Recombinant halomonas strain, and construction method therefor and use thereof

Abstract

Provided are a recombinant Halomonas strain, and a construction method therefor and the use thereof. The recombinant Halomonas strain expresses or overexpresses a channel protein of an osmoregulatory system by using Halomonas as a starting strain. By means of modifying the channel protein of the osmoregulatory system in Halomonas, the modified recombinant strain has unique capabilities in terms of osmotic tolerance and substrate metabolism. The addition of nutrient feedstocks can be achieved without using a fed-batch process, thereby eliminating the strict monitoring of concentrations of various nutrient feedstocks, without causing adverse effects on the cell growth, synthesis amount of products, etc. Moreover, a single-batch addition of all nutrient feedstocks for fermentation production can even be achieved, thereby improving the stability of the production process, significantly reducing production labor costs, reducing the rate of equipment depreciation and preventing tank running.

Description

Halomonas sp. recombinant bacteria and construction method and application thereof

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. 2024107243544 entitled "Halomonas sp. recombinant bacteria and construction method and application thereof" filed on June 5, 2024, the entire disclosure of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0003] The present application relates to the field of bioengineering technology, in particular to a Halomonas sp. recombinant bacteria and construction method and application thereof. BACKGROUND

[0004] Currently, when using microorganisms to produce various products on a large scale in industry, the addition of nutrients required by microorganisms in the culture medium (such as nitrogen source urea, phosphorus source potassium hydrogen phosphate, carbon source glucose, etc.) is often carried out in the way of "consumption and addition at the same time". On the one hand, too high concentration of nutrients in the culture medium may inhibit the growth rate of microorganisms, resulting in reduced yield. On the other hand, a large amount of nutrients will be consumed during the entire production process, and one-time addition will cause the osmotic pressure of the culture medium to exceed the tolerable range of microorganisms. Moreover, the consumption of some substances is relatively high, and it is impossible to achieve one-time liquid feeding, while a large amount of solid feeding will accelerate equipment depreciation and slow down the growth rate of microorganisms, which is not worth the cost.

[0005] Halomonas sp. is a kind of moderately halophilic bacteria isolated from the Aiding Salt Lake in Xinjiang, China, which has an optimal growth salt concentration of 6% and an optimal growth pH of 9.0. During the growth process, a large amount of glucose is consumed to synthesize the dominant product polyhydroxyalkanoate (PHA). The current solution is to detect the residual sugar concentration every hour during the entire fermentation period (36-48 hours), and when the sugar concentration is too high, the sugar feeding is stopped, and when the sugar concentration is too low, the sugar concentration is fed to about 10 g / L for fermentation.

[0006] The existing fermentation process requires a large amount of manual work, and three shifts of personnel are required to complete the fermentation in rotation. Moreover, since judgment needs to be made every hour according to the fermentation situation, if there is a mistake in judgment, the production process will be delayed, resulting in yield loss. In addition, the gradual increase in volume during the fermentation process has an adverse effect on the estimation of tank volume and liquid level, which may cause the tank to run out, which is dangerous and causes waste of manpower and material resources. SUMMARY

[0007] To solve the above technical problems, the application intervenes in the expression of the osmotic pressure regulation system permease protein on the basis of Halomonas, and constructs a Halomonas recombinant bacteria. Since the recombinant bacteria has unique osmotic pressure tolerance and substrate metabolism capacity, the fermentation production can be carried out without adding nutrients raw materials in the form of flow addition, that is, the strict monitoring of the concentration of various nutrients raw materials is omitted, and at the same time, there is no adverse effect on cell growth, product synthesis amount, etc., and even one-time addition of nutrients raw materials for fermentation production is realized.

[0008] Based on this, the following technical scheme is proposed.

[0009] Firstly, the application provides a Halomonas recombinant bacteria, which expresses or overexpresses the osmotic pressure regulation system permease protein based on Halomonas as the starting bacteria.

