CN117417874B - Engineering strain HC6-MT and application thereof in low-temperature production of trehalose - Google Patents
Engineering strain HC6-MT and application thereof in low-temperature production of trehalose Download PDFInfo
- Publication number
- CN117417874B CN117417874B CN202311745292.7A CN202311745292A CN117417874B CN 117417874 B CN117417874 B CN 117417874B CN 202311745292 A CN202311745292 A CN 202311745292A CN 117417874 B CN117417874 B CN 117417874B
- Authority
- CN
- China
- Prior art keywords
- trehalose
- strain
- seq
- primer
- low
- 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.)
- Active
Links
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 title claims abstract description 96
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 title claims abstract description 96
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 title claims abstract description 96
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 23
- 229920002488 Hemicellulose Polymers 0.000 claims abstract description 43
- 240000008042 Zea mays Species 0.000 claims abstract description 25
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims abstract description 25
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims abstract description 25
- 235000005822 corn Nutrition 0.000 claims abstract description 25
- 230000015556 catabolic process Effects 0.000 claims abstract description 19
- 238000006731 degradation reaction Methods 0.000 claims abstract description 19
- 239000013604 expression vector Substances 0.000 claims abstract description 18
- 230000002018 overexpression Effects 0.000 claims abstract description 15
- 238000012224 gene deletion Methods 0.000 claims abstract description 7
- 241000894006 Bacteria Species 0.000 claims description 19
- 238000000855 fermentation Methods 0.000 claims description 19
- 230000004151 fermentation Effects 0.000 claims description 19
- 239000007788 liquid Substances 0.000 claims description 19
- 230000001580 bacterial effect Effects 0.000 claims description 16
- 101100068894 Bacillus subtilis (strain 168) glvA gene Proteins 0.000 claims description 15
- 101100207422 Bacillus subtilis (strain 168) treA gene Proteins 0.000 claims description 15
- 101100207423 Escherichia coli (strain K12) treC gene Proteins 0.000 claims description 15
- 101000795130 Homo sapiens Trehalase Proteins 0.000 claims description 15
- 230000000593 degrading effect Effects 0.000 claims description 15
- 102100029677 Trehalase Human genes 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 12
- 238000011144 upstream manufacturing Methods 0.000 claims description 7
- 230000015572 biosynthetic process Effects 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 5
- 238000003786 synthesis reaction Methods 0.000 claims description 5
- 101100373342 Botryotinia fuckeliana (strain B05.10) xyn11A gene Proteins 0.000 claims description 2
- 101100506054 Cellulomonas fimi cex gene Proteins 0.000 claims description 2
- 101150034999 otsA gene Proteins 0.000 claims description 2
- 101150021205 xlnB gene Proteins 0.000 claims description 2
- 101150031048 xynB gene Proteins 0.000 claims description 2
- 229910052799 carbon Inorganic materials 0.000 abstract description 8
- 238000012216 screening Methods 0.000 abstract description 7
- 239000002154 agricultural waste Substances 0.000 abstract description 4
- 241000889980 bacterium HC6 Species 0.000 abstract description 3
- 238000011161 development Methods 0.000 abstract description 3
- 230000000813 microbial effect Effects 0.000 abstract description 3
- 108090000623 proteins and genes Proteins 0.000 description 33
- 238000012795 verification Methods 0.000 description 25
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 24
- 239000013612 plasmid Substances 0.000 description 23
- 239000013598 vector Substances 0.000 description 21
- 108010049152 Cold Shock Proteins and Peptides Proteins 0.000 description 16
- 108020004414 DNA Proteins 0.000 description 13
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 12
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 12
- 239000000758 substrate Substances 0.000 description 12
- 239000003550 marker Substances 0.000 description 11
- 238000003752 polymerase chain reaction Methods 0.000 description 11
- 239000010902 straw Substances 0.000 description 11
- 238000009825 accumulation Methods 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 9
- 238000001962 electrophoresis Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 238000010276 construction Methods 0.000 description 8
- 238000001514 detection method Methods 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 244000005700 microbiome Species 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 229920001221 xylan Polymers 0.000 description 7
- 150000004823 xylans Chemical class 0.000 description 7
- 101710120163 Cold shock-like protein Proteins 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 230000002950 deficient Effects 0.000 description 6
- 230000003993 interaction Effects 0.000 description 6
- 238000012163 sequencing technique Methods 0.000 description 6
- 230000035939 shock Effects 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- 229910019142 PO4 Inorganic materials 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- 238000009630 liquid culture Methods 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 4
- 239000010452 phosphate Substances 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- 238000010200 validation analysis Methods 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000001976 enzyme digestion Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- MYKOKMFESWKQRX-UHFFFAOYSA-N 10h-anthracen-9-one;sulfuric acid Chemical compound OS(O)(=O)=O.C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 MYKOKMFESWKQRX-UHFFFAOYSA-N 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000005265 energy consumption Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 238000002329 infrared spectrum Methods 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 238000009629 microbiological culture Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- 241001135756 Alphaproteobacteria Species 0.000 description 1
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 108010005054 Deoxyribonuclease BamHI Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 238000001157 Fourier transform infrared spectrum Methods 0.000 description 1
- RGHNJXZEOKUKBD-SQOUGZDYSA-N Gluconic acid Natural products OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 1
- 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 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 240000000220 Panda oleosa Species 0.000 description 1
- 235000016496 Panda oleosa Nutrition 0.000 description 1
- 230000036579 abiotic stress Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000003287 bathing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000013256 coordination polymer Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000001120 cytoprotective effect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000037149 energy metabolism Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 238000000556 factor analysis Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000003197 gene knockdown Methods 0.000 description 1
- 239000000174 gluconic acid Substances 0.000 description 1
- 235000012208 gluconic acid Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 108020004418 ribosomal RNA Proteins 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000011232 storage material Substances 0.000 description 1
- 239000010907 stover Substances 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/248—Xylanases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/12—Disaccharides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01015—Alpha,alpha-trehalose-phosphate synthase (UDP-forming) (2.4.1.15)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01037—Xylan 1,4-beta-xylosidase (3.2.1.37)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of microbial engineering. The invention provides an engineering strain HC6-MT and application thereof in low-temperature production of trehalose, wherein the engineering strain HC6-MT is obtained by introducing a promoter enhanced over-expression vector PBBR1-PD-AB into a trehalose degradation gene deletion strain HC 6-M. The invention also provides a Raoult bacterium HC6 medium-low Wen Jiangqi promoter PcspDThe screening of the low Wen Jiangqi promoter P obtained by the screeningcspDIs used for constructing a promoter enhanced over-expression vector PBBR1-PD-AB. The engineering strain HC6-MT constructed by the technical scheme can degrade corn stalk hemicellulose to produce trehalose at low temperature, and can also remarkably improve the yield of the trehalose, thereby providing an effective scheme for low-heat energy, low-cost and low-pollution production of the trehalose and a new idea for low-carbon economic development of agricultural wastes.
Description
Technical Field
The invention relates to the technical field of microbial engineering, in particular to an engineering strain HC6-MT and application thereof in low-temperature production of trehalose.
