Method for increasing growth rate of Cupriavidus hookworm strain with carbon dioxide as carbon source and engineering strain

Abstract

The invention relates to the technical field of biology, in particular to a method for increasing the growth rate of a Cupriavidus hookworm strain with carbon dioxide as a carbon source and an engineering strain. Glucokinase genes participating in glucose metabolism in the Cupriavidus hookworm strain are knocked out, alkaline phosphatase genes and haloid acid dehalogenase family (HAD) phosphatase genes are knocked out on the basis, and the growth rate of the Cupriavidus hookworm strain under the condition that carbon dioxide serves as a carbon source is increased through the operation. The bacterial strain with the increased proliferation rate can be used for producing single-cell protein by taking carbon dioxide as a raw material and is beneficial to fixation of greenhouse gas carbon dioxide. Meanwhile, the production efficiency can be remarkably improved by utilizing the chassis cell to construct a metabolic engineering-based product synthesis strain.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for improving the growth rate of a Cupria obliquus strain using carbon dioxide as a carbon source and an engineered strain. Background Art

[0002] Cupriella necator H16 is a facultative chemoautotrophic Gram-negative strain capable of utilizing a wide range of organic matter as a carbon source. More importantly, it can directly utilize carbon dioxide as a carbon source for proliferation through the Calvin-Benson-Bassham (CBB) cycle. Due to its metabolic flexibility, light-independent autotrophic growth, and efficient genetic manipulation tools, Cupriella necator H16 has great potential as a chassis cell for the production of single-cell proteins, biofuels, and bioactive substances using carbon dioxide as a feedstock. Currently, metabolically engineered Cupriella necator H16 has successfully achieved the synthesis of single-cell proteins, isobutanol, methyl ketones, trehalose, inositol, and mannose using carbon dioxide as a carbon source.

[0003] However, the high production cost resulting from the slow growth rate of C. necator is a major factor limiting its industrial application. Currently, research on increasing the growth rate of C. necator primarily focuses on enhancing the carbon fixation pathway by overexpressing key enzymes in the Calvin cycle. Studies have shown that overexpressing ribulose-1,5-bisphosphate carboxylase (RuBisCO) and carbonic anhydrase, both from the cyanobacterium Synechococcus, has a positive effect on increasing the growth rate of the strain. However, this strategy faces numerous limitations and still faces significant challenges. First, the Calvin cycle requires a large amount of ATP and reducing power, making optimization of this reaction pathway susceptible to energy and cofactor supply constraints. Furthermore, RuBisCO, the key rate-limiting enzyme in the Calvin cycle, has low catalytic efficiency. Oxygen has a higher affinity for RuBisCO, competitively inhibiting its carboxylase activity. This resulting in inefficient cycling further reduces CO2 fixation efficiency. Attempts to improve the catalytic efficiency of RuBisCO by modifying its catalytic efficiency have been limited. Therefore, it is particularly important to explore non-Calvin cycle-related carbon metabolism gene target genes in H. hookworm that help increase the growth rate of the strain, and then improve the growth rate of the strain based on metabolic engineering to reduce production costs, but related research is scarce.

[0004] Due to the lack of a glucose transporter, C. necrotica cannot grow on glucose as a carbon source. Literature reports indicate that nonspecific phosphatases in C. necrotica can catalyze the conversion of glucose from glucose-1-phosphate and glucose-6-phosphate to glucose. Therefore, the function of the glucokinase GLK in C. necrotica may be to recycle this lost carbon flux. The glucose metabolism reaction catalyzed by GLK is an energy-intensive process. Although knockout of the glucokinase gene glk reduces intracellular ATP consumption, it inevitably results in a loss of carbon flux in the glycolytic pathway. Intermediate metabolites in glycolysis, such as fructose-6-phosphate, are important precursors of ribulose-5-phosphate, a carbon fixation reaction in the Calvin cycle. Currently, there are no reports demonstrating that deletion of the glucokinase-encoding gene enhances microbial growth under autotrophic conditions, nor how this conflict is balanced. Consequently, the growth characteristics of strains affected by glk gene deletion remain uncertain.

[0005] Endogenous nonspecific phosphatases (alkaline phosphatase and HAD phosphatase, etc.) of the hookworm Copperworm fungus can catalyze the dephosphorylation of various metabolic intermediates in the central carbon metabolism pathway, such as glucose-6-phosphate and glucose-1-phosphate, to produce glucose, thereby causing a loss of carbon flux in strains deficient in the glucokinase gene glk. The deletion of genes encoding nonspecific phosphatases has the potential to reduce the loss of carbon flux in the central carbon metabolism of glk-deficient strains and thus promote the proliferation of the strain. However, the loss of phosphatases may also lead to the inability to carry out the reactions that they mainly catalyze, such as signal transduction and metabolic regulation, which are necessary for the growth of the strain, thereby having a negative impact on the growth of the strain. Currently, there are no reports on knocking out nonspecific phosphatases based on strains deficient in the glucokinase encoding gene to increase the growth rate of microorganisms under autotrophic conditions. Therefore, there is no basis for predicting the autotrophic growth characteristics of a strain deficient in alkaline phosphatase or HAD phosphatase. Summary of the Invention

[0006] In order to increase the proliferation rate of Copperbacterium nematodes when carbon dioxide is used as a carbon source, the present invention provides a method for increasing the growth rate of Copperbacterium nematodes strains.

