Genetically engineered bacterium of overexpressed lipB gene and method for producing L-histidine by using genetically engineered bacterium

Abstract

The invention belongs to the technical field of biology, and particularly relates to genetically engineered bacteria of overexpressed lipB genes and a method for producing L-histidine by using the genetically engineered bacteria. Specifically, the genetically engineered bacterium belongs to Escherichia coli, and more specifically, the genetically engineered bacterium is obtained by modifying the Escherichia coli in a manner of overexpressing a lipB gene. Compared with an original strain, the L-histidine yield of the genetically engineered bacterium modified in a lipB gene overexpression mode is improved by at least 16.4%-19.3%, and the conversion rate is also improved by at least 25% compared with the original strain.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and specifically relates to overexpression lipB Genetic engineering bacteria and method for producing L-histidine. Background Art

[0002] Due to its special physiological properties, L-histidine is widely used in the fields of feed, nutritional supplements, cosmetics and medicine. At present, there are three main methods for producing L-histidine: chemical synthesis, protein hydrolysis and microbial fermentation. Among them, the chemical synthesis method and protein hydrolysis method have cumbersome processes and complex raw material sources. In comparison, the microbial fermentation method has the advantages of being environmentally friendly, simple raw material sources, and simple production processes, and has now become the mainstream production method. The highest level of L-histidine production reported in China still has room for improvement, and the conversion rate is low, which is not conducive to large-scale industrial production.

[0003] The key to producing L-histidine by microbial fermentation is the selection of strains. Currently, there are two aspects for the selection of L-histidine production strains: irrational mutagenesis screening and rational metabolic engineering transformation. Irrational breeding methods are to induce strains through traditional mutagenesis and then screen a large number of mutants, including physical or chemical methods to induce wild-type strains. After multiple rounds of mutagenesis and screening, excellent strains with the ability to synthesize L-histidine are finally obtained. Among them, Kino et al. induced and screened wild-type Escherichia coli with nitrosoguanidine and obtained an L-histidine production strain with excellent performance. After batch fed fermentation, the L-histidine concentration can reach 46.5 g / L. Rational metabolic engineering transformation mainly includes relieving feedback inhibition, enhancing the supply of precursors, strengthening the expression of key genes and cofactor balance, etc. With the rapid development of gene editing technology, people have gradually enhanced the production of L-histidine by different host cells using rational metabolic engineering strategies. Among them, Wu et al. modified wild-type Escherichia coli by removing the feedback regulation of histidine synthesis, enhancing the histidine synthesis pathway, and promoting the extracellular secretion of histidine. They constructed an efficient L-histidine production strain. After fermentation in a 5 L fermenter for 48 hours, the L-histidine concentration reached 66.5 g / L, and the conversion rate was 0.22 g L-histidine / g glucose. The host strain was genetically modified from the perspective of rational metabolic engineering to achieve the effect of efficient production of L-histidine. Although the yield and conversion rate of L-histidine were further improved compared with irrational breeding methods, it still did not meet the requirements of industrial production intensity. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention aims to provide a genetically modified Escherichia coli and a method for producing L-histidine.

[0005] The technical solution of the present invention is summarized as follows: In a first aspect, the present invention provides a genetically engineered bacterium for producing L-histidine, specifically, it belongs to Escherichia coli, and more specifically, the genetically engineered bacterium is an Escherichia coli that overexpresses lipB The genes are modified.

[0006] As in the genetically engineered bacteria provided by the present invention in the first aspect, the Escherichia coli has been modified to enable it to have the ability to produce L-histidine.

[0007] In a second aspect, the present invention provides a method for producing L-histidine using the genetically engineered bacteria as described above, comprising: culturing the genetically engineered bacteria in a culture medium to produce L-histidine; and collecting the L-histidine from the genetically engineered bacteria and / or the culture medium.

[0008] As in the method provided by the second aspect of the present invention, the genetically engineered bacteria have overexpressed lipB The gene was modified in such a way that it was compared to the unmodified E. coli lipB The expression intensity of the gene is enhanced or the expression level is increased.

