Production method for improving content of odd-chain fatty acid in grease by modifying schizochytrium limacinum and optimizing substrate and application

Abstract

The invention belongs to the technical field of biochemical engineering, and discloses a production method for improving the content of odd-chain fatty acid in grease by modifying schizochytrium limacinum and optimizing a substrate and application of the method. According to the method, schizochytrium limacinum HX-308 serves as a receptor strain, high-yield odd-chain fatty acid engineering bacteria are obtained through gene overexpression and inhibition, and the high-yield odd-chain fatty acid is obtained. Accumulation of a propionyl CoA precursor pool for synthesizing odd-chain fatty acid is improved, endogenous methylmalonyl-CoA mutase coding gene MUT is inhibited, so that a competitive pathway of the odd-chain fatty acid is inhibited, and then through substrate optimization, the content of fermented grease and the content of the odd-chain fatty acid are obviously improved. The expression of related enzymes in schizochytrium limacinum is adjusted through a genetic engineering strategy to improve the odd-chain fatty acid synthesis, the synthesis and utilization of the key intermediate propionyl coenzyme A in the odd-chain fatty acid synthesis process are increased, the metabolic pathway of the propionyl coenzyme A is inhibited, and substrate optimization is assisted to improve the odd-chain fatty acid yield.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological chemical industry, and particularly relates to a method for improving the content of odd-chain fatty acids in oil by modifying a schizochytrium and optimizing a substrate and application thereof. BACKGROUND

[0002] Odd-chain saturated fatty acids (OCFAs), i.e., saturated fatty acids with an odd number of carbon atoms. OCFAs are a class of trace active fatty acids that can regulate the fluidity of biological membrane lipids and enhance their antioxidant capacity. Their food sources mainly include dairy products, ruminant fats, marine fish, etc. Intake of dairy products is the main way for humans to obtain OCFAs from food. Odd-chain fatty acids (OCFAs) naturally exist in bacteria, higher animals and plants, but the content is extremely low. At present, odd-chain fatty acid oil, as a non-conventional oil, has shown various application prospects in food, medical and chemical industries. Odd-chain fatty acids and their derivatives have attracted attention due to their beneficial physiological effects and potential as a substitute for premium fuels. OCFAs have been detected in human serum, liver, brain tissue, fat, red blood cells and other sites. A number of studies have shown that increased dietary intake and blood levels of OCFAs are associated with a reduced risk of obesity, chronic inflammation, cardiovascular disease, metabolic syndrome, type 2 diabetes, non-alcoholic steatohepatitis (NASH), chronic obstructive pulmonary disease, pancreatic cancer and other conditions. Different chain lengths of OCFAs exhibit a wide range of functions and applications. It has been reported that a new drug using medium-chain OCFAs has been approved for the treatment of fatty acid oxidation disorders. As a long-chain OCFAs, C17:1 has been used to treat allergies and autoimmune diseases due to its anti-inflammatory effects. Similarly, another long-chain OCFAs, C15:0 is believed to inhibit the migration and invasion ability of human breast cancer stem cells. In addition, due to the effects of OCFAs on health, serum levels of C15:0 and C17:0 have been used to assess the risk of coronary heart disease and type II diabetes. Not only that, but the biological properties of OCFAs have also been more widely studied, including antibiotic, cholesterol-lowering and anti-thrombotic effects.

[0003] Schizochytrium limacinum is a DHA-rich heterotrophic marine protist that has been widely used in scientific research and commercial production due to its fast growth, high lipid content, and ease of cultivation. Oily microorganisms have received increasing attention due to their unique characteristics. Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid (PUFA) that is essential for the development of the human nervous system and retinal tissue. Epidemiological studies have shown that DHA intake from diet has many health benefits, so DHA has been widely used in infant formula, nutritional products, food supplements, and pharmaceuticals. DHA is traditionally extracted from marine fish oil, but this method is not sustainable due to environmental pollution and overfishing, and the yield cannot meet the growing market demand for DHA. To date, Schizochytrium limacinum has been found to accumulate fatty acids mainly composed of DHA, DPA (docosapentaenoic acid), C16:0, and C14:0. Foods containing omega-3 polyunsaturated fatty acids (PUFAs) such as DHA and EPA (eicosapentaenoic acid) are considered important factors in promoting the development of retinal and brain function and reducing the risk of cardiovascular disease. Given the enormous benefits and growing demand for omega-3 fatty acids, the proportion of omega-3 fatty acids, particularly DHA, in the total fatty acids of Schizochytrium limacinum has become a focus of attention.

[0004] Currently, the production methods of DHA and OCFAs mainly include chemical synthesis, natural product extraction and microbial fermentation. Chemical synthesis and natural product extraction methods need to use a large number of chemicals reagents, which are high in cost and low in efficiency. Microbial fermentation method is one of the most potential industrial production methods. The microbial fermentation method for producing high value-added products has the advantages of low cost, short production cycle and environmental friendliness. Combined with genetic engineering and fermentation control process, the performance and fermentation state of microorganisms can be accurately controlled, so as to achieve the purpose of high performance production and high density fermentation. It has been reported that OCFAs are produced by yeasts, Escherichia coli and the like, and the synthesis of OCFAs is improved by engineering the precursors, but the yield is very small. Schizochytrium as an oleaginous microorganism has FAS (fatty acid synthase) pathway and PKS (polyketide synthase) pathway, and the two systems respectively dominate the synthesis of different types of fatty acids. Schizochytrium synthesizes DHA in a more energy-saving way (PKS), and its high-yield DHA characteristics have been proven and applied in the industrial field. It is found that Schizochytrium can also synthesize OCFAs through the fatty acid synthase pathway (FAS). Importantly, the PKS pathway cannot use propionyl coenzyme A, an intermediate product in the synthesis of OCFAs, as a substrate to generate OCFAs, which provides an opportunity to accumulate OCFAs without affecting DHA. So far, the industrial application of Schizochytrium has been focused on the field of DHA, and the research tends to be perfect, and the potential of using it to produce OCFAs is huge, which is expected to realize the joint production of DHA and OCFAs. Therefore, under the condition of improving the production of odd-chain fatty acids by Schizochytrium, the effective production of DHA is maintained. It is expected to realize the efficient production of OCFAs by taking advantage of the advantages of Schizochytrium in oil production.

[0005] DHA and OCFAs are important health products and industrial intermediates. The present application helps to improve the commercial potential of Schizochytrium, and provides valuable reference for the production of high value-added products of microalgae. Under the premise of maintaining the DHA production capacity of Schizochytrium, the production of OCFAs in Schizochytrium is greatly improved by inhibiting the decomposition pathway of precursors, increasing the precursor pool and optimizing the substrate combination. OCFAs, as a class of trace active fatty acids, can regulate the fluidity of biological membrane lipids and enhance their antioxidant capacity to reduce oxidative damage. Therefore, the production of OCFAs will be very meaningful to improve the quality and production efficiency of oil during the fermentation process of Schizochytrium. SUMMARY

[0006] The purpose of the present application is to overcome the deficiencies in the prior art, and to provide a method for improving the content of odd-chain fatty acids in oil by modifying Schizochytrium and optimizing substrates and applications.

