Method for producing Porphyra-334 and / or Shinorine and genetically modified host cell

Abstract

The present invention provides a genetically modified host cell capable of producing Porphyra-334 and / or Shinorine, which comprises (a) (i) a 2-demethyl-4-deoxycod's alcohol synthase (DDGS), (ii) an O-methyltransferase (O-MT or OMT), (iii) an ATP dependent carboxylic acid-amine ligase, and (iv) a non-ribosomal peptide synthetase (NRPS) or a D-ala-D-ala ligase, MysD or MysE; or (b) MysA, MysB, MysC or MysD, MysE or NRPS; wherein one or more of the foregoing enzymes are their homologous enzymes.

Description

[0001] Inventors: I.S. Hunus, Li Zhaichun

[0002] Cross-reference to related applications

[0003] This application claims priority to U.S. Provisional Application No. 63 / 493,636, filed March 31, 2023, which is incorporated herein by reference in its entirety.

[0004] Government Support Statement

[0005] This invention was developed with government support under contract number DE-AC02-05CH11231 granted by the U.S. Department of Energy. The government holds certain rights to this invention. Technical Field

[0006] This invention pertains to the field of producing Prophyra-334 and / or Shinorine.

[0007] About sequence lists

[0008] This application contains a sequence list electronically submitted in XML format, and its entire contents are incorporated herein by reference. The XML copy was created in _______, named "_______", and is _______ bytes in size. Background of the Invention

[0010] Prolonged warm periods (Oliver et al., 2018), global warming (Johnson et al., 2022), and overuse of chemical sunscreens (Roberto et al., 2008) are associated with global coral bleaching. Due to concerns about their adverse effects on human health and the environment, some countries and regions have banned the use of commercial sunscreens containing oxybenzone, octinoxate, ZnO, and TiO2. This has led to an increasing demand for sunscreens from environmentally friendly sources. Shinorine is a mycotoxin-like amino acid (MAA) commonly produced by the red algae *Porphyra umbilicalis*. Figure 19 ), has been used in commercial sun protection products (such as Helioguard) TMShinorine is the active ingredient in 365 Helionori®. It has also shown other benefits, including anti-aging properties, antioxidant activity, promoting wound healing, and inhibiting skin inflammation caused by UV radiation (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 *Porphyra umbilicalis* is relatively low (3.27 mg / g cell dry weight) (Becker et al., 2016), and its production is affected by the slow growth of *Porphyra umbilicalis*. With the continued growth in global demand for sunscreens, the bioproduction of shinorine in rapidly growing microorganisms has become an attractive solution.

[0011] To meet the growing demand for shinorine, various host organisms, including *Saccharomyces cerevisiae* (Jin et al., 2021; Kim et al., 2023, 2022; Park et al., 2019), *Corynebacterium glutamicum* (Tsuge et al., 2018), *Synostemma pentaphyllum* PCC 6803 (Yang et al., 2018), *Streptomyces avermitilis* (Miyamoto et al., 2014), and more recently, *Yarrowia lipolytica* (Jin et al., 2023), have been genetically engineered for shinorine production. However, most metabolic engineering efforts in these host organisms have focused primarily on coupling the shinorine production pathway with the xylose utilization pathway to enhance the xylulose 5-phosphate library (Jin et al., 2023; Kim et al., 2023, 2022; Park et al., 2019). While this approach has been shown to improve the production titers of shinorine, challenges remain. First, in fed-batch fermenters, *Saccharomyces cerevisiae* exhibits the highest productivity (12.75 mg / L / h) and yield (3.66 mg / g glucose; assuming a total consumption of 420 g glucose and 160 g xylose) (Kim et al., 2023). Second, providing a mixture of sugars (e.g., hexoses and pentoses) to the culture also adds complexity to production experiments (Park et al., 2019), as the microbial host typically exhibits a preference for consuming hexoses (e.g., glucose) relative to pentoses (e.g., xylose) (Aidelberg et al., 2014; Dvořák and de Lorenzo, 2018; Wang et al., 2019). Third, most of the shinorine produced by *Saccharomyces cerevisiae* and *Yarrowia lipolytica* tends to accumulate intracellularly (Jin et al., 2023; Kim et al., 2023), complicating extraction and purification.

[0012] Currently, shinorine is produced by red algae (3.2 mg / g CDW) and cyanobacteria (2.37 mg / g CDW). Park et al. disclosed the introduction of a shinorine biosynthesis gene from the cyanobacterium *Nostoc punctiforme* into *Saccharomyces cerevisiae* to construct a yeast strain that produces 31.0 mg / L shinorine in an optimized medium containing 8 g / L xylose and 12 g / L glucose (ACS Synthetic Biol. 8:346-357, 2019).

[0013] Hahn et al. (PCT International Patent Application WO 2021 / 133101) disclosed an engineered brewing yeast that uses xylose as a carbon source to produce shinorine.

[0014] Kotagiri et al. (US Patent Application Publication No. 2022 / 0275411) disclosed a genetically modified strain of the symbiotic bacterium Staphylococcus epidermis containing a plasmid comprising four Anabaena variabilis genes to produce a mycosporin amino acid (MAA). Summary of the Invention

[0015] This invention provides a genetically modified host cell capable of producing Porphyra-334 and / or Shinorine. The genetically modified host cell comprises: (a) (i) 2-demethyl 4-deoxygadusol synthase (DDGS), (ii) O-methyltransferase (O-MT or OMT), (iii) ATP-dependent carboxylic acid-amine (ATP-grasp) ligase, and (iv) non-ribosomal peptide synthase (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 foregoing enzymes are homologous enzymes thereof.

[0016] In some embodiments, 2-demethyl-4-deoxycodone alcohol synthase (DDGS) is cyanobacterial DDGS or its homolog. In some embodiments, O-methyltransferase (O-MT or OMT) is cyanobacterial OMT or its homolog. In some embodiments, ATP-dependent carboxylic acid-amine ligase is cyanobacterial ATP-dependent carboxylic acid-amine ligase or its homolog. In some embodiments, non-ribosomal peptide synthase (NRPS) or D-ala-D-ala ligase is cyanobacterial non-ribosomal peptide synthase (NRPS) or D-ala-D-ala ligase or its homolog. In some embodiments, MysA, MysB, MysC, MysD and / or MysE are independently derived from or obtained from *Anabaena ATCC 29413*, *Nostoc ATCC 29133*, *Porphyra yezoensis*, and / or *Chondrus crispus*. The cyanobacterial MysD or D-ala-ala ligases exhibit relaxed substrate specificity, wherein threonine replaces serine in the condensation onto spore-glycine to produce Porphyra-334. In some embodiments, one or more or all of the enzymes are wild-type enzymes.

[0017] In some embodiments, DDGS, OMT, ATP-dependent carboxylic acid-amine ligase and / or non-ribosomal peptide synthase (NRPS) or D-ala-D-ala ligase are each independently obtained from or derived from *Porphyra yezoensis*, *Nostocpunctiforme*, and / or *Anabaena variegata*.

[0018] The amino acid sequences of DDGS from *Nostoc punctata* or *Anabaena variegata* are as follows:

[0019] MSNVQASFEATEAEFRVEGYEKIEFSLVYVNGAFDISNREIADSYEKFGRCLTVIDANVNRLYGKQIKSYFRHYGIDLTVVPIVITEPTKTLATFEKIVDAF SDFGLIRKEPVLVVGGGLTTDVAGFACAAYRRKSNYIRVPTTLIGLIDAGVAIKVAVNHRKLKNRLGAYHAPLKVILDFSFLQTLPTAQVRNGMAELVKIAVV ANSEVFELLYEYGEELLSTHFGYVNGTKELKAIAHKLNYEAIKTMLELETPNLHELDLDRVIAYGHTWSPTLELAPMIPLFHGHAVNIDMALSATIAARRGYITSGERDRILSLMSRIGLSIDHPLLDGDLLWYATQSISLTRDGKQRAAMPKPIGECFFVNDFTREELDAALAEHKRLCATYPRGGDGIDAYIETQEESKLLGV (SEQ ID NO:1)

[0020] The amino acid sequences of OMT from *Nostoc punctata* or *Anabaena variegata* are as follows:

[0021] MTSILGRDTARPI TPHSILVAQLQKT LRMAEESNIPSEI LTSLRQGLQLAAG LDPYLDDCTTPESTALTALAQKTSIE DWSKRFSDGETVR QLEQEMLSGHLEG QTLKMFVHITKAK SILEVGMFTGYSALAMAEALPDDGRL IACEVDSYVAEFA QTCFQESPHGRKI VVEVAPALETLHK LVAKKESFDLIFIDADKKEYIEYFQI ILDSHLLAPDGLI CVDNTLLQGQVYL PSEQRTANGEAIA QFNRIVAADPRVEQVLLPIRDGITLI RRLV (SEQ ID NO:2)

[0022] The amino acid sequences of the ATP-dependent carboxylic acid-amine ligases of *Nostoc punctata* or *Anabaena variegata* are as follows:

[0023] MAGSISLSLPQST TPSKGVRLKIAAL LKTIGTLILLLIA LPLNALIVLISLM CRPFTKKPAVATHPQNILVSGGKMTK ALQLARSFHAAGH RVILIEGHKYWLS GHRFSNSVSRFYT VPAPQDDPEGYTQALLEIVKREKIDV YVPVCSPVASYYD SLAKSALSEYCEV FHFDADITKMLDD KFAFTDRARSLGLSAPKSFKITDPEQ VINFDFSKETRKY ILKSISYDSVRRL NLTKLPCDTPEET AAFVKSLPISPEKPWIMQEFIPGKEL CTHSTVRDGELRL HCCSNSSAFQINY ENVENPQIQEWVQ HFVKSLRLTGQISLDFIQAEDGTAYA IECNPRTHSAITM FYNHPGVAEAYLG KTPLAAPLEPLAD SKPTYWIYHEIWRLTGIRSGQQLQTW FGRLVRGTDAIYR LDDPIPFLTLHHW QITLLLLQNLQRL KGWVKIDFNIGKLVELGGDYSRSEE (SEQ ID NO:3)

[0024] The amino acid sequence of Anabaena NRPS is as follows:

[0025] MQTIDENIRKLLVEWNAT HRDYDLSQSLHELIVAQV ERTPEAIVTFDKQQLTVQELNHKANQLGHYLQTLG VQPETLVGVCLERSLEMV ICLLGILEAGGAYVPIDP EYPQERIAYMLEDSQVEVLLTQEKLLNQIPHHQAQT ICVDREWEKISTQANTNP KSNIKTDNLAYVIYTSGS TGKPKGAMNTHKGICNRLLWMQEAYQIDSTDSILQK TPFSFDVSVWEFFWTLLT GARLVIAKPGGHKDSAYL IDLITQEQITTLHFVPSMLQVFLQNRHVSKCSSLKR VICSGEALSIDLQNRFFQ HLQCELHNLYGPTEAAID VTFWQCRKDSNLKSVPIGRPIANTQIYILDADLQPV NIGVTGEIYIGGVGVARG YLNKEELTKEKFIINPFP NSEFKRLYKTGDLARYLPDGNIEYLGRTDYQVKIRG YRIEIGEIENVLSSHPQV REAVVIARDDNAQEKQII AYITYNSIKPQLDNLRDFLKARLPDFMIPAAFVMLE HLPLTPSGKVDRKALPKP DLFNYSEHNSYVAPRNEV DLFNYSEHNSYVAPRNEVEEKLVQIWSNILHLPKVG VTENFFAIGGNSLKALHL ISQIELFAKEISLATLL TNPVIADLAKVIQANNQIHNSPLVPIQPQGKQQPFF CIHPAGGHVLCYFKLAQY IGTDQPFYGLQAQGFYGD EAPLTRVEDMASLYVKTIREFQPQGPYRVGGWSFGG VVAYEVAQQLHRQGQEVS ILAILDSYVPILLDKQKP IDDVYLVGVLSRVFGGMFGQDNLVTPEEIENLTVEE KINYIIDKARSARIFPPG VERQNNRRILDVLVGTLK ATYSYIRQPYPGKVTVFRAREKHIMAPDPTLVWVEL FSVMAAQEIKIIDVPGNH YSFVLEPHVQVLAQRLQD CLENSES (SEQ ID NO:4)

[0026] The amino acid sequence of the Nostoc punctata D-ala-D-ala ligase is as follows:

[0027] MPVLNILHLVGSA HDKFYCDLSRLYA QDCLAATADPSLY NFQIAYITPDRQW RFPDSLSREDIALTGQNIDMMLPQMF CIPGMTQYRALFD LLKIPYIGNTPDI MAIAAHKARAKAI VEAAGVKVPRGELLRQGDIPTITTPPA VKVSSDNSLGV VLVKDVTEYDAAL KKAFEYASEVIVE AFIELGREVRCGIIVKDGELIGLPLE EYLVDPHDKPIRN YADKLQQTDDGDL DPNDPITQKVQQV AKRCHQALGCRHYSLFDFRIDPKGQP WFLEAGLYCSFAP KSVISSMAKAAGI PLNDLLITAINET LGSNKKVLQN (SEQ IDNO:5)

[0028] In some embodiments, the host cell is genetically modified to metabolize one or more compounds that the unmodified host cell would not normally be able to metabolize. In some embodiments, the host cell is a Pseudomonas host cell. In some embodiments, one or more compounds are aromatic compounds obtained from lignocellulose hydrolysis products. Examples of such aromatic compounds are ferulic acid, 4-hydroxybenzoic acid, protocatechuic acid, vanillic acid, salicylic acid, syringic acid, p-coumaric acid, vanillin, catechol, eugenol, and phenol. Examples of suitable genetic modifications that enable the host cell to metabolize such aromatic compounds include overexpression of erucic acid-CoA synthase (Fcs), enoyl-CoA hydratase / aldolase (Ech), vanillin dehydrogenase (Vdh), p-hydroxybenzoic acid hydroxylase (PobA), and vanillic acid O-demethylase oxygenase (VanAB).

