A microbial fermentation process for high-yield rhamnolipid

Abstract

The invention discloses a microbial fermentation process for high-yield rhamnolipid. The process adopts a high inoculation amount to enable the bacterial body to quickly pass through a slow period and quickly enter a stable period to produce rhamnolipid; and combines a low-temperature fermentation method to extend the rhamnolipid synthesis cycle, so that the bacterial body stable period is maintained at 11 to 16 days. The optimized fermentation process increases the fermentation yield of rhamnolipid by 49.8 times compared with the initial fermentation process, reaching 19.32 g / L; in order to further increase the yield of rhamnolipid, the invention adopts a genetic engineering transformation strategy, that is, simultaneously overexpressing a precursor dTDP-L-rhamnose synthesis gene rmlABCD and a rhamnolipid synthesis gene rhlABRI in Pseudomonas aeruginosa, and the strategy further increases the fermentation yield of rhamnolipid to 1.5 times that of the wild type, reaching 28.47 g / L, and the product yield Y P / S (Y product / substrate ) reached 0.569, effectively improving the atomic utilization of carbon sources and reducing production costs. Therefore, the high-yield rhamnolipid fermentation process and its engineered bacteria have great industrial application potential.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a microbial fermentation process for high-yield rhamnolipid. Background Art

[0002] Rhamnolipids are a class of anionic biosurfactants produced by microorganisms. Their structures contain hydrophilic and hydrophobic groups, of which the hydrophilic group is composed of 1 to 2 molecules of rhamnosyl and the hydrophobic group is composed of 1 to 2 molecules of β-hydroxy fatty acids with different carbon chain lengths. Rhamnolipids not only have good surface activity and emulsification, but also show strong tolerance to extreme pH values, temperature and salinity. In addition, they also have excellent antibacterial and anti-biofilm activity, so they have broad application potential in the fields of cosmetics, biomedicine, food, pesticides, environmental remediation and oil extraction.

[0003] Rhamnolipids are mainly produced by fermentation of Pseudomonas aeruginosa, but the low synthetic yield leads to high production costs of rhamnolipids, which limits the industrial application of rhamnolipids. Therefore, how to improve the yield of rhamnolipids by optimizing the fermentation process and combining it with the transformation to obtain an engineered strain with high rhamnolipid yield is the key to promoting the industrial application of rhamnolipids.

[0004] At present, the production of rhamnolipid is mainly improved by genetic engineering and fermentation process optimization. Among them, the optimization of fermentation process mainly includes the research on the components of fermentation medium and the optimization of process parameters. For example, Gong Zhijin et al. used the response surface method to optimize the components of fermentation medium, but because the influence of process parameters on rhamnolipid production was not considered, the production was only increased by 14.43%; Mariam Hassan et al. used Plackett-Burman design to find that low temperature (30℃), high pH (pH 8.0), and high speed (250rpm) can improve the production of rhamnolipid to a certain extent, but because the interaction between various factors was not considered, the production increase was not obvious, only 1.6 times before optimization. In summary, the above studies have the problem of not comprehensively considering the interaction of various factors, which makes the experimental design have a "blind spot" and makes it impossible to obtain the best experimental plan.

[0005] At present, the research on genetic engineering mainly uses the method of increasing the number of gene copies to increase the production of rhamnolipids. For example, Zhao et al. overexpressed the rhlAB gene in P. aeruginosa SG, increasing the production of rhamnolipids to 1.9 times. Huang Chao overexpressed the key genes rhlAB and rhlC for rhamnolipid synthesis in Pseudomonas aeruginosa DN1, respectively, increasing the production of rhamnolipids to 1.35 times and 1.58 times, respectively. In summary, genetic engineering is an effective method to increase the production of rhamnolipids.

