Method for obtaining strains of phaeodactylum tricornutum that overproduce fucoxanthin

Description

[0001] METHOD FOR OBTAINING STRAINS OF PHAEODACTYLUM TRICORNUTUM THAT OVERPRODUCE FUCOXANTHIN

[0002] TECHNICAL SECTOR

[0003] This technology is related to the pharmaceutical industry, particularly the production of nutraceuticals, cosmetics, drugs, and functional foods. This method allows for meeting the growing global demand for fucoxanthin (FX) without the need to process large quantities of algal biomass.

[0004] STATE OF THE ART

[0005] Fucoxanthin (FX) is a lipophilic carotenoid pigment, orange-brown in color, belonging to the xanthophyll class, with a number of beneficial properties for human health, including potent antioxidant, anti-obesity, anti-inflammatory, anti-diabetic, dermatoprotective and hepatoprotective activity, anti-angiogenic, cerebrovascular and ocular protection 1 ' 2-5FX is used particularly in the pharmaceutical industry as a dietary supplement to promote weight loss. Due to its various health benefits, its demand and application in nutraceuticals, cosmetics, pharmaceuticals, and functional foods is constantly growing. Global sales increased from 396 tons in 2013 to 469 tons in 2017, with a projected 593 tons by 2025. 5 In the US, the European Union, and Japan, FX has been shown to be safe for consumption in animals, including humans. 7 and therefore has the potential to be used as a bioactive molecule for the prevention or treatment of diseases in the same x .

[0006] Currently, commercially available FX is primarily extracted from marine brown macroalgae; however, its use as a natural source has considerable limitations, including its low growth rate, low yield, and potential negative environmental impacts, such as the overexploitation of natural seagrass beds. x ' 8 Current limitations for obtaining FX from brown macroalgae are such that, in Southeast Asia and some European countries, its production is not commercially viable. This is because the algae present in the region, in addition to being prioritized as food and sources of hydrocolloids, contain very low concentrations of the compound (390-870 pg*g). 1 dry weight) 9 ' 10 12This concentration is also subject to the seasonality of these species and their respective life cycles, which can range from months to years, in addition to the environmental impact that the annual extraction of tons of brown algae from natural seagrass meadows or the use of large areas in the sea for their industrial cultivation could constitute.

[0007] Furthermore, the chemical synthesis of FX is complex, significantly increasing its cost and making it an economically unfeasible process. 1 This necessitates the development of new, more efficient, sustainable, and above all, profitable methods for FX production. In this context, microalgae present an excellent opportunity for FX production, as they could be up to 40 times more productive than brown macroalgae. 13 ' 14 .

[0008] Specifically, the diatom Phaeodactylum tricornutum has been extensively studied, and there are multiple reports in the literature of its use as a model microalga for algal biotechnology, primarily for the production of omega fatty acids. Furthermore, P. tricornutum is known to naturally produce omega fatty acids (FX) in the range of 0.2–40 mg / g. 1 dry weight. 15 ' 15 This microalga is ideal for industrial production processes, as it is well known for its robust growth in large-scale culture systems and its tolerance to low light and high pH levels. Given its well-characterized genome, the currently available bioengineering tools make P. tricornutum an interesting candidate as a cellular platform for the commercial-scale production of high-value biological compounds. 17 19 thus generating new industries based on sustainable biotechnology.

[0009] FX is also recognized as a GRAS (Generally Recognized As Safe) compound by various entities dedicated to the safety of bioactive compounds, such as the U.S. Food and Drug Administration (FDA), the European Food Safety Authority (EFSA), and Japan's Food for Specified Health Uses (FOSHU). 20 .

[0010] Products currently on the market containing fucoxanthin use the brown macroalga Undaria pinnatifida as a raw material. This macroalga only grows in the Northern Hemisphere. In Chile, there is no industry for the production of nutraceuticals from algae. Because P. tricornutum is a microalga widely used in the aquaculture industry, especially as mussel feed, it is an excellent candidate for the production of high-value secondary metabolites such as fucoxanthin.

[0011] Currently, there is still a need to supply the growing global demand for FX for the nutraceutical industry, which requires processing large quantities of algal biomass. As mentioned previously, various studies have reported the poor FX yield in various macroalgae, not usually exceeding 1 mg of FX per gram of biomass. 21 ' 22 making its extraction an inefficient and unprofitable process.

[0012] References

[0013] 1. Cornelli, U. Antioxidant use in nutraceuticals. Clin. Dermatol. 27, 175-194 (2009).