Background
Trehalose is widely available in organisms and often exists as a structural component of organisms, energy metabolism, storage material and stress metabolites. Trehalose is a disaccharide with cytoprotective effect, can effectively maintain the integrity of cell membranes and the tertiary structure of proteins, and improves the tolerance of cells to abiotic stress.
In previous studies, although various methods have been found that can produce trehalose, such as: the trehalose is successfully synthesized by utilizing the gluconic acid conversion, the trehalose is synthesized by utilizing a chemical method, the trehalose is produced by utilizing an enzyme conversion method, and the like. However, the chemical method has the problems of more byproducts, low yield and high energy consumption, and the enzymatic conversion method has the problems of complex process, low catalytic efficiency of enzyme, low substrate utilization rate and the like. In addition, the biological trehalose preparation method adopted at present adopts commercial glucose, maltose and the like as substrates, so that the production cost is higher. Therefore, the search for lower cost and low energy consumption synthetic substrates and synthetic methods becomes the key for refining trehalose.
The straws are the most abundant biomass resources on the earth, waste is caused by improper treatment mode at present, and the enzyme activity of microorganisms is limited by temperature under the low-temperature condition, so that the degradation rate of the straws is low, and the microbial biosynthesis generally requires a temperature of more than 30 ℃, so that the straws in the low-temperature agricultural planting area are accumulated in a large amount and are burnt, and the pollution of the atmosphere and the incomplete utilization of resources are caused. The microorganisms obtained by screening in the low temperature region can be metabolized to produce Cold-active enzyme (Cold-active enzyme) so as to adapt to the low temperature environment. The cold-resistant microorganism has higher biological activity in the temperature range of 0-40 ℃, and can allow the biocatalysis reaction in industrial production to be carried out at low temperature, thereby reducing the energy loss in the industrial production process. Therefore, the novel cold-resistant microorganism is provided for producing the trehalose by taking the corn straw as the raw material, which is very necessary for reducing the cost of trehalose production enterprises for utilizing the agricultural waste in low-temperature areas.
Disclosure of Invention
The invention aims to provide an engineering strain HC6-MT and application thereof in low-temperature production of trehalose, wherein the engineering strain HC6-MT can degrade corn straw hemicellulose to produce trehalose under a low-temperature condition, remarkably improve the yield of the trehalose, provide an effective scheme for low-heat, low-cost and low-pollution production of the trehalose, and provide a new idea for low-carbon economic development of agricultural wastes.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an engineering strain HC6-MT, which is constructed by introducing a promoter enhanced over-expression vector PBBR1-PD-AB into a trehalose degradation gene deletion strain HC 6-M.
Preferably, the promoter enhanced over-expression vector PBBR1-PD-AB comprises a low Wen Jiangqi promoter P separated from Raoulet bacteria HC6cspDKey degradation gene of hemicellulosexynBAnd trehalose synthesis baseBecause ofotsA。
Preferably, the trehalose degrading gene-deleted strain HC6-M is obtained by: taking Raoul HC6 as an initial strain, knocking out trehalose degradation genes in Raoul HC6 genomeglvA、treCAndTREHone or more of the following.
The invention provides a Raoult bacterium HC6 medium-low Wen Jiangqi promoter PcspDComprises the following steps: PCR (polymerase chain reaction) is utilized to amplify a promoter sequence of Raoult HC6, a promoter fluorescence intensity expression vector is constructed to carry out recombinant bacterium expression, and recombinant bacterium OD is detected 600nm And fluorescence intensity, screening for fluorescence intensity/OD in the detection range 600nm The promoter with the largest ratio is a low Wen Jiangqi promoter PcspD。
Preferably, the original vector used for constructing the promoter fluorescence intensity expression vector is a pET-RFP vector.
The invention also provides application of the engineering strain HC6-MT in low-temperature production of trehalose.
Preferably, the engineering strain HC6-MT is mixed with the corn stalk hemicellulose, and the trehalose is produced by fermentation.
Preferably, the engineering strain HC6-MT is applied in the form of bacterial liquid, and the OD600nm of the engineering strain HC6-MT bacterial liquid is 1.7-2.3.
Preferably, the temperature of the fermentation is 18-23 ℃, and the time of the fermentation is 40-52h.
By adopting the technical scheme, the invention has the following beneficial effects:
1. according to the invention, the engineering strain HC6-MT is obtained by jointly knocking out the trehalose degrading gene strain HC6-M and the successfully constructed low-temperature promoter enhanced over-expression vector PBBR1-PD-AB, and the engineering strain HC6-MT can degrade hemicellulose extracted from corn stalks under a low-temperature condition to produce trehalose, so that the operation of providing heat from the outside is omitted, and meanwhile, compared with a wild strain, the engineering strain HC6-MT constructed after the wild strain HC6 is imported by the strong promoter has the advantages that the utilization rate of hemicellulose and the efficiency of producing trehalose are enhanced, so that the strain inoculation amount is reduced and the production cost is reduced; in addition, the yield of toxic and harmful byproducts is effectively reduced.
2. The embodiment of the invention shows that the engineering strain HC6-MT can produce the trehalose by degrading the hemicellulose of the corn straw at the low temperature of 20 ℃ so that the maximum yield of the trehalose reaches 1.96g/L, and is improved by 54 times compared with a wild strain. After that, culture conditions are optimized, and the highest yield of trehalose reaches 2.28g/L in 48 hours.
3. The invention converts the low-temperature microorganism fermentation straw into a product with high added value, improves the straw bioavailability, reduces the environmental pollution pressure and provides a new idea for the low-carbon economic development of agricultural wastes.
