A halomonas recombination bacterium, a construction method and application thereof

Abstract

The present application relates to the technical field of bioengineering, and particularly relates to a halomonas recombinant bacterium, a construction method and application thereof. The halomonas recombinant bacterium expresses or overexpresses a permease of an osmotic pressure regulation system by taking halomonas as a starting bacterium. The present application makes the recombinant bacterium after modification of the permease of the osmotic pressure regulation system of halomonas have a unique osmotic pressure tolerance and substrate metabolic capacity, so that the recombinant bacterium can not add nutrient raw materials in a feeding mode, that is, the strict monitoring of the concentration of various nutrient raw materials is omitted, and meanwhile, the recombinant bacterium will not have adverse effects on cell growth, product synthesis and the like, and even can realize one-time addition of nutrient raw materials for fermentation production, so as to improve the stability of the production process, greatly reduce the production labor cost, reduce the equipment depreciation speed, avoid the occurrence of a run-out tank situation, and have great industrial application value.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a recombinant halogenated bacterium, its construction method, and its application. Background Technology

[0002] Currently, in industrial production using microorganisms to mass-produce various products, the nutrients required by microorganisms in the culture medium (such as nitrogen source urea, phosphorus source dipotassium hydrogen phosphate, and carbon source glucose) are often added in a "consumption-as-you-go" manner. On the one hand, excessively high nutrient concentrations in the culture medium may inhibit microbial growth, leading to reduced yield. On the other hand, the entire production process consumes a large amount of nutrients, and adding them all at once would cause the osmotic pressure of the culture medium to exceed the tolerance range of the microorganisms. Moreover, the consumption of certain substances is high, making it impossible to achieve one-time liquid feeding, while large-scale solid feeding would accelerate equipment depreciation and slow down microbial growth, resulting in more harm than good.

[0003] Halomonas sp. is a moderately halophilic bacterium isolated from the Aiding Salt Lake in Xinjiang Uygur Autonomous Region. Its optimal growth salt concentration is 6%, and its optimal growth pH is 9.0. During growth, it consumes a large amount of glucose to synthesize its dominant product, polyhydroxyalkanoates (PHA). The current solution involves monitoring the residual sugar concentration hourly throughout the fermentation cycle (36-48 hours), using 10 g / L as a reference concentration. Sugar supplementation is stopped when the concentration is too high, and supplemented to approximately 10 g / L when the concentration is too low to continue fermentation.

[0004] The existing fermentation process is highly manual, requiring three shifts of personnel to monitor the fermentation process. Furthermore, since hourly assessments of the fermentation progress are necessary, misjudgments can disrupt production and lead to reduced output. Additionally, the gradual increase in volume during fermentation negatively impacts tank volume and level estimation, potentially causing tank overflows, which is dangerous and wastes manpower and resources. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention, based on Halomonas, intervenes in the expression of permeable proteins in the osmolarity regulation system to construct a recombinant Halomonas bacterium. Due to the unique osmolarity tolerance and substrate metabolism capabilities of this recombinant bacterium, nutrient raw materials can be added without a feed-feed method during fermentation production, thus eliminating the need for strict monitoring of the concentration of various nutrient raw materials. At the same time, it does not adversely affect cell growth, product synthesis, or other aspects, and can even achieve fermentation production by adding nutrient raw materials in a single step.

[0006] Based on this, the following technical solution is proposed.

[0007] First, the present invention provides a recombinant halomonas bacterium that expresses or overexpresses a permeation protein of the osmolarity regulation system, using halomonas as the starting bacterium.

[0008] Preferably, the amino acid sequence of the permeable protein of the osmotic pressure regulation system is shown in SEQ ID No. 1 and SEQ ID No. 2.

