CN114561309B - Yarrowia lipolytica engineering bacterium for producing itaconic acid by subcellular compartmentalization and application - Google Patents
Yarrowia lipolytica engineering bacterium for producing itaconic acid by subcellular compartmentalization and application Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular biology, and particularly relates to construction and application of yarrowia lipolytica engineering bacteria for compartmentalized production of itaconic acid. The genetically engineered bacterium takes yarrowia lipolytica as an original strain, and is obtained by carrying out positioning expression on cis-aconitic acid decarboxylase coding genes in peroxisomes through enhanced peroxisome positioning signals ePTS 1. After carrying out compartmentalization expression on itaconic acid in a peroxisome by introducing ePTS < 1 >, the yield of itaconic acid is obviously improved by about 47 times; the waste oil in kitchen (YPO) is used as a culture medium, so that the yield of itaconic acid is improved by about 108 times. Therefore, the genetically engineered strain after positioning and transformation is more suitable for industrial application, the production cost can be reduced, and the production efficiency can be improved; the kitchen waste oil is used as a culture medium, so that the waste utilization is realized, the cost is reduced, and the yield is far higher than that of a common culture medium.
Description
Technical field:
The invention belongs to the technical field of molecular biology, and particularly relates to construction and application of yarrowia lipolytica engineering bacteria for compartmentalized production of itaconic acid.
The background technology is as follows:
Yarrowia lipolytica (Yarrowia lipolytica) is an unconventional oleaginous yeast suitable for the production of various downstream compounds derived from acetyl-coa due to its abundant precursor supply. However, when glucose is used as a carbon source, acetyl-CoA is produced and carbon dioxide (CO 2) is released, resulting in a loss of carbon and a decrease in yield. When the fatty acid is used as a substrate, the fatty acid is converted into acyl-coa and enters a peroxisome, and acetyl-coa is generated through beta-oxidation, so that no CO 2 is generated in the process, no loss of carbon source exists, and the higher carbon conversion rate is achieved. More and more studies in recent years have shown that subcellular localization of specific enzymes or metabolic pathways not only helps to increase product conversion efficiency, but also can act to eliminate competitive metabolic inhibition.
The compartmentalization process is to use the unique physiological and biochemical functions of the organelles in the cells to perform metabolic engineering, synthesis biology research and the like. Meanwhile, due to the blocking of the organelle membrane, the organelle or subcellular localization of the metabolic pathway can avoid metabolic competition of cytoplasmic environment and improve the synthesis efficiency of the product. Organelles have many unique physiological and metabolic environments, such as endoplasmic reticulum associated with lipid formation, peroxisomes are involved in beta-oxidation of fatty acids and have high protein concentrations, nuclei are rich in DNA, etc.
Itaconic acid (C 5H6O4) is known as methylene succinic acid and itaconic acid is an unsaturated dibasic organic acid. It contains unsaturated double bond, has active chemical property, can be polymerized among itself, and can also be polymerized with other monomers such as acrylonitrile, etc., and is slightly soluble in benzene, chloroform, diethyl ether, petroleum ether, carbon disulfide, water, ethanol and acetone; can perform various addition reactions, esterification reactions and polymerization reactions, is an important raw material for the chemical synthesis industry and is also an important raw material for chemical production. The modified starch has stable properties under acidic, neutral and medium alkaline conditions, and can be used for synthesizing industrial products such as resin, fiber, detergent, cleaning agent, bioactive component and the like. The industrial production of itaconic acid has two modes of chemical synthesis and biological synthesis, the chemical synthesis is mainly prepared from petrochemical products by using a chemical method, however, the method has the problems of low raw material utilization rate and difficult separation and purification of products, so that the method is limited in large-scale industrial production. The biosynthesis method of itaconic acid refers to the preparation of the target product through genetic modification of the metabolic pathway of microorganisms. Wherein, fungi such as aspergillus terreus, corn black fungus, candida, pichia and the like have stronger capability of naturally producing itaconic acid. However, fungi have a hyphal phenotype because of their poor tolerance to shear stress. Highly branched hyphal filaments create high broth viscosity during fermentation, which presents a significant challenge for mixing and aeration processes in conventional stirred tank fermenters. Furthermore, their fermentation requires the addition of alkali to maintain neutral pH conditions, which also increases the likelihood of bacterial contamination during culture. Yarrowia lipolytica does not produce itaconic acid by itself, but it is highly tolerant to itaconic acid. In recent years, therefore, the heterologous production of itaconic acid by using metabolic engineering and synthetic biology engineering yarrowia lipolytica has become a hotspot for related research.
