Methods useful for generating genetically modified host cells and Porphyra-334 and / or synolin

Abstract

The present invention provides a genetically modified host cell capable of producing Porphyra-334 and / or synolin, comprising (a) (i) 2-demethyl-4-deoxygazol synthase (DDGS), (ii) O-methyltransferase (O-MT or OMT), (iii) ATP-grasp ligase, and (iv) non-ribosomal peptide synthetase (NRPS) or D-ala-D-ala ligase, MysD or MysE; or (b) MysA, MysB, MysC, or MysD, MysE, or NRPS, wherein one or more of the aforementioned enzymes are homologous enzymes. TIFF2026518832000029.tif128159

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 63 / 493,636, filed Mar. 31, 2023, which is hereby incorporated by reference in its entirety.

[0002] Statement of Government Support This invention was made with government support under Contract No. DE - AC02 - 05CH11231 awarded by the U.S. Department of Energy. The government has certain rights in this invention.

[0003] Field of the Invention This invention pertains to the field of production of porphyrar - 334 and / or cynaurine.

[0004] Reference to a Sequence Listing This application includes a sequence listing submitted electronically in XML format, which is hereby incorporated by reference in its entirety. The XML copy was created on Mar. 28, 2024, named "2021 - 119 - 02 Sequence Listing.xml", and is 16 kilobytes in size.

Background Art

[0005] Background of the Invention The prolonged warm period (Oliver et al., 2018), global warming (Johnson et al., 2022), and the excessive use of chemical sunscreens (Roberto et al., 2008) have been associated with global coral bleaching. Due to concerns about the harmful effects of these on both human health and the environment, the use of commercial sunscreens containing oxybenzone, octinoxate, ZnO, and TiO2 has been prohibited in several countries and regions. This has led to an increasing demand for sunscreens derived from environmentally friendly raw materials. Shinorine is a mycosporine-like amino acid (MAA) typically produced by the red alga Porphyra umbilicalis (Figure 19) and is used as an active ingredient in commercial sunscreen products (e.g., Helioguard (商標) 365, Helionori (登録商標) ). Additional benefits of shinorine, including anti-aging properties, antioxidant effects, promotion of wound healing, and suppression of skin inflammation by ultraviolet rays, have also been demonstrated (Choi et al., 2015; Hartmann Johanna; Fuchs Julian E.; Chaita Eliza; Aligiannis Nektarios; Skaltsounis Leandros; Ganzera Markus, 2015; Orfanoudaki et al., 2020; Suh et al., 2014; Torres et al., 2018). However, the yield of shinorine from P. umbilicalis is relatively low (3.27 mg per g of cell dry weight) (Becker et al., 2016), and its production is adversely affected by the slow growth of P. umbilicalis. With the increasing demand for sunscreens worldwide, the bioproduction of shinorine by fast-growing microorganisms has emerged as an attractive solution.

[0006] To meet the growing demand for synolin, various host organisms have been genetically engineered to produce synolin, including Saccharomyces cerevisiae (Jin et al., 2021; Kim et al., 2023, 2022; Park et al., 2019), Corynebacterium glutamicum (Tsuge et al., 2018), Synechocystis sp. PCC 6803 (Yang et al., 2018), Streptomyces avermitilis (Miyamoto et al., 2014), and more recently, Yarrowia lipolytica (Jin et al., 2023). However, most of the aforementioned metabolic manipulation attempts in host organisms have focused primarily on enhancing the xylulose 5-phosphate pool by linking the synoline production pathway with the xylose utilization pathway (Jin et al., 2023; Kim et al., 2023, 2022; Park et al., 2019). While this approach has proven to improve synoline production titer, challenges remain. Firstly, the best productivity (12.75 mg / L / h) and yield (3.66 mg / g glucose; assuming total consumption of 420 g glucose and 160 g xylose) were achieved in Saccharomyces cerevisiae in fed-batch fermenters (Kim et al., 2023). Secondly, since microbial hosts typically prefer consuming hexoses (e.g., glucose) over pentoses (e.g., xylose) (Aidelberg et al., 2014; Dvorak and de Lorenzo, 2018; Wang et al., 2019), supplying sugar mixtures (e.g., hexoses and pentoses) to the culture medium also adds complexity to the production experiments (Park et al., 2019).Thirdly, most of the cinolines produced from Saccharomyces cerevisiae and Yarowia liporitica tend to accumulate intracellularly (Jin et al., 2023; Kim et al., 2023), complicating extraction and purification.

[0007] Currently, cinolin is produced from red algae (3.2 mg / g CDW) and cyanobacteria (2.37 mg / g CDW). Park et al. have disclosed the construction of a yeast strain that produces 31.0 mg / L of cinolin in an optimized medium containing 8 g / L xylose and 12 g / L glucose by introducing a cinolin biosynthesis gene from the cyanobacterium Nostoc punctiforme into Saccharomyces cerevisiae (ACS Synthetic Biol. 8:346-357, 2019).

[0008] Hahn et al. (PCT international patent application WO 2021 / 133101) disclose a modified Saccharomyces cerevisiae that produces synorine using xylose as a carbon source.

[0009] Kotagiri et al. (U.S. Patent Application Publication No. 2022 / 0275411) disclose a genetically modified strain of the commensal bacterium Staphylococcus epidermis containing a plasmid containing four Anabaena variabilis genes for the production of mycosporine-like amino acids (MAAs). [Overview of the Initiative]

[0010] The present invention provides a genetically modified host cell capable of producing Porphyra-334 and / or synolin. This genetically modified host cell comprises (a) (i) 2-demethyl-4-deoxygazol synthase (DDGS), (ii) O-methyltransferase (O-MT or OMT), (iii) ATP-grasp ligase, and (iv) non-ribosomal peptide synthetase (NRPS) or D-ala-D-ala ligase, MysD or MysE, or (b) MysA, MysB, MysC, or MysD, MysE, or NRPS; where one or more of the aforementioned enzymes are their homologous enzymes.

[0011] In some embodiments, 2-demethyl-4-deoxyguzzol synthase (DDGS) is cyanobacteria DDGS or its homologous enzyme. In some embodiments, O-methyltransferase (O-MT or OMT) is cyanobacteria OMT or its homologous enzyme. In some embodiments, ATP-grasp ligase is cyanobacteria ATP-grasp ligase or its homologous enzyme. In some embodiments, non-ribosomal peptide synthetase (NRPS) or D-ala-D-ala ligase is cyanobacteria non-ribosomal peptide synthetase (NRPS) or D-ala-D-ala ligase, or their homologous enzymes. In some embodiments, MysA, MysB, MysC, MysD, and / or MysE are independently derived from or obtained from Anabaena ATCC 29413, Nostoc ATCC 29133, Porphyra umbilicalis, and / or Chondrus crispus. The cyanobacterial MysD or D-ala-ala ligase has loose substrate specificity and condenses threonine instead of serine on myosporine-glycine to produce Porphyra-334. In some embodiments, one or more or all of the enzymes are wild-type enzymes.

[0012] In some embodiments, DDGS, OMT, ATP-grasp ligase, and / or non-ribosomal peptide synthetase (NRPS) or D-ala-D-ala ligase are each independently obtained from or derived from Porphyra umbilicalis, Nostoc punctiforme, and / or Anabaena variabilis.

[0013] The amino acid sequence of DDGS of Nostoc punctiforme or Anabaena variabilis is as follows: TIFF2026518832000002.tif47158

[0014] The amino acid sequence of OMT from Nostoc punctiforme or Anabaena variabilis is as follows: TIFF2026518832000003.tif39140

[0015] The amino acid sequence of the ATP-grasp ligase of *Nostoc punctiforme* or *Anabaena variabilis* is as follows: TIFF2026518832000004.tif68140

[0016] The amino acid sequence of NRPS from Anabaena variabilis is as follows: TIFF2026518832000005.tif119147

[0017] The amino acid sequence of the D-ala-D-ala ligase from Nostoc punctiforme is as follows: TIFF2026518832000006.tif47140

[0018] In some embodiments, host cells are genetically modified to metabolize one or more compounds that unmodified host cells cannot normally metabolize. In some embodiments, the host cells are Pseudomonas host cells. In some embodiments, one or more compounds are aromatic compounds obtained from hydrolysates of lignocellulose. Examples of such aromatic compounds include ferulic acid, 4-hydroxybenzoic acid, protocatechuic acid, vanillic acid, salicylic acid, syringic acid, p-coumaric acid, vanillin, catechol, syringaldehyde, and phenol. Examples of genetic modifications suitable for enabling host cells to metabolize such aromatic compounds are listed below: overexpression of erloyl-CoA synthetase (Fcs), enoyl-CoA hydratase / aldolase (Ech), vanillin dehydrogenase (Vdh), p-hydroxybenzoic acid hydroxylase (PobA), and vanillic acid O-demethylase oxygenase (VanAB).

[0019] In some embodiments, MysA and MysB form a fusion protein. In some embodiments, MysC and MysD form a fusion protein.

[0020] The amino acid sequence of the MysA-MysB fusion of Porphyra umbilicaris is as follows: (MysA-MysB fusion >tr|A0A1X6NP84|A0A1X6NP84_PORUM 3-dehydroquinate synthase domain-containing protein OS=Porphyra umbilicalis OX=2786 GN=BU14_0783s0002 PE=3 SV=1) TIFF2026518832000007.tif59129

[0021] The amino acid sequence of the MysC-MysD fusion of Porphyra umbilicaris is as follows: (MysC-MysD fusion >tr|A0A1X6NNV6|A0A1X6NNV6_PORUM ATP-grasp domain-containing protein OS=Porphyra umbilicalis OX=2786 GN=BU14_0783s0001 PE=4 SV=1) TIFF2026518832000008.tif84170

[0022] Heterologous expression of the MAA biosynthesis gene derived from Actinosynnema mirum DSM 43827 in the manipulated host Streptomyces abermitilis SUKA22 resulted in the production of cinolin as the main product and porphyra-334 (Miyamoto et al., “Discovery of Gene Cluster for Mycosporine-Like Amino Acid Biosynthesis from Actinomycetales Microorganisms and Production of a Novel Mycosporine-Like Biosynthesis from Actinomycetales Microorganisms and Production of a Novel Mycosporine-Like Amino Acid by Heterologous Expression,” Appl. Env. Microbiol. 80(16):5028-5038, 2014).

[0023] Synorin, a mycosporine-like amino acid (MAA) commonly produced by the red alga Porphyra umbilicalis, is used as an active ingredient in two commercially available sunscreen products (Helioguard 365 and Helionori). However, the yield of synorin from P. umbilicalis is relatively low (3.25 mg / g CDW), and its production is negatively affected by the slow growth rate of P. umbilicalis. To overcome this problem, several organisms, including E. coli, Saccharomyces cerevisiae, the Synechocystis species PCC 6803, Corynebacterium glutamicum, and Streptomyces abermitilis SUKA22, have been metabolically engineered for synorin production. In contrast to previously described methods, the present invention provides a novel process for producing synolin by metabolically manipulating host cells of the genus Pseudomonas, such as Pseudomonas putida KT2440, which can utilize various carbon sources, including aromatic compounds from lignocellulose hydrolysates, as raw materials for synolin production.