[0007] Another object of the present invention is to provide an engineered strain of Cupribotium hookworm with an increased growth rate using carbon dioxide as the sole carbon source.

[0008] According to the present invention, the growth rate of the Cupribotium hookworm engineered strain with improved carbon dioxide as the sole carbon source is improved, and the engineered bacteria is a mutant Cupribotium hookworm with the glucokinase gene knocked out and the alkaline phosphatase gene and HAD phosphatase gene knocked out on this basis.

[0009] The glucokinase gene to be knocked out is the glucokinase encoding gene glk (H16_B2564), and its nucleotide sequence is shown in SEQ ID NO: 1;

[0010] The alkaline phosphatase gene is the alkaline phosphatase encoding gene H16_A2182, and its nucleotide sequence is shown in SEQ ID NO: 2;

[0011] The HAD phosphatase genes are haloacid dehalogenase family phosphatase encoding genes H16_A0174, serB3 (H16_B1164), cbbY2 (H16_B1392) and cbbYp (PHG424), and their nucleotide sequences are shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0012] The method for constructing an engineered bacterium of Cupribotium hookworm with an improved proliferation rate according to the present invention comprises the following steps:

[0013] Knockout of the glucokinase gene in Cupriphora hookworm;

[0014] Knockout of the alkaline phosphatase gene or HAD phosphatase.

[0015] Wherein, the glucokinase gene is the glucokinase encoding gene glk (H16_B2564), and its nucleotide sequence is shown in SEQ ID NO: 1;

[0016] The alkaline phosphatase gene is the alkaline phosphatase encoding gene H16_A2182, and its nucleotide sequence is shown in SEQ ID NO: 2;

[0017] The HAD phosphatase genes are haloacid dehalogenase family phosphatase encoding genes H16_A0174, serB3 (H16_B1164), cbbY2 (H16_B1392) and cbbYp (PHG424), and their nucleotide sequences are shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0018] SEQ ID NO: 1 (glk)

[0019]

[0020] SEQ ID NO:2(H16_A2182)

[0021]

[0022] SEQ ID NO:3(H16_A0174)

[0023] Atgactgcagcaacccgtggccattccggccgattcgattgcgtgatcttcgactgcgacggcgtgctcgtcgacagcgagcccatcgtcaaccgcgtgctcaaccagatgctgaacgagcttggcatcgagatctcgctggaagactccacgcgcctgtttctcggccgcgccgtgcgcgaagagctggagatgatcgagcgcatgcgcggcgcgccgctgcccgagaactggctgtcgacctggctggcgcgccgcaacgcggtgctggaagccgaagtcgctgcggtcgcccatgtgcgcgaggcgattggcaagatcgccgccaccggcatgccggtgtgcgtggcgtcgggtgcggaccgcgtcaaggtcaagctgcaactgaccaggaccggcctggtcgagctgttccagcaggatgagcgcgagcatatcttctccgccaccgaagtggcacgcagcaagcccgcgcccgatgtgtacctgctggccgcgcgcaccatgggcgttgagccggcgcgctgcgccgtggtggaagacagcccgaccggcgtgaccgcgggcgttgccgccggcatgacggtgttcggctacgccgagcgcaacgatgccgcgctgctgcgcgaggccggcgcgggcaccatcttcaccgatatgcgcgacctgccggagctggtgggatga。

[0024] SEQ ID NO:4(serB3(H16_B1164))

[0025] Atgcccgctgccccccgcctcgccctgtttgacctcgaccacaccctgctgccgctcgacagcgagtatgaatgggctcgctacctcgttgccgtgggcggcgccgaggctgccgaaatcgatgcgcacaacacgcgctggctggcggcctaccaggcggggcggctggactttgccgcccatgcgcgctttgccctgggcctgctggcgcgccatccgcgtgaacgactgacgcagtggcgcgcggacttcatgcgcaaagtgatcgtgcccgccatccagccggcggcgcgcgacctgctggcccgccacctctccgccggcgacctgtgctgcatcgtcaccgccacctgccgttttgtcaccgagccgatcgcgcgccagctgggcgtgccgcacctgctggcggtggaggcggaacacgatgcgcacggcgagttcaccggcgcgctggccggcgtgccggcctttgggcctggcaaggtgctgcgcgtgctggcgtggctcgacacgctgggcattgcgcacacggcgctggcacaggccaccttctatagcgactcccgcaacgatctgccgctgctagaaagcgttggccacccggtcgccgtcaatccggattcgacgctgcgggacgcggccgaggcacgcggctggccggtcttgcacctgttcggcacggccggcgtgccgaccgcctga。

[0026] SEQ ID NO:5(cbbY2(H16_B1392))

