A recombinant bacteria and method for reducing fermentation foaming

Abstract

The present invention relates to the field of microbial technology, and in particular to a recombinant bacterium and a method for reducing fermentation foaming. The present invention significantly reduces foaming and liquid escape during fermentation production with oil as the main substrate by overexpressing a lipase gene within a specific intensity range; the foaming and liquid escape of the recombinant bacterium constructed in this way are significantly reduced during the fermentation process, which is beneficial to improving the production application value of the recombinant bacterium and promoting fermentation amplification and stable production increase, and has good application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a recombinant bacterium and a method for reducing fermentation foaming. Background Art

[0002] During the fermentation process of most microorganisms, due to the presence of protein surfactants in the culture medium, foam will form in the culture medium under ventilation conditions, which is fermentation foaming. Foaming will bring many unfavorable factors, such as reduced charging coefficient of the fermentation tank and reduced oxygen transfer coefficient; excessive foam may even cause a large amount of liquid to escape, and the fermentation liquid will escape from the exhaust pipe or shaft seal, increasing the chance of contamination. In severe cases, ventilation and stirring cannot be carried out, and bacterial respiration is hindered, leading to abnormal metabolism or bacterial autolysis. Therefore, controlling fermentation foaming is a basic condition to ensure normal fermentation and production practice.

[0003] Polyhydroxyalkanoates (PHA) are a class of high molecular weight polyesters synthesized by microorganisms. They have good biodegradability and can replace traditional plastics in a variety of scenarios. Ralstonia eutropha , also known as Cupriavidus necator ) is a bacterium of the genus Ralstonia, an important model bacterium for studying PHA synthesis, and also the most studied strain for PHA industrial production. Commonly used E. rhodochii H16 and its derivative strains can be used as a platform chassis to achieve high cell density batch fermentation under industrial conditions, using biomass raw materials such as sugars and oils as substrates to efficiently produce molecular products including different types of PHA.

[0004] In the late fermentation stage of E. rothenbergii using oil as substrate, a large number of bubbles will be generated in the fermentation system, which has a high risk of liquid escape and spray tank. Therefore, it is necessary to control the fermentation foaming to ensure normal fermentation and that the fermentation strain can be effectively used in production practice. For E. rothenbergii, there are currently few reports on the use of genetic engineering to modify strains to improve the problem of fermentation foaming. If the fermentation foaming can be reduced from the strain itself, it is of great significance to reduce the risk of liquid escape and ensure the normal progress and stability of fermentation production. Summary of the invention

[0005] The invention provides a recombinant bacterium and a method for reducing fermentation foaming.

[0006] Specifically, the present invention provides the technical solution described below.

[0007] In a first aspect, the present invention provides a recombinant bacterium, wherein the recombinant bacterium is modified so that the expression level of lipase therein is increased to 2-12 times that before modification; The recombinant bacteria is a Ralstonia bacterium.

[0008] The foaming and / or liquid escape of the recombinant bacteria during the fermentation process is reduced due to the modification; wherein the fermentation substrate includes oil.

[0009] The present invention found that when fermentation production is carried out with oil as the main substrate, a large number of bubbles will be generated in the fermentation system in the late fermentation period. This may be due to the large biomass in the late fermentation period, the relatively viscous fermentation liquid, the slow fatty acid metabolism and the overall fat metabolism, and the accumulation of a large amount of fatty acids in the fermentation system. In response to this problem, the present invention attempts to reduce fermentation foaming and liquid escape by genetically modifying the strain, and unexpectedly found that fermentation foaming and liquid escape can be significantly reduced by increasing the lipase expression level of the strain. Moreover, the higher the expression level of lipase is, the better. On the contrary, excessive lipase expression will lead to a significant increase in foaming during the fermentation process (especially in the early stage of fermentation); therefore, it is necessary to maintain the expression level of lipase at the above-mentioned specific level. Lipase is an enzyme that catalyzes fat hydrolysis reactions and can decompose fat into glycerol and fatty acids. Increasing the expression of lipase will promote the production of fatty acids. However, according to common knowledge in the art, the more fatty acids accumulate in the fermentation system, the more serious the fermentation foaming phenomenon will be. However, the present invention has found through experimental research that increasing the expression of lipase in the strain within a specific range will still promote the decomposition of fat in the fermentation system into fatty acids, thereby increasing the production of fatty acids, but it can significantly reduce the occurrence of fermentation foaming and liquid escape, achieving unexpected results.

