CN105063000A - Escherichia coli 2-deoxidation-D-ribose-5-phosphoric acid aldolase mutants and preparation method thereof - Google Patents

Escherichia coli 2-deoxidation-D-ribose-5-phosphoric acid aldolase mutants and preparation method thereof Download PDF

Info

Publication number
CN105063000A
CN105063000A CN201510548574.7A CN201510548574A CN105063000A CN 105063000 A CN105063000 A CN 105063000A CN 201510548574 A CN201510548574 A CN 201510548574A CN 105063000 A CN105063000 A CN 105063000A
Authority
CN
China
Prior art keywords
mutant
dera
enzyme
mutants
gene
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
Application number
CN201510548574.7A
Other languages
Chinese (zh)
Other versions
CN105063000B (en
Inventor
许�鹏
杨为华
徐斌
张雪锋
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Anhui BBCA Fermentation Technology Engineering Research Co Ltd
Original Assignee
Anhui BBCA Fermentation Technology Engineering Research Co Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Anhui BBCA Fermentation Technology Engineering Research Co Ltd filed Critical Anhui BBCA Fermentation Technology Engineering Research Co Ltd
Priority to CN201510548574.7A priority Critical patent/CN105063000B/en
Publication of CN105063000A publication Critical patent/CN105063000A/en
Application granted granted Critical
Publication of CN105063000B publication Critical patent/CN105063000B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88Lyases (4.)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y401/00Carbon-carbon lyases (4.1)
    • C12Y401/02Aldehyde-lyases (4.1.2)
    • C12Y401/02004Deoxyribose-phosphate aldolase (4.1.2.4)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention provides an escherichia coli 2-deoxidation-D-ribose-5-phosphoric acid aldolase mutants and a preparation method thereof. According to the method, a random mutant library of DERA enzyme is constructed through error-prone PCR; then enzyme activity is tested, and mutant strains highest in enzyme activity are screened. According to enzyme activity testing, the DERA enzyme activity of the provided escherichia coli DERA mutants can be improved by 3.8 activity units compared with wild mutants. Due to large-scale fermentation of escherichia coli engineering bacteria containing genes encoding the DERA mutants, volume production of the escherichia coli DERA mutants can be achieved.