[0024] The following is a theoretical yield calculation for synoline production using glucose as a precursor. It is obtained from the formal condensation of the amino group of L-serine with the keto group of (5S)-5-hydroxy-5-(hydroxymethyl)-2-methoxycyclohexa-2-en-1-one, where the 3-position hydrogen of the cyclohexenone moiety is replaced by the amino group of glycine. See panel A in Figure 3. The compound shown has the following chemical formula and molecular weight: Shinorin: C 13 H 20 N2O8(332.3); Glucose:C6H 12 O6(180). The final equation is as follows (assuming 1 glucose → 1 serine and 1 glucose → 1 glycine): 1. Hydrocarbon core Carbon balance: 4 / 3 C6H 12O6 + 2H2 → C8H 12 O4 + 4H2O Redox balance: C6H 12 O6 + 6H2O → 6CO2 + 12H2 ⇒ 9C6H 12 O6 → 6C8H 12 O4 + 6CO2 + 18H2O ⇒ 1.5 glucose → C8H 12 O4 + CO2 + 3H2O As 1 serine or 1 glycine is required for 2.1 sinoline, each serine or glycine requires 1 glucose. 1 glucose → 1 serine 1 glucose → 1 serine → 1 glycine

[0025] The final equation is 3.5C6H 12 O6 → 1 sinoline (C 13 H 20 N2O8). Therefore, the theoretical yield is 0.52746 g of sinoline / 1 g of glucose ⇒ approximately 0.53 g of sinoline / 1 g of glucose.

[0026] In some embodiments, DDGS, OMT, ATP-grasp ligase, and / or non-ribosomal peptide synthetase (NRPS) or D-ala-D-ala ligase are homologous enzymes, variants, or mutant enzymes of those enzymes, which are any wild-type enzymes described herein. The homologous enzyme, variant, or mutant enzyme has the same enzymatic activity as its corresponding wild-type enzyme and has an amino acid sequence having 70%, 80%, 90%, 95%, or 99% or more sequence identity with the corresponding wild-type enzyme, and optionally has one or more conserved amino acid sequences and / or residues, such as conserved sequences and / or residues that are important with respect to the structure of the enzyme or catalytic residues / pockets. In some embodiments, wild-type DDGS, OMT, ATP-grasp ligase, and / or non-ribosomal peptide synthetase (NRPS) or D-ala-D-ala ligase are obtained from or derived from Porphyra umbilicalis, Nostoc punctiforme, Anabaena variabilis.

[0027] The present invention provides a method for producing Porphyra-334 and / or synolin in a much faster manner and with higher product potency and yield. Furthermore, by utilizing lignocellulose hydrolysates as raw materials, competition for the use of agricultural plants as raw materials, as is commonly used in other similar technologies (e.g., production of Porphyra-334 and / or synolin in E. coli, yeast, etc.), can be eliminated, resulting in lower costs.

[0028] The present invention provides a method for producing porphyra-334 and / or synolin, comprising the steps of: (a) providing genetically modified host cells of the present invention; (b) culturing or growing the genetically modified host cells in a suitable culture or medium so that porphyra-334 and / or synolin are produced; (c) optionally extracting or separating porphyra-334 and / or synolin from the host cells and / or culture or medium to form isolated or purified porphyra-334 and / or synolin; and (d) optionally mixing porphyra-334 and / or synolin with a lotion, oil, or water to form a composition that blocks or filters ultraviolet (UV) light.

[0029] In some embodiments, the mixing step further comprises mixing Porphyra-334 and / or Synolin, or a UV-blocking or filtering composition, with another active UV filtering agent, such as oxybenzone, octinoxate, octisalate, and avobenzone, zinc oxide, and / or titanium dioxide, or a mixture thereof.

[0030] In some embodiments, UV-blocking or filtering compositions are suitable for application to human skin, for example, as a suntan lotion. In some embodiments, UV-blocking or filtering compositions do not contain any ingredients or components that cause coral bleaching.

[0031] In some embodiments, step (a) comprises introducing one or more nucleic acids encoding (i) 2-demethyl-4-deoxyguzzol synthase (DDGS), (ii) O-methyltransferase (O-MT or OMT), (iii) ATP-grasp ligase, and (iv) non-ribosomal peptide synthetase (NRPS) or D-ala-D-ala ligase enzymes into host cells, each enzyme being functionally linked to a promoter capable of expressing each enzyme within the host cells. In some embodiments, step (b) of culturing or growing comprises host cells that grow by the proliferation of respiratory cells. In some embodiments, step (b) of culturing or growing is carried out in a batch process or fed-batch process, for example, a high-gravity fed-batch process.

[0032] In some embodiments, the culture comprises biomass such as lignocellulose biomass, or a hydrolysate thereof. In some embodiments, the biomass is obtained from softwood raw materials (such as poplar), hardwood raw materials, herbaceous raw materials, and / or agricultural raw materials, or mixtures thereof.

[0033] In some embodiments, the culture medium includes a nutrient-rich medium, such as LB (lysogenic broth), or one or more components of LB, such as tryptone and / or yeast extract. In some embodiments, the culture medium includes hydrolysates derived from or obtained therefrom biomass, such as lignocellulose biomass. In some embodiments, the culture medium includes one or more aromatic compounds, such as aromatic compounds obtained from hydrolysates of lignocellulose. In some embodiments, the culture medium includes one or more carbon sources, such as sugars like glucose, xylose, or galactose, or glycerol, or mixtures thereof. In some embodiments, the carbon source is fermentable. In some embodiments, the carbon source is non-fermentable. In some embodiments, the culture medium includes urea as a nitrogen source. In some embodiments, the culture medium includes amino acids such as serine and / or glycine. In some embodiments, the culture medium includes an ionic liquid (IL).

[0034] In some embodiments, genetically modified host cells utilize xylose as a carbon source, either naturally or through manipulation or construction. Increased xylose utilization results in an increase in the X5P pool used in the porphyra-334 and / or synolin biosynthesis pathway.

[0035] In some embodiments, genetically modified host cells exhibit increased S7P production via isomerase pathways, etc., either naturally or through manipulation or construction. In some embodiments, genetically modified host cells have the naturally occurring or endogenous KDPG aldolase-encoding gene PP_1024, or any gene encoding KDPG aldolase, reduced or knocked out, either naturally or through manipulation or construction, to downregulate or minimize the conversion of 2KDPG to pyruvate. In some embodiments, such downregulation results in growth retardation, which is compensated for by supplying the culture with aromatic compounds that ultimately become part of the acetyl-CoA pool. In some embodiments, this co-utilization strategy enables the conversion of glucose to G6P, xylose to X5P, and aromatic compounds to acetyl-CoA, which enhances the titer of porphyra-334 and / or synolin.

[0036] In some embodiments, this method results in genetically modified host cells that produce porphyra-334 and / or synolin at concentrations equal to or exceeding approximately 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, 60 mg / L, 70 mg / L, 80 mg / L, 90 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, 250 mg / L, 300 mg / L, 350 mg / L, or 400 mg / L. In some embodiments, this method is equivalent to approximately 4.5 mg / g DCW, 5.0 mg / g DCW, 5.5 mg / g DCW, 6.0 mg / g DCW, 6.5 mg / g DCW, 7.0 mg / g DCW, 7.5 mg / g DCW, 8.0 mg / g DCW, 8.5 mg / g DCW, 9.0 mg / g DCW, 9.5 mg / g DCW, or 10 mg / g DCW, or approximately 4.5 mg / g DCW, 5.0 mg / g DCW, 5.5 mg / g DCW, 6.0 mg / g DCW, 6.5 mg / g DCW, 7.0 mg / g DCW, 7.5 mg / g DCW, 8.0 mg / g DCW, 8.5 mg / g DCW, 9.0 mg / g DCW, 9.5 mg / g DCW, or 10 mg / g This results in genetically modified host cells that produce porphyra-334 and / or synolin superior to DCW.

[0037] In some embodiments, the present invention involves the use of heterogeneous codon-optimized versions of the nucleic acids encoding the enzymes described herein, which are optimized for host cells. [Brief explanation of the drawing]

[0038] The aforementioned aspects and other details will be readily understood by those skilled in the art from the following description of exemplary embodiments, when read in conjunction with the attached drawings.