[0027] Atgcaagccctgattttcgatgtcgacggcaccctggccgataccgaaagcgcgcacctgcaagccttcaacgccgccttcgccgaggtcggcctggactggtactgggacgcgccgctctacacgcgcctgctcaaggtggccggcggcaaggagcgcctgatgcattactggcgcatggtcgacccggaagaggcccgcggctgcaaggtgaaggaaaccatcgacgccgtgcacgccatcaagacccgccactatgccgagcgcgtcggggcgggcggcctgccgctgcgcccgggcattgcccgcctgatcgacgaggccggcgaggccgggctcccgctggcgattgccaccaccaccacgccggccaacctcgacgcgctgctgcaggcgccgcttggcgccgactggcgccgtcgctttgccgccatcggcgacgccggcaccacggccatcaagaagccggcgcccgatgtctacctggcggtgctggagcggctgggcctggaaggcggtgactgcctggcgatcgaggactcggcgaacggcctgcgcgccgcccgggcggccggcattcccaccgtggtcacgcccaccgcgttcagcgcgcaggactccttcgagggcgcgctgctggtgctgccgcatcttggcgatcccggcgagcccatgccccagcacgtgcccggcgcggcaaaccgctgggccgaccttgccgcgttgcgcgcctggcaccacggcaccctgatcgaggcaacctga。

[0028] SEQ ID NO:6(cbbYp(PHG424))

[0029] .

[0030] The present invention obtains a strain of Copperobacterium necrotizingum with an enhanced proliferation rate, which can be used for single-cell protein production using carbon dioxide as a raw material and helps fix the greenhouse gas carbon dioxide. Furthermore, using this chassis cell to construct a metabolically engineered product-synthesizing strain can significantly improve production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the knockout plasmid vector map of the glucokinase encoding gene glk;

[0032] Figure 2Shows the growth of the Cupriavidus necrotica H16 strain with the glucokinase gene knocked out;

[0033] Figure 3 This is the knockout plasmid vector map of the alkaline phosphatase encoding gene H16_A2182;

[0034] Figure 4 This is the knockout plasmid vector map of the alkaline phosphatase encoding gene H16_B0842;

[0035] Figure 5 This is the knockout plasmid vector map of the alkaline phosphatase encoding gene H16_A2183;

[0036] Figure 6 Shows the growth of the H16 strain of Cupria necrotica with the glucokinase gene and alkaline phosphatase gene knocked out;

[0037] Figure 7 This is the knockout plasmid vector map of the HAD phosphatase encoding gene H16_A2218;

[0038] Figure 8 This is the knockout plasmid vector map of the HAD phosphatase encoding gene H16_A0174;

[0039] Figure 9 This is the knockout plasmid vector map of the HAD phosphatase encoding gene serB3;

[0040] Figure 10 This is the knockout plasmid vector map of the HAD phosphatase encoding gene cbbY2;

[0041] Figure 11 This is the knockout plasmid vector map of the HAD phosphatase encoding gene cbbYp;

[0042] Figure 12 This is the knockout plasmid vector map of the HAD phosphatase encoding gene H16_A2331;

[0043] Figure 13 Shows the growth of the H16 strain of Cupriavida hookworm with the glucokinase gene and HAD phosphatase gene superimposedly knocked out. DETAILED DESCRIPTION

[0044] The bacterial strain used in the following examples is the H16 strain of Copperbacterium truncatum, and the knockout plasmid vector is constructed with the pK18mobSacB plasmid that can replicate in Escherichia coli but cannot replicate in Copperbacterium truncatum H16 as the skeleton. The restriction enzymes and other biochemical reagents used were purchased from biochemical reagent companies. The molecular biology experimental methods not specifically described are all carried out with reference to the specific methods listed in the book "Molecular Cloning Experiment Guide" (3rd edition) J. Sambrook, or are carried out according to the kit and product instructions. The transformation process of Copperbacterium truncatum H16 in this application is shown in Table 1:

[0045] Table 1

[0046] strains Traits Cuprix hookworm H16 wild type strain Cupriella necator H16Δglk Knockout of glk gene based on H16 Cupriella necrotica H16ΔglkΔA2182 Knockout of the H16_A2182 gene based on H16Δglk of the hookworm Copperworm Cupriella necrotica H16ΔglkΔB0842 Knockout of the H16_B0842 gene based on H16Δglk of the hookworm Copperworm Cupriella necrotica H16ΔglkΔA2183 Knockout of the H16_A2183 gene based on H16Δglk of the hookworm Copperworm Cupriella necrotica H16ΔglkΔA2218 Knockout of the H16_A2218 gene based on H16Δglk of the hookworm Copperworm Cupriella necrotica H16ΔglkΔA0174 Knockout of the H16_A0174 gene based on H16Δglk of the hookworm Copperworm Cupriella necator H16ΔglkΔserB3 Knockout of serB3 gene based on H16Δglk of Copperworm Cupriella hookworm H16ΔglkΔcbbY2 Knockout of cbbY2 gene based on H16Δglk of Copperworm Cupriella necator H16ΔglkΔcbbYp Knockout of the cbbYp gene based on H16Δglk of Copperworm Cupriella necrotica H16ΔglkΔA2331 Knockout of the H16_A2331 gene based on H16Δglk of the hookworm Copperworm

[0047] Example 1 Effect of knocking out the glucokinase encoding gene glk on the proliferation rate of hookworms

