CN120683092A

CN120683092A - A polyhydroxyalkanoate engineered bacteria and its preparation method and application

Info

Publication number: CN120683092A
Application number: CN202510813454.9A
Authority: CN
Inventors: 钟近艺; 钟奶才; 潘文锋; 陈缘; 廖景文; 翟英雷
Original assignee: Guangzhou Institute Of Advanced Technology
Current assignee: Guangzhou Institute Of Advanced Technology
Priority date: 2025-06-18
Filing date: 2025-06-18
Publication date: 2025-09-23

Abstract

The invention relates to the technical field of microorganisms, in particular to polyhydroxyalkanoate engineering bacteria, a preparation method and application thereof. The preparation method comprises the steps of 1, selecting PHA production strains, inoculating the PHA production strains to a culture medium, carrying out shake amplification culture, 2, carrying out mutation breeding by adopting a low-temperature plasma mutagenesis instrument after the culture is completed, 3, screening and separating excellent PHA-producing mutant strains to construct a high-yield PHA mutant strain library, and 4, carrying out compound screening on the mutagenized mutant strain library, and selecting strains with high yield of PHA. The invention provides a method for constructing engineering bacteria for producing PHA by adopting a novel small plasma mutagenesis instrument and application thereof, and the engineering bacteria for high-yield PHA are constructed and screened by low-temperature plasma mutagenesis so as to improve PHA yield and provide a new path for producing PHA as a degradable biological material.

Description

Polyhydroxyalkanoate engineering bacterium, and preparation method and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to polyhydroxyalkanoate engineering bacteria, a preparation method and application thereof.

Background

Polyhydroxyalkanoates (PHA) are a class of degradable polymeric polyesters synthesized by a variety of microorganisms under conditions of sufficient carbon source and a lack of other essential nutrients. Similar to the biosynthesis of rhamnolipids produced by pseudomonas or burkholderia, PHA materials are excellent in biocompatibility and environmental friendliness, and can be classified into short-chain and/or medium-long chain types, including homopolymers and copolymers, and are typically stored in the cytoplasm in the form of insoluble particles of about 0.2 to 0.7 μm in diameter.

By virtue of biodegradability and mechanical properties, PHA is not only regarded as an environment-friendly substitute for traditional petroleum-based plastics, but also has wide application prospect in the biomedical field. Industrially, PHA-enriched strains are obtained by adopting a microbial fermentation technology, and then the PHA-enriched strains are refined and purified to prepare high-purity products. The medical property is mainly characterized in the following aspects:

PHA material can be used for constructing biocompatible scaffold, supporting cell adhesion and proliferation, and providing ideal microenvironment for tissue repair and regeneration.

The controlled release and carrier system of the medicine benefits from the controllable degradation characteristic, the PHA can be prepared into a slow release medicine carrier, so that the accurate and continuous release of the medicine in the body is realized, the curative effect is ensured, and the side effect is reduced.

Polyhydroxybutyrate (PHB) is an important member of PHA family, and its excellent biocompatibility and slow release property can make it widely used in absorbable suture lines, implants and other medical instruments, and its degradation products are harmless to human body and environment.

In a word, by virtue of excellent biological safety, the PHA material has wide application prospect in the fields of medicine, tissue engineering, medical equipment, medicine controlled release and the like, and provides powerful technical support for the green sustainable development of modern medical materials.

At present, PHA production is mainly realized through strain fermentation engineering and metabolic engineering, and a plurality of methods and processes are provided. The key to PHA production is the strain performance, which directly determines the production efficiency, process stability, and quality of the final product. Screening and breeding of high PHA-producing strains has been a key step in improving process performance. Common methods include strategies such as genetic engineering, artificial physical mutagenesis, chemical mutagenesis, etc., to improve PHA synthesis efficiency by optimizing metabolic pathways and regulating gene expression. The production strains commonly used at present (such as the genus Ralstonia, bacillus, pseudomonas and Salmonella). For example, CN 113583922A discloses a comprehensive method for constructing efficient PHA engineering bacteria by utilizing genetic engineering transformation and mutation screening, which mainly utilizes ARTP to mutagenize to obtain a halomonas with low salt concentration growth, and then carries out combined genetic molecular transformation to improve PHA production.