[0039] [Figure 1]Metabolic flux distribution of Pseudomonas putida KT2440 given glucose. All values ​​in the box are shown as relative flux normalized to a specific glucose uptake rate. Data were referenced from the following three major studies: (Kukurugya et al., 2019) (top), (Nikel et al., 2015) (middle), and (Kohlstedt and Wittmann, 2019) (bottom). The width of the arrows is proportional to the mean of the three values. Abbreviations: G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; FBP, fructose-1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; 6PG, 6-phosphogluconate; KDPG, 2-keto-3-deoxy-6-phosphogluconate; Ri5P, ribulose 5-phosphate; R5P, ribose 5-phosphate; X5P, xylulose 5-phosphate; S7P, sedoheptulose 7-phosphate; E4P, erythrose 4-phosphate; G3P, glyceraldehyde 3-phosphate; 3PG, 3-phosphoglycerate; PEP, phosphoenolpyruvic acid; Ac-CoA, acetylcoenzyme A; L-ser, L-serine; TCA, tricarboxylic acid; PYR, pyruvate. [Figure 2-1]Figure 2. Development of genetic tools for gene expression in Pseudomonas petida KT2440. (A) Modular cloning assembly method as described in a previously published study (Storch et al., 2015). Gene parts were cloned into pJET1.2 blunt-ended DNA plasmids. These parts were then digested with BsaI restriction enzyme, ligated into prefix and suffix linkers, and subsequently separated and purified using magnetic beads. Finally, the purified gene parts and linkers were assembled in a one-pot reaction at 50°C for 1 hour. (B) Schematic diagrams of plasmids with different skeletons. RFP fluorescence levels measured across various plasmid skeletons (C), different inducible promoters (D), constitutive promoters (E), and ribosome binding site sequences (F). Cultures were grown in M9 minimal medium. RFP fluorescence was measured using a flow cytometer at 48 hours post-seeding. Inducers were added at 0 hours. The error bars represent the standard deviation of the three biological replicas. [Figure 2-2] See the explanation in Figure 2-1. [Figure 2-3] See the explanation in Figure 2-1. [Figure 3-1]Figure 3. Synoline production experiments and confirmation by nuclear magnetic resonance (NMR) spectroscopy in manipulated P. petida KT2440. (A) Schematic diagram of the central carbon metabolism and synoline biosynthesis pathway of P. petida KT2440. The first biosynthetic step involves the synthesis of desmethyl-4-deoxygassol (DDG) via sedoheptulose 7-phosphate (Balskus and Walsh, 2010; Pope et al., 2015). Desmethyl-4-deoxygassol synthase (DDGS) catalyzes the production of DDG. Subsequently, O-methyltransferase (O-MT) converts DDG to 4-deoxygassol (4-DG). 4-DG incorporates glycine via ATG-grasp ligase, yielding mycosporine-glycine (MG). In the final stage, MG is converted to synorine by a non-ribosomal peptide synthetase (NRPS)-like enzyme (Balskus and Walsh, 2010; Portwich and Garcia-Pichel, 2003), which includes adenylation, thiolation, and thioesterase domains. The adenylation domain plays a crucial role in serine binding to the C1 position of myosporine-glycine, which produces synorine. (B) Schematic diagram of plasmid pIY456 used in the synthesis experiment in Figure 3 (Panel C). (C) Synorine production from P. putida KT2440 strain. Chromatograms obtained from P. putida (D) and extracted Helioguard® (E). (F) Assigned 1H and 13C resonances for purified synorine. (G) Correlations (1H-1H NOESY and 1H-13C HMBC) used to clearly attribute all resonances for purified synolines. P. putida KT2440 was grown in M9 medium, and samples were extracted at 48 hours. Error bars represent the standard deviation from three biological replicas: SAM, S-adenosyl-L-methionine; SAH, S-adenosylhomocysteine. Other abbreviations are shown in the explanation of Figure 1. [Figure 3-2] See the explanation in Figure 3-1. [Figure 3-3] See the explanation in Figure 3-1. [Figure 4-1]Figure 4. CRISPRi gene downregulation for improved sinoline production. (A) Schematic diagram of genes involved in 6PG, G3P, L-serine, glycine, and S-adenosyl-L-methionine (SAM) metabolism. (B) Schematic diagram of the plasmids used for gene expression and CRISPRi-mediated gene downregulation of sinoline biosynthesis genes. (C) Titer of sinoline produced by different strains targeting 21 genes. (D) Relative expression levels of target genes in control vs. sample (y-axis; ×10⁶). (E) Volcano plot showing the top 20 downregulated (purple) and upregulated (dark blue) genes in strain PP_1444. Error bars represent the standard deviation from three biological replicas. [Figure 4-2] See the explanation in Figure 4-1. [Figure 4-3] See the explanation in Figure 4-1. [Figure 5-1] Figure 5. Improved cinorine production by RBS optimization. (A) Diagram of a combinatorial modular plasmid assembly consisting of promoters and genes assembled to different RBSs. Two promoters and three different RBS elements were used. (B) Schematic diagrams of 21 different cinorine-producing plasmids. (C) Cinorine titers from strains with the plasmids shown in Figure 5 (Panel B). White and black bar graphs represent strains JBx_250483 and JBx_250497, respectively. (D) Heatmaps of shotgun proteomics analysis of DDGS, O-MT, ATP-grasp ligase, and NRPS. Expression levels of DDGS (E), O-MT (F), ATP-grasp ligase (G), and NRPS (H) from the control strain (JBx_250483) versus the best-producing cinorine-producing strain (JBx_250497). Samples were grown on M9 minimal medium. Sinoline was extracted from the entire liquid culture at 72 hours. Error bars represent the standard deviation from the three biological replicas. [Figure 5-2] See the explanation in Figure 5-1. [Figure 6-1]Figure 6. Effects of glycine and L-serine supplementation on synorin production. (A) Schematic diagram of the genetic construct used in this study. P. petida KT2440 (JBx_250497) with a synorin biosynthesis pathway plasmid was transformed with a CRISPRi plasmid containing either PP_1444 sgRNA (JBx_249619) or non-target sgRNA (JBx_249575). (B, C) Comparison of synorin production, glucose consumption, and growth profiles over a 114-hour period without glycine and L-serine supplementation. (D) Experimental conditions used in B, C, E, and F. (E, F) Comparison of synorin production, glucose consumption, and growth profiles over a 114-hour period with glycine and L-serine supplementation. All strains were cultured in 25 mL of M9 medium containing 20 g / L glucose at an initial OD600 of 0.1. Four hours after seeding, cultures were induced with or without the addition of 10 mM equimolar concentrations of glycine and L-serine using 0.2% L-arabinose. At 18 hours, all cultures were supplemented with an additional 20 g / L of glucose. For samples (D) and (F), an additional 20 g / L of glucose was introduced into the cultures at 42 hours. The Venn diagram was created using the webpage interactivenn.net / index.html (Heberle et al., 2015). Error bars represent the standard deviation from the three biological replicas. (G) Percentage of cinorine observed in the supernatant and cell pellet, measured at 114 hours from the samples in Figure 6 (Panel F). Error bars represent the standard deviation from the three biological replicas. [Figure 6-2] See the explanation in Figure 6-1. [Figure 6-3] See the explanation in Figure 6-1. [Figure 7]Table 1 summarizes the metabolic flux distribution from (A) Bacillus megaterium QM B1551 and (B) the remaining microorganisms (excluding P. putida KT2440 and Bacillus megaterium QM B1551). The flux for Bacillus megaterium QM B1551 was adapted from (Wushensky et al., 2018). In this microorganism, glucose can be oxidized to gluconic acid and then phosphorylated to 6P-gluconic acid, or glucose can be directly phosphorylated to G6P. In other microorganisms, glucose is directly phosphorylated to G6P (B). [Figure 8] Characterization of inducible promoters. (A) Ptet, (B) PA1lacO-1, (C) Pm, (D) PLtet-O1, (E) PnagAa, and (F) PBAD. Cultures were grown in M9 minimal medium. Cultures were induced at 2 hours post-seeding, and fluorescence was measured at 48 hours. Error bars represent the standard deviation from the three biological replicas. [Figure 9-1] Figure 9. Confirmation of extracted cinolin by LC-MS / MS. (A) m / z ratio of cinolin extracted from the sample. (B) m / z ratio of extracted Helioguard® 365. (C) Tandem mass spectrum of cinolin from the extracted sample. (D) Tandem mass spectrum of cinolin from extracted Helioguard® 365. [Figure 9-2] See the explanation in Figure 9-1. [Figure 9-3] See the explanation in Figure 9-1. [Figure 10] Structural numbering for synolin. The multiplicity and / or coupling constants, along with the assigned 1H and 13C chemical shifts, can be found in Table 2. [Figure 11] 1H NMR spectra of heterogeneous synolines. All NMR experiments were acquired in methanol-d4 at 500 MHz. [Figure 12] 13C NMR spectrum of heterologously synthesized synolines acquired at 500 MHz. [Figure 13]Expression levels of DDGS, O-MT, ATP-grasp ligase, and NRPS in different CRISPRi strains. Error bars represent the standard deviation from three biological replicas. The Y-axis represents the number of proteins. [Figure 14] Expression levels of gentamicin (A), kanamycin (B) selection markers, and dCas9 (C) protein in different CRISPRi strains, WT, and control (CRISPRi non-targeted). Protein counts were measured by shotgun proteomics analysis. Error bars represent the standard deviation from the three biological replicas. [Figure 15] (A) OD600 of CRISPRi strains measured at 24, 48, and 72 hours. Gluconate concentration was measured from the supernatant (B) and cell pellet (C) at different time points. Error bars represent the standard deviation from the three biological replicas. [Figure 16] Protein expression levels of O-MT (A) and ATP-grasp ligase (B) plotted against sinoline titer. [Figure 17] Comparison of synoline production, glucose utilization, and growth profiles over time in the PP_1444 strain and non-target strains. Cultures were incubated in 25 mL of M9 medium at an initial glucose concentration of 20 g / L. Error bars represent the standard deviation from three biological replicas. [Figure 18] Extracted cinolines in the supernatant and cell pellet. Error bars represent the standard deviation from three biological replicas. [Figure 19]Conserved gene clusters involved in MAA biosynthesis in P. umbilicalis and related red algae. (A) Biosynthetic pathway from sedoheptulose 7-phosphate to cinolin in cyanobacteria, and proposed pathway to Porphyra-334 in red algae. (B) Comparison of gene clusters and gene fusions in the cyanobacteria genera Anabaena and Nostoc, and the red algae P. umbilicalis and C. crispus. (Brawley et al., “Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta),” Proc. Natl. Acad. Sci. USA, Early Ed.: 1-10, 2017.) [Modes for carrying out the invention]

[0040] Detailed description of the invention Before describing the present invention in detail, it should be understood that, unless otherwise specified, the present invention is not limited to any particular sequence, expression vector, enzyme, host microorganism, or process, and can therefore be modified. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit them.

[0041] In this specification and the subsequent claims, references are made to several terms which are defined as follows:

[0042] As used herein, the terms “optional” or “optional” mean that the features or structures described thereafter may or may not exist, or that the events or situations described thereafter may or may not occur, and that the descriptions include cases where certain features or structures exist and cases where they do not exist, or where events or situations occur and cases where they do not.

[0043] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless the context clearly indicates otherwise. For example, a reference to “expression vector” includes not only a single expression vector but also multiple expression vectors of the same (e.g., the same operon) or different ones; a reference to “cell” includes not only a single cell but also multiple cells.

[0044] In this specification and the subsequent claims, references are made to several terms which are defined as follows:

[0045] As used herein, the terms “optional” or “optional” mean that the features or structures described thereafter may or may not exist, or that the events or situations described thereafter may or may not occur, and that the descriptions include cases where certain features or structures exist and cases where they do not exist, or where events or situations occur and cases where they do not.

[0046] Where a range of values ​​is provided, unless the context clearly indicates otherwise, each intermediate value between the upper and lower limits of that range is also understood to be specifically disclosed to one-tenth of the lower limit. Any smaller range between any stated value or intermediate value within a stated range and any other stated value or intermediate value within such stated range is included in the present invention. The upper and lower limits of these smaller ranges may be independently included in or excluded from the range, and each range that includes one, neither, or both of the limit values ​​within a smaller range is also included in the present invention, subject to any specific excluded limits within the stated range. Where a stated range includes one or both of the limit values, a range that excludes one or both of those included limit values ​​is also included in the present invention.

[0047] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless the context clearly indicates otherwise. For example, a reference to “expression vector” includes not only a single expression vector but also multiple expression vectors of the same (e.g., the same operon) or different ones; a reference to “cell” includes not only a single cell but also multiple cells.

[0048] The term "approximately" refers to a value that includes values ​​that are 10% more or 10% less than the stated value.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention, but preferred methods and materials are described herein. All publications referenced herein are incorporated herein by reference to disclose and describe methods and / or materials in relation to those cited herein.

[0050] In this specification, the term “host cell” is used to refer to a living biological cell that can be transformed by the insertion of an expression vector.

[0051] As used herein, the term “heterogeneous” means a material or nucleotide or amino acid sequence that is found in or related to another material or nucleotide or amino acid sequence, and these materials or nucleotide or amino acid sequences are heterogeneous (i.e., they are not found together or related in nature).

[0052] The terms “expression vector” or “vector” refer to compounds and / or compositions that transduce, transform, or infect host cells, thereby causing the cells to express non-native nucleic acids and / or proteins, or express them in a manner not native to the cells. An “expression vector” contains a nucleic acid sequence (usually RNA or DNA) to be expressed by a host cell. Optionally, an expression vector also includes materials that facilitate the entry of the nucleic acid into the host cell, such as viruses, liposomes, or protein coatings. Expression vectors intended for use in this invention include those into which the nucleic acid sequence can be inserted along with any preferred or required operational elements. Furthermore, the expression vector must be introduced into a host cell and replicated therein. Certain expression vectors are plasmids, in particular those that are well-described and have restriction sites containing preferred or required operational elements for transcription of the nucleic acid sequence. Such plasmids, and other expression vectors, are well known to those skilled in the art.

[0053] The terms “polynucleotide” and “nucleic acid” are used interchangeably and refer to a polymer of single- or double-stranded deoxyribonucleotides or ribonucleotide bases read from the 5' end to the 3' end. The nucleic acids of the present invention generally contain phosphodiester bonds, but in some cases, nucleic acid analogs may be used that may have alternative backbones including, for example, phosphoramidates, phosphorothioates, phosphorodithioates, or O-methylphosphoromidite bonds (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press); positive backbones; nonionic backbones; and non-ribodic backbones. Therefore, nucleic acids or polynucleotides may also include modified nucleotides that enable accurate read-through by polymerases. “Polynucleotide sequences” or “nucleic acid sequences” include both the sense and antisense strands of the nucleic acid as separate single- or double-stranded units. As will be understood by those skilled in the art, the description of a single strand also defines the sequence of the complementary strand; therefore, sequences described herein also provide complementary sequences of that sequence. Unless otherwise specified, a given nucleic acid sequence implicitly includes not only the explicitly indicated sequence but also its variants (e.g., degenerate codon substitutions) and complementary sequences. A nucleic acid may be DNA, RNA, or a hybrid, which is both genomic DNA and cDNA, and may include combinations of deoxyribonucleotides and ribonucleotides, as well as combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, isoguanine, etc.

[0054] As used herein, the term “promoter” refers to a polynucleotide sequence capable of driving the transcription of a DNA sequence within a cell. Therefore, the promoters used in the polynucleotide constructs of the present invention include cis and trans transcriptional regulatory elements and regulatory sequences involved in regulating or modulating the timing and / or rate of gene transcription. For example, a promoter may be a cis transcriptional regulatory element, including enhancers, promoters, transcriptional terminators, origins of replication, chromosomal integration sequences, 5' and 3' untranslated regions, or intron sequences involved in transcriptional regulation. These cis sequences typically interact with proteins or other biomolecules to perform gene transcription (e.g., switching on / off, regulating, or modulating). The promoter is located on the 5' side of the gene being transcribed, and as used herein, includes a sequence 5' from the translation start codon (i.e., including the 5' untranslated region of mRNA, typically containing 100–200 bp). Most frequently, the core promoter sequence is within 1–2 kb of the translation start site, more frequently within 1 kbp, and most frequently within 500 bp of the translation start site. By convention, promoter sequences are typically provided as sequences on the coding strand of the gene they control. In the context of this application, promoters are typically referred to by the name of the gene whose expression they naturally control. Promoters used in the expression constructs of the present invention are referred to by the name of the gene. Nominal references to promoters include wild-type, native promoters, and variants of promoters that retain their expression-inducing ability. Nominal references to promoters are not limited to a specific species and also include promoters derived from the corresponding gene in other species.