[0029] In some embodiments, MysA and MysB form a fusion protein. In some embodiments, MysC and MysD form a fusion protein.

[0030] The amino acid sequence of the MysA-MysB fusion protein from *Porphyra yezoensis* is as follows:

[0031] (MysA-MysB fusion)

[0032] >tr|A0A1X6NP84|A0A1X6NP84_PORUM Protein containing 3-dehydroquinic acid synthase domain OS=Porphyra yezoensis OX=2786 GN=BU14_0783s0002 PE=3 SV=1)

[0033] MHITLDKSAGVQVVQPQHFAAFVTDVKEAGSVVPDGDAKTAVETVLALPALSTFLLRCSR

[0034] ADVFDGSVAADFSSNTKDLCMLRSLKQLKYFYNLPAGQLMQLLSGVVASDDVVAAKPWTA

[0035] LAERVFADEELCTMLIKHLIIGKTARESLKDIYEENAQYLLENDPHALYPTSIYRQGNGH

[0036] VTSSDDSSRIEGVMSTTITTSIEIVRDVLNPKNHQLRDMYKPLKRCVAVVDERLDKLYGQ

[0037] KLCDYFVSHDIPFEKMSYRCMEVDKDISTVEKMLVDLKGRRVSRNEPVLIVGGGVMADVG

[0038] GFACALYHRNTPYVMLCTSIVSGIDAGPSPRTCCDGLGYKNVFGAYHPPVLTLTDRYFFR

[0039] TLEKGWIRHGIAEIIKMAVTKDHTLFEALEEAGQKLVDTKFGIEGVEAGSRFDTLSESII

[0040] AKAMDGYVRSEYGNLWETHQCRPHAYGHTWSPGFELQAGLLHGHAVSIGMGYGAYLSFLE

[0041] GWISVTDFHRILNVISTVGLSLVHPILDNPNSLWQSQVKMTEKRGGNLCAPLPRGSIGKC

[0042] GYLNDLTRARLEATLIDYKELCKAYPRGGAGIEPHCRDVGLEDPSTVKQHADHAVHAIAT

[0043] IGPAASDEVPEANGNGNGVEANGVEANGNGNSNVYNDWIAKQQVSRNQGATSSSLMASMV

[0044] QIQAKDTEAPPAFPHTTLFHEGGEEYAQAMTTAASADFVNIAKETVAADLFAPCMVGAIE

[0045] GQFLKMMAGMTKSKRVLDIGTFTGYSALAFAGGVPADGEVVTIEADKKCADVAQKCFDAS

[0046] ADGSKIKLVRGDAMKVVSEMADSGEKFDIVFLDADKVNYAHYYEAGLKMLAEGGLIMADN

[0047] ALCSLVYADDDPVRLALHAFAQKVRADTRVEQVMLTVREGILLVRRV (SEQ ID NO:6)

[0048] The amino acid sequence of the MysC-MysD fusion protein from *Porphyra yezoensis* is as follows:

[0049] (MysC-MysD fusion)

[0050] >tr|A0A1X6NNV6|A0A1X6NNV6_PORUM Protein containing ATP-dependent carboxylic acid-amine ligase domain OS=Porphyra yezoensis OX=2786 GN=BU14_0783s0001 PE=4 SV=1)

[0051] MASPAIPPANCNLVQIVLRLLDAFFWSLLVALRGALRMATRPLAGVPHGVAAGKTVLVTT

[0052] GRQAKTVHVIRALKAVGATVVVTDYDHVSASAVSTACDHFVVLPSLDVNALDAWVDAFGK

[0053] LLVEYAVDMVVPVSTINEALFLGVAKDRLSPKLPHVEWLCTGLDNVLRLDDRERFSATCR

[0054] QYGVPAPASGLVTRREQVPHSASQPHGIIVKRLESSVNRDEEIVPVAPSDPLPDFVKPSP

[0055] TDAWQWQQMIRGAEYSVWYVCVNGKVTFSACYLSQPDLVHFDHVPTPADVDKPLRDLIAG

[0056] MHLSGQYAFDFIRDQDSGVPYVIECNPRASSILETVSCTPLWGESFFGIDVSARCRTQDV

[0057] GFVFHGNCWPWTARTEGYLCLSDPLPFFAAEIAWPLHAIQTSGMSEAGYRKIDVNICKII

[0058] IDGPSAPRSIGTFENAAQGAKLAVLDRALQYVETIYIDAAVPNVGAVVAVARKASCHPVL

[0059] FQLGAQAGLLSGVSNSLYADLPPIQFVASPEELSGLVAASEAVAPTRLLTGDAASVALGF

[0060] DSELTLLAPRARATPYTYYRIPLRRLKVLHVMGSCTSKYYETVSSYYGFDCISSVTDDVR

[0061] YEHVIGYVHLTGEWSVAVGKDEVYMREKAPRLSLAQALAAIESLAIDLMIPHMFDYAGLT

[0062] AYRSVFSMLDLPTIGCSGEALALSTNKARTKACAAMDGVQVPKSQLLRRGDTHSMPFPIV

[0063] IKPTEEDNSMGVEVVHNEAQVAAALESAFQFGDEVMVEQYIPLGRELRVAVIETPDGGME

[0064] MLPVVEYFLPAEKPIRKSGDKITTDSSGQPSGYAPVDRKVPADIDPVLHDKLYKLAVVSH

[0065] RATDCADYSIYDVRVDPDGEPYMLESCLYCSFSFRSVLVLMADAAGKKHPQLFVEFSERS

[0066] VGRKSAAIAAQAHGKKAPQEFGMKVRR (SEQ ID NO:7)

[0067] Heterologous expression of the MAA biosynthesis gene from *Actinosynnema mirum* DSM 43827 in the engineered host *Streptomyces avermitilis* SUKA22 resulted in the production of Shinorine as the major product, as well as 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 Amino Acid by Heterologous Expression"), Appl. Env. Microbiol. 80(16):5028-5038, 2014).

[0068] Shinorine, a mycosporin-like amino acid (MAA) typically produced by the red algae *Porphyra umbilicalis*, has been used as an active ingredient in two commercial sunscreens (Helioguard 365 and Helionori). However, the yield of shinorine from *Porphyra umbilicalis* is relatively low (3.25 mg / g CDW), and its production is affected by the slow growth of *Porphyra umbilicalis*. To overcome this problem, several organisms have been metabolically engineered, such as *Escherichia coli*, *Saccharomyces cerevisiae*, *Syntrophus umbilicalis* PCC 6803, *Corynbacterium glutamicum*, and *Streptomyces avermitilis* SUKA22, for the production of shinorine. Unlike previously described methods, this invention provides a novel method for producing shinorine by metabolically engineering host cells, such as Pseudomonas species, like Pseudomonas putida KT2440, which can utilize a variety of carbon sources, including aromatic compounds from lignocellulose hydrolysates, as feedstocks to produce shinorine.

[0069] The following are theoretical yield calculations for the production of shinorine using glucose as a precursor. It is produced by the condensation of the amino group of L-serine with the keto group of (5S)-5-hydroxy-5-(hydroxymethyl)-2-methoxycyclohexane-2-en-1-one, wherein the hydrogen at the 3-position of the cyclohexene moiety is replaced by the amino group of glycine. See also Figure 3 Figure A shows a compound with the following chemical formula and molecular weight: Shinorine: C 13 H 20 N2O8 (332.3); Glucose: C6H 12 O6 (180). The final formula is as follows (assuming 1 glucose → 1 serine, 1 glucose → 1 glycine):

[0070] 1. Hydrocarbon core

[0071] Carbon balance: 4 / 3 C6H 12 O6 + 2 H2 → C8H 12 O4 + 4 H2O

[0072] Redox equilibrium: C6H 12 O6 + 6 H2O → 6 CO2 + 12 H2

[0073] ⇒ 9 C6H 12 O6 → 6 C8H 12O4 + 6 CO2 + 18 H2O

[0074] ⇒ 1.5 glucose molecules → C8H 12 O4 + CO2 + 3 H2O

[0075] 2. Because one shinorine requires one serine or one glycine, and each serine or glycine requires one glucose.

[0076] 1 glucose molecule → 1 serine molecule

[0077] 1 glucose molecule → 1 serine molecule → 1 glycine molecule

[0078] The final formula is: 3.5 C6H 12 O6 → 1 Shinorine (C 13 H 20 (N2O8). Therefore, the theoretical yield is 0.52746 g Shinorine / g glucose ⇒ approximately 0.53 g / g glucose.

[0079] In some embodiments, DDGS, OMT, ATP-dependent carboxylic acid-amine ligases and / or non-ribosomal peptide synthases (NRPS) or D-ala-D-ala ligases are homologous, variant, or mutant enzymes of any wild-type enzyme described herein. Homologous, variant, or mutant enzymes have the same enzymatic activity as their corresponding wild-type enzymes and have an amino acid sequence with equal or greater than 70%, 80%, 90%, 95%, or 99% sequence identity with the corresponding wild-type enzyme, and optionally have one or more conserved amino acid sequences and / or residues, such as conserved sequences and / or residues important for the enzyme's structure or catalytic residue / pocket. In some embodiments, wild-type DDGS, OMT, ATP-dependent carboxylic acid-amine ligases and / or non-ribosomal peptide synthases (NRPS) or D-ala-D-ala ligases are obtained from or derived from *Porphyra yezoensis*, *Nostoc punctiforme*, or *Anabaena variegata*.

[0080] This invention provides a method for producing Porphyra-334 and / or Shinorine in a much faster manner, with higher product titers and yields. Furthermore, by utilizing lignocellulose hydrolysates as raw materials, it avoids the reliance on agricultural plants as raw materials, as is often the case in other similar technologies (e.g., the production of Porphyra-334 and / or Shinorine in E. coli, yeast, etc.), thereby reducing costs.

[0081] The present invention provides a method for producing Porphyra-334 and / or Shinorine, comprising: (a) providing the genetically modified host cell of the present invention; (b) culturing or growing the genetically modified host cell in a suitable culture or medium to produce Porphyra-334 and / or Shinorine; (c) optionally extracting or isolating Porphyra-334 and / or Shinorine from the host cell and / or culture or medium to form isolated or purified Porphyra-334 and / or Shinorine; and (d) optionally mixing Porphyra-334 and / or Shinorine with an emulsion, oil, or water to form an ultraviolet (UV) blocking or filtering composition.

[0082] In some embodiments, the mixing step further includes mixing Porphyra-334 and / or Shinorine or the UV blocking or filtering composition with another active UV filter (e.g., hydroxybenzophenone, octyl p-methoxycinnamate, octyl salicylate and avobenzone, zinc oxide and / or titanium dioxide or mixtures thereof).

[0083] In some embodiments, the UV-blocking or filtering composition is suitable for application to human skin, such as in sunscreen lotions. In some embodiments, the UV-blocking or filtering composition does not contain any ingredients or components that cause coral bleaching.

[0084] In some embodiments, step (a) includes introducing into the host cell one or more nucleic acids encoding (i) 2-demethyl-4-deoxycodone alcohol synthase (DDGS), (ii) O-methyltransferase (O-MT or OMT), (iii) ATP-dependent carboxylic acid-amine ligase, and (iv) non-ribosomal peptide synthase (NRPS) or D-ala-D-ala ligase, wherein each enzyme is operatively linked to a promoter capable of expressing each enzyme in the host cell. In some embodiments, the culture or growth step (b) includes growing the host cell in a manner consistent with respiratory cell growth. In some embodiments, the culture or growth step (b) occurs in a batch process or a fed-batch process, such as a high-gravity fed-batch process.

