A formic acid-metabolizing halomonas and its construction method and application

Abstract

The present invention discloses a formic acid-metabolizing Halomonas, a construction method and an application thereof. The Halomonas expresses ftl gene, folD gene, gcvT gene, gcvH gene, gcvP gene, lpdA gene, glyA gene and sdaA gene from Vibrio natrii. The present invention enables Halomonas to acquire the ability to utilize formic acid through genetic engineering, and can ferment and grow with formate as the only carbon source, thereby replacing traditional starch raw materials and sugar-based raw materials, greatly reducing the cost of raw materials, and applying formic acid as a carbon source to the fermentation industry of Halomonas, thereby promoting the development of low-carbon biomanufacturing processes.

Description

Technical Field

[0001] The invention relates to the field of metabolic engineering, and in particular to a formic acid-metabolizing Halomonas bacterium and a construction method and application thereof. Background Art

[0002] The selection of carbon raw materials has evolved from the use of expensive, nutrient-related carbon raw materials (first generation) to industrial waste (second generation), and finally to the simplest form of C1 carbon source (third generation). The first generation of carbon sources directly uses glucose or edible oil, which causes competition between raw materials and food, and the cost is extremely high. The second generation of carbon sources is waste biomass that is not suitable for food or feed, such as textile waste, lignocellulose, crude glycerol, and byproducts of corn deep processing. However, considering that the available amount of carbon sources in waste biomass is usually limited, product quality may be affected. Therefore, the third generation of carbon sources directly uses CO2 as a raw material. The chemical and biological conversion of CO2 as a carbon source has broad application prospects and is conducive to the construction of a new CO2 circular economy industry chain. At present, CO2 can be fixed and converted by organisms. Among them, the technology of fixing CO2 using microalgae has great development and application potential. The carbon fixation capacity of microalgae and the speed of synthesizing biofuels through photosynthesis are much higher than traditional crops. However, biological conversion technology often has limited carbon fixation capacity and is subject to many unstable factors of the organisms themselves. It is still difficult to achieve large-scale conversion and utilization of CO2. The chemical conversion of CO2 mainly produces basic chemical products such as methanol and olefins through technologies such as catalytic hydrogenation, and derives various intermediate products and more than 10,000 terminal products. The chemical conversion and utilization path of CO2 has effectively promoted the high value-added utilization of CO2 and the green, low-carbon and negative carbonization process of the chemical industry, but the technical bottlenecks such as high energy consumption, low catalytic efficiency, and high manufacturing cost (hydrogen production and catalyst production) have further restricted the large-scale industrial promotion of this technology. At present, there are studies that use electrochemical methods to generate formic acid, acetic acid, etc. with CO2 as raw material, and reuse these electrochemical products as fermentation carbon sources. However, the widely used microbial chassis include Escherichia coli, Alcaligenes eutrophus, and Pseudomonas, most of which can only grow under neutral pH, sterile and low osmotic pressure conditions. Therefore, they are intolerant to carbon dioxide electrochemical derivatives and are prone to bacterial contamination. In actual industrial production, a large amount of energy consumption is required to ensure a sterile environment. Halomonas is an engineered strain that can grow rapidly under high salt, high alkali and non-sterile conditions, and can naturally synthesize high value-added products such as PHB. At the same time, engineered Halomonas can directly utilize the electrochemical products produced by CO2 conversion. However, wild-type Halomonas have poor bacterial growth and product production in high-concentration formate. Therefore, a strain that eats formic acid has important practical significance for achieving carbon neutrality by using CO2 electrochemical derivatives as the sole carbon source for large-scale industrial production. Summary of the invention

[0003] In view of the defects in the prior art, the present invention provides a formic acid-metabolizing Halomonas bacterium and a construction method and application thereof.

[0004] The present invention provides a formic acid-metabolizing Halomonas, wherein the Halomonas expresses ftl gene, folD gene, gcvT gene, gcvH gene, gcvP gene, lpdA gene, glyA gene and sdaA gene from Sodium Vibrio. The ftl gene sequence is shown in SEQ ID No.35, the folD gene sequence is shown in SEQ ID No.36, the gcvT gene sequence is shown in SEQ ID No.38, the gcvH gene sequence is shown in SEQ ID No.39, the gcvP gene sequence is shown in SEQ ID No.40, the lpdA gene sequence is shown in SEQ ID No.37, the glyA gene sequence is shown in SEQ ID No.41, and the sdaA gene sequence is shown in SEQ ID No.42.

