CN111635866A - Method for inhibiting cell aggregation of pichia kudriavzevii strain, construction and application - Google Patents
Method for inhibiting cell aggregation of pichia kudriavzevii strain, construction and application Download PDFInfo
- Publication number
- CN111635866A CN111635866A CN202010522228.2A CN202010522228A CN111635866A CN 111635866 A CN111635866 A CN 111635866A CN 202010522228 A CN202010522228 A CN 202010522228A CN 111635866 A CN111635866 A CN 111635866A
- Authority
- CN
- China
- Prior art keywords
- primers
- cell aggregation
- pichia
- strain
- primer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000004220 aggregation Methods 0.000 title claims abstract description 31
- 230000002776 aggregation Effects 0.000 title claims abstract description 30
- 241000235645 Pichia kudriavzevii Species 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 20
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 18
- 238000010276 construction Methods 0.000 title abstract description 9
- 239000004475 Arginine Substances 0.000 claims abstract description 18
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims abstract description 18
- 238000000855 fermentation Methods 0.000 claims abstract description 11
- 230000004151 fermentation Effects 0.000 claims abstract description 11
- 241000235648 Pichia Species 0.000 claims abstract description 7
- 241000235058 Komagataella pastoris Species 0.000 claims description 17
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 239000012634 fragment Substances 0.000 claims description 9
- 230000003321 amplification Effects 0.000 claims description 6
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 6
- 239000013612 plasmid Substances 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 101150008942 J gene Proteins 0.000 claims description 4
- 239000007222 ypd medium Substances 0.000 claims description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 101150050255 PDC1 gene Proteins 0.000 claims description 3
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 108010084455 Zeocin Proteins 0.000 claims description 3
- 108010083912 bleomycin N-acetyltransferase Proteins 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 238000010367 cloning Methods 0.000 claims description 3
- 238000001976 enzyme digestion Methods 0.000 claims description 3
- 239000013604 expression vector Substances 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 230000010354 integration Effects 0.000 claims description 3
- XIXADJRWDQXREU-UHFFFAOYSA-M lithium acetate Chemical compound [Li+].CC([O-])=O XIXADJRWDQXREU-UHFFFAOYSA-M 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC[C@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 230000002441 reversible effect Effects 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- 230000001131 transforming effect Effects 0.000 claims description 3
- 239000013598 vector Substances 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 239000002609 medium Substances 0.000 claims description 2
- 230000002018 overexpression Effects 0.000 claims description 2
- 235000015097 nutrients Nutrition 0.000 abstract description 8
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 abstract description 7
- 239000001301 oxygen Substances 0.000 abstract description 7
- 229910052760 oxygen Inorganic materials 0.000 abstract description 7
- 239000000126 substance Substances 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 6
- 239000002253 acid Substances 0.000 abstract description 5
- 230000003834 intracellular effect Effects 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 241000894006 Bacteria Species 0.000 abstract description 2
- 239000002028 Biomass Substances 0.000 abstract description 2
- 241000269331 Ambystoma Species 0.000 description 22
- 241000205573 Jeffersonia Species 0.000 description 11
- 239000004816 latex Substances 0.000 description 11
- 229920000126 latex Polymers 0.000 description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 150000007524 organic acids Chemical class 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 3
- ALRHLSYJTWAHJZ-UHFFFAOYSA-N 3-hydroxypropionic acid Chemical compound OCCC(O)=O ALRHLSYJTWAHJZ-UHFFFAOYSA-N 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 238000003892 spreading Methods 0.000 description 2
- QXKAIJAYHKCRRA-UHFFFAOYSA-N D-lyxonic acid Natural products OCC(O)C(O)C(O)C(O)=O QXKAIJAYHKCRRA-UHFFFAOYSA-N 0.000 description 1
- QXKAIJAYHKCRRA-FLRLBIABSA-N D-xylonic acid Chemical compound OC[C@@H](O)[C@H](O)[C@@H](O)C(O)=O QXKAIJAYHKCRRA-FLRLBIABSA-N 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 235000021107 fermented food Nutrition 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 235000021110 pickles Nutrition 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 150000005837 radical ions Chemical class 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 230000032895 transmembrane transport Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Mycology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Botany (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method, construction and application for inhibiting cell aggregation of a pichia kudriavzevii strain, which can remarkably promote the growth of the pichia kudriavzevii strain under an extreme acid condition by adding arginine, relieve the cell aggregation, facilitate the transfer of oxygen and nutrient substances and improve the production performance of the strain during low-pH fermentation. The invention also constructs the pichia kudriana for inhibiting cell aggregation, which is a recombinant bacterium for over-expressing the ARGJ, the intracellular arginine content is improved by 26.3 percent, the biomass is obviously improved under the condition of pH2.0, the cell aggregation effect is obviously relieved, and the transfer of nutrient substances and oxygen is facilitated.