[0014] 2. Seth, K. et al. Bioprospecting of fucoxanthin from diatoms — Challenges and perspectives. Algal Res. 60, 102475 (2021)

[0015] 3. Pereira, A. G. et al. Xanthophylls from the Sea: Algae as Source of Bioactive Carotenoids. Mar. Drugsl9, 1-31 (2021). Lourengo-Lopes, C. et al. Biological action mechanisms of fucoxanthin extracted from algae for application in food and cosmetic industries. Trends Food Sci. Technol. 117, 163-181 (2021). Ganesan, A. R., Tiwari, U. & Rajauria, G. Seaweed nutraceuticals and their therapeutic role in disease prevention. Food Sci. Hum. Wellness 8, 252-263 (2019). Hitoe, S. & Shimoda, H. Seaweed Fucoxanthin Supplementation Improves Obesity Parameters in Mild Obese Japanese Subjects. Funct. Foods Heal. Dis. 7, 246 (2017). Sun, H. et al. Fucoxanthin from marine microalgae: A promising bioactive compound for industrial production and food application. https: / / doi.org / 10.1080 / 10408398.2022.2054932 1-17 (2022). doi:10.1080 / 10408398.2022.2054932 Kanda, H., Kamo, Y., Machmudah, S., Wahyudiono & Goto, M.Extraction of fucoxanthin from raw macroalgae excluding drying and cell wall disruption by liquefied dimethyl ether. Mar. Drugs 12, 2383-2396 (2014). Pajot, A., Hao Huynh, G., Picot, L., Marchal, L. & Nicolau, E. Fucoxanthin from Algae to Human, an Extraordinary Bioresource: Insights and Advances in up and Downstream Processes. Mar. Drugs 20, 222 (2022). Bux, F. & Chisti, Y. Algae biotechnology. Products and processes. Green Energy Technol. 344 (2016).doi:10.1007 / 978-3-319-12334-9 Hasunuma, T. et al. Single-Stage Astaxanthin Production Enhances the Nonmevalonate Pathway and Photosynthetic Central Metabolism in Synechococcus sp. PCC 7002. ACS Synth. Biol. 8, 2701-2709 (2019). McClure, D. D., Luiz, A., Gerber, B., Barton, G. W. & Kavanagh, J. M. An investigation into the effect of culture conditions on fucoxanthin production using the marine microalgae Phaeodactylum tricornutum. Algal Res. 29, 41-48 (2018) Neumann, U. et al.Fucoxanthin, a carotenoid derived from Phaeodactylum tricornutum exerts antiproliferative and antioxidant activities in vitro. Antioxidants 8, 1-11 (2019). He, L, Han, X. & Yu, Z. A rare Phaeodactylum tricornutum cruciform morphotype: Culture conditions, transformation and unique fatty acid characteristics. PLoS One 9, 1- 8 (2014). Shannon, E. & Abu-Ghannam, N. Optimisation of fucoxanthin extraction from Irish seaweeds by response surface methodology. J. Appl. Phycol. 29, 1027-1036 (2017). 16. Butler, T., Kapoore, R. V. & Vaidyanathan, S. Phaeodactylum tricornutum: A Diatom Cell Factory .Trends Biotechnol. 1-17 (2020). doi:10.1016 / j.tibtech.2019.12.023.

[0016] 17. Hallmann, A. Grand Challenges in Algae Biotechnology. (2020). doi:https: / / doi.org / 10.1007 / 978-3-030-25233-5

[0017] 18. Rasul, I. et al. Algae Biotechnology: A Green Light for Engineered Algae. Algae Based Polymers, Blends, and Composites: Chemistry, Biotechnology and Materials Science (Elsevier Inc., 2017). doi:10.1016 / B978-0-12-812360-7.00008-2

[0018] 19. Fabris, M. et al. Extrachromosomal Genetic Engineering of the Marine Diatom Phaeodactylum tricornutum Enables the Heterologous Production of Monoterpenoids. ACS Synth. Biol. (2020). doi:10.1021 / acssynbio.9b00455

[0019] 20. Ikeda, K. et al. Effect of lindaría pinnatifida (Wakame) on the development of cerebrovascular diseases in stroke-prone spontaneously hypertensive rats. Clin. Exp. Pharmacol. Physiol. 30, 44-48 (2003).

[0020] 21. Airanthi, M. K. W. A., Hosokawa, M. & Miyashita, K. Comparative Antioxidant Activity of Edible Japanese Brown Seaweeds. J. Food Sci. 76, (2011).