[0005] Furthermore, the Halomonas includes Halomonas sp., preferably Halomonas sp. LY01, Halomonas sp. LY02, Halomonas sp. LY03, Halomonas sp. LY04, Halomonas sp. LY08, and Halomonas bluephagenesis TD.

[0006] Halomonas sp.LY01, Halomonas sp.LY02, Halomonas sp.LY03, Halomonas sp.LY04, and Halomonas sp.LY08 used in the present invention are all preserved in Guangdong Microbiological Culture Collection Center (GDMCC for short, address: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, Postal Code 510070), and are classified and named Halomonas sp.

[0007] The deposit date of Halomonas sp.LY01 is July 19, 2022, and the deposit number is GDMCC NO: 62635; the deposit date of Halomonas sp.LY02 is April 23, 2023, and the deposit number is GDMCC NO: 63381; the deposit date of Halomonas sp.LY03 is April 23, 2023, and the deposit number is GDMCC NO: 63382; the deposit date of Halomonas sp.LY04 is April 23, 2023, and the deposit number is GDMCC NO: 63383; the deposit date of Halomonas sp.LY08 is May 21, 2024, and the deposit number is GDMCC NO: 64665.

[0008] Furthermore, the expression of the ftl gene and folD gene used the pRE112 vector.

[0009] Furthermore, the pRE112 vector comprises a promoter, and the promoter is porin58.

[0010] The present invention also provides a method for constructing a formic acid-metabolizing Halomonas, the construction method comprising introducing a ftl-folD nucleotide sequence, a gcvT-gcvH-gcvP-lpdA nucleotide sequence, and a glyA-sdaA nucleotide sequence into Halomonas;

[0011] Among them, the nucleotide sequence of ftl-folD is shown in SEQ ID No.43; the nucleotide sequence of gcvT-gcvH-gcvP-lpdA is shown in SEQ ID No.44; and the nucleotide sequence of glyA-sdaA is shown in SEQ ID No.45.

[0012] The present invention also provides a method for culturing the Halomonas, comprising the following steps: activating the recombinant Halomonas, performing shake flask seed culture, and then performing fermentation culture in a fermentation medium with formic acid as the sole carbon source.

[0013] Furthermore, the fermentation temperature is 35-37°C.

[0014] Furthermore, the pH of the fermentation is 8.0-9.0.

[0015] Furthermore, the fermentation time is 36 to 48 hours.

[0016] The invention also provides the use of the Halomonas in catalyzing the degradation of formic acid.

[0017] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0018] The present invention enables Halomonas to acquire the ability to utilize formic acid through genetic engineering, and can ferment and grow with formate as the sole carbon source. The cell dry weight is high, and the traditional starch raw materials and sugar-based raw materials can be replaced, which greatly reduces the cost of raw materials. The use of formic acid as a carbon source in the fermentation industry of Halomonas can promote the process of low-carbon biomanufacturing. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 This is a statistical chart of dry weight of Halomonas formicus cells at different concentrations in Example 1 of the present invention;

[0021] Figure 2 pSEVA321 used in Example 2 of the present invention porin58-ftl-folD Plasmid map;

[0022] Figure 3 This is the electrophoresis diagram of the ftl-folD target fragment in Example 2 of the present invention;

[0023] Figure 4 pRE112 used in Example 2 of the present invention G7-porin58-ftl-folD Plasmid map;

[0024] Figure 5 This is the electrophoresis diagram of the ftl-folD target fragment in Example 2 of the present invention;

[0025] Figure 6 pSEVA321 used in Example 3 of the present invention porin226-gcvT-gcvH-gcvP-lpdA Plasmid map;

[0026] Figure 7 pRE112 used in Example 3 of the present invention G49-porin226-gcvT-gcvH-gcvP-lpdA Plasmid map;

