Method for optimizing strains of probiotic microorganisms capable of mono- and co-expression of heterologous proteins, with modification of fermentation and expression conditions

Abstract

FIELD: biotechnology; molecular biology.SUBSTANCE: method for optimizing lactobacilli capable of co-expressing heterologous proteins by modifying fermentation and expression conditions is proposed. The method consists of optimizing the expression of recombinant proteins IL-10, IL-22 and includes: preparing the inoculum using a modified MRS medium, three-stage expression with the prevention of foaming, monitoring using control sensors for temperature, pH, pO2 and maintaining the parameters of temperature, pH, pO2, measuring the optical density and counting viable cells, determining the concentration of glucose and lactic acid in the substrates, lysis of cells, purification of recombinant proteins.EFFECT: improvement in the method for optimizing lactobacilli capable of co-expressing heterologous proteins and allows for a significant increase in the yield of target proteins IL-10 and IL-22, as well as improving their biological activity.1 cl, 3 dwg, 3 ex

Description

[0001] Field of technology to which the invention relates

[0002] The invention relates to the field of biotechnology, genetic engineering and microbiological synthesis, namely to technologies for constructing and optimizing expression systems based on probiotic strains of microorganisms and can be used to increase the final yield of recombinant proteins, with a frequency of >95%, as well as stable mono- and co-expression.

[0003] Technology Level

[0004] Optimization of recombinant probiotic microorganism strains is a key step in their development and application in various fields of biotechnology, medicine, and agriculture. This process aims to improve the characteristics of microorganisms to enhance their efficacy, stability, and safety during use. Probiotic microorganisms are live microorganisms that, when used correctly, have a positive impact on human and animal health by improving the balance of microflora and exerting a range of other beneficial effects. Recombinant strains of such microorganisms are created through genetic modification to introduce beneficial genes into their genomes, such as those encoding antioxidants, enzymes, antimicrobial peptides, or even vaccine antigens.However, at this stage, it is important that the strains be stable and retain their properties over a long period of time, exhibit high productivity during cultivation, interact effectively with the host organism to demonstrate probiotic activity, and be safe, without causing side effects. Strain optimization is essential to achieving these goals. One of the most important aspects is increasing the productivity of microorganisms during cultivation. Optimizing culture conditions (temperature, pH, nutrient medium composition, oxygen levels) can significantly increase the production of target products, such as enzymes, vitamins, or antibacterial substances. Furthermore, it is important to select parameters that stabilize the production of these substances at all stages of cultivation.For industrial use of recombinant strains, it is necessary to ensure that they retain their recombinant structure over a long period of time and under various conditions. Modified strains can lose or alter introduced genes during cell division, which reduces their effectiveness. Strain optimization aims to enhance genetic stability, including the use of strategies for fixing recombinant genes or the creation of systems for regulating their expression. It is important that recombinant probiotics can survive challenging conditions, such as the acidic pH of the stomach, high temperatures, or the effects of antibiotics. This requires optimizing bacterial defense mechanisms, such as resistance to acids or osmotic pressure. Incorporating such genes into the strain or improving their expression is an important aspect of optimization.Recombinant probiotics should possess improved properties, such as the ability to adhere to intestinal epithelial cells, activate immune mechanisms, or inhibit the growth of pathogenic microorganisms. These aspects can be improved by genetically modifying strains to enhance their adhesion to the epithelium or enhance their immunomodulatory properties.

[0005] Optimization of recombinant probiotic strains is a necessary step in creating high-quality and effective products that can be used to treat diseases, improve digestion, support the immune system, and for other purposes. Without this step, it is impossible to guarantee the stability and safety of probiotics for long-term use, making optimization a critical process for the successful use of recombinant probiotic microorganisms.

[0006] There is an optimization method described in a patent for the creation of a recombinant strain of Bacillus subtilis bacteria - a producer of phospholipase C [RU Patent No. RU 2500811 C1], based on the use of a process for cultivating the strain in flasks and laboratory fermenters using LB medium.

[0007] The disadvantages are as follows:

[0008] This method uses LB (Luria-Bertani) culture medium, which is not optimized for maximum recombinant protein yield. Furthermore, the strains are cultivated in flasks, which means there is no automated control of parameters such as pH, dissolved oxygen, and temperature. This leads to cell stress and reduced productivity.

