid vector p-kana (shown as SEQ ID NO. 10) was amplified using Ku70-VEC-F/Ku70-VEC-R to obtain a p-kana vector fragment.
The primer sequences were as follows:
Ku70-VEC-F:TGGCACACCATCAGGCATTTGAGAAGCACACGGTCAC;SEQ ID NO.11。
Ku70-VEC-R:CAAGCCCAAGCGCAACGCAATTAATGTAAGTTAGCTCACTC;SEQ ID NO.12。
(4) The amplified products were added to PCR tubes at a ligation temperature of 50℃for a ligation time of 15min, and a total system of 20. Mu.L, 2. Mu.L of the Ku70-up fragment, 2. Mu.L of the Ku70-down fragment, 3. Mu.L of the 3HA-URA-3HA fragment 1, 3. Mu. L p-kana vector fragment and 10. Mu.L of Gibson ligase.
Gibson-ligated 20. Mu.l ligation was transformed into E.coli Trans10 commercial competence (Beijing full gold Biotechnology Co., ltd.) for cultivation. The transformation procedure was strictly followed by 37℃culture of 1 h followed by plating onto LB plates (containing 50. Mu.g/mL kanamycin), followed by 37℃culture of 12: 12 h, and selection of 10-20 single colonies for colony PCR amplification and DNA sequencing verification. The colony PCR amplification and DNA sequencing primer is Ku70-up-F/Ku70-down-R.
A correct single colony was selected and designated as E.coli EC001, and the plasmid was designated as pkana-Ku70, and the plasmid was extracted by propagation to obtain pkana-Ku70 plasmid. The pkana-Ku70 plasmid is used as a template, ku70 linearization-F/R is used as a primer, and a linearization integration fragment Ku70up-3HA-URA-3HA-Ku70down is amplified.
The primer sequences were as follows:
ku70 linearization-F TTGGGCTTGGGGCACTTCTGC, SEQ ID NO.13.
Ku70 linearization-R: ATGGTGTGCCAGGAGGTGGAC, SEQ ID NO.14.
The linearized integrated fragment Ku70up-3HA-URA-3HA-Ku70down was transformed into the strain yarrowia lipolytica ATCC MYA-2613 by means of chemical transformation.
The transformation method of yarrowia lipolytica ATCC MYA-2613 is as follows:
Yarrowia lipolytica kit transformation using a Frozen-EZ Yeast Transformation II Kit.
The plates were picked and grown overnight in 10mL YPD medium to OD600 =0.5-0.7 (not too high) and 500 μl of inoculum was required for each competence. Taking 1mL as an example, two competence were made, the steps were as follows:
① 1mL of the bacterial liquid is centrifuged at 5000rpm for 3min, and the supernatant is discarded (sucked clean as much as possible).
② An equal volume (1 mL) Bhffer S of 1 was added and mixed well, centrifuged at 5000rpm for 3min, and the supernatant was discarded.
③ 100. Mu.L Buffer S2 was added per 1mL of bacterial solution and gently mixed and dispensed into two clean centrifuge tubes (50. Mu.L each) for immediate use or frozen to-80℃for later use.
④ Plasmid or linearized DNA fragments (the addition volume should be less than 5. Mu.L, DNA addition is about 1. Mu.g) were added to competent cells.
⑤ 500. Mu.L Buffer S3 was added to each competence and resuscitated at 30℃for 1h. Each piece of competent coating was coated with two plates, each directly with 250. Mu.L. The coated plate was SC-URA-.
Verification was performed after incubation at 30 ℃ for 36 h.
The single colony of the gene knockout strain, which is obtained by picking a plurality of the plates, is verified by using a verification primer Ku70-F/R to verify the KU70 gene, if the corresponding band can be amplified to prove that the knockout is successful, if the corresponding band can not be amplified to prove that the knockout is successful, the verification primer is as follows:
Ku70-F:TGCTGGAAATCGAGGACTACAAGG;SEQ ID NO.15。
Ku70-R:CAACCCAGTCCTTCTTCAACTTGCTA;SEQ ID NO.16。
the strain which is verified to be correct is inoculated in YPD medium, cultured for 12h, and the glycerol tube is preserved to obtain the strain ATCC MYA-2613 DeltaKu 70-URA, which is named YL01.
(5) Recovering the screening marker to obtain the strain ATCC MYA-2613 delta Ku70-URA-:
Strain YL01 was inoculated into YPD medium and cultured for 12 h, after the bacterial liquid became turbid, the strain was spread on YPD-5FOA solid medium, and since uracil-containing strain could not survive on YPD-5FOA solid medium, the colony grown on YPD-5FOA solid medium was strain ATCC MYA-2613 Δku70-URA- with loss of URA, designated YL02.
2) Starting from strain ATCC MYA-2613 DeltaKu 70-URA- (YL 02), three exogenous genes are required for the synthesis of delta-tocotrienol, namely the gene encoding 4-hydroxyphenylpyruvate dioxygenase (4-hydroxyphenylpyruvate dioxygenase), the gene encoding homogentisate phytyltransferase (Homogentisate phytyltransferase) and the gene encoding tocopherol cyclase (Tocopherol cyclase).
(1) The gene encoding 4-hydroxyphenylpyruvate dioxygenase is selected from the gene HPD (shown as SEQ ID NO. 17) encoding 4-hydroxyphenylpyruvate dioxygenase which is derived from Pseudomonas putida (Pseudomonas putida) KT2440 and is codon optimized.
(2) The gene encoding homogentisate phytyltransferase is selected from the codon optimized gene HPT encoding homogentisate phytyltransferase (as shown in SEQ ID NO. 18) derived from the genus Synechocystis PCC 6803.
(3) The gene encoding tocopherol cyclase is selected from the group consisting of a codon-optimized gene TC encoding tocopherol cyclase derived from Arabidopsis thaliana (Arabidopsis thaliana) (as shown in SEQ ID NO. 19).
In order to integrate the three exogenous genes described above into strain YL02, the following plasmids need to be constructed:
Firstly, constructing a plasmid pintE-HPD-HPT, wherein the vector skeleton is pintE-TEFin-HPT-xpr 2-EXP-HPD-lip2-3HA-URA-3HA.
The construction method comprises the following steps:
Plasmid pCfB4778 is used as an amplification template, pCfB4778 is purchased from EasyCloneYALI Collection (Kit #1000000140, 1000000141), the plasmid skeleton p-intE1vec is amplified, and the amplification primer is intE1vec-F/R. The primer sequences were as follows:
intE1vec-F:CTACGGCTACccgagcgtcgacaagcatacagc;SEQ ID NO.20。
intE1vec-R:GTGAGTGAATTGagcactatcctctgctgcgtc;SEQ ID NO.21。
the TEFin promoter synthesized by Huada gene (shown as SEQ ID NO. 22) is used as a template, and a primer TEFin-F1/TEFin-R1 is used for amplifying TEFin promoter to obtain TEFin promoter fragment 1.
The primer sequences were as follows:
TEFin-F1:gatagtgctCAATTCACTCACTCTCCCGACTATCC;SEQ ID NO.23。
TEFin-R1:gatggtggcctgcggttagtactgcaaaaagtgc;SEQ ID NO.24。
The xpr2 terminator was amplified using primers xpr 2-F1/xpr 2-R1 using yarrowia lipolytica ATCC MYA-2613 genomic DNA as template (as shown in SEQ ID NO. 25) to obtain xpr2 terminator fragment 1.
The primer sequences were as follows:
xpr2-F1:ccatcttctaaGATCCAACTACGGAACTTGTGTTGATGTCTTTG;SEQ ID NO.26。
xpr2-R1:CCAAACTCGACACGGGCATCTCACTTGCATATG;SEQ ID NO.27。
EXP promoter fragment 1 was obtained by amplifying the EXP promoter (shown as SEQ ID NO. 28) using the yarrowia lipolytica ATCC MYA-2613 genomic DNA as a template and the primers EXP-F1/EXP-R1.