[0048] 1. Construction of the H16Δglk strain of Copperbacterium necrotica

[0049] The targeted knockout vector pK18-glk, encoding the glucokinase gene glk, was introduced into the Cupriella necrotica H16 host using conjugative transfer. A first round of screening using kanamycin identified a host strain in which the knockout vector had successfully integrated into the strain genome. A second round of screening using sucrose yielded the Cupriella necrotica H16Δglk strain in which the glucokinase gene glk was successfully knocked out. The specific method is as follows:

[0050] 1. Construction of knockout plasmid pK18-glk with upstream and downstream homology arms of glk gene sequence

[0051] According to the sequence of glucokinase encoding gene glk (H16_B2564), synthetic primers were designed. Using the H16 genome of Copper nematodes as a template, the primers designed above were used to amplify the upstream and downstream homologous arms of the glk gene, respectively, and the fragment size was about 500bp. The obtained fragment was connected to the EcoRI / SmaI site of the pK18mobSacB plasmid using Gibson Assembly connection. The recombinant plasmid was electroporated into the Escherichia coli TransI cloning host, coated with LB solid plates (kanamycin 50μg / mL), and cultured at 37°C overnight. Positive clones were screened by colony PCR, and the target fragment was 1000bp. The successfully constructed vector was named pK18-glk, and its map is shown below. Figure 1 shown.

[0052] 2. Construction of the H16 strain of Cuprilobacter necrotica with the pK18-glk plasmid integrated into the genome

[0053] The knockout vector pK18-glk was transformed into Escherichia coli S17-1 by electroporation and plated on LB plates containing 50 μg / mL kanamycin. The plates were then cultured overnight at 37°C. Positive clones were selected and plated in LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C in a shaker at 200 rpm.

[0054] Escherichia coli S17-1 and C. necrotica H16 carrying the pK18-glk plasmid were inoculated into LB liquid medium and supplemented with the corresponding antibiotics (kanamycin 50 μg / mL or gentamicin 10 μg / mL). The strains were cultured overnight at 37°C and 30°C, respectively, in a shaker at 200 rpm. The cells were collected by centrifugation at 4600 rpm for 8 minutes and washed three times with LB medium. Escherichia coli S17-1 carrying the pK18-glk plasmid and C. necrotica H16 were mixed in a ratio of 3:1 and spotted on LB plates, incubated at 30°C overnight. The mixed cells were washed with LB and spread onto solid LB plates (kanamycin 200 μg / mL and gentamicin 10 μg / mL). After incubation at 30°C for 48 hours, positive clones were screened using colony PCR. The target fragment was a 500-bp kanamycin resistance gene fragment. Positive clones were cultured in 1.5 mL EP tubes containing LB liquid medium at 30° C. and 200 rpm in a shaking incubator overnight.

[0055] 3. Construction of the H16 strain of Cuprix necrotica with the glucokinase encoding gene glk successfully knocked out

[0056] Dilute the bacterial solution to 10 -2 The strain was plated onto LB plates supplemented with 100 g / L sucrose and incubated at 30°C for 48 hours. Colony PCR was performed to screen for clones with successful knockout of the target gene, with a target fragment size of 1000 bp. The successfully constructed strain was inoculated into LB liquid medium and named Cupriella necator H16Δglk.

[0057] 2. Effect of knockout of the glucokinase encoding gene glk on the autotrophic proliferation rate of Cupribotrys hookworms

[0058] The wild type of Copperworm H16 and the Copperworm H16Δglk bacterial liquid were inoculated into 40 mL of LB medium at an inoculum size of 1‰, and cultured overnight in a shaking incubator at 30°C and 200 rpm for rejuvenation.

[0059] Prepare fermentation medium: Accurately weigh 3.5 g Na2HPO4, 1.5 g KH2PO4, 1.0 g (NH4)2SO4, 80 mg MgSO4·7H2O, 1 mg CaSO4·2H2O, 0.56 mg NiSO4·7H2O, 0.4 mg ferric citrate and 200 mg NaHCO3, dilute to 1 L with water as the solvent, and sterilize by high pressure wet heat at 115°C for 30 min.

[0060] The rejuvenated wild-type and H16Δglk strains of C. necator were centrifuged at 4600 rpm for 8 min to collect the cells, which were then washed three times with PBS buffer solution and inoculated into anaerobic bottles containing 50 mL of fermentation medium. Three replicates were set for each strain. To ensure that the number of cells in each anaerobic bottle after inoculation was the same, the initial OD value of the bacterial solution was adjusted. 600 ≈0.1. The anaerobic flask was placed in a shaker at 30°C and 200 rpm for shake flask fermentation and continuously filled with mixed gas (H2:O2:CO2=8:1:1) to supplement the carbon source. The bacterial liquid OD was measured regularly. 600 .

[0061] Bacterial liquid OD 600 Determination: The absorbance of the bacterial solution was measured at a wavelength of 600 nm using a visible light spectrophotometer.