[0009] There is an optimization method described in the patent for the construction of the plasmid vector pRh15A for producing methionine-free interferon alpha-2b, the Escherichia coli BL21 DE3 bacterial strain - a producer of methionine-free interferon alpha-2b and a method for producing methionine-free interferon alpha-2b [Russian Federation Patent No. RU 2697375 C2].

[0010] The disadvantages are as follows:

[0011] The patent does not detail the optimization of fermentation conditions, such as temperature, pH, nutrient medium composition, and oxygen levels, which could lead to inefficient production of interferon alpha-2b. Also, under fermentation conditions, E. coli strains may lose the stability of their inserted genes, especially during long-term cultivation.

[0012] There is a method for culturing the strain described in the patent for the construction of recombinant microorganisms for the production of useful metabolites [RU Patent No. RU 2658770 C2], based on the use of 250 ml flasks containing 200 ml of MSP medium with glucose and chloramphenicol.

[0013] The disadvantages are as follows:

[0014] When using flasks to cultivate recombinant bacterial strains, there is no automatic control of parameters such as pH, oxygen, and temperature, which reduces the stability and reproducibility of the process. Furthermore, when culturing in flasks, precise aeration and mixing are impossible, and oxygen often becomes a limiting factor, especially with high biomass.

[0015] The prototype is based on the optimization method described in the patent for the production of a recombinant strain of Bacillus licheniformis bacteria producing thermostable lipase [Russian Federation Patent No. RU 2500812 C1]. This method is based on fermentation of the resulting strain in a 3-liter laboratory fermenter; the enzyme activity in the culture fluid reaches 500 U / ml.

[0016] The disadvantages are as follows:

[0017] The patent lacks control over critical cultivation parameters, making it impossible to reproduce the process on an industrial scale with a stable enzyme yield. The optimal composition of the nutrient medium is also not specified.

[0018] Disclosure of invention

[0019] The objective of the proposed invention is to develop an effective and reproducible method for optimizing the processes of expression of heterologous proteins, ensuring high productivity of the synthesis of recombinant proteins, stable mono- and co-expression of target proteins in probiotic systems, and maintaining the viability and metabolic activity of microorganisms.

[0020] The technical result that can be achieved with the help of the proposed invention consists in improving the method for optimizing strains of probiotic microorganisms capable of mono- and co-expression of heterologous proteins, with modification of fermentation and expression conditions, which will significantly increase the yield of target proteins IL-10 and IL-22, as well as improve their biological activity.

[0021] The technical result is achieved by optimizing the expression of recombinant IL-10 and IL-22 proteins using a bioreactor system. Expression is carried out in a three-stage mode: 1) the initial growth phase (0-12 hours) at 37°C, pH 6.8, stirring speed of 200 rpm and aeration of 1.5 rpm (0.5 vvm); 2) the induction phase (12-48 hours) - when OD600 reaches 0.6, 1 mM IPTG is added, the temperature is reduced to 30°C and the stirring speed is increased to 400 rpm; 3) the production phase (48-72 hours) - pO2 is maintained at 30% of saturation by cascade regulation of the stirring speed (300-500 rpm) and aeration (0.5-1.0 vvm). Process monitoring includes hourly recording of parameters (temperature, pH, pO2) and sampling every 4 hours for comprehensive analysis.

[0022] This method provides precise monitoring and control of key parameters (pH, dissolved oxygen, temperature, stirring speed) to create optimal conditions for microorganism growth and protein synthesis.