[0010] In addition, increasing the lipase expression level of the strain within the above range can also significantly improve the PHA fermentation production performance of the recombinant bacteria.

[0011] Preferably, the recombinant bacteria is modified so that the expression level of lipase therein is increased to 5-12 times, more preferably 8-12 times, and more preferably 9-11.5 times that before modification.

[0012] In some specific embodiments of the present invention, the recombinant bacteria is modified so that the expression level of lipase therein is increased to 11 times that before modification.

[0013] Preferably, the PHA fermentation production performance of the above-mentioned recombinant bacteria is improved due to the modification.

[0014] Wherein, the PHA fermentation production performance includes PHA yield, PHA production intensity, etc.

[0015] In the present invention, the fermentation preferably uses fat as the main substrate. Using fat as the main substrate preferably means that fat accounts for at least 50% of the substrate, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%.

[0016] In some embodiments of the present invention, the fermentation uses oil as a substrate.

[0017] The present invention has no special restrictions on the types of oils and fats, and all oils and fats that can be metabolized by Eutropha rosea are applicable, including but not limited to vegetable oils, waste cooking oils, edible oils, etc. The vegetable oils include but are not limited to a mixture of one or more of palm oil, palm kernel oil, peanut oil, soybean oil, linseed oil, rapeseed oil, cottonseed oil, castor oil, and corn oil.

[0018] In the present invention, the increase in the expression level of the lipase is achieved by one or more of the following methods: (1) Increasing the copy number of lipase encoding genes; (2) Enhance the activity of transcriptional and / or translational regulatory elements of lipase encoding genes.

[0019] The above-mentioned transcription regulatory elements include promoters, enhancers, etc. Translation regulatory elements include ribosome binding sites, etc.

[0020] In the above (1), increasing the copy number of the lipase encoding gene can be achieved by introducing an expression plasmid containing the lipase encoding gene into the strain, or by increasing the copy number of the lipase encoding gene on the chromosome.

[0021] In the above (2), enhancing the activity of the transcription and / or translation regulatory elements of the lipase encoding gene can be achieved by replacing the transcription and / or translation regulatory elements of the lipase encoding gene with transcription and / or translation regulatory elements with stronger activity. For example, the promoter of the lipase encoding gene is replaced with a promoter with stronger activity.

[0022] The above-mentioned increase in the expression level of lipase can be achieved by gene editing methods commonly used in the art.

[0023] In some specific embodiments of the present invention, the increased expression level of the lipase is achieved by initiating the transcription of the lipase encoding gene with a medium strength constitutive promoter.

[0024] Those skilled in the art will appreciate that, for a specific bacterium, the range of activity strength of a medium-strength promoter is clear, and those skilled in the art can determine which promoters are medium-strength promoters based on the activity of currently known naturally existing or artificially synthesized promoters for Eutropha rosea.

[0025] In some specific embodiments of the present invention, the nucleotide sequence of the medium strength constitutive promoter is shown as SEQ ID NO.3.

[0026] The above-mentioned initiation of transcription of the lipase encoding gene with a medium strength constitutive promoter can be achieved by inserting a medium strength constitutive promoter before the lipase encoding gene, or replacing the original promoter of the lipase encoding gene with a medium strength constitutive promoter.

[0027] The lipase described in the present invention may be an endogenous lipase of the recombinant bacteria, or may be a lipase derived from other species, such as a lipase derived from bacteria of the same genus.

[0028] Preferably, the lipase is a lipase derived from a Ralstonia bacterial source.

