Polyhydroxyalkanoate engineering bacterium as well as preparation method and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to a polyhydroxyalkanoate engineering bacterium as well as a preparation method and application thereof. The preparation method comprises the following steps: step 1, selecting a PHA production strain, inoculating the PHA production strain to a culture medium, and performing amplification culture on a shaking table; 2, after culture is completed, a low-temperature plasma mutagenesis instrument is adopted for mutation breeding; 3, screening and separating the excellent mutant strains capable of producing PHA, and constructing a high-yield PHA mutant strain library; and 4, carrying out composite screening on the mutated mutant strain library, and selecting a high-yield PHA strain. According to the method for constructing the engineering bacteria capable of producing the PHA by adopting the novel small plasma mutagenesis instrument and the application of the engineering bacteria, the engineering bacteria capable of producing the PHA at high yield are constructed and screened through low-temperature plasma mutagenesis, so that the yield of the PHA is increased, and a new path is provided for production of a degradable biological material PHA.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a polyhydroxyalkanoate engineering bacterium and a preparation method and application thereof. Background Art

[0002] Polyhydroxyalkanoates (PHAs) are a class of biodegradable polymers synthesized by various microorganisms under conditions of sufficient carbon source and lack of other essential nutrients. Similar to the biosynthesis of rhamnolipids produced by Pseudomonas or Burkholderia, PHA materials possess excellent biocompatibility and environmental friendliness. They can be classified as short-chain and / or medium-chain, including homopolymers and copolymers, and are typically stored in the cytoplasm as insoluble particles with a diameter of approximately 0.2 to 0.7 μm.

[0003] Due to its biodegradability and mechanical properties, PHA is not only considered an environmentally friendly alternative to traditional petroleum-based plastics, but also shows broad application prospects in the biomedical field. In industry, microbial fermentation technology is used to obtain strains rich in PHA, which are then carefully purified to produce high-purity products. Its medical applications are mainly reflected in the following aspects:

[0004] Tissue engineering and regenerative medicine: PHA materials can be used to construct biocompatible scaffolds, support cell adhesion and proliferation, and provide an ideal microenvironment for tissue repair and regeneration.

[0005] Drug controlled release and carrier system: Thanks to its controllable degradation characteristics, PHA can be made into a sustained-release drug carrier to achieve precise and sustained release of drugs in the body, ensuring efficacy while reducing side effects.

[0006] Absorbable medical devices: As an important member of the PHA family, polyhydroxybutyrate (PHB) has been widely used in absorbable sutures, implants and other medical devices due to its excellent biocompatibility and sustained-release properties. Its degradation products are harmless to the human body and the environment.

[0007] In short, with its excellent biosafety, PHA materials have shown broad application prospects in medicine, tissue engineering, medical devices, and drug controlled release, providing strong technical support for the green and sustainable development of modern medical materials.

[0008] At present, PHA production is mainly achieved through strain fermentation engineering and metabolic engineering, with a variety of methods and processes. The key to PHA production lies in the strain performance, which directly determines the production efficiency, process stability and quality of the final product. Screening and cultivating high-yield PHA strains has always been a key step in improving process efficiency. Common methods include strategies such as genetic modification, artificial physical mutagenesis, and chemical mutagenesis, which improve the synthesis efficiency of PHA by optimizing metabolic pathways and regulating gene expression. Currently commonly used production strains (such as Ralstonia, Bacillus, Pseudomonas and Halomonas). For example, CN113583922A discloses a comprehensive method for constructing efficient PHA engineered bacteria using genetic engineering and mutagenesis screening, which mainly uses ARTP to induce a Halomonas that grows at low salt concentrations and then combines genetic molecular modification to improve PHA production.

[0009] CN118516416A discloses a method for reducing the expression of the cytochrome d oxidase complex gene cydA or the activity of its encoded protein, which can significantly reduce the dissolved oxygen requirement of Halomonas bacteria in the late fermentation stage. This allows the strain to efficiently synthesize PHA under anoxic or anaerobic conditions, such as low fermentation speed or even no agitation, in the late fermentation stage, thereby effectively increasing PHA yield. However, these methods primarily focus on promoting PHA production through genetic engineering, and the use of novel methods also focuses on reducing growth adaptation requirements rather than using plasma or other methods as the primary means of improving strain performance.