[0021] 22. Maeda, H., Hosokawa, M., Sashima, T., Funayama, K. & Miyashita, K. Fucoxanthin from edible seaweed, lindaria pinnatifida, shows antiobesity effect through UCP1 expression in white adipose tissues. Biochem. Biophys. Commun. Res. 332, 392-397 (2005).

[0022] BRIEF DESCRIPTION OF THE FIGURES

[0023] Figure 1: Schematic of the pPT-FX expression vector. The asterisk (*) shows the reading frame of the coding sequence associated with the promoter.

[0024] Figure 2: Colonies transformed with the pPT-FX vector.

[0025] Figure 3: Chromatogram of fucoxanthin samples from native (a), overproducing (b) and standard (c) strains of P. tricornutum.

[0026] Figure 4: Fucoxanthin concentration results from the native strain (white) and overproducing strain (gray). DESCRIPTION OF THE INVENTION

[0027] A method is presented for obtaining Phaeodactylum tricornutum strains that overproduce Fucoxanthin (FX), by transformation with an expression vector specially designed for this purpose.

[0028] The expression vector is designed as an operon to jointly overexpress the coding sequences of the genes required to enhance the biosynthesis of FX, 1-deoxy-xylulose 5-phosphate synthase (DXS), and phytoene synthase (PSY) in P. tricornutum. These sequences are tandemly regulated by a single transcription promoter.

[0029] It should be noted that the coding sequences of DXS and PSY are chloroplastidial, therefore a signal peptide for transport to the chloroplast is included.

[0030] Figure 1 shows the schematic of the designed pPT-FX expression vector, which features the fcpA promoter (SEQ ID No. 1), a chloroplast transport signal peptide (SEQ ID No. 2), followed by the coding sequence for the 1-deoxyxylulose 5-phosphate synthase (DXS) enzyme (SEQ ID No. 3), then the FLAG epitope (SEQ ID No. 4), followed by peptide 2A (SEQ ID No. 5), then the coding sequence for the phytoene synthase (PSY) enzyme (SEQ ID No. 6), and ending with the HA (hemagglutinin) epitope (SEQ ID No. 7), finally terminating with the fcpA terminator (SEQ ID No. 8). Additionally, zeocin resistance (BleoR) (SEQ ID No. 9) was included in this vector as a selection marker for transformation into P. tricornutum.

[0031] The method for obtaining P. Tricornutum strains transformed with the expression vector (pPT-FX) by electroporation comprises at least the following steps: a) Preparation of P. Tricornutum from growth medium: centrifugation of 2 to 10 x10 5cells in the late exponential state, washing and resuspension of the cells with electroporation solution (sorbitol 375 mM), with a final volume of 0.5 to 5 ml; b) Incubate the cells with 10 to 30 pg of the expression vector (pPT-FX) for 5 minutes at 4°C; c) Transfer to an electroporation cuvette and electroporate between 0.5 kV and 1 kV, performing 1, 3 and 5 pulses; d) Transfer the biomass to a sterile 15 mL tube with 10 mL of Guillard Medium F / 2 (F / 2), incubate with shaking at a light intensity of 10 - 50 pE overnight; e) Centrifuge and streak the biomass onto F / 2 agar plates with zeocin as a selection label at a concentration of 10 - 35 mg / mL; f) Incubate with a light intensity of 80-120 pE at 15-25 °C and with a photoperiod of 16 hours light and 8 hours dark; g) Select colonies that show resistance to the zeocin selection marker; and h) Check the fucoxanthin biosynthesis capacity.

[0032] The transformed strains of P. tricornutum showed a fucoxanthin productivity that was twice as high as that of the base strain.

[0033] A fucoxanthin (FX) overproducing strain of Phaeodactylum tricornutum, designated pt-2FX, is also presented. This strain was obtained through transformation using the designed expression vector and the method described above. It is deposited in the Spanish Bank of Algae (BEA).

[0034] Obtaining fucoxanthin from overproducing P. tricornutum offers several competitive advantages over the current industrial FX source. Table 1 shows some of the advantages of the developed technology. Table 1: Advantages of the current method versus the new technology.

[0035] Industry Problem Proposed Solution

[0036] Low concentration of fucoxanthin High concentration of fucoxanthin Low availability, used in other industries Industrial scalability Model microalgae

[0037] Seasonal variability No seasonal variability

[0038] Low profitability High profitability

[0039] Slow growth Fast growth

[0040] High environmental impact Low environmental impact

[0041] Unsustainable industry Sustainable industry

[0042] Low number of FX producers

[0043] Geographic independence worldwide

[0044] APPLICATION EXAMPLES Example 1: Vector construction using an operon-type modular system.

