CN115960920B - Coroxepin FHb, recombinant bacterium X33-pPICZ alpha A-102C300C-FHb2 and application thereof - Google Patents
Coroxepin FHb, recombinant bacterium X33-pPICZ alpha A-102C300C-FHb2 and application thereof Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Abstract
The invention relates to a coroxepin FHb, recombinant bacteria X33-pPICZ alpha A-102C300C-FHb and application thereof, belonging to the technical field of genetic engineering, wherein the nucleotide sequence of a gene of the coroxepin FHb is shown as SEQ ID N0.1. The invention also provides an amino acid sequence of the coroxgen FHb as shown in SEQ ID N0.2. The invention also provides a recombinant expression vector comprising the algin lyase gene and the gene of the coronetin FHb and recombinant bacteria X33-pPICZ alpha A-102C300C-FHb2, wherein the recombinant bacteria improve the utilization rate of the thalli to oxygen in a low-oxygen environment in the later fermentation period, and finally improve the yield of the algin lyase.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a coroxepin FHb, a recombinant bacterium X33-pPICZ alpha A-102C300C-FHb and application thereof.
Background
Pichia pastoris is the most commonly used protein expression system following E.coli, and it is currently widely used for laboratory-scale protein preparation, characterization and structural analysis. In the field of biopharmaceuticals, various proteins, such as insulin, hepatitis b surface antigen, human serum albumin, epidermal growth factor, etc., can be produced by pichia pastoris expression. Compared with the escherichia coli and bacillus subtilis expression systems, the method has a plurality of advantages, for example, effective secretion expression can be realized, the workload required for separating and purifying target proteins in the later period is reduced, the exogenous genes are integrated into the genome to realize stable expression, and the method is simple, convenient and low in cost and has high-density biomass accumulation.
Pichia pastoris X33 belongs to a wild strain, methanol can be rapidly utilized to integrate a recombinant vector into the genome of the strain, and the expression of a foreign protein in a host is controlled by combining an alcohol oxidase promoter (AOX) on the vector. At present, in the fermentation process, since pichia pastoris is a high aerobic microorganism, the cell density and the rate of oxidative degradation of methanol are often limited due to the limited oxygen supply capacity of equipment, so that the expression amount of target proteins is limited.
Flavohemoglobins (FHb) are a class of hemoglobin found in single cell organisms such as bacteria, yeast and the like. It comprises an n-terminal hemoglobin domain and a c-terminal oxidoreductase domain, which have potential binding sites for NAD (P) H and FAD. FHb is considered to be at O 2 Plays a role in sensing, storage and delivery.
Aiming at the problem of oxygen utilization rate in the high-density fermentation process of pichia pastoris, the invention constructs the high-expression strain for producing the algin lyase by cloning the flavohemoglobin gene FHb, improves the utilization rate of thalli to oxygen in a low-oxygen environment in the later fermentation period, and finally improves the yield of the algin lyase.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a co-oxygen protein FHb and recombinant engineering bacteria X33-pPICZ alpha A-102C300C-FHb-2 thereof and application, wherein the co-oxygen protein can improve the efficiency of secreting algin lyase by pichia pastoris engineering bacteria.
The invention is realized by the following technical scheme:
a gene of a COX FHb, the nucleotide sequence of which is shown in SEQ ID N0.1.
The amino acid sequence of the coroxgen FHb is shown as SEQ ID N0.2.
A recombinant expression vector, which comprises the algin lyase gene and the gene of the coronein FHb, wherein the nucleotide sequence of the algin lyase gene is shown as SEQ ID N0.3.
A recombinant bacterium X33-pPICZ alpha A-102C300C-FHb comprises the recombinant expression vector, and the gene of the co-oxygen protein FHb is connected to the tail end of an algin lyase gene to form a tandem expression cassette.
The gene of the coronetin FHb is applied to improving the secretion efficiency of the algin lyase, and the algin lyase is secreted and expressed in a eukaryotic expression system.
Preferably a pichia X33 expression system.
Gene of the coronexin FHb:
atgactgccccagaaatctaccagatccaggatttgactgaggaccaaaagaccttcatcagaaactccgtgccaatcttga
aggacgctggtgaaactttgaccgccaagttctaccagtacatgatctccaactacgacgaggtcaagccatacttcaacg
agactaaccagaagctgctgaagcagccaagaattttggccttcgccttgttgaagtacgccgagaacattgacgacttgtc
cccattgactgctttcgttcaccagatcgttgttaagcacgttggtctgcagattaaggctgagcactacccaatcgttggtgg
ttccttgcttaagactatggccgagttgttgggtccagaattggctactccagctttcttgaaggcttgggctactgcttacggt
aacttggcccagattttgatcaacgctgaatgggccgagttccaaaagcaaaagtggcacgacttcaaagagttcaaggtc
accaagatcgagaacgagtgtgacgacgttaagtccgtttacttcactccagttgagggtgagatcgctaagccattggatg
gtcagtacgtctgcatcagatggaagttgccaggtgagaagttcgagaagtctagagagtactccttgtcctccagaccaa
acaacaacacctacagaatctccgtcagactgttggagaacggtaagatttccaccttcgtccacaaccagttgaaggtcg
gtgacatcattactgttgctcctccagctggtcagttgttgtacgaagaatctcagaaggacgccgttttcttcatcggtggtat
tggtatcaccccagtcgtgtccattatggaaactgctttggagagaggtcagagggtcactttgttctactccaacagaactt
ccaagtccaccgctttcagaggttggttgaaagagctgaagtccaagttcaacttgcagctgaccgtgaaagagttcgtttc
cgaggaacaagttactgagggtgtcgaccagattaactccgctcaattgcagagatccgacatccaaactgtttccccaga
gaacgaggtttacttggttggtccagttccatatatgcagttcgtgtcctccgagttgaacaagttgggtgttcagaacatccactccgagttcttcggtccaactgttgttgcttaa.
amino acid sequence of the coronexin FHb:
MTAPEIYQIQDLTEDQKTFIRNSVPILKDAGETLTAKFYQYMISNYDEVKPYFN
ETNQKLLKQPRILAFALLKYAENIDDLSPLTAFVHQIVVKHVGLQIKAEHYPIV
GGSLLKTMAELLGPELATPAFLKAWATAYGNLAQILINAEWAEFQKQKWHDF
KEFKVTKIENECDDVKSVYFTPVEGEIAKPLDGQYVCIRWKLPGEKFEKSREY
SLSSRPNNNTYRISVRLLENGKISTFVHNQLKVGDIITVAPPAGQLLYEESQKD
AVFFIGGIGITPVVSIMETALERGQRVTLFYSNRTSKSTAFRGWLKELKSKFNL
QLTVKEFVSEEQVTEGVDQINSAQLQRSDIQTVSPENEVYLVGPVPYMQFVSSELNKLGVQNIHSEFFGPTVVA.
alginate lyase gene:
actgaatctggttctggttcttcttctggtggttcttcctccggatcttcttcttcctcatcctcttccggtggatcatcctctggtg
gatcaggtggtagtagttcaggtggatctttggacccaaacttgccaccatcttccaacttcgatttgtccgcttggtacttgtc
cgttccaactgataacaacggtgacggtaaggccgactccatcaaagaaaacgatttgaacgctggttacgccgacggta
cttacttttacactgctgctgatggtggtatggtgttcagatgtccaatctgtggttacaagacttctaccaacacctcctacac
cagaaccgagttgagagaaatgctgagaagaggtgacacctccattgctactcaaggtgtcaacggtaacaactgggtttt
cggttctgctccagcttccgctagagaagctgctggtggtgtagatggtgttttgagagctactttggccgttaaccacgttac
tactactggtgactctggtcaggttggtagagttatcgttggtcagatccacgctaacaacgacgaaccattgagactgtact
acagaaagttgccaggtcactccaagggttccgtttacattgctcatgaacctaacggtggttccgactcttggtacgacatg
attggttctagatcctcctctgcttctgacccatctgacggtattgctttggacgaagtttggtcctacgaggttaaggttgtcg
gtaacactttgaccgtgaccatcttcagagctggtaaggacgacgttgttcaggttgttgacatgggtaactccggttacgat
gttgctgaccagtaccagtacttcaaggccggtgtttacaaccagaacaatactggtaactgttccgactacgttcaggttactttctacgctttggagcaatctcacgatcatcatcaccatcaccactaa.
compared with the prior art, the invention has the following beneficial effects:
1. according to the invention, the secretory expression of the algin-producing lyase engineering bacteria in a low-oxygen environment is improved by constructing an expression vector combining the co-oxygen protein gene FHb and the algin lyase coding gene at the tail end of the original algin lyase gene with the nucleotide sequence of SEQ ID N0.3.
2. The invention provides a gene of a co-oxygen protein FHb, which can improve the utilization rate of thalli to oxygen in a low-oxygen environment, realize high expression of algin lyase and particularly provide an effective element for expressing the algin lyase gene by pichia pastoris.
Drawings
FIG. 1 is a gene electrophoresis diagram of amplified protocol FHb in example 1; wherein lane M: marker DNA; lane 1 is a gene fragment of the protocol oxygen protein FHb;
FIG. 2 is a schematic diagram of the construction of the recombinant plasmid pPICZ αA-102C300C-FHb of example 1;
FIG. 3 is a graph showing the comparison of the dry weight of the engineering bacteria constructing the gene of the protocol oxygen protein FHb in example 2 with that of the control group before transformation;
FIG. 4 is a graph showing comparison of enzyme activities of alginate lyase in the control group before transformation and engineering bacteria constructing the gene of the protocol oxygen protein FHb in example 2.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto.
The molecular biology experimental techniques used in the examples below include PCR amplification, plasmid extraction, DNA fragment ligation, gel electrophoresis, etc., as described in detail in the "guidelines for molecular cloning experiments" (third edition) (Sambrook J, russell DW, janssen K, argentine J. Huang Peitang et al, 2002, beijing: science Press).
EXAMPLE 1 construction and screening of recombinant engineering bacteria of the coronexin
By means of one-step cloning, an expression vector combining the gene of the COX FHb and the alginate lyase gene is constructed, and a high-throughput screening method is combined to obtain positive clones with highest alginate lyase activity from the clones, wherein the method specifically comprises the following steps:
(1) Modulation and purification of FHb gene: the original sequence of the coronexin is downloaded from a National Center for Biotechnology Information website, is shown as SEQ ID N0.6, is artificially synthesized after codon optimization, and is connected to a pUC57 plasmid to construct a pUC57-FHb strain. Extracting plasmid of pUC57-FHb strainPlasmid DNAMini Kit D6942 kit), PCR amplifying the fishing FHb gene from pUC57-FHb plasmid genome by using two artificially synthesized primer fragments SEQ ID N0.4 and SEQ ID N0.5; nucleic acid electrophoresis was verified (see FIG. 1) and purified (++>Gel Extraction Kit) recovering the desired fragment for use. The nucleotide sequence of the optimized gene of the coronexin FHb is shown as SEQ ID N0.1, and the amino acid sequence is shown as SEQ ID N0.2.
Forward primer: 5'-caccatcaccactaagcggccgccaagcttgatgactgcccca-3' the number of the individual pieces of the plastic,
reverse primer 5'-tgttctagaaagctggcggccgcttaagcaacaacagttggaccg-3'.
Original sequence of the gene of the coronexin FHb:
atgaccgctccagaaatctatcagattcaagacctcactgaggaccagaagactttcattaggaactcagttcctattt tgaaggacgctggagaaactttgactgctaagttttatcagtacatgatttctaattatgatgaagtcaagccatactttaatgagacaaatcaaaaactgttgaaacagcccaggattttagcgtttgctctcctcaagtatgcagaaaacattgacgatttgagcccgctaactgctttcgttcaccagatcgttgtaaagcatgttggactgcagattaaagctgagcactacccaattgtcggtggatcgttgttgaagacaatggctgaactcttgggacctgagcttgctacacctgcgtttctcaaggcatgggcaaccgcttacggaaatcttgctcaaatcctgataaacgctgaatgggcagagttccagaagcagaagtggcatgatttcaaagagtttaaagtgaccaaaatcgaaaatgaatgtgacgatgtgaagtctgtttatttcactccagtagaaggagagatagcaaaacctttggatgggcaatacgtctgcatcagatggaaattacctggggaaaagtttgagaaatctagggagtactctctttcttccaggccaaacaacaatacctacagaatctctgtcaggttgttggaaaatggtaaaatttcgacatttgttcacaatcaactgaaggtgggagacataatcactgtagcaccacccgctggacaattgttatacgaagaatctcaaaaggatgctgttttttttatcggaggtattggtatcactccagtagtatcaatcatggaaactgcccttgaaaggggtcagagagttacattattttactccaatagaacctcaaagagtactgcctttagaggttggttgaaagaactaaagagtaaattcaatttgcaactgacagtgaaggaattcgtctctgaagaacaagttactgaaggtgtagaccaaatcaattctgctcaattacagagatcagacatccaaacggtgtcgccggagaatgaagtttatctggttggacctgttccatacatgcagtttgttagctctgagctcaacaaactgggagtccaaaatattcattccgagttctttggaccaactgtggttgcttga.
(2) Enzyme tangentially pPICZαA-102C300C plasmid: the pPICZαA-102C300C plasmid was digested singly with Not I (TaKaRa) to prepare a linearized vector fragment. The linearized carrier is digested by Fast DigestDpn I, and the treated fragments are purified by a purifying kitCycle Pure Kit D6492) is recovered for later use after purification.
(3) Construction and verification of pPICZ alpha A-102C300C-FHb plasmid: and (3) performing appropriate dilution and recombination connection according to the DNA concentration of the recombinant gene and the linearized pPICZ alpha A-102C300C fragment by using a DNA seamless cloning (Clon Express II One Step Cloning Kit kit), converting E.coli DH5 alpha by using a connected system heat shock method, and then coating an LB (Zeocin) plate for screening positive recombinants. And (3) carrying out monoclonal sequencing verification on the constructed vector by using a pPICZ alpha A vector universal primer pair. The construction process of pPICZαA-102C300C-FHb plasmid is shown in FIG. 2.
(4) Construction of Pichia pastoris Strain X33-pPICZ alpha A-102C300C-FHb 2: extracting plasmid after activating correctly sequenced transformantPlasmid DNAMini Kit D6942 kit) to yield a cloning vector containing FHb. The cloning vector was transformed into Pichia X33 strain by electrotransformation to construct Pichia X33-pPICZ alpha A-102C300C-FHb2 strain.
(5) High throughput screening of recombinant strains: positive transformants were selected for culture in 48-well deep well plates, each well being supplemented with 1mL of BMGY liquid medium. 30 ℃,200rpm, 1% methanol was added every 24h, and induction was continued for 3 days. Centrifuging at 4000rpm for 5min, collecting supernatant, detecting enzyme activity of algin lyase, and screening out strain with high enzyme activity.
Example 2 evaluation of growth and enzyme production levels of the strains containing the COX
(1) The strain constructed in example 1 and the recombinant strain constructed without the gene of the protocol oxygen protein FHb were inoculated as a control group into 20mL of liquid YPD medium, and shake-cultured at 37℃at 200r/min for 24 hours, as seed solutions.
(2) 1mL of seed solution is sucked to 20mL of BMGY culture medium, 1% methanol is added every 24h for induction and enzyme production, the total induction is carried out for 5 times, the first four days of shaking culture is carried out at 30 ℃ for 200r/min, and the second two days of shaking culture is carried out at 30 ℃ for 50 r/min. Every 24 hours, 2mL of bacterial liquid is taken and centrifuged for 5min at 5000r/min, the dry weight is measured by precipitation, and the enzyme activity is measured by supernatant.
(3) And (3) taking a flat weighing bottle made of clean glass, placing the flat weighing bottle in a 105 ℃ drying box, obliquely supporting a bottle cap on the bottle edge, heating for 2 hours, taking out the bottle cap, placing the bottle cap in a dryer for cooling for 0.5 hour, weighing, and repeatedly drying until the difference of the quality of the bottle cap and the bottle cap is not more than 2mg, thus obtaining the constant weight.
(4) The enzyme activity of algin lyase is determined by DNS method, and the unit definition of enzyme activity is: 1 enzyme activity unit (U) is defined as the amount of enzyme required to produce 1. Mu. Mol of reducing sugar per 1 min.
(5) Determination of Pichia pastoris cell dry weight and algin lyase enzymatic activity: compared with the control group (13.05 g/L), the dry weight of the engineering bacteria of the alginate lyase producing Pichia pastoris containing the coroxepin is 13.25g/L when the engineering bacteria are fermented for 144 hours, and the bacterial body quantity of the control group is in a descending trend, as shown in figure 3. The gene of the COX FHb can increase the yield of algin lyase from 7.76U/mL to 10.46U/mL in the low-oxygen environment by 34.8% (see FIG. 4).
Claims (3)
1. The recombinant expression vector is characterized by comprising an algin lyase gene and a gene of a COX FHb shown as SEQ ID N0.1, wherein the nucleotide sequence of the algin lyase is shown as SEQ ID N0.3.
2. A recombinant bacterium X33-ppiczαa-102C300C-FHb2, wherein the recombinant bacterium X33-ppiczαa-102C300C-FHb2 comprises the recombinant expression vector of claim 1; specifically, the gene of the COX FHb is connected to the tail end of the algin lyase gene to form a tandem expression cassette.
3. The use of the gene of the coronexin FHb in increasing the secretory expression of algin lyase, wherein the secretory expression of algin lyase is in a eukaryotic expression system, and the eukaryotic expression system is a pichia pastoris X33 expression system.
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| Over-expression of Vitreoscilla hemoglobin (VHb) and flavohemoglobin (FHb) genes greatly enhances pullulan production;Si-Jia Xue等;International Journal of Biological Macromolecules;第132卷;第701-709页 * |
| XP_002490302.1;无;NCBI_GenPept;序列信息 * |
| 乳糖诱导大肠杆菌产褐藻胶裂解酶的条件优化;唐玮等;食品工业科技(第15期);第134-139页 * |
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