[0055] A polynucleotide is "heterogeneous" to a host cell or a second polynucleotide sequence if it originates from an alien species, or if, even if it originates from the same species, it has been modified from its original form. For example, when a polynucleotide encoding a polypeptide sequence is said to be operably linked to a heterogeneous promoter, this means that the coding sequence of the polynucleotide encoding the polypeptide originates from one species, while the promoter sequence originates from another different species; or, if both originate from the same species, the coding sequence, which is not naturally associated with the promoter (e.g., it is a genetically engineered coding sequence originating from a different gene of the same species, or an allele from a different ecotype or variant).

[0056] The term "functionally linked" refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, this refers to the functional relationship of a transcriptional regulatory sequence to a transcriptional sequence. For example, a promoter or enhancer sequence is functionally linked to a DNA or RNA sequence if it stimulates or modulates the transcription of that DNA or RNA sequence in a suitable host cell or other expression system. Generally, promoters and transcriptional regulatory sequences that are functionally linked to a transcriptional sequence are physically contiguous with the transcriptional sequence; i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, do not need to be physically contiguous or located in close proximity to the coding sequence that promotes transcription.

[0057] In some embodiments, the host cell contains nucleic acids encoding one or more enzymes, functionally linked to promoters that can express one or more enzymes within the host cell. In some embodiments, encoding one or more enzymes in the nucleic acid is codon-optimized for the host cell. In some embodiments, the nucleic acid is a vector or replicon that can stably reside within the host cell. In some embodiments, the nucleic acid is stably integrated into the chromosome of the host cell.

[0058] In some embodiments, step (a) includes introducing nucleic acids encoding one or more enzymes into a host cell, which are functionally linked to a promoter capable of expressing one or more enzymes in the host cell.

[0059] The present invention provides a method for constructing a genetically modified host cell, comprising the step of (a) introducing into a host cell a nucleic acid encoding one or more enzymes, which is functionally linked to a promoter capable of expressing one or more enzymes in the host cell.

[0060] The expression of a gene encoding any of the enzymes taught herein can be modified in various ways according to the methods of the present invention. Those skilled in the art will recognize that increasing gene copy number, ribosome binding site strength, promoter strength, and various transcriptional regulators can be used to alter enzyme expression levels.

[0061] host cell Genetically modified host cells can be any prokaryotic or eukaryotic cell having any genetic modification that can produce isoprenol according to the method of the present invention. Suitable eukaryotic host cells include, but are not limited to, fungal cells. Suitable fungal cells are yeast cells, for example, Saccharomyces yeast cells. Generally, but not necessarily, the host cell is yeast or bacteria. Any prokaryotic or eukaryotic host cell can be used in the method as long as it remains viable after transformation with nucleic acid sequences. In some embodiments, the host cell is not adversely affected by transduction of the required nucleic acid sequence, subsequent protein (i.e., enzyme) expression, or the intermediates necessary for the performance of the resulting steps related to the mevalonate pathway. For example, it is preferable that there is minimal "crosstalk" (i.e., interference) between the host cell's own metabolic processes and processes involved in the mevalonate pathway.

[0062] In some embodiments, the host cell is genetically modified in that a heterologous nucleic acid has been introduced into the host cell, and therefore the genetically modified host cell does not exist in nature. A suitable host cell is one that can express a nucleic acid construct encoding one or more enzymes as described herein. The gene encoding the enzyme may be heterologous to the host cell, or the gene may be native to the host cell but functionally ligated to a heterologous promoter and one or more regulatory regions that result in higher expression of the gene in the host cell.

[0063] Enzymes can be native to a host cell or heterologous. If the enzyme is native to the host cell, the host cell is genetically modified to regulate the expression of the enzyme. This modification may involve altering the chromosomal gene encoding the enzyme in the host cell, or introducing a nucleic acid construct encoding the enzyme gene into the host cell. One effect of the modification is that enzyme expression is regulated in the host cell, for example, increased enzyme expression in the host cell compared to enzyme expression in an unmodified host cell.

[0064] Suitable yeasts for the present invention include, but are not limited to, cells of the genera Yarowia, Candida, Bebaromyces, Saccharomyces, Schizosaccharomyces, and Pichia. In some embodiments, the yeast is Saccharomyces cerevisiae. In some embodiments, the yeast is a species of the genus Candida, including but not limited to C. tropicalis, C. maltosa, C. apicola, C. paratropicalis, C. albicans, C. cloacae, C. guillermondii, C. intermedia, C. lipolytica, C. panapsilosis, and C. zeylenoides. In some embodiments, the yeast is Candida tropicalis. In some embodiments, the yeast is a non-oily yeast. In some embodiments, the non-oily yeast is a species of the genus Saccharomyces. In some embodiments, the species of the genus Saccharomyces is Saccharomyces cerevisiae. In some embodiments, the yeast is oily yeast. In some embodiments, the oily yeast is a species of the genus Rhodosporidium. In some embodiments, the species of the genus Rhodosporidium is Rhodosporidium toruloides.

[0065] In some embodiments, the host cell is Rhodosporidia tolloides or Pseudomonas putida. In some embodiments, the host cell is a Gram-negative bacterium. In some embodiments, the host cell belongs to the phylum Proteobactera. In some embodiments, the host cell belongs to the class Gammaproteobacteria. In some embodiments, the host cell belongs to the order Enterobacteriales. In some embodiments, the host cell belongs to the family Enterobacteriaceae. Suitable examples of bacteria include, but are not limited to, species belonging to the taxonomic genera Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsielia, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, and Paracoccus.

[0066] Suitable bacterial host cells for the present invention include, but are not limited to, the genera Escherichia, Corynebacterium, Pseudomonas, Streptomyces, and Bacillus. In some embodiments, Escherichia cells are E. coli, E. albertii, E. fergusonii, E. hermanii, E. marmotae, or E. vulneris. In some aspects, Corynebacterium cells include Corynebacterium glutamicum, Corynebacterium kroppenstedtii, Corynebacterium alimapuense, Corynebacterium amycolatum, Corynebacterium diphtheriae, Corynebacterium efficiens, Corynebacterium jeikeium, Corynebacterium macginleyi, and Corynebacterium matsuruschotti. These include Corynebacterium matruchotii, Corynebacterium minutissimum, Corynebacterium renale, Corynebacterium striatum, Corynebacterium ulcerans, Corynebacterium urealyticum, or Corynebacterium uropygiale.In some aspects, Pseudomonas cells are P. putida, P. aeruginosa, P. chlororaphis, P. fluorescens, P. pertucinogena, P. stutzeri, P. syringae, P. cremoricolorata, P. entomophila, P. fulva, P. monteilii, P. mosselii, P. oryzihabitans, P. parafluva, or P. plecoglossicida. In some embodiments, Streptomyces cells are S. coelicolor, S. lividans, S. venezuelae, S. ambofaciens, S. avermitilis, S. albus, or S. scabies. In some embodiments, Bacillus cells are B. subtilis, B. megaterium, B. licheniformis, B. anthracis, B. amyloliquefaciens, or B. pumilus.

[0067] In some embodiments, the bacterial host cell is a proteobacterial cell. In some embodiments, the proteobacterial cell is a gammaproteobacterial cell. In some embodiments, the gammaproteobacterial cell is a cell of the order Pseudomonadales or Enterobacterales. In some embodiments, the gammaproteobacterial cell is a cell of the order Pseudomonadales, which is a cell of the family Pseudomonadaceae. In some embodiments, the Pseudomonadaceae cell is a cell of the genera Pseudomonas, Azotobacter, Mesophilobacter, Oblitimonas, Permianibacter, Rugamonas, or Thiopseudomonas. In some embodiments, Pseudomonas cells are P. putida, P. erginosa, P. chlororaffis, P. fluorescein, P. pertukinogena, P. stutzelli, P. syringe, P. cremocolorata, P. entomophila, P. fluva, P. monteirii, P. mossellii, P. origihabitans, P. parafluva, or P. plecoglossus. In some embodiments, Gammaproteobacteria cells are Enterobacterales cells, which are Enterobacteraceae cells. In some embodiments, Enterobacteraceae cells are Escherichia, Enterobacillus, Enterobacter, Klebsiella, Salmonella, or Sigella cells. In some embodiments, the Escherichia cells are E. coli, E. albertii, E. fergusonii, E. hermannii, E. marmotae, or E. brunelis. In some embodiments, the host cells are Gram-negative bacteria. In some embodiments, the host cells are bacteria of the genera Azotobacter, Escherichia, Salmonella, Vibrio, Pasteurella, Haemophilus, or Pseudomonas.In some embodiments, the host cells are bacteria derived from the species Escherichia coli, Salmonella enterica, Vibrio cholerae, Pasteurella multocida, Haemophilus influenzae, Pseudomonas putida, or Pseudomonas aeruginosa.

[0068] Biomass can be obtained from one or more raw materials, such as softwood raw materials, hardwood raw materials, herbaceous raw materials, and / or agricultural raw materials, or mixtures thereof.

[0069] Softwood raw materials include the genus Araucaria (e.g., A. cunninghamii, A. angustifolia, A. araucana); softwood cedar (e.g., Juniperus virginiana, Thuja plicata, Thuja occidentalis, Chamaecyparis thyoides, Callitropsis nootkatensis); cypress (e.g., Chamaecyparis, Cupressus Taxodium, Cupressus arizonica) arizonica), Taxodium distichum, Chamaecyparis obtusa, Chamaecyparis lawsoniana, Cupressus semperviren); Rocky Mountain Douglas fir; European Yew; Fir (e.g., Abies balsamea, Abies alba, Abies procera, Abies amabilis); Hemlock (e.g., Tsuga canadensis, Tsuga mertensiana, Tsuga heterophylla) heterophylla); Kauri pine; Kaya; Larch (e.g., Larix decidua, Larix kaempferi, Larix laricina, Larix occidentalis);Pine trees (for example, Pinus nigra, Pinus banksiana, Pinus contorta, Pinus radiata, Pinus ponderosa, Pinus resinosa, Pinus sylvestris, Pinus strobus, Pinus monticola, Pinus lambertiana, Pinus taeda, Pinus palustris, Pinus rigida, Pinus echinata) echinata); redwood; rimu; spruce (e.g., Picea abies, Picea mariana, Picea rubens, Picea sitchensis, Picea glauca); cedar; and combinations / hybrids of these, but not limited to these.

[0070] For example, softwood raw materials that may be used herein include cedar, fir, pine, spruce, and combinations thereof. Softwood raw materials for the present invention may be selected from pine (Pinus taeda), radiata pine, jack pine, spruce (e.g., white, interior, black), grass fir, Pinus silvestris, Picea abies, and combinations / hybrids thereof. Softwood raw materials for the present invention may be selected from pine (e.g., Pinus radiata, Pinus taeda); spruce; and combinations / hybrids thereof.

