EP4689143A2 - Genetically modified host cells and methods useful for producing porphyra-334 and/or shinorine - Google Patents

Abstract

The present invention provides for a genetically modified host cell capable of producing porphyra-334 and/or shinorine comprising (a) (i) 2-demethyl 4-deoxygadusol synthase (DDGS), (ii) (O-m ethyl transferase (O-MT or OMT), (iii) ATP-grasp ligase, and (iv) nonribosomal peptides 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 preceding enzymes is a homologous enzyme thereof.

Description

Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory Genetically modified host cells and methods useful for producing porphyra-334 and/or shinorine Inventors: Ian Sofian Yunus, Taek Soon Lee CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the priority benefit of U.S. Provisional Application Nos. 63/493,636, filed March 31, 2023, which is hereby incorporated by reference in its entirety. STATEMENT OF GOVERNMENTAL SUPPORT [0002] The invention was made with government support under Contract No. DE-AC02- 05CH11231 awarded by the U.S. Department of Energy. The government has certain rights in the invention. FIELD OF THE INVENTION [0003] The present invention is in the field of producing porphyra-334 and/or shinorine. REFERENCE TO SEQUENCE LISTING [0004] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on 28 March 2024, is named “2021-119-02 Sequence Listing.xml” and is 16 kilobytes in size. BACKGROUND OF THE INVENTION [0005] Extended warm periods (Oliver et al., 2018), global warming (Johnson et al., 2022), and excessive use of chemical sunscreens (Roberto et al., 2008) have been linked to world- wide coral bleaching. Due to concerns about their adverse effects on both human health and the environment, several countries and regions have prohibited the use of commercial sunscreens containing oxybenzone, octinoxate, ZnO, and TiO2. This has led to a rising demand for sunscreens derived from environmentally friendly sources. Shinorine, a mycosporine-like amino acid (MAA) typically produced by red algae Porphyra umbilicalis (Figure 19), has been used as an active ingredient in commercial sunscreen products (e.g., Helioguard^TM 365, Helionori^®). Shinorine has also demonstrated additional benefits including Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory anti-aging properties, antioxidant effects, promotion of wound healing, and inhibition of UV radiation-induced skin inflammation (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). The yield of shinorine from P. umbilicalis, however, is relatively low (3.27 mg/g cell dry weight) (Becker et al., 2016), and its production suffers from the slow growth of P. umbilicalis. With the continuous rise in global demand for sunscreen, the bioproduction of shinorine in a fast-growing microbe emerges as an attractive solution. [0006] To meet the growing demand of 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), Synechocystis sp. PCC 6803 (Yang et al., 2018), Streptomyces avermitilis (Miyamoto et al., 2014), and recently Yarrowia lipolytica (Jin et al., 2023), have been genetically engineered for shinorine production. Most metabolic engineering efforts within the aforementioned host organisms, however, have primarily focused on coupling the shinorine production pathway with the xylose utilization pathway to enhance the xylulose 5-phosphate pool (Jin et al., 2023; Kim et al., 2023, 2022; Park et al., 2019). While this approach has been proven to improve the shinorine production titer, challenges persist. First, the highest productivity (12.75 mg/L/h) and yield (3.66 mg/g glucose; assuming total consumption of 420 g glucose and 160 g xylose) were achieved in Saccharomyces cerevisiae in a fed-batch fermenter (Kim et al., 2023). Second, supplying sugar mixtures (e.g., hexoses and pentoses) into cultures has also introduced complexities to production experiments (Park et al., 2019), as microbial hosts typically exhibit a preference for hexose (e.g., glucose) consumption over pentose (e.g., xylose) (Aidelberg et al., 2014; Dvořák and de Lorenzo, 2018; Wang et al., 2019). Third, a large portion of shinorine produced from Saccharomyces cerevisiae and Yarrowia lipolytica tends to accumulate within the cells (Jin et al., 2023; Kim et al., 2023), complicating extraction and purification. [0007] Currently, shinorine is produced from red algae (3.2 mg/g CDW) and cyanobacteria (2.37 mg/g CDW). Park et al. disclose introducing shinorine biosynthetic genes from cyanobacteria Nostoc punctiforme into Saccharomyces cerevisiae to construct a yeast strain that produces 31.0 mg/L of shinorine in an optimized medium containing 8 g/L of xylose and 12 g/L of glucose (ACS Synthetic Biol.8:346-357, 2019). [0008] Hahn et al. (PCT International Patent Application WO 2021/133101) disclose an Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory engineered Saccharomyces cerevisiae that produces shinorine using xylose as a carbon source. [0009] Kotagiri et al. (U.S. Patent Application Pub. No.2022/0275411) disclose a genetically modified strain of commensal bacteria Staphylococcus epidermis which contains a plasmid containing four Anabaena variabilis genes to produce a mycosporine-like amino acid (MAA). SUMMARY OF THE INVENTION [0010] The present invention provides for 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-grasp ligase, and (iv) nonribosomal peptides 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 preceding enzymes is a homologous enzyme thereof. [0011] In some embodiments, the 2-demethyl 4-deoxygadusol synthase (DDGS) is a cyanobacteria DDGS, or homologous enzyme thereof. In some embodiments, the O- methyltransferase (O-MT or OMT) is a cyanobacteria OMT, or homologous enzyme thereof. In some embodiments, the ATP-grasp ligase is a cyanobacteria ATP-grasp ligase, or homologous enzyme thereof. In some embodiments, the nonribosomal peptides synthetase (NRPS) or D-ala-D-ala ligase is a cyanobacteria nonribosomal peptides synthetase (NRPS) or D-ala-D-ala ligase, or homologous enzyme thereof. In some embodiments, the MysA, MysB, MysC, MysD, and/or MysE are independently derived or obtained from Anabaena ATCC 29413, Nostoc ATCC 29133, Porphyra umbilicalis, and/or Chondrus crispus. Cyanobacterial MysD or D-ala-ala ligase have a relaxed substrate specificity, with condensation of threonine instead of serine onto mycosporine-glycine to yield porphyra-334. In some embodiments, one or more, or all, of the enzymes are a wild-type enzyme. [0012] In some embodiments, the DDGS, OMT, ATP-grasp ligase, and/or nonribosomal peptides synthetase (NRPS) or D-ala-D-ala ligase are each independently obtained or derived from Porphyra umbilicalis, Nostoc punctiforme, and/or Anabaena variabilis. [0013] The amino acid sequence of Nostoc punctiforme or Anabaena variabilis DDGS is as follows: Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory MSNVQASFEATEAEFRVEGYEKIEFSLVYVNGAFDISNREIADSYEKFGRCLTVIDANVNRL YGKQIKSYFRHYGIDLTVVPIVITEPTKTLATFEKIVDAFSDFGLIRKEPVLVVGGGLTTDV AGFACAAYRRKSNYIRVPTTLIGLIDAGVAIKVAVNHRKLKNRLGAYHAPLKVILDFSFLQT LPTAQVRNGMAELVKIAVVANSEVFELLYEYGEELLSTHFGYVNGTKELKAIAHKLNYEAIK TMLELETPNLHELDLDRVIAYGHTWSPTLELAPMIPLFHGHAVNIDMALSATIAARRGYITS GERDRILSLMSRIGLSIDHPLLDGDLLWYATQSISLTRDGKQRAAMPKPIGECFFVNDFTRE ELDAALAEHKRLCATYPRGGDGIDAYIETQEESKLLGV (SEQ ID NO:1) [0014] The amino acid sequence of Nostoc punctiforme or Anabaena variabilis OMT is as follows: MTSILGRDTARPI TPHSILVAQLQKT LRMAEESNIPSEI LTSLRQGLQLAAG LDPYLDDCTTPES TALTALAQKTSIE DWSKRFSDGETVR QLEQEMLSGHLEG QTLKMFVHITKAK SILEVGMFTGYSA LAMAEALPDDGRL IACEVDSYVAEFA QTCFQESPHGRKI VVEVAPALETLHK LVAKKESFDLIFI DADKKEYIEYFQI ILDSHLLAPDGLI CVDNTLLQGQVYL PSEQRTANGEAIA QFNRIVAADPRVE QVLLPIRDGITLI RRLV (SEQ ID NO:2) [0015] The amino acid sequence of Nostoc grasp ligase is as follows: MAGSISLSLPQST TPSKGVRLKIAAL LKTIGTLILLLIA LPLNALIVLISLM CRPFTKKPAVATH PQNILVSGGKMTK ALQLARSFHAAGH RVILIEGHKYWLS GHRFSNSVSRFYT VPAPQDDPEGYTQ ALLEIVKREKIDV YVPVCSPVASYYD SLAKSALSEYCEV FHFDADITKMLDD KFAFTDRARSLGL SAPKSFKITDPEQ VINFDFSKETRKY ILKSISYDSVRRL NLTKLPCDTPEET AAFVKSLPISPEK PWIMQEFIPGKEL CTHSTVRDGELRL HCCSNSSAFQINY ENVENPQIQEWVQ HFVKSLRLTGQIS LDFIQAEDGTAYA IECNPRTHSAITM FYNHPGVAEAYLG KTPLAAPLEPLAD SKPTYWIYHEIWR LTGIRSGQQLQTW FGRLVRGTDAIYR LDDPIPFLTLHHW QITLLLLQNLQRL KGWVKIDFNIGKL VELGGDYSRSEE (SEQ ID NO:3) [0016] The amino acid sequence of Anabaena variabilis NRPS is as follows: MQTIDENIRKLLVEWNAT HRDYDLSQSLHELIVAQV ERTPEAIAVTFDKQQLTV QELNHKANQLGHYLQTLG VQPETLVGVCLERSLEMV ICLLGILEAGGAYVPIDP Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory EYPQERIAYMLEDSQVEV LLTQEKLLNQIPHHQAQT ICVDREWEKISTQANTNP KSNIKTDNLAYVIYTSGS TGKPKGAMNTHKGICNRL LWMQEAYQIDSTDSILQK TPFSFDVSVWEFFWTLLT GARLVIAKPGGHKDSAYL IDLITQEQITTLHFVPSM LQVFLQNRHVSKCSSLKR VICSGEALSIDLQNRFFQ HLQCELHNLYGPTEAAID VTFWQCRKDSNLKSVPIG RPIANTQIYILDADLQPV NIGVTGEIYIGGVGVARG YLNKEELTKEKFIINPFP NSEFKRLYKTGDLARYLP DGNIEYLGRTDYQVKIRG YRIEIGEIENVLSSHPQV REAVVIARDDNAQEKQII AYITYNSIKPQLDNLRDF LKARLPDFMIPAAFVMLE HLPLTPSGKVDRKALPKP DLFNYSEHNSYVAPRNEV DLFNYSEHNSYVAPRNEV EEKLVQIWSNILHLPKVG VTENFFAIGGNSLKALHL ISQIEELFAKEISLATLL TNPVIADLAKVIQANNQI HNSPLVPIQPQGKQQPFF CIHPAGGHVLCYFKLAQY IGTDQPFYGLQAQGFYGD EAPLTRVEDMASLYVKTI REFQPQGPYRVGGWSFGG VVAYEVAQQLHRQGQEVS ILAILDSYVPILLDKQKP IDDVYLVGVLSRVFGGMF GQDNLVTPEEIENLTVEE KINYIIDKARSARIFPPG VERQNNRRILDVLVGTLK ATYSYIRQPYPGKVTVFR AREKHIMAPDPTLVWVEL FSVMAAQEIKIIDVPGNH YSFVLEPHVQVLAQRLQD CLENNS (SEQ ID NO:4) [0017] The amino acid sequence of Nostoc punctiforme D-ala-D-ala ligase is as follows: MPVLNILHLVGSA HDKFYCDLSRLYA QDCLAATADPSLY NFQIAYITPDRQW RFPDSLSREDIAL TGQNIDMMLPQMF CIPGMTQYRALFD LLKIPYIGNTPDI MAIAAHKARAKAI VEAAGVKVPRGEL LRQGDIPTITPPA VVKPVSSDNSLGV VLVKDVTEYDAAL KKAFEYASEVIVE AFIELGREVRCGI IVKDGELIGLPLE EYLVDPHDKPIRN YADKLQQTDDGDL DPNDPITQKVQQV AKRCHQALGCRHY SLFDFRIDPKGQP WFLEAGLYCSFAP KSVISSMAKAAGI PLNDLLITAINET LGSNKKVLQN (SEQ ID NO:5) [0018] In some embodiments, the host cell is genetically modified to be capable of metabolizing one or more compounds that the unmodified host cell is incapable of metabolizing in nature. In some embodiments, the host cell is a Pseudomonas host cell. In some embodiments, the one or more compounds is an aromatic compound obtained from lignocellulosic hydrolysate. Examples of such aromatic compounds are ferulic acid, 4- Hydroxybenzoic acid, protocatechuic acid, vanillic acid, salicylic acid, syringic acid, p- Coumaric acid, vanillin, catechol, syringaldehyde, and phenol. Examples of suitable genetic modifications to enable the host cell to metabolize such aromatic compounds are taught in the following: overexpression of eruloyl-CoA synthetase (Fcs), enoyl-CoA hydratase/aldolase Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory (Ech), vanillin dehydrogenase (Vdh), p-hydroxybenzoate hydroxylase (PobA), and vanillic acid O-demethylase oxygenase (VanAB). [0019] In some embodiments, the MysA and MysB form a fusion protein. In some embodiments, the MysC and MysD form a fusion protein. [0020] An amino acid sequence of Porphyra umbilicalis MysA-MysB fusion is as follows: (MysA-MysB fusion >tr|A0A1X6NP84|A0A1X6NP84_PORUM 3-dehydroquinate synthase domain-containing protein OS=Porphyra umbilicalis OX=2786 GN=BU14_0783s0002 PE=3 SV=1) MHITLDKSAGVQVVQPQHFAAFVTDVKEAGSVVPDGDAKTAVETVLALPALSTFLLRCSR ADVFDGSVAADFSSNTKDLCMLRSLKQLKYFYNLPAGQLMQLLSGVVASDDVVAAKPWTA LAERVFADEELCTMLIKHLIIGKTARESLKDIYEENAQYLLENDPHALYPTSIYRQGNGH VTSSDDSSRIEGVMSTTITTSIEIVRDVLNPKNHQLRDMYKPLKRCVAVVDERLDKLYGQ KLCDYFVSHDIPFEKMSYRCMEVDKDISTVEKMLVDLKGRRVSRNEPVLIVGGGVMADVG GFACALYHRNTPYVMLCTSIVSGIDAGPSPRTCCDGLGYKNVFGAYHPPVLTLTDRYFFR TLEKGWIRHGIAEIIKMAVTKDHTLFEALEEAGQKLVDTKFGIEGVEAGSRFDTLSESII AKAMDGYVRSEYGNLWETHQCRPHAYGHTWSPGFELQAGLLHGHAVSIGMGYGAYLSFLE GWISVTDFHRILNVISTVGLSLVHPILDNPNSLWQSQVKMTEKRGGNLCAPLPRGSIGKC GYLNDLTRARLEATLIDYKELCKAYPRGGAGIEPHCRDVGLEDPSTVKQHADHAVHAIAT IGPAASDEVPEANGNGNGVEANGVEANGNGNSNVYNDWIAKQQVSRNQGATSSSLMASMV QIQAKDTEAPPAFPHTTLFHEGGEEYAQAMTTAASADFVNIAKETVAADLFAPCMVGAIE GQFLKMMAGMTKSKRVLDIGTFTGYSALAFAGGVPADGEVVTIEADKKCADVAQKCFDAS ADGSKIKLVRGDAMKVVSEMADSGEKFDIVFLDADKVNYAHYYEAGLKMLAEGGLIMADN ALCSLVYADDDPVRLALHAFAQKVRADTRVEQVMLTVREGILLVRRV (SEQ ID NO:6) [0021] An amino acid sequence of Porphyra umbilicalis MysC-MysD fusion is as follows: (MysC-MysD fusion >tr|A0A1X6NNV6|A0A1X6NNV6_PORUM ATP-grasp domain-containing protein OS=Porphyra umbilicalis OX=2786 GN=BU14_0783s0001 PE=4 SV=1) MASPAIPPANCNLVQIVLRLLDAFFWSLLVALRGALRMATRPLAGVPHGVAAGKTVLVTT GRQAKTVHVIRALKAVGATVVVTDYDHVSASAVSTACDHFVVLPSLDVNALDAWVDAFGK LLVEYAVDMVVPVSTINEALFLGVAKDRLSPKLPHVEWLCTGLDNVLRLDDRERFSATCR QYGVPAPASGLVTRREQVPHSASQPHGIIVKRLESSVNRDEEIVPVAPSDPLPDFVKPSP TDAWQWQQMIRGAEYSVWYVCVNGKVTFSACYLSQPDLVHFDHVPTPADVDKPLRDLIAG MHLSGQYAFDFIRDQDSGVPYVIECNPRASSILETVSCTPLWGESFFGIDVSARCRTQDV GFVFHGNCWPWTARTEGYLCLSDPLPFFAAEIAWPLHAIQTSGMSEAGYRKIDVNICKII IDGPSAPRSIGTFENAAQGAKLAVLDRALQYVETIYIDAAVPNVGAVVAVARKASCHPVL FQLGAQAGLLSGVSNSLYADLPPIQFVASPEELSGLVAASEAVAPTRLLTGDAASVALGF DSELTLLAPRARATPYTYYRIPLRRLKVLHVMGSCTSKYYETVSSYYGFDCISSVTDDVR YEHVIGYVHLTGEWSVAVGKDEVYMREKAPRLSLAQALAAIESLAIDLMIPHMFDYAGLT AYRSVFSMLDLPTIGCSGEALALSTNKARTKACAAMDGVQVPKSQLLRRGDTHSMPFPIV IKPTEEDNSMGVEVVHNEAQVAAALESAFQFGDEVMVEQYIPLGRELRVAVIETPDGGME MLPVVEYFLPAEKPIRKSGDKITTDSSGQPSGYAPVDRKVPADIDPVLHDKLYKLAVVSH RATDCADYSIYDVRVDPDGEPYMLESCLYCSFSFRSVLVLMADAAGKKHPQLFVEFSERS VGRKSAAIAAQAHGKKAPQEFGMKVRR (SEQ ID NO:7) [0022] Heterologous expression of the MAA biosynthetic genes from Actinosynnema mirum DSM 43827 in an engineered host, Streptomyces avermitilis SUKA22, resulted in the production of shinorine as a main product, as well as porphyra-334 (Miyamoto et al., Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory “Discovery of Gene Cluster for Mycosporine-Like Amino Acid Biosynthesis from Actinomycetales Microorganisms and Production Actinomycetales Microorganisms and Production of a Novel Mycosporine-Like Biosynthesis from Actinomycetales Microorganisms and Production of a Novel Mycosporine-Like Amino Acid by Heterologous Expression,” Appl. Env. Microbiol.80(16):5028-5038, 2014). [0023] Shinorine, a mycosporine-like amino acid (MAA) commonly produced by red algae Porphyra umbilicalis, has been used as an active ingredient in two commercial sunscreen products (Helioguard 365 and Helionori). However, the yield of shinorine from P. umbilicalis is relatively low (3.25 mg/g CDW) and its production suffers from the slow growth of P. umbilicalis. To overcome this issue, several organisms, such as E. coli, Saccharomyces cerevisiae, Synechocystis sp. PCC 6803, Corynbacterium glutamicum, and Streptomyces avermitilis SUKA22 have been metabolically engineered for shinorine production. The present invention provides for a novel process to produce shinorine as opposed to the previously described methods by metabolically engineering a host cell, such as a Pseudomonas species, such as Pseudomonas putida KT2440, which is capable of utilizing various carbon sources, including aromatic compounds from lignocellulose hydrolysate as feedstocks, to produce shinorine. [0024] The following is a theoretical yield calculation for the production of shinorine using glucose as precursor. Resulting from the formal condensation of the amino group of L-serine with the keto group of (5S)-5-hydroxy-5-(hydroxymethyl)-2-methoxycyclohex-2-en-1-one and in which the hydrogen at position 3 of the cyclohexenone moiety has been replaced by the amino group of glycine. See Fig.3, Panel A. The indicated compounds have the following chemical formula and molecular weight: Shinorine: C₁₃H₂₀N₂O₈ (332.3); Glucose: C6H12O6 (180). The final equation is as follows (assuming 1 glucose → 1 serine, 1 glucose → 1 glycine): [0025] 1. Hydrocarbon core Carbon balance: 4/3 C6H12O6 + 2 H2 → C8H12O4 + 4 H2O Redox balance: C6H12O6 + 6 H2O → 6 CO2 + 12 H2 ⇒ 9 C6H12O6 → 6 C8H12O4 + 6 CO2 + 18 H2O Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory ⇒ 1.5 Glucose → C₈H₁₂O₄ + CO₂ + 3 H₂O [0026] 2. As 1 shinorine needs 1 serine or 1 glycine, and each serine or glycine needs 1 glucose. 1 glucose → 1 serine 1 glucose → 1 serine → 1 glycine [0027] The final equation is: 3.5 C6H12O6 → 1 Shinorine (C13H20N2O8). Therefore, theoretical yield is 0.52746 g Shinorine / g glucose ⇒ about 0.53 g / g glucose. [0028] In some embodiments, the DDGS, OMT, ATP-grasp ligase, and/or nonribosomal peptides synthetase (NRPS) or D-ala-D-ala ligase are homologous, variant, or mutant enzymes thereof, of any wild-type enzymes described herein. The homologous, variant, or mutant enzyme has the same enzymatic activity of its corresponding wild-type enzyme, and has an amino acid sequence having equal to or more than 70%, 80%, 90%, 95%, or 99% sequence identity of the corresponding wild-type enzyme, and optionally has one or more conserved amino acid sequence(s) and/or residue(s), such as conserved sequence(s) and/or residue(s) important for a structure or catalytic residue/pocket of the enzyme. In some embodiments, the wild-type DDGS, OMT, ATP-grasp ligase, and/or nonribosomal peptides synthetase (NRPS) or D-ala-D-ala ligase, are obtained or derived from Porphyra umbilicalis, Nostoc punctiforme, Anabaena variabilis. [0029] The present invention provides for a method of producing porphyra-334 and/or shinorine with a much faster manner and with a higher product titer and yield. Additionally, by utilizing lignocellulose hydrolysate as the feedstock, one can eliminate the competition of using agriculture plants as feedstocks as commonly used by other similar technologies (e.g., production of porphyra-334 and/or shinorine in E. coli, yeast, etc) which results in a lower cost. [0030] The present invention provides for a method for producing porphyra-334 and/or shinorine comprising: (a) providing a genetically modified host cell of the present invention, (b) culturing or growing the genetically modified host cell in a suitable culture or medium such that porphyra-334 and/or shinorine is produced, (c) optionally extracting or separating the porphyra-334 and/or shinorine from the host cells, and/or culture or medium to form an Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory isolated or purified porphyra-334 and/or shinorine, and (d) optionally mixing the porphyra- 334 and/or shinorine with a lotion, oil or water to form a ultra-violet light (UV) blocking or filtering composition. [0031] In some embodiments, the mixing step further comprises mixing the porphyra- 334 and/or shinorine or the UV blocking or filtering composition with another active UV filtering agent, such as oxybenzone, octinoxate, octisalate and avobenzone, zinc oxide, and/or titanium dioxide, or a mixture thereof. [0032] In some embodiments, the UV blocking or filtering composition is suitable for application on human skin, such as as a suntan lotion. In some embodiments, the UV blocking or filtering composition does not contain an ingredient or component that causes coral bleaching. [0033] In some embodiments, the providing step (a) comprises introducing one or more nucleic acid(s) encoding the (i) 2-demethyl 4-deoxygadusol synthase (DDGS), (ii) O- methyltransferase (O-MT or OMT), (iii) ATP-grasp ligase, and (iv) nonribosomal peptides synthetase (NRPS) or D-ala-D-ala ligase enzymes, wherein each enzyme is operatively linked to a promoter capable of expressing each enzyme in the host cell into the host cell. In some embodiments, the culturing or growing step (b) comprises the host cell growing by respiratory cell growth. In some embodiments, the culturing or growing step (b) takes place in a batch process or a fed-batch process, such as a high-gravity fed-batch process. [0034] In some embodiments, the culture comprises a biomass, such as a lignocellulosic biomass, or hydrolysate thereof. In some embodiments, the biomass is obtained from softwood feedstock (such as poplar), hardwood feedstock, grass feedstock, and/or agricultural feedstock, or mixture thereof. [0035] In some embodiments, the culture or medium comprises a rich medium, such as LB (Lysogeny-Broth) or comprising one or more ingredients of LB, such as tryptone and/or yeast extract. In some embodiments, the culture or medium comprises hydrolysates derived or obtained from a biomass, such as a lignocellulosic biomass. In some embodiments, the culture or medium comprises one or more aromatic compound(s), such as aromatic compounds obtained from a lignocellulosic hydrolysate. In some embodiments, the culture or medium comprises one or more carbon sources, such as a sugar, such as glucose, xylose, or galactose, or glycerol, or a mixture thereof. In some embodiments, the carbon source is Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory fermentable. In some embodiments, the carbon source is non-fermentable. In some embodiments, the culture or medium comprises urea as a nitrogen source. In some embodiments, the culture or medium comprises an amino acid, such as serine and/or glycine. In some embodiments, the culture or medium comprises an ionic liquid (IL). [0036] In some embodiments, the genetically modified host cell is natively or is engineered or constructed to utilize xylose as a carbon source. An increased xylose utilization results in an increase in the X5P pool used in the porphyra-334 and/or shinorine biosynthetic pathway. [0037] In some embodiments, the genetically modified host cell is natively or is engineered or constructed to increase S7P production, such as via the isomerase pathway. In some embodiments, the genetically modified host cell is natively or is engineered or constructed to decrease, or knock out, a native or endogenous PP_1024 encoding KDPG aldolase, or any gene encoding KDPG aldolase, in order to downregulate or to minimize the conversion of 2KDPG to pyruvate. In some embodiments, such a downregulation results in growth defect which is compensated by feeding the culture with aromatics that ended up in the acetyl-CoA pool. In some embodiments, this co-utilization strategy allows conversion of glucose to G6P, xylose to X5P, and aromatics to acetyl-CoA, which improves the porphyra-334 and/or shinorine titer. [0038] In some embodiments, the method results in the genetically modified host cell producing equal to or more 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 of porphyra-334 and/or shinorine. In some embodiments, the method results in the genetically modified host cell producing equal to or more 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 of porphyra-334 and/or shinorine. [0039] In some embodiments, the invention comprises the use of a heterologous codon- optimized version of the nucleic acid encoding the enzyme(s) described herein which are optimized to the host cell. BRIEF DESCRIPTION OF THE DRAWINGS [0040] The foregoing aspects and others will be readily appreciated by the skilled artisan Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory from the following description of illustrative embodiments when read in conjunction with the accompanying drawings. [0041] Figure 1. Metabolic flux distribution of glucose-fed Pseudomonas putida KT2440. All values in boxes are given as relative fluxes normalized to the specific glucose uptake rate. Data are referenced from three primary studies: (Kukurugya et al., 2019) (top), (Nikel et al., 2015) (middle), and (Kohlstedt and Wittmann, 2019) (bottom). Arrow widths are proportional to the average of the three values. Abbreviations: G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; FBP, fructose-1,6-biphosphate; DHAP, dihydroxyacetone phosphate; 6PG, 6-phosphogluconate; KDPG, 2-keto-3-deoxy-6-phosphogluconate; Ri5P, ribulose 5-phosphate; R5P, ribose 5-phosphate; X5P, xylulose 5-phosphate; S7P, sedoheptulose 7-phosphate; E4P, erythrose 4-phosphate; G3P, glyceraldehyde 3-phosphate; 3PG, 3-phosphoglycerate; PEP, phosphoenolpyruvate; Ac-CoA, acetyl-coenzyme A; L-ser, L-serine; TCA, tricarboxylic acid; PYR, pyruvate. [0042] Figure 2. Development of genetic tools for gene expression in Pseudomonas putida KT2440. (A) Modular cloning assembly method as described in a previously published work (Storch et al., 2015). Genetic parts were cloned into a pJET1.2 blunt DNA plasmid. Subsequently, these parts were digested with BsaI restriction enzyme and ligated with prefix and suffix linkers, followed by separation and purification using magnetic beads. Finally, purified genetic parts and linkers were assembled in one-pot reaction at 50 °C for 1 hr. (B) Schematic diagrams of plasmids with different backbones. RFP fluorescence levels measured across various plasmid backbones (C), under different inducible promoters (D), constitutive promoters (E), and ribosome-binding site sequences (F). Cultures were grown in M9 minimal media. RFP fluorescence was measured using a flow cytometer at 48 hr post-inoculation. Inducer was added at 0 hr. Error bars represent the standard deviation of three biological replicates. [0043] Figure 3. Production experiment of shinorine in engineered P. putida KT2440 and confirmation by nuclear magnetic resonance (NMR) spectroscopy. (A) Schematic diagram of P. putida KT2440 central carbon metabolism and shinorine biosynthetic pathway. The initial biosynthetic step involves the synthesis of desmethyl 4-deoxygadusol (DDG) through sedoheptulose 7-phosphate (Balskus and Walsh, 2010; Pope et al., 2015). Desmethyl-4- deoxygadusol synthase (DDGS) catalyzes the production of DDG. Subsequently, O- methyltransferase (O-MT) transforms DDG to 4-deoxygadusol (4-DG).4-DG incorporates Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory glycine through an ATG-grasp ligase to yield mycosporine-glycine (MG). In the final step, MG is converted to shinorine by a non-ribosomal peptide synthetase (NRPS)-like enzyme (Balskus and Walsh, 2010; Portwich and Garcia-Pichel, 2003), which encompasses adenylation, thiolation, and thioesterase domains. The adenylation domain plays a crucial role in attaching serine to the C1 position of mycosporine-glycine, generating shinorine. (B) Schematic diagram of plasmid pIY456 used for production experiments in Fig.3 (Panel C). (C) Production of shinorine from P. putida KT2440 strains. Chromatogram obtained from P. putida (D) and extracted Helioguard^TM (E). (F) Assigned ¹H and ¹³C resonances for purified shinorine. (G) Correlations (¹H-¹H NOESY and ¹H-¹³C HMBC) used to unambiguously assign all resonances for purified shinorine. P. putida KT2440 were grown in M9 medium and samples were extracted at 48 hr. Error bars represent standard deviations from three biological replicates. SAM, S-adenosyl-L-methionine; SAH, S-adenosylhomocysteine. Other abbreviations are shown in Figure 1 caption. [0044] Figure 4. CRISPRi gene downregulation for improved shinorine production. (A) Schematic diagram of genes involved in 6PG, G3P, L-serine, glycine, and S-adenosyl-L- methionine (SAM) metabolism. (B) Schematic diagram of plasmids used for expression of shinorine biosynthetic genes and CRISPRi-mediated gene downregulation. (C) Shinorine titer produced by different strains targeting 21 genes. (D) Relative expression levels (y-axis; x10⁶) of target genes in the control vs sample. (E) Volcano plot depicting top 20 downregulated (purple) and upregulated (navy) genes in the PP_1444 strain. Error bars represent standard deviation from three biological replicates. [0045] Figure 5. Improvement of shinorine production by RBS optimization. (A) Illustration of combinatorial modular plasmid assembly consisting of promoters and genes assembled to different RBS. Two types of promoters and three different RBS elements were used. (B) Schematic diagram of twenty-one different shinorine-producing plasmids. (C) Shinorine titers from strains carrying plasmid shown in Fig.5 (Panel B). White bar and black bar charts represent JBx_250483 and JBx_250497 strain, respectively. (D) Heatmap of shotgun proteomics analysis of DDGS, O-MT, ATP-grasp ligase, and NRPS. Expression levels of DDGS (E), O-MT (F), ATP-grasp ligase (G), and NRPS (H) from the control strain (JBx_250483) vs the highest shinorine producing strain (JBx_250497). Samples were grown on M9 minimal media. Shinorine was extracted from the whole liquid culture at 72 hr. Error bars represent standard deviations from three biological replicates. Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory [0046] Figure 6. Effects of glycine and L-serine supplementation on shinorine production. (A) Schematics of genetics constructs used in the study. P. putida KT2440, carrying the shinorine biosynthetic pathway plasmid (JBx_250497), was transformed with a CRISPRi plasmid containing either PP_1444 sgRNA (JBx_249619) or nontarget sgRNA (JBx_249575). (B,C) Comparison of shinorine production, glucose consumption, and growth profiles over a 114-hour period without glycine and L-serine supplementation. (D) Experimental conditions used in B, C, E, and F. (E,F) Comparison of shinorine production, glucose consumption, and growth profiles over a 114-hour period with glycine and L-serine supplementation. All strains were cultivated in 25 mL M9 media containing 20 g/L glucose with a starting OD₆₀₀ of 0.1. At 4 hr post inoculation, cultures were induced with 0.2% L- arabinose, with or without the addition of 10 mM equimolar concentration of glycine and L- serine. At 18 hr, all cultures received an additional 20 g/L glucose. For samples in (D) and (F), an extra 20 g/L glucose was introduced to the cultures at the 42-hr time point. Venn Diagram was generated using the webpage at: interactivenn.net/index.html (Heberle et al., 2015). Error bars represent standard deviations from three biological replicates. (G) Fraction of shinorine found in supernatant and cell pellet measured at 114 hr from samples in Fig.6 (Panel F). Error bars represent standard deviations from three biological replicates. [0047] Figure 7. Metabolic flux distribution from (A) Bacillus megaterium QM B1551 and (B) the rest of the microbes (except for P. putida KT2440 and Bacillus megaterium QM B1551) summarized from Table 1. Bacillus megaterium QM B1551 fluxes are adapted from (Wushensky et al., 2018). In this microbe, glucose can be oxidized to gluconate followed by phosphorylation to 6P-gluconate or glucose can be directly phosphorylated to G6P. In other microbes, glucose is directly phosphorylated to G6P (B). [0048] Figure 8. Characterization of inducible promoters. (A) Ptet, (B) PA1lacO-1, (C) Pm, (D) PLtet-O1, (E) PnagAa, and (F) PBAD. Cultures were grown in M9 minimal media. Cultures were induced at 2 hr post-inoculation and the fluorescences were measured at 48 hr. Error bars represent standard deviation from three biological replicates. [0049] Figure 9. LC-MS/MS confirmation of extracted shinorine. (A) m/z ratio of extracted shinorine from sample. (B) m/z ratio of extracted Helioguard^TM 365. (C) Tandem mass spectra of shinorine from extracted sample. (D) Tandem mass spectra of shinorine from extracted Helioguard^TM 365. Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory [0050] Figure 10. Structural numbering for shinorine. Assigned ¹H and ¹³C chemical shifts along with multiplicity and/or coupling constants may be found in Table 2. [0051] Figure 11.¹H NMR spectrum of heterologously produced shinorine. All NMR experiments were acquired in methanol-d4 at 500 MHz. [0052] Figure 12.¹³C NMR spectrum of heterologously produced shinorine acquired at 500 MHz. [0053] Figure 13. Expression levels of DDGS, O-MT, ATP-grasp ligase, and NRPS in different CRISPRi strains. Error bars represent standard deviations from three biological replicates. Y-axis represents protein counts. [0054] Figure 14. Expression levels of gentamicin (A), kanamycin (B) selection markers, and dCas9 (C) protein in different CRISPRi strains, WT, and control (CRISPRi_Nontarget). Protein counts were measured by shotgun proteomics analysis. Error bars represent standard deviations from three biological replicates. [0055] Figure 15. (A) OD600 of CRISPRi strains measured at 24 hr, 48 hr, and 72 hr. Gluconate concentrations were measured from the supernatant (B) and cell pellet (C) at different time points. Error bars represent standard deviations from three biological replicates. [0056] Figure 16. Protein expression levels of O-MT (A) and ATP-grasp ligase (B) plotted against shinorine titer. [0057] Figure 17. Comparison of shinorine production, glucose utilization, and growth profile over time in PP_1444 and nontarget strains. Cultures were cultivated in 25 mL M9 media with initial glucose concentration of 20 g/L. Error bars represent standard deviations from three biological replicates. [0058] Figure 18. Extracted shinorine in the supernatant and cell pellet. Error bars represent standard deviations from three biological replicates. [0059] Figure 19. Conserved gene clusters involved in MAA biosynthesis in P. umbilicalis and related red algae. (A) Biosynthetic pathway from sedoheptulose 7-phosphate to shinorine in cyanobacteria and proposed pathway to porphyra-334 in red algae. (B) Comparison of Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory gene clusters and gene fusions in the cyanobacteria Anabaena and Nostoc and the red algae P. umbilicalis and C. crispus. (Brawley et al., “Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta),” Proc. Natl. Acad. Sci. USA, Early Ed.: 1-10, 2017.) DETAILED DESCRIPTION OF THE INVENTION [0060] Before the invention is described in detail, it is to be understood that, unless otherwise indicated, this invention is not limited to particular sequences, expression vectors, enzymes, host microorganisms, or processes, as such may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. [0061] In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings: [0062] The terms "optional" or "optionally" as used herein mean that the subsequently described feature or structure may or may not be present, or that the subsequently described event or circumstance may or may not occur, and that the description includes instances where a particular feature or structure is present and instances where the feature or structure is absent, or instances where the event or circumstance occurs and instances where it does not. [0063] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an "expression vector" includes a single expression vector as well as a plurality of expression vectors, either the same (e.g., the same operon) or different; reference to "cell" includes a single cell as well as a plurality of cells; and the like. [0064] In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings: [0065] The terms "optional" or "optionally" as used herein mean that the subsequently described feature or structure may or may not be present, or that the subsequently described event or circumstance may or may not occur, and that the description includes instances where a particular feature or structure is present and instances where the feature or structure Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory is absent, or instances where the event or circumstance occurs and instances where it does not. [0066] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. [0067] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an "expression vector" includes a single expression vector as well as a plurality of expression vectors, either the same (e.g., the same operon) or different; reference to "cell" includes a single cell as well as a plurality of cells; and the like. [0068] The term “about” refers to a value including 10% more than the stated value and 10% less than the stated value. [0069] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. [0070] The terms “host cell” is used herein to refer to a living biological cell that can be transformed via insertion of an expression vector. [0071] The term "heterologous" as used herein refers to a material, or nucleotide or amino acid sequence, that is found in or is linked to another material, or nucleotide or amino acid Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory sequence, wherein the materials, or nucleotide or amino acid sequences, are foreign to each other (i.e., not found or linked together in nature). [0072] The terms "expression vector" or "vector" refer 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 other than those native to the cell, or in a manner not native to the cell. An "expression vector" contains a sequence of nucleic acids (ordinarily RNA or DNA) to be expressed by the host cell. Optionally, the expression vector also comprises materials to aid in achieving entry of the nucleic acid into the host cell, such as a virus, liposome, protein coating, or the like. The expression vectors contemplated for use in the present invention include those into which a nucleic acid sequence can be inserted, along with any preferred or required operational elements. Further, the expression vector must be one that can be transferred into a host cell and replicated therein. Particular expression vectors are plasmids, particularly those with restriction sites that have been well documented and that contain the operational elements preferred or required for transcription of the nucleic acid sequence. Such plasmids, as well as other expression vectors, are well known to those of ordinary skill in the art. [0073] 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. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, nucleic acid analogs may be used that may have alternate backbones, comprising, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press); positive backbones; non- ionic backbones, and non-ribose backbones. Thus, nucleic acids or polynucleotides may also include modified nucleotides that permit correct read-through by a polymerase. "Polynucleotide sequence" or "nucleic acid sequence" includes both the sense and antisense strands of a nucleic acid as either individual single strands or in a duplex. As will be appreciated by those in the art, the depiction of a single strand also defines the sequence of the complementary strand; thus the sequences described herein also provide the complement of the sequence. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The nucleic acid Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc. [0074] The term "promoter," as used herein, refers to a polynucleotide sequence capable of driving transcription of a DNA sequence in a cell. Thus, promoters used in the polynucleotide constructs of the invention include cis- and trans- acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene. For example, a promoter can be a cis- acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3' untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) gene transcription. Promoters are located 5' to the transcribed gene, and as used herein, include the sequence 5' from the translation start codon (i.e., including the 5' untranslated region of the mRNA, typically comprising 100-200 bp). Most often the core promoter sequences lie within 1-2 kb of the translation start site, more often within 1 kbp and often within 500 bp of the translation start site. By convention, the promoter sequence is usually provided as the sequence on the coding strand of the gene it controls. In the context of this application, a promoter is typically referred to by the name of the gene for which it naturally regulates expression. A promoter used in an expression construct of the invention is referred to by the name of the gene. Reference to a promoter by name includes a wildtype, native promoter as well as variants of the promoter that retain the ability to induce expression. Reference to a promoter by name is not restricted to a particular species, but also encompasses a promoter from a corresponding gene in other species. [0075] A polynucleotide is "heterologous" to a host cell or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, when a polynucleotide encoding a polypeptide sequence is said to be operably linked to a heterologous promoter, it means that the polynucleotide coding sequence encoding the polypeptide is derived from one species whereas the promoter sequence is derived from another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g., is a Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory genetically engineered coding sequence, e.g., from a different gene in the same species, or an allele from a different ecotype or variety). [0076] The term "operatively linked" refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a DNA or RNA sequence if it stimulates or modulates the transcription of the DNA or RNA sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance. [0077] In some embodiments, the host cell comprises a nucleic acid encoding the one or more enzymes operatively linked to a promoter capable of expressing the one or more enzymes in the host cell. In some embodiments, the encoding of the one or more enzymes to the nucleic acid is codon optimized to the host cell. In some embodiments, the nucleic acid is vector or replicon that can stably reside in the host cell. In some embodiments, the nucleic acid is stably integrated into the chromosome of the host cell. [0078] In some embodiments, the providing step (a) comprises introducing a nucleic acid encoding the one or more enzymes operatively linked to a promoter capable of expressing the one or more enzymes in the host cell into the host cell. [0079] The present invention provides for a method for constructing a genetically modified host cell of the present invention, comprising (a) introducing a nucleic acid encoding the one or more enzymes operatively linked to a promoter capable of expressing the one or more enzymes in the host cell into the host cell. [0080] One can modify the expression of a gene encoding any of the enzymes taught herein by a variety of methods in accordance with the methods of the invention. Those skilled in the art would recognize that increasing gene copy number, ribosome binding site strength, promoter strength, and various transcriptional regulators can be employed to alter an enzyme Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory expression level. Host cells [0081] The genetically modified host cell can be any prokaryotic or eukaryotic cell, with any genetic modifications, capable of production of the isoprenol in accordance with the methods 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 Saccharomyces genus. Generally, although not necessarily, the host cell is a yeast or a bacterium. Any prokaryotic or eukaryotic host cell may be used in the present method so long as it remains viable after being transformed with a sequence of nucleic acids. In some embodiments, the host cell is not adversely affected by the transduction of the necessary nucleic acid sequences, the subsequent expression of the proteins (i.e., enzymes), or the resulting intermediates required for carrying out the steps associated with the mevalonate pathway. For example, it is preferred that minimal "cross-talk" (i.e., interference) occur between the host cell’s own metabolic processes and those processes involved with the mevalonate pathway. [0082] In some embodiments, the host cells are genetically modified in that heterologous nucleic acid have been introduced into the host cells, and as such the genetically modified host cells do not occur in nature. The suitable host cell is one capable of expressing a nucleic acid construct encoding one or more enzymes described herein. The gene(s) encoding the enzyme(s) may be heterologous to the host cell or the gene may be native to the host cell but is operatively linked to a heterologous promoter and one or more control regions which result in a higher expression of the gene in the host cell. [0083] The enzyme can be native or heterologous to the host cell. Where the enzyme is native to the host cell, the host cell is genetically modified to modulate expression of the enzyme. This modification can involve the modification of the chromosomal gene encoding the