[0010] Preferably, the amino acid sequence of the osmotic pressure regulation system permease protein is shown in SEQ ID No. 1 and SEQ ID No. 2.

[0011] SEQ ID No. 1:

[0012] SEQ ID No. 2:

[0013] By letting Halomonas express or overexpress the osmotic pressure regulation system permease protein, the nutrients raw materials can be added without using the form of flow addition, that is, the strict monitoring of the concentration of various nutrients raw materials is omitted, and at the same time, there is no adverse effect on cell growth, product synthesis amount, etc., and even one-time addition of nutrients raw materials (referred to as "one-pot method") for fermentation production is realized.

[0014] In some embodiments, the nutrients raw materials can also be added in batches (without real-time monitoring of the concentration for flow addition), for example, in the case that the total concentration of the carbon source (such as glucose) to be invested exceeds the solubility.

[0015] In the application, the expression or overexpression includes but is not limited to the realization by means of genetic engineering and protein engineering.

[0016] Preferably, the nucleotide sequence of the opuA gene is shown in SEQ ID No. 3, and the nucleotide sequence of the opu BD gene is shown in SEQ ID No. 4.

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

[0018] SEQ ID No. 3:

[0019] SEQ ID No. 4:

[0020] The above sequences are endogenous sources of Halomonas.

[0021] More preferably, the recombinant Halomonas overexpresses the osmosis regulation system permeable proteins shown as SEQ ID No. 1 and SEQ ID No. 2 simultaneously.

[0022] By overexpressing the above two osmosis regulation system permeable proteins simultaneously, cell growth and product synthesis can be promoted under the fermentation method of "one-pot".

[0023] Preferably, the Halomonas is any unmodified Halomonas capable of synthesizing polyhydroxyalkanoates.

[0024] More preferably, the Halomonas is selected from any one or more of Halomonas bluephagenesis or Halomonas campaniensis.

[0025] More preferably, the Halomonas is at least one of Halomonas bluephagenesis TD01, Halomonas campaniensis LS21, Halomonas bluephagenesis WZY254, Halomonas bluephagenesis TD68-194, Halomonas bluephagenesis TDH4.

[0026] Preferably, the osmosis tolerance range of the recombinant Halomonas 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, 90-100 g / L NaCl).

[0027] Preferably, the glucose metabolic consumption rate of the recombinant Halomonas is 10-50 g / L / h (more preferably 25-50 g / L / h, 30-50 g / L / h, 35-50 g / L / h, 40-50 g / L / h, 45-50 g / L / h).

[0028] Further, the present application provides a method for constructing the recombinant Halomonas sp. in any of the above-mentioned solutions, comprising: using a promoter to drive the expression or overexpression of a gene encoding the osmostress-regulated transport protein in the Halomonas sp.

[0029] Preferably, the gene sequence encoding the osmostress-regulated transport protein is shown as SEQ ID No. 3 and SEQ ID No. 4.

[0030] Preferably, the promoter is a constitutive promoter or an inducible promoter.

[0031] Preferably, the promoter is P porin203 , P porin221 , P porin278 , P porin42 , P porin58 , P porin68 or P porin194 .

[0032] Preferably, the promoter is a promoter driving the fluorescence intensity of green fluorescent protein (GFP) in the range of 5000-10000.

[0033] Under the driving of the above-mentioned promoter, the expression amount of the tandem opuA gene and opuBD gene is further improved synchronously.

[0034] Preferably, the promoter is a promoter driving the expression amount of the gene to be increased by 1-5 times of the original gene expression amount.

[0035] Further, the present application provides the application of the recombinant Halomonas sp. in any of the above-mentioned solutions or obtained by any of the construction methods in the fermentation production.

[0036] Further, the present application provides a fermentation method, comprising: using the recombinant Halomonas sp. in any of the above-mentioned solutions or obtained by any of the construction methods for fermentation.

[0037] Preferably, the addition mode of the nutrient raw material in the fermentation method is a one-time addition mode.

[0038] Preferably, the addition mode of the nutrient raw material in the fermentation method is a flow addition mode.

[0039] Preferably, the product obtained by the fermentation method comprises polyhydroxyalkanoate.

[0040] Preferably, the purpose of the fermentation method comprises synthesizing polyhydroxyalkanoate.

[0041] Preferably, the nutrient raw material includes all the nutrient raw materials required for synthesizing polyhydroxyalkanoate, including but not limited to nitrogen source, phosphorus source, carbon source.

[0042] Preferably, the fermentation adopts MM medium as the base medium, and glucose with a final concentration of 10-30 g / L and sodium chloride with a final concentration of 5-100 g / L are added, and the pH is 8.5-9.5.

[0043] Preferably, the fermentation temperature is 36-38℃.

[0044] Preferably, the fermentation time is 36-48 h. Beneficial effects:

[0045] The present application has unique osmotic pressure tolerance and substrate metabolism capacity after the permeation protein of the osmotic pressure regulation system of Halomonas is modified, and the modified recombinant bacteria can not use the way of adding nutrient raw material by flow adding, that is, the strict monitoring of the concentration of various nutrient raw materials is omitted, and at the same time, there is no adverse effect on cell growth, product synthesis amount, etc., and even one-time addition of nutrient raw material for fermentation production is realized, thereby improving the stability of the production process, greatly reducing the production labor cost, reducing the equipment depreciation speed, avoiding the occurrence of tank running, and having great industrial application value. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme in the present application will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0047] If the specific technology or condition is not specified in the embodiments, it is carried out according to the conventional method or the technology or condition described in the literature in the art, or according to the product instruction. If the manufacturer of the reagent and instrument is not specified, it is a conventional product that can be purchased through a regular channel.

[0048] The correspondence between the English abbreviations and Chinese full names in the present application is shown in the following table.

[0049] Halomonas bluephagenesis TD01 in the following examples was deposited at China General Microbiological Culture Collection Center on November 19, 2010, with the accession number of CGMCC No. 4353, and was named as Halomonas sp. TD01, also known as Halomonas bluephagenesis TD01. It was described in patent application CN102120973A, and the public can obtain the strain from Tsinghua University. Halomonas campaniensis LS21 was 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 was 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 was disclosed in “Stimulus response-based fine-tuning of polyhydroxyalkanoate pathway in Halomonas” (C J Y AB, AD H, F J Y, et al. Metabolic Engineering, 2020, 57: 85-95.).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 osmotic pressure tolerance range of the above-mentioned starting strain is 10-30 g / L of sodium chloride concentration, and the glucose utilization rate range is 10-20 g / L / h.

[0050] The specific formula of the MM medium for culturing Halomonas in the following examples is as follows:

[0051] Urea 0.5 g / L; MgS04 0.2 g / L; KH2P04 1.5 g / L; and 0.05 g / L Fe(III)-NH4-Citrate, 0.02 g / L CaCl2·2H2O, 0.1 mg / L ZnS04·7H2O, 0.03 mg / L MnCl2·4H2O, 0.3 mg / L H3B03, 0.2 mg / L CoCl2·6H2O, 0.01 mg / L CuS04·5H2O, 0.02 mg / L NiCl2·6H2O, 0.03 mg / L NaMo04·2H2O.

[0052] In the following examples, the cell dry weight (CDW, g / L) refers to the ratio of the mass of dry bacterial cells to the volume of fermentation product; the calculation method of sugar conversion rate is as follows: the fermentation volume multiplied by the cell dry weight per liter multiplied by the PHA content (wt%) divided by the percentage of total sugar consumption mass is the sugar conversion rate.

[0053] In the following examples, the PHA content is detected by gas chromatography, and the specific steps are as follows:

[0054] The oven temperature is set to 80°C, the injector temperature is 200°C, the detector temperature is 220°C, the column head pressure is 0.25 Mpa, and the programmed temperature conditions are: 80°C for 1.5 minutes, then increased to 140°C at a rate of 30°C / min, then increased to 220°C at a rate of 40°C / min and kept at this temperature for 0.5 minutes. The sample injection volume is 1 μL, and a micro-injector produced by Agilent Company is used.