[0009] SEQ ID No. 1:

[0010] MIRLDNLTKVFDTPKGAVVAADHINMEVPTGEICILLGPSGCGKTTTLKMINRIVRPTSGKVFIDDEDTSSLDTQTLRRNIGYVIQQIGLFPNMTIEENITVVPKLLGWDKAKYKERARELMRMIALEPDAFLKRYPSELSGGQQQRIGVARALAADPPVMLMDEPFGAIDPINRAVIQDEFLKMQQELKKTI MFVSHDIDEAIKMGDRIAIFRDGHLVQYATPDTLLAAPKDSFVESFLGEDRALKRLNLISVREVISTNFETVSPRDSLESALQKLDKCGYQHAIVMVNEERQPVGIIPRSVAQMSQGSCHDYAQKVPAVVNVNDDLRKAVSLMLAHDITWLPCVDDEGRLSGHITQRAMTHHLGARFRTQQAAAELADPMAEE

[0011] SEQ ID No. 2:

[0012] MPIQSLKGVFRLLLLVAVFVAGAWSYATGVIEEFIFYLPDIQFLTMEHLWLTVISGSLAILVAIPLGIVLSRPSMARWAESGMQVLNIGTTIPTLAVLALSMSLLGIGIVPAVFALFVATLLP ITRNTYTGLKGVSPALKEAASGIGMSPAQRLIRVELPNALYVIFAGIRTALAINVGTVPLAFLIGAGGLGELIFTGIDLYDPLMMLAGAIPTAILAILVDAAVASIAFLVVPRGVNPARRAANA

[0013] By allowing Halomonas to express or overexpress the osmolarity-regulating permeable protein, nutrient raw materials can be added without a fed-batch method, thus eliminating the need for strict monitoring of the concentration of various nutrient raw materials. This also avoids adverse effects on cell growth, product synthesis, and other aspects, and even enables fermentation production with the addition of nutrient raw materials in a single batch (referred to as "one-pot method").

[0014] In some implementation schemes, nutrient raw materials can also be added in batches (without real-time monitoring of concentration for continuous addition), for example, when the total concentration of the carbon source (e.g., glucose) to be added exceeds the solubility.

[0015] In this invention, the expression or overexpression includes, but is not limited to, the use of genetic engineering or protein engineering techniques.

[0016] The amino acid sequence shown in SEQ ID No. 1 is encoded by the opuA gene, and the amino acid sequence shown in SEQ ID No. 2 is encoded by the opuBD gene.

[0017] Preferably, the nucleotide sequence of the opuA gene is shown in SEQ ID No. 3, and the nucleotide sequence of the opuBD gene is shown in SEQ ID No. 4.

[0018] SEQ ID No. 3:

[0019]

[0020] SEQ ID No.4:

[0021] atgcccattcaaagtcttaaaggggtcttccgcttactgctcctggtggctgtctttgttgccggcgcctggagctacgcgacaggcgttattgaagaattcatattttattgcccgatattcagtttttaacaatggagcacctatggctcactgttatctctggatcgctggcaattctagtc gcgatacctttaggcattgtgctctcccgtccgagtatggcgcgttgggcagaatctggtatgcaagtgcttaatattgggacaacgattccaacgctagcagtacttgcgttatcaatgagcttgctaggtataggtatagtacctgctgtatttgccttgtttgttgccacactactgcctatt actcgcaatacttatacggggctaaaaaggcgtgtctccggcattaaaagaggctgcttcaggtatcggtatgtcgcctgcacagcggctaatacgcgtcgaattacccaacgcactgtatgttatttttgcgggcatccgaacagctctcgcaatcaatgttggcaccgtaccgttggcattttta atcggcgcaggagggctaggagagttaatttttaccggtattgatctttacgaccctctgatgatgctcgctggtgctatcccgactgccatactagctattttggttgatgccgcagtcgccagtattgcttttcttgtcgtaccccgcggggttaatcccgctcgccgagcagcgaatgcgtag

[0022] The above sequence is of endogenous origin to Halomonas.

[0023] More preferably, the recombinant halomonas strain uses halomonas as the starting strain and simultaneously overexpresses the permeabilizing protein of the osmolarity regulation system as shown in SEQ ID No. 1 and SEQ ID No. 2.

[0024] By simultaneously overexpressing the two permeabilizing proteins of the above-mentioned osmotic pressure regulation system, cell growth and product synthesis can be promoted in the "one-pot" fermentation method.