The invention comprises the following steps:
the technical problem to be solved by the invention is to solve the problem of low yield of the heterologous synthesis of the itaconic acid in yarrowia lipolytica, and provide a genetically engineered strain of yarrowia lipolytica introduced with cis-aconitate decarboxylase (CAD) gene and the application of the strain in the heterologous synthesis of the itaconic acid, which can synthesize the itaconic acid and improve the yield of the itaconic acid through metabolic engineering or synthetic biology means.
One of the technical schemes provided by the invention is as follows: is a yarrowia lipolytica genetically engineered bacterium which is obtained by introducing a cis-aconitate decarboxylase encoding gene into a yarrowia lipolytica starting strain.
The nucleotide of the cis-aconitic acid decarboxylase coding gene is shown as a sequence table SEQ ID NO. 1; is obtained by fusing two gene fragments which do not contain introns on the genome of aspergillus terreus HAT 418.
Further, the CAD gene is expressed in the host cell by a plasmid;
Still further, the plasmid is pYLEX1;
further, the CAD gene is subjected to localization expression in peroxisomes through an enhanced peroxisome localization signal (ePTS 1);
further, a ePTS coding sequence is connected to the rear of the CAD gene sequence, a recombinant plasmid pYLEX-CAD-ePTS 1 is constructed, and the recombinant plasmid is introduced into a yarrowia lipolytica starting strain for expression;
Further, the ePTS1 coding sequence is shown in a sequence table SEQ ID NO.2 (CTGGGCCGAGGACGACGATCCAAGCTG);
Preferably, the yarrowia lipolytica starting strain is yarrowia lipolytica (Yarrowia lipolytica) Po1g ku70 Δ.
The second technical scheme provided by the invention is the application of the yarrowia lipolytica genetically engineered bacterium, in particular to the application in the production of itaconic acid;
further, the method for producing itaconic acid by fermenting yarrowia lipolytica genetically engineered bacteria comprises the following steps:
Inoculating the engineering bacteria seed liquid into YPD culture medium according to 1% inoculum size, culturing for 15-18h at 28-30deg.C under 200-220r/min, transferring into YPO culture medium to make OD 600 reach about 0.1, fermenting at 28-30deg.C under 200-220r/min for 6 days.
The YPD medium comprises the following components: 20g/L peptone, 10g/L yeast extract powder, 20g/L glucose, and the balance of water, and sterilizing for 20min at 115-121 ℃.
The YPO culture medium comprises the following components: 18-22g/L peptone, 8-12g/L yeast extract powder, 1.1-1.2% waste kitchen oil, 0.18-0.22% Tween 80, and the balance water, sterilizing at 115-121 ℃ for 20min.
The beneficial effects are that:
The present invention demonstrates the potential for itaconic acid production (host yarrowia lipolytica Po1g ku70Δ itself cannot produce itaconic acid) by heterologous introduction of CAD gene into yarrowia lipolytica, with a yield of 33.12mg/L. The present invention also demonstrates that enhanced peroxisome localization signal ePTS1 plays a role in yarrowia lipolytica by utilizing the lipophilic nature of green fluorescent protein and nile red. Meanwhile, the invention carries out heterologous localization expression on CAD genes in the peroxisome of yarrowia lipolytica for the first time, and produces itaconic acid by consuming fatty acid of waste kitchen oil.
The result shows that the yield of itaconic acid is obviously improved compared with the strain which only expresses CAD genes in a heterologous way after carrying out compartmentalization expression on itaconic acid in peroxisome by introducing ePTS < 1 >, and is improved by about 47 times, wherein the yield is 1.58 g/L; and compared with the common YPD culture medium, the yield of itaconic acid is improved by about 108 times by adopting the kitchen waste oil (YPO) as the culture medium. Therefore, the genetically engineered strain after positioning and transformation is more suitable for industrial application, the production cost can be reduced, and the production efficiency can be improved; the kitchen waste oil is used as a culture medium, so that the waste utilization is realized, the cost is reduced, and the yield is far higher than that of a common culture medium.