The primer sequences were as follows:
EXP-F1:GATGCCCGTGTCGAGTTTGGCGCCCGTTTTTTCG;SEQ ID NO.29。
EXP-R1:gatgtcggccatTGCTGTAGATATGTCTTGTGTGTAAGGGGG;SEQ ID NO.30。
The lip2 terminator was amplified using primers lip 2-F1/lip 2-R1 using yarrowia lipolytica ATCC MYA-2613 genomic DNA as template (as shown in SEQ ID NO. 31) to obtain lip2 terminator fragment 1.
The primer sequences were as follows:
lip2-F1:cgtgctgtctactgattaagctatttatcactctttacaacttctacctcaactatc;SEQ ID NO.32。
lip2-R1:tcgttttacaaccatttgccattcgtaacgctggtag;SEQ ID NO.33。
The HPT gene synthesized by Huada gene (shown as SEQ ID NO. 18) is used as a template, and the primer HPT-F/HPT-R is used for amplifying the HPT gene to obtain an HPT gene fragment.
The primer sequences were as follows:
HPT-F:tactaaccgcaggccaccatccaggccttctg;SEQ ID NO.34。
HPT-R:CCGTAGTTGGATCttagaagatggtgttagaaaaattaggcagccac;SEQ ID NO.35。
the HPD gene synthesized by the Huada gene (shown as SEQ ID NO. 17) is used as a template, and the primer HPD-F/HPD-R is used for amplifying the HPD gene to obtain an HPD gene fragment.
The primer sequences were as follows:
HPD-F:TCTACAGCAatggccgacatcttcgagaaccccat;SEQ ID NO.36。
HPD-R:tagcttaatcagtagacagcacgcctcgtc;SEQ ID NO.37。
The plasmid pkana-Ku70 constructed as described above was used as a template to amplify 3HA-URA-3HA using primers URA-F2/URA-R2 to obtain 3HA-URA-3HA fragment 2.
The primer sequences were as follows:
URA-F2:ggcaaatggttgtaaaacgacggccagtcgaacc;SEQ ID NO.38。
URA-R2:gacgctcggGTAGCCGTAGGTCTCGTACTGCTTGAC;SEQ ID NO.39。
The gene amplification system and the gene amplification procedure are the same as those described above.
The amplified products were added to PCR tubes at a ligation temperature of 50℃for 15 min, a total system of 20. Mu.L, transformation protocol as above, and cultured at 37℃for 1 h and plated on LB plates (containing 100. Mu.g/mL ampicillin) with 2. Mu. L p-intE vec, 1. Mu. L TEFin promoter fragment 1,1. Mu.L EXP promoter fragment 1,1. Mu.L xpr2 terminator fragment 1,1. Mu.L 3HA-URA-3HA fragment 2,1. Mu.L HPT gene fragment, 1. Mu.L HPD gene fragment and 10. Mu.L Gibson ligase.
The colony PCR amplification and DNA sequencing primer is HPT-F/HPD-R, and is correct if a band of 3519bp can be PCR-amplified.
A correct single colony was selected and designated as E.coli EC002, and the plasmid designated pintE-HPD-HPT was propagated and subjected to plasmid extraction to obtain pintE-HPD-HPT plasmid.
Next, pintE-HPD-HPT-TC plasmid was constructed, the vector backbone was pintE-TEFin-HPT-xpr 2-lip2-TC-TEFin-EXP-HPD-lip2-3HA-URA-3HA.
The construction method comprises the following steps:
The pintE-HPD-HPT plasmid was used as an amplification template, and the primer intE-HPD-HPT-F/intE 1-HPD-HPT-R was used to amplify the pintE-HPD-HPTvec fragment.
The primer sequences were as follows:
intE1-HPD-HPT-F:GAGTGAGTGAATTGGAGTTTGGCGCCCGTTTTTTCG;SEQ ID NO.40。
intE1-HPD-HPT-R:cgaatggcaaatgGACACGGGCATCTCACTTGCATATG;SEQ ID NO.41。
The TEFin promoter (shown as SEQ ID NO. 22) was amplified using the pintE-HPD-HPT plasmid as an amplification template and primers TEFin-F2/TEFin-R2 to obtain TEFin promoter fragment 2.
TEFin-F2:atcggatctcctgcggttagtactgcaaaaagtgc;SEQ ID NO.42。
TEFin-R2:GCCAAACTCCAATTCACTCACTCTCCCGACTATCC;SEQ ID NO.43。
The lip2 terminator is amplified by using pintE-HPD-HPT plasmid as an amplification template and primers lip 2-F2/lip 2-R2 (shown as SEQ ID NO. 31) to obtain lip2 terminator fragment 2.
lip2-F2:CCGTGTCcatttgccattcgtaacgctggtag;SEQ ID NO.44。
lip2-R2:cccggcctgtaagctatttatcactctttacaacttctacctcaactatc;SEQ ID NO.45。
The TC gene synthesized by Huada gene (shown as SEQ ID NO. 19) is used as a template, and a primer TC-F/TC-R is used for amplifying the TC gene to obtain a TC gene fragment.
The primer sequences were as follows:
TC-F:gataaatagcttacaggccgggaggtttgaagaaagg;SEQ ID NO.46。
TC-R:gtactaaccgcaggagatccgatctctgatcgtgtctatga;SEQ ID NO.47。
into the PCR tube were added 3. Mu. L pintE1 of 1-HPD-HPTvec, 2. Mu. L TEFin of promoter fragment 2, 2. Mu.L of lip2 terminator fragment 2, 3. Mu.L of TC gene and 10. Mu.L of Gibson ligase at 50℃for 15min with a total of 20. Mu.L of the total system, and the transformation method was the same as above, and after culturing at 37℃for 1 h, the mixture was plated on LB plates (containing 100. Mu.g/mL of ampicillin).
Colony PCR amplification and DNA sequencing primer are HPT-F/HPD-R. It is correct if a band of 5808bp size can be PCR.
A correct single colony is selected, named as escherichia coli EC003, and a plasmid is named as pintE-HPD-HPT-TC, and is subjected to propagation and plasmid extraction to obtain a pintE-HPD-HPT-TC plasmid.
The pintE-HPD-HPT-TC plasmid is used as a template, intE1 linearization-F/R is used as a primer, and a linearization integration fragment is amplified, wherein intE up-TEFin-HPT-xpr2-lip2-TC-TEFin-EXP-HPD-lip2-3HA-URA-3HA-intE1Down.
The primer sequences were as follows:
intE1 linearization-F. TTGGGCTTGGGGCACTTCTGC; SEQ ID NO.48.
IntE1 linearization-R. ATGGTGTGCCAGGAGGTGGAC; SEQ ID NO.49.
The strain YL02 is taken as an original strain, and the linearized integrated fragment is transformed into YL02 to obtain the strain ATCC MYA-2613ΔKu70-intE1 (HPD-HPT-TC) -URA, which is named YL03.
The transformation method is the same as above.
The screening marker was recovered to give strain ATCC MYA-2613. DELTA. Ku70-intE1 (HPD-HPT-TC) -URA-, designated as YL04.
The method for recovering the screening marker is the same as above.
Strain YL03 was fermented with SC medium for 144h and assayed for HGA, delta-tocotrienol at an OD600 of 20.62 and an HGA yield of 2.52mg/L and delta-tocotrienol yield of 0.119mg/L.
3) Strengthening shikimic acid pathway
The strengthening of the precursor homogentisate HGA was performed based on YL04 strain. The enhanced related genes of the precursor homogentisate HGA include the gene ARO4 encoding 3-deoxy-D-arabinoheptulose 7-phosphate synthase (Phospho-2-dehydro-3-deoxyheptonate aldolase) and the gene ARO7 encoding shikimate mutase (Chorismate mutase).
The gene encoding 3-deoxy-D-arabinoheptulose 7-phosphate synthase is selected from the group consisting of genes scARO andK229L (shown in SEQ ID NO. 50) derived from Saccharomyces cerevisiae (Saccharomy cerevisiae) BY4741 encoding 3-deoxy-D-arabinoheptulose 7-phosphate synthase, in which the amino acid at position 229 is mutated from lysine to leucine.
The gene ARO7 encoding shikimate mutase is selected from gene ylARO7G139S (shown as SEQ ID NO. 51) derived from yarrowia lipolytica ATCC MYA-2613 encoding shikimate mutase and the mutation of amino acid 139 from glycine to threonine.