Description
Technical Field
The invention relates to the technical field of yeast, and particularly relates to a method for inhibiting cell aggregation of pichia kudriavzevii strains, construction and application.
Background
When organic acid is produced by a fermentation method, along with accumulation of products, the organic acid enters microbial cells in a simple diffusion mode and is dissociated into acid radical ions and protons with charges, so that the intracellular pH (pHi) is reduced, the conformation of protein and the intracellular enzymatic reaction are influenced, active transmembrane transport using the protons as the driving force is also interfered, the absorption of nutrient substances by the microbial cells is influenced, and the fermentation performance is sharply reduced. The development of the low pH fermentation technology depending on an acid tolerant host can remarkably reduce or even abandon the use of a neutralizer, simplify the production process, reduce the risk of contamination, and save energy and material consumption, thereby realizing the construction of resource-saving and environment-friendly biological manufacturing. Pichia pastoris (Pichia kudriavzevii) is a common non-traditional yeast in food and plays an important role in the formation of flavor substances of external traditional fermented food in white spirit, pickles, cheese and the like. It has multiple tolerance to high temperature, osmotic pressure, ethanol, especially organic acid and low pH, and is considered as a potential new biotechnology host. In recent years, genetically engineered pichia kudriana has been reported in low pH fermentation of organic acids such as succinic acid, lactic acid, xylonic acid, and 3-hydroxypropionic acid and ethanol. However, Pichia kudriavzevii grows in a pseudo hyphal form under acidic conditions at pH less than 2.5 and aggregates with each other (as shown in FIG. 1), which is disadvantageous in the transfer of oxygen and nutrients and seriously affects the productivity of the strain in low pH fermentation.
Disclosure of Invention
The invention aims to provide a method, construction and application for inhibiting cell aggregation of pichia kudriavzevii strains. The invention can inhibit the cell aggregation of the pichia kudriavzevii strain and improve the fermentation performance.
In order to solve the technical problems, the technical scheme provided by the invention is as follows: a method for inhibiting the cell aggregation of a Pichia kudriavzevii strain is disclosed, wherein arginine is added to promote the growth of the Pichia kudriavzevii strain under the condition of low pH and relieve the cell aggregation.
The method for inhibiting the cell aggregation of the pichia kudriavzevii strain comprises the following components in mass concentration: glucose 20g/L, peptone 20g/L and yeast extract 10g/L, adjusting YPD medium pH to 2.5 or less with HCl or NaOH, autoclaving, and adding 5mmol/L arginine to the medium.
A Pichia pastoris for inhibiting cell aggregation, wherein a Pichia pastoris strain is a Pichia kudriavzevii N-X and over-expression ARG J gene construction recombinant strain; pichia kudriavzevii N-X is deposited in the China Center for Type Culture Collection (CCTCC); address: eight-path Lojia mountain in Wuchang district, Wuhan city, Hubei province, with the preservation number: CCTCC M2017759.
The application of the pichia kudriavzevii inhibiting cell aggregation in low-pH fermentation is provided.