[0071] Hardwood raw materials include: Acacia; Afzelia; Synsepalum duloificum; Albizia; Alder (e.g., Alnus glutinosa, Alnus rubra); Apple wood; Arbutus; Ash (e.g., F. nigra, F. quadrangulata, F. excelsior, F. pennsylvanica lanceolata, F. latifolia, F. profunda, F. americana); Aspen (e.g., P. grandidentata). P. grandidentata), P. tremula, P. tremuloides); Australian red cedar (Toona ciliata); Ayna (Distemonanthus benthamianus); Balsa (Ochroma pyramidale); Basswood (e.g., T. americana, T. heterophylla); Beech (e.g., F. sylvatica, F. grandifolia); Birch (e.g., Betula populifolia, B. nigra, B. papilifera (B. Papyrifera), B. lenta, B. alleghaniensis / B. lutea, B. pendula, B. pubescens); Blackbean; Blackwood; Bocote; Boxelder;Boxwood; Brazilwood; Bubinga; Horse chestnut (e.g., Aesculus hippocastanum, Aesculus glabra, Aesculus flava / Aesculus octandra); Butternut; Catalpa; Chemy (e.g., Prunus serotina, Prunus pennsylvanica, Prunus avium); Crabwood; Chestnut; Coachwood; Cocobolo; Corkwood; Cottonwood (e.g., Populus balsamifera) balsamifera), Populus deltoides, Populus sargentii, Populus heterophylla); Cucumber tree; Dogwood (e.g., Cornus florida, Cornus nuttallii); Ebony (e.g., Diospyros kurzii, Diospyros melanida, Diospyros crassiflora); Elm (e.g., Ulmus americana, Ulmus procera, Ulmus tomasii) thomasii), Ulmus rubra, Ulmus glabra; Eucalyptus; Greenheart; Grenadilla;Rubber tree (e.g., Nyssa sylvatica, Eucalyptus globulus, Liquidambar styraciflua, Nyssa aquatica); Hickory (e.g., Carya alba, Carya glabra, Carya ovata, Carya laciniosa); Hornbeam; Hophornbeam; Ipe; Iroko; Ironwood (e.g., Bangkirai, Carpinus caroliniana, Casuarina equisetifolia) equisetifolia), Choricbangarpia subargentea, species of the genus Copaifera, Eusideroxylon zwageri, Guajacum officinale, Guajacum sanctum, Hopea odorata, Ipe, Krugiodendron ferreum, Lyonothamnus lyonii (L. floribundus), Mesua ferrea, species of the genus Olea, Olneya tesota Tesota, Ostrya virginiana, Parrotia persica, Tabebuia serratifolia); Jacaranda; Jotoba; Lacewood; Laurel; Limba; Lignum vitae;Locust (e.g., Robinia pseudacacia, Gleditsia triacanthos); Mahogany; Maple (e.g., Acer saccharum, Acer nigrum, Acer negundo, Acer rubrum, Acer saccharinum, Acer pseudoplatanus); Meranti; Mpingo; Oak (e.g., Quercus macrocarpa, Quercus alba, Quercus stellata, Quercus bicolor) Quercus bicolor), Quercus virginiana, Quercus michauxii, Quercus prinus, Quercus muhlenbergii, Quercus chrysolepis, Quercus lyrata, Quercus robur, Quercus petraea, Quercus rubra, Quercus velutina, Quercus laurifolia, Quercus falcata, Quercus nigra, Quercus ferrus (Quercus Phellos, Quercus texana; Obeche; Okoume; Oregon Myrtle; California Bay Laurel; Pear;Poplar (e.g., P. balsamifera, P. nigra, hybrid poplar (Populus × canadensis)); Ramin; Red cedar; Rosewood; Sal; Sandalwood; Sassafras; Satinwood; Silky Oak; Silver Wattle; Snakewood; Sourwood; Spanish cedar; American sycamore; Teak; Walnut (e.g., Juglans nigra, Juglans regia); Willow (e.g., Salix nigra, Salix alba) Examples include, but are not limited to, alba; yellow poplar (Liriodendron tulipifera); bamboo; palmwood; and combinations / hybrids thereof.

[0072] For example, hardwood raw materials for the present invention may be selected from the genera Acacia, Aspen, Beech, Eucalyptus, Maple, Birch, Rubber tree, Oak, Poplar, and combinations / hybrids thereof. Hardwood raw materials for the present invention may be selected from species of the genus Populus (e.g., Populus tremuloides), species of the genus Eucalyptus (e.g., Eucalyptus globulus), species of the genus Acacia (e.g., Acacia dealbata), and combinations thereof.

[0073] Herbaceous raw materials include, but are not limited to, C4 or C3 plants, among other species known in the art, such as switchgrass, Indian grass, big blue stem, little blue stem, Canadian wildry, Virginia wildry, and goldenrod wildflower.

[0074] Agricultural raw materials include, but are not limited to, agricultural by-products such as grain husks, stover, and leaves. Such agricultural by-products may be derived from crops for human consumption, livestock consumption, or other non-consumption purposes. Such crops may include, for example, maize, wheat, rice, soybeans, hay, potatoes, cotton, or sugarcane.

[0075] Raw materials can be obtained by harvesting crops resulting from practices such as intercropping, mixed cropping, row cropping, and relay cropping.

[0076] References cited in this specification: TIFF2026518832000009.tif252160TIFF2026518832000010.tif245158TIFF2026518832000011.tif245159TIFF20265188320 00012.tif245159TIFF2026518832000013.tif237159TIFF2026518832000014.tif245160TIFF2026518832000015.tif136158

[0077] Other objects, features, and advantages of the present invention will be apparent to those skilled in the art from the following detailed description and figures.

[0078] While the present invention has been described in conjunction with its preferred specific embodiments, it should be understood that the foregoing description is intended to be illustrative of the invention and not to limit its scope. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

[0079] All patents, patent applications, and publications described herein are incorporated herein by reference in their entirety.

[0080] Although the present invention has been described, the following examples are provided as illustrations of the present invention and are not intended to limit it. [Examples]

[0081] Example 1 Systematic manipulation for the production of anti-aging sunscreen compounds in Pseudomonas putida Sunscreen has been used for thousands of years to protect skin from ultraviolet radiation. However, the recent use of commercially available sunscreens containing oxybenzone, ZnO, and TiO2 has raised concerns due to their adverse effects on human health and the environment. The objective of this study is to establish an efficient microbial platform for the production of synolin, an ultraviolet-absorbing compound with anti-aging properties. First, suitable hosts for synolin production are methodologically selected by analyzing central carbon flux distribution data from previous studies, along with predictions from genome-scale metabolic models (GEMs). Synolin productivity is enhanced by CRISPRi-mediated downregulation, and shotgun proteomics is used to identify potential competitive pathways. Simultaneously, the synolin biosynthesis pathway is improved by refining its design, optimizing promoter use, and altering the strength of ribosome binding sites. Finally, amino acid supply experiments are performed under various conditions to identify key limiting factors in synolin production. In this study, to improve synolin production... 13 By combining meta-analyses of C metabolic flux analysis, GEM, synthetic biology, CRISPRi-mediated gene downregulation, and omics analysis, we demonstrated the potential of Pseudomonas ptida KT2440 as a platform for synolin production.

[0082] This study methodologically selects a suitable host for synolinogenesis by analyzing central carbon flux distribution data from previous studies, along with predictions from genome-scale metabolic models (GEMs). Pseudomonas putida KT2440 was identified as a promising host for synolinogenesis. Synthetic biological tools were developed for use in this microorganism and in heterologously expressed synolin biosynthesis gene clusters (BGCs) derived from Anabaena variabilis ATCC 29413. Synolin productivity is enhanced by CRISPRi-mediated downregulation of potential competitive pathways. Simultaneously, the synolin biosynthesis pathway is improved by refining its design, optimizing promoter use, and altering the strength of ribosome binding sites. Finally, amino acid supply experiments under various conditions and shotgun proteomics are utilized to identify limiting factors in synolinogenesis. Overall, our research provides a comprehensive framework for manipulating Pseudomonas putida KT2440 as an efficient chassis for synolin production.

[0083] Results and Discussion Selection of P. putida KT2440 as a suitable host for synoline production To select a host suitable for synoline production, we examined the reported metabolic flux distributions from various microorganisms (Table 1), along with predictions from genome-scale metabolic models (GEMs). We focused on analyzing the flux toward 6-phosphogluconate (6PG), given that this intermediate metabolite plays a crucial role in the synoline biosynthesis pathway. 6PG functions as an intermediate molecule in both the pentose phosphate pathway and the Entner-Dudoroff pathway. In the pentose phosphate pathway, 6PG undergoes decarboxylation to produce ribulose 5-phosphate (Ri5P), which is further metabolized by ribulose-5-phosphate epimerase into ribose 5-phosphate (R5P) and xylulose 5-phosphate (X5P). R5P acts as a fundamental component of nucleic acid synthesis, while X5P is converted by transketolase to glyceraldehyde 3-phosphate (G3P) and sedoheptulose 7-phosphate (S7P), which functions as a precursor in synoline biosynthesis. G3P is converted to 3-phosphoglycerate (3PG) via either glycolysis (Emden-Mayerhof-Parnass (EMP) pathway) or the Entner-Dudorov pathway. 3PG is used in the biosynthesis of L-serine and glycine, essential amino acids incorporated into the synoline biosynthesis pathway.

[0084] (Table 1) Carbon fluxes from G6P to F6P and from G6P to 6PG in various organisms. All values ​​are given as relative fluxes normalized to specific glucose uptake rate. TIFF2026518832000016.tif97153TIFF2026518832000017.tif231154

[0085] In most organisms, with the exception of Pseudomonas putida KT2440 and Bacillus megatherium QM B1551, glucose is directly phosphorylated by glucokinase to produce glucose 6-phosphate (G6P) (Figures 1 and 7). G6P can be isomerized to fructose 6-phosphate (F6P) in the glycolysis pathway or oxidized through the oxidative pentose phosphate pathway to form 6PG. 13C metabolic flux analysis ( 13 The carbon flux distribution from G6P to F6P, or from G6P to 6PG, as determined by C-MFA, differs among different microorganisms and is influenced by factors such as fermentation conditions and genetic diversity (Table 1). Nevertheless, 13 Meta-analysis of C-MFAs and predictions from GEM (Table 2) revealed that only a small number of microorganisms can allocate more than 50% of their carbon flux to 6PG.

[0086] (Table 2) Carbon flux distribution predicted by genome-scale metabolic models TIFF2026518832000018.tif84144

[0087] Zymomonas mobilis converts approximately 99% of its glucose supply to 6PG (Table 1). However, because Z. mobilis lacks the 6PG dehydrogenase enzyme, the oxidative branching of the pentose phosphate pathway in Z. mobilis appears to be inactive (De Graaf et al., 1999; Jacobson et al., 2019). Instead of using the oxidative branching of the pentose phosphate pathway, Z. mobilis uses non-oxidative branching, converting F6P to R5P and S7P through a series of enzymatic reactions. Only 0.8% of the carbon flux is distributed from G6P to F6P, leaving only a small pool of F6P for S7P synthesis. On the other hand, in silico metabolic network analysis of Rhodosporidia tolloides revealed that glucose-fed R. tolloides allocates 89.7% of its carbon flux to 6PG, forming a significant pool of S7P in the non-oxidative pentose phosphate pathway (Bommareddy et al., 2015). R. tolloides is also an attractive host for the industrial-scale production of many chemicals (Liu et al., 2023, 2020; Schultz et al., 2022; Wehrs et al., 2019). It can readily co-consume C5 and C6 sugars from lignocellulose biomass and grow to very high cell densities. However, manipulating R. tolloides is difficult (Wen et al., 2020), and there are few synthetic biological tools available (Brink et al., 2023).

[0088] Pseudomonas putida KT2440 exhibits a unique central carbon metabolism (Figure 1). In this microorganism, glucose can be phosphorylated to G6P by direct phosphorylation in the cytoplasm, or it can be oxidized to gluconic acid in the periplasm by glucose dehydrogenase. Gluconic acid then follows one of two pathways: it is phosphorylated to 6PG by gluconokinase in the cytoplasm, or oxidized to 2-ketogluconic acid (2-KG) in the periplasm. The latter compound, 2-KG, is then transported to the cytoplasm and converted to 2-keto-6-phosphogluconic acid (2K6PG) by 2KG kinase, which is further reduced to 6PG by 2K6PG reductase. Although the majority of glucose is oxidized to gluconic acid, regardless of the initial step in glucose processing, these pathways converge on the production of 6PG. Furthermore, in contrast to other microorganisms, P. plutida KT2440 prefers the conversion of F6P to 6PG rather than the reverse reaction. As a result, P. plutida KT2440 converts over 90% of its glucose supply to 6PG, creating a substantial pool of 6PG for the biosynthesis of Ri5P and S7P in the non-oxidative pentose phosphate pathway. In addition, genome editing and synthetic biology tools for P. plutida KT2440 are widely available and well-characterized (Nikel and de Lorenzo, 2018). This unparalleled metabolic trait positions P. plutida KT2440 as a promising host for synorin production.