[0025] Preferably, the halomonas is any unmodified halomonas capable of synthesizing polyhydroxy fatty acid esters.

[0026] More preferably, the halomonas is selected from one or more of Halomonas bluephagenesis or Halomonascampaniensis.

[0027] More preferably, the halomonas is at least one of Halomonas bluephagenesis TD01, Halomonas campaniensis LS21, Halomonas bluephagenesis WZY254, Halomonas bluephagenesis TD68-194, and Halomonas bluephagenesis TDH4.

[0028] Preferably, the osmotic pressure tolerance range of the recombinant halometazobacter is 1–100 g / L NaCl (more preferably 20–100 g / L NaCl, 30–100 g / L NaCl, 40–100 g / L NaCl, 50–100 g / L NaCl, 60–100 g / L NaCl, 70–100 g / L NaCl, 80–100 g / L NaCl, or 90–100 g / L NaCl).

[0029] Preferably, the rate at which the recombinant Halomonas bacteria consume glucose is 10 g / L / h to 50 g / L / h (more preferably 25 to 50 g / L / h, 30 to 50 g / L / h, 35 to 50 g / L / h, 40 to 50 g / L / h, or 45 to 50 g / L / h).

[0030] Furthermore, the present invention provides a method for constructing the recombinant halomonas bacteria in any of the above schemes, comprising: using a promoter in halomonas bacteria to drive the expression or overexpression of a gene encoding the permeation protein of the osmolarity regulation system;

[0031] Preferably, the gene sequence encoding the permeable protein of the osmotic pressure regulation system is shown in SEQ ID No. 3 and SEQ ID No. 4.

[0032] Preferably, the promoter is a constitutive promoter or an inducible promoter.

[0033] Preferably, the promoter is Pporin203 P porin221 P porin278 P porin42 P porin58 P porin68 or P porin194 .

[0034] Starter number Fluorescence intensity (FI) sequence Serial Number 203 89 tcccagtatcccttttgac SEQ ID No. 5 221 863 tcccagtatgctatttgac SEQ ID No. 6 194 4289 tcccagtatctaatttgac SEQ ID No.7 278 9371 tcccagtattaaatttgac SEQ ID No. 8 68 14801 tcccagtatgactttgac SEQ ID No. 9 42 38472 tcccagtatctatttgac SEQ ID No. 10 58 80291 tcccagtatatatttgac SEQ ID No. 11

[0035] Preferably, the promoter is a promoter whose fluorescence intensity is in the range of 5000-10000 when driving green fluorescent protein (GFP).

[0036] Driven by the aforementioned promoter, it is more conducive to the simultaneous increase in the expression levels of the tandem opuA and opuBD genes.

[0037] Preferably, the promoter is a promoter that drives the gene expression level to increase to 1 to 5 times the original gene expression level.

[0038] Furthermore, the present invention provides the application of the recombinant halometa bacteria in any of the above-described schemes or the recombinant halometa bacteria obtained by any of the construction methods in fermentation production.

[0039] Furthermore, the present invention provides a fermentation method comprising: fermenting using recombinant halometazobia from any of the above schemes or recombinant halometazobia obtained by any of the construction methods.

[0040] Preferably, the nutrient raw materials in the fermentation method are added in a one-time manner;

[0041] Preferably, the nutrient raw materials are added in the fermentation method by feed-in method.

[0042] Preferably, the product obtained by the fermentation method includes polyhydroxy fatty acid esters.

[0043] Preferably, the purpose of the fermentation method includes the synthesis of polyhydroxy fatty acid esters.

[0044] Preferably, the nutrient raw materials include all the nutrient raw materials required for the synthesis of polyhydroxyalkanoates, including but not limited to nitrogen sources, phosphorus sources, and carbon sources.

[0045] Preferably, the fermentation medium is based on MM medium, with added glucose at a final concentration of 10-30 g / L and sodium chloride at a final concentration of 5-100 g / L, and the pH is 8.5-9.5.

[0046] Preferably, the fermentation temperature is 36–38°C.

[0047] Preferably, the fermentation time is 36 to 48 hours.