The construction method of pichia kudriana for inhibiting cell aggregation comprises the following steps:
a. amplifying by using primers Pdc1F, Pdc1R and pichia pastoris N-X genomic DNA as templates to obtain a fragment containing a PDC1 gene promoter, a reading frame and a terminator, and connecting the fragment into a pMD19T vector; the primer Pdc1F is CGCGGATCCGCGTTTAAGTGGTGGTTGTA, and the primer Pdc1R is CGG GGTACCTGGT GAGATTGATGGATTGT;
b. reverse amplification is carried out by using primers 0-F and 0-R; the primers 0-F are ACAGATAAGTACCTAGGGAGATT, and the primers 0-R are TGACATCT GAATGTAAAATGAAC;
c. primers 1-F and 1-R and a plasmid pGAPZB are used as templates for amplification to obtain bleomycin resistance gene BLE and a CYC1 terminator; the primers 1-F are acacagcaaaacacaaaaatATG GCCAAGTTGACCAGTGC; the primers 1-R are tatcgaaatctagcccGCAAATTAAAGCCTTCGA GC;
d. amplifying by using primers 2-F and 2-R and genome DNA as a template to obtain a pGAP promoter; the primer 2-F is tcgaaggctttaatttgcGGGCTAGATTTCGATATGGAT; the primers 2-R are gtagatt gcggaggacatTTTTTGTAATTGTGTTTGTTTGTGT;
e. amplifying by using primers 3-F and 3-R to obtain a target gene ARGJ; the primer 3-F is caaacacaattacaaaaaATGTTAAGACATGTTGCA; the primer 3-R is cattttacattcagatgtcaTCACGACCTGTAGTCTCCA;
f. using a Clon express Multi S One Step Cloning Kit to perform One-Step connection on the fragments to construct and obtain a recombinant integration expression vector pARGJ; and finally, transforming the recombinant plasmid subjected to BamH I enzyme digestion into an N-X strain by using a lithium acetate method, and screening positive transformants on a YPD plate containing 180 mu g/ml Zeocin to obtain the recombinant strain for over-expressing the ARG J.
Compared with the prior art, the invention can obviously promote the growth of the pichia kudriana under the extreme acid condition by adding the arginine, relieve the cell aggregation, facilitate the transfer of oxygen and nutrient substances and improve the production performance of the strain during low pH fermentation. The invention also constructs the pichia kudriana for inhibiting cell aggregation, which is a recombinant strain for over-expressing the ARGJ, the intracellular arginine content of the recombinant strain is improved by 26.3 percent, the biomass is obviously improved under the condition of pH2.0, the cell aggregation effect is obviously relieved, and the transfer of nutrient substances and oxygen is facilitated.
Drawings
FIG. 1 is a schematic representation of Pichia pastoris growth and inter-aggregation in pseudohyphal form under acidic conditions at pH less than 2.5;
FIG. 2 is a schematic diagram showing the cell spreading effect of Pichia kudriavzevii with arginine addition;
FIG. 3 is a schematic diagram showing the spreading effect of the Pichia pastoris strain suppressor constructed according to the present invention.
Detailed Description
The invention is further illustrated by the following figures and examples, which are not to be construed as limiting the invention.
Example 1: a method for inhibiting cell aggregation of Pichia pastoris strain comprises preparing 200mM arginine mother liquor, filtering, and sterilizing; the pichia kudriavzevii growth YPD culture medium comprises the following components in mass concentration: 20g/L of glucose, 20g/L of peptone and 10g/L of yeast extract; further, pH of YPD medium was adjusted by HCl or NaOH, and 5mmol/L arginine was added after autoclaving, and arginine was exogenously added to YPD medium under different pH conditions, as shown in FIG. 2. The results show that the addition of 5mmol/L arginine can significantly promote the growth of Pichia pastoris at low pH pressure. Meanwhile, pHi is remarkably improved in the presence of arginine, which shows that arginine has a positive effect on protecting Pichia pastoris against low pH pressure stress, and the cell aggregation effect is remarkably reduced when arginine is added.