[0029] In some embodiments of the present invention, the Ralstonia bacterium is Ralstonia eutropha.

[0030] In some embodiments of the present invention, the lipase is a lipase derived from Eutropha rosea.

[0031] For example, the accession number of the lipase of Eutropha rosea is H16_A1322 (lipA), and H16_A1323 (lipB) is used as a molecular chaperone protein, and H16_A1322 and H16_A1323 are co-expressed.

[0032] In some embodiments of the present invention, the amino acid sequence of the lipase is shown as SEQ ID NO.1.

[0033] The present invention overexpresses the endogenous lipase gene of Eutropha rosea and controls the expression level of the lipase gene within the above-mentioned specific range, thereby effectively reducing fermentation foaming and liquid escape in the process of producing PHA by fermentation with oil as the main substrate, and at the same time improving the yield and production intensity of PHA.

[0034] In some embodiments of the present invention, a recombinant bacterium producing PHA is provided, wherein the promoter of the lipase encoding gene shown in SEQ ID NO.1 on the chromosome of the recombinant bacterium is replaced by the promoter shown in SEQ ID NO.3.

[0035] In a second aspect, the present invention provides the use of the recombinant PHA-producing bacteria in PHA fermentation production or construction of PHA-producing bacteria.

[0036] (1) Fermentation production using oil as substrate; (2) Constructing production bacteria that use oil as substrate for fermentation production; (3) PHA fermentation production; (4) Construction of PHA-producing bacteria.

[0037] In the applications described in (1) and (2) above, there is no special restriction in principle on the target product produced by fermentation with oil as substrate. The strategy of reducing foaming and liquid escape in fermentation with oil as substrate by increasing the expression level of lipase within a specific range in the present invention can be applied to different target products, not limited to the exemplary verified PHA.

[0038] The recombinant bacteria provided by the present invention can be directly used as a fermentation strain for fermentation production using oil as a substrate, and can also be used as a starting strain for breeding production bacteria for fermentation production using oil as a substrate, and the production bacteria can be constructed through further genetic engineering modification, mutagenesis and other strain breeding techniques.

[0039] In a third aspect, the present invention provides an application of increasing the expression level of lipase in reducing foaming and / or liquid escape in fermentation production of Ralstonia bacteria using fat as a substrate; The application comprises: modifying the bacteria of the genus Ralstonia so that the expression amount of lipase therein is increased to 2-12 times of that before modification.

[0040] Preferably, the Ralstonia bacterium is modified so that the expression level of lipase therein is increased to 5-12 times, more preferably 8-12 times, and more preferably 9-11.5 times that before modification.

[0041] In some specific embodiments of the present invention, the Ralstonia bacterium is modified so that the expression level of lipase therein is increased to 11 times that before modification.

[0042] In a fourth aspect, the present invention provides an application of increasing the expression level of lipase in improving the fermentation production performance of Ralstonia bacteria in producing PHA using oil as a substrate; The application comprises: modifying the bacteria of the genus Ralstonia so that the expression amount of lipase therein is increased to 2-12 times of that before modification.

[0043] Preferably, the fermentation production performance includes the yield and / or production intensity of PHA.

[0044] Preferably, the amino acid sequence of the lipase is as shown in SEQ ID NO.1; Preferably, the Ralstonia bacterium is Ralstonia eutropha.

[0045] In a fifth aspect, the present invention provides a method for reducing fermentation foaming and / or liquid escape, the method comprising: modifying a fermentation strain so that the expression level of lipase therein is increased to 2-12 times that before modification; the fermentation strain is a Ralstonia bacterium.

[0046] Preferably, the fermentation strain is modified so that the expression level of lipase therein is increased to 5-12 times, more preferably 8-12 times, and more preferably 9-11.5 times of that before modification.

[0047] In some specific embodiments of the present invention, the fermentation strain is modified so that the expression level of lipase therein is increased to 11 times that before modification.