[0023] Lactobacillus spp. is a genus of Gram-positive, anaerobic or microaerophilic bacteria that are widespread in nature and play a key role in various biotechnological and medical processes. They are part of the normal intestinal microflora of humans and other mammals and are also found in various fermented foods such as yogurts, kefir, kvass, and other dairy and plant fermented foods. [Electronic resource] URL: https: / / www.academia.edu / 109403651 / Lactic_Acid_Bacteria_in_Fermented_Foods]. Lactobacilli are lactic acid bacteria that convert carbohydrates (mainly sugars such as lactose) into lactic acid, which helps to lower the pH of the environment. These bacteria are anaerobic, meaning they can grow in low oxygen conditions but can also function in small amounts.Lactobacilli play a vital role in maintaining gut health, helping to maintain a balanced microflora by suppressing the growth of pathogenic microorganisms. They participate in the fermentation process, converting carbohydrates into lactic acid, which contributes to the preservation of foods and improves their flavor. Lactobacilli can be used as probiotics—microorganisms that, when consumed in appropriate doses, have a beneficial effect on health, improving digestive function, strengthening the immune system, and even influencing metabolism. In genetic engineering and biotechnology, lactobacilli are often used as vector systems for the production of recombinant proteins. This is due to their ability to express foreign genes and their safety. They can also be used to create vaccines or other biotherapeutics due to their ability to produce various proteins, including antibodies and cytokines.Lactobacilli are safe and non-pathogenic, making them ideal candidates for use as biofactories for the production of beneficial substances. They are highly resistant to fermentation conditions and can produce large quantities of target proteins [Electronic resource] URL: https: / / www.researchgate.net / publication / 270762494_The_Health_Benefits_of_Probiotics].

[0024] Interleukin-10 is a cytokine that plays an important role in regulating the immune response. It is known for its anti-inflammatory properties, as it helps suppress the activity of immune cells that can cause inflammation. IL-10 regulates the function of T cells, macrophages, and other immune cells, suppressing the production of proinflammatory cytokines and enhancing immune suppression. This makes IL-10 an important molecule for controlling immune diseases such as autoimmune disorders and inflammatory bowel disease, and as a therapy for various inflammatory conditions [Electronic resource]. [resource] URL: https: / / pubmed.ncbi.nlm.nih.gov / 31611251 / ].The main functions of IL-10 include anti-inflammatory activity, where IL-10 suppresses the activity of macrophages, T cells, and other cells of the immune system, which reduces the production of proinflammatory cytokines (e.g., TNF-α, IL-1β, IL-6) and reduces inflammation.IL-10 also regulates the immune response, maintaining a balance between protective and suppressed immune responses, which helps prevent overactivation of the immune system, which can lead to autoimmune diseases. IL-10 also has antibacterial and antifungal activity, participating in the body's defense against infections and limiting inflammation that can cause tissue damage [Electronic resource] URL: https: / / pubmed.ncbi.nlm.nih.gov / 12773629 / ].

[0025] Interleukin-22 is a cytokine that plays a key role in maintaining epithelial barrier homeostasis and regulating inflammation. It was first described as a factor produced by T cells and plays a crucial role in the body's defense responses. IL-22 belongs to a group of cytokines that act on epithelial cells, stimulating their proliferation and secretion of various molecules, such as antimicrobial peptides, which contributes to an enhanced immune response at sites of inflammation and damage. IL-22 does not directly affect immune cells such as macrophages or neutrophils, but it does influence epithelial cells, making it important in protecting mucous membranes and barrier tissues such as the skin, intestines, and respiratory tract. IL-22 is produced by a variety of cells, including T helper 17 (Th17) cells, cytotoxic T cells (Tc), and specific T cell subtypes such as Th22.It activates receptors on epithelial cells, inducing a response that includes the activation of molecules that mediate immune control and tissue repair. One of its main mechanisms is the increased production of antimicrobial peptides, which helps the body defend itself against infections such as bacterial or fungal ones [Electronic resource] URL: https: / / pubmed.ncbi.nlm.nih.gov / 17030002 / ].

[0026] Drawings and other materials

[0027] Fig. 1 - Electrophoregram (SDS-PAGE).

[0028] Fig. 2 - Output and specific activity of IL-10 and IL-22.

[0029] Fig. 3 - Graphs of OD600 (optical density), IL-10 / IL-22 expression level (mg / L), and activity over time.

[0030] Implementation of the invention

[0031] A method for optimizing probiotic microorganism strains capable of mono- and co-expression of heterologous proteins, with modification of fermentation and expression conditions, involves optimizing the expression of recombinant IL-10 and IL-22 proteins. This process begins with the preparation of an inoculum of 50 ml of an overnight culture of transformed lactobacilli in an MRS medium supplemented with erythromycin and chloramphenicol. This inoculum is then added to 500 ml of a modified MRS medium (supplemented with 1% glucose, 0.1% yeast extract, and 0.01% Tween-80) in a 1 L glass bioreactor with a working volume of 600 ml. The bioreactor system is pre-sterilized by autoclaving at 121°C for 20 minutes and equipped with control sensors.