[0089] Development of synthetic biological tools for gene expression in P. petida KT2440 To realize the synorin biosynthesis pathway, we first developed a modular plasmid assembly tool for use with P. petida KT2440 (Figure 2, Panel A). Here, we used the BASIC (Biopart Assembly Standard for Idempotent Cloning) (Storch et al., 2015) plasmid assembly method to enable multipart DNA assembly using standard reusable parts in a single-pot assembly reaction. The resulting expression plasmid was then used to evaluate the functionality of gene elements such as promoters and ribosome binding sites. The primary objective was not to comprehensively characterize the gene elements, but rather to investigate the performance of the expression plasmids and individual gene elements used in this study.

[0090] Six plasmids with different skeletons, copy numbers, and origins of replication (pBBR1, pBBR1-B5, pRK2, pRSF1010, pRO1600 / p15a, and pVS1 / p15a) were constructed, and their characteristics were elucidated by measuring the fluorescence intensity of red fluorescent protein (RFP) (Figure 2, Panel B). The plasmids selected for construction offer diverse characteristics. Firstly, pBBR1, isolated from Bordetella bronchiseptica S87 (Antoine and Locht, 1992), is of a broad host range plasmid origin commonly used for gene expression in metabolic engineering studies of P. putida (Niu et al., 2020; Tiso et al., 2016; X. Wang et al., 2022; Wohlers et al., 2021). Although generally considered a low-to-medium copy number plasmid, the copy number of pBBR1 can be altered by mutations in the rep gene. Secondly, pBBR1-B5, a variant of the pBBR1 plasmid with an early stop codon in the rep gene, exhibits a higher copy number than the original pBBR1 plasmid. Thirdly, plasmid pRK2 is a member of the IncP incompatibility group and requires a replication origin sequence and a replication initiation protein encoded by trfA to function. It has been shown to be stably maintained in P. petida and has been measured to be a low copy number plasmid in Escherichia coli (De Bernardez and Dhurjati, 1987). Fourthly, RSF1010 is a high-copy-number, broad-host-range plasmid and is part of the IncQ incompatibility group of plasmids. It is routinely used for gene expression in P. petida (Aparicio et al., 2019). Finally, pRO1600 (Farinha and Kropinski, 1990) and pVS1 (Itoh and Haas, 1985) are plasmids isolated from species of the genus Pseudomonas and require a host-specific origin (e.g., p15a which replicates in E. coli) to construct a shuttle vector for P. putida.In this study, plasmid characterization revealed that pBBRR1-B5 showed the highest RFP fluorescence level, followed by pBBR1 > pRSF1010 = pRO1600 / p15a > pRK2 > pVS1 / p15a plasmids (Figure 2, Panel C).

[0091] Considering its moderate RFP fluorescence, plasmid pRK2 was then selected as the plasmid backbone for characterizing different inducible (Figure 2, panel D) and constitutive (Figure 2, panel E) promoters. Seven different inducible promoters (P tet (Cook et al., 2018), P tet* (Tian et al., 2019), P A1lacO-1 (Liu et al., 2019), P BAD (Calero et al., 2016), P NagA (Husken et al., 2001), P lacUV5 (Noel Jr. and Reznikoff, 2000), and P XylS / Pm (Calero et al., 2016), six types of Anderson promoters with different strengths (Anderson et al., 2010) (P J23119 , P J23100 , P J23101 , P J23102 , P J23107 , P J23104 ), and P trc1-O The characteristics of (Bagdasarian et al., 1983; Calero et al., 2016) (without LacI repressor) were clarified. tet and P A1lacO-1 The promoter showed a higher basal fluorescence level compared to other promoters (Figure 8). Among the inducible promoters, the arabinose-inducible promoter P BAD The highest RFP fluorescence level was observed. On the other hand, under the conditions tested, the constitutive promoter P trc-1O However, he was the strongest promoter among all the promoters evaluated.

[0092] Finally, three BioBrick (Shetty et al., 2008) ribosome binding site elements. TIFF2026518832000019.tif19153, Shine d'Algarno E. Coli Consensus TIFF2026518832000020.tif4128, and the anti-Shine-Dalgarno complementary sequence The properties of TIFF2026518832000021.tif4128 were revealed based on their different intensities in E. coli. Panel F in Figure 2 shows that selected ribosome binding sites exhibited different RFP fluorescence levels. The use of ribosome binding sites with different intensities has been adopted in metabolic engineering research and has demonstrated to be a useful strategy for pathway optimization.

[0093] Synoline production in Pseudomonas putida KT2440 After evaluating the performance and functionality of the plasmid backbone, promoter, and ribosome binding site in Pseudomonas ptyda KT2440, the synorin biosynthesis pathway was constructed. The synoline biosynthesis gene cluster (BGC) has been identified in various microorganisms, including Anabaena variabilis ATCC 29413 (Balskus and Walsh, 2010), Nostoc punctiforme ATCC 29133 / PCC 73102 (Qunjie and Ferran, 2011), Actinocinema mirum DSM 43827 (Miyamoto et al., 2014), Chlorogloeopsis fritschii PCC 6912 (Llewellyn et al., 2020; Portwich and Garcia-Pichel, 2003), and species of the genus Fischerella (Fischerella sp.) PCC 9339 (Yang et al., 2018). In this study, the synorine biosynthesis pathway was selected from Anabaena variabilis ATCC 29413 because it is one of the earliest pathways characterized for synorine biosynthesis. S7P, an intermediate metabolite of the pentose phosphate pathway, is converted to synorine through a series of four enzymatic steps. First, S7P is converted to 4-deoxygassol (4-DG) by 2-demethyl-4-deoxygassol synthase (DDGS) and O-methyltransferase (O-MT). Subsequently, glycine is bound to 4-DG by ATP-grasp ligase to form myosporine-glycine (MG). Finally, the serine moiety is bound to MG by a non-ribosomal peptide synthetase (NRPS)-like enzyme (Figure 3, Panel A).

[0094] To synthesize cinorine in Pseudomonas petida KT2440, this was initiated by amplifying the entire cinorine BGC (MIBiG accession number: BGC0000427) derived from Anabaena variabilis ATCC 29413 without altering the intergenic regions. The amplified gene cluster was then subjected to strong constitutive P trc1-OCloning into an RK-based vector under promoter control (Figure 3, Panel B). Heterologous expression of the cinorine biosynthesis pathway in Pseudomonas ptyda KT2440 resulted in the production of approximately 60 mg / L of cinorine at 48 hours, with over 80% of the cinorine accumulating in the supernatant. Chromatographic analysis of the sample (Figure 3, Panel D) showed a cinorine peak (1) relative to the reference Helioguard. (商標) The synoline peak obtained from the standard material was shown to co-elute (Figure 3, Panel E). The accuracy of the mass spectrum was confirmed by LC-MS / MS (Figure 9), and the results were consistent with those previously reported in a previous study (Kim et al., 2022). To further investigate the chemical structure of synoline, an extract from 1 L of liquid culture was purified and subjected to nuclear magnetic resonance (NMR) spectroscopy. 1 H- 1 H COSY, 1 H- 1 H NOESY, 1 H- 13 C HSQC, and 1 H- 13 The correlation obtained by C HMBC is all 1 H and 13 This allowed for a clear assignment of the 1C resonance (Figure 3, panels F and G, and Table 3). The observed resonance and its assignment were in very good agreement with previous NMR data reported for synorin (Miyamoto et al., 2014) (Figures 10-12). To the best of our knowledge, this is the first report of synorin formation in Pseudomonas ptyda KT2440.

[0095] (Table 3) Observations in heterogeneized cinorin 1 H and 13 Atomic assignment of C resonances. Atomic numbers can be found in Figure 4. TIFF2026518832000022.tif78128

[0096] Improvement of cinolin production titer through gene downregulation via CRISPRi To further enhance synorine titer, we used CRISPRi-mediated gene downregulation to suppress competing metabolic pathways and direct carbon flux toward synorine production. The use of CRISPRi has the potential to improve the yield of desired metabolites by selectively suppressing the expression of specific genes in metabolic pathways. This approach has been successfully demonstrated in improving the production of various compounds, including isoprenol (Tian et al., 2019; Wang et al., 2022), free fatty acids (Fang et al., 2021), propane (Yunus et al., 2022), fatty alcohols (Kaczmarzyk et al., 2018), and many others (Zhao et al., 2021). In this study, we applied CRISPRi to downregulate a set of 21 genes involved in the central carbon metabolism and biosynthesis of L-serine and glycine, two amino acids essential to the synorine biosynthesis pathway (Figure 4, Panel A). The catalytically inactive Cas9 protein is activated by a salicylic acid-inducible promoter (P nagA The RSF1010 plasmid is expressed under the control of the constitutive promoter P J23119 It was placed below (Figure 4, Panel B). This plasmid was co-expressed with the cinorin-producing RK2 plasmid.

[0097] Our findings demonstrate that knockdown of the PP_1444 gene leads to a significant increase in cinorin titer. P. putida KT2440 strain with downregulated PP_1444 produced approximately 400 mg / L of cinorin at 72 hours, a 160% increase compared to the control strain (i.e., the strain with non-target sgRNA) (Figure 4, Panel C). It is noteworthy that the titer of the control strain shown here is approximately three times higher at 48 hours compared to the strain shown in Panel C of Figure 3. The presence of the CRISPRi plasmid appeared to affect the expression levels of DDGS, O-MT, ATP-grasp ligase, and NRPS (Figure 13). These changes may also be attributable to the increased expression of the aph gene (Figure 14, Panel B), which may suggest that the copy number of the RK2 plasmid was altered in the presence of the second plasmid.

[0098] PP_1444 encodes quinoprotein glucose dehydrogenase, an enzyme responsible for the conversion of glucose to gluconic acid. Previous studies have shown that P. putida KT2440 tends to accumulate gluconic acid and 2-ketogluconic acid, and that the accumulation of these two compounds is effectively resolved by knocking out PP_1444 (Teresa et al., 2007). Interestingly, knockout of PP_1444 was associated with growth failure in previous studies (Bentley et al., 2020). However, no growth failure was observed in this study (Figure 15, Panel A). This discrepancy may be due to the partial suppression of PP_1444 by CRISPRi (Figure 4, Panel D), resulting in a decrease rather than complete elimination of gluconic acid in both the supernatant and cell pellet fractions (Figure 15).

[0099] The downregulation of PP_1444 appeared to be the only example resulting in an increase in sinoline titer, so we further investigated it to confirm the downregulation of other target genes. Of the 21 target genes, 10 were observed to be actually downregulated, 3 did not show downregulation, and 8 could not be confirmed because the protein was undetectable in both the sample and control strains (Figure 4, Panel D). Of the three genes that were not downregulated (PP_1010, PP_2930, and PP_4677), their respective dCas9 expression levels were low, except for PP_4677 (Figure 14, Panel C), but the expression levels of the gentamicin selection marker were consistently maintained in these strains (Figure 14, Panel A). In the case of PP_4677, the dCas9 expression level was similar to that of the other strains. This suggests that the lack of downregulation observed in PP_4677 may be due to the inefficiency of the designed sgRNA for PP_4677. This highlights the importance of designing more effective sgRNAs in future experiments.