Description

Intestinal bacteria DRI-5-phosphate aldolase mutant and preparation method thereof
Technical field
The present invention relates to genetically engineered field, specifically, relate to a kind of intestinal bacteria DRI-5-phosphate aldolase mutant and preparation method thereof.
Background technology
Intestinal bacteria DRI-5-phosphate aldolase (DERA), is numbered EC4.1.2.4, can resolve into 3-phosphoric acid-D-Glycerose and acetaldehyde by catalysis 5-phosphoric acid-DRI, and can its reversed reaction of catalysis.
Fallibility PCR refers to and adjusts mutation frequency in PCR reaction by changing PCR reaction conditions, reduce the proneness of the intrinsic mutant nucleotide sequence of polysaccharase, improve the diversity of mutation spectrum, false bases is made to be incorporated in the gene of amplification with certain frequency randomly, thus obtain the DNA colony of random mutation, finally with suitable carrier cloning mutator gene.
The DERA mutant obtaining enzyme activity higher by fallibility PCR method becomes a kind of effective means transforming DERA enzyme.
Summary of the invention
The object of this invention is to provide a kind of intestinal bacteria DRI-5-phosphate aldolase (DERA) mutant and preparation method thereof.
In order to realize the object of the invention, intestinal bacteria DRI-5-phosphate aldolase mutant provided by the invention, it is the albumen 1) be made up of the aminoacid sequence shown in SEQIDNo.1; Or 2) aminoacid sequence shown in SEQIDNo.1 be substituted, lack and/or add one or several amino acid and same function by 1) derivative albumen.
The present invention also provides the gene of described DERA mutant enzyme of encoding, and its nucleotide sequence is as shown in SEQIDNo.2.
The present invention also provides the carrier of gene, host cell and transgenic engineered bacteria containing the described DERA mutant enzyme of coding.
The present invention also provides the colibacillus engineering of the gene containing the described DERA mutant enzyme of coding.
The present invention further provides the preparation method of described DERA mutant enzyme, the colibacillus engineering of the gene containing the described DERA mutant enzyme of coding is inoculated in LB substratum, 37 DEG C, 180rpm cultivates 8-10h, inoculum size by 3% is inoculated in the fermentor tank filling 3L fermention medium, fermenter volume is 5L, at 37 ± 1 DEG C, 300rpm, after cultivating 8-10h under the condition of air flow quantity 4L/min, add 60% glycerine as feed supplement, when OD600 reaches 30, be cooled to 30 DEG C, add 0.1mMIPTG to induce, after induction 10h, put tank, centrifugal fermented liquid, collect thalline, add the 50mM sodium bicarbonate buffer liquid of 3 times of volume pH7.5, ice-water bath ultrasonication, 8000rpm centrifuging and taking supernatant, obtain the crude enzyme liquid containing described DERA mutant.
Described fermention medium is: glycerine 4g/L, dipotassium hydrogen phosphate 12.8g/L, potassium primary phosphate 3g/L, ammonium sulfate 0.5g/L, calcium chloride 0.0152g/L, magnesium chloride 0.41g/L.
Through enzyme activity determination, intestinal bacteria DERA mutant enzyme provided by the invention comparatively wild-type DERA enzyme work can improve 3.8 unit of activity.By carrying out large scale fermentation to the colibacillus engineering of the gene containing the described DERA mutant of coding, the batch production of intestinal bacteria DERA mutant enzyme can be realized.
Accompanying drawing explanation
Fig. 1 is the electrophoresis detection result of pcr amplification DERA gene in the embodiment of the present invention 1; Wherein, M is DNAMarker, DERA gene for the purpose of 1 and 2.
Fig. 2 is the restriction enzyme digestion and electrophoresis figure of plasmid pET-DERA in the embodiment of the present invention 1; Wherein, M is DNAMarker, 1 for enzyme cut after pET-DERA.
Fig. 3 is the SDS-PAGE electrophoresis detection figure of DERA mutant in the embodiment of the present invention 3; Wherein, M is albumen Marker, and 1 is bacterial lysate.
Fig. 4 is DERA enzyme activity determination principle schematic in the embodiment of the present invention 4.
Fig. 5 is the GC analytical results of DERA mutant enzyme catalytic reaction products in the embodiment of the present invention 7.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, embodiment is experiment condition all conveniently, as Sambrook equimolecular Cloning: A Laboratory Manual (SambrookJ & RussellDW, Molecularcloning:alaboratorymanual, 2001) condition of, or according to manufacturer's specification sheets advising.
The construction process of embodiment 1 plasmid pET-DERA
1, pcr amplification DERA gene
With 1 μ g genome of E.coli DNA for PCR reaction template, design primer A1:5 '-AAAAACATATGACTGATCTGAAAGCAAGCA-3 ' and primer A2:5 '-AAAAACTCGAGTTAGCTGCTGGCGCTCTTAC-3 ', carries out pcr amplification.PCR reaction is carried out in 50 μ l cumulative volumes, and reaction conditions is: 94 DEG C of sex change 5min; 94 DEG C of sex change 50s, 55 DEG C of annealing 1min, 72 DEG C extend 2min, totally 30 circulations; Last 72 DEG C extend 10min.Amplified production 1% agarose gel electrophoresis detects, and the results are shown in Figure 1.
2, the structure of DERA expression plasmid
Gel reclaims pcr amplification product, with Nde I and Xho I double digestion, reclaim endonuclease bamhi, connect (connecting 16h in 16 DEG C) with the same pET-42a carrier with Nde I and Xho I double digestion, transformation of E. coli DH5 α competent cell, selects positive colony, double digestion analysis verification is carried out after extracting plasmid, result as shown in Figure 2, and carries out DNA sequencing qualification, the plasmid called after pET-DERA that insertion sequence is correct.
The aminoacid sequence of DERA is as shown in SEQIDNo.3, and nucleotide sequence is as shown in SEQIDNo.4.
Embodiment 2 builds DERA mutant enzyme library
Use Random Mutagenesis Kit, by changing MnCl 2concentration and dNTPs concentration carry out multiple reaction, thus multiple point mutation are introduced in E.coliDERA gene.