[0007] The technical scheme adopted by the present application to solve its technical problems is: The application discloses an odd-chain fatty acid high-yield engineering bacterium, which is obtained by gene overexpression and inhibition based on a Schizochytrium sp. HX-308 as a receptor strain, wherein the overexpression genes are derived from a Schizochytrium sp. endogenous propionyl-CoA synthetase encoding gene PRPE, an exogenous propionyl-CoA synthetase encoding gene SACE-1780 from S. erythraea propionylation insensitivity and a propionyl-CoA synthetase encoding gene ScPPs of Salmonella enterica, so as to improve accumulation of propionyl-CoA precursor pool for synthesizing odd-chain fatty acids and inhibit an endogenous methylmalonyl-CoA mutase encoding gene MUT. The sequence of the gene PRPE is shown in SEQ ID No. 1, the sequence of the gene SACE-1780 is shown in SEQ ID No. 3, and the sequence of the gene ScPPs is shown in SEQ ID No. 2.

[0008] The application further discloses a method for improving odd-chain fatty acid content in oil by modifying Schizochytrium sp. and optimizing substrates, which is obtained by gene overexpression and inhibition based on a Schizochytrium sp. HX-308 as a receptor strain, so as to improve accumulation of propionyl-CoA precursor pool for synthesizing odd-chain fatty acids, inhibit an endogenous methylmalonyl-CoA mutase encoding gene MUT, thereby inhibiting an odd-chain fatty acid competitive pathway, and then optimize substrates, so that the content of fermented oil and odd-chain fatty acids is obviously improved.

[0009] Further, the method comprises the following steps: The high-yield odd-chain fatty acid engineering bacterium is inoculated into a seed culture medium to be activated, and then a fermentation engineering bacterium seed is obtained; the fermentation engineering bacterium seed is inoculated into a fermentation culture medium to be cultured, 10 mM sodium propionate is added into the fermentation culture medium at 0 h of fermentation, and then the fermentation culture medium is shaken at 25-30 DEG C and 150-250 r / min for 48-120 h to obtain a fermentation liquid; and oil is extracted from the collected bacterium.

[0010] Further, the fermentation engineering bacterium seed is obtained by the following method: the high-yield odd-chain fatty acid engineering bacterium is inoculated into a seed culture medium to obtain a first-level seed; the first-level seed is inoculated into a seed culture medium to obtain a second-level seed; and the second-level seed is inoculated into a seed culture medium to obtain a third-level seed as the fermentation engineering bacterium seed.

[0011] Further, the culture condition is that the culture is carried out at 25-30 DEG C and 150-250 r / min, and the inoculation amount is 2%.

[0012] Further, the inoculation volume ratio of the fermentation engineering strain inoculated in the fermentation medium is 1:9; or the whole cycle of the fermentation culture is 48h, 60h, 72h, 84h, 96h or 120h.

[0013] Further, the method for collecting the bacterial cells to extract oil includes: 1) After the fermentation culture is finished, NaOH solution is added to adjust the pH to 10-13, and then 0.01-0.5% of a cell wall breaking enzyme is added, and the mixture is oscillated at 40-60°C and 100-200r / min for 5-15h; 2) The mixture is cooled to room temperature, and anhydrous ethanol is added to inactivate the cell wall breaking enzyme; 3) n-hexane is added for extraction, and the upper organic phase is collected; 4) Step 3) is repeated several times, the organic phases are combined, and the solvent is evaporated to obtain the oil.

[0014] Further, the high-odd-chain fatty acid-producing engineering strain is inoculated in a plate medium, and after resistance plate culture, a single colony is picked and inoculated in a seed culture medium for activation, and then inoculated in a seed culture medium for culture to obtain a primary seed; wherein the pH value of the plate medium is 6.0-6.5, and the plate medium includes agar 15-20g / L, glucose 30-60g / L, yeast extract powder 8-15g / L, sodium sulfate 10-15g / L, magnesium sulfate 2-4g / L, ammonium sulfate 6-12g / L, potassium chloride 1-2g / L, calcium chloride 0.1-0.2g / L, potassium sulfate 0.5-1g / L, potassium dihydrogen phosphate 0.5-2g / L, sodium glutamate 8-12g / L, zinc sulfate heptahydrate 1-5mg / L, cobalt chloride hexahydrate 0.01-0.1mg / L, copper sulfate pentahydrate 2-6mg / L, nickel sulfate hexahydrate 1-2mg / L, iron sulfate heptahydrate 8-15mg / L, calcium pantothenate 2-4mg / L, manganese chloride tetrahydrate 3-5mg / L, sodium molybdate dihydrate 0.04mg / L, vitamin B64-10mg / L, vitamin B 12 0.1-1.5mg / L; The seed culture medium has a pH value of 6.0-6.5 and comprises: 40-60 g / L of glucose, 4-6 g / L of yeast extract powder, 5-8 g / L of sodium sulfate, 2-4 g / L of magnesium sulfate, 4-8 g / L of ammonium sulfate, 1-2 g / L of potassium chloride, 0.1-0.2 g / L of calcium chloride, 0.5-1 g / L of potassium sulfate, 0.5-2 g / L of potassium dihydrogen phosphate, 8-12 g / L of sodium glutamate, 1-5 mg / L of zinc sulfate heptahydrate, 0.01-0.1 mg / L of cobalt chloride hexahydrate, 2-6 mg / L of copper sulfate pentahydrate, 1-2 mg / L of nickel sulfate hexahydrate, 8-15 mg / L of iron sulfate heptahydrate, 2-4 mg / L of calcium pantothenate, 3-5 mg / L of manganese chloride tetrahydrate, and 0.04 mg / L of sodium molybdate dihydrate.

[0015] The fermentation culture medium has a pH value of 6.0-6.5 and comprises: 60-100 g / L of glucose, 5-15 g / L of yeast extract powder, 5-12 g / L of sodium sulfate, 2-4 g / L of magnesium sulfate, 4-8 g / L of ammonium sulfate, 1-2 g / L of potassium chloride, 0.1-0.2 g / L of calcium chloride, 0.5-1 g / L of potassium sulfate, 0.5-2 g / L of potassium dihydrogen phosphate, 15-20 g / L of sodium glutamate, 1-5 mg / L of zinc sulfate heptahydrate, 0.01-0.1 mg / L of cobalt chloride hexahydrate, 2-6 mg / L of copper sulfate pentahydrate, 1-2 mg / L of nickel sulfate hexahydrate, 8-15 mg / L of iron sulfate heptahydrate, 2-4 mg / L of calcium pantothenate, 3-5 mg / L of manganese chloride tetrahydrate, 0.04 mg / L of sodium molybdate dihydrate, 4-10 mg / L of vitamin B6, 0.1-0.5 mg / L of vitamin B12, 0.1-0.5 mg / L of biotin, and 0.1-0.5 mg / L of folic acid. 12 0.1-0.5 mg / L.