[0006] However, the synthesis of rhamnolipids is not only regulated by the expression of rhamnosyltransferase (rhlAB), but also affected by its synthetic precursors dTDP-L-rhamnose and β-hydroxy fatty acids (HAAs). At present, the improvement of precursor β-hydroxy fatty acids is usually achieved by knocking out genes to block the bypass metabolic flow, but the effect is not significant. For example, Lei et al. knocked out the key gene phaC1DC2 for the synthesis of polyhydroxyalkanoates (PHA) in Pseudomonas aeruginosa, which only increased the production of rhamnolipids by 25.3%. There are few reports on the improvement of the precursor dTDP-L-rhamnose. Therefore, increasing the expression of the rmlABCD gene by increasing the number of gene copies is expected to be an effective method to increase the accumulation of dTDP-L-rhamnose and the production of rhamnolipids. Summary of the invention

[0007] In view of the shortcomings of the prior art described above, the object of the present invention is to provide a microbial fermentation process for high-yield rhamnolipid, that is, to comprehensively investigate the culture medium components and process parameters of rhamnolipid fermentation by Pseudomonas aeruginosa by using Plackett-Burman, steepest climbing test and Box-Behnken comprehensive design, to obtain the optimal process for rhamnolipid fermentation, and to further improve the rhamnolipid yield by using genetic engineering transformation strategy, thereby effectively improving the yield of rhamnolipid and the product yield to solve the problem of low rhamnolipid yield in the prior art.

[0008] One aspect of the present invention provides a microbial fermentation process for high-yield rhamnolipid, which comprises the following steps: inoculating Pseudomonas aeruginosa seed liquid into a fermentation medium with an inoculation amount of 14% to 24%, and fermenting and culturing at 21 to 29° C. for 11 to 16 days to obtain rhamnolipid.

[0009] Furthermore, the inoculation amount is 22%, and the culture is carried out at 24.2°C for 15 days.

[0010] Furthermore, the fermentation culture stirring speed is 180-220 rpm, more preferably 180 rpm.

[0011] Furthermore, the steps include:

[0012] 1) Inoculating Pseudomonas aeruginosa colonies into a culture medium to obtain a fermentation seed solution;

[0013] 2) The fermentation seed liquid is inoculated into the fermentation medium at an inoculation amount of 14% to 24%, and the fermentation is cultured at 21 to 29° C. for 11 to 16 days.

[0014] Furthermore, the carbon source content in the fermentation medium is 25-50 g / L, and the carbon source is selected from at least one of glucose, sunflower oil, soybean oil, rapeseed oil, corn oil, and olive oil, preferably soybean oil.

[0015] Furthermore, the nitrogen source content in the fermentation medium is 5-25 g / L, and the nitrogen source is selected from at least one of NaNO3, NH4Cl, (NH4)2SO4, urea, and L-tryptophan, preferably NaNO3.

[0016] Furthermore, the phosphorus source content in the fermentation medium is: 0.1-0.5 g / L, and the phosphorus source is selected from at least one of K2HPO4, KH2PO4, Na2HPO4, and NaH2PO4, preferably K2HPO4.

[0017] The fermentation medium also includes KCl, trace elements, and MgSO4. The content of KCl is 0.4-0.6 g / L, the content of the trace elements is 0.05-0.15%, the content of MgSO4 is 0.1-1.0 g / L, and the pH is 6.0-8.0.

[0018] Furthermore, the trace elements are selected from at least one of trisodium citrate, MnSO4·H2O, ZnSO4·7H2O, CoCl2·6H2O, CuSO4·5H2O, or FeCl3·6H2O. For example, 2.0 g / L trisodium citrate, 0.8 g / L MnSO4·H2O, 1.4 g / L ZnSO4·7H2O, 1.2 g / L CoCl2·6H2O, 1.2 g / L CuSO4·5H2O, 0.1-0.5 g / L FeCl3·6H2O.

[0019] Furthermore, the Pseudomonas aeruginosa is an engineered bacterium that simultaneously overexpresses rmlABCD and rhlABRI.

[0020] Furthermore, the construction method of the Pseudomonas aeruginosa is: simultaneously transferring an expression vector containing rmlABCD and an expression vector containing rhlABRI into the Pseudomonas aeruginosa.

[0021] Another aspect of the present invention provides Pseudomonas aeruginosa, which is an engineered bacterium that simultaneously overexpresses rmlABCD and rhlABRI.