Example 2: a constructing method of recombinant Pichia pastoris for inhibiting cell aggregation, which is to obtain a recombinant strain for over-expressing ARG J genes in Pichia pastoris strain N-X; wherein, the Pichia kudriavzevii N-X is preserved in China Center for Type Culture Collection (CCTCC); address: eight-path Lojia mountain in Wuchang district, Wuhan city, Hubei province, with the preservation number: CCTCC M2017759, gene accession number MT499446 of the ARG J gene; the method specifically comprises the following steps:
a. amplifying by using primers Pdc1F, Pdc1R and pichia pastoris N-X genomic DNA as templates to obtain a fragment containing a PDC1 gene promoter, a reading frame and a terminator, and connecting the fragment into a pMD19T vector; the primer Pdc1F is CGCGGATCCGCGTTTAAGTGGTGGTTGTA, and the primer Pdc1R is CGG GGTACCTGGT GAGATTGATGGATTGT;
b. reverse amplification is carried out by using primers 0-F and 0-R; the primers 0-F are ACAGATAAGTACCTAGGGAGATT, and the primers 0-R are TGACATCT GAATGTAAAATGAAC;
c. primers 1-F and 1-R and a plasmid pGAPZB are used as templates for amplification to obtain bleomycin resistance gene BLE and a CYC1 terminator; the primers 1-F are acacagcaaaacacaaaaatATG GCCAAGTTGACCAGTGC; the primers 1-R are tatcgaaatctagcccGCAAATTAAAGCCTTCGA GC;
d. amplifying by using primers 2-F and 2-R and genome DNA as a template to obtain a pGAP promoter; the primer 2-F is tcgaaggctttaatttgcGGGCTAGATTTCGATATGGAT; the primers 2-R are gtagatt gcggaggacatTTTTTGTAATTGTGTTTGTTTGTGT;
e. amplifying by using primers 3-F and 3-R to obtain a target gene ARGJ; the primer 3-F is caaacacaattacaaaaaATGTTAAGACATGTTGCA; the primer 3-R is cattttacattcagatgtcaTCACGACCTGTAGTCTCCA;
f. using a Clon express Multi S One Step Cloning Kit to perform One-Step connection on the fragments to construct and obtain a recombinant integration expression vector pARGJ; and finally, transforming the recombinant plasmid subjected to BamH I enzyme digestion into an N-X strain by using a lithium acetate method, and screening positive transformants on a YPD plate containing 180 mu g/ml Zeocin to obtain the recombinant strain for over-expressing the ARG J, wherein the intracellular arginine content is improved by 26.3%.
The ARG J gene sequence:
ATGTTAAGACATGTTGCAAAGAGATCATTTGGATCGCTAGGAATCCCACAAAATAAGCTAAAGTATATTCCTAAAGGAGGATTCTACCCTAAGGGTTTCAAAGTAGGATCAGTAGCTTCCCATGTGAAGAAAACAGGAGCACCCGATCTAGCTTTAATTCATTCAGCCAAACCATGCACAGCTGCAGGTGTCTTTACGACTAACAAGTTCAAAGCAGCACctgttattgttgataaGGAAAACTTAACGCTCAAGAAAAACGAAGACTTCCATTCGATTATAATTAATTCAGGCTGTGCCAATGCTGTCACTGGAAACGGTGGGTTGTCGGATGCAAAGGACATCATAAACTACGTTGATAAAGCATTGCATGGAACAACATACCCTGTATCTAAAACACTGACAATGTCAACGGGTGTCATTGGACAGAGACTACAAGTGGATAAGATCAAGTCAGGTATTGACAACATAGTGACGGAAATTGACTCACAGCATGAAGACTGGCTCAAATGTGCCAAGGGAATAATGACAACCGACACGTTTCCTAAATTAATATCTagaaatttcaagattAATGGAATTGAGTACAATATTGCGGGTCTGGTCAAAGGTGCCGGTATGATTTGTCCCAACATGGCGACTCTGTTGGGTCTGATTATCACAGATGCCCCAATAGAGTCAATCACCTTACAGAATTTGCTTAGTAAGTCTGTTGataaatctttcaattgTATATCGGTGGATGGTGATATGTCTACCAACGATACCATTTTGTCATTATCAAATGGTCAATCTGGTGGAGAATTGATCACCGAGCAGTCTGGAGAGGTTTACGAggtttttgaaaagaacTTCAAAGAGATTGCCATTGAATTGGCAAAACTAGTTGTGAGAGACGGAGAAGGTGCTACCAAATTCATCACAATCAAGGTGAAGAATGCCAAAAACGATGAAGAGGCGAAACAAGCGGCAAACTCTGTCTCTAACTCTGCATTGGTCAAGACTGCCATGTTTGGTAAAGACGCCAATTGGGGTAGAATTTTATGTGCAATTGGATATTCTGAAATCGATGTTGAGCCTACGAAAACCAACGTTTCCTTCGTTCCAAGTGATGGAAGTGCAGAACTGAAGTTGTTAGTCAATGGAGAGCCGCAACTAGTTGACGAAAATAGAGCAAGTGAAATCCTCGAGCACGAAGACCTTGAAATTGCCATCGATCTTGGTCTCGAAGGAAAAGGTGAGTGCACCTTCTGGACATGTGACTTGACTCACGACTACGTGACTATTAATGGAGACTACAGGTCGTGA。
as shown in FIG. 3, the Pichia pastoris constructed by the method of the invention can obviously relieve the cell aggregation effect of the recombinant bacteria under a microscope, and is beneficial to the transfer of nutrients and oxygen.