[0016] The method as described above is applied to increasing the content of odd-chain fatty acids in oil.

[0017] The high-yield odd-chain fatty acid engineering bacteria as described above are applied to increasing the content of odd-chain fatty acids in oil.

[0018] The application has the following advantages and positive effects: 1、The present application is based on the traditional fermentation of Schizochytrium sp., through genetic engineering modification and substrate optimization, the oil yield, oil quality and the proportion of odd-chain fatty acids in oil are significantly improved. The traditional fermentation oil yield is 13.9g / L, under the optimal sodium propionate addition concentration of 10mM fermentation, the oil yield is 18.67g / L, the yield is increased by 34%, the DHA content is increased, the final production of DHA oil yield is 9.2g / L, compared with the traditional fermentation without adding sodium propionate, it is increased by 41.1%, the most significant is that the content of odd-chain fatty acids is increased from 0.44g / L to 2.2g / L, the yield is increased by 4 times. After genetic engineering modification, under the condition of adding the optimal concentration of sodium propionate, the oil and DHA do not occur further significant change, but the proportion of OCFAs is significantly increased from 11.9% to 28.9%, the OCFAs yield is as high as 5.7g / L, compared with the yield of 2.09g / L of wild type added sodium propionate fermentation, it is increased by 1.7 times.

[0019] 2、The present application compares the differences between the substrate optimization fermentation and the traditional fermentation of Schizochytrium sp. of the genetically engineered strain, and determines that after genetic engineering modification, the up-regulation of propionyl CoA synthetase in odd-chain fatty acid synthesis increases the synthesis of propionyl CoA, promotes the effective participation of propionyl CoA in the synthesis pathway of odd-chain fatty acid, inhibits the competitive pathway of propionyl CoA, thereby improving the utilization rate of OCFAs, increasing the synthesis of oil and the production of odd-chain fatty acids.

[0020] 3、The present application adjusts the concentration of the added substrate sodium propionate, and successfully realizes the accumulation of oil and odd-chain fatty acids by batch-continuous feeding Schizochytrium sp., and the content of DHA is not significantly affected.

[0021] 4、The present application regulates the expression of related enzymes in Schizochytrium sp. (such as) by genetic engineering strategy to improve the synthesis of odd-chain fatty acids, increase the synthesis and utilization of key intermediate propionyl coenzyme A in the synthesis process of odd-chain fatty acids, inhibit the metabolic pathway of propionyl coenzyme A, and assist with substrate optimization to improve the yield of odd-chain fatty acids.

[0022] 5、The present application uses Schizochytrium sp. Schizochytrium HX-308 modified engineering strain, regulates the synthesis of odd-chain fatty acids in Schizochytrium sp. by gene, increases the synthesis of oil and the production of odd-chain fatty acids, and lays a foundation for industrial production of products. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 DHA and odd-chain fatty acid difference chart in the fermentation of Schizochytrium sp. HX-308 in the present application at different times of adding sodium propionate; Figure 2 Biomass (CDW) chart of Schizochytrium sp. HX-308 in the present application under different concentrations of sodium propionate in substrate fermentation; Figure 3 OD of different concentrations of sodium propionate in fermentation of the Schizochytrium sp. HX-308 in the presence of the substrate sodium propionate 600 Case diagram Figure 4 OD of different concentrations of sodium propionate in fermentation of the Schizochytrium sp. HX-308 in the presence of the substrate sodium propionate Figure 5 OD of different concentrations of sodium propionate in fermentation of the Schizochytrium sp. HX-308 in the presence of the substrate sodium propionate Figure 6 OD of different concentrations of sodium propionate in fermentation of the Schizochytrium sp. HX-308 in the presence of the substrate sodium propionate Figure 7 OD of different concentrations of sodium propionate in fermentation of the Schizochytrium sp. HX-308 in the presence of the substrate sodium propionate Figure 8 OD of different concentrations of sodium propionate in fermentation of the Schizochytrium sp. HX-308 in the presence of the substrate sodium propionate DETAILED DESCRIPTION

[0024] The present application is further described below in conjunction with examples, which are illustrative and not limiting, and cannot be used to limit the protection scope of the present application.

[0025] The various experimental operations involved in the specific examples are conventional techniques in the art, and the parts not specifically noted herein can be implemented by referring to various conventional toolbooks, scientific literature or related instructions, manuals, etc. before the filing date of the present application.

[0026] The equipment, reagents, processes, parameters, etc. involved in the present application are conventional equipment, reagents, processes, parameters, etc. unless otherwise specified, and will not be described in the examples.

[0027] All ranges listed in the present application include all point values in the range.

[0028] In the present application, unless otherwise indicated or specified, all percentages are mass percentages, and the ratios are mass ratios. The units of mass are, for example, grams, kilograms or tons.

[0029] In the present application, the "room temperature" is the conventional ambient temperature, which can be 10-30°C.

[0030] The application discloses an odd-chain fatty acid high-yield engineering bacterium, which is obtained by gene overexpression and inhibition based on a Schizochytrium sp. HX-308 as a receptor strain, wherein the overexpression genes are derived from a Schizochytrium sp. endogenous propionyl-CoA synthetase encoding gene PRPE, an exogenous propionyl-CoA synthetase encoding gene SACE-1780 from S. erythraea propionylation insensitivity and a propionyl-CoA synthetase encoding gene ScPPs of Salmonella enterica, so as to improve accumulation of propionyl-CoA precursor pool for synthesizing odd-chain fatty acids and inhibit an endogenous methylmalonyl-CoA mutase encoding gene MUT. The sequence of the gene PRPE is shown in SEQ ID No. 1, the sequence of the gene SACE-1780 is shown in SEQ ID No. 3, and the sequence of the gene ScPPs is shown in SEQ ID No. 2.

[0031] The application further discloses a method for improving odd-chain fatty acid content in oil by modifying Schizochytrium sp. and optimizing substrates, which comprises the following steps: obtaining the odd-chain fatty acid high-yield engineering bacterium by gene overexpression and inhibition based on a Schizochytrium sp. HX-308 as a receptor strain, improving accumulation of propionyl-CoA precursor pool for synthesizing odd-chain fatty acids, inhibiting an endogenous methylmalonyl-CoA mutase encoding gene MUT, so as to inhibit an odd-chain fatty acid competitive pathway, and optimizing substrates.