[0055] Gas phase sample preparation: 40-60 mg of dry cells of the sample to be tested (the bacterial solution was centrifuged at 10000 rpm at room temperature for 10 minutes, the obtained cell precipitate was washed once with water, then dried on ice to obtain dry cells, and the polymer was produced in the cells) was added with 2 mL of chloroform and 2 mL of esterification solution (3% (v / v) concentrated sulfuric acid and 1 g / L benzoic acid as an internal standard in pure methanol) in an esterification tube, which was sealed and heated at 100°C for 4 hours. After cooling, 1 mL of distilled water was added, and after thorough shaking, the sample was allowed to stand until the chloroform phase and the water phase were completely separated. Then, 1 μL of the lower chloroform phase was injected into a gas chromatograph (Hewlett Packard 6890) for chromatographic analysis. The gas chromatograph was operated according to the instructions of the Hewlett Packard 6890 gas chromatograph.

[0056] Standard sample preparation: 10-20 mg of the standard sample was added to an esterification tube, 2 mL of chloroform and 2 mL of esterification solution were added, and the tube was sealed and esterified at 100°C.

[0057] Result analysis: using the standard sample as a control, if the esterification sample of the cell to be tested (the sample to be tested) has a clear peak at the standard sample, then the mass of each monomer can be calculated according to the peak area, and then the molar ratio can be calculated according to the mass fraction of each monomer. According to the amount of sample added, the proportion of polymer in the dry weight of the cell (wt%) can be calculated.

[0058] Example 1

[0059] This example provides a recombinant Halomonas bluephagenesis TD01 strain, which overexpresses opuA and opuBD genes, and the specific steps are as follows:

[0060] (1) Construction of pSEVA321-P porin42 -opuA-opuBD expression plasmid:

[0061] The promoter P porin42 (SEQ ID No. 10) and the opuA gene (SEQ ID No. 3) and the opuBD gene (SEQ ID No. 4) were sequentially inserted into the foreign gene expression site of the pSEVA321 plasmid;

[0062] The pSEVA321 plasmid is disclosed in Durante-Rodríguez G, de Lorenzo V, Martínez-García E. The Standard European Vector Architecture (SEVA) plasmid toolkit. Methods Mol Biol. 2014; 1149: 469-78. doi: 10.1007 / 978-1-4939-0473-0_36. PMID: 24818926.

[0063] (2) The plasmid is transformed into the Halomonas bluephagenesis TD01 host by conjugation.

[0064] (3) Through transcriptome sequencing result analysis, the expression amounts of opuA and opuBD genes in the host before and after transformation are 1048 / 4910 and 998 / 3910, respectively, and the unit is fpkm. At the same time, the fluorescence intensity of green fluorescent protein (GFP) is detected by using a fluorescence spectrophotometer to be 6843.

[0065] Further, the embodiment provides a fermentation production method of polyhydroxyalkanoate, and the specific steps are as follows:

[0066] The MM medium is used as the basic medium, and 20 g / L of glucose and 10 g / L of sodium chloride are added to perform the fermentation tank experiment. The fermentation tank used this time is a 7L fermentation tank, the fermentation liquid volume is 3L, the pH is set to 9.0, the temperature is kept at 37°C, the above-mentioned Halomonas recombinant bacteria are inoculated for fermentation production, the inoculation amount is 10% (v / v), and all the above-mentioned nutrient raw materials are put into the fermentation tank at one time before fermentation. During the fermentation process, the temperature and pH indicators are monitored, and the cell dry weight, sugar conversion rate, PHA content and artificial cost consumption are detected and calculated after 48 hours of fermentation. Further detection of the osmotic pressure tolerance range of the Halomonas recombinant bacteria in the embodiment is 20-60 g / L of sodium chloride concentration, and the glucose utilization rate is in the range of 30-40 g / L / h.

[0067] At the same time, a control experiment is set, that is, a fed-batch glucose fermentation production is used, the total amount of added glucose is kept consistent, the residual sugar concentration is detected every hour, 10 g / L is used as the reference concentration, the glucose supplement is stopped when the sugar concentration is too high, and the fermentation is carried out by supplementing 10 g / L when the sugar concentration is too low.

[0068] The results are shown in Table 1.

[0069] Table 1

[0070] It can be seen that the fermentation method of "one-pot" can slightly improve the cell dry weight and PHA content, greatly reduce the production labor cost, and slightly improve the sugar conversion rate, which has great industrial application value.

[0071] Example 2

[0072] This embodiment provides a Halomonas recombinant bacterium, which overexpresses opuA and opuBD genes with Halomonas campaniensis LS21 as the starting bacterium, and the specific steps are the same as the construction method in Example 1. Further detection of the osmotic pressure tolerance range of the Halomonas recombinant bacterium in this embodiment is 20-60 g / L of sodium chloride concentration, and the glucose utilization rate is in the range of 30-40 g / L / h.

[0073] Further, this embodiment provides a fermentation production method of polyhydroxyalkanoate, which is different from the fermentation production method of Example 1 only in that the Halomonas recombinant bacterium constructed in the above embodiment is used for fermentation production.

[0074] The results are shown in Table 2.

[0075] Table 2

[0076] It can be seen that the fermentation method of "one-pot" can slightly improve the cell dry weight and PHA content, greatly reduce the production labor cost, and slightly improve the sugar conversion rate, which has great industrial application value.

[0077] Example 3

[0078] This embodiment provides a Halomonas recombinant bacterium, which overexpresses opuA and opuBD genes with Halomonas bluephagenesis WZY254 as the starting bacterium, and the specific steps are the same as the construction method in Example 1. Further detection of the osmotic pressure tolerance range of the Halomonas recombinant bacterium in this embodiment is 20-60 g / L of sodium chloride concentration, and the glucose utilization rate is in the range of 30-40 g / L / h.

[0079] Further, this embodiment provides a fermentation production method of polyhydroxyalkanoate, which is different from the fermentation production method of Example 1 only in that the Halomonas recombinant bacterium constructed in the above embodiment is used for fermentation production.

[0080] The results are shown in Table 3.

[0081] Table 3

[0082] It can be seen that the fermentation method of "one-pot" can slightly improve the cell dry weight and PHA content, greatly reduce the production labor cost, and slightly improve the sugar conversion rate, which has great industrial application value.

[0083] Example 4

[0084] The present example provides a Halomonas recombinant bacterium, which overexpresses opuA and opuBD genes with Halomonas bluephagenesis TD68-194 as the starting bacterium, and the specific steps are the same as the construction method in Example 1. Further detection of the osmotic pressure tolerance range of the Halomonas recombinant bacterium in the present example is 20-60 g / L of sodium chloride concentration, and the glucose utilization rate is in the range of 30-40 g / L / h.

[0085] Further, the present example provides a fermentation production method of polyhydroxyalkanoate, which is different from the fermentation production method of Example 1 only in that the Halomonas recombinant bacterium constructed in the above example is used for fermentation production.

[0086] The results are shown in Table 4.

[0087] Table 4

[0088] It can be seen that the fermentation method of "one-pot" can slightly improve the cell dry weight and PHA content, greatly reduce the production labor cost, and slightly improve the sugar conversion rate, which has great industrial application value.

[0089] Example 5

[0090] The present example provides a Halomonas recombinant bacterium, which overexpresses opuA and opuBD genes with Halomonas bluephagenesis TDH4 as the starting bacterium, and the specific steps are the same as the construction method in Example 1. Further detection of the osmotic pressure tolerance range of the Halomonas recombinant bacterium in the present example is 20-60 g / L of sodium chloride concentration, and the glucose utilization rate is in the range of 30-40 g / L / h.