[0048] In a sixth aspect, the present invention provides a method for improving the fermentation production performance of PHA, the method comprising: modifying a PHA-producing bacterium so that the expression level of lipase therein is increased to 2-12 times that before modification; the PHA-producing bacterium is a Ralstonia bacterium.

[0049] In a seventh aspect, the present invention provides a method for reducing fermentation foaming and / or liquid escape while improving the fermentation production performance of PHA, the method comprising: modifying the PHA-producing bacteria so that the expression level of lipase therein is increased to 2-12 times that before modification; the PHA-producing bacteria is a Ralstonia bacterium.

[0050] In the above method, the bacterium of the genus Ralstonia is preferably Eutropha rhodochii. The amino acid sequence of the lipase is preferably as shown in SEQ ID NO.1.

[0051] The beneficial effects of the present invention include at least: the present invention significantly reduces the foaming and liquid escape during fermentation production with oil as the main substrate by overexpressing the lipase gene within a specific intensity range; the foaming and liquid escape of the recombinant bacteria constructed in this way are significantly reduced during the fermentation process, which is beneficial to improving the production and application value of the recombinant bacteria and promoting fermentation amplification and stable production increase. In addition, the biomass, PHA yield and production intensity of the recombinant bacteria are also significantly improved, which is beneficial to reducing the cost of PHA industrial production, and is of great significance for improving the competitiveness of PHA in the traditional plastic and bio-based degradable plastic markets and its commercial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0053] Figure 1 The results of thin layer chromatography of the recombinant strain L01 and the control strain H16 in the embodiment of the present invention; wherein, the standard solution is 0.5% palm oil, 1% palm oil, 0.5% oleic acid, and 1% oleic acid, and the fermentation broth of the H16 strain and the L01 strain are both taken after 54h of fermentation.

[0054] Figure 2 The lipase expression of the recombinant strain L01 in the example of the present invention relative to the control strain H16; t-test statistical test: p<0.05, indicated by *; p<0.01, indicated by **; p<0.001, indicated by ***, p<0.0001, indicated by ****. DETAILED DESCRIPTION

[0055] In a specific embodiment of the present invention, a recombinant strain L01 is provided, which is a medium-strength constitutive promoter added before the gene coding region of Eutropha roqueforti H16_A1322. The lipase expression of the L01 strain is stably increased to 11 times the original expression at the genome level, thereby increasing the utilization of the oil substrate during the fermentation process, significantly improving the stability of the fermentation liquid during the fermentation process, effectively reducing the fermentation foaming in the late fermentation process, and reducing the risk of spray tanks caused by fermentation foaming, which is conducive to fermentation amplification, production application and stable production increase. In addition, the recombinant strain L01 has outstanding performance in biomass, PHA yield, and production intensity indicators, which is conducive to reducing the cost of PHA production.

[0056] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0057] The materials and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources. The kit used to extract plasmids was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., the kit used to extract RNA was purchased from Shenggong Biotechnology (Shanghai) Co., Ltd., the kit used to reverse transcribe RNA into cDNA and the fluorescent quantitative PCR reagents were purchased from Takara bio, and the corresponding operation steps were strictly carried out in accordance with the product instructions. All culture media were prepared with deionized water unless otherwise specified.

[0058] The culture medium formula used in the following examples is as follows: Seed medium I: 10 g / L peptone, 5 g / L yeast extract, 3 g / L glucose.

[0059] Seed medium II: 0.15% palm oil, 10 g / L peptone, 5 g / L yeast extract.

[0060] Production medium: 1.0% palm oil, 9.85g / L Na(2)HPO(4)·12H(2)O, 1.5g / L KH(2)PO(4), 3.0g / L NH(4)Cl, 10mL / L trace element solution I and 1mL / L trace element solution II. The composition of trace element solution I is: 20g / L MgSO(4), 2g / L CaCl(2). The composition of trace element solution II is: 100 mg / L ZnSO(4)·7H(2)O, 30 mg / L MnCl(2)·4H(2)O, 300 mg / L H(3)BO(3), 200 mg / L CoCl(2)·6H(2)O, 10 mg / L CuSO(4)·5H(2)O, 20 mg / L NiCl(2)·6H(2)O, 30 mg / L NaMoO(4)·2H(2)O. All the above reagents were purchased from Sinopharm Chemical Reagent Company.

