CN113564064B - Genetic engineering improvement method for improving carbon source conversion rate of pichia pastoris - Google Patents
Genetic engineering improvement method for improving carbon source conversion rate of pichia pastoris Download PDFInfo
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Abstract
The invention discloses a genetic engineering improvement method for improving the carbon source conversion rate of pichia pastoris, and belongs to the technical field of genetic engineering. The invention knocks out or silences and expresses 1, 3-beta-D-glucan synthase related gene PAS_chr2-1_0661 and alpha-1, 2-mannose transferase related gene PAS_chr3_0370 in Pichia pastoris GS115, so that the carbon source conversion rate of the constructed genetically engineered bacterium is improved by 2.23-2.41 times compared with that of the original strain. Under the same fermentation condition, the chassis cells Pichia pastoris GS delta PAS_chr3_0370delta PAS_chr2-1_0661 constructed by the method consume less carbon source compared with Pichia pastoris GS, so that the growth rate of thalli is faster, the expression quantity of exogenous proteins is not influenced, and the method is more suitable for high-density fermentation.
Description
Technical Field
The invention relates to a genetic engineering improvement method for improving the carbon source conversion rate of pichia pastoris, belonging to the technical field of genetic engineering.
Background
Commonly used protein expression systems include E.coli expression systems, mammalian cells, insect expression systems, saccharomyces cerevisiae expression systems, and Pichia expression systems. Coli expression systems are incapable of expressing complex proteins and have low exogenous expression yields. The operation of the mammalian cell and insect expression system is complex, the expression level is low, the production cost is high, and the method is not suitable for large-scale popularization. The Saccharomyces cerevisiae expression system is not stable enough, the exogenous plasmid is easy to lose in the expression process, and the secretion efficiency is poor. Pichia expression systems have significant advantages over other expression systems in expressing foreign proteins. Pichia pastoris (Pichia pastoris) exogenous gene expression system is a widely used expression system, and P.pastoris is a methylotrophic yeast capable of utilizing methanol as the sole carbon and energy source. Pichia pastoris is a single-cell eukaryote, is easy to carry out molecular genetic manipulation on the Pichia pastoris, has strong adaptability and is easy to culture. When the exogenous gene is introduced into P.pastoris, the exogenous gene can be integrated into the P.pastoris genome, and the exogenous gene is copied and inherited together with the chromosome, so that the loss rate of the exogenous gene is low. The pastoris has alcohol oxidase gene promoter, strong inducibility and strong promoter, can grow fast in culture medium to reach high cell concentration, and is suitable for high level induction expression of exogenous gene. And P.pastoris expresses the exogenous protein, and simultaneously has little protein quantity secreted by self, thereby being beneficial to the subsequent purification of the exogenous protein, and simultaneously has little toxic and side effect on host bacteria due to the accumulation of self products, and being suitable for large-scale high-density fermentation.
In recent decades, the biopharmaceutical field has been the large-scale cultivation of pichia pastoris as one of the key technologies, which is continuously driving the development of the bio-industry. Currently, more and more researchers prefer pichia pastoris as an expression system of the foreign protein, more than 5000 foreign proteins such as interleukin, interferon, antibody and the like are successfully expressed in the pichia pastoris expression system, and more than 70 foreign proteins are commercially produced. Increasing the commercial value of pichia pastoris expression systems is a current research hotspot. Lipase surface display efficiency was studied by knocking out P.pastoris GS 115. Alpha. -1, 2-mannosyltransferase PAS_chr3_0370 in university of North China university-related laboratories, but strain growth and carbon source yield were not studied.
Disclosure of Invention
[ technical problem ]
The conversion rate of the carbon source of the pichia pastoris is low, so that the growth rate of cells and the yield of target products are influenced.
Technical scheme
The first object of the present invention is to provide a genetically engineered bacterium which uses Pichia pastoris (Pichia pastoris) as an initial strain and does not express 1, 3-beta-D-glucan synthase and alpha-1, 2-mannose transferase.
In one embodiment, the Pichia is Pichia Pichia pastoris GS or Pichia pastoris x33.
In one embodiment, the nucleotide sequence of the gene encoding 1, 3-beta-D-glucan synthase is shown in SEQ ID NO.1 and the nucleotide sequence of the gene encoding alpha-1, 2-mannose transferase is shown in SEQ ID NO. 2.
In one embodiment, the genetically engineered bacterium further expresses a target gene.