[0062] The results are as follows Figure 2 As shown in the figure, compared with the wild-type strain of H16, the growth rate of H16Δglk strain of H16 was significantly improved. After 6 days of culture, the OD 600 The OD of the two strains reached 6.08, which was 1.58 times that of the H16 strain of Copperobacterium necrotica (3.84). 600 The maximum OD value of the H16Δglk strain of Copperworm nematodes was reached after 10 days of culture. 600 The glucokinase gene glk increased the growth rate of C. necrotizingus H16 and its final biomass, reaching 9.30, 1.69 times that of the wild-type strain (5.49). This suggests that the deletion of the glucokinase gene glk not only increases the growth rate of C. necrotizingus H16 but also increases its final biomass. This is likely because the deletion of the glucokinase-encoding gene glk reprograms the metabolic network and rebalances energy in C. necrotizingus H16, thereby promoting its growth.

[0063] Example 2 Effect of superimposed knockout of glucokinase and alkaline phosphatase genes on the proliferation rate of Cupria obliquus

[0064] 1. Construction of an engineered strain with alkaline phosphatase knockout based on the H16Δglk strain of Copperobacterium necrotizingum

[0065] The targeted knockout vectors pK18-A2182, pK18-B0842, and pK18-A2183 encoding the alkaline phosphatase genes H16_A2182, H16_B0842, and H16_A2183 were introduced into the H16Δglk host strain using conjugative transfer. A first round of screening using kanamycin yielded a host strain in which the knockout vector had successfully integrated into the strain genome. A second round of screening using sucrose yielded H16ΔglkΔA2182, H16ΔglkΔB0842, and H16ΔglkΔA2183, all of which contained knockouts of the glucokinase gene glk and the alkaline phosphatase genes H16_A2182, H16_B0842, and H16_A2183, respectively. The specific methods are as follows:

[0066] 1. Construct knockout plasmids pK18-A2182, pK18-B0842 and pK18-A2183 with upstream and downstream homology arms of alkaline phosphatase encoding genes H16_A2182, H16_B0842 and H16_A2183, respectively

[0067] Synthetic primers were designed based on the alkaline phosphatase encoding gene sequence (H16_A2182, H16_B0842 and H16_A2183). The H16 genome of Copper Turbulence H16 was used as a template, and the upstream and downstream homologous arms of the alkaline phosphatase encoding gene were amplified using the primers designed above, with a fragment size of about 500bp. The obtained fragment was connected to the EcoRI / SmaI site of the pK18mobSacB plasmid using the Gibson Assembly connection method. The recombinant plasmids were electroporated into the Escherichia coli TransI cloning host, coated with LB solid plates (kanamycin 50μg / mL), and screened for positive clones after overnight culture at 37°C. The target fragment was 1000bp. The successfully constructed vectors were named pK18-A2182, pK18-B0842 and pK18-A2183, respectively, and their maps are shown as follows. Figure 3 、 Figure 4 and 5 shown.

[0068] 2. Construction of a strain with knockout plasmid integrated into the genome of C. necrotica H16Δglk

[0069] The knockout vectors pK18-A2182, pK18-B0842, and pK18-A2183 were transformed into E. coli S17-1 by electroporation, plated on solid LB plates containing 50 μg / mL kanamycin, and cultured overnight at 37°C. Positive clones were selected and cultured in liquid LB medium containing 50 μg / mL kanamycin at 30°C, shaking at 200 rpm.

[0070] Escherichia coli S17-1 strains carrying pK18-A2182, pK18-B0842, and pK18-A2183 plasmids and H16Δglk strain of H. necator were inoculated into LB liquid medium and the corresponding antibiotics (kanamycin 50 μg / mL or gentamicin 10 μg / mL) were added. The strains were cultured overnight at 37°C and 30°C in a shaker at 200 rpm. The cells were collected by centrifugation at 4600 rpm for 8 minutes and washed three times with LB medium. Escherichia coli S17-1 strains carrying pK18-A2182, pK18-B0842, and pK18-A2183 plasmids and H16Δglk strain of H. necator were mixed in a ratio of 3:1 and spotted on LB plates and incubated at 30°C overnight. The mixed cells from the overnight incubation were washed with LB and plated onto solid LB plates (200 μg / mL of kanamycin and 10 μg / mL of gentamicin). After incubation at 30°C for 48 hours, positive clones were screened using colony PCR. The target fragment was a 500-bp kanamycin resistance gene fragment. Positive clones were cultured overnight in 1.5 mL EP tubes containing LB liquid medium at 30°C, shaken at 200 rpm.

[0071] 3. Construction of the H16ΔglkΔA2182, H16ΔglkΔB0842, and H16ΔglkΔA2183 strains

[0072] The bacterial solution that has integrated the knockout plasmid was diluted to 10 -2 The strains were plated onto LB plates supplemented with 100 g / L sucrose and incubated at 30°C for 48 hours. Colony PCR was performed to screen for clones with successful knockout of the target gene, with a target fragment size of 1000 bp. The successfully constructed strains were inoculated into LB liquid medium and named H16ΔglkΔA2182, H16ΔglkΔB0842, and H16ΔglkΔA2183, respectively.

[0073] Effects of combined knockout of glucokinase and alkaline phosphatase genes on the autotrophic proliferation rate of C.

[0074] The H16Δglk, H16ΔglkΔA2182, H16ΔglkΔB0842 and H16ΔglkΔA2183 strains of H. nematophila were inoculated into 40 mL of LB medium at an inoculum size of 1‰, and cultured overnight in a shaker at 30°C and 200 rpm for rejuvenation.