[0061] The experimental data in the following examples were obtained from 3 or more parallel group experiments.

[0062] The calculation formula for the PHA yield described in the following examples is as follows: PHA yield = CDW × PHA%; where CDW is cell dry weight (biomass) and PHA% is the percentage of PHA in cell dry weight.

[0063] The calculation formula of the production intensity described in the following examples is as follows: Production intensity = PHA output / time; the PHA output is calculated as described above, and the time is the whole fermentation process time of 54h.

[0064] Example: Construction and performance testing of a recombinant strain with a medium-strength promoter inserted in front of the lipase gene In this embodiment, the H16 strain of Eutropha roeblingii (H16 for short) was used as the starting strain, and a medium-strength constitutive promoter (SEQ ID NO: 31 of patent CN108977890 B, hereinafter referred to as p31) was added in front of the gene coding region of H16_A1322 (sequence as shown in SEQ ID NO.2) using a common gene editing method in the art. The promoter element obtained by the above construction was inserted into the recombinant strain Re-p31-H16_A1322 and named Eutropha roeblingii L01 (recombinant strain L01), and the recombinant strain was further tested for performance. The specific strain construction process and experimental results are described as follows.

[0065] Step 1: Construction of Eutropha rosea L01 1.1 The upstream homology arm A1322-H1 and downstream homology arm A1322-H2 of H16_A1322 were obtained by PCR amplification using the genome of Eutropha rosenbergii H16 as a template; the vector fragment was obtained by PCR amplification using the modified plasmid pK18mob (Orita I, Iwazawa R, Nakamura S, et al. Identification of mutation points in Cupriavidus necatorNCIMB 11599 and genetic reconstitution of glucose-utilization ability in wildstrain H16 for polyhydroxyalkanoate production[J]. Journal of Bioscience&Bioengineering, 2012, 113(1):63-69) as a template. A1322-H1 and A1322-H2 were connected to the vector fragment by the Gibson Assembly method to obtain the basic plasmid pKO-A1322. The subcloning construction of the edited plasmid was completed by WuXi Biologics Co., Ltd. The sequences of homology arms A1322-H1 and A1322-H2 are shown in SEQ ID NO.4 and SEQ ID NO.5.

[0066] 1.2 Gene synthesis The constitutive promoter p31 (SEQ ID NO.3) was synthesized, and GGTCTCATCGT (SEQ ID NO.7) was added upstream and ACGCAGAGACC (SEQ ID NO.8) downstream of the synthesized DNA sequence to facilitate subsequent operations.

[0067] 1.3 The above basic plasmid pKO-A1322 and the gene-synthesized constitutive promoter p31 were assembled using the Goldengate method to obtain the editing plasmid pKO-p31-A1322. The subcloning construction of the editing plasmid was completed by WuXi Biologics Co., Ltd. The editing plasmid pKO-p31-A1322 was transformed into Escherichia coli S17-1, and then transferred into E. rothrips H16 by conjugation transfer. Taking advantage of the fact that the suicide plasmid cannot replicate in the host bacteria, the positive clones were screened using LB plates containing 250 μg / mL kanamycin and 100 μg / mL apramycin. The recombinant plasmid carrying the homologous fragment in the positive clone was integrated into the specific position of the genome, thereby obtaining the first homologous recombinant strain. The first homologous recombinant strain was streaked onto an LB plate containing 100 mg / mL sucrose to culture single clones. Clones without kanamycin resistance were screened from these single clones and identified by PCR. Sequencing confirmed that the recombinant strain was correctly edited. The recombinant strain obtained was E. roeblingii Re-p31-H16_A1322 ， The present invention named Eutropha rosea L01 (recombinant strain L01).