[0048] Beneficial effects:

[0049] This invention modifies the permeable protein of the osmotic pressure regulation system of Halomonas, giving the recombinant bacteria unique osmotic pressure tolerance and substrate metabolism capabilities. This allows for the addition of nutrients without a feed-feed method, eliminating the need for strict monitoring of nutrient concentrations and avoiding adverse effects on cell growth and product synthesis. It even enables fermentation production with a single addition of nutrients, thereby improving the stability of the production process, significantly reducing labor costs, slowing down equipment depreciation, and preventing tank overflow. It has significant industrial application value. Detailed Implementation

[0050] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0051] Unless otherwise specified, all methods used in the examples were conventional or performed according to techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents and instruments used without specified manufacturers were all conventional products that could be purchased from legitimate channels.

[0052] The table below shows the correspondence between the English abbreviations and the full Chinese name of this invention.

[0053] English abbreviations Chinese name PHA Polyhydroxy fatty acid esters GFP Green fluorescent protein

[0054] The Halomonas bluephagenesis TD01 in the following examples was deposited on November 19, 2010, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 4353, and classified as Halomonas sp. TD01, also known as Halomonas bluephagenesis TD01. It is described in patent application CN102120973A, and the public can obtain this bacterium from Tsinghua University. Halomonas campaniensis LS21 is disclosed in "Engineering self-flocculating Halomonas campaniensis for wastewaterless open and continuous fermentation" (Chen, Ling, Guan-Qing, et al. Biotechnology and Bioengineering, 2018. DOI:10.1002 / bit.26897.). Halomonas bluephagenesis WZY254 is disclosed in "Hyperproduction of PHA copolymers containing high fractions of 4-hydroxybutyrate (4HB) by outer membrane-defected Halomonas bluephagenesis grown in bioreactors" (Wang Z, Zheng Y, Ji M, et al. Microbial Biotechnology. DOI:10.1111 / 1751-7915.13999.). Halomonas bluephagenesis TD68-194 is disclosed in "Stimulus response-based fine-tuning of polyhydroxyalkanoate pathway in Halomonas" (CJYAB, ADH, FJY, et al. Metabolic Engineering, 2020, 57:85-95.).The strain *Halomonas bluephagenesis* TDH4 was disclosed in *Effective production of Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by engineered *Halomonas bluephagenesis* grown on glucose and 1,4-Butanediol* (Zhang L, Ye JW, Zhang X, et al. *Bioresource technology*, 2022, 355:127270. DOI:10.1016 / j.biortech.2022.127270.). The osmolar tolerance range of this starting strain is 10-30 g / L sodium chloride concentration, and the glucose utilization rate range is 10-20 g / L / h.

[0055] The specific formulation of MM medium used for culturing Halomonas in the following examples is as follows:

[0056] Urea 0.5 g / L; MgSO4 0.2 g / L; KH2PO4 1.5 g / L; and 0.05 g / L Fe(III)-NH4-Citrate, 0.02 g / L CaCl2·2H2O, 0.1 mg / L ZnSO4·7H2O, 0.03 mg / L MnCl2·4H2O, 0.3 mg / L H3BO3, 0.2 mg / L CoCl2·6H2O, 0.01 mg / L CuSO4·5H2O, 0.02 mg / L NiCl2·6H2O, 0.03 mg / L NaMoO4·2H2O.

[0057] In the following examples, cell dry weight (CDW, g / L) refers to the ratio of the mass of dried cells to the volume of fermentation product; the sugar conversion rate is calculated as follows: fermentation volume multiplied by cell dry weight per liter multiplied by PHA content (wt%) divided by the percentage of total sugar consumed.

[0058] In the following examples, the PHA content was detected by gas chromatography, and the specific steps are as follows:

[0059] The furnace temperature was set to 80℃, the injector temperature to 200℃, the detector temperature to 220℃, and the column head pressure to 0.25 MPa. The programmed temperature rise conditions were: 80℃ for 1.5 minutes, then ramped up to 140℃ at a rate of 30℃ / min, followed by ramping up to 220℃ at a rate of 40℃ / min and holding at this temperature for 0.5 minutes. The sample injection volume was 1 μL, using a microsyringe manufactured by Agilent Technologies.