In conclusion, the invention utilizes arginine to obviously promote the growth of pichia kudriana under extreme acid conditions, relieve cell aggregation, facilitate the transfer of oxygen and nutrient substances and improve the production performance of the strain during low pH fermentation.
Sequence listing
<110> university of Wenzhou
<120> method, construction and application for inhibiting cell aggregation of pichia kudriavzevii strains
<160>11
<170>SIPOSequenceListing 1.0
<210>1
<211>29
<212>DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400>1
cgcggatccg cgtttaagtg gtggttgta 29
<210>2
<211>29
<212>DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400>2
cggggtacct ggtgagattg atggattgt 29
<210>3
<211>23
<212>DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400>3
acagataagt acctagggag att 23
<210>4
<211>23
<212>DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400>4
tgacatctga atgtaaaatg aac 23
<210>5
<211>40
<212>DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400>5
acacagcaaa acacaaaaat atggccaagt tgaccagtgc 40
<210>6
<211>36
<212>DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400>6
tatcgaaatc tagcccgcaa attaaagcct tcgagc 36
<210>7
<211>39
<212>DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400>7
tcgaaggctt taatttgcgg gctagatttc gatatggat 39
<210>8
<211>43
<212>DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400>8
gtagattgcg gaggacattt tttgtaattg tgtttgtttg tgt 43
<210>9
<211>36
<212>DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400>9
caaacacaat tacaaaaaat gttaagacat gttgca 36
<210>10
<211>39
<212>DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400>10
cattttacat tcagatgtca tcacgacctg tagtctcca 39
<210>11
<211>1308
<212>DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400>11
atgttaagac atgttgcaaa gagatcattt ggatcgctag gaatcccaca aaataagcta 60
aagtatattc ctaaaggagg attctaccct aagggtttca aagtaggatc agtagcttcc 120
catgtgaaga aaacaggagc acccgatcta gctttaattc attcagccaa accatgcaca 180
gctgcaggtg tctttacgac taacaagttc aaagcagcac ctgttattgt tgataaggaa 240
aacttaacgc tcaagaaaaa cgaagacttc cattcgatta taattaattc aggctgtgcc 300
aatgctgtca ctggaaacgg tgggttgtcg gatgcaaagg acatcataaa ctacgttgat 360
aaagcattgc atggaacaac ataccctgta tctaaaacac tgacaatgtc aacgggtgtc 420
attggacaga gactacaagt ggataagatc aagtcaggta ttgacaacat agtgacggaa 480
attgactcac agcatgaaga ctggctcaaa tgtgccaagg gaataatgac aaccgacacg 540
tttcctaaat taatatctag aaatttcaag attaatggaa ttgagtacaa tattgcgggt 600
ctggtcaaag gtgccggtat gatttgtccc aacatggcga ctctgttggg tctgattatc 660
acagatgccc caatagagtc aatcacctta cagaatttgc ttagtaagtc tgttgataaa 720
tctttcaatt gtatatcggt ggatggtgat atgtctacca acgataccat tttgtcatta 780
tcaaatggtc aatctggtgg agaattgatc accgagcagt ctggagaggt ttacgaggtt 840
tttgaaaaga acttcaaaga gattgccatt gaattggcaa aactagttgt gagagacgga 900
gaaggtgcta ccaaattcat cacaatcaag gtgaagaatg ccaaaaacga tgaagaggcg 960
aaacaagcgg caaactctgt ctctaactct gcattggtca agactgccat gtttggtaaa 1020
gacgccaatt ggggtagaat tttatgtgca attggatatt ctgaaatcga tgttgagcct 1080
acgaaaacca acgtttcctt cgttccaagt gatggaagtg cagaactgaa gttgttagtc 1140
aatggagagc cgcaactagt tgacgaaaat agagcaagtg aaatcctcga gcacgaagac 1200
cttgaaattg ccatcgatct tggtctcgaa ggaaaaggtg agtgcacctt ctggacatgt 1260
gacttgactc acgactacgt gactattaat ggagactaca ggtcgtga 1308
Claims (5)
1. A method for inhibiting cell aggregation in a Pichia pastoris strain, characterized by: arginine is added into the YPD culture medium to promote the growth of Pichia kudriana under the condition of low pH and relieve cell aggregation.