[0009] As in the method provided by the present invention in the second aspect, the Escherichia coli has been modified to enable it to have the ability to produce L-histidine.

[0010] The beneficial effects of the present invention are as follows: lipB The gene encodes octanoyltransferase, which catalyzes octanoyl transfer. The relationship and role of this gene in the L-histidine synthesis pathway are still unclear. The present invention found that this gene can be stably inherited and expressed on the genome, and can promote the production of L-histidine in the expanded fermentation culture. The present invention found that by overexpression lipB The level of L-histidine production by genetically modified Escherichia coli is significantly improved, which is easier to implement than the existing technology. For example, the L-histidine high-producing strain ALE-HIS-CL (CGMCC NO:31770) is used as the starting strain to overexpress lipB The L-histidine production of the genetically modified bacteria was increased by at least 16.4%-19.3% compared to the original strain, and the conversion rate was also increased by at least 25% compared to the original strain.

[0011] The present invention relates to the deposit information of biological materials: Deposit date: August 28, 2024; Depository: China General Microbiology Center (CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit number: CGMCC No. 31770. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 : In Example 1 lipB Integration fragment construction and verification electropherogram.

[0013] Figure 2 : Fermentation process curve of ALE-HIS-CL strain in Example 3.

[0014] Figure 3 : Fermentation process curve of HIS-lipB strain in Example 3. DETAILED DESCRIPTION

[0015] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0016] In a first aspect, the present invention provides a genetically engineered bacterium for producing L-histidine, specifically, it belongs to Escherichia coli, and more specifically, the genetically engineered bacterium is an Escherichia coli that overexpresses lipB The genes are modified.

[0017] According to the first aspect of the present invention, the genetically engineered bacteria are Escherichia coli that overexpresses lipB The gene was modified in such a way that it was compared to the unmodified E. coli lipB Increased gene expression levels, or lipB The total enzyme activity of the gene expression product, such as octanoyl transferase, is increased, for example, by 150% or more, 200% or more, or 300% or more.

[0018] According to the first aspect of the present invention, the E. coli has been modified to have the ability to produce L-histidine, for example, the E. coli has been modified to release the feedback regulation of the HisG enzyme.

[0019] As a specific embodiment of the present invention, the Escherichia coli can be selected from E. coli WHY2-3 (CN202010151245.X) E. coli WHY3-1(CN202010151245.X) or E. coli ALE-HIS-CL and other modified L-histidine production engineered bacteria.

[0020] Said E. coli WHY2-3 is based on E. coli W3110 is the starting strain overexpressing the histidine operon gene of Escherichia coli hisD , hisB , hisC , hisH , hisA , hisF and hisI , and overexpressing the gene encoding the mutant of Corynebacterium glutamicum ATP phosphoribosyltransferase HisG whose nucleotide sequence is shown in SEQ ID No.3 hisG * and obtain.

[0021] Said E. coli WHY3-1 is in E. coli WHY2-3 overexpressed the gene encoding the arginine / lysine transporter of Corynebacterium glutamicum lysE and the gene encoding glutamate dehydrogenase from Bacillus subtilis rocG .

[0022] Said E. coli ALE-HIS-CL is achieved by E. coli WHY2-3 was screened and screened for high concentration of L-histidine tolerance (tolerance concentration 40 g / L). The strain was deposited in the General Microbiology Center of China Microbiological Culture Collection (CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit number: CGMCC No. 31770, deposit date: August 28, 2024, classification name: Escherichia coli Escherichia coli .

[0023] According to the first aspect of the present invention, overexpression pyrB Genes can be introduced and / or increased in the bacterial genome pyrB Gene copy number (e.g., increased by autonomously replicating plasmids such as pET28a, pTrc99a, pSTV28, etc.) pyrB The number of copies of a gene can be increased by increasing the number of copies of a gene in a bacterial chromosome through gene editing or other methods. pyrB gene copy number), or modification pyrB Gene expression regulatory sequences (e.g., promoters, ribosome binding sites, etc.), or a combination of the above methods.

