LacI mutants and uses thereof

Abstract

The application discloses a LacI mutant and application thereof, and belongs to the technical field of biotechnology.The amino acid sequence of the LacI mutant is shown as SEQ ID NO.2, 3 or 4.In the application, the LacI mutant is obtained by screening based on the LacIQ mutant through directional evolution, the mutant can enhance the binding capacity with the DNA sequence, thereby reducing the background leakage expression (lower than 5% of the leakage expression of the wild type), and meanwhile, the mutant can also achieve good effect when used for preparing a high-efficiency biosensor.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and particularly relates to LacI mutants and their applications. Background Technology

[0002] As one of the most extensively studied microorganisms, *Escherichia coli* possesses two significant characteristics: ease of operation and the ability to achieve high-density culture in inexpensive media. Therefore, the *E. coli* expression system is the most commonly used prokaryotic expression system for efficiently expressing heterologous proteins. Despite the numerous advantages of *E. coli*, not every gene can be effectively expressed within it. This is attributed to factors such as the unique structure of each gene, the stability and translation efficiency of its mRNA, the ease of protein folding, the degradation of proteins by host cell proteases, the foreign gene, key differences in codon utilization, and the potential toxicity of the protein to the host.

[0003] The lactose promoter (Lac) is the earliest discovered and most widely used promoter in *E. coli*. Its mechanism of action involves the regulatory protein LacI binding to the operator gene *lacO* in the absence of a small molecule inducer (such as lactose or its analogues), thereby inhibiting transcription initiation. In the presence of an inducer, it binds to LacI, dissociating from the *lacO* sequence and initiating transcription. The lactose promoter has also spawned other promoters such as Tac and Trc, which are used in constructing T7 high-expression systems. However, one problem with the Lac series promoters is that without an inducer (such as isopropyl thiogalactoside (IPTG)), the system exhibits high basal leakage expression. This drawback can burden the host cell's metabolism and hinder the expression of foreign proteins, especially toxic proteins. Furthermore, when using this system to construct metabolic pathways, leakage expression leads to imprecise regulation of metabolic flux, wasting intracellular resources and making it unsuitable for optimizing metabolic module ratios.

[0004] Current methods involve mutating the promoter region of the LacI regulatory protein to obtain the LacIQ mutant, which enhances promoter strength and significantly reduces basal leakage expression. However, the inherent characteristics of the LacI protein, such as its binding to lacO DNA sequences, cannot be addressed simply by increasing its expression level. Summary of the Invention

[0005] One of the objectives of this invention is to provide a LacI mutant, the amino acid sequence of which is shown in SEQ ID NO.2, 3 or 4.

[0006] A second objective of this invention is to provide an expression cassette containing the aforementioned LacI mutant.

[0007] A third objective of this invention is to provide an expression vector containing the aforementioned LacI mutant or expression cassette.

[0008] Furthermore, the expression vector is a recombinant plasmid.

[0009] Furthermore, the recombinant plasmids are pET-22a(+), pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a(+), pET-14b, pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-31b(+), pET-32a(+), and pET-35b(+). (+), pET-38b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+), pET-42a(+) , pET-43a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE31, pQE 32. pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX- 6p2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-8, pUC-18 or pUC-19.

[0010] The fourth objective of this invention is to provide a recombinant host cell containing the aforementioned LacI mutant.

[0011] Furthermore, the recombinant host cell is Escherichia coli BW25113ΔlacI, Corynebacterium glutamicum, Pseudomonas putida, or Bacillus subtilis.

[0012] A fifth objective of this invention is to provide a method for enhancing the expression of a target gene, the method comprising operatively linking the aforementioned LacI mutant to the target gene.

[0013] The sixth objective of this invention is to provide the application of the above-mentioned LacI mutant in the preparation of biosensors.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] Based on the LacIQ mutant, this invention obtains a LacI regulatory protein mutant through directed evolution screening. This mutant can enhance the binding ability to DNA sequences, thereby reducing basal leakage expression (less than 5% of wild-type leakage expression). Attached Figure Description

[0016] Figure 1a The graph shows the induction curves of LacI mutants (M7, N246S, and I79T) and wild-type LacI in Example 1.

[0017] Figure 1b The bar chart shows the LacI mutants (M7, N246S, and I79T) and wild-type LacI induction in Example 1.

[0018] Figure 2 This is a comparison diagram showing the regulation of the expression of two fluorescent proteins, RFP and GFP, by LacI wild-type (WT) and mutant N246S in Example 2.

[0019] Figure 3 This is a comparison of the expression of heterologous proteins by the pET28a T7 promoter regulated by wild-type (WT) LacI and mutant M7 in Example 3.