In one embodiment, the gene of interest is selected from, but not limited to: genes encoding immunoglobulins, antibody fragments, kringles domain of human plasminogen, erythropoietin, cytokines, clotting factors, soluble IgE receptor alpha-chains, urokinase, chymotrypsin, urease inhibitors, IGF-binding proteins, epidermal growth factor, growth hormone-releasing factor, annexin V fusion protein, angiogenesis inhibitor, vascular endothelial growth factor-2, bone marrow precursor inhibitor-1, osteoprotegerin, alpha-1 antitrypsin, dnase II, alpha fetoprotein, insulin, fc-fusion or HSA-fusion.
The second object of the present invention is to provide an application of the above genetically engineered bacterium in expressing a target protein.
In one embodiment, the protein of interest is selected from, but not limited to: immunoglobulins, antibody fragments, kringle domains of human plasminogen, erythropoietin, cytokines, clotting factors, soluble IgE receptor alpha-chains, urokinase, chymotrypsin, urease inhibitors, IGF-binding proteins, epidermal growth factor, growth hormone-releasing factor, annexin V fusion proteins, angiogenesis inhibitors, vascular endothelial growth factor-2, bone marrow precursor inhibitor-1, osteoprotegerin, alpha-1 antitrypsin, dnase II, alpha fetoprotein, insulin, fc-fusions and HSA-fusions.
In one embodiment, the immunoglobulin comprises IgG, an IgG fragment, an IgG fusion, igM,
In one embodiment, the cytokines include interferon alpha, interferon beta, and interferon gamma.
It is a third object of the present invention to provide a method for increasing the conversion rate of a carbon source of Pichia pastoris by silencing or knocking out a 1, 3-beta-D-glucan synthase-related gene and/or an alpha-1, 2-mannosyltransferase-related gene on the Pichia pastoris genome.
In one embodiment, the pichia is pichia pastoris GS115 and or pichia X33.
In one embodiment, the nucleotide sequence of the gene encoding the 1, 3-beta-D-glucan synthase is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the alpha-1, 2-mannose transferase is shown in SEQ ID NO. 2.
In one embodiment, the method of knockout is Red homologous recombination technology, zinc finger nuclease technology, transcription activator-like effector technology, or clustered regularly interspaced short palindromic repeats/CRISPR-associated protein systems; silencing is by RNA interference or antisense oligonucleotide technology.
In one embodiment, the knockout is a method of using Crisper-Cas9 to knock out the gene encoding the 1,3- β -D-glucan synthase and the gene encoding the α -1, 2-mannosyltransferase on the Pichia genome. .
In one embodiment, the silencing is a method of silencing using antisense mRNA such that the gene encoding the 1, 3-beta-D-glucan synthase and the gene encoding the alpha-1, 2-mannosyltransferase are silenced and not translated normally.
The invention has the beneficial effects that:
1. the invention discovers that the synthesis of glucan and mannoprotein is reduced, the consumption of cells per se is reduced, the conversion rate of carbon sources is improved, and ideal chassis cells are constructed by knocking out the related genes PAS_chr2-1_0661 of 1, 3-beta-D-glucan synthase and the related genes PAS_chr3_0370 of alpha-1, 2-mannoprotein in pichia pastoris.
2. The genetically engineered bacterium constructed by the invention has high growth speed and high carbon source yield, and the carbon source conversion rate is improved by 2.23-2.41 times compared with the original strain. Under the same fermentation condition, the chassis cells Pichia pastoris GS delta PAS_chr3_0370delta PAS_chr2-1_0661 constructed by the method consume less carbon source compared with Pichia pastoris GS, so that the growth rate of thalli is faster, the expression quantity of exogenous proteins is not influenced, and the method is more suitable for high-density fermentation.
Drawings
FIG. 1 shows the results of nucleic acid electrophoresis of a successful construction of Pichia pastoris GS DeltaPAS_chr2-1_0661 knockout strain in Pichia pastoris;
FIG. 2 shows the results of nucleic acid electrophoresis of a strain which was successfully constructed as Pichia pastoris GS DeltaPAS_chr3_0370 DeltaPAS_chr2-1_0661 knockdown in Pichia pastoris;
FIG. 3 Pichia cell dry weight and OD 600 Is a standard curve of (2).
Detailed Description
YPD liquid medium: 1% of yeast extract, 2% of peptone and 2% of glucose.