[0045] The expression vector was designed as an operon to jointly overexpress the coding sequences of the genes necessary to enhance the biosynthesis of FX, 1-deoxy-xylulose 5-phosphate synthase (DXS) (SEQ ID N°3) and phytoene synthase (PSY) (SEQ ID N°6) of P. tricornutum. These sequences are tandemly regulated by a single transcription promoter (SEQ ID N°1) and include a chloroplast transport signal peptide corresponding to SEQ ID N°2.

[0046] The expression vector was designed using as a basis the vector present in the article by Taparía, Y., Zarka, A., Leu, S. et al. k novel endogenous selection marker for the diatom Phaeodactylum tricornutum based on a unique mutation in phytoene desaturase 1. Sci Rep 9, 8217 (2019). Figure 1 shows the schematic of the designed pPT-FX expression vector. This vector was designed with a DNA fragment containing the fcpA promoter (SEQ ID No. 1), followed by the coding sequence for the 1-deoxyxylulose 5-phosphate synthase (DXS) enzyme (SEQ ID No. 3), then the FLAG epitope (SEQ ID No. 4), followed by peptide 2A (SEQ ID No. 5), then the coding sequence for the phytoene synthase (PSY) enzyme (SEQ ID No. 6), and ending with the HA (hemagglutinin) epitope (SEQ ID No. 7), finally terminating with the fcpA terminator (SEQ ID No. 8). Additionally, zeocin resistance (BleoR) (SEQ ID No. 9) was included in this vector as a selection marker for transformation into P. tricornutum.

[0047] Example 2: P. Tricornutum transformation method with the expression vector (pPT-FX)

[0048] P. tricornutum strains were transformed using the expression vector (pPT-FX) prepared in Example 1, by electroporation. A total of 4 million cells in the late exponential stage were centrifuged and resuspended in 1 mL of electroporation solution (375 mM sorbitol). The biomass was washed five times with 2 mL of electroporation solution each time, resuspended in electroporation solution, and centrifuged at 1500 xG for 10 minutes. The supernatant was removed, and the cells were resuspended in electroporation solution. The washed cells were incubated with 30 pg of plasmid DNA for 5 minutes on ice. The plasmid DNA cell solution is transferred to a 2 mm electroporation cuvette and electroporated with 0.5 kV with 25 pF capacitance and 400 Ohm resistance.After electroporation, the biomass was transferred to a sterile 15 mL tube containing 10 mL of F / 2 and incubated with shaking at a light intensity of 30 pE overnight. The tube was then centrifuged, and the biomass was plated onto F / 2 agar plates containing zeocin as a selection marker at a concentration of 20 mg / mL. The plates were incubated at 18 °C with a light intensity of 100 pE and a photoperiod of 16 hours light and 8 hours dark. Colonies exhibiting resistance to the zeocin selection marker were selected (Figure 2).

[0049] Example 3: Selection of colonies and determination of fucoxanthin biosynthesis.

[0050] Colonies were grown on F / 2 culture medium with and without zeocin, and fucoxanthin biosynthesis capacity was studied. Selected cells were grown on F / 2 at 22 °C for one week. Five scale-up stages were performed (5 ml, 50 ml, 250 ml, 1000 ml, and 5000 ml) with one week of culture between stages. Each new scale-up stage was initiated with 30,000 cells / ml.

[0051] To determine fucoxanthin productivity, a fucoxanthin extraction was performed. This was done by weighing 0.02 g of wet P. tricornutum biomass into 2 mL microcentrifuge tubes containing zirconium beads and 1 mL of analytical-grade ethanol. The mixture was then vortexed at maximum power for 1 min and subsequently ultrasonically centrifuged for 20 min at 35°C to extract the compound from the microalga. Finally, the sample was centrifuged for 2 min at 14,000 g, and the supernatant was recovered and stored at -20°C until analysis. This process was repeated until the supernatant was colorless. Once all the extract was collected from the sample, the extraction solvent was evaporated with nitrogen, and the extract was resuspended in 3 mL of mobile phase. (Kim, 2014).

[0052] To quantify the FX present in the samples, after extraction, they were filtered and injected into a Kromasil C18 column (250 x 4.6 mm) with a flow rate of 1 mL min⁻¹ for 30 min. Acetonitrile and water were used as the mobile phase. Chromatography was performed using a Young in Chromass binary pump (model: YL911X) coupled to a PDA detector (model: YL9160) (Figure 3). The FX concentration in the sample was calculated by interpolating from a calibration curve using linear regression and the equation of the line with an analytical standard. The transformed strains of P. tricornutum showed a fucoxanthin productivity that was twice that of the base strain (Figure 4; Table 2).