[0032] Expression is carried out in a three-step mode: 1) the initial growth phase (0-12 hours) at 37°C, pH 6.8, stirring speed of 200 rpm and aeration of 1.5 rpm (0.5 vvm); 2) the induction phase (12-48 hours) - when OD600 reaches 0.6, 1 mM IPTG is added, the temperature is reduced to 30°C and the stirring speed is increased to 400 rpm; 3) the production phase (48-72 hours) - pO2 is maintained at 30% of saturation by cascade regulation of stirring speed (300-500 rpm) and aeration (0.5-1.0 vvm). Process monitoring includes hourly recording of parameters (temperature, pH, pO2) and sampling every 4 hours for comprehensive analysis.

[0033] After fermentation, the biomass is separated by centrifugation at 8000 g for 20 min at 4°C (Sorvall RC-6 Plus centrifuge). The supernatant is concentrated 10-fold by tangential ultrafiltration on a Pellicon 2 system (Millipore) with 10 kDa membranes at 4°C and 1.5 bar. The cell pellet is resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mg / ml lysozyme) and incubated for 30 min at 37°C, followed by sonication (Soniprep 150, 10 cycles of 30 sec at 10 μm amplitude).

[0034] The final yield of IL-10 is 120±15 mg / L, purity>95%, molecular weight 18.5 kDa, specific activity 1.2×10 6 U / mg; IL-22 – yield 95±10 mg / l, purity >93%, molecular weight 20.3 kDa, specific activity 8.5×10 5 U / mg.

[0035] Example 1. To optimize probiotic microorganism strains capable of mono- and co-expressing heterologous proteins, traditional cell cultures were used. This involved small culture volumes and a lack of automated culture control. The medium used was traditional MRS medium for lactobacilli, without the use of additional components to maintain stable pH and oxygen levels during fermentation.

[0036] Thus, the method demonstrated a lack of automated control over environmental parameters such as pH, dissolved oxygen, and temperature, which led to frequent fluctuations in parameters and, in turn, impacted the stability of cell growth and recombinant protein expression. Cells grown in flasks did not reach the same density as those in the bioreactor, resulting in lower yields of IL-10 and IL-22 proteins.

[0037] Example 2. To optimize strains of probiotic microorganisms capable of mono- and co-expression of heterologous proteins, a method of inducing expression with a low concentration of IPTG was used.

[0038] However, the low IPTG concentration did not provide the required activation of the expression system, resulting in a decrease in the yield of target proteins. This was due to the fact that active protein expression required a higher concentration of the inducer, which stimulates the promoter and initiates the synthesis of target proteins. Using a lower IPTG concentration failed to achieve this level of activation, which in turn reduced the efficiency of the process.

[0039] Example 3. To optimize strains of probiotic microorganisms capable of mono- and co-expression of heterologous proteins, a bioreactor with automatic parameter control and an optimal concentration of IPTG (1 mM) were used.

[0040] The method involved combining several factors that ensured higher efficiency in recombinant protein production. A three-step process, including an induction phase and a production phase, with precise control of temperature, pH, and dissolved oxygen, enabled the expression process to be initiated most efficiently and stable conditions to be maintained throughout the process. The use of 1 mM IPTG, despite the higher stress on the cells, resulted in a significant increase in protein yield, which is not achievable with lower IPTG concentrations. This approach significantly increased efficiency and yielded proteins in greater quantities and with improved purity and activity.

[0041] The proposed method for optimizing strains of probiotic microorganisms capable of mono- and co-expression of heterologous proteins, with modification of fermentation and expression conditions, compared to the prototype and other known solutions, has the following advantages:

[0042] 1. A modified MRS environment with additional components is used.

[0043] 2. A three-stage process including growth phase, induction phase and production phase.

[0044] 3. High-precision sensors are used to control pH, temperature and dissolved oxygen during the fermentation process.

[0045] 4. Provides recombinant proteins with a purity of over 95% and high yield (e.g. 120±15 mg / L for IL-10).