[0060] Gas chromatograph sample preparation: Take 40-60 mg of stem cells from the sample to be tested (centrifuge the bacterial culture at 10000 rpm at room temperature for 10 minutes, wash the resulting cell pellet once with water, and then freeze-dry to obtain stem cells; the homopolymer is produced in the cells), add 2 mL of chloroform and 2 mL of esterification solution (pure methanol containing 3% (v / v) concentrated sulfuric acid and 1 g / L benzoic acid as an internal standard) to the esterification tube, cap and seal, and heat at 100℃ for 4 hours. After cooling, add 1 mL of distilled water, shake thoroughly, and let stand until the chloroform phase and aqueous phase completely separate. Inject 1 μL of the lower chloroform phase into the gas chromatograph (HP Hewlett Packard 6890) for chromatographic analysis. Operate the gas chromatograph according to the HP Hewlett Packard 6890 gas chromatograph instruction manual.

[0061] Preparation of standard samples: Take 10-20 mg of standard sample into an esterification tube, add 2 mL of chloroform and 2 mL of esterification solution, seal the tube, and carry out esterification at 100℃.

[0062] Results analysis: Using the standard sample as a control, if the esterified sample of the cells to be tested (the sample to be tested) has a significant peak at the standard sample, the mass of each monomer can be calculated based on the peak area, and then the molar ratio can be calculated based on the mass fraction of each monomer; the proportion of polymer in the dry weight of the cells (wt%) can be calculated based on the amount of sample added.

[0063] Example 1

[0064] This embodiment provides a recombinant Halomonas bacterium that simultaneously overexpresses the opuA and opuBD genes using Halomonas bluephagenesis TD01 as the starting strain. The specific steps are as follows:

[0065] (1) Construct pSEVA321-P porin42 -opuA-opuBD expression plasmid:

[0066] Promoter P porin42 (SEQ ID No. 10) and opuA gene (SEQ ID No. 3) and opuBD gene (SEQ ID No. 4) were sequentially tandemly inserted into the foreign gene expression site of pSEVA321 plasmid;

[0067] The pSEVA321 plasmid is disclosed in Durante-Rodríguez G, de Lorenzo V, Martínez-García E. The Standard European Vector Architecture (SEVA) plasmid toolkit. Methods MolBiol. 2014; 1149:469-78. doi:10.1007 / 978-1-4939-0473-0_36. PMID:24818926.

[0068] (2) The plasmid was transformed into the host of Halomonas bluephagenesis TD01 via conjugation transformation.

[0069] (3) The expression levels of opuA and opuBD genes in the host before and after transfection were detected to be 1048 / 4910 and 998 / 3910, respectively, in fpkm. At the same time, the fluorescence intensity of green fluorescent protein (GFP) was detected to be 6843 by culturing the recombinant halomonas using a fluorescence spectrophotometer.

[0070] Furthermore, this embodiment provides a fermentation production method for polyhydroxyalkanoates, the specific steps of which are as follows:

[0071] MM medium was used as the basal medium, with glucose and sodium chloride added to a final concentration of 20 g / L and 10 g / L respectively. A 7L fermenter was used, with a fermentation broth volume of 3L, pH set at 9.0, and temperature maintained at 37°C. The aforementioned recombinant halometabolite strain was inoculated at a rate of 10% (v / v). All nutrients were added to the fermenter at once before fermentation. During fermentation, temperature and pH were monitored. After 48 hours of fermentation, cell dry weight, sugar conversion rate, PHA content, and labor costs were measured and calculated. Further testing showed that the osmotic pressure tolerance range of the recombinant halometabolite strain in this embodiment was 20-60 g / L sodium chloride concentration, and the glucose utilization rate was within the range of 30-40 g / L / h.

[0072] Meanwhile, a control experiment was set up, in which glucose was produced by fed-batch fermentation. The total amount of glucose added was kept constant, and the residual sugar concentration was measured every hour. 10 g / L was used as the reference concentration. If the sugar concentration was too high, the sugar was stopped and if the sugar concentration was too low, it was added to 10 g / L for fermentation.

[0073] The results are shown in Table 1.

[0074] Table 1

[0075] Fermentation method Cell dry weight Sugar conversion rate PHA content Number of personnel Human resource costs Control experiment 89.39g / L 31％ 78％ 3 people 150 yuan / hour One-pot method 90.77g / L 32％ 81％ 2 people 100 yuan / hour

[0076] It is evident that the "one-pot" fermentation method can slightly increase cell dry weight and PHA content, significantly reduce production labor costs, and slightly increase sugar conversion rate, thus possessing significant industrial application value.

[0077] Example 2

[0078] This embodiment provides a recombinant Halomonas bacterium, which uses Halomonas campaniensis LS21 as the starting strain and simultaneously overexpresses the opuA and opuBD genes. The specific steps are the same as the construction method in Example 1. Further testing showed that the osmotic pressure tolerance range of this recombinant Halomonas bacterium is 20-60 g / L sodium chloride concentration, and the glucose utilization rate is in the range of 30-40 g / L / h.

[0079] Furthermore, this embodiment provides a fermentation production method for polyhydroxy fatty acid esters, which differs from the fermentation production method in Example 1 only in that the fermentation production is carried out using the recombinant halometazobacterium constructed in this embodiment.

[0080] The results are shown in Table 2.

[0081] Table 2

[0082] Fermentation method Cell dry weight Sugar conversion rate PHA content Number of personnel Human resource costs Control experiment 78.66g / L 30％ 65％ 3 people 150 yuan / hour One-pot method 81.27g / L 31％ 77％ 2 people 100 yuan / hour

[0083] It is evident that the "one-pot" fermentation method can slightly increase cell dry weight and PHA content, significantly reduce production labor costs, and slightly increase sugar conversion rate, thus possessing significant industrial application value.

[0084] Example 3

[0085] This embodiment provides a recombinant Halomonas bacterium that simultaneously overexpresses the opuA and opuBD genes using Halomonas bluephagenesis WZY254 as the starting strain. The specific steps are the same as the construction method in Example 1. Further testing revealed that the osmotic pressure tolerance range of this recombinant Halomonas bacterium is 20-60 g / L sodium chloride concentration, and the glucose utilization rate is within the range of 30-40 g / L / h.

[0086] Furthermore, this embodiment provides a fermentation production method for polyhydroxy fatty acid esters, which differs from the fermentation production method in Example 1 only in that the fermentation production is carried out using the recombinant halometazobacterium constructed in this embodiment.

[0087] The results are shown in Table 3.

[0088] Table 3

[0089] Fermentation method Cell dry weight Sugar conversion rate PHA content Number of personnel Human resource costs Control experiment 94.59g / L 32％ 79％ 3 people 150 yuan / hour One-pot method 98.71g / L 34％ 81％ 2 people 100 yuan / hour

[0090] It is evident that the "one-pot" fermentation method can slightly increase cell dry weight and PHA content, significantly reduce production labor costs, and slightly increase sugar conversion rate, thus possessing significant industrial application value.

[0091] Example 4

[0092] This embodiment provides a recombinant Halomonas bacterium that simultaneously overexpresses the opuA and opuBD genes using Halomonas bluephagenesis TD68-194 as the starting strain. The specific steps are the same as the construction method in Example 1. Further testing revealed that the osmotic pressure tolerance range of this recombinant Halomonas bacterium is 20-60 g / L sodium chloride concentration, and the glucose utilization rate is within the range of 30-40 g / L / h.

[0093] Furthermore, this embodiment provides a fermentation production method for polyhydroxy fatty acid esters, which differs from the fermentation production method in Example 1 only in that the fermentation production is carried out using the recombinant halometazobacterium constructed in this embodiment.

[0094] The results are shown in Table 4.