Drawings
FIG. 1 is an electrophoretogram of plasmid PKO3-knoc (M in FIG. 1 represents 5000bp DNA Marker,1 represents plasmid PKO3-glvA2 represents plasmid PKO3-treC3 represents plasmid PKO3-TREH);
FIG. 2 shows the knock-out verification electrophoretogram of different trehalose gene-deficient strains (FIG. 2 (a) is HC6-g (glvA) M in (a) represents 2000bp DNA Marker,1 which represents a geneglvAKnockout verification strip, 2 denotes suicide plasmid verification strip; fig. 2 (b) shows HC6-t (treC) M in (b) represents 2000bp DNA Marker,1, 2 represents a suicide plasmid verification band, and 2 represents a genetreCKnocking out the verification strip; fig. 2 (c) shows HC6-T (TREH) M in (c) represents 2000bp DNA Marker,1 which represents a geneTREHKnockout verification strip, 2 denotes suicide plasmid verification strip; FIG. 2 (d) shows HC6-Tg (TREH▲glvA) M in (d) represents 2000bp DNA Marker,1 representing a geneglvAKnockout verification band, 2 represents a geneTREHKnockout verification strip, 3 denotes suicide plasmid verification strip; the (e) in FIG. 2 is HC6-tg (treC▲glvA) M in (e) represents 2000bp DNA Marker,1, 2 represents a suicide plasmid verification band, and 2 represents a geneglvAKnockout verification band, 3 represents genetreCKnocking out the verification strip; fig. 2 (f) is HC6-Tt (TREH▲treC) M in (f) represents 2000bp DNA Marker,1 which represents a genetreCKnockout verification band, 2 represents a geneTREHThe verification strip is knocked out,3 represents a suicide plasmid verification band; the (g) in FIG. 2 is HC6-M (glvA▲treC▲TREH) M in (g) represents 2000bp DNA Marker,1, 2 represents a suicide plasmid verification band, and 2 represents a geneglvAKnockout verification band, 3 represents geneTREHKnockout verification band, 4 represents genetreCA knockout verification strip);
FIG. 3 shows trehalose gene-deficient strain HC6-M (FIGS.)glvA▲treC▲TREH) Is a construction process diagram of (1);
FIG. 4 is a graph showing the growth conditions of the trehalose degrading gene-deficient strain and the HC6 strain (WT) and the accumulation amount of trehalose (FIG. 4 (a) shows the growth curves of the trehalose degrading gene-deficient strain and the HC6 strain (WT) and the accumulation of trehalose, FIG. 4 (b) shows the growth conditions of the trehalose degrading gene-deficient strain and the HC6 strain (WT) at different dilution factors, the difference in significance is expressed in different lower case letters,p<0.05);
FIG. 5 is an electrophoresis chart of different promoters in the strain HC6 (M in FIG. 5 represents 2000bp DNA Marker,1)cspAGene promoter, 2 representscspBGene promoter, 3 denotescspCGene promoter, 4 meanscspDGene promoter, 5 meanscspEGene promoter, 6 representscspGA gene promoter);
FIG. 6 is an electrophoretogram of pET-master-RFP vector (M in FIG. 6 represents 5000bp DNA Marker,1 represents pET-P)cspARFP validation, 2 represents pET-PcspBRFP validation, 3 represents pET-PcspCRFP validation, 4 represents pET-PcspDRFP validation, 5 represents pET-PcspERFP verification, 6 represents pET-PcspG-RFP);
FIG. 7 is a graph showing the expression of recombinant bacteria with different promoter fluorescence intensities (FIG. 7 (a) shows the color change of the culture medium of recombinant bacteria with different promoter fluorescence intensities, and FIG. 7 (b) shows the fluorescence intensity of recombinant bacteria with different promoter fluorescence intensities);
FIG. 8 is an electrophoretogram of different PBBR1-PD-AB recombinant vectors (M in FIG. 8 represents 2000bp DNA Marker,1)xynBVerification, 2 denotes PcspD+xynB3 represents PBBR1,4 representsotsA5 represents PcspD+otsA);
Fig. 9 shows corn stalk hemicellulose and fourier infrared spectrum detection and fermentation results (fig. 9 (a) shows intermediate products in the corn stalk hemicellulose extraction process, (a) shows dry stalk powder, (a) shows stalk precipitation after alkali dissolution, (a) shows hemicellulose solution, (a) shows hemicellulose precipitation at 3 and (a) shows hemicellulose precipitation at 4, fig. 9 (b) shows FTIR spectra of xylan standard (upper curve) and corn stalk hemicellulose extraction (lower curve), and fig. 9 (c) shows growth curve of strain HC6 and xylose yield);
FIG. 10 is a graph showing the accumulation of xylose and trehalose, which are the conditions of the wild strain HC6 and the engineering strain HC6-MT, on the substrate hemicellulose (FIG. 10 (a) shows the conditions of the wild strain HC6 and FIG. 10 (b) shows the conditions of the engineering strain HC 6-MT);
FIG. 11 is a graph showing the effect of substrate concentration versus cold shock time ratio interaction on HC6-MT trehalose production;
FIG. 12 is a graph showing the effect of pH-to-cold shock time ratio interaction on HC6-MT trehalose production;
FIG. 13 is a graph showing the effect of phosphate concentration versus cold shock time duty cycle interaction on HC6-M trehalose production;
FIG. 14 is a graph showing the effect of substrate concentration and pH interaction on HC6-MT trehalose production;
FIG. 15 is a graph showing the effect of substrate concentration interaction with phosphate concentration on HC6-MT trehalose production;
FIG. 16 is a graph showing the effect of phosphate concentration and pH interaction on HC6-MT trehalose production.
Description of biological preservation
The invention relates to Raoult fungusRaoultellasp.) HC6 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on 09/07 th year 2020, and has a preservation address of No.3 of North Chen Xiyu No.1 in the Chaoyang area of Beijing city, and a preservation number of CGMCC No.20607.
Detailed Description
The invention provides an engineering strain HC6-MT, which is constructed by introducing a promoter enhanced over-expression vector PBBR1-PD-AB into a trehalose degradation gene deletion strain HC 6-M.
In the invention, the promoter enhanced over-expression vector PBBR1-PD-AB comprises a low Wen Jiangqi promoter P separated from Raoulet bacteria HC6cspDKey degradation gene of hemicellulosexynBAnd trehalose synthesis genesotsA。
In the invention, the Raoult fungus @ isRaoultella sp.) HC6 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on 09/07 of 2020, and has a preservation address of CGMCC No.20607, namely, no.3 of North West Lu 1, the Korean region of Beijing. The Raoult HC6 belongs to cold-resistant bacteria, can be used for growth and metabolism under low temperature, and contains hemicellulose key degradation genesxynB、Trehalose synthesis geneotsATherefore, hemicellulose can be used as a substrate for producing trehalose.
In the invention, the trehalose degrading gene deletion strain HC6-M is obtained by the following method: taking Raoul HC6 as an initial strain, knocking out trehalose degradation genes in Raoul HC6 genomeglvA、treCAndTREHone or more of the following. Further preferably, the trehalose degrading gene deletion strain HC6-M of the invention takes Raoult HC6 as an initial strain, and knocks out trehalose degrading genes in Raoult HC6 genomeglvA、treCAndTREH。
the invention provides a Raoult bacterium HC6 medium-low Wen Jiangqi promoter PcspDComprises the following steps: PCR (polymerase chain reaction) is utilized to amplify a promoter sequence of Raoult HC6, a promoter fluorescence intensity expression vector is constructed to carry out recombinant bacterium expression, and recombinant bacterium OD is detected 600nm And fluorescence intensity, screening for fluorescence intensity/OD in the detection range 600nm The promoter with the largest ratio is a low Wen Jiangqi promoter PcspD。
In the invention, the original vector used for constructing the promoter fluorescence intensity expression vector is a pET-RFP vector.
The invention also provides application of the engineering strain HC6-MT in producing trehalose under a low-temperature condition.