The pPpT4 pHTX1-PARS1-hsCas9 plasmid: is disclosed in article Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris, DOI:10.1016/j.jbiotec.2016.03.027.
pPICZ alpha A plasmid: plasmid sequence and map reference to pprizαa (or ppriz (alpha) a) in snapge.
The method for measuring the carbon source yield comprises the following steps: taking pichia pastoris GS115 bacterial liquid, diluting to the corresponding OD 600 (1, 5, 10, 20), 10mL of the extract is taken from the extract and placed in a beaker, the extract is dried and weighed (the mass of the beaker is weighed in advance), the dry weight of bacteria is calculated, and the extract is drawnPichia pastoris cell dry weight and OD 600 Is a standard curve of (2). The glucose content in the initial medium of pichia pastoris GS115, gs115Δpas_chr3_0370Δpas_chr2-1_0661 was measured, and the residual sugar content in the medium was measured after 36 hours of cultivation, thereby calculating the concentration of the consumed carbon source. Based on pichia pastoris cell dry weight and OD 600 Standard curve formula of (2) and OD of 36h 600 And calculating the dry weight of the bacterial liquid, and finally calculating according to a carbon source yield formula to obtain the carbon source yield. Carbon source yield equation (Y) X/S Is the yield g/g of carbon source; Δx is the cell increment g; Δs is the consumed carbon source amount g) as follows:
Y X/S ＝ΔX/ΔS (1)
the relation between the carbon source yield and the cell increment and the carbon source consumption is described in the formula (1).
Example 1: construction of high carbon source yield Pichia pastoris genetic engineering bacteria
Taking the genome of pichia pastoris Pichia pastoris GS as a template, respectively utilizing the PAS_chr3_0370 upstream positive and negative primer and the PAS_chr3_0370 downstream positive and negative primer for PCR amplification to obtain the PAS_chr3_0370 upstream and downstream homologous arms, and after sequencing verification is successful, utilizing the PAS_chr3_0370 upstream positive primer and the PAS_chr3_0370 downstream negative primer for PCR amplification to construct an overlapped arm by taking the upstream and downstream homologous arms mixed in equal proportion concentration as the template; the pPpT4 pHTX1-PARS1-hsCas9 plasmid is used as a template, an upstream primer and a downstream primer are constructed by using a plasmid with a 20bp guide sequence carried at the 5' end for PCR amplification to obtain a linearized plasmid, the obtained linearized plasmid is connected and transformed into escherichia coli Escherichia coli JM by using T4 ligase at 4 ℃ overnight for amplification, and the plasmid is extracted and sequenced for verification.
The plasmid with correct sequence and the overlapped arm are selected and electrically transferred to P.pastoris GS115, the electric transformation method is well known in the art, the transformant is coated on a YPD plate, the YPD plate is cultured for 36 hours at 30 ℃, a monoclonal is selected and is subjected to colony PCR verification by using a recombinant plasmid to test an upstream primer and a downstream primer (the result of nucleic acid electrophoresis is shown in figure 1), and finally the genetically engineered bacterium P.pastoris GS115 delta PAS_chr3_0370 with alpha-1, 2-mannose transferase PAS_chr3_0370 deleted is obtained.
The primer is as follows:
TABLE 1 primers for knocking out PAS_chr3_0370 Gene
TABLE 2 primers for knocking out PAS_chr2-1_0661 Gene
The overlapping arm and the knockout plasmid of PAS_chr2-1_0661 are constructed by adopting a similar method, the plasmid and the overlapping arm which are properly selected and sequenced are transformed into P.pastoris GS115 delta PAS_chr3_0370 for secondary knockout, and the recombinant strain Pichia pastoris GS delta PAS_chr3_0370 delta PAS_chr2-1_0661 is obtained, and the result of nucleic acid electrophoresis is shown in figure 2.
Example 2: determination of the carbon source yield of Pichia pastoris GS DeltaPAS_chr3_0370 DeltaPAS_chr2-1_0661
Taking pichia pastoris Pichia pastoris GS and Pichia pastoris GS delta PAS_chr3_0370 delta PAS_chr2-1_0661 constructed in example 1 as production strains, inoculating single colony into 5mL YPD culture medium, and culturing at 30 ℃ under 200rmp condition for overnight to obtain seed culture solution; seed culture was inoculated into 50mL of YPD medium to give an initial OD 600 =0.4, cultured at 30 ℃,200rmp for 48h. After the culture is finished, the corresponding bacterial liquid is taken and diluted to the corresponding OD 600 (1, 5, 10, 20), 10mL of the extract is taken from the extract in a beaker, the extract is dried and weighed (the mass of the beaker is weighed in advance), the dry weight of bacteria is calculated, and the dry weight and OD of pichia pastoris cells are drawn 600 And gives the standard curve formula y=0.0036 x-0.0002, r 2 =0.99 (fig. 3).