[0075] Prepare fermentation medium: Accurately weigh 3.5 g Na2HPO4, 1.5 g KH2PO4, 1.0 g (NH4)2SO4, 80 mg MgSO4·7H2O, 1 mg CaSO4·2H2O, 0.56 mg NiSO4·7H2O, 0.4 mg ferric citrate and 200 mg NaHCO3, dilute to 1 L with water as the solvent, and sterilize by high pressure wet heat at 115°C for 30 min.

[0076] The rejuvenated H16Δglk, H16ΔglkΔA2182, H16ΔglkΔB0842, and H16ΔglkΔA2183 strains were centrifuged at 4600 rpm for 8 min to collect the cells, washed three times with PBS buffer solution, and inoculated into anaerobic bottles containing 50 mL of fermentation medium. Three replicates were set for each strain. To ensure that the number of cells in each anaerobic bottle after inoculation was the same, the initial OD value of the bacterial solution was adjusted. 600 ≈0.1. The anaerobic flask was placed in a shaker at 30°C and 200 rpm for shake flask fermentation and a mixed gas (H2:O2:CO2=8:1:1) was continuously added to supplement the carbon source. The bacterial solution OD was measured at regular intervals. 600 .

[0077] Bacterial liquid OD 600 Determination: The absorbance of the bacterial solution was measured at a wavelength of 600 nm using a visible light spectrophotometer.

[0078] The results are as follows Figure 6 As shown in the figure, compared with the H16Δglk strain of H. nematophila, the proliferation rate of the H16ΔglkΔA2182 strain of H. nematophila was significantly improved. However, the knockout of the H16_B0842 and H16_A2183 genes had a negative impact on the proliferation rate of the H16Δglk strain of H. nematophila. After 4 days of culture, the OD of the H16ΔglkΔA2182 strain of H. nematophila was 0. 600 The OD value of the H16Δglk strain of Copperworm was 5.07, which was 1.57 times that of the H16Δglk strain of Copperworm (3.22). 600 The OD of the H16ΔglkΔA2182 strain of Copperobacterium necrotica reached its maximum value after 10 days of cultivation. 600 The maximum value was reached after 12 days of cultivation. The highest OD of the H16ΔglkΔA2182 strain of Copperworm 600 The OD value of H16Δglk strain was 12.29, which was 1.32 times that of H16Δglk strain (9.30). The knockout of H16_B0842 gene could significantly inhibit the growth of H16Δglk strain. After 10 days of growth, the OD value of H16Δglk strain was 12.29, which was 1.32 times that of H16Δglk strain (9.30). 600 Only 4.12. Highest OD 600The OD value of H16Δglk was 4.34, which was 53.33% lower than that of H16Δglk. In the first 6 days, H16ΔglkΔA2183 strain had similar growth rate to H16Δglk strain. 600 No longer increases significantly, maximum OD 600 It was 6.99, which was 24.84% lower than that of H16Δglk of Copperworm. Alkaline phosphatase is a non-specific phosphatase that can catalyze the dephosphorylation of various metabolic intermediates (such as glucose-6-phosphate, glucose-1-phosphate, etc.) in the central carbon metabolic pathway to produce glucose. However, the glucose kinase gene glk-deficient strain cannot recycle the lost carbon flow, resulting in a loss of carbon flow within the strain. The deletion of the alkaline phosphatase encoding gene H16_A2182 can reduce the loss of carbon flow in the central carbon metabolic pathway of the Copperworm H16Δglk strain, thereby having a positive promoting effect on the proliferation of the strain. The deletion of the H16_B0842 and H16_A2183 genes may cause the reactions necessary for the growth of the strain (such as signal transduction, metabolic regulation, etc.) that they mainly catalyze to be unable to proceed, thereby having a negative impact on the growth of the strain.

[0079] Example 3 Effect of superimposed knockout of glucokinase gene and HAD phosphatase gene on the proliferation rate of Cupria obliquus

[0080] 1. Construction of a strain with HAD phosphatase knockout based on the H16Δglk strain of Copperophilus necrotizingus

[0081] The targeted knockout vectors pK18-A2218, pK18-A0174, pK18-serB3, pK18-cbbY2, pK18-cbbYp, and pK18-A2331 encoding the HAD phosphatase genes H16_A2218, H16_A0174, serB3 (H16_B1164), cbbY2 (H16_B1392), cbbYp (PHG424), and H16_A2331, respectively, were introduced into the Cupria necrotica H16Δglk host by conjugative transfer. A first round of selection using kanamycin identified host strains in which the knockout vectors had successfully integrated into the genome. On this basis, a second round of screening was performed using sucrose, and the strains H16ΔglkΔA2218, H16ΔglkΔA0174, H16ΔglkΔserB3, H16ΔglkΔcbbY2, H16ΔglkΔcbbYp, and H16ΔglkΔA2331, in which the glucokinase gene glk was superimposedly knocked out with the HAD phosphatase genes H16_A2218, H16_A0174, serB3 (H16_B1164), cbbY2 (H16_B1392), cbbYp (PHG424), and H16_A2331, were obtained. The specific methods are as follows:

[0082] 1. Construction of knockout plasmid vector with upstream and downstream homology arms of HAD phosphatase encoding gene sequence