In order to integrate the two exogenous genes into strain YL04, plasmid pintB-scARO,K229L-ylARO7G139S needs to be constructed, and the vector skeleton is pintB1-TEFin-scARO, 4K229L-xpr2-EXP-ylARO7G139S -lip2-3HA-URA-3HA.
The construction method comprises the following steps:
The plasmid skeleton p-intB vec synthesized by Huada gene (shown as SEQ ID NO. 52) is used as a template, and the primer intB vec-F/R is used for amplification to obtain a plasmid skeleton p-intB vec fragment.
The primer sequences are as follows:
intB1vec-F:GAGACCTACGGCTACgtgaacttcttatgggaagtcaagttgagattgtg;SEQ ID NO.53。
intB1vec-R:GAGTGAGTGAATTGacctgctcctgcacctaagttcgt;SEQ ID NO.54。
the TEFin promoter (shown as SEQ ID NO. 22) was amplified using the pintE-HPD-HPT plasmid as an amplification template and primers TEFin-F3/TEFin-R3 to obtain TEFin promoter fragment 3.
The primer sequences were as follows:
TEFin-F3:aggagcaggtCAATTCACTCACTCTCCCGACTATCCAACAACG;SEQ ID NO.55。
TEFin-R3:ggagattcactctgcggttagtactgcaaaaagtgctgg;SEQ ID NO.56。
The xpr2-EXP was amplified using the pintE-HPD-HPT plasmid as an amplification template and the primer xprEXP-F1/xprEXP-R1 to obtain the xpr2-EXP fragment 1.
xprEXP-F1:caagaaatagGATCCAACTACGGAACTTGTGTTGATGTCTTTG;SEQ ID NO.57。
xprEXP-R1:ctttagtgaagtccatTGCTGTAGATATGTCTTGTGTGTAAGGGGG;SEQ ID NO.58。
The lip2 terminator (shown as SEQ ID NO. 31) was amplified using the pintE-HPD-HPT plasmid as an amplification template and the primers lip 2-F3/lip 2-R3 to obtain lip2 terminator fragment 3.
lip2-F3:gcggttggagtaggctatttatcactctttacaacttctacctcaactatc;SEQ ID NO.59。
lip2-R3:ccgtcgttttacaaccatttgccattcgtaacgctggtagacagg;SEQ ID NO.60。
The ylARO7G139S gene synthesized by Huada gene (shown as SEQ ID NO. 51) is used as a template, and primer ylARO7G139S-F/ylARO7G139S -R is used for amplifying ylARO7G139S gene to obtain ylARO7G139S gene fragment.
The primer sequences were as follows:
ylARO7G139S-F:CTACAGCAatggacttcactaaagccgacaccgttctg;SEQ ID NO.61。
ylARO7G139S-R:tgataaatagcctactccaaccgccggagcag;SEQ ID NO.62。
The scARO geneK229L was amplified by using the scARO4K229L gene synthesized by Huada gene (shown as SEQ ID NO. 50) as a template and using the primer scARO4K229L-F/scARO4K229L -R to obtain a scARO4K229L gene fragment.
scARO4K229L-F:ctaaccgcagagtgaatctccaatgttcgctgccaac;SEQ ID NO.63。
scARO4K229L-R:CCGTAGTTGGATCctatttcttgttaacttctcttctttgtctgacagc;SEQ ID NO.64。
The plasmid pkana-Ku70 constructed as described above was used as a template to amplify 3HA-URA-3HA using primers URA-F3/URA-R3 to obtain 3HA-URA-3HA fragment 3.
URA-F3 is as shown in SEQ ID NO.38.
URA-R3: gaagttcacGTAGCCGTAGGTCTCGTACTGCTTGAC;SEQ ID NO.65。
The gene amplification system and the gene amplification procedure are the same as those described above.
1 Mu L p-intB vec fragment, 1 mu L TEFin promoter fragment 3, 1 mu L xpr2-EXP fragment 1,1 mu L lip2 terminator fragment 3,2 mu L3 HA-URA-3HA fragment 3,2 mu L scARO4K229L gene fragment, 2 mu LylARO7G139S gene fragment and 10 mu L Gibson ligase were added to the PCR tube at 50℃for a ligation time of 15 min with a total system of 20. Mu.L, transformation procedure as above, and after culturing 1h at 37℃the plates were plated onto LB plates (containing 50. Mu.g/mL kanamycin).
The colony PCR amplification and DNA sequencing primer was scARO.sup.4K229L-F/ ylARO7G139S -R, which was correct if a 3399bp band could be PCR-generated.
A correct single colony is selected, named as Escherichia coli EC004, and the plasmid is named pintB-scARO 4K229L-ylARO7G139S, and plasmid extraction is carried out by expanding propagation, so as to obtain pintB1-scARO4K229L-ylARO7G139S plasmid.
The linearized integrated fragment IntB up-TEFin-scARO4K229L-xpr2-EXP-ylARO7G139S -lip2-3HA-URA-3HA-IntB1Down was amplified using intB1 linearization-F/R as primer.
The primer sequences were as follows:
intB1 linearization-F. CATAAGACGCCTCGTTGCTCGGG; SEQ ID NO.66.
IntB1 linearization-R. GAATGCGTGCGATCCCACAGTTCTCA; SEQ ID NO.67.
The strain YL04 is taken as an original strain, and the fragment is transformed into YL04 to obtain the strain ATCC MYA-2613 delta Ku70-intE1 (HPD-HPT-TC) -intB1 (scARO 4K229L-ylARO7G139S) -URA named YL05.
The transformation method is the same as above.
The screening marker was recovered to give strain ATCC MYA-2613. DELTA. Ku70-intE (HPD-HPT-TC) -intB1 (scARO, 4,K229L-ylARO7G139S)URA-, designated as YL06.
The method for recovering the screening marker is the same as above.
Strain YL05 was fermented with SC medium for 144h and assayed for HGA, delta-tocotrienol with OD600 of 19.26, HGA yield of 116mg/L and delta-tocotrienol yield of 0.802mg/L.
4) Strengthening mevalonate pathway
(1) The strengthening of the precursor geranylgeranyl pyrophosphate GGPP was performed based on strain YL 06. The genes involved in the enhancement of precursor geranylgeranyl pyrophosphate GGPP include genes encoding hydroxymethylglutaryl-CoA (HMG-CoA) reductase and genes encoding geranylgeranyl pyrophosphate (GGPP) synthase.
The gene encoding hydroxymethylglutaryl-CoA (HMG-CoA) reductase is the gene tHMGR (shown in SEQ ID NO. 68) derived from yarrowia lipolytica (Yarrowia lipolytica) ATCC MYA-2613 encoding hydroxymethylglutaryl-CoA reductase and deleting the first 500 amino acids from the N-terminus.
The gene encoding geranylgeranyl pyrophosphate (GGPP) synthase is a codon-optimized gene GGPPsa encoding geranylgeranyl pyrophosphate (GGPP) synthase derived from Acidophilic thermophilic sulfureus (Sulfolobus acidocaldarius) ATCC 33909 (as shown in SEQ ID No. 69).
In order to integrate the two genes into strain YL06, plasmid pintD-tHMGR-GGPPsa needs to be constructed with a vector backbone of pintD1-TEFin-tHMGR-xpr2-EXP-GGPPsa-lip2-3HA-URA-3HA.
The construction method comprises the following steps:
Plasmid pkana-Ku70 is used as an amplification template, the plasmid skeleton p-intD vec is amplified, and the amplification primer is intD vec-F/R.
The primer sequences were as follows:
intD1vec-F:TGTTGTTGCCTCTCACAGGCATTTGAGAAGCACACGGTCAC;SEQ ID NO.70。
intD1vec-R:GATGCGACAGAGGCGCAACGCAATTAATGTAAGTTAGCTCACTC;SEQ ID NO.71。
the TEFin promoter (shown as SEQ ID NO. 22) was amplified using the pintE-HPD-HPT plasmid as an amplification template and primers TEFin-F4/TEFin-R4 to obtain the TEFin promoter fragment 4.