Description of the drawings:
FIG. 1 is a schematic diagram of the itaconic acid production pathway;
FIG. 2 is a construction diagram of CAD gene recombinant plasmids;
FIG. 3 is a gel electrophoresis chart of a fragment of a verification gene
Wherein, figure a is a recombinant plasmid pYLEX-CAD validation drawing; FIG. b is a schematic diagram of the verification of recombinant strain Po1 gku. Delta. -CAD-ePTS 1;
FIG. 4 is a fluorescent image under a microscope
Wherein, a graph a shows green fluorescence of yeast at 488nm, b graph b shows red fluorescence of yeast at 561nm, c graph a and b overlapped and combined graph;
FIG. 5 is a graph showing the comparative yields of itaconic acid produced by different strains and media.
The specific embodiment is as follows:
The technical contents of the present invention are further described below with reference to examples. It should be noted that, in the case of no conflict, the embodiments in the present disclosure are only some embodiments, but not all embodiments in the present disclosure. Based on the embodiments of the present invention, other embodiments that may be obtained by a person of ordinary skill in the art without making any inventive effort are within the scope of the present invention.
The yarrowia lipolytica Po1gKU delta used in the present invention is obtained by knocking out KU70 gene in yarrowia lipolytica Po1g strain purchased from Yeastern Biotech Co. Of Taiwan, china, and the construction method is referred to as Genetic engineering of an unconventional yeast for renewable biofuel and biochemical production.Journal of Visualized Experiments,2016,115,e54371..
The pYLEX1 plasmid used in the present invention was purchased from Yeastern Biotech co. The plasmid has an auxotroph screening gene leucine expression box, a marker gene Amp, a strong promoter hp4d and a terminator XPR2 term.
The method for measuring itaconic acid in fermentation liquor comprises the following steps:
Centrifuging the fermented culture solution, collecting 2mL of fermentation supernatant, and adding 1.5mL of 10%
HCl-CH 3 OH solution, esterified for 3h at 62℃and then added with 2mL of n-hexane, vigorously shaken for 1min to dissolve the dimethyl itaconate. After centrifugation (6000 r/min,5 min), the upper organic phase was poured into another clean bottle for GC-MS detection of the dimethyl itaconate content.
Taking 2mL of 100mg/L itaconic acid standard substance to carry out methyl esterification according to the method, and detecting the content of dimethyl itaconate obtained after the methyl esterification of the itaconic acid standard substance by GC-MS. And then taking 100mg/L of itaconic acid dimethyl ester standard substance to carry out GC-MS detection on the content of itaconic acid dimethyl ester. And finally, converting the content of the dimethyl itaconate of the two to obtain the methyl ester rate of methyl esterification according to the method, and calculating the yield of the itaconic acid in the sample according to the methyl ester rate and the content of the dimethyl itaconate after methyl esterification of the fermentation sample.
The invention is further illustrated by the following examples.
Example 1: construction of yarrowia lipolytica Strain producing itaconic acid
(1) And (3) carrying out gene synthesis according to the CAD gene nucleotide sequence shown in SEQ ID NO.1, and designing a primer pair to amplify the obtained CAD gene fragment.
The PCR reaction system was (recombinase from Nanjinouzan Biotechnology Co., ltd.):
| Component (A) | System of |
| Fragments | 1μL |
| CAD1-F/CAD2-R | 1. Mu.L/min |
| 2×phanta Max Master Mix | 10μL |
| H2O | ～20μL |
PCR reaction conditions: 95 ℃ for 5min;95 ℃ for 30s; 30s at 72 ℃;72 ℃ for 1min; 10min at 72℃for 30 cycles.
(2) And performing gel cutting purification on the amplified CAD gene, performing enzyme cutting on the original plasmid pYLEX by using restriction enzyme Pml1 to obtain a linear plasmid, and integrating the CAD gene fragment into the linear plasmid to obtain a recombinant plasmid pYLEX-CAD, wherein the specific construction schematic diagram is shown in FIG. 2.
(3) PCR was performed on recombinant plasmid pYLEX-CAD using primer PYL-F/R to obtain a fragment size of 1870bp band (FIG. 3 a).
(4) After the recombinant plasmid pYLEX-CAD in (2) was digested and linearized with Spe1, the linearized fragment was transformed into Po1g ku 70. Delta. By lithium acetate transformation, and the recombinant yarrowia lipolytica strain Po1g ku 70. Delta. CAD was obtained by screening on leucine defect plate YNB. The genomic DNA of the transformant strain Po1g ku70Δ -CAD was extracted, and PCR was performed using this as a template, and the primer was identical to that used in (3), and the results obtained were identical. The result shows that the linearized recombinant plasmid is successfully integrated on the chromosome genome of the original strain, and the genetically engineered strain Po1g ku70 delta-CAD is obtained.