[0024] As a specific embodiment of the present invention, overexpression pyrBThe gene is integrated into the E. coli genome by gene editing, wherein the integration site of the gene can be selected according to the conventional knowledge of those skilled in the art to select some pseudogene sites that will not have a significant effect on bacterial growth and basic metabolism, such as mbhA, yeeP , ygiP , yghX, ygaY , yjiT , ycjV , ycgH, ygaY, yeeL, ilvG The gene loci are all selectable; in a specific implementation manner, the gene is also connected to a strong promoter, and the strong promoter can be selected from any one of Ptrc, BBa-J23100, and T7.

[0025] According to the first aspect of the present invention, lipB The gene is not limited to the nucleotide sequence shown in SEQ ID No.1, and may also include a mutant nucleotide sequence of the sequence shown in SEQ ID No.1 or a nucleotide sequence homologous to the sequence shown in SEQ ID No.1, and encoding lipB A gene that is a mutant of a protein. lipB The gene may be a variant nucleotide sequence due to the degeneracy of the genetic code, such as a nucleotide sequence encoding a protein as shown in SEQ ID NO.2.

[0026] In a second aspect, the present invention provides a method for producing L-histidine using the genetically engineered bacteria as described above, comprising: culturing the genetically engineered bacteria in a culture medium to produce L-histidine; and collecting the L-histidine from the genetically engineered bacteria and / or the culture medium.

[0027] According to the second aspect of the present invention, the genetically engineered bacteria have overexpressed lipB The gene was modified in such a way that it was compared to the unmodified E. coli lipB The expression intensity of the gene is enhanced or the expression level is increased.

[0028] According to the second aspect of the present invention, the E. coli has been modified to enable it to produce L-histidine, for example, by introducing gene mutations, gene knockouts, etc. to release the feedback regulation of the HisG enzyme.

[0029] According to the second aspect of the present invention, the L-histidine includes not only free form of L-histidine but also salts or hydrates of L-histidine.

[0030] According to a second aspect of the present invention, the culture of the genetically engineered bacteria can be carried out by conventional methods in the art. The culture medium used to produce L-histidine can be a synthetic or natural culture medium, for example a typical culture medium containing a carbon source, a nitrogen source, a sulfur source, inorganic ions and other organic and inorganic components required.

[0031] As a specific embodiment of the present invention, the culture medium composition is: glucose 20-30 g / L, yeast extract 2-5 g / L, peptone 2-4 g / L, KH 2 PO 4 1-3 g / L, MgSO 4 7H 2 O 1-2 g / L, FeSO 4 7H 2 O 5-20 mg / L, MnSO 4 7H 2 O 5-20 mg / L, VB1, VB3, VB5, VB12, VH 1-3 mg / L each, the rest is water, pH 7.0-7.2.

[0032] According to the second aspect of the present invention, the genetically engineered bacteria can be cultured under aerobic conditions for 16 to 72 hours, or 24 to 60 hours, or 40 to 50 hours; the culture temperature can be controlled within 30 to 45°C, or 30 to 37°C; and the pH can be adjusted between 5.0 and 8.0, or between 6.0 and 7.5, or between 6.8 and 7.2. The pH can be adjusted by using inorganic or organic acidic or alkaline substances, as well as ammonia gas.

[0033] As another specific embodiment, the method further comprises: maintaining the culture temperature at a constant 37° C., and / or maintaining the pH at about 7.0.

[0034] As another specific implementation, the method further includes: maintaining the dissolved oxygen at about 35%.

[0035] As another specific embodiment, the method further comprises: during the culture process, when glucose is exhausted, a glucose solution of a certain concentration needs to be added to maintain the residual glucose concentration in the fermentation medium at 0.1-5 g / L.

[0036] After cultivation, solids such as cells and cell debris can be removed from the liquid culture medium by conventional techniques (e.g., centrifugation, membrane filtration), and L-arginine can then be recovered from the fermentation broth by any combination of conventional techniques (e.g., concentration, ion exchange chromatography, crystallization).