[0032] Preferably, the method comprises the following steps: The high-yield odd-chain fatty acid engineering bacterium is inoculated into a seed culture medium to activate the engineering bacterium seed for fermentation; the engineering bacterium seed for fermentation is inoculated into a fermentation culture medium to perform fermentation culture, 10 mM sodium propionate is added into the fermentation culture medium at 0 h of fermentation, and fermentation is performed at 25-30 DEG C and 150-250 r / min for 48 h-120 h to obtain a fermentation liquor; and oil is extracted from the collected bacterium.

[0033] Preferably, the engineering bacterium seed for fermentation is obtained by the following method: the high-yield odd-chain fatty acid engineering bacterium is inoculated into a seed culture medium to obtain a first-level seed; the first-level seed is inoculated into a seed culture medium to obtain a second-level seed; and the second-level seed is inoculated into a seed culture medium to obtain a third-level seed as the engineering bacterium seed for fermentation.

[0034] Preferably, the culture condition is 25-30 DEG C and 150-250 r / min shaking culture, and the inoculation amount is 2%.

[0035] Preferably, the inoculation volume ratio of the fermentation with the engineering strain in the fermentation medium is 1:9; or the whole cycle of the fermentation culture is 48h, 60h, 72h, 84h, 96h or 120h.

[0036] Preferably, the method for collecting the bacterial cells to extract the oil includes: 1) After the fermentation culture is finished, NaOH solution is added to the fermentation broth to adjust the pH to 10-13, and then 0.01-0.5% of a cell wall breaking enzyme is added, and the mixture is oscillated at 40-60°C and 100-200r / min for 5-15h; 2) The mixture is cooled to room temperature, and anhydrous ethanol is added to inactivate the cell wall breaking enzyme; 3) n-hexane is added for extraction, and the upper organic phase is collected; 4) Step 3) is repeated several times, the organic phases are combined, and the solvent is evaporated to obtain the oil.

[0037] Preferably, the high-odd-chain fatty acid-producing engineering strain is inoculated in a plate medium, and after resistance plate culture, a single colony is picked and inoculated in a seed culture medium for activation, and then inoculated in a seed culture medium to obtain a primary seed; wherein the pH value of the plate medium is 6.0-6.5, and the plate medium includes agar 15-20g / L, glucose 30-60g / L, yeast extract powder 8-15g / L, sodium sulfate 10-15g / L, magnesium sulfate 2-4g / L, ammonium sulfate 6-12g / L, potassium chloride 1-2g / L, calcium chloride 0.1-0.2g / L, potassium sulfate 0.5-1g / L, potassium dihydrogen phosphate 0.5-2g / L, sodium glutamate 8-12g / L, zinc sulfate heptahydrate 1-5mg / L, cobalt chloride hexahydrate 0.01-0.1mg / L, copper sulfate pentahydrate 2-6mg / L, nickel sulfate hexahydrate 1-2mg / L, iron sulfate heptahydrate 8-15mg / L, calcium pantothenate 2-4mg / L, manganese chloride tetrahydrate 3-5mg / L, sodium molybdate dihydrate 0.04mg / L, vitamin B64-10mg / L, vitamin B 12 0.1-1.5mg / L; The seed culture medium has a pH value of 6.0-6.5 and comprises: glucose 40-60 g / L, yeast extract powder 4-6 g / L, sodium sulfate 5-8 g / L, magnesium sulfate 2-4 g / L, ammonium sulfate 4-8 g / L, potassium chloride 1-2 g / L, calcium chloride 0.1-0.2 g / L, potassium sulfate 0.5-1 g / L, potassium dihydrogen phosphate 0.5-2 g / L, sodium glutamate 8-12 g / L, zinc sulfate heptahydrate 1-5 mg / L, cobalt chloride hexahydrate 0.01-0.1 mg / L, copper sulfate pentahydrate 2-6 mg / L, nickel sulfate hexahydrate 1-2 mg / L, iron sulfate heptahydrate 8-15 mg / L, calcium pantothenate 2-4 mg / L, manganese chloride tetrahydrate 3-5 mg / L, and sodium molybdate dihydrate 0.04 mg / L.

[0038] The fermentation culture medium has a pH value of 6.0-6.5 and comprises: glucose 60-100 g / L, yeast extract powder 5-15 g / L, sodium sulfate 5-12 g / L, magnesium sulfate 2-4 g / L, ammonium sulfate 4-8 g / L, potassium chloride 1-2 g / L, calcium chloride 0.1-0.2 g / L, potassium sulfate 0.5-1 g / L, potassium dihydrogen phosphate 0.5-2 g / L, sodium glutamate 15-20 g / L, zinc sulfate heptahydrate 1-5 mg / L, cobalt chloride hexahydrate 0.01-0.1 mg / L, copper sulfate pentahydrate 2-6 mg / L, nickel sulfate hexahydrate 1-2 mg / L, iron sulfate heptahydrate 8-15 mg / L, calcium pantothenate 2-4 mg / L, manganese chloride tetrahydrate 3-5 mg / L, sodium molybdate dihydrate 0.04 mg / L, vitamin B6 4-10 mg / L, vitamin B12 0.1-0.5 mg / L, and vitamin B1 0.1-0.5 mg / L. 12 0.1-0.5 mg / L.

[0039] The method as described above is applied to increasing the content of odd-chain fatty acids in oil.

[0040] The high-yield odd-chain fatty acid engineering bacteria as described above are applied to increasing the content of odd-chain fatty acids in oil.

[0041] Specifically, the preparation and detection are as follows: The culture medium used in the following examples is as follows: The plate culture medium has a pH of 6.6 and includes: agar 15-20 g / L, glucose 40 g / L, yeast extract 10 g / L, sodium sulfate 10 g / L, magnesium sulfate 2 g / L, ammonium sulfate 6 g / L, potassium chloride 1 g / L, calcium chloride 0.1 g / L, potassium sulfate 0.6 g / L, potassium dihydrogen phosphate 1 g / L, monosodium glutamate 10 g / L, 0.1% trace minerals (zinc sulfate heptahydrate 3 g / L, cobalt chloride hexahydrate 0.05 g / L, copper sulfate pentahydrate 5 g / L, nickel sulfate hexahydrate 1 g / L, ferric sulfate heptahydrate 10 g / L, calcium pantothenate 4 g / L, manganese chloride tetrahydrate 5 g / L, sodium molybdate dihydrate 0.04 g / L), vitamin B6 5 mg / L, and vitamin B1. 12 0.5 mg / L.