Example 2 localization of CAD genes in yarrowia lipolytica peroxisomes
(1) EPTS1 sequence: CTGGGCCGAGGACGACGATCCAAGCTG, adding the sequence to a hrGFPO sequence (SEQ ID NO. 3) terminator in the form of a primer to obtain a hrGFPO-ePTS gene fragment, connecting the fragment to a plasmid pYLEX1 to construct a recombinant plasmid pYLEX1-hrGFPO-ePTS1, and transforming a yarrowia lipolytica strain Po1g ku70 delta to obtain a recombinant strain.
After verification, transformants were picked and cultured in a shaker containing 50mL YPD flasks at 30℃for 24 hours, and 1mg/mL acetone-nile red solution was added to the culture and incubated at room temperature in the absence of light for 1 hour. The stained cells were washed with physiological saline and resuspended in potassium phosphate buffer (pH 7.4).
The resuspended cell fluid was plated directly onto a glass slide, and the cover glass was covered under a laser scanning confocal microscope to observe GFP at 488nm and Nile red at 561nm, respectively. Here we chose to use nile red staining to determine the localization of peroxisomes in yarrowia lipolytica, as it is a dry dye that can stain intracellular lipids. The green fluorescence and red fluorescence can be almost overlapped to be yellow, so that ePTS can be proved to play a role in positioning peroxisomes in yarrowia lipolytica (see FIG. 4:a for details, FIG. 488nm for green fluorescence of yeast, FIG. b for red fluorescence of yeast at 561nm, and FIG. c for a-b overlap).
(2) The ePTS sequence was added as a primer to the CAD gene terminator sequence shown in SEQ ID NO.1 to obtain a CAD-ePTS gene fragment, and the CAD-ePTS gene fragment was integrated into pYLEX linear plasmid by the same method as in step (2) of example 1 to construct recombinant plasmid pYLEX-CAD-ePTS 1, and after E.coli amplification, yarrowia lipolytica Po1g ku 70. Delta. Was transformed to verify that the correct strain was designated Po1g ku 70. Delta. -CAD-ePTS1 (FIG. 3 b).
The hrGFPO or CAD coding gene is used as a template to amplify hrGFPO-ePTS1 or CAD-ePTS1 gene fragments, and the primers are as follows:
Example 3 Yeast lipolytic production of itaconic acid
(1) The strains Po1g ku70Δ -CAD-ePTS and Po1g ku70Δ -CAD which are verified to be correct are respectively used as production strains, transformants are selected and cultured in 5mL YPD test tubes for 24 hours, 250 μl of culture solution is further taken and cultured in 25mL YPD triangular flasks for 16 hours, a proper amount of culture solution is taken and cultured in 50mL YPO or YPD culture medium to enable OD 600 to reach 0.1, and fermentation is carried out for 4 days (pH is not controlled and the culture is carried out in a shaking incubator at 30 ℃ C., 220 r/min).
(2) Centrifuging the fermented culture solution, adding 1.5mL 10% HCl-CH 3 OH solution into 2mL of fermentation supernatant, esterifying at 62 ℃ for 3h, adding 2mL of n-hexane, and shaking vigorously for 1min to dissolve dimethyl itaconate. After centrifugation (6000 r/min,5 min), the upper organic phase was poured into another clean bottle for detection. 3 parts of 2mL of 100mg/L itaconic acid standard was methyl esterified as described above, and the area analyzed with 100mg/L dimethyl itaconate standard was converted to the average area analyzed by GC-MS.
(3) 0.6. Mu.L of the organic phase was analyzed by GC-MS using Agilent 7890A GC and 5975C MSD equipped with an HP-5MS chromatography column (30 m 0.25mm 0.25 micron, agilent, santa Clara, calif.). The GC oven temperature was initially held at 60 ℃ for 2 minutes, then raised to 250 ℃ at a rate of 10 ℃/minute and held for 9 minutes. The split ratio was 10:1. Helium was used as the carrier gas and the inlet pressure was 13.8psi. The injector temperature was maintained at 250 ℃ and the ion source temperature was set at 220 ℃. Final data analysis was performed using enhanced data analysis software (Agilent, SANTA CLARA, CA, USA). The results are shown in the following table and fig. 5:
production of itaconic acid (mg/L) on day 4 using different strains and media
The invention confirms the possibility of producing itaconic acid by introducing CAD gene in yarrowia lipolytica in a heterologous way, and the yield is 33.12mg/L. Meanwhile, the CAD gene is subjected to heterologous localization expression in the peroxisome of yarrowia lipolytica for the first time, and itaconic acid is produced by consuming fatty acid of waste kitchen oil, so that compared with a bacterial strain which only expresses the CAD gene in a heterologous way, the yield of itaconic acid is obviously improved, and the yield is 1.58g/L and is improved by 48 times. The specific results are shown in FIG. 5, in which the second row of dataThe strain Po1g ku70Δ -CAD-ePTS1 was fermented in YPO medium, data on line three/>The strain Po1g ku70Δ -CAD-ePTS1 was fermented in YPD medium, data on line four/>The strain Po1g ku70Δ -CAD was fermented in YPO medium.
The use of kitchen waste oil compartmentalized localization of peroxisomes was determined to provide a significant increase in itaconic acid yield in yarrowia lipolytica, demonstrating that this approach both reduces cost and increases yield. It is believed that by further development of the above studies, yarrowia lipolytica must play an increasingly important role in the production of carboxylic acids, ultimately becoming the best microbial cell factory for the industrial production of carboxylic acids.
The above examples merely represent a few embodiments of the present invention, which are described in more detail and are not to be construed as limiting the scope of the patent. It should be noted that, for a person skilled in the art, the above embodiments may also make several variations, combinations and improvements, without departing from the scope of the present patent. Therefore, the protection scope of the patent is subject to the claims.
SEQUENCE LISTING
<110> University of Tianjin science and technology
<120> Engineering strain of yarrowia lipolytica for producing itaconic acid by subcellular compartmentalization and application thereof
<130> 1
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 1473
<212> DNA
<213> Artificial sequence
<400> 1
atgaccaaac aatctgcgga cagcaacgca aagtcaggag ttacggccga aatatgccat 60
tgggcatcca acctggccac tgacgacatc ccttcggacg tattagaaag agcgaaatac 120
ctgattctcg atggtattgc atgtgcctgg gttggtgcaa gagtgccttg gtcagagaag 180
tatgtgcagg caacaatgag ctttgagccg ccaggagcct gcagggtgat tggatatgga 240
cagaaactgg ggcctgttgc agcagccatg accaattccg ctttcataca ggccacagag 300
cttgacgact accacagcga agccccccta cactctgcaa gcatcgtcct ccctgcggtc 360
tttgcagcaa gtgaggtctt agccgagcaa ggcaaaacaa tttctggtat agatgtcatt 420
ctagccgcca ttgtggggtt tgaatctggc ccgcggatcg gcaaagcaat ttacggatcg 480
gacctcttga acaacggctg gcattgtgga gccgtgtatg gtgctccagc tggtgcgctg 540
gccacaggaa agctcctcgg tctaactcca gactccttgg aagatgctct cggaatcgca 600
tgcacgcaag cctgtggttt aatgtcggcg caatacggag gcatggtcaa gcgcgtgcaa 660
catggattcg cagcgcgtaa tggtcttctt gggggactgt tggcctatgg tgggtacgag 720
gccatgaagg gtgtcctgga gagatcttat ggcggtttcc tcaaaatgtt caccaagggc 780
aatggcagag agcctcccta caaagaggag gaagtggtgg ccggtctcgg ttcattctgg 840
cataccttta ctattcgcat caagctctat gcctgctgcg gacttgtcca tggtccagtc 900
gaagctatcg aaaagcttca gaggagatac cccgagctct tgaatagagc caacctcagc 960
aacattcgcc atgtttatgt acagctttca acagcctcga acagtcactg tggatggata 1020
ccagaggaga gacccatcag ttcaatcgca gggcagatga gtgtcgcata catcctcgcc 1080
gtccagctgg tcgaccagca atgtcttctg gctcagtttt ctgagtttga tgacaacttg 1140
gagagaccag aagtgtggga tctggccagg aaggttactc catctcatag cgaagagttt 1200
gatcaagacg gcaactgtct cagtgcgggt cgcgtgagga ttgagttcaa cgatggctct 1260
tctgttacgg aaactgtcga gaagcctctt ggagtcaaag agcccatgcc aaacgaacgg 1320
attctccaca aataccgaac ccttgcgggt agcgtgacgg acgaatcccg ggtgaaagag 1380
attgaggatc ttgtcctcag cctggacagg ctcaccgaca ttagcccatt gctggagctg 1440
cttaattgtc ccgtgaaatc gccactggta taa 1473
<210> 2
<211> 27
<212> DNA
<213> Artificial sequence
<400> 2
ctgggccgag gacgacgatc caagctg 27
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<211> 738
<212> DNA
<213> Artificial sequence
<400> 3
atggtgtcta agcagatcct gaagaacact ggcctgcaag agatcatgtc tttcaaggtg 60
aacctggagg gggtggtgaa caaccatgtg ttcaccatgg agggctgtgg caagggcaac 120
atcctgtttg gcaatcagct ggtgcagatc agagtgacca agggagcccc cctgcccttt 180
gcctttgaca tcctgtctcc tgcctttcag tatggcaaca gaaccttcac caagtaccct 240
gaggacatct ctgacttctt cattcagtct ttccctgctg gctttgtgta tgagagaacc 300
ctgagatatg aggatggagg cttggtggag atcagatctg acatcaatct gattgaggag 360
atgtttgtgt acagagtgga gtacaagggc agaaacttcc ccaatgatgg ccctgtgatg 420
aagaagacca tcactggcct gcagccctct tttgaggtgg tgtacatgaa tgatggagtg 480
ctggtgggcc aagtgatcct ggtgtacaga ctgaactctg gcaagttcta ctcttgtcac 540
atgagaaccc tgatgaagtc taagggagtg gtgaaggact tccctgagta ccacttcatt 600
cagcacagac tggagaagac ctatgtggag gatggaggct ttgtagagca gcatgagact 660
gccattgctc agctgacctc tctgggcaag cccctgggct ctctgcatga gtgggtgcac 720
caccaccacc accactaa 738
Claims (7)
1. A yarrowia lipolytica genetically engineered bacterium, which is characterized in that the genetically engineered bacterium is obtained by taking yarrowia lipolytica as an original strain and introducing a cis-aconitic acid decarboxylase encoding gene; the nucleotide of the cis-aconitic acid decarboxylase coding gene is shown as a sequence table SEQ ID NO. 1; the cis-aconitate decarboxylase coding gene is subjected to positioning expression in a peroxisome through an enhanced peroxisome positioning signal ePTS;
The yarrowia lipolytica starting strain is yarrowia lipolytica (Yarrowia lipolytica) Po1gku delta.
2. The genetically engineered yarrowia lipolytica of claim 1, wherein the cis-aconitate decarboxylase encoding gene is expressed in a host cell by a plasmid.
3. The genetically engineered yarrowia lipolytica of claim 2 wherein said plasmid is pYLEX a.
4. The genetically engineered yarrowia lipolytica strain of claim 1, wherein the recombinant plasmid pYLEX-CAD-ePTS 1 is constructed by ligating ePTS a coding gene to a cis-aconitate decarboxylase coding gene and the recombinant plasmid is introduced into a yarrowia lipolytica starting strain for expression.
5. The genetically engineered yarrowia lipolytica strain of claim 1, wherein the ePTS's 1 coding sequence is shown in sequence table SEQ ID No. 2.
6. Use of a yarrowia lipolytica genetically engineered bacterium of any one of claims 1-5 for the production of itaconic acid.
7. The use according to claim 6, wherein the method for producing itaconic acid by using the engineering bacteria comprises the following steps:
Inoculating the engineering bacteria seed liquid into YPD culture medium according to 1% inoculum size, culturing for 15-18h at 28-30deg.C under 200-220r/min, transferring into YPO culture medium to make OD 600 reach 0.1, fermenting at 28-30deg.C under 200-220r/min for 6 days;
The YPO culture medium comprises the following components: 18-22g/L peptone, 8-12g/L yeast extract powder, 1.1-1.2% waste kitchen oil, 0.18-0.22% Tween 80, and the balance water, sterilizing at 115-121 ℃ for 20min.
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| CN110106209A (en) * | 2019-05-09 | 2019-08-09 | 山东大学 | A method of synthesis terpenoid is positioned using Yarrowia lipolytica approach |
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| CN110106209A (en) * | 2019-05-09 | 2019-08-09 | 山东大学 | A method of synthesis terpenoid is positioned using Yarrowia lipolytica approach |
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| 无.Aspergillus terreus var. terreus strain HAT418 cis-aconitic acid decarboxylase gene,complete cds.《GENBANK》.2021,序列部分. * |
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