[0010] Low-temperature plasma, which can produce a large number of high-energy reactive particles (RONS) at room temperature, is a promising physical mutagenesis method. Plasma, also known as the fourth state of matter, is the state when a gas is partially or fully ionized after being excited by high-voltage electricity. It is mainly composed of free electrons, atoms, and molecules in neutral, ionized, and excited states. It can effectively act on the DNA molecules of microorganisms, causing DNA chain breaks or base damage to trigger gene mutations. This in turn triggers the strain's SOS repair mechanism, produces a variety of mismatch sites, forms mutations, and ultimately generates a rich mutation library through stable inheritance. Summary of the Invention

[0011] In order to comprehensively solve the above problems, the present invention provides a method for constructing PHA-producing engineered bacteria using a new small plasma mutagenizer and its application. By constructing and screening high-yielding PHA-engineered bacteria through low-temperature plasma mutagenesis, PHA production can be increased, providing a new path for the production of degradable biomaterial PHA.

[0012] In order to achieve the above object, the present invention provides a method for preparing polyhydroxyalkanoate engineering bacteria in the first aspect, comprising:

[0013] Step 1: Select a PHA-producing strain and inoculate it into the culture medium for amplification culture on a shaking platform;

[0014] Step 2: After the culture is completed, a low-temperature plasma mutagenizer is used for mutation breeding;

[0015] Step 3: Screen and isolate excellent PHA-producing mutant strains to construct a high-yield PHA mutant strain library;

[0016] Step 4: Perform composite screening on the mutant bacterial library after mutagenesis to select strains with high PHA production.

[0017] Preferably, the PHA producing strain in step 1 is any one of the genus Ralstonia, Bacillus, Pseudomonas or Halomonas.

[0018] Preferably, the mutagenesis conditions in step 2 are: 10-150s under the conditions of a low-temperature plasma mutagenizer with a power of 6-8W, a frequency of 20-40Khz, and an electrode distance of 3-5cm from the bacterial liquid slide.

[0019] Preferably, in step 3, a strain with a mortality rate of 96-99% is selected.

[0020] Preferably, it is characterized in that, in step 4, Nile red fluorescence staining is combined with 96-well plate high-throughput screening during the composite screening.

[0021] The second aspect of the present invention provides polyhydroxyalkanoate engineered bacteria prepared by the above method.

[0022] The third aspect of the present invention provides the use of polyhydroxyalkanoate engineering bacteria in the production of polyhydroxyalkanoate.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The present invention adopts a new type of small plasma mutagenesis equipment in the mutagenesis process, and clarifies key parameters such as power (6-8W), frequency (20-40KHz), action distance (3-5cm) and action time (10-150s).

[0025] These specific mutagenesis conditions, carried out in atmospheric conditions, allow for precise control of mutagenesis intensity and duration, ensuring high mutation rates while maintaining optimal strain activity. This results in higher mutagenesis efficiency and reproducibility.

[0026] 2. This invention groups strains according to different mutagenesis intensities, calculates their mortality rates, and selects groups with mortality rates between 96% and 99% for the construction of a high-yielding PHA mutant library. This strategy strikes a balance between achieving a sufficiently large mutation amplitude and retaining viable individuals, significantly increasing the probability of subsequent high-yielding strains. Existing techniques often rely solely on experience or simple adjustments to the mutagenesis time, lacking precise screening for mortality, resulting in inefficient acquisition of high-yielding mutants.

[0027] 3. During the composite screening stage, the present invention employed Nile Red fluorescence staining and, with the help of the fluorescence detection function of a microplate reader, performed high-throughput quantitative screening of strains in 96-well plates. Compared to the traditional "cultivation followed by extraction and then PHA content detection" approach, this method allows for rapid, semi-quantitative evaluation of a large number of strains at an earlier stage and on a smaller culture scale (96-well plates), significantly saving screening time and costs. This "fluorescence + high-throughput" composite screening technology provides an important guarantee for quickly and accurately identifying high-yielding strains.

[0028] 4. This method not only considers PHA production but also considers growth rate within the first 12 and 48 hours as a key indicator. This comprehensive assessment of growth rate and PHA fluorescence allows the selection of mutants that exhibit both rapid growth and high PHA production, making them more valuable for subsequent fermentation scale-up. Compared to existing screening methods that rely solely on PHA production, this combined screening approach significantly improves strain adaptability in industrial production environments.

[0029] 5. After fluorescent detection identifies candidate strains, the present invention uses GC for final quantitative confirmation of PHA content and composition through centrifugation, lyophilization, and esterification. This "dual detection" model (fluorescence pre-screening + GC quantification) ensures the efficiency of high-throughput screening while accurately confirming PHA production and composition through chemical analysis, minimizing the risk of false positives or missed results.