[0022] As described above, the production process of the present invention for preparing rhamnolipid by fermentation of strains has the following beneficial effects:

[0023] A higher inoculation amount is used to allow the bacteria to quickly pass through the slow phase and quickly enter the stable phase to produce rhamnolipid; and the low-temperature fermentation method is combined to extend the rhamnolipid synthesis cycle, so that the bacteria stable phase is maintained at 11 to 16 days. The optimized fermentation process increases the rhamnolipid fermentation yield by 49.8 times compared with the initial fermentation process, reaching 19.32 g / L; in order to further increase the yield of rhamnolipid, the present invention adopts a genetic engineering transformation strategy, namely: simultaneously overexpressing the precursor dTDP-L-rhamnose synthesis gene rmlABCD and the rhamnolipid synthesis gene rhlABRI in Pseudomonas aeruginosa, so that the rhamnolipid fermentation yield is further increased to 1.5 times that of the wild type, reaching 28.47 g / L, and the product yield Y P / S (Y product / substrate ) reached 0.569, which effectively improved the atomic utilization of the carbon source and reduced the production cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a graph showing the effect of different carbon sources on rhamnolipid production by Pseudomonas aeruginosa ATCC 27853.

[0025] Figure 2 This is a graph showing the effect of different nitrogen sources on rhamnolipid production by Pseudomonas aeruginosa ATCC 27853.

[0026] Figure 3 This is a graph showing the effect of different phosphorus sources on rhamnolipid production by Pseudomonas aeruginosa ATCC 27853.

[0027] Figure 4 This is a three-dimensional response surface diagram of the effects of inoculum size and temperature (A), inoculum size and fermentation time (B), and temperature and fermentation time (C) on the rhamnolipid production of Pseudomonas aeruginosa ATCC 27853.

[0028] Figure 5 This is the PCR map of cloning rmlABCD gene and rhlABRI gene.

[0029] Figure 6 This is the colony PCR image for verifying the pUCP18-rmlABCD expression vector.

[0030] Figure 7 This is the colony PCR image for verifying the pBBR1MCS5-rhlABRI expression vector.

[0031] Figure 8The plasmid maps are pUCP18-rmlABCD and pBBR1MCS5-rhlABRI.

[0032] Fig. 9 It is a comparison chart of rhamnolipid production of RL01 strain and control strains ATCC 27853 / rmlABCD and ATCC 27853 / rhlABRI. DETAILED DESCRIPTION

[0033] The following is an explanation of the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the process equipment or devices not specifically noted in the following embodiments all adopt conventional equipment or devices in the art. In addition, it should be understood that one or more method steps mentioned in the present invention do not exclude the presence of other method steps before and after the combination step or the insertion of other method steps between these explicitly mentioned steps, unless otherwise specified; it should also be understood that the combined connection relationship between one or more devices / devices mentioned in the present invention does not exclude the presence of other devices / devices before and after the combination device / device or the insertion of other devices / devices between these explicitly mentioned two devices / devices, unless otherwise specified. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, rather than limiting the order of arrangement of each method step or limiting the scope of the present invention. The change or adjustment of its relative relationship, without substantial change in the technical content, should also be regarded as the scope of the present invention.

[0034] Culture medium:

[0035] The seed culture medium included 10.0 g / L peptone, 5.0 g / L yeast powder, 10.0 g / L NaCl, the pH was adjusted to 7.0, and sterilized by high pressure steam at 121°C for 20 min.

[0036] The initial fermentation medium includes 37.5 g / L soybean oil, 15.0 g / L NaNO3, 0.5 g / L MgSO4·7H2O, 0.5 g / L KCl, 0.3 g / L K2HPO4, pH is adjusted to 7.0, sterilized by high pressure steam at 121°C for 20 min, and 0.1% trace element solution is added before use. The initial trace element solution includes 2.0 g / L trisodium citrate, 0.8 g / L MnSO4·H2O, 1.4 g / L ZnSO4·7H2O, 1.2 g / L CoCl2·6H2O, 1.2 g / L CuSO4·5H2O, 0.28 g / L FeCl3·6H2O, and sterilized by 0.22 μm filter membrane.

[0037] Determination of rhamnolipid content by orcinol method: Inoculate 1% of the fermentation seed liquid in a 250mL triangular flask containing 40mL of the initial fermentation medium and culture at 37°C and 220rpm for 72h. After the fermentation is completed, take 100μL of the fermentation liquid, extract the fermentation liquid acidified with hydrochloric acid with 300μL of ethyl acetate, dry the organic phase at 70°C, add 100μL of ddH2O to dissolve and dilute appropriately. Add 100μL of the sample to be tested, 100μL of 1.6% orcinol aqueous solution and 800μL of 60% concentrated sulfuric acid to a 1.5mL EP tube, and react at 80°C and 1000rpm for 30min. After the reaction is completed, measure the absorbance value at a wavelength of 421nm, and calculate the content of rhamnosyl according to the regression equation of the rhamnose standard curve, and then multiply it by the mass ratio coefficient of rhamnolipid to rhamnosyl 3.4 to calculate the content of rhamnolipid in the fermentation liquid.