[0083] Synthetic primers were designed based on the HAD phosphatase-encoding gene sequences (H16_A2218, H16_A0174, serB3, cbbY2, cbbYp, and H16_A2331). Using the H16 genome of C. necrotica as a template, the designed primers were used to amplify the upstream and downstream homology arms of the HAD phosphatase-encoding gene, respectively, with a fragment size of approximately 500 bp. The obtained fragments were ligated into the EcoRI / SmaI sites of the pK18mobSacB plasmid using Gibson Assembly ligation. The recombinant plasmids were electroporated into the Escherichia coli TransI cloning host, coated with LB solid plates (kanamycin 50 μg / mL), and cultured overnight at 37°C. Positive clones were screened by colony PCR. The target fragment was 1000 bp. The successfully constructed vectors were named pK18-A2218, pK18-A0174, pK18-serB3, pK18-cbbY2, pK18-cbbYp and pK18-A2331, respectively. Figure 7 、 Figure 8 、 Figure 9 、 Figure 10 、 Figure 11 and Figure 12 shown.

[0084] 2. Construction of a strain with knockout plasmid vector integrated into the genome of C. necrotica H16Δglk

[0085] The knockout vectors were transformed into E. coli S17-1 by electroporation, plated on solid LB plates containing 50 μg / mL kanamycin, and cultured overnight at 37°C. Positive clones were picked and cultured in liquid LB medium containing 50 μg / mL kanamycin at 37°C, shaking at 200 rpm overnight.

[0086] Escherichia coli S17-1 strain carrying the knockout plasmid and C. necrotica H16Δglk strain were inoculated into LB liquid medium supplemented with the corresponding antibiotics (kanamycin 50 μg / mL or gentamicin 10 μg / mL). The cultures were incubated overnight at 37°C and 30°C, respectively, in a shaker at 200 rpm. The cells were harvested by centrifugation at 4600 rpm for 8 minutes and washed three times with LB medium. Escherichia coli S17-1 strain carrying the knockout plasmid and C. necrotica H16Δglk strain were mixed in a 3:1 ratio and spotted onto LB plates. The cells were incubated overnight at 30°C. The mixed cells were washed with LB and plated onto solid LB plates (kanamycin 200 μg / mL and gentamicin 10 μg / mL). After incubation at 30°C for 48 hours, positive clones were screened using colony PCR for the 500-bp kanamycin resistance gene fragment. Positive clones were cultured in 1.5 mL EP tubes supplemented with LB liquid medium at 30°C and 200 rpm in a shaking incubator overnight.

[0087] 3. Construction of an engineered strain of Copperbacterium necrotum with both the glucokinase gene and the HAD phosphatase gene knocked out

[0088] The above bacterial solutions were diluted to 10 -2 The strains were plated onto LB plates supplemented with 100 g / L sucrose and incubated at 30°C for 48 hours. Colony PCR was performed to screen clones with successful knockout of the target gene, with a target fragment size of 1000 bp. Successfully constructed strains were inoculated into LB liquid medium and named H16ΔglkΔA2218, H16ΔglkΔA0174, H16ΔglkΔserB3, H16ΔglkΔcbbY2, H16ΔglkΔcbbYp, and H16ΔglkΔA2331.

[0089] Effects of superimposed knockout of the glucokinase and HAD phosphatase genes on the autotrophic proliferation rate of Cupribotrys nematodes

[0090] The H16Δglk, H16ΔglkΔA2218, H16ΔglkΔA0174, H16ΔglkΔserB3, H16ΔglkΔcbbY2, H16ΔglkΔcbbYp and H16ΔglkΔA2331 strains were inoculated into 40 mL of LB medium at an inoculum size of 1‰ and cultured overnight in a shaker at 30°C and 200 rpm for rejuvenation.

[0091] Prepare fermentation medium: Accurately weigh 3.5 g Na2HPO4, 1.5 g KH2PO4, 1.0 g (NH4)2SO4, 80 mg MgSO4·7H2O, 1 mg CaSO4·2H2O, 0.56 mg NiSO4·7H2O, 0.4 mg ferric citrate and 200 mg NaHCO3, dilute to 1 L with water as the solvent, and sterilize by high pressure wet heat at 115°C for 30 min.

[0092] The rejuvenated H16Δglk, H16ΔglkΔA2218, H16ΔglkΔA0174, H16ΔglkΔserB3, H16ΔglkΔcbbY2, H16ΔglkΔcbbYp and H16ΔglkΔA2331 strains were centrifuged at 4600 rpm for 8 min to collect the bacteria, and then washed three times with PBS buffer solution and inoculated into anaerobic bottles containing 50 mL of fermentation medium. Three parallels were set for each strain. To ensure that the number of bacteria in each anaerobic bottle after inoculation was the same, the initial OD of the bacterial solution was adjusted. 600 ≈0.1. The anaerobic flask was placed in a shaker at 30°C and 200 rpm for shake flask fermentation and continuously filled with mixed gas (H2:O2:CO2=8:1:1) to supplement the carbon source. The bacterial liquid OD was measured regularly. 600 .

[0093] Bacterial liquid OD 600 Determination: The absorbance of the bacterial solution was measured at a wavelength of 600 nm using a visible light spectrophotometer.