In the invention, the engineering strain HC6-MT can be used for producing trehalose by fermenting corn stalk hemicellulose. The invention mixes the engineering bacterial strain HC6-MT with the corn stalk hemicellulose, the engineering bacterial strain HC6-MT is mixed and applied in the form of bacterial liquid, and the OD of the bacterial liquid of the engineering bacterial strain HC6-MT 600nm Preferably 1.7 to 2.3, more preferably 1.8 to 2.2, and still more preferably 1.9 to 2.1. The fermentation according to the invention is preferably a shake flask fermentation, the rotation speed of the shake flask preferably being 170-250rpm, more preferably 185-220rpm, still more preferably 200rpm; the fermentation temperature is preferably 18-23 ℃, further preferably 19-21 ℃, still further preferably 20 ℃; the fermentation time is preferably 40 to 52 hours, more preferably 45 to 50 hours, still more preferably 48 hours.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
EXAMPLE 1 obtaining trehalose degrading Gene-deleted Strain HC6-M
Sequencing of the HC6 genome of Raouli Strain
Sequencing of the HC6 genome of Raouli strain shows that the genome (accession number is CP 093276.1) has the total length of 6243766bp, wherein the total length of chromosome is 595116 bp and the GC content is 58.42%. The strain HC6 has 3 plasmids, the size of the plasmid 1 is 167204bp, and the GC content is 51.75%; plasmid 2 was 100650bp in size and had a GC content of 51.54%; plasmid 3 had a size of 20796bp and a GC content of 57.56%. The sequencing result of the 16SrDNA gene sequence of the strain HC6 is compared with the data in the NCBI ribosomal RNA sequence database, and the result shows that the sequencing result is matched with the genus of the strain HC6Raoultella The sp and similarity reaches 100%, which indicates that the strain HC6 has no species variation and can be subjected to subsequent experiments.
(II) trehalose degrading Gene excision
The suicide plasmid PKO3-km (obtained by conventional purchase) is utilized to carry out enzyme digestion for 30min under the condition of 35 ℃ and the action of endonuclease BamHI, and the enzyme digestion reaction system is as follows: PKO3-km 5.0. Mu.L, 10×Lication Buffer 2.0. Mu.L, quick cut BamHI 1.0. Mu.L, sterilized ddH 2 O Up to 20.0μL。
The PCR amplification reaction (PCR amplification system: 5 XPrimeSTAR Buffer (Mg) 2+ Plus) 10μL、dNTP Mixture(2.5 mM each) 4μL、Primer F 10-15pmol、Primer R 10-15pmol、Template <200ng, 2. Mu.L of template DNA, sterilized water up to 50. Mu.L. The PCR amplification procedure was: denaturation at 98℃for 10s, annealing at 60℃for 5s, and circulation at 72℃for 1min for 30 times. ) Obtaining the target gene to be knocked out (trehalose degradation gene)glvA、treCAndTREH) The sequences of the primers are shown in Table 1, wherein the homologous arm sequences of the upstream and downstream 500bp sequences of the target gene sequence to be knocked out are used as homologous arms.
TABLE 1 PCR primer sequences of the upstream and downstream homology arms of the target Gene to be knocked out
After the amplification reaction was completed, the target gene fragment was purified and recovered using FastPure Gel DNA Extraction Mini Kit kit (available from Nanjinozan Biotechnology Co., ltd.). The purified product is directly subjected to a ligation reaction. The connection reactant is the trehalose degradation gene to be knocked out obtained by the stepsglvA、treCAndTREHthe upstream and downstream homology arms of the sequence are then ligated to linearized PKO3-km plasmids using an In-Fusion seamless cloning ligation technique to obtain plasmids PKO3-knock, in particular PKO3-glvA、PKO3-treC、PKO3-TREHThe recombinant plasmid vector is knocked out. The In-Fusion seamless cloning ligation system is shown In Table 2.
Table 2 In-Fusion seamless cloning ligation System
(the term "Target gene fragment" means homologous arms upstream and downstream of the corresponding gene)
Electrophoresis was performed to verify whether plasmid PKO3-knock was constructed successfully. For the plasmid obtained abovePKO3-knock was digested with BamHI and SalI to give 1.0kb and 5.6kb bands, which were PKO 3-compatibleglvA、PKO3-treC、PKO3-TREHThe size of the bands indicated that these recombinant plasmids were constructed correctly (see FIG. 1).
The bacterial strain HC6 is used as host bacteria, the plasmid PKO3-knock is electrically transformed into the host bacteria, and is cultured for 24 hours in an SOC culture medium at 30 ℃, and at the moment, the plasmid PKO3-knock can undergo homologous recombination with a host genome, so that a target gene is knocked out. The specific bands were then amplified again by PCR to verify that the gene knockdown was successful, and the primers used are shown in Table 3. The results are shown in FIG. 2.
TABLE 3 PCR primer sequences for checking deletion of target genes
(III) verification of growth of trehalose Gene-deficient Strain
Based on the above operations, 7 trehalose degrading gene defect strains (shown in FIG. 3) of HC6-g (, respectively) are constructed in sequenceglvA)、HC6-t(▲treC)、HC6-T(▲TREH)、HC6-Tg(▲TREH▲glvA)、HC6-tg(▲treC▲glvA)、HC6-Tt(▲TREH▲treC)、HC6-M(▲glvA▲treC▲TREH)。
Inoculating the constructed trehalose degrading gene defect strain and HC6 strain (WT) into LB liquid culture medium respectively for overnight activation, and regulating bacterial liquid OD 600nm =2.0±0.1, and the bacterial liquids were diluted to 10 respectively -1 -10 -6 2.0 mu L of bacterial liquid is taken from each dilution multiple, and the growth detection and the trehalose accumulation amount measurement are carried out in a hemicellulose liquid culture medium shaking flask for fermentation (20 ℃,80h,200 rpm). Similarly, 2.0. Mu.L of the bacterial liquid was taken for each dilution factor, and cultured in an incubator at a constant temperature of 20℃on an inorganic salt solid medium containing trehalose as a sole carbon source, and the colony growth state was observed. The results are shown in FIG. 4.
Example 2 obtaining Low temperature promoter enhanced overexpression vector PBBR1-PD-AB
Screening of cold shock protein low-temperature strong promoter and prediction of functional region
A total of 6 strains HC6cspGene, 6 are obtained from the whole genome of the strain HC6 for screening low-temperature high-expression promoterscspThe promoter sequence of the gene was analyzed on line using the prokaryotic promoter to predict the site BPROM (http:// linux1. Software. Com /) predicted promoter-10 region, -35 region and spacer distance.