[0020] Figure 4 This is a comparison of the expression of fluorescent protein regulated by wild-type LacI (light gray) and mutant M7 (dark gray) in Corynebacterium glutamicum in Example 4.

[0021] Figure 5 This is a comparison of the expression of fluorescent protein regulated by wild-type LacI (light gray) and mutant M7 (dark gray) in *Pseudomonas putida* in Example 4.

[0022] Figure 6 This is a comparison of the expression of fluorescent protein regulated by wild-type LacI (light gray) and mutant M7 (dark gray) in Bacillus subtilis in Example 4.

[0023] Figure 7 Example 5 shows the screening of LacS transglycosylation activity mutants using a biosensor constructed based on the LacI mutant M7. In Example 5, (A) several LacS mutants and wild-type (WT) were screened and compared with the fluorescent protein GFP values ​​and the content of galactooligosaccharides (GOS) generated under conditions of no addition of substrate lactose (-) and addition of substrate lactose (+). The results of comparing the enzyme activity of the optimal mutant LasS S5-11 with wild-type (WT) in vivo (B) and in vitro (C) at 37℃ and 70℃ were also presented. Detailed Implementation

[0024] Example 1

[0025] LacI wild-type sequence SEQ ID NO.1:

[0026] MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ*.

[0027] Sequence of LacI M7 mutant SEQ ID NO.2:

[0028] MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQ T VAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVA S DQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ*.

[0029] Sequence of LacI N246S mutant SEQ ID NO.3:

[0030] MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGL IINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVA S DQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ*.

[0031] LacI I79T mutant sequence SEQ ID NO.4:

[0032] MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQ T VAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDW SAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ*.

[0033] The horizontal lines in the above sequence represent mutation sites.

[0034] The induction curves of the above mutants were tested using the external inducer IPTG. The results are shown below. Figure 1a and 1bThe specific experimental method was as follows: pLac7-RFP plasmid (reference: Wu, J., Jiang, P., Chen, W., Xiong, D., Huang, L., Jia, J., Chen, Y., Jin, J.-M., Tang, S.-Y. 2017. Design and application of a lactulose biosensor. Scientific Reports, 7) and other LacI mutant plasmids were transformed into E. coli BW25113ΔlacI (i.e., chromosomal lacI gene knocked out) host bacteria and plated on ampicillin-resistant plates. Single colonies were picked and transferred to LB medium containing the corresponding antibiotics and cultured at 37°C with shaking for 14 hours. This was used as seed culture and transferred to LB medium containing the corresponding antibiotics at a 1% (v / v) inoculation rate. When the cell concentration reached about 0.6, 0-10 mM IPTG inducer was added and cultured at 37°C with shaking for another 12 hours before detecting the fluorescent protein content. Compared with wild-type LacI (WT), all three mutants significantly reduced P. tac Promoter basal leakage expression was observed. Without the addition of an inducer, the RFP expression levels of LacI(WT), LacI-M7, LacI-N246S, and LacI-I79T were 3005, 150, 183, and 405 A.U., respectively. The highest expression levels after induction in both wild-type and mutants were around 30,000 A.U., with mutants exhibiting approximately 5-15% of the wild-type basal leakage expression level. Mutant M7 showed the lowest leakage level, thus achieving the highest induction fold (approximately 200-fold), but also requiring the highest IPTG concentration (approximately 1 mM) to reach half of the highest induction level. Mutant N246S achieved a maximum induction fold of approximately 150-fold, and required an IPTG concentration of 0.2 mM to reach half of the highest induction level. The mutant I79T exhibits a relatively gradual induction trend. Although the highest induction level is only 66% of that of the wild type (the final induction level of I79T is about 24708 A.U.), the induction system it regulates shows a gradual upward trend over a wide range of inducer concentrations. Therefore, it is suitable for applications that respond to a wide range of concentrations.

[0035] Example 2

[0036] Using the pLac7 plasmid, the expression of two fluorescent proteins, RFP and GFP, was regulated by wild-type (WT) LacI and the mutant N246S, respectively. Flow cytometry was used to monitor the fluorescence intensity of different fluorescent cell components at various IPTG inducer concentrations. The specific experimental method was as follows: The plasmid was transformed into *E. coli* BW25113ΔlacI (i.e., the chromosomal lacI gene knocked out) host bacteria and plated on ampicillin-resistant plates. Single colonies were picked and cultured in LB medium containing the corresponding antibiotic at 37°C for 14 hours. This was then used as seed culture for inoculation into LB medium containing the corresponding antibiotic at a 1% (v / v) inoculation rate. When the cell concentration reached approximately 0.6, 0-2 mM of the specified IPTG inducer was added, and the cells were cultured for another 12 hours at 37°C. The fluorescent protein content was then measured. The cells were collected by centrifugation at 4,000g for 5 minutes and resuspended in PBS buffer. Detection was performed using a flow cytometer (FACSAria II sorter (manufactured by BD, San Jose, USA)). RFP (red fluorescent protein) was detected using an excitation wavelength of 561 nm and a detection wavelength of 610 nm; GFP (green fluorescent protein) was detected using an excitation wavelength of 395 nm and a detection wavelength of 509 nm. Data were analyzed and plotted using FlowJo software. See [link to results]. Figure 2 .Depend on Figure 2 It is known that under the regulation of the LacI mutant N246S, fluorescent proteins in both independent reading frames can undergo fluorescence changes under the regulation of the inducer IPTG. Both wild type and mutant can reach the same maximum induction level (IPTG 2mM), but the mutant can regulate a wider range of inducer concentrations.

[0037] Example 3

[0038] The T7 system is a commonly used and efficient system for regulating the expression of heterologous proteins. pET28a is a frequently used plasmid expression system. This paper uses pET28a as an example to illustrate the effect of the LacI mutant on the induction of T7 system expression. The pET28a plasmid contains the LacI regulatory protein. The LacI protein in the pET28a plasmid was mutated to an M7 mutant, and the effect on the induction of the heterologous protein RFP by this system was observed and verified using a gel electrophoresis. The specific experimental method was as follows: the wild-type and mutant LacI-regulated pET28a-RFP plasmids were transformed into *E. coli* BL21(DE3) host bacteria and plated on kanamycin-resistant plates. Single clones were picked and cultured in LB medium containing the appropriate antibiotic at 37°C for 14 hours using a shaker. This culture was then used as a seed culture for inoculation with 1% (v / v) of LB medium containing the appropriate antibiotic. The cells were cultured at 37°C until the cell concentration reached approximately 0.6. Then, 0-0.02% of the specified concentration of lactose, the inducer, was added. The cells were cultured at 37°C for another 12 hours using a shaker, and the fluorescent protein content was then measured. Figure 3As shown, compared with the wild type, the pET28a system regulated by the LacI mutant has lower background expression, and the highest induction level is at the same level as the wild type.

[0039] Example 4

[0040] The mutant LacI was used to replace the wild-type LacI in regulating the induction expression systems of other strains (Corynebacterium glutamicum, Pseudomonas putida, and Bacillus subtilis). Results are shown in [link to results]. Figure 4-6 This shows that it can also effectively reduce background expression.

[0041] Corynebacterium glutamicum, Pseudomonas putida KT2440, and Bacillus subtilis 168 are three microbial host bacteria commonly used for biosynthesis. Therefore, this study also involves experiments on the induction of protein expression using the mutant LacI in the above three microorganisms.

[0042] Corynebacterium glutamicum commonly uses the plasmid pMXJ119, which is linked to the GFP fluorescent protein (i.e., pMEJ119-GFP), and a mutation site is introduced into the lacI gene on the plasmid. After pMEJ119-GFP is introduced into the host bacteria, it is cultured at 30°C, and a specified concentration of the inducer IPTG is added. After culturing for another 12 hours, the fluorescent protein content is tested.

[0043] The plasmid p2015a-p23107-LacI-Ptac-sfGFP (plasmid source: Y. Xue; T. Qiu; Z. Sun; X. Liu; B. Yu, Mercury bioremediation by engineered Pseudomonasputida KT2440 with adaptationally optimized biosecuritycircuit. Environ. Microbiol. 24(7), 3022-3036(2022).) was introduced with a mutation site and then transformed into Pseudomonas putida. A specified concentration of the inducer IPTG was added, and the fluorescent protein content was tested after culturing for 12 hours.

[0044] Bacillus subtilis commonly uses plasmid pHT01, which is linked to GFP fluorescent protein (i.e., pHT01-GFP), and a mutation site is introduced into the lacI gene on the plasmid. After pHT01-GFP is introduced into the host bacteria, it is cultured at 30°C, and a specified concentration of the inducer IPTG is added. After culturing for another 24 hours, the content of fluorescent protein is tested.

[0045] Example 5

[0046] Biosensors based on transcriptional regulatory proteins can correlate the concentration of a target small molecule compound with the expression level of a reporter gene, thereby indirectly characterizing the concentration of the small molecule compound by monitoring the reporter gene expression level. Therefore, they are suitable for constructing dynamic regulatory systems or high-throughput screening tools. The LacI mutant has the effect of low background expression and can be induced by inducers over a wide range, making it more suitable for application in high-efficiency biosensors.

[0047] The biosensor constructed in this way was used to screen for the β-glycosidase LacS transglycosylase activity of the thermophilic archaea *Lactobacillus sulfideus*, obtaining LacS mutants S4, S5, S19, and S21. Subsequently, a fault-prone mutant library was constructed based on S5, and mutants S5-1, S5-11, and S5-34 were obtained through screening. Figure 7 As shown in Figure A, the transglycosylation activities of the LacS mutants were all higher than those of the wild type, ranging from 2.6 to 8.4 times that of wild-type LacS. The optimal mutants 5-11 were further compared with the wild type in both in vivo reaction and after purification. Since the optimal enzyme activity of LacS is at 70℃, but the screening of the LacS mutant library was conducted at the optimal growth temperature of E. coli (37℃), the enzyme activities of the mutants and wild type were compared at both temperatures. Figure 7 As shown in B and 7C, the mutant exhibits superior activity compared to the wild type under various conditions.

[0048] The specific experimental protocol was as follows: Under error-prone PCR conditions, the plasmid pBADLacS (Jiang PX, Mu SS, LiH., Li YH, Feng CM, Jin JM, Tang SY, 2015. Design and application of a novel high-throughput screening technique for 1-deoxynojirimycin. Sci. Rep.-UK5, 8563.) encoding the LacS gene was amplified. The LacS gene with a random mutation site was then re-ligated to the pBAD plasmid to obtain a mutant library. The pBADLacS mutant library was transformed into the BW25113(ΔlacIΔgalKΔrecA) / LacI-M7 plasmid strain, and an appropriate amount was plated on LB agar plates containing 1 μM lactose and 5 mM L-arabinose. After static incubation at 37°C for 12 hours, the intensity of the fluorescent protein in the single clone strain was observed. Single clones of mutants expressing high levels of fluorescent protein were selected and cultured overnight in LB tubes at 37°C. They were then transferred to fresh LB medium. When the bacterial concentration reached approximately 0.6, arabinose was added as an inducer to induce LacS mutant protein expression. After culturing at 30°C with a shaker for 12 hours, the bacterial cells were collected. While testing for fluorescent protein expression, the cells were also sonicated to disrupt the cell wall. 50 μl of the crude LacS mutant enzyme solution was then dissolved in phosphate buffer (50 mM, pH 6.5) at 150 g / L. -1 The product GOS content was determined by HPLC after incubation at 37℃ and 70℃ for 12 hours on lactose substrate. Specifically, the detection system was an Agilent 1200 system (Agilent Technologies, Palo Alto, USA); separation conditions were a Cosmosil sugar-D column (250×4.6mm, 5μm) (Nacalaitesque, Japan); column temperature was 60℃; and elution conditions were 80% acetonitrile 1.5 mL / min. -1 Constant gradient, visual detector. The unit enzyme activity (U) of LacS is defined as the amount of pure enzyme required to catalyze the production of 1 μmol of GOS per hour.

[0049] The LacS protein purification method is as follows: The pET28a-LacS plasmid (Jiang PX, Mu SS, Li H., Li YH, Feng CM, Jin JM, Tang SY, 2015. Design and application of a novel high-throughput screening technique for 1-deoxynojirimycin. Sci. Rep.-UK 5,8563.) was transformed into Escherichia coli BL21(DE3) host bacteria and plated on kanamycin-resistant plates. Single colonies were picked and transferred to LB medium containing the appropriate antibiotic and cultured at 37°C with shaking for 14 hours. This was used as seed culture and inoculated into LB medium containing the appropriate antibiotic at a 1% (v / v) inoculum rate. When the cell concentration reached approximately 0.6, 0.4 mM IPTG was added and cultured at 37°C with shaking for another 12 hours. Afterward, the bacterial cells were collected, the cell walls were disrupted by sonication, and the recombinant protein LacS was purified using nickel column affinity chromatography.

[0050] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A LacI mutant, characterized in that, The amino acid sequence of the LacI mutant is shown in SEQ ID NO.

2.

2. An expression box, characterized in that, The expression cassette contains a nucleic acid sequence encoding the LacI mutant of claim 1.

3. An expression carrier, characterized in that, The expression vector contains a nucleic acid sequence encoding the LacI mutant of claim 1.

4. The expression vector according to claim 3, characterized in that, The expression vector is a recombinant plasmid.

5. The expression vector according to claim 4, characterized in that, The recombinant plasmid is pET-28a(+).

6. A recombinant host cell, characterized in that, The recombinant host cell contains the expression vector according to any one of claims 3-5.

7. The recombinant host cell according to claim 6, characterized in that, The recombinant host cell is Corynebacterium glutamicum, Pseudomonas putida, or Bacillus subtilis.

8. The use of the LacI mutant as described in claim 1 in the preparation of biosensors for screening LacS.

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