[0038] Example 1 Optimization of carbon source, nitrogen source and phosphorus source of rhamnolipid fermentation medium by single factor method

[0039] Screening of the best carbon source: Glucose, sunflower oil, soybean oil, rapeseed oil, corn oil, and olive oil were used as carbon sources, and 1% of the seed solution was inoculated in a 250 mL Erlenmeyer flask containing 40 mL of the initial fermentation medium. The culture was carried out at 37 ° C and 220 rpm for 144 h, and the rhamnolipid concentration in the fermentation broth was determined. The results of the effects of different carbon sources on the yield of rhamnolipids synthesized by Pseudomonas aeruginosa ATCC 27853 are shown in Figure 2. Figure 1 As shown by Figure 1 It can be seen that the best carbon source for fermentation is soybean oil.

[0040] Screening of the best nitrogen source: NaNO3, NH4Cl, (NH4)2SO4, urea, and L-tryptophan were used as nitrogen sources, respectively. 1% seed solution was inoculated in a 250mL Erlenmeyer flask containing 40mL of initial fermentation medium, and the culture was carried out at 37°C and 220rpm for 144h. The rhamnolipid concentration in the fermentation broth was determined.

[0041] The results of the effects of different nitrogen sources on the yield of rhamnolipids synthesized by Pseudomonas aeruginosa ATCC 27853 are shown in Figure 2 Figure 2 As shown by Figure 2 It can be seen that although the final yield of urea as a nitrogen source is higher, the bacteria only entered the stable period at 72 hours, while Pseudomonas aeruginosa using NaNO3 as a nitrogen source entered the stable period and produced rhamnolipids at 24 hours. Therefore, NaNO3 was finally selected as the fermentation nitrogen source.

[0042] Screening of the best phosphorus source: K2HPO4, KH2PO4, Na2HPO4, and NaH2PO4 were used as phosphorus sources, and 1% of the seed solution was inoculated in a 250mL Erlenmeyer flask containing 40mL of the initial fermentation medium. The culture was carried out at 37°C and 220rpm for 144h, and the rhamnolipid concentration in the fermentation broth was determined. The results of the effects of different phosphorus sources on the yield of rhamnolipids synthesized by Pseudomonas aeruginosa ATCC 27853 are shown in Figure 2. Figure 3 As shown by Figure 3 It can be seen that the best phosphorus source is K2HPO4.

[0043] In summary, soybean oil, NaNO3 and K2HPO4 were finally determined as the carbon, nitrogen and phosphorus sources for rhamnolipid fermentation medium.

[0044] Example 2 Plackett-Burman (PB) combination design to screen key factors of fermentation process conditions

[0045] According to the above carbon, nitrogen and phosphorus source optimization experimental results, the Design-expert 10 software was used to perform Plackett-Burman combination design on the 10 factors of rhamnolipid fermentation process conditions, including carbon source concentration, nitrogen source concentration, phosphorus source concentration, MgSO4 concentration, FeCl3 concentration, pH, temperature, rotation speed, inoculation amount and fermentation time. The factors and level designs are shown in Table 1, and the rhamnolipid yields of each experimental ratio are shown in Table 2.

[0046] Table 1 Levels of each factor in Plackett-Burman combination design of key factors of fermentation process conditions

[0047]

[0048] Table 2 Plackett-Burman experimental design and experimental results of key factors of fermentation process conditions

[0049]

[0050]

[0051] The results were analyzed by variance analysis using Design-expert 10 software. The results are shown in Table 3. Table 3 shows that the order of the effects of various factors on the production of rhamnolipids by Pseudomonas aeruginosa ATCC 27853 is as follows: temperature > fermentation time > inoculation amount > MgSO4 concentration > carbon source concentration > nitrogen source concentration > phosphorus source concentration > FeCl3 concentration > pH > rotation speed, among which temperature (p = 0.011), fermentation time (p = 0.027) and inoculation amount (p = 0.031) are the three most important influencing factors. Therefore, these three significant factors were selected to conduct the steepest climbing experiment and response surface analysis to determine their optimal levels.