Fallibility PCR the primer is A1:5 '-AAAAACATATGACTGATCTGAAAGCAAGCA-3 ' and A2:5 '-AAAAACTCGAGTTAGCTGCTGGCGCTCTTAC-3 ', fallibility PCR is totally 50 μ l, and reaction conditions is: 94 DEG C of sex change 4min; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 1min, totally 30 circulations; Last 72 DEG C extend 10min.By fallibility PCR fragment Nde I and Xho I double digestion, reclaim endonuclease bamhi, connect with the same pET-42a carrier with Nde I and Xho I double digestion, be built into expression library, choose 10 mono-clonals.Then by expression library Plastid transformation E.coliBL21 (DE3) competent cell, for expressing the DERA gene of sudden change.
The expression of embodiment 3DERA mutant enzyme
The expression of sudden change DERA gene: by the mutant bacteria obtained in embodiment 2, picking mono-clonal is seeded to (kantlex containing 50 μ g/ml) in 5mlLB substratum, and 37 DEG C, 200rpm cultivates 6-8h, OD 600during=0.6-0.8, add the IPTG that final concentration is 0.5mM, the broken thalline of ice-bath ultrasonic after 30 DEG C of abduction delivering 10h, bacterial lysate is through SDS-PAGE electrophoresis detection, and result as shown in Figure 3.The expression method of the pET-DERA do not suddenlyd change is the same.
The enzyme activity determination of embodiment 4DERA mutant
Medium centrifugal in embodiment 3 is collected thalline, and add the 50mM sodium bicarbonate buffer liquid of 3 times of volume pH7.5, ice-water bath ultrasonication, 8000rpm centrifuging and taking supernatant, obtains the crude enzyme liquid containing DERA mutant.
DERA enzyme activity determination principle as shown in Figure 4, pH value of reaction system is 7.5, containing 50mmol triethanolamine hydrochloride solution 195 μ l in reaction system, NADH wherein containing 0.3mmol, the substrate DRP of 30mmol, triosephosphate isomerase and glycerolphos phate dehydrogenase (GPD/TPI) 1.7U/ml, add 5 μ l crude enzyme liquids in 25 DEG C and start reaction, detect the change of 340nm place absorbancy.
Unit enzyme is lived and is defined: under these conditions, the enzyme amount needed for per minute cracking 1 μm of ol substrate.
Enzyme calculation formula: U (μm ol/min)=Ew × V × 10 alive 6/ (6220 × L)
The change of 340nm place absorbancy in Ew:1min; V: the cumulative volume (L) of reaction solution; 6220:NADH is at the molar extinction coefficient (Lmol of 340nm -1cm -1); L: optical path length (cm).
Enzyme activity determination the results are shown in Table 1.
Table 1 wild-type and mutant DERA enzyme live (U)
DERA enzyme Enzyme lives (U)
Wild-type 11.8
Mutant 1 14.4
Mutant 2 14.0
Mutant 3 14.5
Mutant 4 15.6
Mutant 5 12.1
Mutant 6 14.4
Mutant 7 15.3
Mutant 8 11.3
Mutant 9 14.2
Mutant 10 13.1
Wherein, the enzyme activity of mutant 4 is the highest, and mutant 7 takes second place.
Embodiment 5DERA mutant enzyme is to the tolerance of acetaldehyde
Enzyme work is cultivated 8h respectively higher than the Recombinant organism strain (mutant strain) containing mutant enzyme of wild-type in the LB substratum that with the addition of 50mM and 100mM acetaldehyde, contrasts with the wild-type of not suddenling change, measure OD 600, after wherein mutant strain 4 cultivates 8h under these two groups different acetaldehyde concentrations, OD 600all the highest, the LB be inoculated in by mutant strain 4 again containing 100mM acetaldehyde cultivates 8h, so 3 times repeatedly, again it is carried out to the test of 200mM acetaldehyde tolerance, the bacterial strain finally choosing acetaldehyde tolerance the highest checks order, through order-checking, the aminoacid sequence of DERA mutant 4 is as shown in SEQIDNo.1, and nucleotide sequence is as shown in SEQIDNo.2.
The fermentation of embodiment 6DERA mutant enzyme is standby
DERA mutant Escherichia coli genetic engineering bacterium embodiment 5 obtained is inoculated in LB substratum, 37 DEG C, 180rpm cultivates 8-10h, and the inoculum size by 3% is inoculated in (3L substratum) in 5L fermentor tank, after inoculation, at 37 ± 1 DEG C, cultivate under the condition of 300rpm, air flow quantity 4L/min, after cultivating 8-10h, add 60% glycerine as feed supplement, work as OD 600when reaching 30, be cooled to 30 DEG C, add 0.1mMIPTG and induce.After induction 10h, put tank.
Fermention medium is: glycerine 4g/L, dipotassium hydrogen phosphate 12.8g/L, potassium primary phosphate 3g/L, ammonium sulfate 0.5g/L, calcium chloride 0.0152g/L, magnesium chloride 0.41g/L.
The analysis of the conversion of embodiment 7DERA mutant enzyme and substrate and product
Embodiment 6 puts the fermented liquid of tank, collected by centrifugation thalline, adds the sodium bicarbonate buffer liquid (pH7.5) of 3 times of volume 50mM, ice-water bath ultrasonication 180 times, 8000rpm centrifuging and taking supernatant, is DERA mutant crude enzyme liquid, for enzymic catalytic reaction.Catalystic converter system is 1L, containing 0.5M acetaldehyde 600mL, and DERA mutant enzyme liquid 400mL.Acetaldehyde is dropped in enzyme liquid, dropwise in half an hour, temperature of reaction 25 DEG C, catalyzed reaction 2h.Add 2 times of vol acetone precipitating proteins after reaction, the centrifugal 10min of 8000rpm removes protein; Rotary evaporation removes acetone, then extracts 3 times with 2 times of volume of ethylacetate, merges organic phase, except desolventizing obtains product crude product.
Vapor-phase chromatography is adopted to detect product: be interior mark with diethyl malonate, chromatographic column is non-polar column, and stationary phase is 100% polydimethylsiloxane; Column temperature: 50 DEG C (stopping 2min), is warmed up to 240 DEG C with 10 DEG C/min; Carrier is helium; Injector temperature: 300 DEG C; Ion source temperature: 250 DEG C; Obtain two peaks respectively at 9.267min, 16.74min, first peak is that acetaldehyde remains, and second peak is 6-chloro-2,4,6 three deoxyhexamethylose generated after enzyme reaction, and result as shown in Figure 5.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.