The 6 cold shock protein gene sequences and promoters thereof are as follows:
cold shock protein gene Hc6_00172 Cold shock proteinCspAThe sequence is shown as SEQ ID NO.21, and specifically comprises the following steps: ATGTCCGGTAAAATGACTGGTATCGTAAAATGGTTCAACGCTGATAAAGGTTTCGGCTTCATTACTCCTGATGACGGTTCTAAAGATGTATTCGTACACTTCTCCGCTATCATGAACGATGGCTACAAATCTCTGGATGAAGGTCAGAAAGTTTCCTTCACCATCGAAAGCGGCGCTAAAGGCCCGGCAGCTGGCAACGTTACCAGCCTGTAA;
promoter Hc6_00172 Cold shock protein of cold shock protein geneCspAThe sequence of the promter is shown as SEQ ID NO.22, and specifically comprises the following steps: AGTTCTATAATGTTATTGGACACTGATTACGGTCCTGATTAGGGACCGTTTTAATTTCTATGAAAAAAACGTTTGCTATCGCACCTCAAAAATGAGTTAATGCTCGCAGGTTTGATGTACAGACCACAGAGCATTAGTATAGAGTAGTCCTTGAGCGGTTATCCTAGATACCCCGGTAGTGAACTTTCCCTTTATAGCTTCAAATCTGTAGTCCAGACCGTATCGCCGAAAGGTTCATTTATTATTTAAAGGTATTTCGCT;
cold shock protein gene Hc6_02655 Cold shock-like proteinCspBThe sequence is shown as SEQ ID NO.23, and specifically comprises the following steps: ATGTCTGGTAAGATGATTGGTTTAGTAAAATGGTTTAATGAAGGTAAAGGTTTCGGCTTTATTTCTCCAGTAGACGGCAGTAAAGATGTTTTCGTGCATTTTTCTGCGCTGCAGGGCGATGGCTTTAAAACTTTATTTGAAGGGCAGAAGGTCGAATTCACTATCGAGAGCGGCGCCAAAGGCCCTGCTGCCGGTAATGTCGTTCTGCGCGACTAA;
promoter Hc6_02655 Cold shock-like protein of Cold shock protein geneCspBThe sequence of the promter is shown as SEQ ID NO.24, and specifically comprises the following steps: TTATTCACGGCTTCTCTGTGGGGGCAGAAAATAATGCTTGCTATCAATGGTTATCTATGTGATCAATAGCAGGTCGGTTTGGTACACAGAACCAATAAAGCGGTTTAGTAAAGCAGTCCTCATCTCAAGCGTTATTCCTAAATAATCCTTTTTTCGAGCCTCCTTATCGTTATTAATTAATTTCTGTGATGCGAACTTTTTCGCCGCAAGGCTTTGAACATTGGGATAATACTT;
cold shock protein gene Hc6_02212 Cold shock-like protein CspCThe sequence is shown as SEQ ID NO.25, and specifically comprises the following steps: ATGGCAAAGATTAAAGGTCAAGTTAAGTGGTTCAACGAGTCTAAAGGTTTTGGCTTTATTACTCCGGCTGATGGCAGCAAAGACGTGTTCGTACACTTCTCCGCTATCCAGGGTAATGGCTTCAAAACTCTGGCTGAAGGCCAGAACGTTGAGTTCGAAATTCAGGACGGCCAGAAAGGTCCGGCAGCAGTTAACGTAACTGCTATCTGA;
promoter Hc6_02212 Cold shock-like protein of Cold shock protein geneCspCThe sequence of the promter is shown as SEQ ID NO.26, and specifically comprises the following steps: CTGATAAACGGGTGTACACCGCGCTGGTGTACACCTGCCTCATCGCCTTACGCAATAACTTATTCTTATCCTTCCGCGCTTATTGCCACATTTCTTATGTCACTTAATGTGTTAGCGATCGCAATTTTATCCAATAAGCTATGGATTTTTGAGCTATTCAAATAAAATAATAACATCGTGATTTAATTGCATGATGAAAGTGTGTGTCATCGAAATTTCATTATTAAGATGCAATATAAACACTATTTGTCGCGTGGCTAACAGCATTTAATTAACCCTGCATATTCTCGGCCTAAGCCAATTTTCAACTATCAATATGCGCGTTTATGCGCATCGTTATGCAGCTATGCGCACGTAACCCCTCAAAATTGGCAGGGTGCATGCATGGCGGGCTGGACGGAGCGAAGTTGTTGAGCTGTGGTGAAAAAGTTGCAGCTATCATCCATGCATAATCGGATGTTTGCCGCGCGCCCCGGGATTAACAGCGGGCGAGAAAAGGCGCATAAACGTGCAGTTGGTCAAATGATTCCCTTATATTTTGTGCGAAGGATCGAGAGCCGTTTAAAAATGGCTTGCCATTATTAACGTTGTATGTGATAACACCTTTCGGGTTAAACGAGGTACAGTTCTGTTTATGTGTGGCATTTTCAGTAAAGAAGTCCTGAGTAAACACGTTGTCGTTGAATACCGCTTCTCTGCCGAACCTTATATTAGTGCCTCATGCAGTAGTGTGTCAGTTTTATCTATGTAAGCGCCTGCGGGCGAAGAAAACAGTCTAAGGAATTTTGCAA;
cold shock protein gene Hc6_03855 Cold shock-like proteinCspDThe sequence is shown as SEQ ID NO.27, and specifically comprises the following steps: ATGGAAATGGGTACTGTTAAGTGGTTCAACAATGCCAAAGGGTTTGGTTTTATTTGCCCTGAGGGCGGAGGCGAAGACATTTTCGCCCATTACTCCACCATCCAGATGGATGGTTACAGAACGCTAAAAGCCGGACAAGCCGTTCGGTTTGATGTTCACCAGGGACCGAAAGGTAATCACGCCAGCGTGATTGTTCCTGTAGAAGCGGAAGCGGCTGCATAA;
promoter Hc6_03855 Cold shock-like protein of Cold shock protein geneCspDThe sequence of the promter is shown as SEQ ID NO.28, and specifically comprises the following steps: CGTCAGTATTCATCATCGGTTGCTGTTGCCAACAGGCGGTGGCCTGTCGATGACCAAAAGTTATGCCCATCACAAATCTACAATAGATCATAGATAACTATCATCTATTACTTCCATCCGCGACGTCTGTCACATTCCCCGGCAATAGCGTTAACTGCTTCAAATTTTGACGCATTTTTCGCCTTCCCCTACCGTCAATCGCTTGACGCCTTTTCGTATTTCTCTAAATTGTACTGGCGAGAGTTGGCGAGCATTTGAACAACTCGTCACTCCACTACCGGTTCATTCCATCTTACTTATAAAGAATTACGAAGGATGTCGAAGT;
Cold shock protein gene Hc6_04170 Cold shock-like proteinCspEThe sequence is shown as SEQ ID NO.29, and specifically comprises the following steps: ATGTCTAAGATTAAAGGTAACGTTAAGTGGTTTAATGAGTCCAAAGGATTCGGTTTCATTACTCCGGAAGATGGCAGCAAAGATGTATTCGTACATTTCTCTGCAATCCAGTCCAACGGTTTCAAAACTCTGGCTGAAGGTCAGCGTGTAGAGTTCGAAATCACTAACGGTGCCAAAGGCCCTTCTGCTGCAAACGTAAACGCTATCTAA;
promoter HC 6-04170 Cold shock-like protein of Cold shock protein geneCspEThe sequence of the promter is shown as SEQ ID NO.30, and specifically comprises the following steps: TCCGCACTAGCTTAGTGATAAAAGAGCTGAGCATTATGTTATGTGGAAAAACAATAACTAAAGCGCAACCACTAAAAAAGATAGCGACTTTTGTCACTTTTTAGCAAAGTTCGACTGGACAAAAGGCACCACAATTGATGTACTGGATCGCGACACAGTATCAGTGTCTTTTTTTCATATAAAGGTAATTTTG;
cold shock protein gene Hc6_02543 Cold shock-like proteinCspGThe sequence is shown as SEQ ID NO.31, and specifically comprises the following steps: TTGTTCCAAAAAATGACAGGCATTGTCAAAAGCTTTGATAATAAAACCGGCAGAGGCCTTATCGTCCCTTCCGACGGTCGTAAAGACGTTCAGGTTCATATTTCCGCATTATCCCCCAACGAGTCCACGCCAATGACGCCCGGTATTCGCGTTGAGTTTCGTCGGGTTAACGGCCTGCGCGGGCCAACCGCGGCAAACGTCTATACCTGCTAG;
promoter Hc6_02543 Cold shock-like protein of Cold shock protein geneCspGThe sequence of the promoter is shown as SEQ ID NO.32, and is specifically CCGTCGCGGGGCGGAGGACGCGGCTGCCGCATCGTCCTCCCGCCCCGCCGGGATTGCGTAAATAGCCTTGACCAATCAGACGCTTATTTGCCCGCGGCGAAATCATTTCGAGCTATTGATAGCAGAATGATGTATGGTTGCCGGCCATTACATCAGGAAAAAATCATATGTCAGATCGAAAAGACTCGAAAACTCGCCGTAATTACCTTGTAAAATGCTCCTGCCCTAACTGCTCCCAGGAATCAGAACATAGCTTTAGCCGAGTACAAAAAGGCGCCCAGCTCATCTGCCCTTACTGCCACAAACTCTTCCAGTCATCACCCAGAAACGCCGCCTAATCCCTAAATGCCCGACGCTGATTGTGCCTTGCCCCGTTATTTAGATAATTCTTATTATTGGGTCGAATGAGACAATCGGTTTTATTCCGTTCAGGCAAATAGAAAACCCGCACTCCGGCGGGTTTTAAATAGTTCAATATTGTATTTCTTTGCATAACCCAGGTTATCGCATTCCTGTTTTTACCAGGAGTTTAT.