[0052] Table 3 Plackett-Burman variance analysis of key factors of fermentation process conditions

[0053]

[0054] Example 3 Steepest climbing experiment and Box-Behnken (BBD) response surface experimental design to determine the optimal fermentation process of rhamnolipid

[0055] The steepest climb experiment approaches the optimal value area: According to the Plackett-Burman experimental results of the above fermentation process conditions, the inoculum, temperature and fermentation time were comprehensively selected as the research objects for optimization. According to the estimated coefficients of these three factors, the inoculum and fermentation time have positive effects, the temperature has a negative effect, and the other non-significant factors take the maximum or minimum value in the PB design according to the positive or negative estimated coefficients in Table 3. The experimental results are shown in Table 4. It can be seen from Table 4 that the optimal fermentation conditions for rhamnolipid production are between Experiment 4 and Experiment 6, so Experiment 5 was selected as the center point for response surface analysis.

[0056] Table 4 Steepest climbing experiment design and results

[0057]

[0058] Box-Behnken (BBD) response surface experimental design: According to the main factors and levels determined by the Plackett-Burman experiment and the steepest climbing experiment, the Box-Behnken experiment was used to conduct a response surface analysis experiment with three factors and three levels. The experiment included 12 factorial experiments and 5 central experiments. The coding levels are shown in Table 5. The BBD response surface combination design and the corresponding rhamnolipid production are shown in Table 6.

[0059] Table 5 Levels of factors in BBD response surface experimental design

[0060]

[0061] Table 6 BBD response surface combination design and experimental results

[0062]

[0063] The experimental results were fitted with a quadratic polynomial regression using the Design-expert 10 software. The statistical significance was determined by the F test. The optimal point was predicted using differential equations, and a quadratic response surface regression model was established to find the optimal factor level. The variance analysis results of the quadratic response surface regression model are shown in Table 7.

[0064] Table 7 Analysis of variance for BBD response surface experimental design

[0065]

[0066]

[0067] As shown in Table 7, the P value of the regression model is less than 0.0001, indicating that the model is correct and the actual measured value of rhamnolipid production is significantly correlated with the equation prediction value. The results of the effects of inoculation amount and temperature, inoculation amount and fermentation time, and temperature and fermentation time on the rhamnolipid production of Pseudomonas aeruginosa ATCC 27853 are shown in Figure 7. Figure 4 As shown. Figure 4 It can be seen that there is a certain interaction between temperature and inoculation amount, and between fermentation time and temperature, and the best prediction point is within the experimental investigation range. Through calculation, the quadratic polynomial regression equation of rhamnolipid production on inoculation amount, temperature, and fermentation time was obtained, and the optimal conditions were obtained as inoculation amount, temperature, and fermentation time of 22%, 24.2℃, and 15 days, respectively. At this time, the predicted response value of rhamnolipid production can reach 19.031g / L.

[0068] Example 4 Experimental verification of fermentation optimization process

[0069] Based on the optimal fermentation process conditions screened in this study (50 g / L soybean oil, 25 g / L NaNO3, 0.5 g / L K2HPO4, 1.0 g / L MgSO4, 0.5 g / L KCl, 0.1 mg / L FeCl3, medium pH 6.0, inoculation amount: 22%, temperature: 24.2 °C, fermentation time: 15 days, rotation speed: 180 rpm), a shake flask fermentation experiment was carried out. The rhamnolipid produced by Pseudomonas aeruginosa ATCC 27853 was 19.32 g / L, and the product yield was Y P / S (Y product / substrate ) was 0.386, and the rhamnolipid yield was increased by 49.8 times compared with the initial fermentation process (the rhamnolipid yield of the initial fermentation process was 0.38 g / L). Further, we compared the fermentation process of this embodiment with the fermentation process reported in the literature, and the results are shown in Table 8.

[0070] Table 8 Fermentation process comparison

[0071]

[0072]

[0073] As shown in Table 8, the current fermentation process generally adopts a low inoculation amount (≤10%) and a high fermentation temperature (≥30°C), which shortens the rhamnolipid synthesis cycle and leads to a low final rhamnolipid yield and product yield. Compared with the existing technology level, this study adopts a higher inoculation amount (14% to 24%) to enable the bacteria to quickly pass through the slow period and quickly enter the stable period to produce rhamnolipid; and combined with low-temperature fermentation (21 to 29°C) to extend the rhamnolipid synthesis cycle, so that the bacteria stable period is maintained at 11 to 16 days, thereby achieving a higher rhamnolipid yield. This fermentation process is suitable for Pseudomonas aeruginosa and its engineered bacteria, which can effectively improve the atomic utilization of carbon sources, reduce production costs, and help promote the industrial application of rhamnolipids.