2. The method of inhibiting cell aggregation of a pichia pastoris strain according to claim 1, wherein: the pichia kudriavzevii growth YPD culture medium comprises the following components in mass concentration: 20g/L of glucose, 20g/L of peptone and 10g/L of yeast extract; adjusting the pH of YPD medium to 2.5 or less with HCl or NaOH, autoclaving, and adding 5mmol/L arginine to the medium.
3. A pichia kudriavzevii that inhibits cell aggregation, characterized by: the Pichia pastoris strain is a recombinant strain constructed by overexpression of an ARG J gene in Pichia kudriavzevii N-X; pichia kudriavzevii N-X is deposited in the China Center for Type Culture Collection (CCTCC); address: eight-path Lojia mountain in Wuchang district, Wuhan city, Hubei province, with the preservation number: CCTCC M2017759.
4. The use of pichia kudriavzevii inhibiting cell aggregation according to claim 3 in low pH fermentation.
5. The method for constructing pichia kudriavzevii that inhibits cell aggregation according to claim 3, wherein: the method comprises the following steps:
a. amplifying by using primers Pdc1F, Pdc1R and pichia pastoris N-X genomic DNA as templates to obtain a fragment containing a PDC1 gene promoter, a reading frame and a terminator, and connecting the fragment into a pMD19T vector; the primer Pdc1F is CGCGGATCCGCGTTTAAGTGGTGGTTGTA, and the primer Pdc1R is CGG GGTACCTGGT GAGATTGATGGATTGT;
b. reverse amplification is carried out by using primers 0-F and 0-R; the primers 0-F are ACAGATAAGTACCTAGGGAGATT, and the primers 0-R are TGACATCT GAATGTAAAATGAAC;
c. primers 1-F and 1-R and a plasmid pGAPZB are used as templates for amplification to obtain bleomycin resistance gene BLE and a CYC1 terminator; the primers 1-F are acacagcaaaacacaaaaatATG GCCAAGTTGACCAGTGC; the primers 1-R are tatcgaaatctagcccGCAAATTAAAGCCTTCGA GC;
d. amplifying by using primers 2-F and 2-R and genome DNA as a template to obtain a pGAP promoter; the primer 2-F is tcgaaggctttaatttgcGGGCTAGATTTCGATATGGAT; the primers 2-R are gtagatt gcggaggacatTTTTTGTAATTGTGTTTGTTTGTGT;
e. amplifying by using primers 3-F and 3-R to obtain a target gene ARGJ; the primer 3-F is caaacacaattacaaaaaATGTTAAGACATGTTGCA; the primer 3-R is cattttacattcagatgtcaTCACGACCTGTAGTCTCCA;
f. using a Clon express Multi S One Step Cloning Kit to perform One-Step connection on the fragments to construct and obtain a recombinant integration expression vector pARGJ; and finally, transforming the recombinant plasmid subjected to BamH I enzyme digestion into an N-X strain by using a lithium acetate method, and screening positive transformants on a YPD plate containing 180 mu g/ml Zeocin to obtain the recombinant strain for over-expressing the ARG J.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010522228.2A CN111635866B (en) | 2020-06-10 | 2020-06-10 | Method for inhibiting cell aggregation of pichia kudriavzevii strain, construction and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010522228.2A CN111635866B (en) | 2020-06-10 | 2020-06-10 | Method for inhibiting cell aggregation of pichia kudriavzevii strain, construction and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111635866A true CN111635866A (en) | 2020-09-08 |
| CN111635866B CN111635866B (en) | 2021-11-23 |
Family
ID=72325466
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010522228.2A Expired - Fee Related CN111635866B (en) | 2020-06-10 | 2020-06-10 | Method for inhibiting cell aggregation of pichia kudriavzevii strain, construction and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111635866B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112877375A (en) * | 2021-03-26 | 2021-06-01 | 温州大学 | Method for continuously producing xylonic acid and ethanol by using recombinant pichia pastoris fermentation |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH09261A (en) * | 1995-06-16 | 1997-01-07 | Green Cross Corp:The | Production of glycoprotein |