The glucose content in the initial medium of pichia pastoris GS115 and gs115Δpas_chr3_0370Δpas_chr2-1_0661 was measured, and the residual sugar content in the medium was measured after 36 hours of cultivation, thereby calculating the concentration of the consumed carbon source. Based on the dry weight of Pichia pastoris cellsAnd OD (optical density) 600 Standard curve formula of (2) and OD of 36h 600 And calculating the dry weight of the bacterial liquid, and finally calculating according to a carbon source yield formula to obtain the carbon source yield.
Tables 3P.pastoris GS115 and Pichia pastoris GS ΔPAS_chr3_0370 ΔPAS_chr2-1_0661 carbon source yield
Example 3 effects of knockdown of PAS_chr3_0370 and PAS_chr2-1_0661 on exogenous Gene expression
The green fluorescent protein gene gfp (nucleotide sequence shown as SEQ ID NO. 3) was synthesized, ligated with EcoR1 endonuclease cut and purified linearized plasmid pPICZαA, and electrotransformed into Pichia pastoris GS115 and Pichia pastoris GS115 ΔPAS_chr3_0370ΔPAS_chr2-1_0661 constructed in example 1, respectively, coated on YPD plates, incubated overnight at 30℃and single colonies were picked for sequencing verification. Inoculating the single colony which is verified to be correct into 5mL YPD culture medium, and culturing at 30 ℃ and 200rmp for overnight to obtain seed culture solution; seed culture was inoculated into 50mL of YPD medium to give an initial OD 600 After incubation at 30℃and 200rmp for 48h, 2mL of methanol was added for 24h of induction, and 1mL of the bacterial liquid was taken for fluorescence intensity measurement. The comparison of the fluorescence intensities of the thalli measured by an enzyme-labeled instrument shows that the GFP expression levels of Pichia pastoris GS and Pichia pastoris GS delta PAS_chr3_0370delta PAS_chr2-1_0661 of the overexpressed GFP are not obviously different, and the knocked-out of the related genes PAS_chr2-1_0661 of the 1, 3-beta-D-glucan synthase and the related genes PAS_chr3_0370 of the alpha-1, 2-mannose transferase does not influence the expression level of exogenous genes.
Example 4: antisense mRNA silencing
The antisense RNA sequence of the non-coding sequence of the gene 5' UTR can be specifically combined with mRNA to form dsRNA, and the ribosome cannot translate dsRNA, so that translation cannot be performed normally. The principle of antisense RNA interference of 5' UTR is utilized to silence the related gene PAS_chr2-1_0661 of 1, 3-beta-D-glucan synthase and the related gene PAS_chr3_0370 of alpha-1, 2-mannose transferase, thus obtaining the target strain A. Antisense mRNA silencing is a conventional molecular biology approach in the art and can be referred to "RNA interference and small RNA analysis" in chapter 18 of the molecular cloning guide, fourth edition, book of molecular biology authorities.
GS115 and the target strain A obtained were cultured in the same manner as in example 2, and the carbon source yields were measured as shown in the following Table:
table 4 P.pastoris GS115 carbon source yield of Strain A
Example 5: high density fermentation Pichia pastoris GS ΔPAS_chr3_0370 ΔPAS_chr2-1_0661
Pichia pastoris GS115 and Pichia pastoris GS 115.DELTA.PAS_chr3_0370ΔPAS_chr2-1_0661 constructed in example 1 were inoculated into 5mL of YPD liquid medium, respectively, and cultured at 30℃for 200mL overnight to obtain seed culture broth, and the seed culture broth was inoculated into 50mL of YPD medium to give an initial OD 600 After culturing at 30deg.C and 200rmp for 24 hr, inoculating 50mL of the whole bacterial liquid into 1L of YPD medium in a 2L fermentation tank to perform high density fermentation, and fermenting for 48 hr to obtain OD of Pichia pastoris GS ΔPAS_chr3_0370ΔPAS_chr2-1_0661 600 Up to about 260 OD of Pichia pastoris GS115 600 About 230% methanol was added to the fermenter, and the OD of the mixture was found to be equal to or greater than about 2% in the methanol induction period Pichia pastoris GS. DELTA.PAS_chr3_0370. DELTA.PAS_chr2-1_0661 600 Stabilized around 240, while the OD of Pichia pastoris GS115 was 600 The stability is about 200, the improvement is about 20%, and the knocking-out of the related genes PAS_chr2-1_0661 of the 1, 3-beta-D-glucan synthase and the related genes PAS_chr3_0370 of the alpha-1, 2-mannose transferase is proved to promote the utilization of the pichia pastoris to the methanol, so that the cell conversion rate is higher and the tolerance is better under the induction of the methanol.