[0027] Figure 8 This is the electrophoresis diagram of the target fragment gcvT-gcvH-gcvP-lpdA of Example 3 of the present invention;

[0028] Fig. 9 pSEVA321 used in Example 4 of the present invention porin140-glyA-sdaA Plasmid map;

[0029] Fig.10 pRE112 used in Example 4 of the present invention G43-porin140-glyA-sdaA Plasmid map;

[0030] Fig.11 pSEVA321 used in Example 5 of the present invention porin68-glyA-sdaA Plasmid map;

[0031] Fig.12 This is the electrophoresis diagram of the glyA-sdaA target fragment in Example 5 of the present invention. DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0033] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used are all commercially available unless otherwise specified.

[0034] Halomonas bluephagenesis TD was obtained from Tsinghua University.

[0035] Halomonas sp.LY01 itself does not have the ability to assimilate formate. In order to achieve the assimilation of synthetic formate, Halomonas sp.LY01 needs to introduce an additional exogenous reduced glycine pathway. The invention introduces a reduced glycine pathway from Vibrio natriegens, a formate-tetrahydrofolate ligase encoded by the ftl gene, a bifunctional formate-tetrahydrofolate cyclohydrolase and a methylene-tetrahydrofolate dehydrogenase encoded by the folD gene, a glycine cleavage system protein encoded by gcvT, gcvH, gcvP and lpdA, a dihydrolipoamide dehydrogenase encoded by glyA, and a serine dehydrogenase encoded by sdaA. The eight genes are all codon-optimized to adapt to Halomonas sp.LY01. The heterologous introduction pathway is first induced to express by a plasmid, thereby achieving a breakthrough in the formate assimilation of Halomonas sp., and then the heterologous gene is integrated into the genome, so that Halomonas sp. can stably grow and produce with formate as a carbon source.

[0036] Example 1 Test of the growth ability of Halomonas formic acid assimilation

[0037] The purpose of this example is to obtain the tolerance of the original untamed strain Halomonas sp.LY01 to formate for subsequent tolerance and metabolic enhancement.

[0038] (1) Preparation of culture medium:

[0039] 60LB medium: 10 g L -1 Tryptone, 5 g L -1 Yeast extract, 60 g L -1 NaCl

[0040] 60MM culture medium:

[0041] Base material: 60g·L -1 NaCl, 1 g L -1 Yeast extract;

[0042] Component I: 0.2 g L -1 MgSO4·7H2O, 9.65g·L -1 Na2HPO4·12H2O, 1.5g·L -1 KH2PO4;

[0043] Component II: 5 g·L -1 Fe(III)-NH4-citrate and 2 g·L -1 CaCl2 was dissolved in 1M concentrated hydrochloric acid;

[0044] Component III: 0.1 g·L -1 ZnSO4·7H2O, 0.03g·L -1 MnCl2·4H2O, 0.3g·L -1 H3BO3, 0.2g·L -1 CoCl2·6H2O, 0.03g·L -1 NaMoO4·2H2O, 0.02g·L -1 NiCl2·6H2O, 0.01g·L -1 CuSO4·5H2O;

[0045] 10 ml / L of component II and 1 mL / L of component III were added to the mixture of the base material and component I, and the pH value of the 60 MMA culture medium was adjusted to 8.5-9.0 using 5 M NaOH.

[0046] (2) Formate tolerance test: Precipitation was performed using 50 mL of 60MMA in a 500 mL conical flask at 37°C. Specifically, a single colony was inoculated in 5 mL of 60LB medium and inoculated at 37°C, 200 rpm for 12 hours to obtain the first seed culture, which was then transferred to 20 mL of 60LB medium at a volume ratio of 1% for secondary seed culture preparation. After incubation for 10 to 12 hours, the secondary seed culture was inoculated into 50 mL of 60MMA (containing 20, 30, 40, and 50 g·L, respectively) at a volume ratio of 2%. -1 The cells were cultured in sodium formate (5% sodium formate) medium at 37°C and 200 rpm for 72 hours, and the cell growth was observed.

