A method for converting gluconic acid (salt) into erythritol using Yarrowia lipolytica

Abstract

The present invention relates to a method for converting gluconic acid (salt) into erythritol by a strain of Yarrowia lipolytica. The yeast is artificially mutated to obtain a mutant strain, and gluconic acid (salt) is converted into erythritol by microbial fermentation. By this method, gluconic acid (salt) can be converted into 7.6% (w / v) erythritol by shaking flask fermentation at a temperature of 30°C for 72 hours, and the conversion rate based on the consumption of gluconic acid can reach more than 54%. The available substrates include gluconic acid, gluconate and ester derivatives, and raw materials such as sodium gluconate crystallization mother liquor can also be used for conversion.

Description

Technical Field

[0001] The invention relates to the transformation and application of industrial microorganisms and belongs to the technical field of bioengineering. Background Art

[0002] Gluconic acid is a non-corrosive, non-toxic, biodegradable weak organic acid (pKa = 3.86). It is found in nature mainly in plants, fruits, wine and honey. Gluconic acid is produced by the oxidation of the aldehyde group (C1) of D-glucose to a carboxyl group. In aqueous solution at neutral pH, gluconic acid forms a gluconate ion in equilibrium with its cyclic ester D-glucono delta-lactone (1,5-gluconolactone). Gluconic acid is used as a flavoring agent in a variety of foods, including meat, wine and dairy products. Due to its ability to form water-soluble complexes (chelates) with divalent and trivalent metal ions, it is often used as a counterion during therapeutic calcium and / or iron administration. For the same reason, it is also used to remove calcium and rust deposits from metals or other surfaces, and sodium gluconate is also used in considerable amounts as a concrete water reducer and in household detergents. Today's annual market capacity for gluconic acid and its derivatives is about 800,000 tons, making it a common bio-based raw material. However, for some special occasions, such as the reduction of acid in edible fruit juice, metal surface treatment, chemical plating waste liquid, and the recovery and reduction of large amounts of gluconic acid (salt) produced as a by-product in the industrial production of gluconate, the recycling of gluconic acid has considerable value both from an environmental perspective and from a resource conservation perspective.

[0003] Lignocellulose is the most abundant renewable biomass in nature. It is very attractive to convert this type of resource into bulk fuels and chemicals. However, in its biological utilization process, the high amount of cellulase used for cellulose degradation remains one of the technical bottlenecks for reducing processing costs. The recently discovered lytic polysaccharide monooxygenase (LPMO) can accelerate cellulose degradation and greatly improve the efficiency of cellulose hydrolysis and sugar yield, opening up a new way for the efficient utilization of lignocellulose resources. However, the oxidative hydrolysis of cellulose catalyzed by LPMOs produces a high proportion of oxidized cellulose degradation products (including cellobionic acid and gluconic acid) in the total hydrolysate. In order to reduce the sugar loss of these degradation products, the utilization of sugar acids including gluconic acid needs to be solved to make this approach more technically advantageous.

[0004] At present, one of the methods for treating gluconic acid by biochemical methods is to remove the carboxyl group in the molecule through the so-called Luffy descent reaction to obtain a pentose with one less carbon atom, that is, calcium gluconate and ferric gluconate are oxidized together with hydrogen peroxide. Strong oxidants are used in the process, and the obtained D-arabinose has no significant practical utilization value. Like gluconic acid, it is also a carbon source that is difficult for microorganisms to use. In addition, it will produce more by-products and lose a carbon atom, so it has little practical value.

[0005] Yarrowia lipolytica Yarrowialipolytica ) is a non-fermentative aerobic yeast strain. Due to its safety and easy cultivation, it is currently widely used in the production of products such as lipase and organic acids and is widely used as a genetic engineering host for the expression of exogenous genes. However, the available carbon source spectrum of Yarrowia lipolytica is very narrow. Generally, it can only use simple sugars such as glucose and hydrocarbons such as n-hydrocarbons, which will bring certain restrictions to the application of this strain. However, relevant studies have shown that the pentose phosphate pathway of Yarrowia lipolytica is very active. This metabolic branch that uses glucose through 6-phosphogluconic acid as an intermediate will produce a large amount of reducing coenzyme NADPH for the organism to use for the synthesis of lipids and sugar alcohols. Therefore, it is expected to transform and develop the ability of this strain to use gluconic acid (salt) as an effective carbon source for the biosynthesis of useful target compounds. At present, Yarrowia lipolytica is the only practical strain that is internationally recognized as safe (GRAS) for the production of erythritol, a natural sweetener with a very broad market. Getting rid of the single existing raw materials and developing new production raw materials are undoubtedly of great significance to improving and enhancing the economy of the erythritol production process.