[0030] Therefore, the present invention incorporates targeted improvements and combinations in multiple areas, including the design of low-temperature plasma mutagenesis equipment and parameters, mortality control to construct a mutant library, high-throughput fluorescence screening methods, and ultimately, precise quantitative detection. These improvements address the low efficiency, long cycle times, and uncontrollable processes associated with traditional mutagenesis and screening. These "differentiated structures or steps" collectively form the core innovation of the present invention and contribute significantly to the rapid and efficient production of engineered bacteria with stable, high-yield PHAs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0032] In the attached figure:

[0033] Figure 1 It is the observation table of mortality rate of coated plate;

[0034] Figure 2 is the death curve;

[0035] Figure 3 This is the 45-h growth curve;

[0036] Figure 4 This is the result of Nile red fluorescence detection;

[0037] Figure 5 This is the GC test result diagram;

[0038] Figure 6 Flow chart of the method of the present invention. DETAILED DESCRIPTION

[0039] The following combination Figures 1-6 The preferred embodiments of the present invention are described herein. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0040] Example 1:

[0041] A polyhydroxyalkanoate engineered bacterium was prepared using the method of Example 2.

[0042] Example 2:

[0043] A method for preparing polyhydroxyalkanoate engineering bacteria, comprising:

[0044] Step 1: Select a PHA-producing strain and inoculate it onto a culture medium for amplification culture on a shaker. The PHA-producing strain is any one of the genus Ralstonia, Bacillus, Pseudomonas, or Halomonas.

[0045] In the embodiment, Halomonas sp. (purchased from China Industrial Microorganism Culture Collection Center, No.: CICC 24456) was inoculated into the culture medium and amplified and cultured on a shaking platform.

[0046] Furthermore, the culture medium is LB broth medium, and the culture medium formula is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0-7.4, and the balance is water. In the case of Halomonas, 5%-7% sodium chloride is additionally added.

[0047] Furthermore, the culture medium was sterilized at 121 degrees for 20 minutes and cooled to room temperature before use.

[0048] Furthermore, the inoculation amount was 5%, the culture was carried out at 30 degrees and 200 r / min for 12 h until the logarithmic phase and the OD was greater than 3.

[0049] Step 2: After the culture is completed, a low-temperature plasma mutagenizer is used for mutation breeding;

[0050] Furthermore, the microorganisms were cultured to OD 600 The shaking culture can be stopped when the value is greater than 3. Dilute the cultured microorganisms with 6% sodium chloride saline to obtain an OD 600 =1

[0051] For mutagenesis, sterilize a 10mm diameter stainless steel disc under an alcohol lamp, then cool and allow to stand for 10 minutes before use. Evenly spread 10µl of the bacterial solution onto the sterile disc, allow it to dry slightly, and then place it directly under the low-temperature plasma source in a plasma mutagenizer for mutagenesis.

[0052] According to the operating procedures of the low-temperature plasma mutagenizer, the breeding conditions are: under the conditions of power 6-8W, frequency 20-40Khz, the electrode is 3-5cm away from the bacterial liquid slide, and the action time is 10-150s.

[0053] During the atmospheric pressure glow discharge process, a large number of active particles (including free electrons, oxygen free radicals and nitrogen free radicals) generated by the low-temperature plasma source will cause diverse damage to the fungal DNA structure, thereby forming a large number of mutation sites.

[0054] Step 3: Screen and isolate excellent PHA-producing mutant strains to construct a high-yield PHA mutant strain library.

[0055] The suspension obtained after mutagenesis in step 2 was diluted to a certain gradient and plated onto LB60 plates. Under these conditions, the lethality and mutation rates of the strain Halomonas sp. were calculated. (Lethality = (number of colonies before glow discharge treatment - number of colonies after glow discharge treatment / number of colonies before glow discharge treatment) * 100%, mutation rate = (number of mutant colonies / number of colonies after glow discharge treatment) * 100%).

[0056] Under the conditions of gradient mutagenesis time, a variety of mutant strains can be obtained by comparing the color depth and radius of the colonies with those of the starting strain. Figure 1 , death curve Figure 2 , we found that the mortality and mutation rates at 100s and 120s were higher than those in other groups, and many colony morphologies were significantly different from those of the starting strain. Therefore, this group was selected as the treatment group to construct a mutant library for screening mutant bacteria.

[0057] Step 4: Perform composite screening on the mutant bacterial library after mutagenesis to select strains with high PHA production.

[0058] 48 mutant strains with obvious morphological differences were selected and placed in a 48-well deep-well plate containing 1 ml of LB60 and cultured at 30 degrees and 200 rpm for 12 h. The mutants were diluted 2*10 2 The mutant strain was then added to a 96-well microplate and incubated at 30-36 degrees with medium or high shaking speed for 45-56 hours in the incubation mode of a microplate reader to detect the growth curve of the mutant strain.

[0059] The growth curve data of 48 mutant bacteria were analyzed and calculated, and the average growth rate of each mutant bacteria in the first 12 hours, the total time growth rate and other indicators shown in Table 1 were obtained. Figure 3 The growth curve itself comprehensively selects 4-8 mutant strains for fermentation culture in the fermentation medium. Here, the strain No. 15 with the best growth in the first 12 hours and the mutant strains No. 26, 27, 30, and 35 with the best growth in 45 hours are selected for fermentation production testing.