Claims (8)

1. intestinal bacteria DRI-5-phosphate aldolase mutant, is characterized in that, it is the albumen 1) be made up of the aminoacid sequence shown in SEQIDNo.1; Or
2) aminoacid sequence shown in SEQIDNo.1 be substituted, lack and/or add one or several amino acid and same function by 1) derivative albumen.
2. the gene of enzyme mutant described in coding claim 1.
3. gene as claimed in claim 2, it is characterized in that, its nucleotide sequence is as shown in SEQIDNo.2.
4. the carrier containing gene described in Claims 2 or 3.
5. the host cell containing carrier described in gene described in Claims 2 or 3 or claim 4.
6. the colibacillus engineering containing gene described in Claims 2 or 3.
7. the preparation method of enzyme mutant described in claim 1, it is characterized in that, colibacillus engineering according to claim 6 is inoculated in LB substratum, 37 DEG C, 180rpm cultivates 8-10h, inoculum size by 3% is inoculated in the fermentor tank filling 3L fermention medium, and fermenter volume is 5L, at 37 ± 1 DEG C, 300rpm, after cultivating 8-10h under the condition of air flow quantity 4L/min, add 60% glycerine as feed supplement, work as OD 600when reaching 30, be cooled to 30 DEG C, add 0.1mMIPTG to induce, after induction 10h, put tank, centrifugal fermented liquid, collect thalline, add the 50mM sodium bicarbonate buffer liquid of 3 times of volume pH7.5, ice-water bath ultrasonication, 8000rpm centrifuging and taking supernatant, obtains the crude enzyme liquid containing described intestinal bacteria DRI-5-phosphate aldolase mutant.
8. the preparation method of mutant as claimed in claim 7, it is characterized in that, described fermention medium is: glycerine 4g/L, dipotassium hydrogen phosphate 12.8g/L, potassium primary phosphate 3g/L, ammonium sulfate 0.5g/L, calcium chloride 0.0152g/L, magnesium chloride 0.41g/L.
CN201510548574.7A 2015-08-28 2015-08-28 Escherichia coli 2-deoxy-D-ribose -5- phosphate aldolase mutant and preparation method thereof Active CN105063000B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201510548574.7A CN105063000B (en) 2015-08-28 2015-08-28 Escherichia coli 2-deoxy-D-ribose -5- phosphate aldolase mutant and preparation method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201510548574.7A CN105063000B (en) 2015-08-28 2015-08-28 Escherichia coli 2-deoxy-D-ribose -5- phosphate aldolase mutant and preparation method thereof

Publications (2)

Publication Number Publication Date
CN105063000A true CN105063000A (en) 2015-11-18
CN105063000B CN105063000B (en) 2018-07-13

Family

ID=54492518

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201510548574.7A Active CN105063000B (en) 2015-08-28 2015-08-28 Escherichia coli 2-deoxy-D-ribose -5- phosphate aldolase mutant and preparation method thereof

Country Status (1)

Country Link
CN (1) CN105063000B (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108559742A (en) * 2018-05-11 2018-09-21 浙江理工大学 A kind of preparation method of modification infusorial earth immobilization aldolase and application