[0100] Further investigation revealed that in the PP_1444 strain, approximately 83 other genes were unexpectedly and significantly downregulated in addition to PP_1444 (Figure 4, Panel E; Table 4). Among these, proteins involved in the metabolism of gluconic acid to 2-ketogluconate and 6PG (Figure 4, Panel A), such as Q88HH4, Q88HH5, and Q88HH6 (gluconate 2-dehydrogenases encoded by PP_3384, PP_3383, and PP_3382, respectively), as well as Q88HI1 (ketogluconate-6-P-reductase encoded by PP_3376) and Q88HH8 (2-ketogluconate epimerase encoded by PP_3379), were downregulated. The enzyme phosphoglucumutase (Q88GY7, encoded by PP_3578), which is responsible for glycogen biosynthesis from G6P, also appeared to be downregulated. This could redirect the G6P pool to 6PG synthesis (Figure 4, Panel A). Furthermore, L-serine dehydratase (Q88P66, ​​encoded by PP_0987), which is involved in the conversion of L-serine to pyruvate, was also downregulated. This could conserve the L-serine pool for synorine biosynthesis. Interestingly, downregulating PP_0987 alone did not improve synorine titer (Figure 4, Panel C). In addition to the large, unexpected gene downregulation, 73 genes were found to be significantly upregulated in strain PP_1444 (Figure 4, Panel E, Table 5). For example, 5-methyltetrahydropteroyltriglutamate-homocysteine ​​methyltransferase (Q88JF1, encoded by PP_2698) was upregulated 28-fold. This enzyme may play a role in maintaining L-methionine synthesis, which is used in the biosynthesis of S-adenosyl-L-methionine (SAM), a methyl donor to O-methyltransferase.

[0101] (Table 4) List of downregulated proteins TIFF2026518832000023.tif246159TIFF2026518832000024.tif244155TIFF2026518832000025.tif168160

[0102] (Table 5) List of upregulated proteins TIFF2026518832000026.tif55170TIFF2026518832000027.tif241166TIFF2026518832000028.tif136170

[0103] These results may demonstrate that downregulation of PP_1444 not only reduces gluconic acid accumulation but also broadly affects the expression of multiple genes in Pseudomonas putida KT2440. Many of the listed proteins appear to be involved in various metabolic pathways, including amino acid metabolism and central carbon metabolism. Their direct roles in synolin production remain unclear. Some of these gene expression changes align with our goal of increasing synolin production by altering the direction of carbon flux and preserving essential metabolites, while others remain unclear and require further investigation. Nevertheless, these findings highlight the potential of CRISPRi as a valuable tool for metabolic engineering and the production of desired compounds in microbial hosts, providing a promising means to further optimize and increase the yield of synolin and other valuable metabolites in biotechnological applications.

[0104] Pathway optimization through improved promoter usage and modification of ribosome binding site strength. In conjunction with a CRISPRi-mediated gene downregulation approach, we sought to enhance synorin titer through improvements in gene construct design, including optimization of promoter use and modification of ribosome binding site strength. Previous studies have demonstrated the effectiveness of adjusting RBS strength in increasing metabolite production (Jeschek et al., 2017; Jones et al., 2015; Rao et al., 2024; Yunus et al., 2020; Yunus and Jones, 2018). However, constructing plasmids for biosynthetic pathways containing multiple genes can be laborious. To streamline this process, we employed a modular linker-based plasmid construction method (Storch et al., 2015) to facilitate the creation of plasmid constructs with various RBSs, thereby regulating the expression levels of proteins involved in the synorin pathway (Figure 5, Panel A).

[0105] Two different promoters (P BAD and P trc1-O Using this method, we successfully assembled 21 different plasmids by reconstructing the cinorin BGC into one or two transcription units and combining them with variations of RBS (Figure 5, Panel B). This combinatorial approach led to a significant increase in cinorin production, raising it from 100 mg / L (Figure 5, Panel C; JBx_250483, represented by white bars) to approximately 467 mg / L of cinorin (Figure 5, Panel C; JBx_250497, represented by black bars) within 72 hours after seeding.

[0106] Our shotgun proteomics analysis revealed that manipulating promoters and RBSs is a feasible strategy for increasing the expression levels of cinorin pathway proteins (Figure 5, Panel D). Arabinose-inducible promoter P BAD is a constructive P trc1-OThe foreign RBS appeared to be superior to the promoter. Replacing native RBS with foreign RBS sequences resulted in a significant increase in protein expression levels, except for the native O-MT RBS. Using foreign RBS achieved substantial improvements in the expression levels of DDGS, O-MT, ATP-grasp ligase, and NRPS in JBx_250497, with increases of 2.1-fold, 2.6-fold, 40.5-fold, and 6.7-fold, respectively, compared to the original strain (JBx_250483) (Figure 5, panels E-H).

[0107] Multiple linear regression analysis was performed to identify the enzyme that has the greatest impact on synoline production. The results of standard least squares (OLS) regression showed that the expression levels of O-MT and ATP-grasp ligase had considerable statistical significance in predicting synoline titer, while DDGS and NRPS did not appear to have a significant effect in this model. This could mean that further increases in the expression levels of O-MT and ATP-grasp ligase could lead to a more substantial increase in synoline titer. However, after thoroughly exploring the available rearrangements of RBS variations, it was found that optimizing the expression levels of these two enzymes beyond a certain point did not result in a significant improvement in synoline production (Figure 16). Therefore, while O-MT and ATP-grasp ligase play an important role in predicting synoline titer, there may be other factors or pathways that need to be considered for further enhancement.

[0108] Supplementation with glycine and L-serine further enhances synoline production. Following successful implementation of CRISPRi-mediated gene downregulation and refinement of gene constructs, these two strategies were combined to further enhance synorin production. The strain with the highest synorin production (JBx_250497) was used as the base strain and co-transformed with a CRISPRi plasmid containing either PP_1444 sgRNA or non-target sgRNA (Figure 6, Panel A). The PP_1444 strain produced approximately 524 mg / L of synorin at 66 hours, while the control non-target strain produced approximately 365 mg / L (Figure 17). Glucose consumption profiles showed that both samples completely consumed glucose after 42 hours. No significant growth or synorin production was observed after 66 hours. These results may indicate that glucose supply limited synorin production under the test conditions.

[0109] To increase the glucose supply, 20 g / L of glucose was added at 18 hours. With the addition of this extra glucose, strain PP_1444 produced approximately 723 mg / L of synorin at 66 hours (Figure 6, Panel B). Synorin titer peaked at 902 mg / L at 90 hours. To investigate whether another set of glucose supplementation would further improve titer, an additional 20 g / L of glucose was added at 42 hours (Figure 6, Panel C). Synorin titer did not peak beyond 114 hours of fermentation, although the highest synorin titer was 900 mg / L achieved at 114 hours. These results suggest that another factor was potentially limiting synorin production.

[0110] The synoline biosynthesis pathway involves the uptake of two amino acids, glycine and L-serine. Glycine and L-serine are incorporated into the synoline molecule by specific enzymatic reactions (Figure 4, Panel A). ATP-grasp ligase, a key enzyme in the synoline biosynthesis pathway, facilitates the uptake of glycine into 4-deoxygassol to form mycosporine-glycine, a precursor of synoline. Subsequently, an NRPS-like enzyme attaches the serine moiety to mycosporine-glycine, resulting in synoline formation. Given the roles of these amino acids in synoline biosynthesis, it is hypothesized that external supplementation of these amino acids could potentially alleviate any metabolic bottlenecks resulting from limited intracellular availability, thereby increasing the overall production of synoline.

[0111] To test this hypothesis, a series of fermentation experiments were conducted with PP_1444 and non-target strains, accompanied by glycine and L-serine supplementation (Figure 6, Panel D). Supplementation with glycine and L-serine resulted in a significant increase in synorin production in both the PP_1444 and non-target strains (Figure 6, Panels E and B compared). In the PP_1444 strain, synorin titer increased rapidly within the first 66 hours and continued to increase steadily thereafter, reaching 1,134 mg / L at 114 hours (Figure 6, Panel E). Approximately 1.7 g / L of glucose remained in the liquid culture at 66 hours. To further enhance synorin titer, an additional 20 g / L of glucose was added at 42 hours (Figure 6, Panel F). Here, a rapid increase in synorin titer was observed over 114 hours. The highest synorin titer of 1,601 mg / L was achieved from strain PP_1444 at 114 hours, when the strain had completely consumed glucose.

[0112] While cinorin was detected only in the supernatant, in the PP_1444 strain, 91% and 9% cinorin were detected in the supernatant and cell pellet, respectively (Figures 7 and 18). From a biotechnological downstream processing perspective, these findings have further significance for the use of P. putida KT2440 as a microbial platform for cinorin production. The accumulation of cinorin in liquid culture medium rather than intracellularly offers several important advantages for industrial biotechnology. Because cell disruption is not required, the product can be more easily recovered from the supernatant, which simplifies downstream processing and reduces costs (Ying Wang et al., 2019). This also reduces the required rigorous purification steps, which can be cost-effective. Furthermore, if the product is secreted into the culture medium, the process can be adapted to a continuous production system that continuously recovers the product while maintaining the culture. When the product accumulates in cells, it often causes stress and eventually leads to cell death. Secretion avoids this problem, maintains cell viability, and potentially increases the overall cinorin yield.

[0113] material and method Strains, plasmids, culture media, and growth conditions All plasmids used in this study were amplified using the E. coli XL 1-Blue strain (Thermo Fisher Scientific). This strain was routinely cultured in lysogenic broth (LB) medium (LB broth, Sigma Aldrich) at 37°C, 180 rpm, and with the addition of appropriate antibiotics (final concentrations: gentamicin 10 μg / mL and kanamycin 50 μg / mL). Plasmids were constructed using a modular plasmid assembly method, specifically BASIC (Biopart Assembly Standard Idempotent Cloning) (Storch et al., 2015), with modifications.

[0114] Self-replicating plasmids (RK2 and RSF1010-based plasmids) were used to transform Pseudomonas putida KT2440 cells by electroporation. The electroporation procedure was modified from that of Choi et al. Briefly, one fresh colony of Pseudomonas putida KT2440 was seeded in 5 mL of LB and incubated overnight at 30°C and 200 rpm. The overnight cultures were centrifuged at 13,000 × g for 1 minute, washed three times with 1 mL of 10% glycerol, and resuspended in 500 μL of 10% glycerol at room temperature. Electroporation was performed by adding approximately 100 ng of DNA to a 100 μL cell aliquot and shocking with a Bio-Rad GenePulser II (USA) using a 1 mm cuvette (1.8 kV, 200 Ω). After electroporation, 1 mL of LB medium was added to the cuvette, and the cell mixture was transferred to a new 1.5 mL microcentrifuge tube. For cell harvesting, the cell mixture was grown at 30°C and 200 rpm for 1 hour. After incubation, 20 μL of the cell mixture was plated onto a selective agar plate containing the appropriate antibiotic and incubated overnight at 30°C.

[0115] Fluorescence measurement For fluorescence measurement using a plate reader, a liquid culture of P. petida KT2440 grown overnight in LB medium containing appropriate antibiotics (final concentrations: gentamicin 10 μg / mL and kanamycin 50 μg / mL), or a separately described preparation, was diluted 1,000-fold in LB medium. 200 μL of liquid culture was grown in a 96-well plate, and RFP fluorescence was measured using a Tecan Infinite F200 PRO instrument at an excitation wavelength of 575 ± 10 nm and an emission wavelength of 620 ± 10 nm for 24 hours, with continuous shaking except during measurement. For measurements using a flow cytometer, the overnight culture (0.1% v / v) was seeded in 2 mL of LB medium, and appropriate antibiotics were added. After 24 hours of incubation, 1–3 μL of the sample was added to 150 μL of 1X phosphate-buffered saline. Using a BD C6 Accuri flow cytometer (BD Bioscience), single-cell RFP and GFP fluorescence were immediately recorded from at least 30,000 cells. GFP and RFP fluorescence were measured using FL1 and FL4 detectors, respectively. Protein fluorescence levels were determined by averaging the fluorescence distribution.