[0068] Step 2: Detection of the main fatty acids (oleic acid) and fermentation performance indicators during the fermentation production process of Eutropha roqueforti L01 2.1 The Roebuck eutropha L01 and control strain H16 (1000 μL) prepared in step 1 above and stored in glycerol tubes were inoculated into seed culture medium I (20 mL) for 12 hours of primary seed culture to obtain seed culture solution I; then, seed culture solution I was inoculated into seed culture medium II (100 mL) at a volume ratio of 1 v / v% for secondary seed culture for 13 hours to obtain seed culture solution II; then, seed culture solution II was inoculated into a 500 mL small fermenter (Dibil Company) containing 250 mL of production culture medium at a volume ratio of 10 v / v%. The operating conditions were: culture temperature 30°C, stirring speed 800 rpm, ventilation (air) 1 L / min, and pH was controlled between 6.7 and 6.8. A 28% ammonia solution was used for pH control. During the culture process, the carbon source palm oil was supplemented by continuous flow feeding, and the variable speed feeding strategy was adopted to control the oil rate. The specific oil feeding strategy was: 0h~10h, 0mL / h; 10h~20h, 1mL / h; 20h~36h, the oil feeding rate increased by 0.2mL / h every 4h, 36h~44h maintained at 1.8mL / h, 44h~54h, every 2h the oil feeding rate decreased by 0.1mL / h. The culture time of the whole process fermentation was 54 hours, and no defoaming agent was added to the fermentation system.

[0069] 2.2 After 1 mL of fermentation liquid fermented for 54 hours was mixed with 0.2 mL of n-hexane by oscillation and centrifugation, the upper liquid was taken as the sample, and 0.5 v / v% palm oil, 1 v / v% palm oil, 0.5 v / v% oleic acid, and 1 v / v% oleic acid were used as standards (the standards were dissolved in n-hexane) for thin layer chromatography. The developing solvent system was 5 mL, the ratio was n-hexane: ethyl acetate: acetic acid = 10:1:1, and the color development method was vanillin sulfate ethanol colorimetric agent. The results are as follows Figure 1 As shown in the figure, the palm oil content in the fermentation broth of the H16 strain is between 0.5% and 1%, and the oleic acid content is less than 0.5%; the palm oil content in the fermentation broth of the L01 strain is less than 0.5%, and the oleic acid content is less than 0.5%, which is less than the oleic acid content in the fermentation broth of the H16 strain. The results show that the fermentation broth of the H16 strain contains a large amount of oleic acid, while the fermentation broth of the L01 strain contains a small amount of oleic acid. The higher the oleic acid content in the fermentation broth, the more serious the foaming may be.

[0070] 2.3 Record the amount of escaped liquid generated during the fermentation process. A large amount of fatty acids will accumulate in the fermentation system in the middle and late stages of fermentation, the fermentation liquid will be relatively viscous, and a large number of bubbles will be generated in the system. The fermentation liquid in the fermenter will be discharged to the escape bottle through the external pipe. The fermentation liquid in the escape bottle is poured into a 50mL measuring cylinder and the escaped liquid volume is recorded. After the fermentation is completed, the fermentation liquid in the fermenter is poured into a 500mL measuring cylinder and the tank volume is recorded. The following data (Table 1) show that the liquid volume of the L01 strain has reached the limit of the liquid volume of the fermenter, and the escaped liquid volume is significantly lower than that of the H16 strain. This result further shows that under the same oil replenishment amount and stirring speed, the bubble level in the L01 strain tank is much lower than that of the H16 strain, and the fermentation foaming of the L01 strain is significantly alleviated.

[0071] Table 1

[0072] Step 3: Determination of lipase expression during fermentation of Eutropha rosea L01 200 μL of the fermentation broth of strain L01 and the fermentation broth of control strain H16 fermented for 32 h in step 2 were taken for RNA extraction, reverse transcription into cDNA, and fluorescence quantitative PCR. The specific operations were performed according to the instructions of the kit. The fluorescence quantitative PCR instrument was purchased from Hangzhou Biori Technology Co., Ltd. The lipase expression of the recombinant strain L01 relative to the control strain H16 is shown in Figure 2 As shown, compared with the starting strain H16, the lipase expression level of the recombinant strain L01 was upregulated to 11 times that of the control strain, indicating that the lipase expression level in the recombinant strain L01 was much higher than that in the control strain H16.

[0073] Step 4: Performance test of PHA production by Eutropha roqueforti L01 4.1 Take the above fermentation liquid and centrifuge it to obtain bacterial cells. Dry the bacterial cells to constant weight. Determine the weight of the dried bacterial cells and record it as dry weight. Add 25mL of chloroform to the obtained dry bacterial cells, stir at room temperature for a day and night, and extract the polyester in the bacterial cells. After filtering out the bacterial residue, use an evaporator to concentrate to a total volume of about 7.5mL, then slowly add about 22.5mL of hexane and let it stand for 1 hour under slow stirring. After filtering out the precipitated polyester, vacuum dry it for 3 hours. Determine the mass of the dried polyester and calculate the polyester content in the bacterial cells.

[0074] 4.2 Results As shown in Table 2, the following three strain performance indicators of the recombinant strain L01 were better than those of the control strain H16. Compared with the control strain H16, the dry weight of strain L01 increased significantly by 11.26%, the PHA yield increased significantly by 13.77%, and the production intensity increased significantly by 11.99%.

[0075] Table 2

[0076] Comparative Example: Construction and performance test of a recombinant strain with a high-strength promoter inserted before the lipase gene In this comparative example, the H16 strain of Eutropha roebuckii (H16 for short) was used as the starting strain, and a high-strength constitutive promoter (SEQ ID NO: 68 of patent CN108977890B, hereinafter referred to as p68) was added in front of the gene coding region of H16_A1322 by using the common gene editing method in the art. According to the results of CN108977890B, the expression intensity of the p68 promoter is about 47 times that of the p31 promoter. The promoter element obtained by the above construction was inserted into the recombinant strain Re-p68-H16_A1322, named Eutropha roebuckii L02 (recombinant strain L02), and the recombinant strain was further tested for performance.

[0077] Step 1: Construction of recombinant strain L02 with high isoenzymatic expression 1.1 Gene synthesis The constitutive promoter p68 (SEQ ID NO. 6) was synthesized, and GGTCTCATCGT was added upstream and ACGCAGAGACC was added downstream of the synthesized DNA sequence to facilitate subsequent operations.

[0078] 1.2 The basic plasmid pKO-A1322 obtained in step 1 above and the constitutive promoter p68 synthesized by gene were assembled by the Goldengate method to obtain the editing plasmid pKO-p68-A1322. The subcloning construction of the editing plasmid was completed by WuXi Biologics Co., Ltd. The editing plasmid pKO-p68-A1322 was transformed into Escherichia coli S17-1, and then transferred into E. rothenbergii H16 by conjugation transfer. The specific steps were the same as step 1.3 of the embodiment. The obtained recombinant strain expressing Lipase with high isostrength was named E. rothenbergii L02 (recombinant strain L02).

[0079] Step 2: Fermentation performance test of E. rothenbergii L02 2.1 The recombinant strain L02 and the control strain H16 (1000 μL) constructed in step 1 of comparative example 1 stored in the glycerol tube were inoculated into seed culture medium I (20 mL) respectively, and the seed primary culture was carried out for 12 hours to obtain seed culture solution I; then, the seed culture solution I was inoculated into seed culture medium II (100 mL) at a volume ratio of 1 v / v%, and the secondary seed culture was carried out for 13 hours to obtain seed culture solution II; then, the seed culture solution II was inoculated into a 500 mL small fermentation tank (Dibil Company) containing 250 mL production culture medium at a volume ratio of 10 v / v%. The operating conditions were a culture temperature of 30°C, a stirring speed of 800 rpm, a ventilation volume (air) of 1 L / min, and the pH was controlled between 6.7 and 6.8. A 28% ammonia solution was used for pH control. During the culture process, the carbon source palm oil was supplemented by continuous flow feeding, and the variable speed feeding strategy was adopted to control the oil rate. The specific oil feeding strategy was: 0h~10h, 0mL / h, 10h~20h, 1mL / h, 20h~36h, the oil feeding rate increased by 0.2mL / h every 4h, 36h~44h maintained at 1.8mL / h, 44h~54h, the oil feeding rate decreased by 0.1mL / h every 2h. The culture time of the whole process fermentation was 54 hours, and no defoaming agent was added to the fermentation system.