[0037] Before inoculation and fermentation, bacteria can be activated and seed cultured according to their storage status. Appropriate conditions and culture media can be selected according to conventional techniques in the art. For example, the seed culture medium can have the same composition as the fermentation medium, or it can be appropriately adjusted on this basis.

[0038] Other specific operation methods such as molecular biology and genetic engineering involved can be implemented according to technical manuals, textbooks or literature reports that are easily available to those skilled in the art, and it is not necessary to describe the operation process in detail here. In addition, specific strains are selected as hosts in the following embodiments, and thus specific gene integration sites, target genes and primers thereof are selected according to the hosts, but it does not mean that the purpose of the present invention can only be achieved through these specific selections, and the scope of the present invention cannot be limited to this. The essence and scope of the present invention are only limited by the claims.

[0039] The present invention is described in more detail below through specific examples.

[0040] Embodiment 1: This embodiment relates to a genetically engineered bacterium and a method for constructing the same. The genetically engineered bacterium is a bacterium that overexpresses lipB The Escherichia coli is selected from E. coli WHY2-3, E. coli WHY3-1 or E. coli ALE-HIS-CL (CGMCC No. 31770).

[0041] The gene editing method used in this example was carried out according to the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR–Cas9 meditated genomeediting. Metabolic engineering, 2015, 31: 13-21.). pREDCas9 carries the elimination system of the gRNA expression plasmid pGRB, the Red recombination system of λ phage and the Cas9 protein expression system, spectinomycin resistance (working concentration: 100 mg / L), and is cultured at 32°C; pGRB uses pUC18 as the backbone, including the promoter J23100, the gRNA-Cas9 binding region sequence and the terminator sequence, ampicillin resistance (working concentration: 100 mg / L), and is cultured at 37°C.

[0042] Construct overexpression lipBThe engineered bacteria HIS-lipB, WHY3-1-lipB and WHY2-3-lipB of the gene were used. Using the MG1655 genome as a template, upstream homology arm primers (ΜP-mbhA-S, ΜP-mbhA-A) and downstream homology arm primers (DN-mbhA-S, DN-mbhA-A) were designed according to the upstream and downstream sequences of its mbhA gene, and PCR amplification of its upstream and downstream homology arm fragments. Using the MG1655 genome as a template, primers (lipB-S, lipB-A) were designed according to its lipB gene sequence (SEQ ID No.1), and PCR amplification of the lipB fragment; promoter P trc Designed in the downstream primer of the upstream homology arm and the upstream primer of the lipB gene. The above fragments were fused by overlapping PCR to obtain the integrated fragment of the lipB gene (upstream homology arm-Ptrc-lipB-downstream homology arm). The DNA fragment containing the target sequence used to construct pGRB-mbhA was obtained by annealing primers gRNA-mbhA-S and gRNA-mbhA-A. Prepare ALE-HIS-CL, WHY3-1, and WHY2-3 competent cells, and according to the CRISPR / Cas9 gene editing method, the pGRB plasmid and the recombinant DNA fragment were simultaneously electrotransformed into competent cells containing the pREDCas9 plasmid, and the positive transformants were screened to finally obtain the recombinant strains HIS-lipB, WHY3-1-lipB or WHY2-3-lipB. During the integration of the Ptrc-lipB fragment, the electrophoresis diagram of the construction of the integrated fragment and the PCR verification of the positive strain is attached. Figure 1 . Among them, M: 1 kb DNA marker; 1: upstream homology arm; 2: lipB fragment; 3: downstream homology arm; 4: overlapping fragment; 5: original bacteria control; 6: positive bacteria identification fragment.

[0043] The primers involved in the above operation are shown in the following table:

[0044] Embodiment 2: This embodiment relates to a method for producing L-histidine using genetically engineered bacteria, wherein the genetically engineered bacteria are selected from ALE-HIS-CL, WHY3-1, WHY2-3, HIS-lipB, WHY3-1-lipB or WHY2-3-lipB strains.