The predicted promoter sequence results are shown in Table 4.
TABLE 4 promoter sequence prediction results
(II) construction of promoter fluorescence intensity expression vector pET-promoter-RFP
PCR amplification of the promoter DNA sequences of the strain HC6 genome DNA serving as a template, wherein the size of each promoter sequence is 200-500 bp. The amplified band gel was recovered by agarose gel electrophoresis and sent to the Shanghai Co., ltd for sequencing, and the sequencing result was consistent with the genome sequence (FIG. 5).
The amplified promoter DNA sequence was ligated to pUCmT vector, and the successfully ligated vector was named pUCmT-promoter and transferred into DH5 alpha bacteria for stable cloning. Extracting pUCmT-promoter vector and pET-RFP vector, respectively performing double enzyme digestion with Quick cut BamHI and Quick cut KpnI enzyme, and connecting the digested pET-RFP vector with strain HC6 promoter, wherein the successfully connected vector is named pET-promoter-RFP, specifically pET-PcspA-RFP、pET-PcspB-RFP、pET-PcspC-RFP、pET-PcspD-RFP、pET-PcspE-RFP、pET-PcspG-RFP。
And (5) carrying out electrophoresis on the constructed carrier, and verifying whether the construction is successful. The pET-promoter-RFP vector (about 5000 bp) and the promoter-RFP gene recombinant fragment (about 1000 bp) bands are respectively consistent with the lengths shown by the electrophoresis results, and the vector construction is successful (as shown in FIG. 6).
(III) determination of a Low temperature Strong promoter
The method for constructing the fluorescence intensity expression vector of the promoter is adopted for the traditional T7 strong promoter to construct pET-PT7-RFP, and then pET-P is respectively carried outcspA-RFP、pET-PcspB-RFP、pET-PcspC-RFP、pET-PcspD-RFP、pET-PcspE-RFP、pET-PcspGRFP and pET-PT7-RFP transferE.coliBL31 (DE 3) and the relative fluorescence intensity (fluorescence intensity/OD) of RFP gene expression is calculated by detecting the biomass and fluorescence intensity of recombinant bacteria 600nm ) And used to characterize the promoter strength.
After culturing in 48-well plate at 37deg.C for 16h, the promoters were ordered in P-strengthcspC<PcspB<PcspG<PT7<PcspA<PcspE<PcspDAfter 8h cold induction, P is removedcspCAnd PcspBThe intensity sequence of the promoters is P in sequence outside the group in which no red fluorescence is detectedcspG<PT7<PcspA<PcspE<PcspD. Meanwhile, a difference in color change of the medium due to accumulation of red mCherry protein was observed during the culture of each strain LB liquid medium (see fig. 7). By combining the results, the cold shock protein gene is determinedcspDPromoter P of (C)cspDIs a low Wen Jiangqi promoter, and is used for constructing a promoter enhanced over-expression vector.
Construction of promoter-enhanced overexpression vector PBBR1-PD-AB
Amplification of hemicellulose Key degradation Gene by PCRxynBSequence, trehalose synthesis geneotsASequence and sequencecspDPromoter sequence, then use In-Fusion ® Snap Assembly Master Mix seamless connection kit (purchased from Takara Bio-engineering (Dalian) Co., ltd.), universal restriction enzymes BmHI and KpnI are connected with linearized universal expression plasmid PBBR1-MCS2, and construction of PBBR1-PD-AB is completed.
TABLE 5 xynB、otsAAndcspDSequence PCR primer sequences
To verify whether the recombinant vector is successfully constructed, the PBBR1-PD-AB vector is used as a template for amplificationxynBGene band (1321 bp), PcspD+xynBFusion band (1624 bp),otsAGene strip (896 bp), PcspD+otsAFusion band (1199 bp) and vector specific band PBBR1 (346 bp). Amplified band electrophoresis gel As shown in FIG. 8, the band size conforms, demonstrating PET-PD-xynBWith PET-PD-otsAThe vector construction was successful.
Example 3
Construction of engineering strain HC6-MT
Taking out the prepared trehalose gene defect strain HC6-M competent cells in a refrigerator at the temperature of-80 ℃, and thawing the competent cells on ice. 10. Mu.L of the extracted overexpression vector (PBBR 1-PD-AB) was added into competent cells, and the mixture was left on ice for 30 min. Adding the mixed solution added with the over-expression vector (PBBR 1-PD-AB) into a precooled electrorotating cup, and setting electrotransformation conditions as follows: 1500KV,800, ꭥ. And (3) adding the bacteria liquid subjected to electrotransformation into 1mL of SOC culture solution for resuscitating and culturing for 1h, coating the bacteria liquid on a Kana antibiotic LB solid culture medium plate with the working concentration of 50ng/mL, culturing overnight at 20 ℃, picking out colonies, and performing PCR (polymerase chain reaction) verification by using an over-expression vector (PBBR 1-PD-AB) verification primer. The strain that was successfully verified was named HC6-MT.
Extraction of corn stalk hemicellulose
Drying corn stalk (from the northeast agricultural university to the sun farm), cutting into 2.0-3.0cm small sections, grinding into powder by using a grinder, sieving the obtained dried stalk powder with a 80-mesh sieve, mixing with 10% NaOH according to a ratio of 1:10, soaking at 70deg.C for 2h, filtering the mixture (stalk precipitation) with gauze, and collecting filtrate. Regulating pH of the filtrate to 7.0+ -0.2 with concentrated hydrochloric acid, mixing with active carbon at a ratio of 10:1 (v/w), water-bathing at 60deg.C for 15min, centrifuging at 10000rpm for 10min, and removing active carbon to obtain hemicellulose solution. And adding 50% of absolute ethyl alcohol of the volume of the hemicellulose solution into the hemicellulose solution to obtain hemicellulose sediment, carrying out solid-liquid separation, collecting filter residues, and drying at 50 ℃ to obtain hemicellulose.