[0085] In some embodiments, the culture comprises biomass, such as lignocellulose biomass or its hydrolysates. In some embodiments, the biomass is obtained from softwood feedstock (e.g., poplar), hardwood feedstock, grass feedstock, and / or agricultural feedstock, or mixtures thereof.

[0086] In some embodiments, the culture or culture medium comprises a medium rich in LB (lysozyme broth) or one or more components containing LB, such as tryptone and / or yeast extract. In some embodiments, the culture or culture medium comprises hydrolysates derived from or obtained from autogenous matter (e.g., lignocellulose biomass). In some embodiments, the culture or culture medium comprises one or more aromatic compounds, such as aromatic compounds obtained from lignocellulose hydrolysates. In some embodiments, the culture or culture medium comprises one or more carbon sources, such as sugars, such as 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 or culture medium comprises urea as a nitrogen source. In some embodiments, the culture or culture medium comprises amino acids, such as serine and / or glycine. In some embodiments, the culture or culture medium comprises an ionic liquid (IL).

[0087] In some implementations, the genetically modified host cells are natural or engineered or constructed to utilize xylose as a carbon source. Increased xylose utilization leads to an increase in the X5P library used in the Porphyra-334 and / or Shinorine biosynthetic pathways.

[0088] In some embodiments, the genetically modified host cells are natural or engineered or constructed to increase S7P production, for example, via an isomerase pathway. In some embodiments, the genetically modified host cells are natural or engineered or constructed to reduce or knock out natural or endogenous PP_1024 encoding KDPG aldolase or any gene encoding KDPG aldolase, downregulating or minimizing the conversion of 2KDPG to pyruvate. In some embodiments, this downregulation results in growth defects, which are compensated for by supplementing the culture with aromatic compounds that ultimately enter the acetyl-CoA pool. In some embodiments, this co-utilization strategy allows glucose to be converted to G6P, xylose to X5P, and aromatics to acetyl-CoA, which increases Porphyra-334 and / or Shinorine titers.

[0089] In some embodiments, the method results in genetically modified host cells producing Porphyra-334 and / or Shinorine at concentrations equal to or greater than about 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, the method results in genetically modified host cells producing Porphyra-334 and / or Shinorine at doses equal to or greater than about 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.

[0090] In some embodiments, the invention includes using a heterocodon-optimized form of the nucleic acid encoding the enzyme described herein, which is optimized for the host cell. Attached Figure Description

[0091] When read in conjunction with the accompanying drawings, those skilled in the art will readily understand the above and other aspects from the following description of illustrative embodiments.

[0092] Figure 1 Metabolic flux distribution of glucose-fed *Pseudomonas putida* KT2440. All values ​​in the boxes are given as relative flux normalized relative to a specific glucose uptake rate. Data are from three preliminary studies: (Kukurugya et al., 2019) (top), (Nikel et al., 2015) (middle), and (Kohlstedt and Wittmann, 2019) (bottom). Arrow width 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-phosphate gluconate; KDPG, 2-keto-3-deoxy-6-phosphate gluconate; Ri5P, ribulose 5-phosphate; R5P, ribose 5-phosphate; X5P, xylulose 5-phosphate; S7P, sedulose 7-phosphate; E4P, erythrose 4-phosphate; G3P, glyceraldehyde 3-phosphate; 3PG, 3-phosphoglycerate; PEP, phosphoenolpyruvate; Ac-CoA, acetyl-CoA; L-serine; TCA, tricarboxylic acid; PYR, pyruvate.

[0093] Figure 2 Development of a genetic tool for gene expression in *Pseudomonas putida* KT2440. (A) Modular cloning assembly method as described in previously published work (Storch et al., 2015). Genetic components were cloned into pJET1.2 insensitive DNA plasmids. These parts were then digested with BsaI restriction enzyme and ligated with prefix and suffix adapters, followed by separation and purification using magnetic beads. Finally, the purified genetic parts and adapters were assembled in a one-pot reaction at 50°C for 1 hour. (B) Schematic diagram of plasmids with different backbones. RFP fluorescence levels measured on various plasmid backbones (C) under different inducible promoters (D), constitutive promoters (E), and ribosome binding site sequences (F). Cultures were grown in M9 basal medium. RFP fluorescence was measured by flow cytometry 48 hours after inoculation. Inducer was added at 0 hours. Error bars represent the standard deviation of three biological replicates.

[0094] Figure 3 Production of Shinorine in engineered *Pseudomonas putida* KT2440 was confirmed by experimental nuclear magnetic resonance (NMR) spectroscopy. (A) Schematic diagram of central carbon metabolism and the Shinorine biosynthetic pathway in *Pseudomonas putida* KT2440. The initial biosynthetic step involves the synthesis of demethyl-4-deoxycodone (DDG) via sedoheptulose-7-phosphate (Balskus and Walsh, 2010; Pope et al., 2015). Demethyl-4-deoxycodone synthase (DDGS) catalyzes the production of DDG. Subsequently, O-methyltransferase (O-MT) converts DDG to 4-deoxycodone (4-DG). 4-DG is incorporated into glycine via an ATG-dependent carboxylic acid-amine ligase to produce spore-glycine (MG). In the final step, MG is converted to shinorine via a nonribosomal peptide synthase (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 linking serine to the C1 position of spore-glycine to produce shinorine. (B) For Figure 3 (Figure C) Schematic diagram of plasmid pIY456 used in the production experiment. (C) Production of Shinorine from *Pseudomonas putida* strain KT2440. Chromatogram and extract of *Helioguard* obtained from *Pseudomonas putida* (D). TM (E). (F) Allocation of purified Shinorine 1 H and 13 C-resonance. (G) Correlation of all resonances used to definitively allocate purified shinorine. 1H- 1 H NOESY and 1 H- 13 C HMBC). *Pseudomonas putida* KT2440 was grown in M9 medium, and samples were extracted after 48 hours. Error bars represent the standard deviation of three biological replicates. SAM, S-adenosyl-L-methionine; SAH, S-adenosylhomocysteine. Other abbreviations include... Figure 1 As indicated in the title.

[0095] Figure 4 : CRISPRi gene downregulation to improve shinorine production. (A) Schematic diagram of genes involved in the metabolism of 6PG, G3P, L-serine, glycine, and S-adenosyl-L-methionine (SAM). (B) Schematic diagram of plasmids used to express shinorine biosynthesis genes and CRISPRi-mediated gene downregulation. (C) Shinorine titers produced by different strains targeting 21 genes. (D) Relative expression levels of target genes in controls and samples (y-axis; x10). 6 (E) The volcano plot depicts the top 20 downregulated (purple) and upregulated (dark blue) genes in strain PP_1444. Error bars represent the standard deviation of three biological replicates.

[0096] Figure 5 Improved Shinorine production through RBS optimization. (A) Illustration of modular plasmid assembly consisting of promoters and genes assembled into different RBSs. Two types of promoters and three different RBS elements were used. (B) Schematic diagram of 21 different plasmids that produce Shinorine. (C) From carrying Figure 5 (Figure B) shows the Shinorine titer of the strains containing the plasmids. White and black bars represent strains JBx_250483 and JBx_250497, respectively. (D) Heatmap of shotgun proteomics analysis of DDGS, O-MT, ATP-dependent carboxylic acid-amine ligases, and NRPS. Expression levels of DDGS (E), O-MT (F), ATP-dependent carboxylic acid-amine ligases (G), and NRPS (H) from the control strain (JBx_250483) and the strain that produced the highest amount of Shinorine (JBx_250497). Samples were grown on M9 basal medium. Shinorine was extracted from the entire liquid culture at 72 hours. Error bars represent the standard deviation of three biological replicates.

[0097] Figure 6Effects of glycine and L-serine supplementation on Shinorine production. (A) Schematic diagram of the genetic constructs used in the study. *Pseudomonas putida* KT2440 carrying the Shinorine biosynthetic pathway plasmid (JBx_250497) was transformed with the CRISPRi plasmid containing either PP_1444 sgRNA (JBx_249619) or non-target sgRNA (JBx_249575). (B, C) Comparison of Shinorine production, glucose consumption, and growth curves over 114 hours without glycine and L-serine supplementation. (D) Experimental conditions used in B, C, E, and F. (E, F) Comparison of Shinorine production, glucose consumption, and growth curves over 114 hours with glycine and L-serine supplementation. All strains were cultured in 25 mL M9 medium containing 20 g / L glucose, with an initial OD... 600 The value was 0.1. Four hours after inoculation, cultures were induced with 0.2% L-arabinose, with or without the addition of 10 mM equimolar concentrations of glycine and L-serine. At 18 hours, all cultures were additionally treated with 20 g / L glucose. For samples in (D) and (F), an additional 20 g / L glucose was introduced into the cultures at the 42-hour time point. The Venn diagram was generated using the following webpage: interactivenn.net / index.html (Heberle et al., 2015). Error bars represent the standard deviation of three biological replicates. (G) in Figure 6 The fraction of shinorine found in the supernatant and cell pellet of the sample measured at 114 hours in Figure F. Error bars represent the standard deviation of three biological replicates.

[0098] Figure 7 The metabolic flux distributions of (A) *Bacillus megaterium* QM B1551 and (B) other microorganisms (excluding *Pseudomonas putida* KT2440 and *Bacillus megaterium* QM B1551) are summarized in Table 1. The flux of *Bacillus megaterium* QM B1551 is adapted from (Wushensky et al., 2018). In this microorganism, glucose can be oxidized to gluconate and then phosphorylated to 6P gluconate, or glucose can be directly phosphorylated to G6P. In other microorganisms, glucose is directly phosphorylated to G6P (B).

[0099] Figure 8 Characterization of inducible promoters. (A)P tet (B)P A1lacO-1 (C)P m (D)P Ltet-O1 (E)P nagAa and (F)PBAD The cultures were grown in M9 basal medium. Cultures were induced 2 hours post-inoculation and fluorescence was measured at 48 hours. Error bars represent the standard deviation of three biological replicates.

[0100] Figure 9 LC-MS / MS confirmed the extracted Shinorine. (A) m / z ratio of Shinorine extracted from the sample. (B) Extracted Helioguard TM (C) Tandem mass spectra of Shinorine obtained from the extracted sample. (D) Extracted Helioguard. TM Tandem mass spectrometry of 365 Shinorine.

[0101] Figure 10 The structural designation of Shinorine can be found in Table 2. 1 H and 13 C chemical shifts and multiplicity and / or coupling constants.

[0102] Figure 11 : Heterogeneous Shinorine 1 1H NMR spectra. All NMR experiments were obtained at 500 MHz in methanol-d4.

[0103] Figure 12 Shinorine generated heterogeneously at 500 MHz 13 C NMR spectrum.

[0104] Figure 13 Expression levels of DDGS, O-MT, ATP-dependent carboxylic acid-amine ligases, and NRPS in different CRISPRi strains. Error bars represent the standard deviation of three biological replicates. The Y-axis represents protein counts.

[0105] Figure 14 Expression levels of gentamicin (A), kanamycin (B) selection markers, and dCas9 (C) protein in different CRISPRi strains, WT, and controls (CRISPRi_Nontarget). Protein counts were measured by shotgun proteomics analysis. Error bars represent the standard deviation of three biological replicates.

[0106] Figure 15 (A) OD of CRISPRi strain measured at 24, 48 and 72 hours 600 Gluconate concentrations were measured from the supernatant (B) and cell pellet (C) at different time points. Error bars represent the standard deviation of three biological replicates.

[0107] Figure 16 Plotting protein expression levels of O-MT (A) and ATP-dependent carboxylic acid-amine ligase (B) against Shinorine titers.

[0108] Figure 17 Comparison of Shinorine production, glucose utilization, and growth curves over time in PP_1444 and non-target strains. Cultures were incubated in 25 mL M9 medium with an initial glucose concentration of 20 g / L. Error bars represent the standard deviation of three biological replicates.

[0109] Figure 18 Shinorine extracted from the supernatant and cell pellet. Error bars represent the standard deviation of three biological replicates.

[0110] Figure 19 Conserved gene clusters involved in MAA biosynthesis in *Porphyra umbilicalis* and related red algae. (A) Biosynthetic pathway from sedulose-7-phosphate to shinorine in cyanobacteria and the proposed pathway of Porphyra-334 in red algae. (B) Comparison of gene clusters and gene fusions in the cyanobacteria *Anabaena* and *Nostoc*, and the red algae *Porphyra umbilicalis* and *Rhodophyta*. (Brawley et al., “Insights into the red algae and eukaryotic evolution from the genome of *Porphyra umbilicalis* (Bangiophyceae, Rhodophyta),” Proc. Natl. Acad. Sci. USA, Preprint: 1–10, 2017.) Detailed Implementation

[0111] Before describing the invention in detail, it should be understood that, unless otherwise stated, the invention is not limited to specific sequences, expression vectors, enzymes, host microorganisms, or processes, and therefore these are variable. It should also be understood that the terminology used herein is for describing particular embodiments only and is not intended to be limiting.

[0112] Many terms will be used in this specification and the appended claims, and these terms shall have the following meanings:

[0113] The terms “optional” or “optionally” as used herein mean that the features or structures subsequently described may or may not be present, or that the events or circumstances subsequently described may or may not occur, and the description includes instances where a particular feature or structure is present and instances where the feature or structure is not present, or instances where an event or circumstance occurs and instances where it does not occur.

[0114] The singular forms “an,” “a,” and “the” used in this specification and the appended claims include multiple indicators unless the context clearly indicates otherwise. Thus, for example, a reference to “expression vector” includes a single expression vector as well as multiple expression vectors that are the same (e.g., the same operon) or different; a reference to “cell” includes a single cell as well as multiple cells; and so on.

[0115] Many terms will be used in this specification and the appended claims, and these terms shall have the following meanings:

[0116] The terms “optional” or “optionally” as used herein mean that the features or structures subsequently described may or may not be present, or that the events or circumstances subsequently described may or may not occur, and the description includes instances where a particular feature or structure is present and instances where the feature or structure is not present, or instances where an event or circumstance occurs and instances where it does not occur.

[0117] When providing a numerical range, it should also be considered as specifically disclosing intermediate values ​​between the upper and lower limits of the range, spaced one-tenth of the lower limit unit, unless the context explicitly states otherwise. This invention also includes smaller ranges between any set value or intermediate value within the set range and any other set value or intermediate value within the set range. Depending on any explicitly excluded limit value within the set range, the range may independently include or exclude the upper and lower limits of these smaller ranges. This invention also includes ranges that do not contain a limit value, or that contain one or both limit values. When a set range contains one or two limit values, this invention also includes ranges that exclude one or both of those limit values.

[0118] The singular forms “an,” “a,” and “the” used in this specification and the appended claims include multiple indicators unless the context clearly indicates otherwise. Thus, for example, a reference to “expression vector” includes a single expression vector as well as multiple expression vectors that are the same (e.g., the same operon) or different; a reference to “cell” includes a single cell as well as multiple cells; and so on.

[0119] The term "about" refers to a value that is 10% more or 10% less than a specified value.

[0120] Unless otherwise stated, all technical and technical terms used herein have the same meaning as understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may be used to practice or test the invention, preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials associated with the cited publications.

[0121] The term "host cell" is used in this article to refer to a living biological cell that can be transformed by inserting an expression vector.

[0122] As used in this article, the term "heterologous" refers to a material or nucleotide or amino acid sequence found or linked to another material or nucleotide or amino acid sequence that is unrelated to each other (i.e., not found or linked together in the natural state).

[0123] The term "expression vector" or "vector" refers to a compound and / or composition that transduces, transforms, or infects a host cell, thereby causing the cell to express nucleic acids and / or proteins that it would not normally express, or to express them in a manner different from what the cell would normally express. An "expression vector" contains a nucleic acid sequence (typically RNA or DNA) expressed by the host cell. Optionally, the expression vector also contains materials that facilitate the entry of nucleic acids into the host cell, such as viruses, liposomes, protein coatings, etc. Expression vectors contemplated for use in this invention include expression vectors into which nucleic acid sequences can be inserted, along with any preferred or desired manipulation elements. Furthermore, the expression vector must be a vector capable of being transferred to and replicated within a host cell. Specific expression vectors are plasmids, particularly those with verifiable restriction sites and containing preferred or desired manipulation elements for nucleic acid sequence transcription. Such plasmids, and other expression vectors, are well known to those skilled in the art.

[0124] The terms “polynucleotide” and “nucleic acid” are used interchangeably and refer to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. The nucleic acids of this invention will generally contain phosphodiester bonds, although in some cases nucleic acid analogs may be used, which may have alternative backbones, including, for example, phosphoramide, thiophosphate, dithiophosphate, or O-methylphosphamide bonds (see Eckstein, Oligonucleotides and Analogs: A Practical Approach, Oxford University Press); positively charged backbones; nonionic backbones; and nonribose backbones. Thus, nucleic acids or polynucleotides may also include modified nucleotides that allow polymerases to read them correctly. A “polynucleotide sequence” or “nucleic acid sequence” includes the sense and antisense strands of a nucleic acid, as a single- or double-stranded entity. 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, the sequences described herein also provide for the complements of the sequence. Unless otherwise stated, specific polynucleotide sequences also implicitly include their variants (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly indicated sequences. Nucleic acids can be DNA, RNA, or hybrids of genome and cDNA. Nucleic acids can contain combinations of deoxyribonucleotides and ribonucleotides, as well as combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine-hypoxanthine, isocytosine, isoguanine, etc.

[0125] As used herein, the term "promoter" refers to a polynucleotide sequence capable of driving the transcription of a DNA sequence in a cell. Therefore, the promoters used in the polynucleotide constructs of this invention include cis- and trans-acting transcriptional control elements as well as regulatory sequences involved in regulating or modulating gene transcription timing and / or rate. For example, promoters can be cis-acting transcriptional control elements, including enhancers, promoters, transcription terminators, origins of replication, chromosomal integration sequences, 5' and 3' untranslated regions, or intron sequences that participate in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to perform (on / off, regulation, etc.) gene transcription. The promoter is located at the 5' end of the transcribed gene and, as used herein, includes a 5' sequence derived from the translation start codon (i.e., the 5' untranslated region of the mRNA, typically containing 100-200 bp). Most commonly, the core promoter sequence is located within 1-2 kb of the translation start site, more commonly within 1 kbp, and typically 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 generally referred to by the name of the gene whose expression is naturally regulated. The promoters used in the expression constructs of this invention are named after the gene. Promoters referred to by name include wild-type, natural promoters, and promoter variants that retain the ability to induce expression. Promoters referred to by name are not limited to a specific species, but also include promoters from corresponding genes in other species.

[0126] If a polynucleotide sequence originates from a foreign species, or is from the same species but modified in its original form, it is "heterologous" relative to the host cell or a second polynucleotide sequence. For example, when a polynucleotide encoding a polypeptide sequence is operatively linked to a heterologous promoter, this means that the polynucleotide coding sequence of 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 and the promoter are unrelated in their natural state (e.g., are genetically engineered coding sequences, such as those from different genes within the same species, or from alleles of different ecotypes or varieties).

[0127] The term "operationally linked" refers to a functional relationship between two or more segments of a polynucleotide (e.g., DNA). Typically, it refers to the functional relationship between a transcriptional regulatory sequence and a transcriptional sequence. For example, a promoter or enhancer sequence is operationally linked to a DNA or RNA sequence if it can stimulate or regulate transcription of a DNA or RNA sequence in a suitable host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operationally linked to a sequence are physically contiguous with the transcriptional sequence; that is, they act in cis. However, some transcriptional regulatory sequences (such as enhancers) do not need to be physically contiguous or adjacent to the coding sequence that enhances transcription.

[0128] In some embodiments, the host cell contains a nucleic acid encoding one or more enzymes, said nucleic acid being operatively linked to a promoter capable of expressing said one or more enzymes in the host cell. In some embodiments, the nucleic acid encoding said one or more enzymes is codon-optimized for the host cell. In some embodiments, said nucleic acid is a vector or replicon capable of stably residing in the host cell. In some embodiments, the nucleic acid is stably integrated into the chromosome of the host cell.

[0129] In some embodiments, step (a) includes introducing a nucleic acid encoding one or more enzymes into a host cell, the nucleic acid being operatively linked to a promoter capable of expressing the one or more enzymes in the host cell.

[0130] The present invention provides a method for constructing the genetically modified host cell of the present invention, comprising (a) introducing a nucleic acid encoding one or more enzymes into the host cell, said nucleic acid encoding one or more enzymes being operatively linked to a promoter capable of expressing said one or more enzymes in the host cell.

[0131] The expression of genes encoding any of the enzymes described herein can be modified by various methods according to the present invention. Those skilled in the art will recognize that enzyme expression levels can be altered by increasing gene copy number, ribosome binding site strength, promoter strength, and various transcriptional regulatory factors.

[0132] host cells

[0133] The genetically modified host cell can be any prokaryotic or eukaryotic cell with any genetic modification, capable of producing isopentenol according to the method of the invention. Suitable eukaryotic host cells include, but are not limited to, fungal cells. Suitable fungal cells are yeast cells, such as yeast cells of the genus *Saccharomyces*. Typically, although not necessarily, the host cell is yeast or bacteria. Any prokaryotic or eukaryotic host cell can be used for this method as long as it remains viable after transformation with the nucleic acid sequence. In some embodiments, the host cell is not adversely affected by the transduction of the essential nucleic acid sequence, the subsequent expression of the protein (i.e., enzyme), or the resulting intermediates required for steps related to the mevalonate pathway. For example, it is preferable to minimize "crosstalk" (i.e., interference) between the host cell's own metabolic processes and those involving the mevalonate pathway.

[0134] In some implementations, the host cell is genetically modified because a heterologous nucleic acid has been introduced into the host cell, thus the genetically modified host cell is not naturally present. A suitable host cell is one capable of expressing a nucleic acid construct encoding one or more enzymes described herein. The gene encoding the enzyme may be a heterologous gene of the host cell, or it may be a gene originally present in the host cell but operatively linked with a heterologous promoter and one or more control regions, resulting in higher expression of the gene in the host cell.

[0135] The enzyme can be either a naturally occurring enzyme in the host cell or a heterologous enzyme. If the enzyme is naturally occurring in the host cell, the host cell is genetically modified to regulate its expression. This modification can involve modifying 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 to regulate enzyme expression in the host cell, for example, by increasing enzyme expression in the host cell compared to unmodified host cells.

[0136] Yeasts suitable for use in this invention include, but are not limited to, *Yarrowia lipolytica*, *Candida*, *Basilospora bühlerii*, *Saccharomyces* species, *Schizosaccharomyces*, and *Pichia pastoris* cells. In some embodiments, the yeast is *Saccharomyces cerevisiae*. In some embodiments, the yeast is *Candida*, including but not limited to *Candida tropicalis*, *Candida maltosa*, *Candida apicola*, *Candida paratropicalis*, *Candida albicans*, *Candida cloacae*, *Candida guillermondii*, *Candida intermedia*, *Candida lipolytica*, *Candida panapsilosis*, and *Candida 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 *Saccharomyces* species. In some embodiments, the yeast species is *Saccharomyces cerevisiae*. In some embodiments, the yeast is an oily yeast. In some embodiments, the oily yeast is a species of the genus *Rhodosporidium*. In some embodiments, the *Rhodosporidium* species is *Rhodosporidium toruloides*.

[0137] In some embodiments, the host cell is *Rhodosporidium toruloides* or *Pseudomonas putida*. In some embodiments, the host cell is a Gram-negative bacterium. In some embodiments, the host cell belongs to the phylum Proteobacteria. In some embodiments, the host cell belongs to the class Gamma-Proteobacteria. In some embodiments, the host cell belongs to the order Enterobacteriaceae. In some embodiments, the host cell belongs to the family Enterobacteriaceae. Examples of suitable bacteria include, but are not limited to, those species assigned to the taxonomic categories of *Escherichia coli*, *Enterobacter*, nitrogen-fixing bacteria, *Erwinia*, *Bacillus*, *Pseudomonas*, *Klebsiella*, *Proteus*, *Salmonella*, *Serratia*, *Shigella*, *Rhizobium*, *Vibrio hygroscopicus*, and *Paracococcus*.

[0138] Bacterial host cells suitable for use in this invention include, but are not limited to, *Escherichia coli*, *Corynebacterium*, *Pseudomonas*, *Streptomyces*, and *Bacillus*. In some embodiments, the Escherichia coli cells are *Escherichia coli*, *E. albertii*, *E. fergusonii*, *E. hermanii*, *E. marmotae*, or *E. vulneris*. In some embodiments, the Corynebacterium cells are *Corynebacterium glutamicum*, *Corynebacterium kroppenstedtii*, *Corynebacterium alimapuense*, *Corynebacterium amycolatum*, *Corynebacterium diphtheriae*, *Corynebacterium efficiens*, *Corynebacterium jeikeium*, *Corynebacterium macginleyi*, *Corynebacterium matruchotii*, *Corynebacterium minutissimum*, *Corynebacterium renale*, *Corynebacterium striatum*, and *Corynebacterium ulcerans*. Ulcers, Corynebacterium urealyticum, or Corynebacterium uropygiale.In some embodiments, the Pseudomonas cells are *Pseudomonas putida*, *Pseudomonas aeruginosa*, *Pseudomonas chlororaphis*, *Pseudomonas fluorescens*, *Pseudomonas pertucinogena*, *Pseudomonas stutzeri*, *Pseudomonas syringae*, *Pseudomonas cremoricolorata*, *Pseudomonas entomophila*, *Pseudomonas fulva*, *Pseudomonas monteilii*, *Pseudomonas mosselii*, *Pseudomonas oryzihabitans*, *Pseudomonas parafluva*, or *Pseudomonas plecoglossicida*. In some embodiments, the Streptomyces cells are *Streptomyces coelicolor*, *Streptomyces lividans*, *Streptomyces venezuelae*, *Streptomyces ambofaciens*, *Streptomyces avermitilis*, *Streptomyces albus*, or *Streptomyces scabies*. In some embodiments, the Bacillus cells are *Bacillus subtilis*, *Bacillus megaterium*, *Bacillus licheniformis*, *Bacillus anthracis*, *Bacillus amyloliquefaciens*, or *Bacillus pumilus*.

[0139] In some embodiments, the bacterial host cell is a Proteobacterium cell. In some embodiments, the Proteobacterium cell is a γ-Proteobacterium cell. In some embodiments, the γ-Proteobacterium cell is a Pseudomonas or Enterobacteriaceae cell. In some embodiments, the γ-Proteobacterium cell is a Pseudomonas cell, specifically a Pseudomonas family cell. In some embodiments, the Pseudomonas family cell is a Pseudomonas, nitrogen-fixing bacterium, neutrophil, Oblitimonas, Permanibacter, Rugamonas, or Thiopeseudomonas cell. In some embodiments, the Pseudomonas cells are *Pseudomonas putida*, *Pseudomonas aeruginosa*, *Pseudomonas chlororaphis*, *Pseudomonas fluorescens*, *Pseudomonas pertucinogena*, *Pseudomonas stutzeri*, *Pseudomonas syringae*, *Pseudomonas cremoricolorata*, *Pseudomonas entomophila*, *Pseudomonas fulva*, *Pseudomonas monteilii*, *Pseudomonas mosselii*, *Pseudomonas oryzihabitans*, *Pseudomonas parafluva*, or *Pseudomonas plecoglossicida*. In some embodiments, the γ-Proteobacterium cells are Enterobacteriaceae cells, which are Enterobacteriaceae cells. In some embodiments, the Enterobacteriaceae cells are Escherichia coli, Enterobacillus, Enterobacter, Klebsiella, Salmonella, or Shigella cells. In some embodiments, the Escherichia coli cells are Escherichia coli, E. albertii, E. fergusonii, E. hermanii, E. marmotae, or E. vulneris. In some embodiments, the host cells are Gram-negative bacteria. In some embodiments, the host cells are bacteria from the genera *Dinosporium*, *Escherichia*, *Salmonella*, *Vibrio*, *Pasteurella*, *Haemophilus*, or *Pseudomonas*.In some implementations, the host cell is a bacterium derived from Escherichia coli, Salmonella enterica, Vibrio cholerae, Pasteurella multocida, Haemophilus influenzae, Pseudomonas putida, or Pseudomonas aeruginosa.

[0140] Biomass can be obtained from one or more raw materials, such as softwood, hardwood, grass and / or agricultural materials or mixtures thereof.

[0141] Cork raw materials include, but are not limited to, Araucaria heterophylla (e.g., Araucaria cunninghamii, Araucaria angustifolia, Araucaria araucana); cork cedar (e.g., Juniperus virginiana, Thuja plicata, Thuja occidentalis, Chamecyparis thyoides, Callitropsis nootkatensis); cypress (e.g., Chamecyparis, Cupressus, Taxodium, Cupressus aizonica, Taxodium distichum, Chamecyparis obtusa, Chamecyparis lawsoniana, Cupressus semperviren); and Rocky Mountain Douglas fir. fir); European yew; fir (e.g., balsam fir, European fir, magnificent fir, Pacific fir); hemlock (e.g., Canada hemlock, long-fruited hemlock, heterophyllous hemlock); kauri; torreya; larch (e.g., European larch, Japanese larch, Japanese larch, bald pine, western larch).Pine trees (e.g., European black pine (Pinus nigra), North American shortleaf pine (Pinus banksiana), twisted-leaf pine (Pinus contorta), Montalcian pine (Pinus radiata), Western yellow pine (Pinus ponderosa), North American red pine (Pinus resinosa), Scots pine (Pinus sylvestris), American white pine (Pinus strobus), silver-leaf five-needle pine (Pinus monticola), Lambert's pine (Pinus lambertiana), loblolly pine (Pinus taeda), longleaf pine (Pinus palustris), rigid pine (Pinus rigida), sprouting pine (Pinus echinata)); redwood; privet juniper; spruce (e.g., European spruce (Picea abies), black spruce (Picea mariana), red spruce (Picea rubens), Sitka spruce (Picea sitchensis), white spruce (Picea glauca); Japanese cedar; and its combinations / hybrids.

[0142] For example, the cork raw materials that may be used herein include cedar; fir; pine; spruce; and combinations thereof. The cork raw materials of this invention may be selected from spruce, Montalcian pine, shortleaf pine, spruce (e.g., white spruce, inland spruce, black spruce), Douglas fir, Scots pine, European spruce, and combinations / hybrids thereof. The cork raw materials of this invention may be selected from pine (e.g., Montalcian pine, spruce); spruce; and combinations / hybrids thereof.

[0143] Hardwood raw materials include, but are not limited to, acacia; *Acacia mangium*; *Miracle fruit*; *Albizia julibrissin*; alder (e.g., *Alnus glutinosa*, *Alnus rubra*); apple wood; bayberry; ash (e.g., black ash (*F. nigra*, *F. quadrangulata*, *F. excelsior*, American red ash (*F. pennsylvanicalanceolata*, *F. latifolia*, *F. profunda*, *F. americana*); poplar (e.g., *P. grandidenta*, *P. tremula*, *P. tremuloides*); Australian red cedar (*Toona ciliata*); sappanwood (*Distemonanthus benthamianus*); tropical American balsa wood (*Ochroma*). Pyramidale); Linden (e.g., American linden (T. americana), heterophylla); Beech (e.g., European beech (F. sylvatica), American beech (F. grandifolia)); Birch (e.g., grey birch (Betula populifolia), water birch (B. nigra), paper birch (B. papyrifera), sweet birch (B. lenta), Canadian yellow birch (B. alleghaniensis) / yellow birch (B. lutea), European white birch (B. pendula), hairy birch (B.pubescens); black bean; blackwood; Mexican golden sandalwood; compound maple; boxwood; Brazilian sappanwood; murica; horse chestnut (e.g., European horse chestnut (Aesculus hippocastanum), glossy horse chestnut (Aesculus glabra), yellow horse chestnut (Aesculus flava / Aesculus octandra)); grey walnut; catalpa; cherry wood (e.g., North American cherry (Prunus serotina), Pennsylvania cherry (Prunus pennsylvanica), European cherry (Prunus avium)); Crabwood; chestnut; hornwood; rosewood; corkwood; cotton poplar (e.g., balsamifera, deltoides, plain cotton poplar (Ppulus sargentii), heterophylla (Ppulus heterophylla)); sharp-leaved magnolia; cornelian cherry (e.g., large-flowered dogwood (Cornus) *Cornus nuttallii*; ebony (e.g., *Diospyros kurzii*, *Diospyros melanida*, *Diospyros crassiflora*); elm (e.g., *Ulmus americana*, *Ulmus procera*, *Ulmus thomasii*, *Ulmus rubra*, *Ulmus glabra*); eucalyptus; green camphor; ebony; viscose (e.g., *Nyssa sylvatica*, *Eucalyptus globulus*, *Liquidambar styraciflua*, *Nyssa aquatica*); hickory (e.g., *Carya alba*, *Carya glabra*, *Carya rubra*). Ovata), Caryalaciniosa); Carpinus; Elm; Ipe; Iroraceae; Ironwood (e.g., Balau, Carpinus caroliniana, Casuarina equisetifolia, Choricbangarpia subargentea, Copaifera spp.).), Eusideroxylon zwageri, Guajacum officinale, Guajacum sanctum, Hopea odorata, Ipe, Krugiodendron ferreum, Lyonnohamnus lyonii (Ceylon ironwood (L.floribundus)), Mesua ferrea, Olea spp.* *Olneyatesota*, *Ostrya virginiana*, *Parrotia persica*, *Tabebuia serratifolia*; Jacaranda; Brazilian cherry; London plane tree; Laurel; West African terminalia; Ironwood (*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 wood; East African blackwood; Oak (e.g., *Quercus macrocarpa*, *Quercus alba*, *Quercus serratifolia*). *Quercus stellata*, *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 phellos*, *Quercus texana*); African balsa wood; *Oregon myrtle*; *California bay laurel*; pear trees; poplar trees (e.g., balsam poplar). balsamifera), black poplar (P. )Poplar (Populus xcanadensis)); white wood; redwood; rosewood; sago palm; sandalwood; sassafras; satinwood; silver birch; silver privet; snakewood; amber; Spanish cedar; American sycamore; teak; walnut (e.g., black walnut (Juglans nigra), walnut (Juglans regia)); willow (e.g., black willow (Salix nigra), white willow (Salix alba)); boxwood (Liriodendron tulipifera); bamboo; palm wood; and their combinations / hybrids.

[0144] For example, the hardwood raw materials of the present invention can be selected from acacia, poplar, beech, eucalyptus, maple, birch, viscose, oak, poplar and combinations / hybrids thereof. The hardwood raw materials of the present invention can be selected from the genera *Populus* (e.g., *Populus tremuloides*), *Eucalyptus* (e.g., blue gum), *Acacia* (e.g., Australian acacia) and combinations thereof.

[0145] Grass feedstocks include, but are not limited to, C4 or C3 grasses such as switchgrass, Indian grass, big bluegrass, little bluegrass, Canadian wild rye, Virginia wild rye, and yellow alfalfa, as well as other species known in the art.

[0146] Agricultural raw materials include, but are not limited to, agricultural byproducts such as husks, straw, and leaves. These byproducts can come from crops intended for human consumption, animal consumption, or other non-consumption purposes. These crops can be agricultural crops such as corn, wheat, rice, soybeans, hay, potatoes, cotton, or sugarcane.

[0147] Raw materials can come from crops harvested using the following planting methods: intercropping, mixed intercropping, inter-row planting, relay cropping, etc.

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[0222] Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description and accompanying drawings.

[0223] It should be understood that although the invention has been described in conjunction with preferred embodiments, the foregoing description is intended to illustrate and not limit the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art.

[0224] All patents, patent applications and publications mentioned in this article are incorporated herein by reference in their entirety.

[0225] Following the description of the invention, the following examples are provided to illustrate the invention by way of description rather than limitation.

[0226] Example 1

[0227] System engineering transformation for the production of anti-aging and sunscreen compounds from Pseudomonas putida.

[0228] Sunscreens have been used to protect skin from ultraviolet radiation for thousands of years. However, the use of modern commercial sunscreens containing oxybenzone, ZnO, and TiO2 has raised concerns due to their negative impacts on human health and the environment. This study aimed to establish an efficient microbial platform for the production of shinorine, a UV-absorbing compound with anti-aging properties. First, suitable hosts were systematically selected for shinorine production by analyzing central carbon flux distribution data from previous studies and predictions from genome-scale metabolic models (GEM). Shinorine productivity was enhanced by CRISPRi-mediated downregulation, and potential competing pathways were precisely identified using shotgun proteomics. Simultaneously, the shinorine biosynthetic pathway was improved by refining its design, optimizing promoter use, and altering the strength of the ribosome binding site. Finally, amino acid feeding experiments were conducted under various conditions to identify key limiting factors in shinorine production. This study combined… 13 Meta-analysis of C-metabolic flux analysis, GEM, synthetic biology, CRISPRi-mediated gene downregulation, and omics analysis to improve shinorine production demonstrates the potential of *Pseudomonas putida* KT2440 as a shinorine production platform.

[0229] In this study, suitable hosts for shinorine production were systematically selected by analyzing central carbon flux distribution data from previous studies and predictions from genome-scale metabolic models (GEM). *Pseudomonas putida* KT2440 was identified as a promising host for shinorine production. Synthetic biology tools were developed for this microorganism, and the shinorine biosynthetic gene cluster (BGC) from *Anabaena variabilis* ATCC 29413 was heterologously expressed. Shinorine productivity was enhanced by downregulation of a potential competing pathway mediated by CRISPRi. Simultaneously, the shinorine biosynthetic pathway was improved by refining its design, optimizing promoter use, and altering the strength of the ribosome binding site. Finally, amino acid feeding experiments were conducted under various conditions, and shotgun proteomics was used to identify limiting factors in shinorine production. In conclusion, our study provides a comprehensive framework for the engineering of *Pseudomonas putida* KT2440 as an efficient chassis for shinorine production.

[0230] Results and discussion

[0231] Pseudomonas putida KT2440 as a suitable host for Shinorine production

[0232] To select suitable hosts for shinorine production, we reviewed published metabolic flux distributions from various microorganisms (Table 1) and predictions from genome-scale metabolic models (GEM). The primary focus was on analyzing fluxes for gluconic acid 6-phosphate (6PG), as this intermediate metabolite plays a crucial role in the shinorine biosynthetic pathway. 6PG acts as an intermediate molecule in both the pentose phosphate pathway and the Entner-Doudoroff pathway. In the pentose phosphate pathway, 6PG undergoes decarboxylation to produce ribulose 5-phosphate (Ri5P), which is further metabolized by ribulose epimerases to ribose 5-phosphate (R5P) and xylulose 5-phosphate (X5P). While R5P is a fundamental building block of nucleic acid synthesis, X5P is converted by transketolases to glyceraldehyde 3-phosphate (G3P) and heptaheptaose 7-phosphate (S7P), serving as precursors in shinorine biosynthesis. G3P is converted to glycerol 3-phosphate (3PG) via glycolysis (Embden-Meyerhof-Parnas (EMP) pathway) or the Entner-Doudoroff pathway. 3PG is used in the biosynthesis of L-serine and glycine, which are essential amino acids incorporated into the shinorine biosynthetic pathway.

[0233] Table 1. Carbon fluxes from G6P to F6P and from G6P to 6PG in various organisms. All values ​​are given as relative fluxes normalized relative to a specific glucose uptake rate.

[0234]

[0235]

[0236]

[0237]

[0238] In most organisms, except for *Pseudomonas putida* KT2440 and *Bacillus megaterium* QM B1551, glucose is directly phosphorylated by glucokinase to produce glucose-6-phosphate (G6P). Figure 1 , Figure 7 G6P can isomerize to fructose-6-phosphate (F6P) in the glycolysis pathway, and can also be oxidized to 6PG via the pentose phosphate oxidase pathway. 13 C metabolic flux analysis ( 13 C-MFA determinations showed that the carbon flux distribution from G6P to F6P or from G6P to 6PG varied among different microorganisms and was influenced by factors such as fermentation conditions and genetic variation (Table 1). Nevertheless, for 13 Meta-analysis of C-MFA and predictions of GEM (Table 2) show that only a few microorganisms are able to allocate more than 50% of the carbon flux to 6PG.

[0239] Table 2. Carbon flux distribution predicted by genome-scale metabolic models.

[0240]

[0241] *Fermentomonas motilityis* converts approximately 99% of its glucose supply to 6PG (Table 1). However, due to the lack of 6PG dehydrogenase, the oxidative branch of the pentose phosphate pathway in *Fermentomonas motilityis* appears to be inactive (DeGraaf et al., 1999; Jacobson et al., 2019). Instead of using the oxidative branch of the pentose phosphate pathway, *Fermentomonas motilityis* utilizes the non-oxidative branch, converting F6P to R5P and S7P via a series of enzymatic reactions. Only 0.8% of the carbon flux is allocated from G6P to F6P, leaving a very small pool of F6P for S7P synthesis. On the other hand, computational metabolic network analysis of *Rhodotorula glutinis* showed that glucose-fed *Rhodotorula glutinis* allocates 89.7% of its carbon flux to 6PG, generating a large amount of S7P in the non-oxidative pentose phosphate pathway (Bommareddy et al., 2015). Rhodotorula glutinis is also an attractive host for the industrial-scale production of many compounds (Liu et al., 2023, 2020; Schultz et al., 2022; Wehrs et al., 2019). It can readily co-consume C5 and C6 sugars from lignocellulosic biomass and grow to very high cell densities. However, engineering Rhodotorula glutinis has been challenging (Wen et al., 2020), with only a few synthetic biology tools available (Brink et al., 2023).

[0242] Pseudomonas putida KT2440 exhibits a unique central carbon metabolism ( Figure 1In this microorganism, glucose can be phosphorylated to G6P in the cytoplasm via direct phosphorylation, or oxidized to gluconate in the periplasm by glucose dehydrogenase. Subsequently, gluconate can follow one of two pathways: it can be phosphorylated to 6PG by gluconate kinase 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 2-KG kinase, and further reduced to 6PG by 2K6PG reductase. Although most glucose is oxidized to gluconate, these pathways converge to the production of 6PG regardless of the initial steps in glucose processing. Furthermore, compared to other microorganisms, *Pseudomonas putida* KT2444 prefers to convert F6P to 6PG rather than the reverse reaction. Therefore, *Pseudomonas putida* KT2440 converts over 90% of its glucose supply to 6PG, creating a significant amount of 6PG for the biosynthesis of Ri5P and S7P in the non-oxidative pentose phosphate pathway. Furthermore, genome editing and synthetic biology tools for *Pseudomonas putida* KT2440 have been widely used and well characterized (Nikel and de Lorenzo, 2018). This unique metabolic trait makes *Pseudomonas putida* KT2440 a promising host for the production of shinorine.

[0243] Development of synthetic biology tools for gene expression in *Pseudomonas putida* KT2440

[0244] To realize the Shinorine biosynthetic pathway, a modular plasmid assembly tool for Pseudomonas putida KT2440 was first developed. Figure 2 (Figure A). Here, the Biopart Assembly Standard (BASIC) plasmid assembly method for idempotent cloning (Storch et al., 2015) was used to assemble multipart DNA in a single-pot assembly reaction using standard, reusable components. The resulting expression plasmids were then used to evaluate the function of genetic elements, such as promoters and ribosome binding sites. The primary objective of this study was not to provide a detailed characterization of the genetic elements, but rather to investigate the performance of the expression plasmids and individual genetic elements used in this study.

[0245] Six plasmids (pBBR1, pBBR1-B5, pRK2, pRSF1010, pRO1600 / p15a, and pVS1 / p15a) with different backbones, copy numbers, and origins of replication were constructed and characterized by measuring the fluorescence intensity of red fluorescent protein (RFP). Figure 2(Figure B). The plasmids selected for construction have several characteristics. First, pBBR1, isolated from *Bordetella bronchiseptica* S87 (Antoine and Locht, 1992), is a broad-host-range plasmid source commonly used for gene expression in *Pseudomonas putida* metabolic engineering studies (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. Second, pBBR1-B5 is a variant of the pBBR1 plasmid with an early stop codon in the rep gene, resulting in a higher copy number than the original pBBR1 plasmid. Third, plasmid pRK2 is a member of the IncP incompatible group and requires an origin of replication sequence and a replication initiation protein encoded by trfA to function. It has been shown to be stably maintained in *Pseudomonas putida* and has been measured as a low-copy plasmid in *Escherichia coli* (De Bernardez and Dhurjati, 1987). Fourth, RSF1010 is a high-copy, broad-host-range plasmid belonging to the IncQ incompatible group. It has been routinely used for gene expression in *Pseudomonas putida* (Aparicio et al., 2019). Finally, pRO1600 (Farinha and Kropinski, 1990) and pVS1 (Itoh and Haas, 1985) are plasmids isolated from *Pseudomonas* species that require a host-specific source (e.g., p15a, which replicates in *E. coli*) to generate shuttle vectors for *Pseudomonas putida*. In this study, plasmid characterization showed that pBBRR1-B5 exhibited the highest RFP fluorescence level, followed by pBBR1 > pRSF1010 = pRO1600 / p15a > pRK2 > pVS1 / p15a plasmids. Figure 2 (Photo set C).

[0246] Given its moderate range of RFP fluorescence, plasmid pRK2 was selected as the plasmid backbone for characterizing different inducible types. Figure 2 (Group D) and compositional type ( Figure 2 Figure E) shows the promoters. It characterizes 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 (Hüsken et al., 2001), P lacUV5 (Noel Jr. and Reznikoff, 2000) and P XylS / Pm(Calero et al., 2016)), six 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 (Bagdasarian et al., 1983; Calero et al., 2016) (without LacI repressor) was... Compared to other promoters, P... tet and P A1lacO-1 The promoter exhibits a high level of basal fluorescence ( Figure 8 In inductive promoters, the arabinose-inducible promoter P... BAD It exhibited the highest RFP fluorescence level. Simultaneously, under the test conditions, the constitutive promoter P... trc-1O It is the strongest promoter among all the evaluated promoters.

[0247] Finally, based on their different intensities in Escherichia coli, three BioBrick (Shetty et al., 2008) ribosome binding site elements (RBS1; ATCACACAGGAC (BBa_B0033) (SEQ ID NO: 8), RBS2; AAAGAGGGGAAA (BBa_B0064) (SEQ ID NO: 9), RBS3; AAAGAGGAGAAA (BBa_B0034) (SEQ ID NO: 10), the Shine-Dalgarno Escherichia coli common sequence (RBS4; ATCACAAGGAGG) (SEQ ID NO: 11), and the anti-Shine-Dalgarno complementary sequence (RBS5: ATTAGGGAGGT) (SEQ ID NO: 12) were characterized. Figure 2 Figure F shows that the selected ribosome binding sites exhibited different levels of RFP fluorescence. The use of ribosome binding sites with varying intensities has been employed in metabolic engineering studies and has proven to be a useful strategy for pathway optimization.

[0248] Shinorine production in Pseudomonas putida KT2440

[0249] The shinorine biosynthetic pathway was constructed after evaluating the performance and function of the plasmid backbone, promoter, and ribosome binding site in *Pseudomonas putida* KT2440. The shinorine biosynthetic gene cluster (BGC) has been identified in various microorganisms, including *Anabaena variegata* ATCC 29413 (Balskus and Walsh, 2010), *Nostoc punctata* ATCC 29133 / PCC73102 (Qunjie and Ferran, 2011), *Actinosynnema mirum* DSM 43827 (Miyamoto et al., 2014), *Chlorogloeopsis fritschii* PCC 6912 (Llewellyn et al., 2020; Portwich and Garcia Pichel, 2003), and *Fritschii* PCC 9339 (Yang et al., 2018). In this study, the shinorine biosynthetic pathway was selected from *Anabaena variegata* ATCC 29413 because it is one of the earliest characterized shinorine biosynthetic pathways. The intermediate metabolite S7P of the pentose phosphate pathway is converted to shinorine via a series of four enzymatic steps. First, S7P is converted to 4-deoxycodone (4-DG) via 2-demethyl-4-deoxycodone synthase (DDGS) and O-methyltransferase (O-MT). Subsequently, glycine is coupled to 4-DG via an ATP-dependent carboxylic acid-amine ligase to form spore-glycine (MG). Finally, the serine moiety is linked to MG via a non-ribosomal peptide synthase (NRPS)-like enzyme. Figure 3 (Photo set A).

[0250] To synthesize Shinorine in *Pseudomonas putida* KT2440, the entire Shinorine BGC (MIBiG accession number: BGC0000427) was first amplified from the mutant *Anabaena ATCC 29413*, while maintaining intergenic regions. Subsequently, in a strongly constitutive P... trc1-O Under the control of the promoter, the amplified gene cluster is cloned into an RK-based vector. Figure 3 (Figure B). Heterologous expression of the Shinorine biosynthetic pathway in *Pseudomonas putida* KT2440 resulted in the production of approximately 60 mg / L of Shinorine within 48 hours, with over 80% of the Shinorine accumulating in the supernatant. Chromatographic analysis of the samples ( Figure 3 Figure D shows that the Shinorine peak (1) is similar to that from the reference Helioguard. TM Standard products ( Figure 3 The Shinorine peak obtained in Figure E was co-eluted. The accuracy of the mass spectrometry was confirmed by LC-MS / MS. Figure 9 The results were consistent with those reported in previous studies (Kim et al., 2022). To further validate the chemical structure of Shinorine, an extract from 1-L 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 can clearly allocate all 1 H and 13 C resonance ( Figure 3 (Figures F, G and Table 3). The observed resonances and their assignments are highly consistent with previously reported NMR data for Shinorine (Miyamoto et al., 2014) Figure 10-12 This is, to our knowledge, the first report of shinorine production in *Pseudomonas putida* KT2440.

[0251] Table 3. Observations in heterologously produced Shinorine 1 H and 13 Atom assignment for C resonance. Atom numbering can be performed... Figure 4 Found it.

[0252]

[0253] Increasing Shinorine Production Titer via CRISPRi-Mediated Gene Downregulation

[0254] To further improve shinorine titers, CRISPRi-mediated gene downregulation was used to suppress competitive metabolic pathways and redirect carbon flux to shinorine production. The use of CRISPRi has the potential to increase the yield of desired metabolites by selectively inhibiting the expression of specific genes in metabolic pathways. This approach has been shown to successfully improve the production of a variety of compounds, including isopentenols (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, CRISPRi was used to downregulate a group of 21 genes involved in central carbon metabolism and the biosynthesis of L-serine and glycine, two amino acids crucial for the shinorine biosynthetic pathway. Figure 4 (Figure A). In the salicylic acid-inducible promoter (PnagA Under the control of ), the catalytically inactivated Cas9 protein was expressed in the RSF1010 plasmid, and a single guide RNA (sgRNA) was placed in the constitutive promoter P. J23119 Down( Figure 4 (See Figure B). This plasmid is co-expressed with the RK2 plasmid that produces shinorine.

[0255] Our results indicate that knocking down the PP_1444 gene leads to a significant increase in shinorine titers. The *Pseudomonas putida* strain KT2440 with downregulated PP_1444 produced approximately 400 mg / L of shinorine within 72 hours, a 160% increase compared to the control strain (i.e., the strain with non-target sgRNA). Figure 4 (See Figure C). It is worth noting that, with Figure 3 Compared to the strain shown in Figure C, the control strain shown here had a titer approximately 3-fold higher at 48 hours. The presence of the CRISPRi plasmid appears to affect the expression levels of DDGS, O-MT, ATP-dependent carboxylic acid-amine ligase, and NRPS. Figure 13 These changes may also be attributed to the high expression of the aph gene. Figure 14 (Figure B) This may indicate that the copy number of the RK2 plasmid changed in the presence of the second plasmid.

[0256] PP_1444 encodes quinoline glucose dehydrogenase, an enzyme responsible for converting glucose to gluconic acid. Previous studies have observed that *Pseudomonas putida* KT2440 tends to accumulate gluconic acid and 2-ketogluconic acid, and that knocking out PP_1444 effectively eliminates the accumulation of these two compounds (Teresa et al., 2007). Interestingly, in previous studies, knocking out PP_1444 was associated with growth defects (Bentley et al., 2020). However, in this study, no growth defects were observed. Figure 15 (Figure A). This difference may be attributed to the partial inhibition of PP_1444 by CRISPRi ( Figure 4 (Figure D) This leads to a reduction rather than complete elimination of gluconic acid in the supernatant and cell sediment fractions. Figure 15 ).

[0257] Since downregulation of PP_1444 appeared to be the only instance leading to an increase in Shinorine titers, further investigation was conducted to validate downregulation of other target genes. Of the 21 target genes, 10 were indeed downregulated, 3 were not downregulated, and 8 could not be validated because the protein was undetectable in both the sample and control strains. Figure 4(Figure D). Among the three genes that were not downregulated (PP_1010, PP_2930, and PP_4677), except for PP_4677, their respective dCas9 expression levels were low. Figure 14 (Figure C) Although the expression level of gentamicin selection markers remained consistent in these strains ( Figure 14 (Figure A). In the case of PP_4677, dCas9 expression levels were comparable to those of other strains. This leads us to speculate that the lack of downregulation observed in PP_4677 may be due to the inefficiency of the sgRNA designed for PP_4677. This underscores the importance of designing more efficient sgRNAs for future experiments.

[0258] Further research revealed that, in addition to PP_1444, approximately 83 other genes were unexpectedly and significantly downregulated in strain PP_1444. Figure 4 Figure E; Table 4). Among them, proteins involved in the metabolism of gluconic acid to 2-ketogluconic acid and 6PG ( Figure 4 (Figure A) Enzymes such as Q88HH4, Q88HH5, and Q88HH6 (glucuronide 2-dehydrogenases, encoded by PP_3384, PP_3383, and PP_3382, respectively), Q88HI1 (ketoglucose-6-P-reductase, encoded by PP_3376), and Q88HH8 (2-ketoglucose epimerase, encoded by PP_3379) were downregulated. Phosphoglucose mutase (Q88GY7, encoded by PP_3578), an enzyme responsible for G6P glycogen biosynthesis, also appears to be downregulated. This may cause the G6P library to be diverted to the synthesis of 6PG. Figure 4 (See Figure A). Furthermore, the L-serine dehydratase (Q88P66, ​​encoded by PP_0987), involved in the conversion of L-serine to pyruvate, was also downregulated. This may preserve the L-serine library for the biosynthesis of shinorine. Interestingly, downregulating PP_0987 alone did not increase the shinorine titer. Figure 4 (Figure C). In addition to a large number of unexpected gene downregulations, 73 genes were found to be significantly upregulated in strain PP_1444. Figure 4 (Figure E, Table 5). For example, 5-methyltetrahydropteroyltriglutamate-homocysteine ​​methyltransferase (Q88JF1, encoded by PP_2698) was upregulated 28-fold. This enzyme is likely responsible for maintaining L-methionine synthesis for the biosynthesis of S-adenosyl-L-methionine (SAM), which is the methyl donor for O-methyltransferases.

[0259] Table 4. List of downregulated proteins.

[0260]

[0261]

[0262]

[0263]

[0264]

[0265] Table 5. List of upregulated proteins.

[0266]

[0267]

[0268]

[0269]

[0270] These results suggest that downregulation of PP_1444 not only reduces gluconic acid accumulation but also has profound implications for 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 role in shinorine production remains unclear. While some of these gene expression changes are consistent with our goal of increasing shinorine production by redirecting carbon flux and conserving key metabolites, other gene expression changes remain unexplained 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 promising pathways for further optimization and enhancement of shinorine and other valuable metabolites in biotechnological applications.

[0271] Optimize the pathway by improving promoter usage and altering the strength of the ribosome binding site.

[0272] Combined with CRISPRi-mediated gene downregulation methods, attempts have been made to enhance shinorine titers by improving the design of genetic constructs, including optimizing promoter use and altering the strength of ribosome binding sites. Previous studies have demonstrated the effectiveness of modulating RBS strength to enhance 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 involving biosynthetic pathways of multiple genes can be labor-intensive. To simplify this process, a modular adapter-based plasmid construction approach has been adopted (Storch et al., 2015) to facilitate the creation of plasmid constructs using various RBSs, thereby modulating the expression levels of proteins involved in the shinorine pathway. Figure 5 (Photo set A).

[0273] By using two different promoters (P BAD and P trc1-O ) and by recombining Shinorine BGC into one or two transcription units, along with variations in RBS, 21 different plasmids were successfully assembled. Figure 5 (Figure B). This combination method resulted in a significant increase in Shinorine production, reducing it from 100 mg / L within 72 hours post-inoculation. Figure 5 Figure C; JBx_250483 (indicated by the white bar) increased to approximately 467 mg / L Shinorine ( Figure 5 (Figure C; JBx_250497, indicated by the black bar).

[0274] Our shotgun proteomics analysis showed that manipulating promoters and RBS proved to be a viable strategy for enhancing the expression levels of proteins in the Shinorine pathway. Figure 5 (Figure D) Arabinose-inducible promoter P BAD It seems to be superior to constitutive P trc1-O Promoter. Replacing the original RBS with an exogenous RBS sequence, in addition to the existing O-MT RBS, significantly increased protein expression levels. By using exogenous RBS, the expression levels of DDGS, O-MT, ATP-dependent carboxylic acid-amine ligase, and NRPS in JBx_250497 were significantly increased, by 2.1-fold, 2.6-fold, 40.5-fold, and 6.7-fold, respectively, compared to the original strain (JBx_250483). Figure 5 (Photo set EH).

[0275] To identify the enzymes with the greatest impact on shinorine production, multiple linear regression analysis was performed. Ordinary least squares (OLS) regression results showed that the expression levels of O-MT and ATP-dependent carboxylic acid-amine ligases were statistically significant in predicting shinorine titers, while DDGS and NRPS appeared to have no significant effect in the model. This may mean that further increases in the expression levels of O-MT and ATP-dependent carboxylic acid-amine ligases could lead to a larger increase in shinorine titers. However, after a thorough exploration of the available permutations of RBS variants, it was found that optimizing the expression levels of these two enzymes beyond a certain point did not significantly improve shinorine production. Figure 16 Therefore, while O-MT and ATP-dependent carboxylic acid-amine ligases play a crucial role in predicting Shinorine titers, there may be other factors or pathways that need to be considered to further improve yield.

[0276] Supplementing with glycine and L-serine can further increase shinorine production.

[0277] After successfully implementing CRISPRi-mediated gene downregulation and genetic construct improvement, these two strategies were then combined to further increase shinorine production. The strain producing the highest shinorine yield (JBx_250497) was used as the base strain, and this strain was co-transformed with a CRISPRi plasmid containing PP_1444 sgRNA or a non-target sgRNA. Figure 6 (Figure A). The PP_1444 strain produced approximately 524 mg / L of Shinorine after 66 hours, while the control non-target strain produced approximately 365 mg / L of Shinorine. Figure 17 The glucose consumption curves showed that both samples completely consumed glucose after 42 hours. No significant growth or shinorine production was observed after 66 hours. These results suggest that glucose supply limited shinorine production under the test conditions.

[0278] To increase glucose supply, 20 g / L glucose was added at 18 hours. Following the additional glucose addition, strain PP_1444 produced approximately 723 mg / L of Shinorine at 66 hours. Figure 6 (Figure B). The shinorine titer reached a peak of 902 mg / L at 90 hours. To investigate whether another glucose supplementation would further increase the titer, an additional 20 g / L of glucose was added at 42 hours. Figure 6(Figure C). Although the shinorine titer did not reach its peak after 114 hours of fermentation, the highest shinorine titer reached at 114 hours was 900 mg / L. These results suggest that another factor may limit shinorine production.

[0279] The biosynthetic pathway of shinorine involves the incorporation of two amino acids, glycine and L-serine. Glycine and L-serine are incorporated into the shinorine molecule through specific enzymatic reactions. Figure 4 (Figure A). ATP-dependent carboxylic acid-amine ligases are key enzymes in the shinorine biosynthesis pathway, promoting the incorporation of glycine into 4-deoxycodone to form spore-glycine, a precursor of shinorine. Subsequently, an NRPS-like enzyme ligates a serine moiety to spore-glycine, resulting in shinorine formation. Considering the role of these amino acids in shinorine biosynthesis, it is hypothesized that their external supplementation may alleviate any metabolic bottlenecks caused by limited intracellular accessibility, thereby increasing the overall yield of shinorine.

[0280] To verify this hypothesis, a series of fermentation experiments were conducted on PP_1444 and non-target strains with the addition of glycine and L-serine. Figure 6 (Figure D). Supplementation with glycine and L-serine led to a significant increase in shinorine production in PP_1444 and non-target strains (comparison). Figure 6 (Figures E and B). For strain PP_1444, the Shinorine titer increased sharply in the first 66 hours, and then continued to increase steadily to 1134 mg / L after 114 hours. Figure 6 (Figure E). At 66 hours, approximately 1.7 g / L of glucose remained in the liquid culture. To further increase the Shinorine titer, an additional 20 g / L of glucose was added at 42 hours. Figure 6 (Figure F). Here, a sharp increase in Shinorine titer was observed within 114 hours. The highest Shinorine titer of 1601 mg / L was reached from strain PP_1444 at 114 hours, where the strain completely consumed glucose.

[0281] Shinorine was found only in the supernatant, while for strain PP_1444, 91% and 9% of shinorine were found in the supernatant and cell pellet, respectively. Figure 7 , Figure 18From a downstream biotechnology processing perspective, these findings increase the importance of *Pseudomonas putida* KT2440 as a microbial platform for shinorine production. When shinorine accumulates in liquid culture media rather than intracellularly, it offers several significant advantages for industrial biotechnology. The product in the supernatant can be harvested more easily because cell disruption is not required, which simplifies downstream processing and reduces costs (Ying Wang et al., 2019). It may also require less stringent purification steps, which could be advantageous from a cost perspective. Furthermore, if the product is secreted into the culture medium, the process can be adapted to continuous production systems where the product is continuously harvested while maintaining the culture. Accumulation of intracellular products typically leads to stress and eventual cell death. Secretion avoids this problem, maintains cell viability, and potentially increases the overall yield of shinorine.