[0006] In theory, in order to obtain Yarrowia lipolytica strains that can utilize gluconate, the strain is required to have a gluconate (salt) transporter ( gnt )、Gluconate kinase( gnt ) and other genes, so that gluconic acid can enter the cell from the matrix and be converted into phosphorylated gluconic acid that can be used by the strain, and then be used after entering the pentose phosphate pathway. In fact, although the wild-type Yarrowia lipolytica strain has the activity of gluconate kinase, the existence of gluconate (salt) transporter has not been verified, so it is generally believed that Yarrowia lipolytica does not have the ability to assimilate this substance.

[0007] At present, in order to expand the substrate utilization ability of Yarrowia lipolytica, the method of constructing strains containing key genes for exogenous special substrate utilization is generally adopted to solve the problem. For example, the sucrase gene from Saccharomyces cerevisiae can be expressed in Yarrowia lipolytica ( Suc) to improve the ability of bacteria to utilize this disaccharide. However, the actual success rate of this method has been low so far, and the impact on the production characteristics of the base bacteria may be large. Global cell biology optimization is still needed to obtain good strains for industrial application. Moreover, in many countries and regions, the application of engineered bacteria obtained by genetic engineering methods in food and its ingredients is still facing regulatory and ethical restrictions, so the use of classical methods for strain selection is a more common technical route.

[0008] Since gluconic acid (salt) is in an ionic state under neutral pH conditions suitable for yeast growth, and the gluconic acid (salt) transporters of most organisms are symporters coupled to hydrogen ions, it is possible to consider establishing a hydrogen ion gradient from the outside to the inside to assist in achieving the transmembrane transport of gluconic acid (salt). For this reason, the transformation route of Yarrowia lipolytica strains with the screening target of variants that can tolerate the high osmotic pressure of high-concentration salt substrates and adapt to the characteristics of a lower pH growth environment may become a technical solution to solve the bottleneck of the strain's utilization of gluconic acid (salt).

[0009] The invention conducts physical and chemical and natural mutagenesis transformation on the hyperosmotic-resistant Yarrowia lipolytica, then selectively screens the mutants with high gluconic acid (salt) assimilation ability, and selectively applies the mutants to the bioconversion of erythritol. Summary of the invention

[0010] The purpose of the present invention is to provide a method for breeding a Yarrowia lipolytica strain that can utilize gluconic acid (salt), and to utilize the strain to carry out fermentation conversion with erythritol as a target product.

[0011] The Yarrowia lipolytica variant 77 of the present invention has a taxonomic name of Yarrowia lipolytica Yarrowia lipolytica , deposited with the General Microbiology Center of China Microorganism Culture Collection Administration, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit number: CGMCC No.25431, deposit date: July 29, 2022.

[0012] To achieve the above purpose, the present invention adopts the following technical solution.

[0013] 1. The Yarrowia lipolytica strain transformation technology of the present invention uses wild-type Yarrowia lipolytica as a starting strain, and uses physical, chemical and molecular biological methods to make it genetically mutate, thereby selecting mutants with strong gluconate conversion ability.

[0014] Further, the operation is performed as follows:

[0015] (1) Wild-type Yarrowia lipolytica cells were treated with chemical and physical mutagenesis methods such as EMS, NTG, ionizing radiation and plasma, and other molecular mutagenesis methods to induce random mutations.

[0016] (2) The yeast cells treated by (1) are cultured in an acidic synthetic medium with gluconate as the sole carbon source for multiple rounds to enrich for yeast mutants that can assimilate gluconate under acidic conditions.