[0045] The strain stored in the -80℃ refrigerator was transferred to the LB shake tube, and then placed in a 37℃ constant temperature incubator for 12 h. The strain was cultured and activated in the LB shake tube for the first generation. The bacterial solution in the shake tube was transferred to a triangular flask containing 27 mL LB medium at an inoculation rate of 10%, and sealed with nine layers of gauze. It was cultured at 37℃ and 220 rpm for 8 h. The strain was cultured and activated in the shake flask for the second generation. The bacterial solution in the triangular flask was transferred to a triangular flask containing 26 mL fermentation medium at an inoculation rate of 10% (1mL60% (m / v) glucose solution was added as the initial culture carbon source), and sealed with nine layers of gauze. It was cultured at 37℃ and 220 rpm for 24 h. During the fermentation process, ammonia water was added through a micro syringe to maintain the pH at 7.0, and 60% (m / v) glucose solution was added to maintain the carbon source during the fermentation process. The composition of the shake flask fermentation medium is: glucose 20 g / L, yeast extract 2 g / L, peptone 2 g / L, KH 2 PO 4 1g / L, MgSO 4 7H 2 O 2 g / L, FeSO 4 7H 2 O 10 mg / L, MnSO 4 7H 2 O 10mg / L, VB1, VB3, VB5, VB12, VH 2mg / L each, the rest is water, pH 7.0-7.2.

[0046] The results are shown in Table 1. After 24 h of fermentation, the OD 600 It is known that enhancement lipB The gene expression intensity did not significantly affect the growth of the engineered strains. However, the L-histidine concentration in the HIS-lipB strain increased from 3.1 g / L to 3.7 g / L, the L-histidine concentration in the WHY3-1-lipB strain increased from 5.1 g / L to 6.0 g / L, and the L-histidine concentration in the WHY2-3-lipB strain increased from 4.3 g / L to 5.1 g / L. This result shows that enhancing the expression of E. coli lipB The gene expression intensity can significantly improve the L-histidine production performance of the engineered strain.

[0047] Table 1 Shake flask fermentation results of engineered strains

[0048] Embodiment 3: This embodiment relates to a method for producing L-histidine using genetically engineered bacteria, wherein the genetically engineered bacteria are selected from ALE-HIS-CL or HIS-lipB strains.

[0049] Take the bacteria preservation tube stored in the -80℃ refrigerator and place it on the ice box to thaw naturally. Use an inoculation loop to take a ring of bacteria and streak it densely on the slant medium, and then put it in a 37℃ constant temperature incubator for 12 hours to get the first generation of seeds. Use an inoculation loop to scrape the bacteria on the slant medium and streak it densely in the eggplant-shaped bottle, and also put it in a 37℃ constant temperature incubator for 12 hours to get the second generation of seeds. Take one-third of the volume of the eggplant-shaped bottle and pour it into the bottle. Resuspend the bacteria in the eggplant-shaped bottle and pour it into the seed fermentation tank for cultivation. During the cultivation process, the pH is stable at about 7.0, the temperature is constant at 37℃, and the dissolved oxygen is maintained at about 35%. When the seed culture reaches the logarithmic growth phase of the cells, it is transferred to the fermentation culture. The seed culture solution is transferred to the fresh fermentation medium according to the inoculation amount of 15%. Similarly, the pH of the fermentation process is stable at about 7.0, the temperature is constant at 37℃, and the dissolved oxygen is maintained at about 35%. When glucose is exhausted during the culture process, 80% (m / v) glucose solution needs to be fed to maintain the residual glucose concentration in the fermentation medium at 0.1-5 g / L.

[0050] The slant culture medium in this embodiment is composed of: 1 g / L glucose, 5 g / L peptone, 5 g / L beef extract, 1 g / L yeast powder, 1 g / L NaCl, 20 g / L agar, and the rest is water, pH 7.0-7.2.

[0051] The seed culture medium in this embodiment is composed of: glucose 15 g / L, yeast extract 5 g / L, peptone 5 g / L, KH 2 PO 4 5 g / L, MgSO 4 7HO 2 5 g / L, FeSO 4 7HO 2 10 mg / L, MnSO 4 HO 2 10 mg / L, VB1 2 mg / L, VH 1mg / L, 2 drops of defoamer, and the rest is water, pH 7.0-7.2.