[0074] Example 5 Genetic Engineering Strategy to Construct Rhamnolipid Engineering Bacteria RL01

[0075] Construction of expression vector pUCP18-rmlABCD: The rmlABCD gene fragment was amplified using the genomic DNA of Pseudomonas aeruginosa ATCC 27853 as a template and Prml-F and Prml-R as primers. The primer sequences are shown in SEQ ID NO.2-3, the gene sequence is shown in SEQ ID NO.1, and the gene amplification results are shown in Figure 5 As shown by Figure 5 It can be seen that the rmlABCD gene fragment was successfully amplified and obtained, with a length of 3869bp.

[0076] SEQ ID NO.1:

[0077]

[0078]

[0079]

[0080] SEQ ID NO.2:

[0081]

[0082] SEQ ID NO.3:

[0083]

[0084] The pUCP18 vector was then digested with EcoR I and BamH I, and the linearized vector fragment was recovered by gel. The linearized pUCP18 vector fragment obtained above was connected with the rmlABCD gene fragment using a one-step cloning ligation kit (Nanjing Novogene Biotech Co., Ltd.), and the obtained expression vector was subjected to colony PCR and sequencing identification. The identification results of colony PCR are as follows: Figure 6 As shown by Figure 6 It can be seen that PCR obtained a band with the same length as the rmlABCD gene fragment, indicating that the expression vector pUCP18-rmlABCD was successfully constructed.

[0085] Construction of expression vector pBBR1MCS5-rhlABRI: The rhlABRI gene fragment was amplified using P. aeruginosa ATCC 27853 genomic DNA as a template and Prhl-F and Prhl-R as primers. The primer sequences are shown in SEQ ID NO. 4 to 5, the gene sequence is shown in SEQ ID NO. 6, and the gene amplification results are shown in Figure 5 As shown by Figure 5 It can be seen that the rhlABRI gene fragment was successfully amplified and obtained, with a length of 4488 bp.

[0086] SEQ ID NO.4

[0087]

[0088] SEQ ID NO.5

[0089]

[0090] SEQ ID NO.6

[0091]

[0092]

[0093]

[0094] The pBBR1MCS5 vector was digested with BamH I and Hind III, and the linearized vector fragment was recovered by electrophoresis. The linearized pBBR1MCS5 vector fragment obtained above was connected with the rhlABRI gene fragment using a one-step cloning kit (Nanjing Novogene Biotech Co., Ltd.), and the expression vector was identified by colony PCR and sequencing. The identification result of colony PCR was as follows: Figure 7 As shown by Figure 7 It can be seen that PCR obtained bands with consistent length of rhlABRI gene fragments, indicating that the expression vector pBBR1MCS5-rhlABRI was successfully constructed.

[0095] Construction of rhamnolipid engineering bacteria RL01: pUCP18-rmlABCD and pBBR1MCS5-rhlABRI plasmids were extracted respectively, and the two plasmids were sequentially transformed into Pseudomonas aeruginosa ATCC 27853 by electroporation to obtain rhamnolipid engineered bacteria P. aeruginosa ATCC 27853 / pUCP18-rmlABCD / pBBR1MCS5-rhlABRI, named RL01. The plasmid map is shown in Figure 8 shown.

[0096] Construction of control strains ATCC 27853 / rmlABCD and ATCC 27853 / rhlABRI: The pUCP18-rmlABCD and pBBR1MCS5-rhlABRI plasmids were extracted respectively, and the two plasmids were transformed into Pseudomonas aeruginosa ATCC 27853 by electroporation to obtain the comparison strains ATCC 27853 / rmlABCD and ATCC 27853 / rhlABRI.

[0097] Example 6 Application of the Optimal Fermentation Process in Rhamnolipid Engineering Bacteria

[0098] Determination of the amount of rhamnolipid synthesis by rhamnolipid engineered bacteria RL01 and control strains: The rhamnolipid engineered bacteria RL01, control strains ATCC 27853 / rmlABCD and ATCC 27853 / rhlABRI and wild bacteria ATCC 27853 were selected and inoculated into the seed culture medium for culture to obtain the fermentation seed liquid. Under the optimal fermentation process conditions screened in this study (50g / L soybean oil, 25g / L NaNO3, 0.5g / L K2HPO4, 1.0g / L MgSO4, 0.5g / L KCl, 0.1mg / L FeCl3, culture medium pH 6.0, inoculation amount: 22%, temperature: 24.2℃, fermentation time: 15 days, rotation speed: 180rpm), a shake flask fermentation culture experiment was carried out. After the fermentation, the content of rhamnolipid was measured, and the results are as follows: Fig. 9 As shown by Fig. 9 It can be seen that under the same fermentation process conditions, the rhamnolipid production of the engineered strain RL01 was further improved compared with the wild type, reaching 28.47 g / L, and the product yield was Y P / S (Y product / substrate ) reached 0.569, which was 1.5 times that of the wild type and 74.92 times that of the initial fermentation process. In addition, the yield of the engineered bacteria RL01 was significantly higher than that of the control strain that overexpressed rmlABCD or rhlABRI alone, indicating that the transformation strategy of simultaneously overexpressing rmlABCD and rhlABRI genes can effectively increase the yield of rhamnolipids.

[0099] In summary, the present invention provides a production process for preparing rhamnolipids by fermentation of strains, which increases the rhamnolipid yield by 73.92 times compared with before optimization, solves the problems of low yield and high cost of rhamnolipid production by existing Pseudomonas aeruginosa fermentation. At the same time, the fermentation process optimization method provided by the patent of the present invention is also applicable to the optimization of other Pseudomonas and their engineered bacteria.

[0100] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limitations of the present invention. In addition, the various modifications listed herein and the variations of methods and compositions in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications obvious to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. A microbial fermentation process for high-yield rhamnolipid, the process comprising the following steps: The seed liquid of Pseudomonas aeruginosa is inoculated into the fermentation medium at an inoculation amount of 14% to 24%, and fermented and cultured at 21 to 29° C. for 11 to 16 days to obtain rhamnolipid; The fermentation medium has the following characteristics: a) the carbon source content in the fermentation medium is: 25-50 g / L, and the carbon source is selected from soybean oil; b) the nitrogen source content in the fermentation medium is: 5-25 g / L, and the nitrogen source is selected from NaNO3; c) the phosphorus source content in the fermentation medium is: 0.1-0.5 g / L, and the phosphorus source is selected from K2HPO4; The Pseudomonas aeruginosa is an engineered bacterium that simultaneously overexpresses rmlABCD and rhlABRI.

2. The fermentation process according to claim 1, characterized in that: The inoculation amount was 22%, and the culture was carried out at 24.2°C for 15 days.

3. The fermentation process according to claim 1, characterized in that: The fermentation culture stirring speed is 180-220 rpm.

4. The fermentation process according to claim 1, characterized in that: The process steps include: 1) Inoculating Pseudomonas aeruginosa colonies into a culture medium to obtain a fermentation seed solution; 2) The fermentation seed liquid is inoculated into the fermentation medium at an inoculation amount of 14% to 24%, and the fermentation is cultured at 21 to 29° C. for 11 to 16 days.

5. The fermentation process according to claim 1, characterized in that: The fermentation medium also includes KCl, trace elements, and MgSO4. The content of KCl is 0.4-0.6 g / L, the content of the trace elements is 0.05-0.15%, the content of MgSO4 is 0.1-1.0 g / L, and the pH is 6.0-8.

0.

6. The fermentation process according to claim 5, characterized in that: The trace elements are selected from one or more of trisodium citrate, MnSO4·H2O, ZnSO4·7H2O, CoCl2·6H2O, CuSO4·5H2O, or FeCl3·6H2O.

7. The fermentation process according to claim 1, characterized in that: The construction method of the Pseudomonas aeruginosa is as follows: an expression vector containing rmlABCD and an expression vector containing rhlABRI are simultaneously transferred into the Pseudomonas aeruginosa.

8. A Pseudomonas aeruginosa, characterized in that: The Pseudomonas aeruginosa is an engineered bacterium that simultaneously overexpresses rmlABCD and rhlABRI.

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