[0017] (3) The yeast culture enriched in step (2) is subjected to a second round of enrichment on a synthetic medium supplemented with erythritol as the sole carbon source to further enrich for Yarrowia lipolytica mutants with erythritol utilization defective traits.

[0018] (4) The yeast culture enriched in step (3) is diluted and single colonies are isolated on YPDA plates to select yeast variants that can synthesize erythritol using gluconic acid (salt).

[0019] 2. Use the obtained Yarrowia lipolytica variant strain to ferment and convert various gluconic acid (salt) substrates into erythritol.

[0020] Further, the specific steps are as follows:

[0021] (1) Activation and cultivation of bacterial strains

[0022] The mutant strain was streaked on a YDPA solid culture medium plate containing gluconate, cultured at 30±1°C for 72 hours, a single colony on the plate was picked, inoculated into a fermentation medium, and cultured in a shaking incubator at 30±1°C for 24 hours to obtain a seed solution.

[0023] (2) Fermentation culture

[0024] The seed liquid was inoculated into the fermentation medium at an inoculation rate of 0.1-20%, and cultured in a shaking incubator at 30±1℃ for 48-96 hours to obtain the fermentation liquid.

[0025] The YPDA medium in step (1) is formulated as follows: 20 g of sodium gluconate, 20 g of glucose, 10 g of yeast powder, 20 g of peptone, 10 mg of cinnamon, and 20 g of agar are added to 1 L of pure water and sterilized at 115° C. for 30 minutes.

[0026] The seed culture medium formula in the above step (1) is as follows: add 20 g sodium gluconate, 3 g yeast extract powder, 5 g peptone, 20 g glucose, 10 g yeast powder, and 20 g peptone to 1 L water, and sterilize at 115°C for 30 minutes.

[0027] The fermentation medium formula in the above step (2) is as follows: add 3 g yeast extract powder, 5 g peptone, and 300 g sodium glucose into 1 L water, and sterilize at 115°C for 30 minutes.

[0028] (4) Detection methods of glucose (acid) and glucose in the fermentation process

[0029] After the fermentation broth was diluted and filtered through a 0.22 μm filter membrane, the contents of glucose (acid) and erythritol in the filtrate were detected by HPLC. The chromatographic conditions were as follows: detector, differential detector; chromatographic column, H-type strong cation exchange column; column temperature, 65 °C; mobile phase, 7 mM H2SO4 aqueous solution; flow rate, 0.7 mL / min; injection volume, 10 μL.

[0030] The present invention successfully achieved the goal of obtaining a variant strain of Yarrowia lipolytica that can efficiently convert gluconate into erythritol by enriching the gluconic acid assimilation function and weakening the erythritol assimilation function of the variant strain library in an acidic environment from the wild strain of Yarrowia lipolytica. The strain can be used to efficiently convert gluconate (ester) and sodium gluconate mother liquor for erythritol fermentation, which has important economic and environmental value.

[0031] The outstanding features of the present invention are:

[0032] (1) The present invention provides a method for obtaining a yeast mutant with the ability to assimilate gluconate by using composite mutagenesis on wild-type Yarrowia lipolytica, combined with antibiotic and acid induction treatments in the trait enrichment process. The method has a high directional screening efficiency.

[0033] (2) The Yarrowia lipolytica variant obtained by the above method can convert 20% of sodium gluconate into more than 10% of erythritol within 96 hours, and the conversion rate can reach more than 50%, allowing the strain to use lower-quality carbon sources to produce useful compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 HPLC profile of sodium gluconate fermentation products of Yarrowia lipolytica variants.

[0035] Figure 2 HPLC graph of the sodium gluconate crystallization mother liquor product from the fermentation of sodium gluconate by a variant strain of Yarrowia lipolytica. DETAILED DESCRIPTION

[0036] The present invention is further illustrated by the following examples, which will help to understand the present invention, but are not limited thereto.

[0037] Example 1. Obtaining a mutant strain of Yarrowia lipolytica

[0038] (1) Inoculate activated Yarrowia lipolytica CGMCC No.1431 into YPD medium. After 24 hours of shaking flask culture, add 1 ml of bacterial solution to a sterile centrifuge tube, centrifuge to remove the culture medium in the supernatant, wash the precipitated yeast cells three times with pH 6.0 phosphate buffer solution, and then adjust the OD with buffer. 600 After reaching about 1.0, 200 ppm of NTG was added and shaken at 30°C for 30 minutes. The reaction was terminated by centrifugation at 1000 g for 10 minutes to remove the mutagen in the supernatant. The precipitated yeast cells were washed three times with a 50 mM phosphate buffer solution at pH 6.0 and inoculated into 100 ml of GnYNB medium and shaken at 28°C until OD 600 After reaching about 0.5, pipette 1ml of bacterial solution into another 100ml bottle of GnYNB medium. Repeat the transfer 5 times, take 1ml of bacterial solution and inoculate it into 100ml MT+1% erythritol nitrogen-free medium for shaking flask culture for 8 hours, then add sterile ammonium sulfate solution with a final concentration of 5g / l, shake culture at 28℃ for 8 hours, then add nystatin to a final concentration of 20μg / ml and continue shaking culture for 3 hours. Appropriately dilute the treated bacterial solution and spread it on the GnYNBA plate, and culture it at 30℃ for 3 days. Select larger single colonies with smooth surfaces for fermentation performance verification.

[0039] The YPD medium formula is: 3.0g yeast extract powder, 5.0g peptone, 50g anhydrous glucose, add water to 1.0 liter, and sterilize at 115°C for 30 minutes.

[0040] The formula of the GnYNB medium is: 6.7 g of YNB (filter sterilized), 15.0 g of sodium gluconate, 20 g of agar, add water to 1.0 liter, adjust the pH to 3.5 with 2M hydrochloric acid, and sterilize at 115°C for 30 minutes.

[0041] The formula of the GnYNBA medium is: 6.7 g of YNB (filter sterilized), 15.0 g of sodium gluconate, 20 g of agar, add water to 1.0 liter, adjust the pH to 3.5 with 2M hydrochloric acid, and sterilize at 115°C for 30 minutes.

[0042] The formula of the MT culture medium is:

[0043] NH4H2PO4 5 g / l

[0044] KH2PO4 2.5 g / l

[0045] MgSO4•7H2O 1 g / l

[0046] Thiamine hydrochloride 0.3mg / l

[0047] Trace element solution 1ml / l

[0048] Trace element solution composition: (mg / 100 ml):

[0049] Ca(NO3)2•4H2O 2000

[0050] FeCl3•6H2O 200

[0051] H3BO3 50

[0052] CuSO4•5H2O 10

[0053] MnSO4•4H20 40

[0054] ZnSO4•7H2O 40

[0055] Na2MoO4 20

[0056] CoCl2 10

[0057] KI 10

[0058] The NTG refers to N-methyl-N'-nitro-N-nitrosoguanidine.

[0059] The phosphate buffer solution is a solution obtained by adjusting a 50 mM phosphoric acid solution to pH 6.0 using a concentrated NaOH solution.

[0060] (2) Pick out a single colony from the plate cultured on the GnYNBA medium for 3 days in step (1), inoculate it into 2.5 ml of YPD medium, culture it with shaking at 30°C overnight, and then inoculate it into a 500 mL Erlenmeyer flask containing 25 ml of GnYS medium. The inoculated Erlenmeyer flask is cultured at 30°C with a rotary shaking of 250 rpm for 72 hours, and then the fermentation product is analyzed by HPLC. The chromatogram is attached. Figure 1 . Based on the analysis results, mutant strains with high efficiency in converting erythritol were selected.

[0061] The formula of the GnYS culture medium is: 5g yeast powder (filter sterilized), 5g ammonium citrate, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 15.0g sodium gluconate, 20g agar, add water to 1.0 liter, and sterilize at 115°C for 30 minutes.

[0062] Example 2. Fermentation of erythritol by Yarrowia lipolytica CGMCC 25431 using sodium gluconate

[0063] In a 500ml Erlenmeyer flask filled with 25ml of sterilized sodium gluconate fermentation medium, 2.5ml of seed solution of Yarrowia lipolytica variant CGMCC 25431 cultured in YPD medium at 30°C for 24h (inoculation ratio 1:10) was inoculated, and fermented at 30±1°C with a rotary shaking speed of 280rpm for 72h. The analysis results are shown in the following table:

[0064] Initial gluconolactone g / l (calculated as gluconic acid) Gluconolactone g / l at 72h (calculated as gluconic acid) Consumption of gluconolactone g / l (calculated as gluconic acid) Erythritol g / l at 72h Erythritol conversion rate% 180 36.5 143.5 73.2 51.0%

[0065] During the 72-hour fermentation, the conversion rate of erythritol based on the consumption of gluconic acid reached 51.0%.

[0066] The components of the sodium gluconate fermentation medium are: 20% (w / v) sodium gluconate, 0.5 (w / v) ammonium citrate, 0.5 (w / v) yeast extract, 0.05% (w / v) potassium dihydrogen phosphate, and 0.025% magnesium sulfate.

[0067] Example 3. Fermentation of erythritol by Yarrowia lipolytica CGMCC 25431 using ammonium gluconate

[0068] In a 500 ml Erlenmeyer flask containing 25 ml of sterile ammonium gluconate medium, 2.5 ml of the seed solution of Yarrowia lipolytica variant CGMCC 25431 cultured in YPD medium at 30°C for 24 h (OD 600 =15), shake flask fermentation was carried out at 30±1℃ and 280rpm for 72h. The amount of ammonium gluconate consumed in the fermentation broth and the amount of erythritol produced are as follows:

[0069] Initial gluconolactone g / l (calculated as gluconic acid) Gluconolactone g / l at 72h (calculated as gluconic acid) Consumption of gluconolactone g / l (calculated as gluconic acid) Erythritol g / l at 72h Erythritol conversion rate% 185 50.3 134.7 61.0 45.3%

[0070] During the 72-hour fermentation, the conversion rate of erythritol based on the consumption of ammonium gluconate reached 45.3%.

[0071] The ammonium gluconate culture medium comprises: 20% (w / v) ammonium gluconate, 0.5% (w / v) yeast powder, 0.025% (w / v) magnesium sulfate and 0.05% (v / v) potassium dihydrogen phosphate.

[0072] Example 4. Erythritol fermentation using gluconolactone

[0073] In a 500 ml Erlenmeyer flask containing 25 ml of sterile gluconolactone medium, 2.5 ml of the seed solution of Yarrowia lipolytica variant CGMCC 25431 cultured in YPD medium at 30°C for 24 h (OD 600 =15), shake flask fermentation was carried out at 30±1℃ and 280rpm for 72h. The amount of gluconolactone consumed in the fermentation broth and the amount of erythritol produced are as follows:

[0074] Initial gluconolactone g / l (calculated as gluconic acid) Gluconolactone g / l at 72h (calculated as gluconic acid) Consumption of gluconolactone g / l (calculated as gluconic acid) Erythritol g / l at 72h Erythritol conversion rate% 198 57.1 140.9 76.1 54.0%

[0075] Gluconolactone is a lactone compound formed by dehydration of gluconic acid molecules. It is strongly acidic when dissolved in water. After neutralization with an appropriate amount of sodium hydroxide to a pH below 4.5, the conversion rate of erythritol based on the consumption of gluconolactone can reach 54.0% after 72 hours of fermentation.

[0076] The gluconolactone culture medium comprises: gluconolactone 18% (w / v), yeast powder 0.5 (w / v), diammonium hydrogen phosphate 0.45%, magnesium sulfate 0.025%, and potassium dihydrogen phosphate 0.05% (v / v).

[0077] Example 5. Fermentation of erythritol using sodium glucose mother liquor

[0078] Sodium gluconate mother liquor is a by-product in the production process of sodium gluconate. Due to the high viscosity and high impurities, it is difficult to separate more sodium gluconate crystals from it. Its main components include: solid content 50%, sodium gluconate 30%, and reducing sugar 15%.

[0079] In a 500ml Erlenmeyer flask containing 25ml of sterilized mother liquid culture medium, 2.5ml of Yarrowia lipolytica CGMCC 25431 seed solution (inoculation ratio 1:10) cultured in YPD medium at 30°C for 24h was added, and fermented at 30±1°C with a rotary shaking speed of 280rpm for 72h. The analysis results are shown in the following table:

[0080] Initial sodium gluconate mother liquor g / l (in terms of gluconic acid) Sodium gluconate g / l at 72h (calculated as gluconic acid) Consumption of sodium gluconate g / l (calculated as gluconic acid) Erythritol g / l at 72h Erythritol conversion rate% 167.5 48.6 118.9 56.6 47.6%

[0081] The mother liquor of sodium gluconate crystallization contains more soluble impurities, and the fermentation efficiency is slightly lower than that of the pure raw material. However, after 72 hours of fermentation, the conversion rate of erythritol based on the consumption of gluconate can reach 47.6%, and the oligosaccharides in the raw material that are difficult for the yeast to assimilate are still retained (see the HPLC chart of the sugar and acid components during the fermentation process in the attached Figure 2 ).

[0082] The mother liquid culture medium comprises: 0.5% (w / v) yeast powder, 10% (w / v) anhydrous glucose, 0.2% (w / v) diammonium phosphate, 0.025% magnesium sulfate, and 30% (v / v) sodium gluconate mother liquid.

[0083] Example 6. Sodium gluconate fermentation of wild-type Yarrowia lipolytica CGMCC 1431 (control)

[0084] In a 500 ml Erlenmeyer flask containing 25 ml of sterilized sodium gluconate fermentation medium, 2.5 ml of the seed solution of Yarrowia lipolytica wild strain CGMCC 1431 cultured in YPD medium at 30°C for 24 h (OD 600 =15), at 30±1℃, with a rotational shaking fermentation speed of 280rpm for 96h. The analysis results are shown in the following table:

[0085] Initial sodium gluconate g / l (calculated as gluconic acid) Sodium gluconate g / l at 96h (calculated as gluconic acid) Consumption of sodium gluconate g / l (calculated as gluconic acid) Erythritol g / l at 96h Erythritol conversion rate% 180 175 5.2 0.2 3.8%

[0086] After 96 hours of fermentation, wild-type Yarrowia lipolytica basically does not use gluconic acid (salt) to produce erythritol, except for a small amount of erythritol brought in by the seeds.

[0087] The components of the sodium gluconate fermentation medium are: 20% (w / v) sodium gluconate, 0.5 (w / v) ammonium citrate, 0.5 (w / v) yeast extract, 0.05% (w / v) potassium dihydrogen phosphate, and 0.025% magnesium sulfate.

Claims

1. Yarrowia lipolytica Yarrowia lipolytica ) A method for producing erythritol using gluconate or gluconolactone by variant 77, characterized in that: The deposit number of the Yarrowia lipolytica variant 77 is CGMCC No. 25431; the gluconate is sodium gluconate or ammonium gluconate.

2. The method for producing erythritol using gluconate or gluconolactone by Yarrowia lipolytica variant 77 according to claim 1, characterized in that: The following steps are involved: (1) The Yarrowia lipolytica variant 77 of claim 1 is used to prepare a seed solution using the following culture medium: 0-20% (w / v) glucose, 0-20% (w / v) sodium gluconate, 0.03-2.5% (w / v) yeast extract, 0.03-2.5% (w / v) peptone, 0.02-0.5% (w / v) potassium dihydrogen phosphate, 0.1-0.75% (w / v) ammonium citrate; 0-0.75% (w / v) diammonium hydrogen phosphate; (2) The above seed liquid is inoculated into a fermentation medium at a ratio of 0.5-50%, wherein the medium has the following composition: 0-20% (w / v) gluconate, 0.03-2.5% (w / v) yeast extract, 0.03-2.5% (w / v) peptone, 0.02-0.5% (w / v) potassium dihydrogen phosphate, 0.1-0.75% (w / v) ammonium citrate; 0-0.75% (w / v) diammonium hydrogen phosphate, and fermentation is carried out in a shaking flask at 25-33°C for 60-120 hours at a shaking speed of 200-400 rpm to obtain a fermentation broth with erythritol as the main product.

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