Compared with Pichia pastoris GS, the Pichia pastoris GS delta PAS_chr3_0370 delta PAS_chr2-1_0661 constructed by the method is more suitable for high-density fermentation, and is mainly characterized in that Pichia pastoris GS delta PAS_chr3_0370 delta PAS_chr2-1_0661 consumes fewer carbon sources, the conversion rate of the carbon sources to the dry weight of cells is higher, the number of cells generated under the same fermentation condition is more, and the tolerance to methanol is stronger.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of Jiangnan
<120> a genetic engineering improvement method for improving the carbon source conversion rate of Pichia pastoris
<130> BAA211030A
<160> 21
<170> PatentIn version 3.3
<210> 1
<211> 5637
<212> DNA
<213> Pichia pastoris
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gcccgacgaa aattcaaatt tgttgtggct atgcaaagat tgtctaagtt caaacctgag 3060
gaattagaaa acgctgagtt cttgctgagg gcttaccctg accttcaaat tgcttacctg 3120
gacgaagaac ctcctcttaa cgagggcgag gagcctaggt tatactcggc tttgattgat 3180
ggacattgtg aaattatgga gaatggtaga agaagaccta aatttagagt tcagctctct 3240
ggtaacccaa ttttgggtga tggtaaatct gacaaccaaa atcattctat catctttact 3300
cgtggtgagt acattcagtt gattgatgcg aaccaggata actatttgga agaatgtttg 3360
aagattagat ctgttttggc tgagtttgag gaactgaaca tcgaccatgt taacccttat 3420
actcctggtt tgaaatctga atttgacggt gttaaacacc ctgtagccat tgtgggtgcc 3480
agagaataca tcttctctgt taactccgga gttctgggtg acgttgctgc aggtaaggaa 3540
caaacctttg gtaccctgtt tgctcgtact ctggcccaga ttggtggtaa gctgcattat 3600
ggacatcccg attttttgaa taacatatac atgactactc gtggtggtgt ttctaaggca 3660
cagaagggac tacatttgaa tgaggatatt tatgctggta tgaccgccat gtgtcgtggt 3720
ggtagaatca agcattgtga gtactaccag tgtggtaaag gtagagatct tggttttggc 3780
tcgattttga atttcacaac caagattggt gccggtatgg gtgagcaaat gctttcccga 3840
gagtattact acctaggtac acaattgcca ctggaccgtt tcctttcatt ctactacgcc 3900
catcctggtt tccatattaa caatttattt attcagttgt cgcttcagat gtttatgttg 3960
actttagtca acctgaactc tttggcccac gagtcaatta tctgtcagta caacagaaat 4020
atacccatta cggatattat gtatcctgtt ggatgttata acctcatgcc taccattgac 4080
tggattagac gttatacgtt gtccattttc atcgttttct tcatttcttt cattccacta 4140
gctgtccaag aattgattga aaggggaatg tggaaggctg ctcagagatt ctgtcgtcat 4200
ttcatttctt tgtctccaat gttcgaagtt tttgttgctc agatctattc gtcatctctt 4260
gtgaacgatt tgaccgttgg tggtgctcgt tacatctcca ctggtcgtgg ctttgcaacc 4320
gcacgtatcc ccttctctgt gctttactca cgttttgctg actcgtcaat ttacatggga 4380
gctaggtcca tgctgctgtt actgttcggt actgtcgctc actggcaacc agcattattg 4440
tggttctggg catcattgtc ggctttgatg ttctctccat tcatcttcaa cccccaccaa 4500
tttgcttggc aagattactt tattgactac cgtgacttca taagatggtt gtctcgtggt 4560
aacaccagat ggcaccgtaa ctcctggatc ggttatgtac gtctttccag atctagaatc 4620
accggtttca agaggaagac cattaacgac gagtctgaaa aggctgctgg agatgcagcc 4680
agagctcatc gttccaacat tcttgttgcc gatttcattc cttccgctat ttacactgct 4740
ggttgcttta cagcttacac attcatgaac gctcagaatg gtgttgctaa tgctactgtt 4800
gtgaactctg tattgcgtat tgtcattgtc tcaattgctc caattgtggt cgatctggcc 4860
gtgttaggat tctgtgctgg tttagcttgt tgtgccggtc ctatgttggg tctgtgttgc 4920
aagaagactg gtgctgtgat ggctggtgta gctcacggta ttgctgtcgt cattcacatc 4980
attgctttca ttggtctatg ggtgttagag ggattcaact tcactaaaac actgatgggt 5040
ctcgcagcaa tgatttatat tcaacgtctg attttcaagt taatgacttt gaccatgttg 5100
actagagaat ttaagaatga tcattcgaac acttcattct ggactggtaa atggtacggt 5160
tctggactag gctggatggc ctggacccaa ccagcccgtg agtttgctgc aaaagttata 5220
gagatgtctg aatttgctgg tgacttcgtt ttaggacata ttctcttgtt ctgtcagttc 5280
cctctactgt gtgtccctct gattgacagg tggcactcaa tgatgttgtt ctggttgaag 5340
cctagtcgtc aaatcagacc accaatctac tcgttgaagc aagctcgtct tagaaagaga 5400
atggtgagaa actacctgac cctctacttc ttggtatttg ccatctttgt tgctatcatt 5460
gcaggtcctg cagctgcagc tggtcaagtg aatgtcgagg aactaactga acatttacca 5520
gagtttgctg aaggtttgat tcaaccaaga cgccaggaca ataacgatac aggaaacaac 5580
aacacctggg gatggactta tacaccatca acatcttcct ggtctaccaa ggcttaa 5637
<210> 2
<211> 1980
<212> DNA
<213> Pichia pastoris
<400> 2
atgtttggaa agaggaggca ggtaagaaaa ttacttatat gggttgtctt actgttgata 60
gtttactttt ttggtttgca atttagagcc aaaaactcag ctcatcagtc ttccatcagg 120
agcttttatg cggataacaa agagtttttt gatcggcaat acagcagata tgacgagtat 180
gatatcatag acaacatgaa tagccataac gagttactac aggaacagtt tcgcaatgga 240
aagctcgctg cgggactcag aggtgtagcc gaagaaccta attccgatga ggttacagat 300
gacactgcca tcgaagaaga cgagcaagct gcaatgatta atttcccgaa gagatctccc 360
cagagagaga agagtctagt tgagttacgc aagttttaca agaacgtgct ttcgattata 420
atcaacaaca aacctgctat gccaattgaa aatcctcgtg atcccacacc taacgaaaac 480
gcactcaaga gaaagttcgg taaaagtgga atcatcaaca ttgctttaca tgacaccgat 540
ccttcgctgc ctattctttc tgaagcgtat ctccgggact cgttgcagtt gagcccctct 600
tttattgcct cattgtccaa atcccacagc gccgtggtaa aggcgtttcc cccttccttt 660
cctgcgaatg catataatgg gacaggaatt gtatttattg gtggtcaaaa gttttcctgg 720
ttatcattac tttctatcga aaaccttagg aaaacaggat ccaaagtacc tgtcgagttg 780
ataatcccat ttgcacatga atatgaacct caattatgtg aagagatact accaaagttg 840
aatgctactt gcgttctttt gcaggagact gtgggaattg atctgcttaa atctggacat 900
ctcaaagggt accagtttaa gtcattagcg cttttggcct cctcttttga gcaggttcta 960
ctagtagact ctgataacat cattgttgaa aatccagacc ccatatttga ttcagaggtt 1020
tttcaacgga cgggattagt gttgtggcct gatttctgga ggcgcgttac ccatcctgat 1080
tactataaga ttgctggcat caagttgggc tctgaacgag taaggcatgt tgttgactcc 1140
tacactgacc cctcgttgta tacctctagt tcggaagatc cctttaccga tattcctttg 1200
catgataggg aaggggcaat tccagatggt tccacagaat ctggccaaat actaatatcc 1260
aagaccaaac attgccaaac gatccttcta tctttgtatt ataacttttt tggaccagat 1320
tactactacc ctctgttcac ccaaggagca agcggagaag gagataaaga gaccttttta 1380
gctgctgcta actactataa actaccattt tacaacatca agaaaggtgt ggacgtcatt 1440
ggatattgga agcctgacca atctgcatat cagggttgcg gtatgctgca atacgaccca 1500
atcgtagatt atcaaaactt acaaacattt ttaaagactc ataaaggctc aagggtcaac 1560
aaacttgaac agtcggagct agacaaaccc ggattactat ccagattaat accaaaattt 1620
ttctttcgca aaacttttga tgagcatcag cttcagagtc atttcaccaa ggacagatct 1680
aagatcatgt ttatacactc aaactttcca aaactagacc catttggatt gaagcttcac 1740
aactatcttt ttgtagatca agacactcat aaaccaagga taagaatgta tgcggaccaa 1800
acaggacttt catttgactt tgagctccgt caatggatta tcattcatga atacttttgt 1860
gagtacccag atttcaactt gaaatatttg gagaacgcta acgtgaagcc tcaagattta 1920
tgtatgttca tcaaggaaga gctaaacttt ttacagaata acccaataca attgacgtga 1980
<210> 3
<211> 660
<212> DNA
<213> artificial sequence
<400> 3
cggtgacgtc aacggccaca agttctccgt ctccggcgag ggtgagggcg acgccaccta 60
cggcaagctg accctgaagt tcatctgcac caccggtaag ctgccggtcc cgtggccgac 120
cctggtcacc accctgacct acggcgtcca gtgcttctcc cgctacccgg accacatgaa 180
gcgccacgac ttcttcaagt ccgccatgcc ggagggttac gtccaggagc gcaccatctc 240
cttcaaggac gacggtaact acaagacgcg tgccgaggtc aagttcgagg gcgacaccct 300
ggtcaaccgc atcgagctga agggcatcga cttcaaggag gacggtaaca tcctgggcca 360
caagctggag tacaactaca actcccacaa cgtctacatc accgcggaca agcagaagaa 420
cggcatcaag gccaacttca agacccgcca caacatcgag gacggtggcg tccagctagc 480
cgaccactac cagcagaaca ccccgatcgg cgacggcccg gtcctgctgc cggacaacca 540
ctacctgtcc acccagtccg ccctgtccaa ggacccgaac gagaagcgcg accacatggt 600
cctgctggag ttcgtcaccg ccgccggcat cacccacggc atggacgagc tgtacaagta 660
<210> 4
<211> 30
<212> DNA
<213> artificial sequence
<400> 4
ctagaaaaat gaaacgaaga accccaaccg 30
<210> 5
<211> 59
<212> DNA
<213> artificial sequence
<400> 5
atcgatcgat cgatcgatcg atcgatcgat cggggaagtt attgtgaatg cagaatgcg 59
<210> 6
<211> 61
<212> DNA
<213> artificial sequence
<400> 6
cgatcgatcg atcgatcgat cgatcgatcg atcttgctac tggtttatca cactaatgac 60
g 61
<210> 7
<211> 30
<212> DNA
<213> artificial sequence
<400> 7
gaacactttg gattgcactg atggaaaagc 30
<210> 8
<211> 20
<212> DNA
<213> artificial sequence
<400> 8
aggtaagaaa attacttata 20
<210> 9
<211> 52
<212> DNA
<213> artificial sequence
<400> 9
aggtaagaaa attacttata gttttagagc tagaaatagc aagttaaaat aa 52
<210> 10
<211> 40
<212> DNA
<213> artificial sequence
<400> 10
tataagtaat tttcttacct tttgatttgt ttaggtaact 40
<210> 11
<211> 33
<212> DNA
<213> artificial sequence
<400> 11
gagtaagctc gtcaggtaag aaaattactt ata 33
<210> 12
<211> 27
<212> DNA
<213> artificial sequence
<400> 12
accttgtcgt attatacgag ccggaag 27
<210> 13
<211> 31
<212> DNA
<213> artificial sequence
<400> 13
cggatgacgt tggttccaag ttcttccaag t 31
<210> 14
<211> 62
<212> DNA
<213> artificial sequence
<400> 14
atagatcaat ctatcgatct agagagctag ctagggaaag gattgcaggg cgtaggaggt 60
at 62
<210> 15
<211> 62
<212> DNA
<213> artificial sequence
<400> 15
agctagctct ctagatcgat agattgatct atggaaccgt tcacagggag aaatcaaatg 60
at 62
<210> 16
<211> 29
<212> DNA
<213> artificial sequence
<400> 16
gctggatgcc cctggagtag ttgatgact 29
<210> 17
<211> 20
<212> DNA
<213> artificial sequence
<400> 17
caatgagagt caagaaatct 20
<210> 18
<211> 52
<212> DNA
<213> artificial sequence
<400> 18
caatgagagt caagaaatct gttttagagc tagaaatagc aagttaaaat aa 52
<210> 19
<211> 41
<212> DNA
<213> artificial sequence
<400> 19
agatttcttg actctcattg gacgagctta ctcgtttcgt c 41
<210> 20
<211> 31
<212> DNA
<213> artificial sequence
<400> 20
gggcatagcc aatcttcccc gatgcgatta a 31
<210> 21
<211> 30
<212> DNA
<213> artificial sequence
<400> 21
acacaaagct gcagtgaaag caatatcttg 30
Claims (6)
1. A genetically engineered bacterium is characterized by using pichia pastorisPichia pastoris) GS115 is an original strain, and does not express 1, 3-beta-D-glucan synthase and alpha-1, 2-mannose transferase;
the nucleotide sequence of the gene for encoding the 1, 3-beta-D-glucan synthase is shown as SEQ ID NO.1, and the nucleotide sequence of the gene for encoding the alpha-1, 2-mannose transferase is shown as SEQ ID NO. 2.