[0282] Materials and methods

[0283] Strains, plasmids, culture media and growth conditions

[0284] All plasmids used in the study were amplified using *Escherichia coli* XL 1-Blue strain (Thermo Fisher Scientific). The strain was routinely cultured in lysogenic broth (LB) medium (LB broth, Sigma-Aldrich), 37°C, 180 rpm, supplemented with appropriate antibiotics (final concentrations: gentamicin 10 μg / mL and kanamycin 50 μg / mL). Plasmids were constructed using a modular plasmid assembly method, namely, modified Biopart assembly of standard idempotent clones (BASIC) (Storch et al., 2015).

[0285] Self-replicating plasmids (based on RK2 and RSF1010) were transformed into *Pseudomonas putida* KT2440 via electroporation. The electroporation procedure was a modification of (Choi et al., 2006). Briefly, a fresh *Pseudomonas putida* KT2440 colony was inoculated into 5 mL LB and incubated overnight at 30°C, 200 rpm. The overnight culture was centrifuged at 13000 xg for 1 min, 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 100 μL aliquots and electroporating using a Bio-Rad GenePulser II (USA) 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 fresh 1.5 mL microtube. To restore the cells, the cell mixture was incubated at 30°C and 200 rpm for 1 hour. After incubation, 20 μL of the cell mixture was plated on a selective agar plate containing an appropriate antibiotic and incubated overnight at 30°C.

[0286] Fluorescence measurement

[0287] For fluorescence measurements using a plate reader, an overnight liquid culture of *Pseudomonas putida* KT2440 grown in LB medium containing appropriate antibiotics (final concentrations: gentamicin 10 μg / mL and kanamycin 50 μg / mL) or other indicated components was diluted 1000-fold in LB medium. 200 μL of the liquid culture was cultured in 96-well plates, and RFP fluorescence was measured using a TecanInfinite F200 PRO instrument with excitation at 575 ± 10 nm and emission at 620 ± 10 nm, with continuous shaking for 24 hours unless measurements were performed. For flow cytometry measurements, the overnight culture (0.1% v / v) was inoculated into 2 mL of LB medium and supplemented with appropriate antibiotics. After 24 hours of incubation, 1–3 μL of sample was added to 150 μL of 1X phosphate-buffered saline. Single-cell RFP and GFP fluorescence from at least 30,000 cells were immediately recorded using a BD C6 Accuri flow cytometer (BD Bioscience). GFP and RFP fluorescence were measured using FL1 and FL4 detectors, respectively. Protein fluorescence levels were determined by averaging the fluorescence distributions.

[0288] Preparation of Shinorine Standards

[0289] At the initial OD 600A strain producing Shinorine was cultured in 250 mL of M9 medium in a 1-L flask with a concentration of 0.2 mL. Forty-eight hours after inoculation, the sample was centrifuged at 4500 × g for 30 minutes at 4 °C. To concentrate and desalt Shinorine 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 eluent was evaporated to dryness on a LabConco SpeedVac. The dried SPE eluent was then reconstituted in MilliQ water and semi-purified using an Agilent 1260 HPLC system equipped with a Macherry-Nagel Nucleosil 100-10 SB strong anion exchange column (250 mm x 4.6 mm, 10 μm particle size) at a flow rate of 1 mL / min using an isocratic mobile phase consisting of 25 mM LC-MS grade ammonium bicarbonate (Fisher Scientific). Fractions were manually collected by monitoring the absorbance of the Shinorine chromophore (λ = 334 nm). The observed Shinorine retention time was approximately 12.4 min. The collected fractions were then evaporated to dryness on a LabConco SpeedVac.

[0290] The dried fraction from the anion-exchange semi-purified fraction was reconstituted in water and injected into an Agilent 1260 HPLC system equipped with a Thermo HyperCarb column (150 mm x 4.6 mm, 5 μm particle size) at a rate of 1.5 mL / min using the following gradient (A = 0.3% ammonium formate, pH 9.0, B = acetonitrile): 0 min 2% B, 20 min 15% B, 26 min 50% B, 27–33 min 90% B, 35–40 min 2% B. The fraction was manually collected by monitoring the absorbance of the shinorine chromophore (λ = 334 nm). The observed shinorine retention time was approximately 9.2 min. The shinorine-containing fraction was then rapidly frozen in liquid nitrogen and placed on a LabConco lyophilizer. The dried fraction was reconstituted in water, frozen, and lyophilized three times to volatilize residual ammonium formate. The resulting purified shinorine dry solid was then used for characterization by NMR spectroscopy.

[0291] NMR spectrum of purified Shinorine

[0292] Approximately 4.6 mg of purified Shinorine was soluble in 400 μL of methanol-d4 containing 0.03% trimethylsilane (>99.8% atomic %D; Sigma-Aldrich). NMR spectroscopy was performed using a 5 mm... 1 Acquired on an H / BB iProbe using a Bruker Avance NEO 500 MHz. Samples were held at 298 K during acquisition. Standard Bruker pulse sequences were used for each of the following experiments: 1 H, 13 C, 1 H- 1 H COSY, 1 H- 1 H NOESY (750 ms mixing time) 1 H- 13 C HSQC and 1 H- 13 CHMBC. Spectra were recorded using Bruker TopSpin 4.0.6 software and analyzed using MestReNova 14.3.2. Chemical shifts (δ, ppm) were determined using trimethylsilane as an internal standard.

[0293] Conventional Shinorine Extraction and Analysis

[0294] For routine shinorine analysis from whole liquid cultures, 100 μL of liquid culture was mixed with 250 μL of methanol and 125 μL of chloroform. The resulting mixture was vortexed at 3000 rpm for 5 min. Next, 125 μL of ultrapure water and 100 μL of chloroform were added, and the sample was vortexed again at 3000 rpm for 5 min, followed by centrifugation at 13000 xg for 1 min. Shinorine was then sampled from the top aqueous layer and measured at 334 nm using a NanoDrop™ 2000 / 2000c spectrophotometer. To determine the concentration of shinorine in the sample, serially diluted purified shinorine standards were prepared and measured using Beer-Lambert's law and the extinction coefficient of ε = 44,700 Mn. −1 cm −1 Its concentration was determined. (Llewellyn et al., 2020; Wada et al., 2015).

[0295] To analyze extracellular shinorine, 100 μL of liquid culture was centrifuged at 13000 x g for 5 minutes, and shinorine was measured directly from the supernatant without extraction. To analyze intracellular shinorine, 100 μL of liquid culture was centrifuged at 13000 × g for 5 minutes. The supernatant was removed, and the cell pellet was washed three times with 500 μL of ultrapure water. Finally, following the extraction method described above for whole liquid cultures, the cell pellet was resuspended in 100 μL of ultrapure water and mixed with 250 μL of methanol and 125 μL of chloroform.

[0296] in conclusion

[0297] The growing interest in sustainable and environmentally friendly sunscreens has spurred scientists to produce shinorine, a natural compound with UV-absorbing properties, in microorganisms. Our research provides a comprehensive approach to the engineering of *Pseudomonas putida* KT2440 as a highly efficient chassis for shinorine production. A comprehensive review of metabolic flux distributions from various microorganisms identified *P. putida* KT2440 as a potential host for shinorine production. By utilizing synthetic biology methods and metabolic engineering strategies, shinorine production was significantly increased, with productivity CRISPRi-mediated gene downregulation (specifically targeting the PP_1444 gene) resulting in a substantial increase in shinorine yield compared to the first engineered strain. Proteomics analysis revealed that the downregulation of PP_1444 had a profound impact on the expression of multiple genes in *P. putida* KT2440. Improvements in gene design, promoter use, and ribosome binding sites also contributed to the increased titer. Feeding studies indicated that the supply of two key amino acids, L-serine and glycine, may be limiting factors in shinorine biosynthesis. Final titers, productivity, and yields of 900 mg / L or 10 mg / L / h or 22.5 mg / g glucose (without glycine and L-serine supplementation) and 1601 mg / L or 14 mg / L / h or 26–28.35 mg / g glucose (with glycine and L-serine supplementation), respectively, represent significant improvements over initial production levels and surpass achievements from earlier studies. Furthermore, the characteristic of Shinorine being completely secreted into the culture medium provides an advantage for downstream processing in industrial applications. These findings highlight the potential of *Pseudomonas putida* KT2440 as a microbial platform for the production of valuable natural compounds. Through continuous optimization and scale-up studies, our work paves the way for the commercialization of Shinorine as a bio-based alternative in the sunscreen industry.

[0298] While the invention has been described with reference to specific embodiments, those skilled in the art will understand that various changes can be made and equivalent substitutions can be used without departing from the true spirit and scope of the invention. Furthermore, many modifications can be made to adapt specific circumstances, materials, composition, processes, or one or more process steps to the purpose, spirit, and scope of the invention. All such modifications fall within the scope of the appended claims.

Claims

1. A genetically modified host cell capable of producing Porphyra-334 and / or Shinorine comprising (a) (i) a 2-demethyl 4-deoxyphloroglucinol synthase (DDGS), (ii) an O-methyltransferase (O-MT or OMT), (iii) an ATP-dependent carboxylate-amine ligase, and (iv) a 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 foregoing enzymes is a homolog thereof.

2. The genetically modified host cell of claim 1, wherein the 2-demethyl 4-deoxyphloroglucinol synthase (DDGS) is a cyanobacterial DDGS or a homolog thereof.

3. The genetically modified host cell of claim 1, wherein the O-methyltransferase (O-MT or OMT) is a cyanobacterial OMT or a homolog thereof.

4. The genetically modified host cell of claim 1, wherein the ATP-dependent carboxylate-amine ligase is a cyanobacterial ATP-dependent carboxylate-amine ligase or a homolog thereof.

5. The genetically modified host cell of 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 homolog thereof.

6. The genetically modified host cell of claim 1, wherein MysA, MysB, MysC, MysD and / or MysE are independently derived from or obtained from Anabaena ATCC 29413, Nostoc ATCC 29133, Porphyra purpurea and / or Irish moss. The cyanobacterial MysD or D-ala-ala ligase has relaxed substrate specificity in which a threonine is condensed onto sporamin-glycine in place of serine to produce Porphyra-334.

7. The genetically modified host cell of claim 1, wherein the DDGS, OMT, ATP-dependent carboxylate-amine ligase and / or non-ribosomal peptide synthetase (NRPS) or D-ala-D-ala ligase are each independently obtained from or derived from Porphyra purpurea, Nostoc commune and / or Anabaena variabilis.

8. The genetically modified host cell of claim 1, wherein the host cell is genetically modified to be capable of metabolizing one or more compounds that the unmodified host cell is not otherwise capable of metabolizing.

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

10. The genetically modified host cell of claim 8, wherein the one or more compounds are aromatic compounds obtained from lignocellulosic hydrolysate.

11. The genetically modified host cell of 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 of claim 1, wherein the MysA and MysB form a fusion protein.

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

6.

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

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

7.

16. A method of producing Porphyra-334 and / or Shinorine, comprising: (a) providing a genetically modified host cell comprising: (1) (i) 2-demethyl 4-deoxymycothiazole synthase (DDGS), (ii) O-methyltransferase (O-MT or OMT), (iii) ATP-dependent carboxylate-amine ligase, and (iv) non-ribosomal peptide synthetase (NRPS) or D-ala-D-ala ligase, MysD or MysE; or (2) MysA, MysB, MysC or MysD, MysE or NRPS; and (b) culturing or growing the genetically modified host cell in a suitable culture or medium to produce Porphyra-334 and / or Shinorine.

17. The method of claim 16, comprising (c) extracting or isolating Porphyra-334 and / or Shinorine from the host cell and / or culture or medium to form isolated or purified Porphyra-334 and / or Shinorine.

18. The method of claim 16, comprising (d) mixing Porphyra-334 and / or Shinorine with an emulsion, oil or water to form an ultraviolet (UV) blocking or filtering composition.

19. The method of claim 18, wherein the mixing step further comprises mixing Porphyra-334 and / or Shinorine or the ultraviolet blocking or filtering composition with another active UV filter.

20. The method of claim 18, wherein the active UV filter is oxybenzone, octinoxate, homosalate, avobenzone, zinc oxide and / or titanium dioxide or mixtures thereof.

21. The method of claim 18, wherein the ultraviolet blocking or filtering composition is suitable for use on human skin.

22. The method of claim 18, wherein the ultraviolet blocking or filtering composition is free of ingredients or components that cause coral bleaching.

23. The method of claim 18, wherein the culturing or growing step (b) occurs in a batch process or a fed-batch process.

24. The method of claim 18, wherein the culture or medium comprises a biomass.

25. The method of claim 24, wherein the biomass is a lignocellulosic biomass or a hydrolysate thereof.

26. The method of claim 25, wherein the biomass is from softwood feedstock (e.g., poplar), hardwood feedstock, grass feedstock, and / or agricultural feedstock or mixtures thereof.

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

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