[0094] The results are as follows Figure 13As shown in the figure, compared with the H16Δglk strain of Copperworm, the HAD phosphatase gene knockout strains (H16ΔglkΔA0174, H16ΔglkΔserB3, H16ΔglkΔcbbY2 and H16ΔglkΔcbbYp) on the basis of the H16Δglk strain were significantly improved. After the strains were cultured for 4 days, the OD values of H16ΔglkΔA0174, H16ΔglkΔserB3, H16ΔglkΔcbbY2 and H16ΔglkΔcbbYp were significantly increased. 600 That is, they reached 6.95, 5.92, 5.41 and 6.65 respectively. At this time, the OD of the H16Δglk strain of Copperworm 600 The OD value of H16Δglk strain of Copperworm was 3.22 after 10 days of culture. 600 The highest value (9.30) was reached. H16ΔglkΔA0174 of H. nematophila reached its maximum value (9.37) after 8 days of culture; while H16ΔglkΔserB3 of H. nematophila, H16ΔglkΔcbbY2 of H. nematophila and H16ΔglkΔcbbYp of H. nematophila reached their highest OD on the 6th day. 600 , 8.86, 9.54 and 9.85 respectively. That is, the superposition of knockout of HAD phosphatase encoding genes (H16_A0174, serB3, cbbY2 and cbbYp) on the H16Δglk strain of H. nematophila with the glucose kinase gene glk deleted can further increase the proliferation rate of the strain. However, compared with the H16Δglk strain of H. nematophila, the growth rate of the strain after the superposition of knockout of H16_A2218 and H16_A2331 genes on its basis decreased. Figure 13 As shown, after 8 days of fermentation of H16ΔglkΔA2218, the OD values of H16ΔglkΔA2218 and H16ΔglkΔA2331 were 600were 7.16 and 5.22, respectively, which were 19.37% and 41.22% lower than those of the H16Δglk strain of Copperworm. Similar to alkaline phosphatase, HAD phosphatase is also a non-specific phosphatase that catalyzes the dephosphorylation of various metabolic intermediates (such as glucose-6-phosphate, glucose-1-phosphate, etc.) in the central carbon metabolism pathway to produce glucose. However, the strain deficient in the glucokinase gene glk cannot recycle the lost carbon flow, resulting in a loss of carbon flow within the strain. The deletion of the HAD phosphatase encoding genes H16_A0174, serB3, cbbY2 and cbbYp can reduce the loss of carbon flow in the central carbon metabolism pathway in the H16Δglk strain of Copperworm, thereby positively promoting the growth rate of the strain. The deletion of the H16_A2218 and H16_A2331 genes may cause the reactions necessary for the growth of the strain (such as signal transduction, metabolic regulation, etc.) that they mainly catalyze to be unable to proceed, thereby having a negative impact on the growth of the strain.

[0095] The above embodiments are only used to explain the technical solutions of the present application and do not limit the scope of protection of the present application.

Claims

1. A method for increasing the growth rate of a Copperobacterium nematophilum strain using carbon dioxide as a carbon source, characterized in that: The method comprises the following steps: Knockout of the glucokinase gene in the H16 strain of Copperobacterium necrotizingum; Stacked knockout of alkaline phosphatase gene.

2. the method for the growth rate of the lifting hookworm greedy copper bacteria strain with carbon dioxide as carbon source according to claim 1, is characterized in that, The method further comprises the following steps: Knockout of phosphatase genes of the haloacid dehalogenase family.

3. the method for the growth rate of the lifting hookworm greedy copper bacteria strain using carbon dioxide as carbon source according to claim 2, is characterized in that, The nucleotide sequence of the haloacid dehalogenase family phosphatase gene is shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO:

6.

4. The method for increasing the growth rate of a Copperobacterium necrotizer strain using carbon dioxide as a carbon source according to any one of claims 1 to 3, characterized in that: The glucokinase gene is the glucokinase gene glk with a nucleotide sequence as shown in SEQ ID NO:

1.

5. The method for increasing the growth rate of a Copperobacterium necrotizingum strain using carbon dioxide as a carbon source according to any one of claims 1 to 3, characterized in that: The nucleotide sequence of the alkaline phosphatase gene is shown in SEQ ID NO:

2.

6. An engineered strain of Copperbacterium hookworm, characterized in that: The engineered strain is a hookworm copper-greedy strain having the following characteristics: Both the glucokinase gene and the alkaline phosphatase gene were knocked out.

7. The engineering strain of Copperobacterium hookworm according to claim 6, characterized in that The phosphatase gene of the haloacid dehalogenase family of the engineered strain is also knocked out.

8. The engineering strain of Copperobacterium hookworm according to claim 7, characterized in that The nucleotide sequence of the haloacid dehalogenase family phosphatase gene is shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO:

6.

9. The engineered strain of Cupriavidus necator according to any one of claims 6 to 8, characterized in that The nucleotide sequence of the glucokinase gene is shown in SEQ ID NO: 1, and the nucleotide sequence of the alkaline phosphatase gene is shown in SEQ ID NO:

2.

10. Use of the engineered strain of Copperobacterium necrotizer according to claim 6 for fermentation production using carbon dioxide as a carbon source.

Images