[0042] The seed culture medium for the seed culture solution had a pH of 6.6 and included: 50 g / L glucose, 5 g / L yeast extract, 5 g / L sodium sulfate, 2 g / L magnesium sulfate, 6 g / L ammonium sulfate, 1 g / L potassium chloride, 0.1 g / L calcium chloride, 0.6 g / L potassium sulfate, 1 g / L potassium dihydrogen phosphate, 10 g / L monosodium glutamate, 0.1% trace minerals, 5 mg / L vitamin B6, and vitamin B1. 12 0.5 mg / L.

[0043] The fermentation medium has a pH of 6.0–7.5 and comprises: 80 g / L glucose, 10 g / L yeast extract, 10 g / L sodium sulfate, 2 g / L magnesium sulfate, 6 g / L ammonium sulfate, 1 g / L potassium chloride, 0.1 g / L calcium chloride, 0.6 g / L potassium sulfate, 1 g / L potassium dihydrogen phosphate, 20 g / L monosodium glutamate, 0.1% trace minerals, 5 mg / L vitamin B6, and vitamin B12. 12 0.5 mg / L.

[0044] Example 1. Sodium propionate as a substrate increases the total amount of propionyl-CoA in Schizochytrium, effectively increasing the synthesis of odd-chain fatty acids, and enhancing lipid synthesis and odd-chain fatty acid production. (Addition time screening) Selecting Schizochytrium ( Schizochytrium A single colony of HX-308 (sp.) was inoculated into a 250 mL Erlenmeyer flask (containing 50 mL of seed culture medium) and cultured at 28°C and 180 rpm for 24 h to obtain the first-generation seed culture. 1 mL of the first-generation seed culture was inoculated into a 250 mL Erlenmeyer flask (containing 50 mL of seed culture medium) and cultured at 28°C and 180 rpm for 24 h to obtain the second-generation seed culture. 1 mL of the second-generation seed culture was inoculated into a 250 mL Erlenmeyer flask (containing 50 mL of seed culture medium) and cultured at 28°C and 180 rpm for 24 h to obtain the third-generation seed culture, which was used as the fermentation strain.

[0045] 1. Shake flask fermentation culture: 10 mL of the third level seed culture was inoculated into a 500 mL conical flask (containing 90 mL of fermentation medium), 10 mM sodium propionate was added at 0 h / 12 h / 24 h / 36 h of fermentation, and the flask was incubated at 28°C, 180 r / min for 120 h. The fermentation broth was collected, and the fatty acid composition was measured.

[0046] 2. Collection of bacterial cells to extract oil, including the following steps: (1) After the fermentation culture was completed, NaOH solution was added to the fermentation broth to adjust the pH to 10-13, and 0.01-0.2% of a cell wall breaking enzyme was added, and the mixture was oscillated at 40-60°C and 100-200 r / min for 5-15 h; (2) The mixture was cooled to room temperature, and anhydrous ethanol was added to inactivate the cell wall breaking enzyme; (3) Oil was extracted by adding n-hexane: The extracted organic phase was allowed to stand for 5 h, and after the upper and lower layers were clearly separated, the upper organic phase was collected. The upper organic phase was taken out and placed in a rotary evaporation flask, which was rotated at 45°C water bath and 120 r / min. After the organic phase was no longer evaporated, the rotary evaporation flask was removed and dried in a 60°C oven until the weight no longer changed. The oil was weighed and obtained; (4) Gas phase detection analysis of fatty acids, the specific procedure is as follows: 20 μL of oil was added to an EP tube containing 1 ml of 1M potassium hydroxide-methanol solution, and oscillated at 20°C and 1000 r / min for 6 h. 50 μL of concentrated sulfuric acid was added to terminate the reaction, 1 ml of n-hexane was added, and the mixture was oscillated at 20°C and 1000 r / min for 0.5 h to extract the oil. The extracted phase was loaded into a liquid phase vial for gas phase detection. GC-2010 (Shimadzu, Japan) gas phase system was used for analysis, equipped with DB-23 capillary column (60 m 0.22 mm) and flame ionization detector (FID). Nitrogen was used as the carrier gas. The injection volume was 1 μL, and the injection temperature was 250°C. The column temperature was increased from 100°C to 200°C at a rate of 25°C / min, and then increased to 230°C at a rate of 4°C / min, and maintained for 9 min. The FID detector temperature was 280°C. By comparing with sigma standard, different fatty acid compositions were identified. Non-endogenous fatty acid (C19:0) was used as an internal standard, and the content of individual fatty acid was calculated from the peak area on the chromatogram.

[0047] The results are as follows: Figure 1As shown, the same amount (10 mM) of sodium propionate was added at different times, and the oil production showed no significant difference, but the data showed that the proportion of DHA and odd-chain fatty acids was significantly different, where the proportion of DHA added at 12, 24, and 36 h was 47.6%, 48.1%, and 47.2%, respectively, and the proportion of OCFAs was 9.9%, 10.1%, and 9.4%, respectively. It was found that the fermentation with sodium propionate added at 0 h showed higher proportions of DHA and OCFAs, with the proportion of DHA being 49.3% and the proportion of OCFAs being 11.8%. Compared with the fermentation without the addition of sodium propionate, the oil composition changed significantly, especially the proportion of OCFAs increased significantly. Therefore, the addition of sodium propionate at different time points of 0, 12, 24, and 36 h, and the addition of sodium propionate at 0 h showed the best fermentation production effect.

[0048] Example 2. Sodium propionate as a three-carbon substrate increases the synthesis of propionyl CoA in Schizochytrium, enters the odd-chain fatty acid synthesis pathway, and increases the production of oil and odd-chain fatty acids Different amounts of sodium propionate were weighed and added to the fermentation medium.

[0049] According to Example 1, the seed liquid culture and fermentation were carried out, except that 0 / 5 / 10 / 20 / 50 / 100 mM sodium propionate was added at 0 h of fermentation. After fermentation, the biomass, OD 600 , residual sugar; sodium propionate residue; oil content; DHA and odd-chain fatty acid yield were detected. Including the following steps: (1) Determination of glucose and sodium glutamate concentration, the specific procedure is as follows: take 1 mL of fermentation liquid at a certain time point, centrifuge at 12000 rpm and take the supernatant gradient dilution to 100 times, dilute the liquid and use SBA-40ES for detection. Glucose and sodium glutamate are determined by different enzyme membranes, which are calibrated in advance with standard solution.

[0050] (2) Determination of cell dry weight (CDW), the specific procedure is as follows: take the fermentation liquid (1 mL) at a certain time point to the EP tube (the mass of the EP tube is determined in advance), centrifuge to remove liquid interference, then wash the bacterial body with deionized water, finally put the EP tube in the oven at 65℃ and dry (about 48h) to constant weight, calculate CDW. Determination of oil content: put the pre-determined mass of the rotary evaporator bottle collecting oil in the oven at 65℃ and dry to constant weight, and weigh after cooling.

[0051] (3) Fatty acid composition detection is as described in Example 1 above.

[0052] (4) Take 5 ml of the fermentation broth that has just finished fermentation, centrifuge it, add an equal volume of methanol to the fermentation broth and soak and extract it several times. Combine the methanol extracts, remove the methanol by rotary vacuum evaporator, add carbon tetrachloride to dissolve the concentrate, and filter it through a 0.22 μm organic filter membrane for later use. The sodium propionate content was determined by high performance liquid chromatography. Chromatographic column: Amethyst C18-H column (4.6X150mm, 5μm), detection wavelength 475nm, column temperature at room temperature, mobile phase flow rate of 1.0 mL / min, gradient elution was used to prepare the corresponding sodium propionate standard sample and obtain the standard curve equation.

[0053] The results are as follows Figures 2-6 As shown, 10 mM sodium propionate was the optimal addition amount for fermentation. At this level, the oil yield was higher and the proportion of odd-chain fatty acids was higher compared to other addition concentrations, reaching 18.67 g / L, a 34% increase compared to the 13.9 g / L yield of fermentation with 0 mM sodium propionate. The proportion of odd-chain fatty acids increased from 3.2% in the 0 mM sodium propionate group to 11.8% (10 mM). Data from other concentration groups showed that their fermentation effects were lower than the 10 mM group. The fermentation data for the remaining groups are shown in Table 1. Table 1 shows that the fatty acid composition underwent significant changes, with a marked increase in the proportion of odd-chain fatty acids compared to fermentation without sodium propionate. Figure 2 , Figure 3 , Figure 4 Analysis showed that, compared with no addition, the addition of an appropriate concentration of sodium propionate (10 mM) resulted in an increase in biomass or OD. 600 No significant inhibition was observed. Adding an appropriate concentration of sodium propionate (10 mM) resulted in virtually no difference in biomass compared to the control group, with normal sugar consumption and stable, slightly increased, oil yield. However, higher concentrations of sodium propionate (50 mM, 100 mM) showed significant growth inhibition, with variations in biomass or OD at different time points. 600 All are at low levels. Figure 5 , Figure 6 The results showed that increasing the proportion of odd-chain fatty acids from 3.2% to 11.8% increased the OCFA yield from 0.44 g / L to 2.2 g / L, a fourfold increase. In other groups, the OCFA proportions at addition levels of 0, 5, 20, 50, and 100 mM were only 3.2%, 10.29%, 10.53%, 10.39%, and 9.81%, respectively, while the OCFA yields were only 0.44 g / L, 1.78 g / L, 1.85 g / L, 1.33 g / L, and 0.40 g / L, respectively. Therefore, in fermentation with different concentrations of 0 / 5 / 10 / 20 / 50 / 100 mM sodium propionate, 10 mM was the optimal addition level.

[0054] Table 1. Composition of oils and fatty acids produced by normal fermentation and fermentation with different concentrations of sodium propionate added by Schizochytrium HX-308.

[0055] Example 3 Other three-carbon substrates valine, isoleucine, n-propanol, glycerol, lactic acid improve the total amount of propionyl CoA in Schizochytrium sp., and screen and optimize substrates to effectively improve the synthesis of odd-chain fatty acids.

[0056] According to Example 1, seed liquid culture and fermentation were carried out in the early stage, except that: at 0 h of fermentation, the same molar mass (10 mM) of sodium propionate, valine, isoleucine, n-propanol, glycerol, and lactic acid were added for fermentation. After fermentation, the main reference parameters DHA and oil yield were detected; DHA and OCFAs composition.

[0057] The specific steps are carried out according to Example 2.

[0058] The results are shown in Figure 7 As shown, the oil biomass produced by adding sodium propionate and isoleucine as substrates for fermentation did not show obvious inhibition, and the oil yield was not affected and slightly increased. The oil yield of the control group (i.e., without adding any additional substrate) was 14.91 g / L, while the oil yield of the sodium propionate fermentation was increased to 18.67 g / L, and the isoleucine fermentation reached 17.99 g / L. The other experimental groups were also increased, but the effect was poor. The valine group, n-propanol group, glycerol group, and lactic acid group were only 16.12 g / L, 15.52 g / L, 14.57 g / L, and 14.32 g / L, respectively. In addition, the fatty acid composition of the sodium propionate group changed significantly compared to the control group. The proportion of odd-chain fatty acids in the sodium propionate and isoleucine fermentation was significantly increased. The OCFAs proportion of the sodium propionate group increased from 3.18% to 11.49%, and the isoleucine group increased to 9.89%. The other experimental groups were lower than these two groups. The valine group, n-propanol group, glycerol group, and lactic acid group were only 7.2%, 8.64%, 9.53%, and 7.95%, respectively, with poor effect. The data showed that sodium propionate showed the best effect in increasing the production of odd-chain fatty acids, and isoleucine also showed outstanding performance in increasing the production of odd-chain fatty acids and oil quality.

[0059] Example 4 Genetic engineering of Schizochytrium sp. HX-308 to construct high oil yield and odd-chain fatty acid content, and effectively produce DHA and odd-chain fatty acid oil engineering strain.

[0060] Synthesis of plasmid containing PRPE, ScPPs, SACE-1780 gene coding expression of propionyl-CoA synthase, design and construct shRNA plasmid (plasmid construction expression gene connected to the vector, the relevant method using the method known in the art can be used.), overexpression of propionyl-CoA synthase containing PRPE, ScPPs, SACE-1780 gene coding expression, improve the synthesis of propionyl-CoA precursor OCFA supply, using RNAi biological tools to inhibit MUT gene coding expression (MUT-RNAi) of methylmalonyl-CoA mutase, so as to inhibit the entry of propionyl-CoA into TCA cycle, reduce the competition way of propionyl-CoA through FAS pathway to synthesize OCFAs. (The required plasmid provides gene sequence, and the biological company provides technical support to synthesize the required plasmid containing specific resistance gene coding sequence). The relevant gene sequence is shown in Table 2. Prepare Agrobacterium tumefaciens strain AGL-1 electroporation, the implementation process is as follows: 1) Pick a single colony of strain AGL-1 from the LB (containing 50 μg / mL carbenicillin) plate into 10 mL of LB liquid medium (containing 50 μg / mL carbenicillin), 28 ℃, 200 rpm shaking bed overnight.

[0061] 2) Take 2 mL of the above bacterial solution into 50 mL of LB (containing 50 μg / mL carbenicillin (Carb) liquid medium, 28 ℃, 200 rpm shaking bed culture to OD600 at about 0.5-0.7, about 8 h.

[0062] 3) Take out the bacterial solution, ice bath for 30 min, then centrifuge at 4 ℃, 5000 rpm for 5 min to collect the bacterial cells.

[0063] 4) Resuspend with 20 mL of pre-cooled 10% (w / v) glycerol, centrifuge at 4 ℃, 4000 rpm for 10 min.

[0064] 5) Repeat step 4).

[0065] 6) Add 2 mL of pre-cooled 10% glycerol, aliquot 100 μL of competent cells in each EP tube, and store at -80 ℃ after aliquoting.

[0066] Subsequently, Agrobacterium tumefaciens strain AGL-1 is electroporated: 1) Take the Agrobacterium competent cells stored at -80 ℃, thaw on ice.

[0067] 2) Add 0.1-1 μg of plasmid containing the specific expression gene mentioned above (PRPE, ScPPs, SACE-1780, MUT-RNAi plasmid), mix well by blowing, and then transfer to a pre-cooled 2 mm electroporation cup.

[0068] 3) After 2500 V (Sc2) electroporation, quickly add 500 μL of LB liquid medium, mix well, and then transfer to a 1.5 mL EP tube. Incubate at 28°C, 150-180 rpm for 3 h.

[0069] 4) After centrifugation at 4000 rpm for 5 min, discard the supernatant, and spread the remaining bacterial solution on solid LB resistance (50 μg / mL carbenicillin and 50 μg / mL kanamycin) plates to screen for the correct Agrobacterium transformation strain.

[0070] Agrobacterium AGL-1 transformation of Schizochytrium sp. steps: 1) The Agrobacterium transformation strain was streaked on LB (50 μg / mL carbenicillin and 50 μg / mL kanamycin) plates and incubated at 28°C for 48 h.

[0071] 2) A single colony was picked and inoculated in 5 mL of LB (50 μg / mL carbenicillin and 50 μg / mL kanamycin) liquid medium and incubated at 28°C, 220 rpm on a shaker for 8-12 h.

[0072] 3) Centrifuge at 4000 rpm for 5 min to collect the bacterial cells, resuspend in 10 mL of IM liquid medium (50 μg / mL carbenicillin, 50 μg / mL kanamycin, 400 μM AS (acetyl-syringone), 0.1 mL MES (2-morpholinoethanesulfonic acid), and adjust the OD600 to 0.5 (dilute 5 times and detect OD600).

[0073] 4) Incubate at 28°C, 220 rpm on a shaker for about 12 h, and adjust the concentration to 1 10^9 cells / mL (sorbitol dilution, OD 0.5).

[0074] 5) At the same time as step 3), inoculate Schizochytrium sp. HX-308, and incubate the fermentation liquid seed solution for about 12 h, then transfer 1 mL of bacterial solution to a new seed culture medium and incubate for about 12 h.

[0075] 6) Centrifuge at 200 rpm for 5 min to collect the bacterial cells, and adjust the bacterial cell concentration to 1 10^8 cells / mL (blood cell counting plate number of grid, for example: after sorbitol dilution 100 times, add 100 uL counting, 30 points, then 1 mL of bacterial liquid centrifugal precipitation add 300 μL sorbitol). Take 100 μL of the above Agrobacterium bacterial liquid and the prepared Schizochytrium HX-308 bacterial liquid, add to a sterile 1.5 mL centrifuge tube, mix thoroughly and evenly, then spread on IM plate, and co-culture at 28 ℃ for 48 h.

[0076] 7) Add 2 mL sorbitol to the above culture dish, and scrape the co-culture with a spreader.

[0077] 8) Take 300 μL of the mixture scraped from the IM plate and spread on GPYS solid medium (containing 100 mg / ml cefotaxime sodium and 300 mg / mL G418, 10 g / L proteose peptone, 5 g / L yeast extract, 50 g / L glucose, 20 g / L sea salt), and incubate at 28 ℃ for 4-5 days, and observe whether resistant colonies (suspected transformants) appear.

[0078] 9) The suspected transformants are picked up with a toothpick and transferred to GPYS solid medium (containing 100 mg / mL cefotaxime sodium and 400 mg / mL G418, 10 g / L proteose peptone, 5 g / L yeast extract, 50 g / L glucose, 20 g / L sea salt), and at least passaged twice before verification.

[0079] Through the above process, the specific genes PRPE, ScPPs, SACE-1780, RNAi-MUT are introduced into Schizochytrium HX-308 through Agrobacterium AGL-1 to obtain the desired engineering strains PRPE engineering bacteria, ScPPs engineering bacteria, SACE-1780 engineering bacteria, RNAi-MUT engineering bacteria, and then replace Schizochytrium HX-308 with PRPE engineering bacteria, ScPPs engineering bacteria, SACE-1780 engineering bacteria as a chassis, and introduce MUT-RNAi into Schizochytrium PRPE engineering bacteria, ScPPs engineering bacteria, SACE-1780 engineering bacteria according to the same Agrobacterium AGL-1 transformation Schizochytrium step, thereby obtaining RNAi-MUT-SACE1780, RNAi-MUT-PRPE, RNAi-MUT-ScPPs engineering strains, and finally further fermentation verification.

[0080] Example 5 Analysis of biomass, oil yield, and fatty acid content of wild-type strains and genetically engineered strains under optimal substrate and conditions.

[0081] The seed liquid culture of wild strain HX-308 and the early stage of the engineered strain was carried out according to Example 1, and the optimal sodium propionate substrate, i.e., 10 mM sodium propionate, was added at the beginning of fermentation, i.e., at 0 h of fermentation, to carry out fermentation, and the biomass, oil yield, oil content, residual sugar, fatty acid composition, and odd-chain fatty acid yield were detected. The implementation process was carried out according to Example 2.

[0082] As shown in Table 3, the oil and odd-chain fatty acid contents of the oil produced by the normal fermentation of the Schizochytrium sp. HX-308 and the fermentation of the engineered strain under the optimal concentration of sodium propionate were compared. Figure 8 As shown in Table 3, the oil and odd-chain fatty acid contents of the oil produced by the normal fermentation of the Schizochytrium sp. HX-308 and the fermentation of the engineered strain under the optimal concentration of sodium propionate were compared.

[0083] Table 2. Required coding gene sequences

[0084] Table 3 is a comparison table of the oil and odd-chain fatty acid contents of the oil produced by the normal fermentation of the Schizochytrium sp. HX-308 and the fermentation of the engineered strain under the optimal concentration of sodium propionate.

[0085] Although the embodiments of the present application are disclosed for illustrative purposes, those skilled in the art can understand that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present application and the appended claims, and therefore, the scope of the present application is not limited to the disclosed content.

Claims

1. A high-yield engineered bacterium producing odd-chain fatty acids, characterized in that: The engineered bacteria are obtained by overexpressing and inhibiting *Schizochytrium* HX-308 as the recipient strain, resulting in high production of odd-chain fatty acids. The overexpressed genes include the endogenous propionyl-CoA synthase encoding gene PRPE from *Schizochytrium*, the exogenous propionyl-CoA synthase encoding gene SACE-1780 from *S. erythraea* which is insensitive to propionylation, and the propionyl-CoA synthase encoding gene ScPPs from *Salmonella enterica*. This process increases the accumulation of the propionyl-CoA precursor pool for the synthesis of odd-chain fatty acids and inhibits the endogenous methylmalonyl-CoA mutase encoding gene MUT. The sequence of the gene PRPE is shown in SEQ ID No. 1, the sequence of the gene SACE-1780 is shown in SEQ ID No. 3, and the sequence of the gene ScPPs is shown in SEQ ID No.

2.

2. A method for increasing the content of odd-chain fatty acids in oils by modifying Schizochytrium and optimizing substrates using engineered bacteria that produce high-yield odd-chain fatty acids as described in claim 1, characterized in that: The method uses Schizochytrium HX-308 as the recipient strain, and obtains an engineered strain that produces high levels of odd-chain fatty acids through gene overexpression and inhibition. This increases the accumulation of propionyl-CoA precursor pools for the synthesis of odd-chain fatty acids, inhibits the endogenous methylmalonyl-CoA mutase encoding gene MUT, thereby suppressing the competitive pathway of odd-chain fatty acids. Furthermore, through substrate optimization, the fermentation oil and the content of odd-chain fatty acids are significantly increased.

3. The method according to claim 2, characterized in that: Includes the following steps: High-yield odd-chain fatty acid engineered bacteria were inoculated into a seed culture medium and activated to obtain an engineered strain for fermentation. The engineered strain for fermentation was inoculated into a fermentation culture medium for fermentation culture. At 0 h of fermentation, 10 mM sodium propionate was added to the fermentation culture medium, and the mixture was shaken at 25-30℃ and 150-250 r / min for 48-120 h to obtain a fermentation broth. The bacterial cells were collected to extract the oil.

4. The method according to claim 3, characterized in that: The engineered microbial strain for fermentation is obtained by the following method: inoculating high-yield odd-chain fatty acid engineered bacteria into a seed culture medium and culturing to obtain a primary seed; taking the primary seed and inoculating it into a seed culture medium and culturing to obtain a secondary seed; taking the secondary seed and inoculating it into a seed culture medium and culturing to obtain a tertiary seed, which is used as the engineered microbial strain for fermentation.

5. The method according to claim 4, characterized in that: The culture conditions were 25–30°C, shaking culture at 150–250 r / min, and the inoculum size was 2%.

6. The method according to claim 3, characterized in that: The inoculation volume ratio of the engineered microorganisms used for fermentation to the fermentation medium is 1:9; or, the entire fermentation cycle is 48h, 60h, 72h, 84h, 96h, or 120h.

7. The method according to claim 3, characterized in that: The method for collecting bacterial cells and extracting oil includes: 1) After fermentation, add NaOH solution to the fermentation broth to adjust the pH to 10-13, then add 0.01-0.5% of cell wall breaking enzyme at a final mass concentration, and shake at 100-200 r / min for 5-15 h at 40-60℃. 2) Cool to room temperature and add an equal volume of anhydrous ethanol to the fermentation broth to inactivate the cell wall-breaking enzyme; 3) Extract with n-hexane and collect the upper organic phase; 4) Repeat step 3) several times, combine the organic phases, evaporate the solvent, and obtain the oil.

8. The method according to any one of claims 3 to 7, characterized in that: Engineered bacteria producing high levels of odd-chain fatty acids were inoculated onto agar plates. After culturing on resistant plates, single colonies were picked, activated, and then inoculated onto a seed culture medium to obtain primary seed culture. The agar plate medium had a pH of 6.0–6.5 and contained: 15–20 g / L agar, 30–60 g / L glucose, 8–15 g / L yeast extract, 10–15 g / L sodium sulfate, 2–4 g / L magnesium sulfate, 6–12 g / L ammonium sulfate, 1–2 g / L potassium chloride, 0.1–0.2 g / L calcium chloride, 0.5–1 g / L potassium sulfate, 0.5–2 g / L potassium dihydrogen phosphate, 8–12 g / L monosodium glutamate, 1–5 mg / L zinc sulfate heptahydrate, 0.01–0.1 mg / L cobalt chloride hexahydrate, 2–6 mg / L copper sulfate pentahydrate, and 1–2 mg / L nickel sulfate hexahydrate. mg / L, ferric sulfate heptahydrate 8~15 mg / L, calcium pantothenate 2~4 mg / L, manganese chloride tetrahydrate 3~5 mg / L, sodium molybdate dihydrate 0.04 mg / L, vitamin B6 4-10 mg / L, vitamin B 12 0.1-1.5 mg / L; The seed culture medium has a pH of 6.0–6.5 and comprises: 40–60 g / L glucose, 4–6 g / L yeast extract, 5–8 g / L sodium sulfate, 2–4 g / L magnesium sulfate, 4–8 g / L ammonium sulfate, 1–2 g / L potassium chloride, 0.1–0.2 g / L calcium chloride, 0.5–1 g / L potassium sulfate, 0.5–2 g / L potassium dihydrogen phosphate, 8–12 g / L monosodium glutamate, 1–5 mg / L zinc sulfate heptahydrate, 0.01–0.1 mg / L cobalt chloride hexahydrate, 2–6 mg / L copper sulfate pentahydrate, 1–2 mg / L nickel sulfate hexahydrate, 8–15 mg / L ferric sulfate heptahydrate, 2–4 mg / L calcium pantothenate, 3–5 mg / L manganese chloride tetrahydrate, and 0.04 mg / L sodium molybdate dihydrate. The fermentation medium has a pH of 6.0–6.5 and comprises: glucose 60–100 g / L, yeast extract 5–15 g / L, sodium sulfate 5–12 g / L, magnesium sulfate 2–4 g / L, ammonium sulfate 4–8 g / L, potassium chloride 1–2 g / L, calcium chloride 0.1–0.2 g / L, potassium sulfate 0.5–1 g / L, potassium dihydrogen phosphate 0.5–2 g / L, monosodium glutamate 15–20 g / L, zinc sulfate heptahydrate 1–5 mg / L, cobalt chloride hexahydrate 0.01–0.1 mg / L, copper sulfate pentahydrate 2–6 mg / L, nickel sulfate hexahydrate 1–2 mg / L, ferric sulfate heptahydrate 8–15 mg / L, calcium pantothenate 2–4 mg / L, manganese chloride tetrahydrate 3–5 mg / L, sodium molybdate dihydrate 0.04 mg / L, vitamin B6 4–10 mg / L, and vitamin B12. 12 0.1-0.5 mg / L.

9. The application of the method according to any one of claims 3 to 8 in increasing the content of odd-chain fatty acids in oils and fats.

10. The application of the engineered bacteria for high production of odd-chain fatty acids as described in claim 1 in increasing the content of odd-chain fatty acids in oils.

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