[0047] (2)OD 600 Determination: Take 1mL of the fermented bacterial solution, centrifuge it at 12000r / min for 10min, discard the supernatant, add an equal volume of deionized water, resuspend the bacteria, and use a spectrophotometer to detect the absorbance at a wavelength of 600nm (if necessary, dilute the bacterial solution to ensure that the reading of the spectrophotometer at a wavelength of 600nm is in the range of 0.3-0.8).

[0048] (3) HPLC detection of sodium formate: 1 mL of the fermentation culture medium was placed in a 1.5 mL centrifuge tube and centrifuged at 9000 rpm for 5 min. The supernatant was diluted 10 times and filtered through a 0.22 μm filter membrane. Quantitative analysis was performed using an Aminex HPX-87H column (300*7.8 mm, 9 μm, Bio-Rad, USA) and a refractive index detector (RID-20A). The mobile phase was 5 mM H2SO4 at a flow rate of 0.8 mL -1 min -1 , the column temperature was maintained at 60 °C.

[0049] The experimental results are as follows Figure 1 As shown, Halomonas sp.LY01 could not grow when sodium formate (0-20 g / L) was the sole carbon source.

[0050] Example 2 Construction of formate to 5,10-methylenetetrahydrofolate pathway

[0051] 1. Construction of ftl and folD gene expression plasmids

[0052] Plasmid construction: The target gene fragments of ftl and folD amplified from Vibrio natriegens ATCC 14048 were cloned into plasmid pSEVA321. porin58-GFP pSEVA321 was amplified as a template porin58, under the action of Gibson ligase, ftl, folD fragment and pSEVA321 porin58 The backbone was recombined to form a new plasmid, named pSEVA321 porin58-ftl-folD , plasmid information such as Figure 2 shown.

[0053] (1) PCR amplification of ftl, folD gene sequences and backbone pSEVA321 porin58 The primer sequences (5'-3') of the backbone are as follows:

[0054] ftl-F: SEQ ID NO.1;

[0055] ftl-R: SEQ ID NO.2;

[0056] folD-F: SEQ ID NO.3;

[0057] folD-R: SEQ ID NO.4;

[0058] pSEVA321 porin58 -F: SEQ ID NO.5;

[0059] pSEVA321 porin58 -R: SEQ ID NO.6.

[0060] The amplification system and amplification procedure are shown in Table 1 and Table 2:

[0061] Table 1 Amplification system

[0062]

[0063] Table 2 Amplification program

[0064]

[0065] After the PCR reaction is completed, prepare agarose gel of corresponding concentration and perform electrophoresis to observe the size of DNA bands. Place the gel under ultraviolet light, quickly cut out the gel of the target DNA fragment, and cut off as much excess gel as possible.

[0066] (2) Gibson Assembly method connection

[0067] The concentration of the recovered DNA was tested, and the proportion of DNA added was calculated according to the length and concentration of the target fragment and the backbone, and the Gibson mixed enzyme was used for connection. The Gibson Assembly connection system and procedure are shown in Tables 3 and 4.

[0068] Table 3 Gibson Assembly connection system

[0069]

[0070] Table 4 Gibson Assembly connection procedure

[0071]

[0072] (3) S17-1 Escherichia coli transformation

[0073] Step 1: Take out the prepared S17-1 E. coli competent cells from -80℃, thaw them on ice, and wait for the bacteria to melt after 5 minutes;

[0074] Step 2: Add 5 μL of ligation product to the competent cells and gently tap the tube wall to mix the reaction solution (do not oscillate to mix). Note: The transformation volume of the ligation product should not exceed 1 / 10 of the volume of the competent cells used;

[0075] Step 3: Place on ice for 30 minutes, heat shock at 42°C for 2 minutes, and immediately cool on ice for 2 minutes. Note: shaking will reduce transformation efficiency.

[0076] Step 4: Add 400 μL LB medium (without antibiotics) to the centrifuge tube, mix well, and place in a 37°C shaker at 200 rpm for 60 min.

[0077] Step 5: Centrifuge at 5000 rpm for 5 min to collect the bacteria, discard 350 μL of the supernatant, keep 100 μL of the suspension, gently blow to resuspend the bacteria, and spread it on LB medium containing the corresponding antibiotics;

[0078] Step 6: Invert the culture medium into a 37°C incubator and culture for 12 to 16 hours.