The result of fourier infrared spectrum detection of the obtained hemicellulose and xylan standard is shown in fig. 9, and the result shows that the absorption peak positions of the corn straw extract and the xylan standard are basically consistent, and the hemicellulose extracted from the corn straw is mainly xylan.
Taking corn stalk hemicellulose as a substrate, inoculating 5% (v/v) Raoult HC6, culturing and fermenting at 20 ℃ for 80 hours, sampling every 6 hours, observing the growth curve of the Raoult HC6, and detecting the xylose yield. The results are shown in fig. 9, which shows that the strain HC6 can grow with hemicellulose extracted from corn stover as the only carbon source and degrade xylan (hemicellulose as the main component) to produce xylose.
Example 4
Preparation of corn stalk hemicellulose liquid culture medium
The corn stalk hemicellulose liquid culture medium is prepared according to the following formula: 10g/L, NH of hemicellulose obtained above 4 NO 3 2g/L、K 2 HPO 4 2g/L、MgSO 4 0.2g/L。
(II)
The ability of Raoult HC6 and the engineering strain HC6-MT prepared in example 3 to produce trehalose was examined by using Raoult HC6 alone as a control to produce trehalose.
Control group: the strain HC6 bacterial liquid (OD 600nm =2.0±0.1) was inoculated into the above-mentioned corn stalk hemicellulose liquid medium in an inoculum size (v/v) of 5%, shake flask fermentation was performed at 20 ℃ and 200rpm for 80 hours, sampling was performed every 6 hours, and the substrate hemicellulose utilization of the strain HC6 and trehalose yield were examined.
Experimental group: engineering bacteria HC6-MT bacterial liquid (OD) 600nm =2.0±0.1) was inoculated into the above-mentioned corn stalk hemicellulose liquid medium in an inoculum size (v/v) of 5%, shake flask fermentation was performed at 20 ℃ and 200rpm for 80 hours, sampling was performed every 6 hours, and the substrate hemicellulose utilization and trehalose yield by the strain HC6-MT were examined.
The trehalose content detection method comprises the following steps: preparing trehalose standard solutions (0.2, 0.1, 0.075, 0.05, 0.025, 0.0125 mg/mL) with different concentrations, respectively taking 2.0mL of standard solution, adding 5.0mL of 0.2% anthrone-sulfuric acid solution, boiling water bath for 8min, cooling, measuring light absorption value at 620nm, and obtaining OD 620nm And drawing a standard curve for the trehalose content. The same procedure was followed for a boiling water bath of 8 minutes with a blank of 2.0mL deionized water added to 5.0mL of a 0.2% anthrone-sulfuric acid solution.
The xylose is accumulated in a certain amount in the earlier stage of the xylan, and the depolymerization rate (namely the xylose generation rate) of the xylan is larger than the xylose degradation rate, so that the xylose is accumulated in the initial stage of depolymerization, and then the xylose is further utilized by the engineering strain HC6-MT to convert the xylose into the trehalose.
The detection results of the accumulation amounts of the wild strain HC6 and the engineering strain HC6-MT trehalose and xylose are shown in figure 10, and the maximum accumulation amount of the engineering strain HC6-MT trehalose is 1.96g/L, which is improved by 54 times compared with the accumulation amount of the wild strain HC6 trehalose; the maximum xylose accumulation of the engineering strain HC6-MT is 3.61g/L, which is enhanced by 2.3 times compared with the wild strain, and the hemicellulose utilization capacity and the trehalose accumulation capacity of the strain HC6 are successfully enhanced.
Example 5 optimization of fermentation conditions for HC6-MT Strain
Four-factor three-level experiments were designed using Minitab2022 software. Based on the result of single factor analysis to significancep<0.05 is a selection standard, and the optimal fermentation conditions of the constructed recombinant strain HC6-MT are explored by taking the cold shock time (A) at 10 ℃ after inoculating a fermentation medium, the concentration (B) of a substrate (corn hemicellulose) of the medium, the pH (C) of the medium and the phosphate (D) in the medium as independent variables and the trehalose yield as a response value. The levels of 4 response surface factors and variables affecting the production of trehalose by fermentation of the strain HC6-MT are shown in Table 6.
TABLE 6 optimization of response surface factor and variable levels for HC6-MT fermentation by strain
As can be seen from FIGS. 11 to 16, the OD was adjusted by sterile water after inoculating the engineering strain HC6-MT into LB liquid medium and performing activation culture at 20℃for 16 hours 600nm =2.0±0.1, and the corn stalk hemicellulose liquid culture medium is inoculated in an inoculation proportion of 5%, and after optimization, the highest yield of trehalose is 2.28g/L in 48 hours under the optimal trehalose production condition.
According to the embodiment, the engineering strain HC6-MT provided by the technical scheme of the invention can be used for producing trehalose by degrading hemicellulose extracted from corn straw under a low-temperature condition, so that the production raw materials are saved, the production cost is reduced, and the yield, the conversion rate and the production rate of the trehalose are improved.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (3)
1. An engineering strain HC6-MT is characterized in that a promoter enhanced over-expression vector PBBR1-PD-AB is introduced into a trehalose degradation gene deletion strain HC6-M, and the engineering strain HC6-MT is constructed;
the promoter enhanced over-expression vector PBBR1-PD-AB comprises a low-temperature strong promoter PcspD separated from Raoult bacteria HC6, a hemicellulose key degradation gene xynB and a trehalose synthesis gene otsA;
the sequence of the low-temperature strong promoter PcspD is shown as SEQ ID NO. 28;
the temperature of the low temperature is 20 ℃;
the trehalose degrading gene deletion strain HC6-M is obtained by the following method: taking Raoult bacteria HC6 as an initial strain, and knocking out trehalose degradation genes glvA, treC and TREH in a Raoult bacteria HC6 genome;
the sequences of the PCR primers of the upstream and downstream homology arms of the trehalose degradation gene glvA are a glvA primer 1 group and a glvA primer 2 group, and the specific sequences of the glvA primer 1 group are shown as SEQ ID NO.1 and SEQ ID NO. 2; the specific sequences of the glvA primer 2 group are shown in SEQ ID NO.3 and SEQ ID NO. 4;
the PCR primer sequences of the upstream and downstream homology arms of the trehalose degradation gene treC are treC primer 1 groups and treC primer 2 groups, and the specific sequences of the treC primer 1 groups are shown as SEQ ID NO.5 and SEQ ID NO. 6; the specific sequences of the treC primer 2 group are shown as SEQ ID NO.7 and SEQ ID NO. 8;
the upstream and downstream homology arm PCR primer sequences of the trehalose degradation gene TREH are a TREH primer 1 group and a TREH primer 2 group, and the specific sequences of the TREH primer 1 group are shown as SEQ ID NO.9 and SEQ ID NO. 10; the specific sequences of the TREH primer 2 group are shown as SEQ ID NO.11 and SEQ ID NO. 12.