| CN106520581A (en) * | 2016-11-22 | 2017-03-22 | 中国海洋大学 | Pichia kudriavzevii mutant strain and application thereof |
| CN110846235A (en) * | 2019-09-23 | 2020-02-28 | 广西科学院 | High-temperature-resistant stress-resistant Pichia kudriavzevii high-yield ethanol and application thereof |
-
2020
- 2020-06-10 CN CN202010522228.2A patent/CN111635866B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH09261A (en) * | 1995-06-16 | 1997-01-07 | Green Cross Corp:The | Production of glycoprotein |
| CN106520581A (en) * | 2016-11-22 | 2017-03-22 | 中国海洋大学 | Pichia kudriavzevii mutant strain and application thereof |
| CN110846235A (en) * | 2019-09-23 | 2020-02-28 | 广西科学院 | High-temperature-resistant stress-resistant Pichia kudriavzevii high-yield ethanol and application thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112877375A (en) * | 2021-03-26 | 2021-06-01 | 温州大学 | Method for continuously producing xylonic acid and ethanol by using recombinant pichia pastoris fermentation |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111635866B (en) | 2021-11-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2016361425B2 (en) | Genetically modified yeasts and fermentation processes using genetically modified yeasts | |
| US12012604B2 (en) | Acetate consuming yeast cell | |
| AU2015212752B2 (en) | Method for obtaining low ethanol-producing yeast strains, yeast strains obtained therefrom and their use | |
| JP2013524797A (en) | Method for producing cells capable of converting arabinose | |
| US10472656B2 (en) | Method for fermenting sugars using genetically engineered yeast | |
| CN109207373B (en) | Microbial strain for high yield of citric acid and method for producing citric acid by fermenting starch sugar through microbial strain | |
| Matsushika et al. | Evaluation of Saccharomyces cerevisiae GAS1 with respect to its involvement in tolerance to low pH and salt stress | |
| CN111635866B (en) | Method for inhibiting cell aggregation of pichia kudriavzevii strain, construction and application | |
| CN105624051B (en) | Wood-sugar fermentation yeast strain and construction method based on the building of evolution engineering | |
| US20180223271A1 (en) | Xylose isomerase-modified yeast strains and methods for bioproduct production | |
| CN105062981A (en) | Pyruvate carboxylase mutant N315F with improved enzymatic activity and application of pyruvate carboxylase mutant N315F | |
| US9719112B2 (en) | Mutant beta-glucosidases having enhanced activity and a method for producing bioethanol using the same | |
| US20220017933A1 (en) | Method for the production of enzymes by a strain belonging to a filamentous fungus | |
| CN105132388A (en) | Pyruvate carboxylase mutant R485P with improved enzymatic activity and application of mutant | |
| US20150167040A1 (en) | Fungal Cells That Produce Decreased Amounts of Peptaibols | |
| CN112625932B (en) | Engineering bacterium of yarrowia lipolytica for tolerating ferulic acid and vanillic acid and construction method thereof | |
| CN104403956A (en) | Construction and application of xylitol high-temperature and high-yield engineered strains | |
| WO2014021163A1 (en) | Method for producing ethanol using recombinant yeast | |
| EP2774980B1 (en) | Novel yeast and method for producing ethanol using same | |
| US20230407348A1 (en) | An Improved Process For Xylitol Production | |
| CN116179382A (en) | Genetic engineering bacterium for high-yield erythritol, construction method and application | |
| US20120115191A1 (en) | Production of fructo-oligosaccharide and derivatives by use of aspergillus spp | |
| BRPI0901254A2 (en) | process for genetically modifying saccharomyces yeasts, and their use in fermentative metabolite production processes | |
| JP2006149403A (en) | Yeast variant for producing alcoholic beverages, and method for producing alcoholic beverages by using the yeast variant |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20211123 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |