CN110885848A - Method for improving protein expression level under stress condition - Google Patents

Method for improving protein expression level under stress condition Download PDF

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CN110885848A
CN110885848A CN201910766130.9A CN201910766130A CN110885848A CN 110885848 A CN110885848 A CN 110885848A CN 201910766130 A CN201910766130 A CN 201910766130A CN 110885848 A CN110885848 A CN 110885848A
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artificially synthesized
synthesized sequence
codon
expression level
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张沨
王新飞
王兴春
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Shandong Huiguan Kangbo Biotechnology Co Ltd
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Abstract

The invention discloses a method for improving protein expression level under a stress condition, which specifically comprises the following steps: according to the codon sensitivity of organisms, high-sensitivity codons are replaced by low-sensitivity codons, so that the protein can still maintain high-level expression under the stress condition, and the strategy is utilized to optimize ilvC and ilvD genes in an isobutanol production path so as to improve the yield of isobutanol. The method provided by the invention verifies the feasibility of the low-sensitivity codon optimization strategy, provides a new strategy for codon optimization, and improves the continuous expression of the protein under the adverse conditions.

Description

Method for improving protein expression level under stress condition
Technical Field
The invention relates to the technical field of bioengineering, in particular to a method for improving protein expression level under a stress condition.
Background
In cell engineering and metabolic engineering, increasing the expression level of a protein is the most effective method for obtaining high levels of a target product. However, in the actual production process, the expression of the protein reaches the maximum value after the cell reaches the stable period, and the accumulation of the target product and some other metabolites can cause the cell to be in a stress environment (such as acid stress, high salt stress, high temperature stress and the like), so that the expression amount of the protein is reduced, and the production is not facilitated. Therefore, how to increase the expression level of proteins under stress has become a major issue.
When the demand for a certain amino acid by the ribosomal translation machinery exceeds its biosynthetic rate in anabolism (i.e., amino acid starvation in the cell), the population of empty corresponding tRNA isoreceptors will increase and the translation process will be compromised. Thus, under starvation conditions, the de novo rate of amino acid synthesis may become a limiting factor for translation. In this case, if a strategy for high frequency codon optimization is used, which is just a poor "robust" property, it is difficult to use even the corresponding high tRNA charge levels of the other synonymous codons, and protein synthesis is severely inhibited. "robust" heteroreceptors respond less to fluctuations in their corresponding amino acid supply, and their loading levels will remain unchanged and their codons will be translated efficiently even under non-optimal metabolic conditions, even at very low intracellular concentrations of the corresponding amino acid, e.g., due to starvation or forced overexpression. The level of loading of the tRNA corresponding to such a codon that changes in response to the concentration of an amino acid in a cell is referred to as codon sensitivity.
Currently, codon optimization is the most commonly used method for the efficient expression of proteins. In the genetic code, 3 contiguous bases on an mRNA constitute one codon, one codon corresponding to one amino acid. There are 64 codons in an organism, 20 amino acids, which results in the degeneracy of the genetic code, and the corresponding multiple codons encoding the same amino acid are called synonymous codons. The usage frequency of synonymous codons between species is not uniform, and most organisms tend to use only a portion of these codons, i.e., species have codon preference. Codon optimization by using codon preference becomes the most direct and effective method for improving the protein expression level, and the method modifies the base sequence of the gene according to the preference of each biological codon, thereby improving the protein expression level. However, one of the drawbacks of this optimization method is that the co-translational folding kinetics are completely neglected and will thus have a considerable impact on protein quality. Especially under stress conditions, the method is more limited, and the accumulation of metabolites over time can cause the protein expression level to be arrested or even reduced. Therefore, if a method capable of stably and continuously expressing protein under the stress condition and in the later fermentation stage can be found, the high-efficiency production of a target product can be promoted, and a new way is opened up for the fermentation industry.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for improving the protein expression level under the stress condition so as to solve the problem that the protein expression level is stagnated or even reduced under the slope environment in the prior art.
In order to solve the technical problems, the technical scheme of the invention is as follows:
a method of increasing the expression level of a protein under stress conditions comprising the steps of:
step 1, selecting a gene sequence needing high-efficiency expression of protein;
step 2, determining a hypo-sensitive codon corresponding to the optimal amino acid according to the sensitivity of the biological codon, completely replacing the corresponding codon in the target protein gene sequence with the hypo-sensitive codon, and re-synthesizing by using a method for artificially synthesizing genes to obtain an artificially synthesized sequence;
step 3, connecting the artificially synthesized sequence obtained in the step 2 with a carrier, transferring the artificially synthesized sequence into a target strain, and expressing the fluorescent protein eGFP;
or constructing the artificially synthesized sequence obtained in the step 2 into a fermentation path for the fermentation production of the isobutanol.
Optionally: the target strain includes Escherichia coli.
Optionally: the step 2 of connecting the artificially synthesized sequence obtained in the step with a vector and transferring the artificially synthesized sequence into a target strain specifically comprises the following steps of:
connecting the artificially synthesized sequence obtained in the step 2 with a carrier, transferring the artificially synthesized sequence into escherichia coli for culture, and sequencing the artificially synthesized sequence by a PCR (polymerase chain reaction) verification method;
the corresponding strain with correct gene sequencing verified by PCR was cultured in M9 medium, and its fluorescence value and OD were measured600。
Optionally: the fermentation pathway comprises two plasmids, pYP69 and pYP 65.
Optionally: the method for constructing the artificially synthesized sequence obtained in the step 2 into the fermentation pathway specifically comprises the following steps: all codons encoding isoleucine in ilvC gene and ilvD gene of pYP69 and pYP65 were replaced with AUA, respectively, to obtain gene sequence Ile-AUA.
By adopting the technical scheme, the feasibility of the low-sensitivity codon optimization strategy is verified, a new strategy is provided for codon optimization, and the continuous expression of the protein under the stress condition is improved.
Drawings
FIG. 1 is a graph showing the relationship between codon frequency and sensitivity in a method for increasing the expression level of a protein under stress conditions according to the present invention;
FIG. 2 is a graph showing fluorescence intensity and OD at different glucose concentrations in the method for increasing protein expression level under stress conditions of the present invention600A ratio relation graph of (1);
FIG. 3 is a diagram of the isobutanol metabolic pathway in the method for increasing the protein expression level under stress conditions according to the present invention;
FIG. 4 is a graph of isobutanol fermentation at different glucose concentrations in the method for increasing the expression level of a protein under stress conditions according to the present invention;
Detailed Description
The following further describes embodiments of the present invention with reference to the drawings. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Example 1
The feasibility of the less sensitive codon optimization strategy was verified with green fluorescent protein eGFP:
as can be seen from FIG. 1, the relationship between codon frequency and sensitivity, UUG, UUA, CUC, CUG, CUU and CUA were used as the leucine-encoding codons in 6, respectively, and the sequences of eGFP-CUC and eGFP-UUG were obtained by replacing all the leucine-encoding codons in the sequence of eGFP with CUC and UUG and artificially synthesizing the genes. Among them, the frequencies of CUC and UUG codons were similar (6.3 and 6.2, respectively), but the sensitivity differences were large (0.59 and 24.8, respectively).
The gene sequences eGFP-CUC and eGFP-UUG are synthesized by using a whole gene synthesis technology. The gene total synthesis system is as follows: 5 × TransStart FastPFFly Buffer 10 μ L, dNTP (2.5mM)4 μ L, primer (10 μ M) premix 2 μ L, TransStart FastPFu Fly DNApolymerase 1 μ L, distilled water 35 μ L, total volume of 50 μ L. The amplification conditions were 95 ℃ denaturation 20s, 55 ℃ annealing 20s, 72 ℃ extension 20s (30 cycles); extension at 72 ℃ for 5min (1 cycle). The synthetic gene sequences eGFP-CUC and eGFP-UUG were ligated to pJET1.2/blunt Cloning Vector (Thermo Scientific), respectively. A connection system: 1.5. mu.L of the gene fragment, 0.5. mu.L of blunt-ended vector, 0.5. mu.L of LT4 ligase, 1. mu.L of 10 XT 4 ligase Buffer, 6.5. mu.L of distilled water, and 10. mu.L of the total. The resulting pJET _ eGFP-CUC and pJET _ eGFP-UUG plasmids were subsequently transferred to XL10-Gold competent cells, ice-bathed for 30min, heat shock at 42 ℃ for 60s, ice-bathed for 2min, 200uL SOC added, incubated at 37 ℃ for 45min, the whole liquid was spread on plates containing ampicillin, overnight at 37 ℃. The verification was performed by PCR and the plasmid verified to be correct was sequenced (Kingy sequencing Co.).
The corresponding strain with correct gene sequencing was cultured in M9 medium (glucose was added at different concentrations, 1.5g/L and 2.0g/L, respectively) at 37 deg.C, and its log-phase fluorescence value and OD were measured600Comparison of fluorescence value/OD600. As shown in FIG. 2, wherein Codon is a Codon; sens is codon sensitivity; usage is codon frequency, and the results show the pJET _ eGFP-CUC (i.e., Leu-LS) fluorescence intensity and OD under 1.5g/L glucose culture conditions600The ratio of the fluorescence intensity of pJET _ eGFP-UUG (namely Leu-HS) to the OD of the sample was 1090.02600361.80; pJET _ eGFP-CUC (Leu-LS) fluorescence intensity and OD under 2.0g/L glucose culture condition600Is 1484.18, pJET _ eGFFluorescence intensity and OD of P-UUG (namely Leu-HS)600759.80, indicating that the eGFP protein level was higher after optimization with the desensitization codon.
The culture medium comprises the following components: m9 medium, 1.5g/L glucose or 2.0g/L glucose, 1mM MgSO4,0.1mM CaCl2,0.1mM VB1。
Example 2
A less sensitive codon optimization strategy was used for the actual production fermentation of isobutanol:
the feasibility of the less sensitive codon optimization strategy was verified by example 1, on the basis of which ilvC and ilvD in the isobutanol production pathway were codon optimized and used for actual fermentation production. The isobutanol fermentation pathway is composed of 2 plasmids, pYP69 (expressing AlsS, ilvC and ilvD genes) and pYP65 (expressing kivD and adhA genes), as shown in FIG. 3, all codons encoding isoleucine on the ilvC and ilvD genes are replaced by AUA, the gene sequence is Ile-AUA, the codon frequency and sensitivity are respectively 0.9 and 2, and the gene sequence is lower than that of other two codons (AUU is 21.4, 15.2; AUC is 36.7, 15.2).
The gene sequence Ile-AUA is obtained by a method of artificially synthesizing genes. The gene total synthesis system is as follows: 5 × TransStart FastPfFy Buffer 10. mu.L, dNTP (2.5mM) 4. mu.L, primer (10. mu.M) premix 2. mu.L, TransStart FastPfFy DNApolymerase 1. mu.L, distilled water 35. mu.L, total volume of 50. mu.L. The amplification conditions were 95 ℃ denaturation 20s, 55 ℃ annealing 20s, 72 ℃ extension 20s (30 cycles); extension at 72 ℃ for 5min (1 cycle). The sequence was ligated to the backbone of pYP69 to give pYP69_ Ile-AUA. A connection system: mu.L of Ile-AUA fragment, 1.5. mu.L of pYP69 backbone, 7.5. mu.L of Gibson Mix, 10. mu.L total, ligated at 50 ℃ for 1 h. Then transferred into XL10-Gold competent cells, ice-cooled for 30min, heat shock at 42 ℃ for 60s, ice-cooled for 2min, added with 200. mu.L of SOC, incubated at 37 ℃ for 45min, taken out of the liquid and spread on a plate containing ampicillin and kanamycin, and cultured overnight at 37 ℃. The verification was performed by PCR and the plasmid verified to be correct was sequenced (Kingy sequencing Co.).
Both pYP65 and pYP69_ Ile-AUA plasmids were co-transferred into JCL260 strain as experimental group, and both the original pYP65 and pYP69 plasmids were transferred into JCL260 strain as control group. The experimental group and the control group obtained above were subjected to fermentation culture under different medium conditions as follows. The yield of isobutanol was measured by GC, and as shown in FIG. 4, A is the result of isobutanol fermentation using 20g/L glucose as a carbon source, and B is the result of isobutanol fermentation using 40g/L glucose as a carbon source
The experimental result shows that under the condition of taking 20g/L glucose as a carbon source, the yield of the isobutanol of the experimental group is 2.90g/L (96h), the yield of the isobutanol of the control group is 2.30g/L (96h), and the yield is improved by 26.09%; under the condition of using 40g/L glucose as a carbon source, the yield of the isobutanol of the experimental group is 4.16g/L (96h), the yield of the isobutanol of the control group is 2.64g/L (96h), and the yield is improved by 57.57%, as shown in figure 4.
Wherein the fermentation medium comprises the following components: m9 medium, 40g/L or 20g/L glucose, 1mM MgSO4,0.1mM CaCl20.1mM VB1 and 0.1mM IPTG;
wherein, the fermentation culture conditions are as follows: 30 ℃ and 250 rpm.
Method for measuring yield of isobutanol: agilent 6890GC chromatograph, DB-FFAP capillary column (30m × 0.32mm × 0.25 μm; Agilent Technologies), sample size 0.2 μ l, and injected at a split ratio of 1:50 using n-pentanol as internal standard. The injector and detector were maintained at 250 ℃ and 280 ℃ respectively. The initial temperature was maintained at 80 deg.C for 3min, followed by 115 deg.C for min-1The gradient of (2) was raised to 230 ℃ and held for 1 min.
Wherein, the formula of the internal standard method is as follows:
(Ci) sample ═ sample (Ai/As) (Ai/As) standard: (Ci) standard
C, substance concentration; a: peak area; i: a component to be tested (isobutanol); s: internal standard (n-pentanol).
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.