[0079] (4) Monoclonal colony positive verification

[0080] Colonies were picked out on the corresponding resistance LB plates and verified by colony PCR. PCR products with the correct band size were sent to a biological company for sequencing.

[0081] (5) Product identification

[0082] The size of the target product was verified by PCR, and the results showed that the ftl and folD gene expression plasmids were successfully constructed. Figure 3 As shown, the target fragment is 3583 bp, which is consistent with the expected result.

[0083] 2. Construction of ftl and folD gene integration plasmids

[0084] Utilizing pSEVA321 porin58-ftl-folDThe porin58-ftl-folD gene fragment was obtained by PCR amplification using the plasmid as a template; the pRE112-G7-Backbone was used as a template to PCR amplify the pRE112-G7 backbone to obtain the plasmid pRE112 G7-porin58-ftl-folD Plasmid map, see Figure 4 .

[0085] The primer sequences used for PCR amplification are as follows:

[0086] Porin58-ftl-folD-F: SEQ ID NO.7;

[0087] Porin58-ftl-folD-R: SEQ ID NO.8;

[0088] pRE112-G7-F: SEQ ID NO.9;

[0089] PRE112-G7-R: SEQ ID NO.10.

[0090] (5) Product identification

[0091] The size of the target product was verified by PCR, and the results showed that the ftl and folD gene integration plasmids were successfully constructed. Figure 5 As shown, the target fragment is 5183 bp, which is consistent with the expected result.

[0092] 3. ftl, folD gene integration:

[0093] The integrated plasmid constructed above was cultured, and after 12-16 hours, it was conjugated with Halomonas sp.LY01 in a 20LB plate. After 8 hours, a small amount of conjugated bacteria was selected and spread on a 60LB plate with corresponding resistance. After 36-48 hours, a single clone colony verification was performed again. After a single colony grows on the plate, resistance verification can be performed. The colonies that have successfully lost resistance are selected for double verification (universal F1 / R and specific F2 / R). The results are as follows: Figure 6 As shown, the length of the fragment amplified by the universal primer band was 5500 bp, which was larger than 2200 bp of the wild type. There was no specific primer band, and the strain was named LY01-F1 strain.

[0094] Example 3 Construction of 5,10-methylenetetrahydrofolate to glycine pathway

[0095] 1. Construction of gcvT-gcvH-gcvP-lpdA gene expression plasmid

[0096] Vibrio natriegens ATCC 14048 was used as a template for amplification to obtain the target gene fragments of gcvT, gcvH, gcvP, and lpdA. porin226-GFP The backbone pSEVA321 was obtained by amplification using porin226 The specific implementation of the plasmid is the same as in Example 2 to obtain plasmid pSEVA321 porin226-gcvT-gcvH-gcvP-lpdA , plasmid information such as Figure 7 shown.

[0097] PCR amplification of gcvT, gcvH, gcvP, lpdA gene sequences and backbone pSEVA321 porin226 The primer sequences (5'-3') are as follows:

[0098] gcvT-F: SEQ ID NO.11;

[0099] gcvT-R: SEQ ID NO.12;

[0100] gcvH-F: SEQ ID NO.13;

[0101] gcvH-R: SEQ ID NO.14;

[0102] gcvP-F: SEQ ID NO.15;

[0103] gcvP-R: SEQ ID NO.16;

[0104] lpdA-F: ​​SEQ ID NO.17;

[0105] lpdA-R: SEQ ID NO.18;

[0106] pSEVA321 porin226 -F: SEQ ID NO. 19;

[0107] pSEVA321 porin226 -R: SEQ ID NO.20.

[0108] 2. Construction of gcvT-gcvH-gcvP-lpdA gene integration plasmid

[0109] Utilizing pSEVA321 porin226-gcvT-gcvH-gcvP-lpdA The porin226-gcvT-gcvH-gcvP-lpdA gene fragment was obtained by PCR amplification using the plasmid as a template; the pRE112 backbone was amplified by PCR using pRE112-G49-Backbone as a template to obtain the plasmid

[0110] pRE112 G49-porin226-gcvT-gcvH-gcvP-lpdA Plasmid map, see Figure 8 .

[0111] The primer sequences (5'-3') used for PCR amplification are as follows:

[0112] porin226-gcvT-gcvH-gcvP-lpdA-F: ​​SEQ ID NO.21;

[0113] porin226-gcvT-gcvH-gcvP-lpdA-R: SEQ ID NO. 22;

[0114] pRE112-G49-F: SEQ ID NO.23;

[0115] pRE112-G49-R: SEQ ID NO. 24.

[0116] 3. gcvT-gcvH-gcvP-lpdA gene integration

[0117] The specific steps refer to Example 2, and the gcvT-gcvH-gcvP-lpdA gene is inserted into the genome of the LY01-E1 strain. The PCR cloning verifies that the band is correct, indicating that the gcvT-gcvH-gcvP-lpdA gene is successfully inserted into the genome of the LY01-F1 strain. Fig. 9 As shown, the target fragment was 7200 bp, which was consistent with the expected result, and the obtained strain was named LY01-F2.

[0118] Example 4 Construction of glycine to pyruvate pathway

[0119] 1. Construction of glyA and sdaA gene expression plasmids

[0120] The glyA and sdaA gene fragments were obtained by amplification using Vibrio natriegens ATCC 14048 as template. porin140-GFP The backbone pSEVA321 was amplified by PCR using 5% paraformaldehyde as a template to obtain plasmid pSEVA321. porin140-glyA-sdaA , plasmid information such as Fig.10 Show.

[0121] The primer sequences (5'-3') for PCR amplification of glyA and sdaA genes are as follows:

[0122] glyA-F: SEQ ID NO.25;

[0123] glyA-R: SEQ ID NO. 26;

[0124] sdaA-F: SEQ ID NO. 27;

[0125] sdaA-R: SEQ ID NO. 28;

[0126] pSEVA321 porin140 -F: SEQ ID NO. 29;

[0127] pSEVA321 porin140 -R: SEQ ID NO.30.

[0128] 2. Construction of glyA and sdaA gene integration plasmid

[0129] Utilizing pSEVA321 porin140-glyA-sdaA The porin140-glyA-sdaA gene fragment was obtained by PCR amplification using the plasmid as a template; the pRE112 backbone was amplified by PCR using pRE112-Backbone as a template; the homology arms G43L and G43R were amplified by PCR using the Halomonas p.LY01 genome as a template to construct pRE112 G43-porin140-glyA-sdaA Plasmid, plasmid information such as Fig.11 shown.

[0130] The primer sequences (5'-3') used for PCR amplification are as follows:

[0131] porin140-glyA-sdaA-F: SEQ ID NO.31;

[0132] porin140-glyA-sdaA-R: SEQ ID NO.32;

[0133] pRE112-F: SEQ ID NO. 33;

[0134] PRE112-R: SEQ ID NO.34.

[0135] 3. Integration of glyA and sdaA genes

[0136] The specific steps refer to Example 2, the porin140-glyA-sdaA sequence was inserted into the genome of the LY01-F2 strain, and the bands were verified by PCR cloning, indicating that the glyA and sdaA genes were successfully inserted into the genome of the LY01-E2 strain. Fig.12 The results showed that the target fragment was 5300 bp, which was in line with the expected result. The obtained strain was named LY01-F3.

[0137] Example 5 Growth ability test using three modified bacteria

[0138] The fermentation test was carried out with reference to Example 1, and the amount of sodium formate added was 10 g / L.

[0139] Cell dry weight measurement: 15 mL of bacterial solution after fermentation was taken into a 50 mL centrifuge tube, centrifuged at 9000 rpm for 10 min, and then the supernatant was discarded. Another 15 mL of deionized water was taken and placed in an oscillator for resuspending for 5 min, and then centrifuged again at 9000 rpm for 5 min, and then the supernatant was discarded again. The bottom bacteria was placed in a -80°C ultra-low temperature refrigerator for precooling for 30-40 min, and then placed in a freeze dryer for freeze drying for 24 h, and the freeze-dried bacteria blocks were taken out, and the freeze-dried mass was weighed on an electronic balance. The fermentation results are shown in Table 5.

[0140] Table 5 Fermentation production results of four recombinant bacteria

[0141]

[0142] According to the fermentation results, the cell dry weight of LY01, LY01-F1, and LY01-F2 strains was low, indicating that LY01, LY01-F1, and LY01-F2 strains could not grow with sodium formate as the sole carbon source. Only LY01-F3 grew successfully with a cell dry weight of 1.18 g / L, indicating that the overexpression of several module exogenous genes at the chromosome level can effectively improve the bacterial sodium formate metabolism capacity.

[0143] Example 6 Testing of sodium formate modules using TD-F, LY02-F, LY03-F, LY04-F, and LY08-F recombinant bacteria

[0144] The above-mentioned genetic modification strategy was used to introduce the ftl, folD, gcvT, gcvH, gcvP, lpdA, glyA and sdaA genes into Halomonas sp. TD, Halomonas sp. LY02, Halomonas sp LY03, Halomonas sp LY04 and Halomonas sp LY08, respectively, and the recombinant strains were named TD-F, LY02-F, LY03-F, LY04-F and LY08-F, respectively.

[0145] The constructed recombinant strain was grown by shake flask fermentation, and the specific implementation method was the same as Example 5.

[0146] Table 6 Fermentation results of four recombinant strains

[0147]

[0148] According to the fermentation results, TD-F, LY02-F, LY03-F, LY04-F and LY08-F strains all successfully grew under the condition of formate as the only carbon source, and the cell dry weights were 1.01g / L, 1.09g / L, 1.16g / L, 1.06g / L, and 1.28g / L, respectively. It shows that the overexpression of several module exogenous genes at the chromosome level can effectively improve the ability of Halomonas to utilize formate. The results of this embodiment show that the method of the present application is not only applicable to Halomonas sp.LY01, but also to other Halomonas. Introducing metabolic genes in other Halomonas can achieve the effect of increasing the ability to assimilate formic acid.

[0149] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A formic acid-metabolizing Halomonas bacterium, characterized in that: The Halomonas needs to express the ftl-folD nucleotide sequence, gcvT-gcvH-gcvP-lpdA nucleotide sequence, and glyA-sdaA nucleotide sequence derived from Sodium Vibrio; Among them, the nucleotide sequence of ftl-folD is shown in SEQ ID No.43; the nucleotide sequence of gcvT-gcvH-gcvP-lpdA is shown in SEQ ID No.44; and the nucleotide sequence of glyA-sdaA is shown in SEQ ID No.

45.

2. The Halomonas according to claim 1, characterized in that The Halomonas include Halomonas sp.

3. The Halomonas according to claim 1, characterized in that The pRE112 vector was used to express the ftl gene and folD gene.

4. The Halomonas according to claim 3, characterized in that The pRE112 vector comprises a promoter, and the promoter is porin58.

5. A method for constructing a formic acid-metabolizing Halomonas bacterium, characterized in that: The construction method comprises introducing ftl-folD nucleotide sequence, gcvT-gcvH-gcvP-lpdA nucleotide sequence, and glyA-sdaA nucleotide sequence into Halomonas; Among them, the nucleotide sequence of ftl-folD is shown in SEQ ID No.43; the nucleotide sequence of gcvT-gcvH-gcvP-lpdA is shown in SEQ ID No.44; and the nucleotide sequence of glyA-sdaA is shown in SEQ ID No.

45.

6. The method for culturing Halomonas according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: activating the recombinant salt mononas, carrying out shaking bottle seed culture, and then carrying out fermentation culture in a fermentation medium with formic acid as the sole carbon source.

7. The culture method according to claim 6, characterized in that: The fermentation temperature is 35-40°C.

8. The culture method according to claim 6, characterized in that: The pH of the fermentation is 8.0-10.

0.

9. The culture method according to claim 6, characterized in that: The fermentation time is 36 to 72 hours.

10. Use of the Halomonas according to any one of claims 1 to 4 in catalyzing the degradation of formic acid.

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