CN118516416a discloses that reducing the expression level of cytochrome d oxidase complex gene cydA or the activity of its coded protein can significantly reduce the requirement of halomonas for dissolved oxygen in the late fermentation stage, and can realize that the strain can still perform PHA synthesis efficiently under the conditions of low rotation speed and even stirring-free hypoxia or anaerobic in the late fermentation stage, thereby effectively improving the yield of PHA. However, these methods have focused on promoting PHA production based on genetic engineering, and the use of novel methods has focused on reducing their growth adaptation requirements rather than modifying strains by using methods such as plasma as a major enhancement means.

Low temperature plasma, which can generate large amounts of high energy active particles (RONS) at room temperature, is a promising physical mutagenesis approach. The plasma is also called a fourth state of a substance, is a state when gas is partially ionized or completely ionized after being excited by high voltage, mainly comprises components such as free electrons, atoms, molecules and the like in neutral, ionized and excited states, can effectively act on DNA molecules of microorganisms, and causes DNA chain breakage or base damage to cause gene mutation. Further triggering SOS repair mechanism of strain, generating various mismatch sites, forming mutation and finally generating rich mutation library through stable inheritance

Disclosure of Invention

In order to comprehensively solve the problems, the invention provides a method for constructing engineering bacteria for producing PHA by adopting a novel small plasma mutagenesis instrument and application thereof, and the engineering bacteria for high-yield PHA are constructed and screened by low-temperature plasma mutagenesis so as to improve the PHA yield and provide a new path for the production of PHA as a degradable biological material.

In order to achieve the above object, a first aspect of the present invention provides a method for producing polyhydroxyalkanoate engineering bacteria, comprising

Step 1, selecting PHA production strains, inoculating the PHA production strains to a culture medium, and performing shake amplification culture;

step2, after the culture is completed, adopting a low-temperature plasma mutagenesis instrument to carry out mutagenesis breeding;

step 3, screening and separating excellent mutant strains for producing PHA, and constructing a high-yield PHA mutant strain library;

And 4, carrying out compound screening on the mutant bacteria library after mutagenesis, and selecting strains with high PHA yield.

Preferably, the PHA-producing strain of step 1 is any one of the genera Ralstonia, bacillus, pseudomonas or Salmonella.

Preferably, the mutagenesis conditions of step 2 are: under the conditions that the power of the low-temperature plasma mutagenesis instrument is 6-8W, the frequency is 20-40Khz, and the distance between an electrode and a bacterial liquid slide is 3-5cm, the action is 10-150s.

Preferably, the strain with the death rate of 96-99% is selected in the step 3.

Preferably, the step 4 compound screening is carried out by combining nile red fluorescent staining with 96-well plate high-throughput screening.

The second aspect of the invention provides polyhydroxyalkanoate engineering bacteria prepared by the method.

The third aspect of the invention provides the application of the polyhydroxyalkanoate engineering bacteria in the production of polyhydroxyalkanoate.

Compared with the prior art, the invention has the beneficial effects that:

1. The novel small plasma mutagenesis equipment is adopted in the mutagenesis ring joint, and key parameters such as power (6-8W), frequency (20-40 KHz), acting distance (3-5 cm), acting time (10-150 s) and the like are defined.

The specific mutagenesis conditions are carried out in the atmospheric environment, so that the mutagenesis intensity and time can be precisely controlled, and better strain activity can be maintained while higher mutation rate is ensured. The mutagenesis efficiency and repeatability are higher.

2. According to the invention, after grouping different mutation intensities, the death rate of the strain is counted, and the group with the death rate of 96-99% is selected for constructing the high-yield PHA mutant bacteria library. The strategy can give consideration to the balance point of 'enough mutation amplitude' and 'still keeping viable individuals', and greatly improves the occurrence probability of subsequent high-yield strains. In the prior art, the mutagenesis time is often adjusted only empirically or simply, but accurate screening aiming at mortality is lacking, so that the acquisition efficiency of high-yield mutant strains is low.

3. In the composite screening stage, the invention adopts a Nile red fluorescent staining method, and carries out high-flux quantitative screening on bacterial strains in a 96-well plate by means of the fluorescent detection function of an enzyme-labeled instrument. Compared with the traditional mode of firstly culturing and then extracting and then detecting PHA content, the method can rapidly and semi-quantitatively evaluate a large number of strains on a culture scale (96-well plate) with relatively early and relatively small volume, and the fluorescent and high-flux compound screening technology which greatly saves screening time and cost provides important guarantee for rapidly and accurately locking high-yield strains.

4. The invention considers PHA synthesis amount and takes the growth speed of the first 12 hours and 48 hours as an important index. Through comprehensive evaluation of growth speed and PHA fluorescence value, mutant strains with rapid growth and high PHA yield can be selected, so that the method has more practical value in subsequent fermentation scale amplification. Compared with the existing single PHA yield index screening, the compound screening mode remarkably improves the adaptability of the strain in the industrialized production environment.

5. After fluorescence detection of the locked candidate strain, final quantitative confirmation of PHA content and composition is carried out by adopting GC detection through the steps of centrifugation, freeze-drying, esterification and the like. The mode of double detection (fluorescence pre-screening and GC quantification) ensures the efficiency of high-throughput screening, and can confirm the yield and the components of PHA through accurate chemical analysis, thereby reducing the risk of false positive or screening omission to the greatest extent.

Therefore, the invention carries out targeted improvement and combination in the aspects of low-temperature plasma mutagenesis equipment and parameter design, mortality control construction of a mutant bacteria library, a high-throughput fluorescence screening method, final accurate quantitative detection and the like, and solves the problems of low mutagenesis and screening efficiency, long period, uncontrollable process and the like of the traditional mutagenesis and screening. It is these "differential structures or steps" that together form the innovative core of the present invention, which makes a decisive contribution to the rapid and efficient acquisition of engineering bacteria that stably produce PHA in high yield.

Drawings

The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention.

In the drawings:

FIG. 1 is a chart of the mortality observations of coated plates;

FIG. 2 is a death curve;

FIG. 3 is a 45h growth curve;

FIG. 4 is a graph of the results of Nile red fluorescence detection;

FIG. 5 is a graph of GC detection results;

fig. 6 is a flow chart of the method of the present invention.

Detailed Description

The following description of the preferred embodiments of the present invention is provided in conjunction with fig. 1-6, it being understood that the preferred embodiments described herein are provided to illustrate and explain the present invention, and are not intended to limit the present invention.

Example 1:

Polyhydroxyalkanoate engineering bacteria are prepared by the method of example 2.

Example 2:

a method for preparing polyhydroxyalkanoate engineering bacteria comprises

Step 1, selecting PHA production strains, inoculating the PHA production strains to a culture medium, and carrying out shake amplification culture. The PHA producing strain is any one of genus Ralstonia, genus Bacillus, genus Pseudomonas or genus Salmonella.

In the examples, halomonas sp (available from China center for type culture Collection of microorganisms, no. CICC 24456) was inoculated onto a medium and subjected to a shaking table amplification culture.

Further, the culture medium is LB broth culture medium, and the formula of the culture medium comprises 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride, pH of 7.0-7.4 and the balance of water, and 5% -7% of sodium chloride is additionally added under the condition of halomonas.

Further, the medium was sterilized at 121℃for 20 minutes, and cooled to room temperature for use.

Further, the inoculation amount is 5%,30 degrees, 200r/min culture is carried out for 12 hours to logarithmic phase, and OD is more than 3.

Further, the shake culture can be stopped when the microorganism is cultured until the OD ₆₀₀ is more than 3. The cultured microorganism was diluted with 6% sodium chloride brine to OD ₆₀₀ =1

When in mutation breeding, a small iron sheet of a circular sheet with the stainless steel sheet phi of 10mm is placed under an alcohol lamp for high-temperature sterilization, and then cooled and kept stand for 10 minutes for standby. And uniformly coating 10ul of bacterial liquid on a small sterile iron sheet, slightly airing, and placing the small sterile iron sheet under a low-temperature plasma source in a plasma mutagenesis instrument for mutagenesis.

According to the operation flow of the low-temperature plasma mutagenesis instrument, the breeding condition is that the electrode is under the condition of 3-5cm away from the bacterial liquid slide glass under the conditions of 6-8W of power and 20-40Khz of frequency, and the action is 10-150s.

In the atmospheric pressure glow discharge process, a low-temperature plasma source generates a large number of active particles (including free electrons, oxygen free radicals, nitrogen free radicals and the like) which can cause the fungal DNA structure to be damaged in a diversity way, so that a large number of mutation sites are formed.

And 3, screening and separating excellent mutant strains for producing PHA, and constructing a high-yield PHA mutant strain library. Specific:

The suspension after mutagenesis in step 2 was diluted to a gradient and plated on LB60 plates, under which conditions the mortality and mutation rate results of the strain Halomonas sp were calculated. (mortality = (number of colonies before glow discharge treatment-number of colonies after glow discharge treatment/number of colonies before glow discharge treatment) = 100%, mutation = (number of mutant colonies/number of colonies after glow discharge treatment) = 100%).

Under the condition of gradient mutagenesis time, a plurality of mutant strains can be obtained by comparing the color depth and the radius of the bacterial colony and the color of the original strain. The results were observed in FIG. 1, mortality graph 2, and mortality and mutation rates were found to be higher at 100s and 120s compared to the other groups, and a significant difference in colony morphology was observed for many compared to the starting strain. The group was selected as a treatment group to construct a mutant library for screening for mutants.

Selecting 48 mutant strains with obvious morphological difference, placing the mutant strains in a 48-hole deep hole plate containing 1ml of LB60, culturing for 12h at 30 degrees and 200r/min, respectively diluting the cultured mutant strains by 2 x 10 ² times, adding the diluted mutant strains into a 96-hole ELISA plate, and detecting the growth curve of the mutant strains under the incubation mode of an ELISA instrument at 30-36 degrees, medium or high shaking speed and 45-56 h.

The growth curve data of 48 mutant bacteria are analyzed and calculated, and indexes such as average growth speed, total time growth speed and the like of each mutant bacteria shown in table 1 are comprehensively selected from 4-8 mutant bacteria and a growth curve of figure 3 for fermentation culture of a fermentation medium, wherein 15 strains with the most excellent growth in the first 12 hours and 26, 27, 30 and 35 mutant strains with the most excellent growth in the second 45 hours are selected for fermentation production detection.

Further, the general formulation of the fermentation medium is :0.1‰-2‰(NH₄)₂CL,0.1‰-1‰MgSO₄,5‰-10‰Na₂HPO₄·12H₂O,0.5‰-2‰KH₂PO₄, to 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 trace) (pH adjusted to about 9.0).

Preferably 0.1% (NH ₄)₂SO₄ or 0.2% urea, 0.02% mgso ₄,

1.0% Na ₂HPO₄·12H₂O,0.15％KH₂PO₄, no more than 0.1% other trace elements (Fe(III)-NH₄-Citrate,CaCl₂·2H₂O,ZnSO₄·7H₂O,MnCl₂·4H₂O,H₃BO₃,CoCl₂·6H₂O,CuSO₄·5H₂O,NiCl₂·6H₂O,NaMoO₄·2H₂O)(pH to about 9.0).

Further, the culture conditions were 30℃and 200r/min for 48 hours.

TABLE 1 growth curve index growth rate Table

In the step 4, after fermentation is completed, 1mL of mutant bacteria fermentation liquor is taken, after a culture medium is removed by centrifugation, clear water is added, 30-50ul of nile red fluorescent staining liquor is additionally added for staining for 30min, after the staining is completed, the obtained product is placed in a fluorescent ELISA plate (96-well plate), and the PHA production value is indirectly represented by detecting the fluorescence intensity under the fluorescent condition of light emission of 530-530nm and light reception of 600-605 nm;

The fluorescence intensity of strains 30 and 35 was found to be significantly improved compared with the starting strain 1. The strain was selected as a subsequent test strain for the next step.

Next, 10-20ml of fermentation broths of the first 2 strains 30 and 35 strains with the highest fluorescence values are selected and subjected to centrifugal freeze-drying, and 10mins at 8000-10000rpm are repeated three times and washed with ddH ₂ O. And (3) performing grinding esterification reaction on 20-40mg of thalli after freeze-drying, determining the final yield by GC detection, and taking the mutant strain with the highest yield as the best mutant strain.

Nile red fluorescence detection results FIG. 4 shows that the fluorescence changes of 26, 30 and 35 strains among the five selected mutant strains are most obvious, and the 30 and 35 strains are selected based on all data for subsequent GC yield detection.

GC test results FIG. 5 shows that the highest yield mutant strain No. 35 is the best mutant. The yield is increased by 27% compared with the yield of the starting strain 1. The method is proved to effectively screen out positive efficient high-yield mutant PHA strains.

The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. A method for preparing polyhydroxyalkanoate engineering bacteria, characterized in that it comprises

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 engineered bacterium according to claim 1, wherein the PHA-producing strain in step 1 is any one of the genus Ralstonia, Bacillus, Pseudomonas or Halomonas.

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

4 . The method for preparing polyhydroxyalkanoate engineered bacteria according to claim 3 , wherein in step 3, a strain with a mortality rate of 96-99% is selected.

5. The method for preparing a polyhydroxyalkanoate engineered bacterium according to claim 4, wherein the composite screening in step 4 is performed by combining Nile red fluorescence staining with 96-well plate high-throughput 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.