2. The application of the engineering strain HC6-MT in the production of trehalose, which is characterized in that the engineering strain HC6-MT is mixed with corn stalk hemicellulose, and the mixture is fermented to produce the trehalose;
the fermentation temperature is 18-23 ℃, and the fermentation time is 40-52h.
3. The use according to claim 2, wherein the engineering strain HC6-MT is used in the form of a bacterial liquid, the OD of the bacterial liquid of the engineering strain HC6-MT 600nm 1.7-2.3.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311745292.7A CN117417874B (en) | 2023-12-19 | 2023-12-19 | Engineering strain HC6-MT and application thereof in low-temperature production of trehalose |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311745292.7A CN117417874B (en) | 2023-12-19 | 2023-12-19 | Engineering strain HC6-MT and application thereof in low-temperature production of trehalose |
Publications (2)
Publication Number | Publication Date |
---|---|
CN117417874A CN117417874A (en) | 2024-01-19 |
CN117417874B true CN117417874B (en) | 2024-04-09 |
Family
ID=89525189
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311745292.7A Active CN117417874B (en) | 2023-12-19 | 2023-12-19 | Engineering strain HC6-MT and application thereof in low-temperature production of trehalose |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117417874B (en) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108531434A (en) * | 2018-04-04 | 2018-09-14 | 江南大学 | A method of improving Raoul bacterium 2,5- furandicarboxylic acid yield |
CN113322250A (en) * | 2021-02-23 | 2021-08-31 | 齐鲁工业大学 | Preparation method of MTSase immobilized enzyme and MTHase immobilized enzyme and application of MTSase immobilized enzyme and MTHase immobilized enzyme in trehalose production |
CN114107358A (en) * | 2020-12-03 | 2022-03-01 | 德州汇洋生物科技有限公司 | Construction method of heat-resistant aspergillus niger engineering bacteria for increasing content of stress trehalose |
CN116286560A (en) * | 2023-05-11 | 2023-06-23 | 东北农业大学 | Raoult bacterium HC6 and application thereof in low-temperature production of 2, 3-butanediol |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2022216614A1 (en) * | 2021-02-05 | 2023-02-23 | Christiana Care Gene Editing Institute, Inc. | Methods of and compositions for reducing gene expression and/or activity |
-
2023
- 2023-12-19 CN CN202311745292.7A patent/CN117417874B/en active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108531434A (en) * | 2018-04-04 | 2018-09-14 | 江南大学 | A method of improving Raoul bacterium 2,5- furandicarboxylic acid yield |
CN114107358A (en) * | 2020-12-03 | 2022-03-01 | 德州汇洋生物科技有限公司 | Construction method of heat-resistant aspergillus niger engineering bacteria for increasing content of stress trehalose |
CN113322250A (en) * | 2021-02-23 | 2021-08-31 | 齐鲁工业大学 | Preparation method of MTSase immobilized enzyme and MTHase immobilized enzyme and application of MTSase immobilized enzyme and MTHase immobilized enzyme in trehalose production |
CN116286560A (en) * | 2023-05-11 | 2023-06-23 | 东北农业大学 | Raoult bacterium HC6 and application thereof in low-temperature production of 2, 3-butanediol |
Non-Patent Citations (4)
Title |
---|
Efficient conversion of hemicellulose into 2, 3-butanediol by engineered psychrotrophic Raoultella terrigena: mechanism and efficiency;Yue Wang 等;《Bioresource Technology》;20220611;第359卷;第1-11页 * |
Enzymatic synthesis of l‑fucose from l‑fuculose using a fucose isomerase from Raoultella sp. and the biochemical and structural analyses of the enzyme;In Jung Kim 等;《Biotechnol Biofuels》;20191205;第12卷(第282期);第1-12页 * |
Wang,Y..CP093276.1 Raoultella sp. HC6 chromosome, complete genome.《GenBank》.2022,第1-2页. * |
大肠杆菌海藻糖的代谢调控;戴秀玉 等;《生物工程进展》;20001231;第20卷(第6期);第26-29页 * |
Also Published As
Publication number | Publication date |
---|---|
CN117417874A (en) | 2024-01-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10577615B2 (en) | Genetically engineered Candida utilis capable of degrading and utilizing kitchen waste and construction method therefor | |
JP2013545491A (en) | Enhancement of ethanol production by xylose-utilizing zymomonas mobilis in biomass hydrolyzate medium | |
CN110484471B (en) | Acid-resistant strain for high yield of bacterial cellulose and method for producing bacterial cellulose by using acid-resistant strain | |
Li et al. | Genome shuffling of Aspergillus niger for improving transglycosylation activity | |
CN103131720A (en) | Fungi xylose isomerase gene and application thereof | |
CN112280700A (en) | Acetic acid and formic acid resistant fermentation strain and construction method thereof | |
CN101469318B (en) | Synthesis of (R)-styrene glycol by coupling acceleration of (R)-carbonyl reduction enzyme and formic dehydrogenase | |
CN117417874B (en) | Engineering strain HC6-MT and application thereof in low-temperature production of trehalose | |
CN114196682B (en) | Method for improving activity of stropharia rugoso-annulata lignocellulose | |
CN104974945A (en) | Saccharomyces cerevisiae for over-expressing MIG1 gene and preparation method and application thereof | |
CN112646797B (en) | Method for heterogeneously expressing stropharia rugoso-annulata beta-glucosidase gene | |
CN111893107A (en) | Pichia pastoris engineering strain for heterologous expression of cellulase gene EG IV and application | |
CN111334446B (en) | High-temperature-resistant saccharifying yeast strain and application thereof | |
CN110804620A (en) | Efficient expression and application of recombinant isoamylase for maltose production | |
CN108949784A (en) | Application of the sporulation related gene sigmaF in producing enzyme | |
CN114015634B (en) | Recombinant escherichia coli for high yield of succinic acid and construction method and application thereof | |
CN108865903B (en) | Trichoderma aquaticum and method for producing cellulase by trichoderma aquaticum | |
CN114874961B (en) | Recombinant zymomonas mobilis for synthesizing acetoin by using acetaldehyde, and construction method and application thereof | |
CN118272397A (en) | Gene combination, genetically engineered bacterium containing same and application of genetically engineered bacterium in L-lysine production | |
CN117987444A (en) | Expression cassette, genetically engineered bacterium containing same and application of genetically engineered bacterium in L-lysine production | |
CN117821527A (en) | Method for synthesizing vanillic acid | |
CN115838747A (en) | Gene of alginate lyase, recombinant cell, construction method and application thereof | |
CN111850027A (en) | Pichia pastoris engineering strain for heterologous expression of cellulase gene CBH II and application | |
CN111893131A (en) | Pichia pastoris engineering strain for heterologous expression of cellulase gene EG II and application | |
CN111893106A (en) | Pichia pastoris engineering strain for heterologous expression of cellulase gene EG V and application |
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 |