Claims (5)

1. A method of increasing protein expression levels under stress conditions, comprising: the method comprises the following steps:
step 1, selecting a gene sequence needing high-efficiency expression of protein;
step 2, determining a hypo-sensitive codon corresponding to the optimal amino acid according to the sensitivity of the biological codon, completely replacing the corresponding codon in the target protein gene sequence with the hypo-sensitive codon, and re-synthesizing by using a method for artificially synthesizing genes to obtain an artificially synthesized sequence;
step 3, connecting the artificially synthesized sequence obtained in the step 2 with a carrier, transferring the artificially synthesized sequence into a target strain, and expressing the fluorescent protein eGFP;
or constructing the artificially synthesized sequence obtained in the step 2 into a fermentation path for the fermentation production of the isobutanol.
2. Method for increasing the expression level of proteins under stress conditions according to claim 1, wherein: the target strain includes Escherichia coli.
3. Method for increasing the expression level of proteins under stress conditions according to claim 2, wherein: the step 2 of connecting the artificially synthesized sequence obtained in the step with a vector and transferring the artificially synthesized sequence into a target strain specifically comprises the following steps of:
connecting the artificially synthesized sequence obtained in the step 2 with a carrier, transferring the artificially synthesized sequence into escherichia coli for culture, and sequencing the artificially synthesized sequence by a PCR (polymerase chain reaction) verification method;
the corresponding strain with correct gene sequencing verified by PCR was cultured in M9 medium, and its fluorescence value and OD were measured600。
4. Method for increasing the expression level of proteins under stress conditions according to claim 1, wherein: the fermentation pathways include pYP69 expressing the alsS, ilvC and ilvD genes and pYP65 expressing the kivD and adhA genes.
5. Method to increase the expression level of proteins under stress conditions according to claim 4, characterized in that: the method for constructing the artificially synthesized sequence obtained in the step 2 into the fermentation pathway specifically comprises the following steps: all codons encoding isoleucine in ilvC gene and ilvD gene of pYP69 and pYP65 were replaced with AUA, respectively, to obtain gene sequence Ile-AUA.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014037912A2 (en) * 2012-09-07 2014-03-13 Cellworks Research India Pvt. Ltd Methods for production of isobutanol through nutrient stress and genetically modified microorganisms thereof
CN108795966A (en) * 2018-03-05 2018-11-13 北京理工大学 A kind of screening technique of branched-chain amino acid superior strain

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014037912A2 (en) * 2012-09-07 2014-03-13 Cellworks Research India Pvt. Ltd Methods for production of isobutanol through nutrient stress and genetically modified microorganisms thereof
CN108795966A (en) * 2018-03-05 2018-11-13 北京理工大学 A kind of screening technique of branched-chain amino acid superior strain

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ELF J, NILSSON D等: "Selective charging", 《SCIENCE》 *
郑丽娟等: "利用双启动子载体构建产异丁醇大肠杆菌", 《中国生物工程杂志》 *

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