[0053] Table 2: Comparison of fucoxanthin productivity.

[0054] Sample PT solvent extraction Concentration FX pg mL -1

[0055] Native 190,386 ± 34,08

[0056] Overproducer 322,094 ± 51.49

Claims

CLAIMS 1. An expression vector CHARACTERIZED because it allows joint overexpression of the coding sequences of the genes l-deoxy-xylulose 5-phosphate synthase (DXS) (SEQ ID N°3) and phytoene synthase (PSY) (SEQ ID N°6), necessary to enhance the biosynthesis of Fucoxanthin (FX).

2. An expression vector, according to claim 1, CHARACTERIZED in that the SEQ ID N°3 and SEQ ID N°6 sequences are in tandem regulated by a single fcpA transcription promoter (SEQ ID N°1).

3. An expression vector, according to claim 1, CHARACTERIZED in that the coding sequences SEQ ID No. 3 and SEQ ID No. 6 are chloroplastidial, whereby the vector includes a chloroplast transport signal peptide (SEQ ID No. 2).

4. An expression vector, according to claim 1, CHARACTERIZED in that the designed expression vector, named pPT-FX, comprises an fcpA promoter (SEQ ID No. 1), a chloroplast transport signal peptide (SEQ ID No. 2), a coding sequence for the enzyme 1-deoxy-xylulose 5-phosphate synthase (DXS) (SEQ ID No. 3), a FLAG epitope (SEQ ID No. 4), a peptide 2A (SEQ ID No. 5), a coding sequence for the enzyme phytoene synthase (PSY) (SEQ ID No. 6), an HA (hemagglutinin) epitope (SEQ ID No. 7), an fcpA terminator (SEQ ID No. 8), and a selection marker for zeocin resistance (BleoR) (SEQ ID No. 9).

5. Use of the expression vector, according to claim 1, CHARACTERIZED in that it serves to obtain Fucoxanthin (FX) overproducing strains.

6. Use of the expression vector, according to claim 5, CHARACTERIZED in that the overproducing strain is a strain of Phaeodactylum tricornutum.

7. A method for obtaining Phaeodactylum tricornutum strains that overproduce Fucoxanthin (FX), CHARACTERIZED in that the strains are transformed with the pPT-FX expression vector, specially designed for this purpose.

8. A method for obtaining Fucoxanthin (FX) overproducing strains of Phaeodactylum tricornutum, according to claim 7, CHARACTERIZED in that it comprises at least the following steps: a) Preparation of P. tricornutum from growth medium: centrifugation from 2 to 10 x10 5cells in the late exponential state, washed and resuspended with electroporation solution (375 mM sorbitol), with a final volume of 0.5 to 5 ml; b) Incubate the cells with 10 to 30 pg of the pPT-FX expression vector for 5 minutes at 4°C; c) Transfer to an electroporation cuvette and electroporate between 0.5 kV and 1 kV, performing 1, 3 and 5 pulses; d) Transfer the biomass to a sterile 15 mL tube with 10 mL of Guillard Medium F / 2 (F / 2), incubate with shaking at a light intensity of 10 - 50 pE overnight; e) Centrifuge and streak the biomass onto F / 2 agar plates with zeocin as a selection label at a concentration of 10 - 35 mg / mL; f) Incubate with a light intensity of 80-120 pE at 15-25 °C and with a photoperiod of 16 hours light and 8 hours dark; g) Select colonies that show resistance to the zeocin selection marker; and h) Check the fucoxanthin biosynthesis capacity.

9. Use of the method for obtaining fucoxanthin (FX) overproducing strains of Phaeodactylum tricornutum, according to claim 7, CHARACTERIZED in that it serves to obtain strains that have an FX productivity twice that of the control strain.

10. A method for obtaining fucoxanthin (FX) overproducing strains of Phaeodactylum tricornutum, according to claim 7, CHARACTERIZED in that it serves to obtain fucoxanthin on an industrial scale, without seasonal variability, with low environmental impact and geographical independence.

11. A Phaeodactylum tricornutum overproducing strain of Fucoxanthin (FX), CHARACTERIZED because it is obtained through the transformation method with the designed expression vector pPT-FX, is called strain pt-2FX and is deposited in the Spanish Bank of Algae (BEA).

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