[0080] 2.2 The results show (Table 3) that after 6 hours of fermentation, the bubble level of the fermentation tank of strain L02 was too high, and a large amount of fermentation liquid sprayed out from the escape tube, about 100 mL, and the escape volume exceeded 1 / 3 of the total volume, ending the fermentation. The results show that high-intensity expression of lipase in E. rothenbergii will cause a large amount of bubbling in the fermentation liquid in the early stage of fermentation, which will lead to fermentation failure. It is speculated that this may be due to the excessive enzyme activity of the highly expressed lipase, which makes it difficult for E. rothenbergii to completely decompose and utilize a large amount of fatty acids after fermentation for 6 hours. It can be seen that only the overexpression of a specific amount of lipase is conducive to the fermentation and production of PHA with oil as the substrate and stable increase in production.

[0081] Table 3

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant bacterium, characterized in that: The recombinant bacteria is modified so that the expression level of lipase therein is increased to 2-12 times that before modification; The recombinant bacteria is a Ralstonia bacterium; The foaming and / or liquid escape of the recombinant bacteria during the fermentation process is reduced due to the modification; wherein the fermentation substrate includes oil.

2. The recombinant bacterium according to claim 1, characterized in that The recombinant bacteria is modified so that the expression level of lipase therein is increased to 5-12 times that before modification; Preferably, the fermentation uses fat as a substrate.

3. The recombinant bacterium according to claim 1 or 2, characterized in that The increased expression of the lipase is achieved by one or more of the following methods: (1) Increasing the copy number of lipase encoding genes; (2) Enhance the activity of transcriptional and / or translational regulatory elements of lipase encoding genes.

4. The recombinant bacterium according to any one of claims 1 to 3, characterized in that The increased expression of the lipase is achieved by initiating the transcription of the lipase encoding gene with a medium strength constitutive promoter; Preferably, the nucleotide sequence of the medium strength constitutive promoter is as shown in SEQ ID NO.

3.

5. The recombinant bacterium according to any one of claims 1 to 4, characterized in that The amino acid sequence of the lipase is shown in SEQ ID NO.1; And / or, the bacterium of the genus Ralstonia is Ralstonia eutropha.

6. Any of the following uses of the recombinant bacteria according to any one of claims 1 to 5: (1) Fermentation production using oil as substrate; (2) Constructing production bacteria that use oil as substrate for fermentation production; (3) PHA fermentation production; (4) Construction of PHA-producing bacteria.

7. Application of increased expression of lipase in reducing foaming and / or liquid escape in fermentation production of Ralstonia bacteria using oil as substrate; The applications include: The Ralstonia bacterium is modified so that the expression level of lipase therein is increased to 2-12 times that before modification.

8. The use according to claim 7, characterized in that: The amino acid sequence of the lipase is shown in SEQ ID NO.1; And / or, the bacterium of the genus Ralstonia is Ralstonia eutropha.

9. A method for reducing fermentation foaming and / or liquid escape, characterized in that: The method comprises: modifying the fermentation strain so that the expression amount of lipase therein is increased to 2-12 times of that before modification; The fermentation strain is a Ralstonia bacterium; Preferably, the bacterium of the genus Ralstonia is Eutropha rhodochii; and / or the amino acid sequence of the lipase is as shown in SEQ ID NO.

1.

10. The method according to claim 9, characterized in that The fermentation strain is modified so that the expression level of lipase therein is increased to 5-12 times that before modification.

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