[0091] Further, the present example provides a fermentation production method of polyhydroxyalkanoate, which is different from the fermentation production method of Example 1 only in that the Halomonas recombinant bacterium constructed in the above example is used for fermentation production.

[0092] The results are shown in Table 5.

[0093] Table 5

[0094] It can be seen that the fermentation method of "one-pot method" can slightly improve the cell dry weight and PHA content, greatly reduce the production labor cost, slightly improve the sugar conversion rate, and has great industrial application value.

[0095] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application. Industrial applicability

[0096] The application provides a recombinant Halomonas sp. strain and a construction method and application thereof. The recombinant Halomonas sp. strain expresses or overexpresses a permeability protein of an osmotic pressure regulation system by taking Halomonas sp. as a starting strain. The recombinant strain after modification of the permeability protein of the osmotic pressure regulation system of the Halomonas sp. has unique osmotic pressure tolerance and substrate metabolism capacity, can not add nutrient raw materials in a feeding mode, that is, strict monitoring of the concentration of various nutrient raw materials is omitted, meanwhile, does not have adverse effects on cell growth, product synthesis amount and the like, even realizes one-time addition of nutrient raw materials for fermentation production, thereby improving the stability of a production process, greatly reducing production labor cost, reducing equipment depreciation speed, avoiding a run-away tank situation, has great industrial application value, has good economic value and application prospect.

Claims

1. A recombinant bacterium of Halomonas sp., characterized in that, The osmotic pressure regulation system permeable protein is expressed or overexpressed by the halomonas bacteria as a starting strain.

2. The recombinant Halomonas saltans bacterium of claim 1, wherein, The amino acid sequence of the osmotic pressure regulation system permeable protein is shown in SEQ ID No. 1 and SEQ ID No.

2.

3. The recombinant Halomonas saltans bacteria of claim 2, wherein, The osmotic pressure regulation system permeable protein as shown in SEQ ID No. 1 and SEQ ID No. 2 is overexpressed by the halomonas bacteria as a starting strain.

4. The recombinant Halomonas saltans bacterium according to any one of claims 1 to 3, characterized in that, The halomonas bacteria is at least one of Halomonas bluephagenesis TD01, Halomonas campaniensis LS21, Halomonas bluephagenesis WZY254, Halomonas bluephagenesis TD68-194, and Halomonas bluephagenesis TDH4. Preferably, the osmotic pressure tolerance range of the halomonas recombinant bacteria is 1-100 g / L NaCl, and / or the metabolic glucose consumption rate is 10 g / L / h-50 g / L / h.

5. The method for constructing a recombinant Halomonas saltans according to any one of claims 1 to 4, characterized in that, The application also provides a fermentation method using the halomonas recombinant bacteria. The gene coding the osmotic pressure regulation system permeable protein is expressed or overexpressed in the halomonas bacteria using a promoter. Preferably, the gene sequence coding the osmotic pressure regulation system permeable protein is shown in SEQ ID No. 3 and SEQ ID No.

4.

6. The construction method of claim 5, wherein, The promoter is P porin203 , P porin221 , P porin278 , P porin42 , P porin58 , P porin68 , or P porin194 .

7. The construction method of claim 5, wherein, The promoter is a promoter driving the green fluorescent protein to have a fluorescence intensity in the range of 5000-10000.

8. The construction method of claim 5, wherein, The promoter is a promoter driving the expression amount of the gene to be increased to 1-5 times of the original gene expression amount.

9. A fermentation process characterized by, The application also provides a fermentation method using the halomonas recombinant bacteria. Preferably, the fermentation method is a batch fermentation method. Preferably, the fermentation method is a fed-batch fermentation method. Preferably, the product obtained by the fermentation method includes polyhydroxyalkanoate. The culture medium used in the fermentation is a MM culture medium as a basic culture medium, and 10-30 g / L of glucose and 5-100 g / L of sodium chloride are added to the culture medium, and the pH is 8.5-9.

5.

10. The fermentation process of claim 9, wherein, ​

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