[0052] The fermentation medium in this example is composed of 20 g / L glucose, 2 g / L yeast extract, 2 g / L peptone, and 2 g / L KH 2 PO 4 1 g / L, MgSO 4 7H 2 O 2 g / L, FeSO 4 7H 2 O 10 mg / L, MnSO 4 7H 2 O 10 mg / L, VB1, VB3, VB5, VB12, VH 2 mg / L each, the rest is water, pH 7.0-7.2.

[0053] After 52 h of cultivation in a 5 L fermenter, the fermentation results of the HIS-lipB strain were as follows: Figure 2 and Figure 3 As shown, this strain ( Figure 3 ) reached a L-histidine production level of 78 g / L, which was 16.4% higher than that of the control strain ALE-HIS-CL. The ALE-HIS-CL strain ( Figure 2 ) reached 67 g / L of L-histidine. In addition, the conversion rate of the HIS-lipB strain reached 0.3 g L-histidine / g glucose, which was 25% higher than that of the control strain ALE-HIS-CL, and the conversion rate of the ALE-HIS-CL strain reached 0.24 g L-histidine / g glucose. This result shows that the HIS-lipB strain can achieve a higher level of L-histidine production in scale-up production, and also shows that lipB Overexpression of the gene can effectively promote the strain to produce L-histidine. According to the above conversion rate level, the strain can greatly reduce costs in large-scale production, thereby having higher economic benefits in business.

[0054] Although the present invention has been disclosed as above in terms of preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various changes, modifications, substitutions and variations in form and details to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A genetically engineered bacterium for producing L-histidine, characterized in that: The genetically engineered bacteria are Escherichia coli that overexpresses lipB The genes are modified.

2. The genetically engineered bacteria according to claim 1, characterized in that: The E. coli has been modified to enable it to have the ability to produce L-histidine, or the E. coli has been modified to release the feedback regulation of HisG enzyme.

3. The genetically engineered bacterium according to claim 1, characterized in that: The E. coli also overexpresses the E. coli histidine operon gene hisD , hisB , hisC , his , hisA , hisF and hisI , and overexpressing the gene encoding the mutant of Corynebacterium glutamicum ATP phosphoribosyltransferase HisG as shown in SEQ ID No.3 his *.

4. The genetically engineered bacterium according to claim 1, characterized in that: The genetically engineered bacteria are E. coli WHY2-3, E. coli WHY3-1 or E. coli ALE-HIS-CL (CGMCC NO:31770) was overexpressed lipB The genes are modified.

5. The genetically engineered bacteria according to claim 1, characterized in that: The overexpression lipB Genes can be added through autonomously replicating plasmids pyrB Gene copy number, and / or increase through gene editing methods pyrB Gene copy number, and / or modification pyrB Gene expression regulatory sequences.

6. The genetically engineered bacterium according to claim 1, characterized in that: Said lipB The nucleotide sequence of the gene is shown as SEQ ID No. 1, or is a nucleotide sequence encoding a protein shown as SEQ ID NO.

2.

7. Use of the genetically engineered bacteria according to any one of claims 1 to 6 in the fermentation production of L-histidine.

8. A method for producing L-histidine, characterized in that: include: Cultivating the genetically engineered bacteria according to claims 1 to 6 in a culture medium to produce L-histidine; And, collecting the L-histidine from the genetically engineered bacteria and / or the culture medium.

9. The method according to claim 8, characterized in that: The method further comprises: maintaining a constant culture temperature of 37° C., and / or maintaining a pH of about 7.0, and / or maintaining dissolved oxygen at about 35%.

10. The method according to claim 9, characterized in that: The method further comprises: during the culture process, when glucose is exhausted, a glucose solution of a certain concentration needs to be added to maintain the residual glucose concentration in the fermentation medium at 0.1-5 g / L.

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