2. The use of the genetically engineered bacterium of claim 1 for expressing a protein of interest.
3. The use according to claim 2, wherein the protein of interest is selected from the group consisting of: immunoglobulins, antibody fragments, kringle domains of human plasminogen, erythropoietin, cytokines, clotting factors, soluble IgE receptor alpha-chains, urokinase, chymotrypsin, urease inhibitors, IGF-binding proteins, epidermal growth factor, growth hormone-releasing factor, annexin V fusion proteins, angiogenesis inhibitors, vascular endothelial growth factor-2, bone marrow precursor inhibitor-1, osteoprotegerin, alpha-1 antitrypsin, dnase II, alpha fetoprotein, insulin, fc-fusions, HSA-fusions.
4. A method for improving the carbon source conversion rate of pichia pastoris is characterized in that the method is that 1, 3-beta-D-glucan synthase related genes and alpha-1, 2-mannose transferase related genes on pichia pastoris GS115 genome are silenced or knocked out, the nucleotide sequence of the genes for encoding the 1, 3-beta-D-glucan synthase is shown as SEQ ID NO.1, and the nucleotide sequence of the genes for encoding the alpha-1, 2-mannose transferase is shown as SEQ ID NO. 2.
5. The method of claim 4, wherein the method of knockout is Red homologous recombination technology, zinc finger nuclease technology, transcription activator-like effector technology or clustered regularly interspaced short palindromic repeats/CRISPR-associated protein systems; silencing is by RNA interference or antisense oligonucleotide technology.
6. The method of claim 5, wherein the knockout is a knockout of the gene encoding 1,3- β -D-glucan synthase and the gene encoding α -1, 2-mannosyltransferase on the pichia pastoris genome using the approach of Crisper-Cas 9; the silencing is that the antisense mRNA silencing method is used to silence the gene encoding 1, 3-beta-D-glucan synthase and the gene encoding alpha-1, 2-mannose transferase, so that the gene cannot be translated normally.
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| CN101195809A (en) * | 2006-12-07 | 2008-06-11 | 中国人民解放军军事医学科学院生物工程研究所 | Pichia pastoris strain with deletion of alpha-1,6-mannose transferase and construction method thereof |
| CN102120967A (en) * | 2010-12-09 | 2011-07-13 | 江南大学 | Preparation and application of OCH1 genetic flaw type P. pastoris X-33 bacterial strain |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101195809A (en) * | 2006-12-07 | 2008-06-11 | 中国人民解放军军事医学科学院生物工程研究所 | Pichia pastoris strain with deletion of alpha-1,6-mannose transferase and construction method thereof |
| CN102120967A (en) * | 2010-12-09 | 2011-07-13 | 江南大学 | Preparation and application of OCH1 genetic flaw type P. pastoris X-33 bacterial strain |
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| Title |
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| 双缺陷型毕赤酵母X33突变株的诱变育种;顾鹏飞等;食品与生物技术学报;第35卷(第2期);第129-135页 * |
| 毕赤酵母细胞壁多糖合成相关基因GAS1和MNN2对毕赤酵母表面展示效率的影响;朱俊;中国优秀硕士学位论文全文数据库基础科学辑(第07期);A006-135 * |
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