TEFin-F4:CTCGACAAGGCAATTCACTCACTCTCCCGACTATCCAACAAC;SEQ ID NO.72。
TEFin-R4:cagactgggtctgcggttagtactgcaaaaagtgctgg;SEQ ID NO.73。
The xpr2-EXP was amplified using the pintE-HPD-HPT plasmid as an amplification template and the primer xprEXP-F2/xprEXP-R2 to obtain the xpr2-EXP fragment 2.
xprEXP-F2:acggtcaTAAGATCCAACTACGGAACTTGTGTTGATGTCTTTGC;SEQ ID NO.74。
xprEXP-R2:GTCGAAGTAAGACATTGCTGTAGATATGTCTTGTGTGTAAGGGGGT;SEQ ID NO.75。
The lip 2-terminator and 3HA-URA-3HA fragment 1 was amplified using the pintE-HPD-HPT plasmid as an amplification template and the primers lip2URA-F1/lip2URA-R1 to obtain lip2-3HA-URA-3HA fragment 1.
lip2URA-F1:CCGACGACGAAAGTAAgctatttatcactctttacaacttctacctcaactatc;SEQ ID NO.76。
lip2URA-R1:TAGAATGAACCGGTAGCCGTAGGTCTCGTACTGCTTGAC;SEQ ID NO.77。
The tHMGR gene (as shown in SEQ ID NO. 68) was amplified using the primers tHMGR-F/tHMGR-R using the yarrowia lipolytica ATCC MYA-2613 genome as a template to obtain a fragment of the tHMGR gene.
tHMGR-F:ctaaccgcagacccagtctgtgaaggtggttgagaag;SEQ ID NO.78。
tHMGR-R:CCGTAGTTGGATCttatgaccgtatgcaaatattcgaaccgttttgtagacg;SEQ ID NO.79。
Using the yarrowia lipolytica ATCC MYA-2613 genome as a template, the upstream homology arm intD up (as shown in SEQ ID NO. 80) was amplified using primer intD up-F/intD1up-R to obtain the intD1up fragment.
The primer sequences were as follows:
intD1up-F:CGTTGCGCCTCTGTCGCATCTCTAGTAGAGGTGGTGAC;SEQ ID NO.81。
intD1up-R:GTGAGTGAATTGCCTTGTCGAGACGCTAACAGACACATGC;SEQ ID NO.82。
using yarrowia lipolytica ATCC MYA-2613 genome as template, primer intD down-F/intD1down-R was used to amplify downstream homology arm intD1down (as shown in SEQ ID NO. 83) to obtain intD down fragment.
The primer sequences were as follows:
intD1down-F:ACCTACGGCTACCGGTTCATTCTAGCACATGTGCCATGT;SEQ ID NO.84。
intD1down-R:TCAAATGCCTGTGAGAGGCAACAACACCCTCGTATCTCAAC;SEQ ID NO.85。
The GGPPsa gene synthesized by Huada gene (shown as SEQ ID NO. 69) is used as a template, and primer GGPPsa-F/GGPPsa-R is used for amplifying GGPPsa gene to obtain GGPPsa gene fragment.
The primer sequences were as follows:
GGPPsa-F:TCTACAGCAATGTCTTACTTCGACAACTACTTCAACGAGATCG;SEQ ID NO.86。
GGPPsa-R:aatagcTTACTTTCGTCGTCGGATGGTGAACTCG;SEQ ID NO.87。
The gene amplification system and the gene amplification procedure are the same as those described above.
2. Mu. L p-intD vec, 1. Mu. L intD1up fragment, 1. Mu. L intD1up fragment, 1. Mu. L TEFin promoter fragment 4, 1. Mu.L xpr2-EXP fragment 2, 2. Mu.L lip2-3HA-URA-3HA fragment 1, 1. Mu.L tHMGR gene fragment, 1. Mu. L GGPPsa gene fragment and 10. Mu.L Gibson ligase were added to the PCR tube at 50℃for 15 min total ligation time of 20. Mu.L, the transformation procedure was the same as above, and after culturing 1 h at 37℃plates were plated on LB plates (containing 50. Mu.g/mL kanamycin).
The colony PCR amplification and DNA sequencing primer was tHMGR-F/GGPPsa-R, which was correct if a 4011bp band could be PCR-amplified.
A correct single colony was selected and designated as E.coli EC005, and the plasmid designated pintD-tHMGR-GGPPsa, which was propagated and subjected to plasmid extraction to obtain the pintD-tHMGR-GGPPsa plasmid.
The linearized integrated fragment IntD up-TEFin-tHMGR-xpr2-EXP-GGPPsa-lip2-3HA-URA-3HA-IntD1Down was amplified using intD1 linearization-F/R as primer.
The primer sequences were as follows:
intD1 linearization-F. CTCTTTCGTAGATGGTTTCCGTAAGACGTTTGAG, SEQ ID NO.88.
IntD1 linearization-R. GCTCCTGCAACTCCTGTCTATGGTCTGTAAC, SEQ ID NO.89.
The strain YL06 is used as an original strain, and the linearization integrated fragment is transformed into YL06 to obtain a strain ATCC MYA-2613ΔKu70-intE1:(HPD-HPT-TC)-intB1:( scARO4K229L-ylARO7G139S)-intD1:(tHMGR-GGPPsa)-URA, named YL07.
The transformation method is the same as above.
The screening marker was recovered to give strain ATCC ATCCMYA-2613ΔKu70-intE1:(HPD-HPT-TC)-intB1:(scARO4K229L-ylARO7G139S)-intD1:(tHMGR-GGPPsa)-URA-, designated YL08.
The method for recovering the screening marker is the same as above.
(2) For a second integration of the two genes, tHMGR, GGPPsa was continued to integrate into the F30 locus starting from strain YL 08.
In order to integrate the two genes into strain YL08, plasmid PintF-tHMGR-GGPPsa needs to be constructed, the vector backbone is pintF-TEFin-tHMGR-xp 2-EXP-GGPPsa-lip2-3HA-URA-3HA.
The construction method comprises the following steps:
Plasmid pkana-Ku70 is used as an amplification template, the plasmid skeleton p-intF vec is amplified, and the amplification primer is intF vec-F/R.
The primer sequences were as follows:
intF30vec-F:CACTCTCCTGTCAGGCATTTGAGAAGCACACGGTCAC;SEQ ID NO.90。
intF30vec-R:AGACAATGTGCGCAACGCAATTAATGTAAGTTAGCTCACTC;SEQ ID NO.91。
The pintD-tHMGR-GGPPsa plasmid was used as an amplification template to amplify the TEFin-tHMGR-xp 2-EXP-GGPPsa-lip2-3HA-URA-3HA fragment using primer TEFin-F5/lip2 URA-R5.
The primer sequences were as follows:
TEFin-F5:ACTAAGGCCagagaccgggttggcggc;SEQ ID NO.92。
lip2URA-R5:CTTCAACCGTGTAGCCGTAGGTCTCGTACTGC;SEQ ID NO.93。
Using the yarrowia lipolytica ATCC MYA-2613 genome as a template, the homology arms intF up (as shown in SEQ ID NO. 94) were amplified using primers intF up-F/intF up-R to obtain the intF up fragment.
The primer sequences were as follows:
intF30up-F:GCGTTGCGCACATTGTCTTCACCTGTTCGGCTCATGAG;SEQ ID NO.95。
intF30up-R:cccggtctctGGCCTTAGTTTCGTCTTGACTCGGC;SEQ ID NO.96。
Using yarrowia lipolytica ATCC MYA-2613 genome as template, primer intF Down-F/intF30Down-R was used to amplify homology arm intF Down (as shown in SEQ ID NO. 97) to obtain intF down fragment.
The primer sequences were as follows:
intF30down-F:CCTACGGCTACACGGTTGAAGCAAAGCTTTAGTGTGTTAGC;SEQ ID NO.98。
intF30down-R:CTCAAATGCCTGACAGGAGAGTGACAAGCCAACTGTGG;SEQ ID NO.99。
The gene amplification system and the gene amplification procedure are the same as those described above.
Into PCR tubes were added 3. Mu. L p-intF vec, 3. Mu. L TEFin-tHMGR-xp 2-EXP-GGPPsa-lip2-3HA-URA-3HA fragment, 2. Mu. L intF30up fragment, 2. Mu. L intF30down fragment, and 10. Mu.l Gibson ligase at 50℃for 15 min total system of 20. Mu.L, transformation procedure was the same as above, cultured at 37℃for 1 h and plated onto LB plates (containing 50. Mu.g/mL kanamycin).
The colony PCR amplification and DNA sequencing primer was tHMGR-F/GGPPsa-R, which was correct if a 4011bp band could be PCR-amplified.
A correct single colony was selected and designated as E.coli EC006, and the plasmid designated PintF-tHMGR-GGPPsa was propagated and subjected to plasmid extraction to obtain a PintF-tHMGR-GGPPsa plasmid.
The linearized integrated fragment intF was amplified using the PintF-tHMGR-GGPPsa plasmid as template and intF30 linearized-F/R as primer, with 30up-TEFin-tHMGR-xpr2-EXP-GGPPsa-lip2-3HA-URA-3HA-intF down.
The primer sequences were as follows:
intF30 linearization-F. GCCAAGTCTAGACCAACGGTCCATGAC, SEQ ID NO.100.
IntF30 linearization-R. CGTCCTTGAAGTCGACGTACACATCCTG, SEQ ID NO.101.
The strain YL08 is taken as an original strain, and the linearization integrated fragment is transformed into YL08 to obtain a strain ATCC MYA-2613ΔKu70-intE1:(HPD-HPT-TC)-intB1:(scARO4K229L-ylARO7G139S)-intD1:(tHMGR-GGPPsa)-intF30:(tHMGR-GGPPsa)-URA, named YL09.
The transformation method is the same as above.
The screening marker was recovered to give strain ATCC ATCCMYA-2613ΔKu70-intE1:(HPD-HPT-TC)-intB1:(scARO4K229L-ylARO7G139S)-intD1:(tHMGR-GGPPsa)-intF30:(tHMGR-GGPPsa)-URA-, designated YL10.
The method for recovering the screening marker is the same as above.
Strains YL07 and YL09 were fermented with SC medium for 144h, respectively, and delta-tocotrienol was assayed. The results showed that the strain YL07 was fermented 144h, the OD600 was 19.26, the delta-tocotrienol was 1.445mg/L, the strain YL09 was fermented 144h, the OD600 was 18.32, and the delta-tocotrienol yield was 1.840mg/L.
5) Introduction of the prenyl alcohol pathway
Based on the strain YL10, a gene encoding choline kinase (choline kinase) and a gene encoding isopentenyl phosphate kinase (isopentenyl phosphate kinase) were introduced.
The gene encoding choline kinase is a codon-optimized gene CK (shown as SEQ ID NO. 102) encoding choline kinase from Saccharomyces cerevisiae (Saccharomy cerevisiae) BY 4741.
The gene encoding isopentenyl pyrophosphate kinase is the codon-optimized gene IPK encoding isopentenyl pyrophosphate kinase from Arabidopsis thaliana (as shown in SEQ ID NO. 103).
In order to integrate the two genes into strain YL10, plasmid pintF-CK-IPK needs to be constructed, and the vector skeleton is pintF-TEFin-CK-xpr 2-EXP-IPK-lip2-3HA-URA-3HA.
The construction method comprises the following steps:
Plasmid PintF-tHMGR-GGPPsa is used as an amplification template, the plasmid skeleton p-intF vec is amplified, and the amplification primer is intF2vec-F/R.
The primer sequences were as follows:
intF2vec-F:CCAAACCAAACCCAGGCATTTGAGAAGCACACGGTCAC;SEQ ID NO.104。
intF2vec-R:GAGGAGAAGTGGCGCAACGCAATTAATGTAAGTTAGCTCACTCA;SEQ ID NO.105。
The TEFin promoter (shown as SEQ ID NO. 22) was amplified using the pintE-HPD-HPT plasmid as an amplification template and primers TEFin-F5/TEFin-R5 to obtain the TEFin promoter fragment 5.
The primer sequences were as follows:
TEFin-F5:GATCTGGGGTTCAATTCACTCACTCTCCCGACTATCC;SEQ ID NO.106。
TEFin-R5:GACTCTTGCACctgcggttagtactgcaaaaagtgctgg;SEQ ID NO.107。
The xpr2-EXP was amplified using the pintE-HPD-HPT plasmid as an amplification template and the primer xprEXP-F3/xprEXP-R3 to obtain the xpr2-EXP fragment 3.
The primer sequences were as follows:
xprEXP-F3:CCTGTAAGATCCAACTACGGAACTTGTGTTGATGTCTTTGC;SEQ ID NO.108。
xprEXP-R3:GATGTTCAGTTCCATTGCTGTAGATATGTCTTGTGTGTAAGGGGGT;SEQ ID NO.109。
The lip2 terminator and 3HA-URA-3HA were amplified using primers lip2URA-F2/lip2URA-R2 to obtain lip2-3HA-URA-3HA fragment 2.
The primer sequences were as follows:
lip2URA-F2:CATCCGATTCTCCAAGTAAgctatttatcactctttacaacttctacctcaactatc;SEQ ID NO.110。
lip2URA-R2:CTATGGGATGGTAGCCGTAGGTCTCGTACTGCTTGAC;SEQ ID NO.111。
Using the yarrowia lipolytica ATCC MYA-2613 genome as a template, the homology arm intF up (as shown in SEQ ID NO. 112) was amplified using primers intF up-F/intF up-R to obtain the intF up fragment.
The primer sequences were as follows:
intF2up-F:CGTTGCGCCACTTCTCCTCAGCTCACGTGAATCACAC;SEQ ID NO.113。
intF2up-R:GTGAGTGAATTGAACCCCAGATCCAAGTCCACACC;SEQ ID NO.114。
using yarrowia lipolytica ATCC MYA-2613 genome as template, primer intF down-F/intF2down-R was used to amplify homology arm intF down (as shown in SEQ ID NO. 115) to obtain intF2down fragment.
The primer sequences were as follows:
intF2down-F:CCTACGGCTACCATCCCATAGTGTTGAAGGTAATACCCGGTAG;SEQ ID NO.116。
intF2down-R:TCAAATGCCTGGGTTTGGTTTGGTTTGATTTGGTGTGCCA;SEQ ID NO.117。
The CK gene synthesized by Huada gene (shown as SEQ ID NO. 102) is used as a template, and a primer CK-F/CK-R is used for amplifying the CK gene to obtain a CK gene fragment.
The primer sequences were as follows:
CK-F:ctaaccgcagGTGCAAGAGTCTCGACCCGGCT;SEQ ID NO.118。
CK-R:GTTCCGTAGTTGGATCTTACAGGTAAGAGGTGTCCAGGAACTTG;SEQ ID NO.119。
The IPK gene synthesized by Huada gene (shown as SEQ ID NO. 103) is used as a template, and the primer IPK-F/IPK-R is used for amplifying the IPK gene to obtain an IPK gene fragment.
The primer sequences were as follows:
IPK-F:TCTACAGCAATGGAACTGAACATCTCTGAGTCTCG;SEQ ID NO.120。
IPK-R:tagcTTACTTGGAGAATCGGATGATGGTGCC;SEQ ID NO.121。
The gene amplification system and the gene amplification procedure are the same as those described above.
2. Mu. L p-intF vec, 1. Mu. L intF2up fragment, 1. Mu. L intF2down fragment, 1. Mu. L TEFin promoter fragment 5, 1. Mu.L xpr2-EXP fragment 3, 2. Mu.L lip2-3HA-URA-3HA fragment 2, 1. Mu.L CK gene fragment, 1. Mu.L IPK gene fragment and 10. Mu.L Gibson ligase were added to the PCR tube at 50℃for a period of 15 min, 20. Mu.L of the total system, and after culturing 1 h at 37℃the plates were plated on LB plates (containing 50. Mu.g/mL kanamycin) as described above.
The colony PCR amplification and DNA sequencing primer was CK-F/IPK-R, which was correct if a band of 4263bp could be PCR-amplified.
A correct single colony is selected, named as Escherichia coli EC007, and a plasmid is named pintF-CK-IPK, and is subjected to propagation and plasmid extraction to obtain a pintF-CK-IPK plasmid.
The linear integration fragment intF up-TEFin-CK-xp 2-EXP-IPK-lip2-3HA-URA-3HA-intF2Down was amplified using pintF-CK-IPK plasmid as a template and intF2 linearization-F/R as a primer.
The primer sequences were as follows:
intF2 linearization-F. CACTTCTCCTCAGCTCACGTGAATCACAC, SEQ ID NO.122.
IntF2 linearization-R. GGTTTGGTTTGGTTTGATTTGGTGTGCCA; SEQ ID NO.123.
The linearized integrated fragment was transformed into YL10 using strain YL10 as starting strain, resulting in strain ATCC MYA-2613ΔKu70-intE1:(HPD-HPT-TC)-intB1:(scARO4K229L-ylARO7G139S)-intD1:(tHMGR-GGPPsa)-intF30:(tHMGR-GGPPsa)-intF2:(CK-IPK)-URA, designated YL11.
The transformation method is the same as above.
The screening marker was recovered to give strain ATCC MYA-2613ΔKu70-intE1:(HPD-HPT-TC)-intB1:(scARO4K229L-ylARO7G139S)-intD1:(tHMGR-GGPPsa)-intF30:(tHMGR-GGPPsa)-intF2:(CK-IPK)-URA-, designated YL12.
The method for recovering the screening marker is the same as above.
Strain YL09 and strain YL11 were fermented with SC medium for 144h, respectively, and delta-tocotrienol was assayed. The results showed that strain YL09 was fermented 144h, OD600 was 18.92, delta-tocotrienol was 1.869mg/L, strain YL11 was fermented 144h, OD600 was 17.26, delta-tocotrienol yield was 2.707mg/L.
The above results indicate that the delta-tocotrienol production can be significantly increased by expressing both CK and IPK genes.
With the strain YL12 as an original strain, the integration of the CK gene and the IPK gene is carried out at a multicopy locus YLT1 locus in order to further increase the yield of tocotrienol.
To integrate CK and IPK at YLT1 site, plasmid PYLT-CK-IPK needs to be constructed, the vector backbone is PYLT-TEFin-CK-xpr 2-EXP-IPK-lip2-3HA-11bpURA-3HA. Compared with 3HA-URA-3HA, 3HA-11bpURA-3HA truncates the original promoter of the URA expression cassette to 11bp promoter, and 3HA-11bpURA-3HA (shown as SEQ ID NO. 124) is synthesized by Huada genes.
The construction method comprises the following steps:
plasmid pintF-CK-IPK is used as an amplification template, the plasmid skeleton p-YLT1vec is amplified, and the amplification primer is YLT1vec-F/R.
The primer sequences were as follows:
YLT1vec-F:agagtcgacaaaggccctccaattgcttctaacatcgcgtg;SEQ ID NO.125。
YLT1vec-R:GAGTGAGTGAATTGCACTGAGGGCTTTGTGAGGAGGTAACG;SEQ ID NO.126。
The plasmid pintF-CK-IPK is used as an amplification template, the amplified fragment TEFin-CK-xpr2-EXP-IPK-lip2 and the amplified primer TEFin-FA/lip2-RA.
The primer sequences were as follows:
TEFin-FA:CCCTCAGTGCAATTCACTCACTCTCCCGACTATCCAACAAC;SEQ ID NO.127。
lip2-RA:cgtcgttttacaaccatttgccattcgtaacgctggtag;SEQ ID NO.128。
3HA-11bpURA-3HA synthesized by Huada gene (shown as SEQ ID NO. 124) is used as a template, and 3HA-11bpURA-3HA is amplified by using a primer 11bpURA-F/11bpURA-R to obtain a 3HA-11bpURA-3HA fragment.
The primer sequences were as follows:
11bpURA-F:ggcaaatggttgtaaaacgacggccagtcg;SEQ ID NO.129。
11bpURA-R:cGTAGCCGTAGGTCTCGTACTGCTTGAC;SEQ ID NO.130。
The homologous arm YLT1up (shown as SEQ ID NO. 131) was amplified using primers YLT1up-F/YLT1up-R using the yarrowia lipolytica ATCC MYA-2613 genome as template to obtain the YLT1up fragment.
The primer sequences were as follows:
YLT1up-F:gtgtaacaatgCCTAGGCATGTGTAACACTCGCTCTGG;SEQ ID NO.132。
YLT1up-R:GAGTGAGTGAATTGCACTGAGGGCTTTGTGAGGAGGTAACG;SEQ ID NO.133。
The homologous arm YLT1Down (shown as SEQ ID NO. 134) was amplified using the yarrowia lipolytica ATCC MYA-2613 genome as template and the primer YLT1Down-F/YLT1Down-R to obtain the YLT1Down fragment.
The primer sequences were as follows:
YLT1down-F:CAGTACGAGACCTACGGCTACgcggccgctgtcgggaa;SEQ ID NO.135。
YLT1down-R:ggagggcctttgtcgactctatctagcaaag;SEQ ID NO.136。
The gene amplification system and the gene amplification procedure are the same as those described above.
Into the PCR tube were added 2. Mu. L p-YLT1vec, 2. Mu.L of YLT1up fragment, 2. Mu.L of YLT1down fragment, 2. Mu. L TEFin-CK-xpr2-EXP-IPK-lip2 fragment, 2. Mu.L of 3HA-11bpURA-3HA fragment and 10. Mu.L of Gibson ligase at 50℃for 15 min total system of 20. Mu.L, the transformation method was the same as above, and after culturing at 37℃for 1h, the plate was plated on LB plates (containing 50. Mu.g/mL kanamycin).
The colony PCR amplification and DNA sequencing primer was CK-F/IPK-R, which was correct if a band of 4263bp could be PCR-amplified.
A correct single colony is selected, named as Escherichia coli EC008, and a plasmid is named as pYLT-CK-IPK, and is subjected to propagation and plasmid extraction to obtain a pYLT-CK-IPK plasmid.
The pYLT-CK-IPK plasmid is used as a template, YLT1 linearization-F/R is used as a primer, and a linearization integrated fragment YLT1up-TEFin-CK-xpr2-EXP-IPK-lip2-3HA-11bpURA-3HA-YLT1down is amplified.
The primer sequences were as follows:
YLT1 linearization-F CCTAGGCATGTGTAACACTCGCTCTGG, SEQ ID No.137.
YLT1 linearization-R: GGCCTTTGTCGACTCTATCTAGCAAAG, SEQ ID NO.138.
The strain YL12 is used as an original strain, and the linearization integrated fragment is transformed into the YL12 to obtain a strain ATCC MYA-2613ΔKu70-intE1:(HPD-HPT-TC)-intB1:(scARO4K229L-ylARO7G139S)-intD1:(tHMGR-GGPPsa)-intF30:(tHMGR-GGPPsa)-intF2:(CK-IPK)-YLT1:(CK-IPK)-URA, named YL13.
The transformation method is the same as above.
The screening marker was recovered to give strain ATCC MYA-2613ΔKu70-intE1:(HPD-HPT-TC)-intB1:(scARO4K229L-ylARO7G139S)-intD1:(tHMGR-GGPPsa)-intF30:(tHMGR-GGPPsa)-intF2:(CK-IPK)- YLT1:(CK-IPK)-URA-, designated YL14.
Strain YL13 was fermented with SC medium for 144h and delta-tocotrienol was assayed. The results showed that the strain YL13 was fermented 144h, OD600 was 16.73 and delta-tocotrienol yield was 16.29mg/L.
6) Protein fusion of HPD and HPT
With the strain YL14 as the starting strain, to further increase the delta-tocotrienol production, integration of the HPD-HPT (RH) fusion protein was performed at the YLT2 site. The HPD-HPT (RH) fusion protein was obtained by fusing 4-hydroxyphenylpyruvate dioxygenase-encoding gene HPD (4-hydroxyphenylpyruvate dioxygenase-encoding gene HPD encoding 4-hydroxyphenylpyruvate dioxygenase-encoding gene HPD which is derived from Pseudomonas putida (Pseudomonas putida) KT2440 and is codon-optimized) and homogentisate phytyltransferase-encoding gene HPT (homogentisate phytyltransferase-encoding gene HPT encoding homogentisate phytyltransferase-encoding gene which is derived from Synechocystis sp.) PCC 6803 and is codon-optimized using linker. The HPD-HPT (RH) gene sequence is shown as SEQ ID NO. 139.
In SEQ ID NO.139, 1-1074bp is HPD gene sequence, 1075-1104bp is linker sequence, 1105-2031bp is HPT gene sequence.
Plasmid pYLT-CK-IPK is used as an amplification template, the plasmid skeleton p-YLT2vec is amplified, and the amplification primer is YLT2vec-F/R.
The primer sequences were as follows:
YLT2vec-F:cttcaatactccaattgcttctaacatcgcgtg;SEQ ID NO.140。
YLT2vec-R:CCGGGTATTACCTTCAACAcattgttacaccatatcaaatcgcacgc;SEQ ID NO.141。
The plasmid pYLT-CK-IPK is used as an amplification template, and a primer TEFin-F6/TEFin-R6 is used for amplifying TEFin promoter (shown as SEQ ID NO. 22) to obtain TEFin promoter fragment 6.
The primer sequences were as follows:
TEFin-F6:CTCTTTTCTGGCAATTCACTCACTCTCCCGACTATCC;SEQ ID NO.142。
TEFin-R6:tgtcggcctgcggttaGTACTGCAAAAAGTGCTG;SEQ ID NO.143。
the plasmid pYLT-CK-IPK is used as an amplification template, and the primer 11bpURA-F1/11bpURA-R1 is used for amplifying the lip2-3HA-11bpURA-3HA to obtain the lip2-3HA-11bpURA-3HA segment 1.
The primer sequences were as follows:
11bpURA-F1:ccatcttctaagctatttatcactctttacaacttctacctcaactatc;SEQ ID NO.144。
11bpURA-R1:gctcattcaGTAGCCGTAGGTCTCGTACTGCTTGAC;SEQ ID NO.145。
the homologous arm YLT2up (shown as SEQ ID NO. 146) was amplified using the yarrowia lipolytica ATCC MYA-2613 genome as template and the primer YLT2up-F/YLT2up-R to obtain the YLT2up fragment.
The primer sequences were as follows:
YLT2up-F:caatgTGTTGAAGGTAATACCCGGTGGGGTA;SEQ ID NO.147。
YLT2up-R:GAGTGAATTGCCAGAAAAGAGATTCTAGATCAGTGGATAAAACTATCTACAGG;SEQ ID NO.148。
The homologous arm YLT2Down (shown as SEQ ID NO. 149) was amplified using primers YLT2Down-F/YLT2Down-R using yarrowia lipolytica ATCC MYA-2613 genome as template to obtain YLT2Down fragment.
The primer sequences were as follows:
YLT2down-F:CCTACGGCTACtgaatgagcaagcacacaccaggg;SEQ ID NO.150。
YLT2down-R:gaagcaattggagtattgaagatcataagtattattgatgtgatatag;SEQ ID NO.151。
The HPD-HPT (RH) gene sequence synthesized by Huada genes (shown as SEQ ID NO. 139) is used as a template, and the primer HPD-HPT (RH) -F/HPD-HPT (RH) -R is used for amplifying HPD-HPT (RH) to obtain the HPD-HPT (RH) fragment.
The primer sequences were as follows:
HPD-HPT(RH)-F:gtactaaccgcaggccgacatcttcgagaaccccat;SEQ ID NO.152。
HPD-HPT(RH):agtgataaatagcttagaagatggtgttagaaaaattaggcagccac;SEQ ID NO.153。
The gene amplification system and the gene amplification procedure are the same as those described above.
Into the PCR tube were added 2. Mu. L p-YLT2vec, 1. Mu.L of YLT1up fragment, 1. Mu.L of YLT1down fragment, 2. Mu.L of HPD-HPT (RH) fragment, 2. Mu.L of lip2-3HA-11bpURA-3HA fragment 1 and 10. Mu.L of Gibson ligase at 50℃for 15 min and 20. Mu.L of the total system, the transformation method was the same as above, and after culturing at 37℃for 1 h, the plate was plated on LB plates (containing 50. Mu.g/mL kanamycin).
The colony PCR amplification and DNA sequencing primer was TEFin-F6/11bpURA-R2, which was correct if a 5068bp band could be PCR-generated.
A correct single colony was selected and designated as E.coli EC009, and the plasmid designated pYLT-HPD-HPT (RH), and plasmid extraction was performed by propagation to obtain pYLT-HPD-HPT (RH) plasmid.
The linear integrated fragment YLT2up-TEFin-HPD-HPT (RH) -lip2-3HA-11bpURA-3HA-YLT2Down was amplified using pYLT-HPD-HPT (RH) plasmid as template and YLT2 linearization-F/R as primer.
The primer sequences were as follows:
YLT2 linearization-F CAATGTGTTGAAGGTAATACCCGGTGGGGTA, SEQ ID NO.154.
YLT2 linearization-R: TATTGAAGATCATAAGTATTATTGATGTGATATAG, SEQ ID No.155.
The linearized integrated fragment was transformed into YL14 using strain YL14 as starting strain, resulting in strain ATCC MYA-2613ΔKu70-intE1:(HPD-HPT-TC)-intB1:(scARO4K229L-ylARO7G139S)-intD1:(tHMGR-GGPPsa)-intF30:(tHMGR-GGPPsa)-intF2:(CK-IPK)-YLT1:(CK-IPK)- YLT2:(HPD-HPT(RH))-URA, designated YL15.
The transformation method is the same as above.
The selection marker was recovered to give strain ATCC ATCCMYA-2613ΔKu70-intE1:(HPD-HPT-TC)-intB1:( scARO4K229L-ylARO7G139S)-intD1:(tHMGR-GGPPsa)-intF30:(tHMGR-GGPPsa)-intF2:(CK-IPK)-YLT1:(CK-IPK)- YLT2:(HPD-HPT(RH))-URA-, designated YL16.
The method for recovering the screening marker is the same as above.
Strain YL15 was fermented with SC medium for 144h and delta-tocotrienol was assayed. The results showed that strain YL15 was fermented for 144h with OD600 of 17.32 and delta-tocotrienol production of 32.58mg/L.
7) Plant-derived signal peptide truncated TC and leucine deficiency in anaplerotic
To further increase delta-tocotrienol production, 46AATC and LEU2 integration was performed at the multicopy position 26s with strain YL16 as starting strain. For the gene encoding tocopherol cyclase selected from the gene TC encoding tocopherol cyclase derived from arabidopsis thaliana (Arabidopsis thaliana) and codon optimized, a truncated signal peptide was performed, 46 amino acids after the N-terminal start codon ATG was truncated, yielding 46AATC (as shown in SEQ ID No. 156). LEU2 is a gene encoding leucine synthesis (shown as SEQ ID NO. 157) derived from yarrowia lipolytica (Yarrowia lipolytica) CLIB 122/E150.
Plasmid pYLT-CK-IPK is used as an amplification template, the plasmid skeleton p-26svec is amplified, and the amplification primer is 26svec-F/R.
The primer sequences were as follows:
26svec-F:cataccgaagCAGGCATTTGAGAAGCACACGGTCAC;SEQ ID NO.158。
26svec-R:-cttaggatcgaGCGCAACGCAATTAATGTAAGTTAGCTCACTC;SEQ ID NO.159。
the plasmid pYLT-CK-IPK is used as an amplification template, and a primer TEFin-F7/TEFin-R7 is used for amplifying TEFin promoter (shown as SEQ ID NO. 22) to obtain TEFin promoter segment 7.
The primer sequences are as follows:
TEFin-F7:taCAATTCACTCACTCTCCCGACTATCCAACAAC;SEQ ID NO.160。
TEFin-R7:gatagaggcctgcggttagtactgcaaaaagtgc;SEQ ID NO.161。
the plasmid pYLT-CK-IPK is used as an amplification template, and the primers xpr2-EXP-F4/xpr2-EXP-R4 are used for amplifying the xpr2-EXP to obtain the xpr2-EXP fragment 4.
The primer sequences are as follows:
xpr2-EXP-F4:ccggcctgtaaGATCCAACTACGGAACTTGTGTTGATGTCTTTG;SEQ ID NO.162。
xpr2-EXP-R4:gtttcgggttccatTGCTGTAGATATGTCTTGTGTGTAAGGGGG;SEQ ID NO.163。
The plasmid pYLT-CK-IPK is used as an amplification template, and the primer 11bpURA-F2/11bpURA-R2 is used for amplifying the lip2-3HA-11bpURA-3HA to obtain the lip2-3HA-11bpURA-3HA segment 2.
The primer sequences were as follows:
11bpURA-F2:gctgctcaagaaggagtaagctatttatcactctttacaacttctacctcaactatc;SEQ ID NO.164。
11bpURA-R2:gatgacgaggcGTAGCCGTAGGTCTCGTACTGCTTGAC;SEQ ID NO.165。
The yarrowia lipolytica ATCC MYA-2613 genome is used as a template, and a primer 26sup-F/26sup-R is used for amplifying a homology arm 26sup (shown as SEQ ID NO. 166) to obtain a 26sup fragment.
The primer sequences were as follows:
26sup-F:GCGTTGCGCtcgatcctaaggggtggcataactgtc;SEQ ID NO.167。
26sup-R:GTCGGGAGAGTGAGTGAATTGtatatgatatggtgtcgactggctaccttaagagagtc;SEQ ID NO.168。
The homology arm 26sdown (shown as SEQ ID NO. 169) was amplified using the yarrowia lipolytica ATCC MYA-2613 genome as template and the primer 26sdown-F/26sdown-R to obtain the 26sdown fragment.
The primer sequences were as follows:
26sdown-F:CTACGGCTACgcctcgtcatctaattagtgacgcg;SEQ ID NO.170。
26sdown-R:CTCAAATGCCTGcttcggtatgataggaagagccgaca;SEQ ID NO.171。
46AATC fragment was obtained by amplifying 46AATC with primer 46AATC-F/46AATC-R using 46AATC (shown as SEQ ID NO. 156) synthesized from Huada gene as a template.
The primer sequences were as follows:
46AATC-F:tactaaccgcaggcctctatctctacccccaactctgaaac;SEQ ID NO.172。
46AATC-R:GTAGTTGGATCttacaggccgggaggtttgaagaaagg;SEQ ID NO.173。
LEU2 gene synthesized by Huada genes (shown as SEQ ID NO. 157) is used as a template, and the LEU2 gene is amplified by using a primer LEU2-F/LEU2-R to obtain an LEU2 gene fragment.
The primer sequences were as follows:
LEU2-F:TCTACAGCAatggaacccgaaactaagaagaccaag;SEQ ID NO.174。
LEU2-R:tagcttactccttcttgagcagctccttgacc;SEQ ID NO.175。
The gene amplification system and the gene amplification procedure are the same as those described above.
1 Mu L p-26svec, 1 mu L of the 26sup fragment, 1 mu L of the 26sdown fragment, 1 mu L TEFin promoter fragment 7, 2 mu L of the 46AATC fragment, 1 mu L of the xpr2-EXP fragment 4, 1 mu L of the LEU2 gene fragment, 2 mu L of lip2-3HA-11bpURA-3HA fragment 2 and 10 mu L of Gibson ligase were added to the PCR tube at a ligation temperature of 50℃for a ligation time of 15 min, the total system of which was 20 mu L, the transformation method was the same as above, and after culturing 1 h at 37℃the plates were plated on LB plates (containing 50. Mu.g/mL kanamycin).
The colony PCR amplification and DNA sequencing primer was 46AATC-F/LEU2-R, which was correct if a 3981bp band could be PCR-generated.
A correct single colony is selected, the single colony is named as escherichia coli EC010, the plasmid is named as p26s-46AATC-LEU2, and plasmid extraction is carried out through propagation, so that the p26s-46AATC-LEU2 plasmid is obtained.
The linearized integrated fragment 26sup-TEFin-46AATC-xpr2-EXP-LEU2-lip2-3HA-11bpURA-3HA-26sdown was amplified using the p26s-46AATC-LEU2 plasmid as template and 26s linearization-F/R as primer.
The primer sequences were as follows:
26s linearization-F TCGATCCTAAGGGGTGGCATAACTGTC, SEQ ID NO.176.
26S linearization-R: CTTCGGTATGATAGGAAGAGCCGACA; SEQ ID NO.177.
The linearized integrated fragment was transformed into YL16 using strain YL16 as starting strain, resulting in strain ATCC MYA-2613ΔKu70-intE1:(HPD-HPT-TC)-intB1:(scARO4K229L-ylARO7G139S)-intD1:(tHMGR-GGPPsa)-intF30:(tHMGR-GGPPsa)-intF2:(CK-IPK)-YLT1:(CK-IPK)-YLT2:(HPD-HPT(RH))-26s:(46AATC-LEU2)-URA, designated YL17.
The transformation method is the same as above.
Strain YL17 was fermented with SC medium for 144h and delta-tocotrienol was assayed. The results showed that the strain YL17 was fermented 144h, OD600 was 15.49 and delta-tocotrienol production was 50.94mg/L.
Example 2L fermenter Magnification optimization
The gene integrated strain YL17 is subjected to amplification culture in a 2L fermentation tank, and the fermentation method is as follows:
1) Culturing seed liquid:
(1) Streaking was performed on the SC solid medium plates, and culturing was performed at 30℃for 36 hours.
(2) 5Ml of SC liquid medium was added to a 50ml sterile test tube, and the lawn was scraped from the plate with 200. Mu.l of yellow gun head, blown and mixed into the liquid medium, and cultured at 30℃for 12 hours.
(3) 50Ml of SC liquid medium was added to a 250ml baffle-less shake flask, and the seed solution was added to a 1ml test tube and cultured for 24 hours.
2) 2L fermentation tank amplification culture:
The initial fermentation volume was 1L, which contained 3.5 g/L Yeast Nitrogen Base (YNB), 1.5g/L yeast powder, 13 g/L ammonium sulfate, 80 g/L glucose, and the OD600 in the fermenter after seed liquid inoculation was 0.8.
The rotation speed during fermentation was 600rpm, the aeration was 1.5vvm, and the pH was set to 5.00 (an alkali feeding bottle for 5mol/L potassium hydroxide was connected to an alkali feeding pump).
Starting from 0h, sampling every 12h to measure the concentration of glucose, starting to feed glucose when the glucose concentration in the initial fermentation medium is consumed to 15g/L, and controlling the glucose to be 5-15g/L during feeding. 10ml of fermentation broth was placed in a 15ml centrifuge tube, centrifuged at 12000rpm for 10min, and the upper aqueous phase was diluted 10 times for glucose detection. The glucose detection method comprises eluting 5mM dilute sulfuric acid, detecting equipment in the liquid phase of Sieimer, and column in the organic acid column of BIO-RAD with elution rate of 0.6mL/min and column temperature of 60deg.C.
Fermenting for 48h, adding 100ml dodecane containing 0.5g of dibutyl hydroxy toluene (BHT) as antioxidant. The samples were taken every 12h, starting at 96h, and the detection of delta-tocotrienol was performed. 10ml of the fermentation broth was placed in a 15ml centrifuge tube, centrifuged at 12000rpm for 10min, and the upper organic phase was diluted 100-fold for detection of delta-tocotrienol.
The sugar feed medium had a formulation of 3.5 g/L Yeast Nitrogen Base (YNB), 1.5g/L yeast powder, 80g/L ammonium sulfate, 600g/L glucose.
Taking 1L of sugar feed medium as an example, weighing 3.5 g Yeast Nitrogen Base (YNB), 1.5g of yeast powder, adding deionized water, sterilizing independently, wherein the volume of the yeast powder is 200ml, weighing 80g of ammonium sulfate, adding deionized water, sterilizing independently, wherein the volume of the ammonium sulfate is 200ml, weighing 600g of glucose, adding deionized water, and sterilizing independently, wherein the volume of the yeast powder is 600 ml. After sterilization, the three separately sterilized liquids were mixed together to obtain 1L of sugar feed medium.
Delta-tocotrienol yields are shown in Table 1.
TABLE 1
The results in Table 1 show that strain YL17 had a delta-tocotrienol yield of 616.45mg/L after 240h fermentation.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.