[0095] Table 4

[0096] Fermentation method Cell dry weight Sugar conversion rate PHA content Number of personnel Human resource costs Control experiment 62.50g / L 29％ 71％ 3 people 150 yuan / hour One-pot method 71.76g / L 32％ 73％ 2 people 100 yuan / hour

[0097] It is evident that the "one-pot" fermentation method can slightly increase cell dry weight and PHA content, significantly reduce production labor costs, and slightly increase sugar conversion rate, thus possessing significant industrial application value.

[0098] Example 5

[0099] This embodiment provides a recombinant Halomonas bacterium that simultaneously overexpresses the opuA and opuBD genes using Halomonas bluephagenesis TDH4 as the starting strain. The specific steps are the same as the construction method in Example 1. Further testing revealed that the osmotic pressure tolerance range of this recombinant Halomonas bacterium is 20-60 g / L sodium chloride concentration, and the glucose utilization rate is within the range of 30-40 g / L / h.

[0100] Furthermore, this embodiment provides a fermentation production method for polyhydroxy fatty acid esters, which differs from the fermentation production method in Example 1 only in that the fermentation production is carried out using the recombinant halometazobacterium constructed in this embodiment.

[0101] The results are shown in Table 5.

[0102] Table 5

[0103] Fermentation method Cell dry weight Sugar conversion rate PHA content Number of personnel Human resource costs Control experiment 90.09g / L 33％ 76％ 3 people 150 yuan / hour One-pot method 93.51g / L 34％ 78％ 2 people 100 yuan / hour

[0104] It is evident that the "one-pot" fermentation method can slightly increase cell dry weight and PHA content, significantly reduce production labor costs, and slightly increase sugar conversion rate, thus possessing significant industrial application value.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant halometazobacterium, characterized in that, It uses Halomonas as the starting strain to simultaneously express or overexpress a permeabilizing protein of the osmolarity regulation system; the amino acid sequence of the permeabilizing protein of the osmolarity regulation system is shown in SEQ ID No. 1 and SEQ ID No.

2.

2. The recombinant halometazoan bacterium according to claim 1, characterized in that, The halometa is Halomonas bluephagenesis TD01 Halomonas campaniensis LS21 Halomonas bluephagenesis WZY254 Halomonas bluephagenesis TD68-194 Halomonas bluephagenesis At least one of TDH4.

3. The recombinant halometazobacterium according to claim 1, characterized in that, The recombinant halometazobacteria have an osmotic pressure tolerance range of 1~100 g / L NaCl and / or a glucose metabolism rate of 10 g / L / h~50 g / L / h.

4. The method for constructing the recombinant halosamonella according to any one of claims 1 to 3, characterized in that, include: In Halomonas, promoters are used to drive the expression or overexpression of genes encoding proteins that permeate the osmolarity regulation system.

5. The construction method according to claim 4, characterized in that, The gene sequences encoding the permeable proteins of the osmotic pressure regulation system are shown in SEQ ID No. 3 and SEQ ID No.

4.

6. The construction method according to claim 4, characterized in that, The promoter is P. porin203 P porin221 P porin278 P porin42 P porin58 P porin68 or P porin194 .

7. The construction method according to claim 4, characterized in that, The promoter is a promoter whose fluorescence intensity is in the range of 5000-10000 when driving green fluorescent protein.

8. The construction method according to claim 4, characterized in that, The promoter is a promoter that drives the expression level of the gene to increase to 1 to 5 times the original gene expression level.

9. A fermentation method, characterized in that, include: Fermentation is carried out using the recombinant halometa bacteria according to any one of claims 1 to 3 or the recombinant halometa bacteria obtained by the construction method according to any one of claims 4 to 8.

10. The fermentation method according to claim 9, characterized in that, The fermentation process uses MM medium as the base medium, with added glucose at a final concentration of 10-30 g / L and sodium chloride at a concentration of 5-100 g / L, and a pH of 8.5-9.

5.

11. The fermentation method according to claim 9, characterized in that, The nutrient raw materials in the fermentation method are added in a one-time manner.

12. The fermentation method according to claim 9, characterized in that, The nutrient raw materials in the fermentation method are added in a fed-batch manner.

13. The fermentation method according to claim 9, characterized in that, The products obtained by the fermentation method include polyhydroxy fatty acid esters.