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1965084A (en) * 2004-06-04 2007-05-16 帝斯曼知识产权资产管理有限公司 Improved 2-deoxy-d-ribose 5-phosphate aldolases for, and use in production of 2, 4, 6-trideoxyhesoses and 6-halo- or 6-cyano-substituted derivatives thereof
CN103409402A (en) * 2013-08-28 2013-11-27 南京博优康远生物医药科技有限公司 Aldolase mutant
CN103881997A (en) * 2014-03-31 2014-06-25 邦泰生物工程(深圳)有限公司 Cubilose acid aldolase mutant as well as coding gene and application thereof
CN104017795A (en) * 2014-05-27 2014-09-03 中国科学院天津工业生物技术研究所 Biosynthesis method of 2-deoxy scarce aldose by using aldolase

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1965084A (en) * 2004-06-04 2007-05-16 帝斯曼知识产权资产管理有限公司 Improved 2-deoxy-d-ribose 5-phosphate aldolases for, and use in production of 2, 4, 6-trideoxyhesoses and 6-halo- or 6-cyano-substituted derivatives thereof
CN103409402A (en) * 2013-08-28 2013-11-27 南京博优康远生物医药科技有限公司 Aldolase mutant
CN103881997A (en) * 2014-03-31 2014-06-25 邦泰生物工程(深圳)有限公司 Cubilose acid aldolase mutant as well as coding gene and application thereof
CN104017795A (en) * 2014-05-27 2014-09-03 中国科学院天津工业生物技术研究所 Biosynthesis method of 2-deoxy scarce aldose by using aldolase

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108559742A (en) * 2018-05-11 2018-09-21 浙江理工大学 A kind of preparation method of modification infusorial earth immobilization aldolase and application

Also Published As

Publication number Publication date
CN105063000B (en) 2018-07-13

Similar Documents

Publication Publication Date Title
Wang et al. Metabolic engineering of thermophilic Bacillus licheniformis for chiral pure D‐2, 3‐butanediol production
Tang et al. Microbial conversion of glycerol to 1, 3-propanediol by an engineered strain of Escherichia coli
Liu et al. Construction and characterization of ack deleted mutant of Clostridium tyrobutyricum for enhanced butyric acid and hydrogen production
US20100137655A1 (en) Process for the biological production of 1,3-propanediol from glycerol with high yield
Wang et al. Improvement of stress tolerance and riboflavin production of Bacillus subtilis by introduction of heat shock proteins from thermophilic bacillus strains
CN101792729B (en) Genetically engineered bacteria for efficiently secreting, expressing and reconstructing cutinase and method for constructing same
CN105802985A (en) Method for achieving bacillus licheniformis gene knockout rapidly
Klijn et al. Construction of a reporter vector for the analysis of Bifidobacterium longum promoters
CN104059913A (en) Temperature-regulated promoter derived from Serratia marcecens strain having high prodigiosin yield
CN105200020B (en) A kind of high substrate specificity bacillus pumilus CotA laccase being transformed by compound point mutation
CN103667163A (en) Recombinant microorganism for producing isobutanol and method thereof
CN105063000A (en) Escherichia coli 2-deoxidation-D-ribose-5-phosphoric acid aldolase mutants and preparation method thereof
Luo et al. Gene cloning, overexpression, and characterization of the nitrilase from Rhodococcus rhodochrous tg1-A6 in E. coli
CN112126615A (en) Butyric acid producing bacillus subtilis and construction method and application thereof
CN104031892A (en) Leucine dehydrogenase and gene for coding same
Zhang et al. Construction of a novel recombinant Escherichia coli strain capable of producing 1, 3–propanediol and optimization of fermentation parameters by statistical design
CN103275918B (en) The production bacterial strain of high yield DL-Alanine and application thereof
CN115806922A (en) Genetically engineered strain of zymomonas mobilis and application thereof
CN111088267B (en) Method for improving cell density of liquid fermentation of clostridium solvolyticum
CN110945013A (en) Promoters for heterologous expression
CN111286481B (en) Pseudomonas stutzeri mutant strain and construction method and application thereof
CN108949784B (en) Application of sporulation-related gene sigmaF in enzyme production
CN106148367A (en) Marine low temperature alpha amylase gene cloning and expression
Su et al. Development and application of a novel screening method and experimental use of the mutant bacterial strain Clostridium beijerinckii NCIMB 8052 for production of butanol via fermentation of fresh cassava
Akentyev et al. Expression level of SOR1 is a bottleneck for efficient sorbitol utilization by yeast Komagataella kurtzmanii

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant