Process for producing epa from a schizochytrium sp.

Abstract

This invention belongs to the field of microbial fermentation technology, specifically a process for producing EPA using Schizochytrium fungi as raw material. It employs a dual-gene modified strain with MAT gene overexpression and CLF gene precise knockout, which is domesticated under high-sugar oxidative stress to obtain a highly stable strain. Through three-stage temperature-controlled, stepwise dissolved oxygen fermentation, and the combined addition of para-aminobenzoic acid and ascorbic acid, EPA synthesis is enhanced while oxidative loss is reduced. Highly efficient EPA enrichment is achieved through low-temperature ultrasonic cell disruption of wet cells, urea inclusion, two-stage molecular distillation, and tandem purification using immobilized composite adsorption decolorization. This invention significantly improves EPA yield and proportion, reduces oxidative loss, and enhances product purity and stability through the synergistic effect of gene modification, metabolic regulation, and purification processes. The process is highly controllable, suitable for industrial production, and yields EPA products with high purity and good stability, which can be directly used in the food, health product, and pharmaceutical fields.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically a process for producing EPA using Schizochytrium as a raw material. Background Technology

[0002] Among omega-3 polyunsaturated fatty acids, eicosapentaenoic acid (EPA) has irreplaceable physiological functions in regulating blood lipids, anti-inflammation, and protecting the cardiovascular system, and has been widely used in the fields of medicine, health food, and special dietary foods. Traditionally, EPA is mainly derived from deep-sea fish oil, but its raw materials are easily affected by marine pollution, seasonality, and fish species. Its composition is complex, and the cost of separation and purification is high, making it difficult to stably supply high-purity products.

[0003] Schizochytrium sp., as an excellent strain for the industrial production of ω-3 polyunsaturated fatty acids, has advantages such as rapid growth, high biomass, rich oil content, and the ability to produce high-purity unsaturated fatty acids through heterotrophic fermentation. It is an ideal microorganism to replace fish oil in the production of high-purity unsaturated fatty acids. Currently, Schizochytrium fermentation mainly focuses on the synthesis of docosahexaenoic acid (DHA), while the level of EPA synthesis is generally low. The core reason for this is the imbalance in the metabolic flux distribution between the polyketide synthase (PKS) pathway and the fatty acid synthase (FAS) pathway within the strain. Carbon flux tends to flow towards the synthesis of C22 long-chain unsaturated fatty acids. However, the metabolic mechanism for targeted regulation of EPA production, the methods for modifying key nodes, and the supporting fermentation and purification processes are still unclear.

[0004] Existing technologies for producing EPA from Schizochytrium mostly achieve yield increases through simple fermentation regulation or optimization of a single extraction process, which has significant shortcomings: 1. The strain was not genetically modified or directed to domestication, resulting in low expression levels of key genes in the PKS pathway, insufficient malonyl-CoA supply, and limited EPA synthesis throughput; 2. The fermentation process involves crude strategies for temperature, dissolved oxygen, and feeding, which fail to achieve a temporal match between cell growth, lipid accumulation, and EPA synthesis, resulting in a high proportion of byproducts. 3. The exogenous additives are singular and lack a combined strategy to synergistically enhance metabolism and antioxidant protection. EPA is easily oxidized and degraded during synthesis and extraction. 4. The cell wall breaking and purification processes mostly adopt drying post-treatment and single-stage separation methods, resulting in large oil loss, low purification efficiency, and product purity and stability that are difficult to meet the requirements of high-end applications; 5. The processes of gene modification, domestication, fermentation, cell wall disruption, and purification are independent of each other and have not formed a systematic coupled process, making it impossible to achieve the synergistic improvement of metabolic flow guidance, efficient transformation, and high-purity enrichment.

[0005] Therefore, developing a new process for producing EPA from Schizochytrium by enhancing EPA synthesis through genetic modification and targeted domestication, improving conversion efficiency through segmented and precise fermentation, using low-temperature and low-oxygen coupling protection, and multi-stage tandem purification and enrichment, is of great practical significance for overcoming the bottlenecks of low EPA yield, insufficient purity, poor stability, and process mismatch in existing technologies. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, a new process for producing EPA from Schizochytrium was developed, which involves enhancing EPA synthesis through genetic modification and targeted domestication, improving conversion efficiency through segmented and precise fermentation, using low-temperature and low-oxygen coupling protection, and employing multi-stage tandem purification and enrichment.

[0007] The technical solution adopted by this invention to solve its technical problem is: a process for producing EPA using Schizochytrium as raw material, comprising: using a dual-gene modified Schizochytrium strain with MAT gene overexpression and CLF gene precise knockout, implementing high sugar and reactive oxygen coupled adaptive domestication on the modified strain, fermenting under three-stage temperature control and stepwise dissolved oxygen control, using wet cell ultrasonic cell disruption treatment, and then purifying by urea inclusion, secondary molecular distillation and immobilized adsorption decolorization to obtain high-purity EPA.

[0008] Furthermore, the Schizochytrium sp. ATCC1381 was identified, and the MAT gene was stably overexpressed by integrating into the 18S rDNA site via homologous recombination. The CLF gene was expressed using CRISPR. Cas9 was sequence-specifically knocked out, causing the strain's carbon flow to be directed toward C20 fatty acid synthesis.

[0009] Furthermore, the domestication process employs a glucose concentration gradient increase coupled with bipyridine-induced oxidative stress through passage, with a domestication cycle of no less than 30 generations, to obtain stable domesticated strains with high glucose tolerance, low malondialdehyde accumulation, and high NADPH regeneration capacity.

[0010] Furthermore, the fermentation process employs a three-stage temperature control mode: 0 Control the temperature at 28℃ for 36 hours. The temperature is controlled at 25℃ for 60 hours, and then at 20℃ after 60 hours, in conjunction with a step-down control mode for dissolved oxygen.

[0011] Furthermore, para-aminobenzoic acid and ascorbic acid were added to the fermentation medium in combination, with para-aminobenzoic acid added at a concentration of 200 mg / L and ascorbic acid added at a concentration of 150 mg / L. Both were added at once during the 24-hour fermentation period.

[0012] Furthermore, the ultrasonic disruption of the wet bacterial cells was performed at a low temperature of 4°C and an ultrasonic power of 300 kW. 400W, 2s operation time, 2s interval, total processing time 10 After 15 minutes of cell wall disruption, the oil is extracted directly without drying.

[0013] Furthermore, the urea-ester mass ratio during the inclusion process was 0.8:1, the inclusion temperature was 4℃, and the inclusion time was 8h. After centrifugation, the oil phase of the inclusion product was directly fed into molecular distillation.

[0014] Furthermore, the secondary molecular distillation adopts a continuous feed mode, with the primary distillation temperature at 120°C. 130℃, vacuum degree 5 10 Pa, secondary distillation temperature 140 150℃, vacuum degree 3 5Pa, feed rate 1 2 mL / min.

[0015] Furthermore, the molecularly distilled heavy phase is directly fed into a decolorization column filled with a composite immobilized adsorbent of attapulgite and activated carbon. The length-to-diameter ratio of the decolorization column is not less than 8:1, and the operating temperature is 35°C. 45℃.

[0016] Furthermore, the composite immobilized adsorbent is composed of attapulgite and activated carbon in a mass ratio of 3:1, and is activated at 350℃ for 2 hours, with the particle size controlled at 80 mm. Between 120 mesh.

[0017] The beneficial effects of this invention are as follows: 1. The present invention describes a process for producing EPA using Schizochytrium fungi as raw material, which involves MAT gene overexpression, precise CLF gene knockout, and high sugar content. The synergistic effect of oxidative stress coupled with domestication significantly enhanced the malonyl content of the strain. ACP supply capacity and block excessive carbon chain extension, while maintaining normal cell growth and total lipid synthesis level, to directionally increase EPA synthesis flux and proportion, achieving a balance between high EPA production and optimal component control.

[0018] 2. The process for producing EPA using Schizochytrium as raw material, as described in this invention, involves three-stage temperature control, stepwise dissolved oxygenation, and para-aminobenzoic acid. The coupled regulation of ascorbic acid complex addition makes the fermentation process highly matched with the EPA synthesis sequence of the PKS pathway, significantly improves NADPH supply and reduces intracellular oxidative stress, greatly reduces EPA oxidative degradation, improves fermentation efficiency, reduces foam generation, and enhances process stability.

[0019] 3. The process for producing EPA using Schizochytrium as raw material described in this invention involves a series of purification steps, including low-temperature ultrasonic cell disruption of wet mycelium, urea inclusion, secondary molecular distillation, and immobilized composite adsorption decolorization. This process effectively removes saturated fatty acids, pigments, odors, and impurities while avoiding high-temperature oxidation of EPA, thus significantly improving the purity, recovery rate, and storage stability of the EPA product. Attached Figure Description

[0020] The invention will now be further described with reference to the accompanying drawings.

[0021] Figure 1 This is a flowchart of the production method of the present invention. Detailed Implementation

[0022] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1: As Figure 1 As shown, a process for producing EPA using Schizochytrium as a raw material according to the present invention includes the following steps: 1. Construction and activation of genetically modified strains 1.1 Starting strain: This invention uses Schizochytrium sp. ATCC1381 as the starting strain. This strain is a publicly available type strain that can rapidly accumulate oil under heterotrophic conditions and is a commonly used production strain in the field.

[0024] 1.2 Construction of strains overexpressing the MAT gene: Using the genome of Schizochytrium as a template, the malonyl-CoA-ACP transacylase (MAT) gene was cloned, and an overexpression cassette driven by a constitutive promoter was constructed. The MAT gene was then site-specifically integrated into the 18S rDNA site of the strain via homologous recombination. The linearized recombinant plasmid was electroporated into competent cells, cultured on bleomycin-containing selection plates, and positive clones were selected and verified by PCR and sequencing. The MAT gene was stably overexpressed at a conserved site on the 18S rDNA, significantly enhancing the intracellular malonyl-ACP supply capacity, providing sufficient precursors for fatty acid synthesis, increasing the total lipid synthesis flux, and strengthening the metabolic basis for EPA synthesis via the PKS pathway.

[0025] 1.3 CLF gene precise knockout: Using the CRISPR-Cas9 system, specific gRNAs were designed targeting the chain-long factor (CLF) gene in the PKS pathway. A knockout vector was constructed and electroporated into the aforementioned MAT-overexpressing strains. The Cas9 protein and gRNA were assembled in vitro into a ribonucleoprotein complex. After electroporation, the complex was cultured on selection plates, and transformants were picked and sequenced to verify the results, obtaining a positive mutant strain with the CLF gene sequence deletion. The CLF gene is responsible for extending the PUFA carbon chain to C22. After knockout, the carbon flux cannot be excessively extended to DHA / DPA. Without significantly affecting cell growth and total lipid synthesis, the metabolic flux is forced to converge towards C20, significantly increasing the proportion of EPA in total fatty acids.

[0026] 1.4. Strain activation: The successfully constructed dual-gene modified strain was streaked onto a solid seed culture medium and incubated at 28°C for 48 hours until milky yellow, round, and plump single colonies grew. This allowed the strain to recover its activity from low-temperature storage, ensuring uniform inoculation activity and stable subsequent fermentation.

[0027] 2. High sugar Oxidative stress coupled adaptive domestication: 2.1 Gradient acclimatization steps: The genetically modified strain was inoculated into liquid seed culture medium and passaged at an initial glucose concentration of 30 g / L, with subculturing every 48 hours at a rate of 10%. After every 10 generations of domestication, the glucose concentration was successively increased to 60 g / L, 90 g / L, 120 g / L, 150 g / L, and 180 g / L, for a total of at least 30 generations. During the domestication process, a low concentration of oxidant was added to the culture medium to apply mild oxidative stress. Through synergistic screening of high glucose stress and oxidative stress, stable mutant strains with high glucose tolerance, rapid glucose absorption, strong NADPH regeneration capacity, and low intracellular reactive oxygen species levels were obtained. The strains exhibited significantly improved stress resistance, reduced foaming during fermentation, stronger cell vigor, and reduced oxidative damage to EPA during synthesis.

[0028] 3. Seed culture 3.1 Seed culture medium: Glucose 30g / L, yeast extract 10g / L, monosodium glutamate 5g / L, artificial sea salt 20g / L, MgSO4・7H2O 2g / L, KH2PO4 1g / L, pH natural.

[0029] 3.2 Cultivation process: Select activated single colonies and inoculate them into seed culture medium. Incubate at 28°C and 180 rpm for 24 hours until OD reaches the target level. 600 Reaching 8–10; obtaining a seed culture with logarithmic growth period, vigorous growth, and high uniformity, which enters the growth stage rapidly after being introduced into the fermenter without a stagnation period.

[0030] 4. Three-stage temperature control and stepped dissolved oxygen fermentation 4.1 Fermentation medium: Glucose 90g / L, ammonium sulfate 5g / L, yeast extract 3g / L, monosodium glutamate 7g / L, artificial sea salt 20g / L, MgSO4・7H2O 1g / L, KH2PO4 2g / L, trace element solution 1mL / L, vitamin solution 1mL / L.

[0031] 4.2. Vaccination: The seed culture was added to the fermenter at a volume fraction of 10%, with the initial liquid volume being 70% of the working volume.

[0032] 4.3 Three-stage temperature control: The first stage lasted 0–36 hours, maintaining a temperature of 28℃, a stirring speed of 300–500 r / min, and an aeration rate of 1.2 vvm. The temperature matched the optimal growth range of the cells, resulting in rapid cell proliferation and biomass accumulation, laying the biomass foundation for subsequent lipid synthesis.

[0033] In the second stage (36–60 hours), the temperature is lowered to 25°C, and the stirring speed is appropriately reduced. Moderate cooling slows down the excessive proliferation of bacteria, guides the carbon flow from growth metabolism to lipid synthesis metabolism, and initiates the large-scale synthesis of PUFA.

[0034] The third stage lasts 60 hours until the end of fermentation. The temperature is then lowered to 20°C and maintained until the product is removed from the tank. Low temperature can reduce the activity of fatty acid elongation enzymes, further weaken the DHA synthesis pathway, strengthen the targeted accumulation of EPA, and achieve a significant increase in the proportion of EPA.

[0035] 4.4 Stepped Dissolved Oxygen Control: Dissolved oxygen levels were gradually reduced throughout the fermentation process: 40% in the early stage (0–36 h), 25% in the middle stage (36–60 h), and 15% after 60 h. The dissolved oxygen level was perfectly matched with the oxygen requirements for cell growth, lipid synthesis, and EPA accumulation. High oxygen levels in the early stage met the needs of rapid growth, moderate oxygen levels in the middle stage facilitated lipid synthesis, and low oxygen levels in the later stage reduced fatty acid β-oxidation and decreased EPA oxidation loss.

[0036] 4.5. Addition of compound additives: When fermentation reaches 24 hours, 200 mg / L para-aminobenzoic acid + 150 mg / L ascorbic acid are added to the tank at once. The additives are dissolved in sterile water, filtered through a filter membrane for sterilization, and then pumped into the fermenter. Para-aminobenzoic acid enhances the pentose phosphate pathway, increases NADPH supply, and provides reducing power for EPA synthesis. Ascorbic acid removes intracellular reactive oxygen species and protects the unsaturated double bonds of EPA. The two work synergistically to promote synthesis and prevent oxidation, thus significantly reducing the EPA oxidation loss rate.

[0037] 4.6 Fermentation endpoint: The total fermentation time was 120 hours, and the residual sugar was maintained at 20–30 g / L before fermentation was stopped. The yield and purity of EPA reached their peak, and the cell activity was good, avoiding the decomposition of oil due to over-fermentation.

[0038] 5. Low-temperature ultrasonic disruption of wet bacterial cells 5.1. Collection of bacterial cells: Cool the fermentation broth to 4°C, centrifuge at 8000 r / min for 10 min, discard the supernatant, and collect the wet cells; collect rapidly at low temperature to avoid cell autolysis and EPA oxidation.

[0039] 5.2 Low-temperature ultrasonic cell disruption: The wet bacterial cells were suspended in a buffer solution and subjected to ultrasonic disruption at a low temperature of 4°C throughout the process. The ultrasonic power was 300–400W, using an intermittent mode of 2 seconds of operation followed by 2 seconds of rest, with a total processing time of 10–15 minutes. After disruption, no drying was performed, and the cells were directly subjected to extraction. Low temperature and intermittent ultrasonication can efficiently disrupt cell walls, resulting in an oil release rate of ≥98%. This avoids the oxidation and denaturation of EPA caused by high-temperature drying, thus preserving the structural integrity of EPA to the greatest extent possible.

[0040] 6. Urea inclusion purification 6.1 Crude oil extraction: Add food-grade n-hexane to the cell wall disruption liquid, shake thoroughly for 30 minutes, allow to stand and separate into layers, take the upper organic phase, evaporate under reduced pressure to remove the solvent, and obtain crude algae oil.

[0041] 6.2 Urea inclusion complexation: Urea was dissolved in anhydrous ethanol at a mass ratio of 0.8:1 to crude oil. The crude oil was then added and heated to 60°C with stirring until completely dissolved. The mixture was slowly cooled to 4°C and allowed to stand for 8 hours to allow the inclusion complex to form. The urea inclusion complex precipitate was removed by centrifugation, and the upper oil phase was collected. Urea molecules selectively formed stable inclusion complexes with saturated fatty acids and monounsaturated fatty acids, which crystallized out and efficiently removed saturated fatty acids. This significantly increased the proportion of polyunsaturated fatty acids in the oil phase and reduced the load on subsequent molecular distillation.

[0042] 7. Secondary molecular distillation enrichment 7.1 First-stage molecular distillation: The oil phase encapsulated with urea is fed into a molecular distillation apparatus, with the temperature controlled at 120–130℃, the vacuum at 5–10Pa, and the feed rate at 1–2mL / min. Light components such as residual solvent, free fatty acids, and small molecule odor substances are removed to obtain an odorless, low-acid-value refined oil.

[0043] 7.2 Secondary Molecular Distillation: The product from the first-stage distillation is transferred to the second-stage molecular distillation, with the temperature controlled at 140–150℃, the vacuum at 3–5Pa, and the feed rate at 1–2mL / min. The heavy phase is collected. By utilizing the difference in boiling points of fatty acids, EPA is efficiently separated from high-carbon chain PUFAs such as DPA and DHA, achieving a high enrichment of EPA.

[0044] 8. Immobilized adsorption decolorization and purification 8.1 Preparation of composite adsorbent: Attapulgite clay and activated carbon were mixed at a mass ratio of 3:1 and activated in a muffle furnace at 350℃ for 2 hours. After cooling, the mixture was pulverized and sieved to control the particle size to 80–120 mesh. High-temperature activation significantly increased the specific surface area and activity of the adsorbent, enhancing its ability to decolorize, deodorize, and remove peroxides and heavy metals.

[0045] 8.2 Decolorization of immobilized columns: The composite adsorbent is packed into a chromatography column, with the column length-to-diameter ratio controlled to be no less than 8:1. The secondary molecular distillation product is passed through the chromatography column at a flow rate of 1–2 mL / min, and the operating temperature is 35–45℃. Activated carbon adsorbs pigments and odor molecules, while attapulgite adsorbs heavy metals and peroxides. The product is a light yellow, clear, oily substance with no fishy smell, low acid value, and high stability, and can be directly used as a raw material for food, health products, and pharmaceuticals.

[0046] 9. Collection and Preservation of Finished Products The decolorized EPA refined oil is sealed with nitrogen and stored in a dark, low-temperature environment. Nitrogen isolates oxygen, further preventing EPA oxidation and extending its shelf life.

[0047] Comparative example: Using the unmodified, undomesticated original Schizochytrium ATCC1381, the traditional process for DHA production by fermentation of Schizochytrium was followed: Strains: Wild-type Schizochytrium, without MAT overexpression, CLF knockout, or high-sugar domestication; Fermentation: Cultured at a constant temperature of 28℃ with a constant dissolved oxygen level of 30%, without the addition of para-aminobenzoic acid and ascorbic acid; Cell wall disruption: After the bacterial cells are dried with hot air, the cell walls are mechanically disrupted; Purification: single urea inclusion and single-stage molecular distillation; Decolorization: Only activated carbon decolorization, no immobilized column, no composite adsorbent; The remaining culture conditions, culture medium, and fermentation time are consistent with those of this invention.

[0048] The following comparison is made between Embodiment 1 of the present invention and a comparative example: Comparison conditions: same fermenter, same culture medium, same culture time (120 h), same extraction and purification equipment, same detection method (GC). (FID determination of fatty acid composition); Comparison table of test results:

[0049] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A process for producing EPA using Schizochytrium fungi as raw material, characterized in that, include: A dual-gene modified Schizochytrium strain with MAT gene overexpression and CLF gene precise knockout was used. The modified strain was subjected to adaptive domestication with high sugar and reactive oxygen species coupling. Fermentation was carried out under three-stage temperature control and stepwise dissolved oxygen control. The wet cell was ultrasonically disrupted, and then purified by urea inclusion, secondary molecular distillation and immobilized adsorption decolorization to obtain high-purity EPA.

2. The process for producing EPA from Schizochytrium as a raw material according to claim 1, characterized in that, The Schizochytrium strain described was *Schizochytrium* sp. ATCC1381. The MAT gene was stably overexpressed by homologous recombination and integration into the 18S rDNA site. The CLF gene was expressed using CRISPR. Cas9 was sequence-specifically knocked out, causing the strain's carbon flow to be directed toward C20 fatty acid synthesis.

3. The process for producing EPA from Schizochytrium as a raw material according to claim 1, characterized in that, The domestication process involves coupled passage of glucose concentration gradients with bipyridine-induced oxidative stress, with a domestication cycle of no less than 30 generations, to obtain stable domesticated strains with high glucose tolerance, low malondialdehyde accumulation, and high NADPH regeneration capacity.

4. The process for producing EPA from Schizochytrium as a raw material according to claim 1, characterized in that, Fermentation adopts a three-stage temperature control mode: 0 Control the temperature at 28℃ for 36 hours. The temperature is controlled at 25℃ for 60 hours, and then at 20℃ after 60 hours, in conjunction with a step-down control mode for dissolved oxygen.

5. The process for producing EPA from Schizochytrium as a raw material according to claim 1, characterized in that, Para-aminobenzoic acid and ascorbic acid were added to the fermentation medium in combination. The amount of para-aminobenzoic acid added was 200 mg / L and the amount of ascorbic acid added was 150 mg / L. Both were added at once after 24 hours of fermentation.

6. The process for producing EPA from Schizochytrium as a raw material according to claim 1, characterized in that, The ultrasonic disruption of the wet bacterial cells was performed at a low temperature of 4°C with an ultrasonic power of 300 kW. 400W, 2s operation time, 2s interval, total processing time 10 After 15 minutes of cell wall disruption, the oil is extracted directly without drying.

7. The process for producing EPA from Schizochytrium as a raw material according to claim 1, characterized in that, During the inclusion process of urea, the mass ratio of urea to ester was 0.8:1, the inclusion temperature was 4℃, and the inclusion time was 8h. After centrifugation, the oil phase of the inclusion product was directly fed into molecular distillation.

8. The process for producing EPA from Schizochytrium as a raw material according to claim 1, characterized in that, The second-stage molecular distillation uses a continuous feed mode, with the first-stage distillation temperature at 120°C. 130℃, vacuum degree 5 10 Pa, secondary distillation temperature 140 150℃, vacuum degree 3 5Pa, feed rate 1 2 mL / min.

9. The process for producing EPA from Schizochytrium as a raw material according to claim 8, characterized in that, The molecularly distilled heavy phase is directly fed into a decolorization column filled with a composite immobilized adsorbent of attapulgite and activated carbon. The aspect ratio of the decolorization column is not less than 8:1, and the operating temperature is 35°C. 45℃.

10. A process for producing EPA from Schizochytrium fungi according to claim 9, characterized in that, The composite immobilized adsorbent is composed of attapulgite and activated carbon in a mass ratio of 3:1, and is activated at 350℃ for 2 hours, with the particle size controlled at 80 mm. Between 120 mesh.

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