[0060] Furthermore, the general formula of the fermentation medium is: 0.1‰-2‰ (NH4)2CL, 0.1‰-1‰ MgSO4, 5‰-10‰ Na2HPO4·12H2O, 0.5‰-2‰ KH2PO4, and no more than 0.1% of other trace elements (Fe(III)-NH4-Citrate, CaCl2·2H2O, ZnSO4·7H2O, MnCl2·4H2O, H3BO3, CoCl2·6H2O, CuSO4·5H2O, NiCl2·6H2O, and trace amounts of NaMoO4·2H2O) (pH adjusted to about 9.0).

[0061] Preferably: 0.1% (NH4)2SO4 or 0.2% urea, 0.02% MgSO4,

[0062] 1.0% Na2HPO4·12H2O, 0.15% KH2PO4, no more than 0.1% of other trace elements (Fe(III)-NH4-Citrate, CaCl2·2H2O, ZnSO4·7H2O, MnCl2·4H2O, H3BO3, CoCl2·6H2O, CuSO4·5H2O, NiCl2·6H2O, NaMoO4·2H2O) (pH adjusted to about 9.0).

[0063] Furthermore, the culture conditions were: 30 degrees, 200 r / min, and 48 hours.

[0064] Table 1 Growth curve index growth rate table

[0065]

[0066]

[0067] In step 4, after the fermentation is completed, 1 mL of the fermentation broth of the mutant bacteria is taken, the culture medium is removed by centrifugation, clean water is added, and 30-50 μL of Nile Red fluorescent staining solution is additionally added for staining for 30 minutes. After the staining is completed, the broth is placed in a fluorescent microplate (96-well plate) and the fluorescence intensity is detected under the conditions of emitting light at 530-530 nm and receiving light at 600-605 nm to indirectly characterize the PHA production value;

[0068] It was found that the fluorescence intensity of strains 30 and 35 was significantly higher than that of starting strain 1. They were selected as subsequent test strains for the next step.

[0069] Next, 10-20 ml of fermentation broth from strains 30 and 35, the top two with the highest fluorescence values, was centrifuged and freeze-dried at 8,000-10,000 rpm for 10 minutes, repeated three times, and rinsed with ddH₂O. After freeze-drying, 20-40 mg of the cells were ground and esterified for GC analysis to determine the final yield. The mutant with the highest yield was designated the optimal mutant.

[0070] Nile red fluorescence test results Figure 4 It was found that the fluorescence changes of strains 26, 30, and 35 were the most obvious among the five selected mutant strains. Based on all the data, strains 30 and 35 were selected for subsequent GC yield detection.

[0071] GC test results Figure 5 The highest-yielding mutant strain, strain 35, was found to be the best mutant. Its yield was 27% higher than that of the starting strain, strain 1. This indicates that this method is effective in screening positive, effective, and high-yielding mutant PHA strains.

[0072] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing polyhydroxyalkanoate engineered bacteria, characterized in that: include Step 1: Select a PHA-producing strain and inoculate it into the culture medium for amplification culture on a shaking platform; Step 2: After the culture is completed, a low-temperature plasma mutagenizer is used for mutation breeding; Step 3: Screen and isolate excellent PHA-producing mutant strains to construct a high-yield PHA mutant strain library; Step 4: Perform composite screening on the mutant bacterial library after mutagenesis to select strains with high PHA production.

2. The method for preparing a polyhydroxyalkanoate engineering bacterium according to claim 1, characterized in that: The PHA production strain in step 1 is any one of the genus Ralstonia, Bacillus, Pseudomonas or Halomonas.

3. The method for preparing a polyhydroxyalkanoate engineering bacterium according to claim 2, characterized in that: The mutagenesis conditions of step 2 are: using a low-temperature plasma mutagenizer with a power of 6-8W, a frequency of 20-40Khz, and an electrode distance of 3-5cm from the bacterial liquid slide for 10-150s.

4. The method for preparing a polyhydroxyalkanoate engineering bacterium according to claim 3, characterized in that: Step 3: Select a strain with a mortality rate of 96-99%.

5. The method for preparing a polyhydroxyalkanoate engineering bacterium according to claim 4, characterized in that: In step 4, Nile red fluorescence staining combined with 96-well plate high-throughput screening was used for compound screening.

6. A polyhydroxyalkanoate engineered bacterium prepared by the method according to any one of claims 1 to 5.

7. Use of the polyhydroxyalkanoate engineered bacteria according to claim 6 in the production of polyhydroxyalkanoate.

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