[0116] Standard preparation of cinolin The cinorin-producing strain was placed in 250 mL of M9 medium in a 1 L flask, with an initial OD of 0.2. 600The cells were cultured in [specimen type]. 48 hours after seeding, the samples were centrifuged at 4,500 × g for 30 minutes at 4°C. To concentrate and desalt the cinorine from the culture supernatant, the supernatant was loaded onto a HyperCarb 2G SPE column (Thermo Scientific), washed with 10 mL of 5% acetonitrile, and eluted with 10 mL of 80% acetonitrile. The eluate was evaporated to dryness using a LabConco SpeedVac. The dried SPE eluate was then reconstituted with MilliQ water and semi-purified using an Agilent 1260 HPLC system equipped with a Machery-Nagel Nucleosil 100-10 SB strong anion exchange column (250 mm × 4.6 mm, particle size 10 μm) operating at a flow rate of 1 mL / min with an isocratic mobile phase (Fisher Scientific) consisting of 25 mM LC-MS grade ammonium bicarbonate. The fraction was collected manually by monitoring the absorbance (λ=334nm) of the sinoline chromophore. The observed retention time of sinoline was approximately 12.4 minutes. The collected fraction was then evaporated to dryness using a LabConco SpeedVac.

[0117] The dried fraction from the anion-exchange semi-purified solution was reconstituted with water and injected into an Agilent 1260 HPLC system equipped with a Thermo HyperCarb column (150 mm × 4.6 mm, particle size 5 μm) operating at 1.5 mL / min using the following gradient (A = 0.3% ammonium formate pH 9.0, B = acetonitrile): 2% B for 0, 15% B for 20, 50% B for 26, 90% B for 27–33, and 2% B for 35–40. The fraction was manually collected by monitoring the absorbance (λ = 334 nm) of the synorine chromophore. The observed retention time of synorine was approximately 9.2 minutes. The synorine-containing fraction was then rapidly frozen in liquid nitrogen and subjected to a LabConco freeze-dryer. The dried fraction was reconstituted with water, frozen, and freeze-dried three times to volatilize residual ammonium formate. The resulting dried solid of purified synoline was then used for characterization by NMR spectroscopy.

[0118] NMR spectroscopy of purified cinoline Approximately 4.6 mg of purified cinolin can be dissolved in 400 μL of methanol-d4 (>99.8% atomic % D; Sigma-Aldrich) containing 0.03% trimethylsilane. The NMR spectrum was obtained using a 5 mm NMR spectrum analyzer. 1 Acquisition was performed using a Bruker Avance NEO 500MHz with an H / BB iProbe. The sample was held at 298K during acquisition. A standard Bruker pulse sequence was used for each of the following experiments: 1 H, 13 C, 1 H- 1 H COSY, 1 H- 1 H NOESY (750ms mixing time), 1 H- 13 C HSQC, and 1 H- 13 C HMBC. Spectra were recorded using Bruker TopSpin 4.0.6 software and analyzed using MestReNova 14.3.2. Chemical shifts (δ, ppm) are based on trimethylsilane as the internal reference.

[0119] Routine synoline extraction and analysis For routine synolin analysis from the entire liquid culture, 100 μL of the liquid culture was mixed with 250 μL of methanol and 125 μL of chloroform. The resulting mixture was vortexed at 3,000 rpm for 5 minutes. Next, 125 μL of ultrapure water and 100 μL of chloroform were added, and the sample was re-vortexed at 3,000 rpm for 5 minutes, followed by centrifugation at 13,000 × g for 1 minute. Synolin was then sampled from the uppermost aqueous layer and subjected to NanoDrop. (商標) Measurements were taken at 334 nm using a 2000 / 2000c spectrophotometer. To measure the synorin concentration in the sample, purified synorin standards were prepared by serial dilution, and ε = extinction coefficient of synorin (ε = 44,700 M) was used. -1 cm -1 The concentrations were determined using the Beer-Lambert law (Llewellyn et al., 2020; Wada et al., 2015).

[0120] For the analysis of extracellular cinolin, 100 μL of liquid culture was centrifuged at 13,000 × g for 5 minutes, and cinolin was measured from the supernatant without extraction. For the analysis of intracellular cinolin, 100 μL of liquid culture was centrifuged at 13,000 × g for 5 minutes. The supernatant was removed, and the cell pellet was washed three times with 500 μL of ultrapure water. Finally, the cell pellet was resuspended in 100 μL of ultrapure water according to the extraction method described above for the entire liquid culture, and mixed with 250 μL of methanol and 125 μL of chloroform.

[0121] conclusion The growing interest in sustainable and environmentally friendly sunscreens has motivated scientists to produce synorines, naturally occurring compounds with UV-absorbing properties, in microorganisms. Our research provides a comprehensive approach to manipulating Pseudomonas putida KT2440 as an efficient chassis for synorine production. Through a comprehensive investigation of the metabolic flux distribution of various microorganisms, Pseudomonas putida KT2440 was identified as a promising host for synorine production. By leveraging synthetic biological approaches and metabolic engineering strategies, synorine yields were significantly increased, and productivity was greatly improved compared to the initial manipulated strain, particularly through CRISPRi-mediated gene downregulation targeting the PP_1444 gene. Proteomic analysis showed that the downregulation of PP_1444 broadly affected the expression of multiple genes in Pseudomonas putida KT2440. Genetic design, promoter use, and improvements to the ribosome binding site also contributed to the increased titer. Supply tests indicated that the supply of two essential amino acids, L-serine and glycine, may be limiting factors in synolin biosynthesis. The final titers, productivity, and yields at 900 mg / L, or 10 mg / L / h, or 22.5 mg / g glucose (without glycine and L-serine supplementation), and at 1,601 mg / L, or 14 mg / L / h, or 26–28.35 mg / g glucose (with glycine and L-serine supplementation), showed substantial improvements over initial production levels, surpassing the results of previous studies. Furthermore, the exclusive secretion of synolin into the culture is advantageous for downstream processing in industrial applications. These findings highlight the potential of P. putida KT2440 as a microbial platform for producing valuable natural compounds. Through continuous optimization and scale-up efforts, our research paves the way for the commercialization of cinolin as a bio-based alternative in the sunscreen industry.

[0122] While the present invention has been described with reference to its specific embodiments, it should be understood by those skilled in the art that various modifications can be made and equivalents can be substituted without departing from the true spirit and scope of the invention. Furthermore, many modifications can be made to adapt specific situations, materials, compositions, processes, or one or more process steps to the object, spirit, and scope of the invention. All such modifications are intended to be included within the scope of the appended claims.

Claims

1. (a) a genetically modified host cell capable of producing porphyra-334 and / or synolin, comprising (i) 2-demethyl-4-deoxyguzzol synthase (DDGS), (ii) O-methyltransferase (O-MT or OMT), (iii) ATP-grasp ligase, and (iv) non-ribosomal peptide synthetase (NRPS) or D-ala-D-ala ligase, MysD or MysE; or (b) MysA, MysB, MysC, or MysD, MysE, or NRPS, wherein one or more of the aforementioned enzymes are homologous enzymes thereof.

2. The genetically modified host cell according to claim 1, wherein 2-demethyl-4-deoxygassol synthase (DDGS) is cyanobacteria DDGS or its homologous enzyme.

3. The genetically modified host cell according to claim 1, wherein the O-methyltransferase (O-MT or OMT) is cyanobacteria's OMT or its homologous enzyme.

4. The genetically modified host cell according to claim 1, wherein the ATP-grasp ligase is a cyanobacteria ATP-grasp ligase or its homologous enzyme.

5. The genetically modified host cell according to claim 1, wherein the non-ribosomal peptide synthetase (NRPS) or D-ala-D-ala ligase is a cyanobacterial non-ribosomal peptide synthetase (NRPS) or D-ala-D-ala ligase, or a homologous enzyme thereof.

6. MysA, MysB, MysC, MysD, and / or MysE are independently derived from or obtained from the genus Anabaena ATCC 29413, the genus Nostoc ATCC 29133, Porphyra umbilicalis, and / or Chondrus crispus, and the cyanobacterial MysD or D-ala-ala ligase has loose substrate specificity and condenses threonine on myosporine-glycine instead of serine to produce porphyra-334, according to claim 1.

7. The genetically modified host cell according to claim 1, wherein the DDGS, OMT, ATP-grasp ligase, and / or non-ribosomal peptide synthetase (NRPS) or D-ala-D-ala ligase are each independently obtained from or derived from Porphyra umbilicalis, Nostoc punctiforme, and / or Anabaena variabilis.

8. The genetically modified host cell according to claim 1, wherein the host cell is genetically modified so that it can metabolize one or more compounds that an unmodified host cell would not normally be able to metabolize.

9. The genetically modified host cell according to claim 8, wherein the host cell is a host cell of the genus Pseudomonas.

10. The genetically modified host cell according to claim 8, wherein the one or more compounds are aromatic compounds obtained from hydrolysates of lignocellulose.

11. The genetically modified host cell according to claim 10, wherein the aromatic compound is ferulic acid, 4-hydroxybenzoic acid, protocatechuic acid, vanillic acid, salicylic acid, syringic acid, p-coumaric acid, vanillin, catechol, syringaldehyde, or phenol.

12. The genetically modified host cell according to claim 1, wherein MysA and MysB form a fusion protein.

13. The genetically modified host cell according to claim 12, wherein the fusion protein has an amino acid sequence containing SEQ ID NO:

6.

14. The genetically modified host cell according to claim 1, wherein MysC and MysD form a fusion protein.

15. The genetically modified host cell according to claim 14, wherein the fusion protein has an amino acid sequence containing SEQ ID NO:

7.

16. (a) (1) (i) 2-demethyl-4-deoxygazol synthase (DDGS), (ii) O-methyltransferase (O-MT or OMT), (iii) ATP-grasp ligase, and (iv) non-ribosomal peptide synthetase (NRPS) or D-ala-D-ala ligase, MysD or MysE; or (2) a step of providing a genetically modified host cell comprising MysA, MysB, MysC, or MysD, MysE, or NRPS; and (b) The step of culturing or growing the genetically modified host cells in a suitable culture medium or culture medium so that Porphyra-334 and / or synolin are produced. A method for producing Porphyra-334 and / or synolin, including the above.

17. (c) The step of extracting or separating Porphyra-334 and / or synolin from the host cells and / or culture or medium in order to form isolated or purified Porphyra-334 and / or synolin. The method according to claim 16, including the method described in claim 16.

18. (d) The step of mixing Porphyra-334 and / or Synolin with lotion, oil, or water to form a composition that blocks or filters ultraviolet (UV) rays. The method according to claim 16, including the method described in claim 16.

19. The method according to claim 18, wherein the mixing step further comprises mixing the porphyra-334 and / or synolin, or the UV-blocking or filtering composition, with another active UV filtering agent.

20. The method according to claim 18, wherein the active UV filtering agent is oxybenzone, octinoxate, octisalate, and avobenzone, zinc oxide, and / or titanium dioxide, or a mixture thereof.

21. The method according to claim 18, wherein the composition for blocking or filtering UV is suitable for application to human skin.

22. The method according to claim 18, wherein the composition for blocking or filtering UV does not contain any components or elements that cause coral bleaching.

23. The method according to claim 18, wherein the step (b) of culturing or growing is carried out in a batch process or a fed-batch process.

24. The method according to claim 18, wherein the culture or culture medium contains biomass.

25. The method according to claim 24, wherein the biomass is lignocellulose biomass or a hydrolyzed product thereof.

26. The method according to claim 25, wherein the biomass is obtained from softwood raw materials (e.g., poplar), hardwood raw materials, herbaceous raw materials, and / or agricultural raw materials, or mixtures thereof.

27. The method according to claim 18, wherein the culture or culture medium comprises one or more aromatic compounds.

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