enzyme in the host cell or a nucleic acid construct encoding the gene of the enzyme is introduced into the host cell. One of the effects of the modification is the expression of the enzyme is modulated in the host cell, such as the increased expression of the enzyme in the host cell as compared to the expression of the enzyme in an unmodified host cell. [0084] Yeasts suitable for the invention include, but are not limited to, Yarrowia, Candida, Bebaromyces, Saccharomyces, Schizosaccharomyces and Pichia cells. In some embodiments, the yeast is Saccharomyces cerevisae. In some embodiments, the yeast is a Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory species of Candida, including but not limited to C. tropicalis, C. maltosa, C. apicola, C. paratropicalis, C. albicans, C. cloacae, C. guillermondii, C. intermedia, C. lipolytica, C. panapsilosis and C. zeylenoides. In some embodiments, the yeast is Candida tropicalis. In some embodiments, the yeast is a non-oleaginous yeast. In some embodiments, the non- oleaginous yeast is a Saccharomyces species. In some embodiments, the Saccharomyces species is Saccharomyces cerevisiae. In some embodiments, the yeast is an oleaginous yeast. In some embodiments, the oleaginous yeast is a Rhodosporidium species. In some embodiments, the Rhodosporidium species is Rhodosporidium toruloides. [0085] 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 is of the phylum Proteobactera. In some embodiments, the host cell is of the class Gammaproteobacteria. In some embodiments, the host cell is of the order Enterobacteriales. In some embodiments, the host cell is of the family Enterobacteriaceae. Examples of suitable bacteria include, without limitation, those species assigned to the Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsielia, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, and Paracoccus taxonomical classes. [0086] Bacterial host cells suitable for the invention include, but are not limited to, Escherichia, Corynebacterium, Pseudomonas, Streptomyces, and Bacillus. In some embodiments, the Escherichia cell is an E. coli, E. albertii, E. fergusonii, E. hermanii, E. marmotae, or E. vulneris. In some embodiments, the Corynebacterium cell is Corynebacterium glutamicum, Corynebacterium kroppenstedtii, Corynebacterium alimapuense, Corynebacterium amycolatum, Corynebacterium diphtheriae, Corynebacterium efficiens, Corynebacterium jeikeium, Corynebacterium macginleyi, Corynebacterium matruchotii, Corynebacterium minutissimum, Corynebacterium renale, Corynebacterium striatum, Corynebacterium ulcerans, Corynebacterium urealyticum, or Corynebacterium uropygiale. In some embodiments, the Pseudomonas cell is a P. putida, P. aeruginosa, P. chlororaphis, P. fluorescens, P. pertucinogena, P. stutzeri, P. syringae, P. cremoricolorata, P. entomophila, P. fulva, P. monteilii, P. mosselii, P. oryzihabitans, P. parafluva, or P. plecoglossicida. In some embodiments, the Streptomyces cell is a S. coelicolor, S. lividans, S. venezuelae, S. ambofaciens, S. avermitilis, S. albus, or S. scabies. In some embodiments, the Bacillus cell is a B. subtilis, B. megaterium, B. licheniformis, B. anthracis, B. amyloliquefaciens, or B. pumilus. Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory [0087] In some embodiments, the bacterial host cell is a proteobacteria cell. In some embodiments, the proteobacteria cell is a Gammaproteobacteria cell. In some embodiments, the Gammaproteobacteria cell is a Pseudomonadales or Enterobacterales cell. In some embodiments, the Gammaproteobacteria cell is a Pseudomonadales cell, which is a Pseudomonadaceae cell. In some embodiments, the Pseudomonadaceae cell is a Pseudomonas, Azotobacter, Mesophilobacter, Oblitimonas, Permianibacter, Rugamonas, or Thiopseudomonas cell. In some embodiments, the Pseudomonas cell is a P. putida, P. aeruginosa, P. chlororaphis, P. fluorescens, P. pertucinogena, P. stutzeri, P. syringae, P. cremoricolorata, P. entomophila, P. fulva, P. monteilii, P. mosselii, P. oryzihabitans, P. parafluva, or P. plecoglossicida. In some embodiments, the Gammaproteobacteria cell is an Enterobacterales cell, which is an Enterobacteriaceae cell. In some embodiments, the Enterobacteriaceae cell is an Escherichia, Enterobacillus, Enterobacter, Klebsiella, Salmonella, or Shigella cell. In some embodiments, the Escherichia cell is an E. coli, E. albertii, E. fergusonii, E. hermanii, E. marmotae, or E. vulneris. In some embodiments, the host cell is a Gram negative bacterium. In some embodiments, the host cell is a bacterium from the Azotobacter, Escherichia, Salmonella, Vibrio, Pasteurella, Haemophilus, or Pseudomonas genus. In some embodiments, the host cell is a bacterium from the species Escherichia coli, Salmonella enterica, Vibrio cholerae, Pasteurella multocida, Haemophilus influenza, Pseudomonas putida, or Pseudomonas aeruginosa. [0088] The biomass can be obtained from one or more feedstock, such as softwood feedstock, hardwood feedstock, grass feedstock, and/or agricultural feedstock, or a mixture thereof. [0089] Softwood feedstocks include, but are not limited to, Araucaria (e.g. A. cunninghamii, A. angustifolia, A. araucana); softwood Cedar (e.g. Juniperus virginiana, Thuja plicata, Thuja occidentalis, Chamaecyparis thyoides Callitropsis nootkatensis); Cypress (e.g. Chamaecyparis, Cupressus Taxodium, Cupressus arizonica, Taxodium distichum, Chamaecyparis obtusa, Chamaecyparis lawsoniana, Cupressus semperviren); Rocky Mountain Douglas fir; European Yew; Fir (e.g. Abies balsamea, Abies alba, Abies procera, Abies amabilis); Hemlock (e.g. Tsuga canadensis, Tsuga mertensiana, Tsuga heterophylla); Kauri; Kaya; Larch (e.g. Larix decidua, Larix kaempferi, Larix laricina, Larix occidentalis); Pine (e.g. Pinus nigra, Pinus banksiana, Pinus contorta, Pinus radiata, Pinus ponderosa, Pinus resinosa, Pinus sylvestris, Pinus strobus, Pinus monticola, Pinus lambertiana, Pinus taeda, Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory Pinus palustris, Pinus rigida, Pinus echinata); Redwood; Rimu; Spruce (e.g. Picea abies, Picea mariana, Picea rubens, Picea sitchensis, Picea glauca); Sugi; and combinations/hybrids thereof. [0090] For example, softwood feedstocks which may be used herein include cedar; fir; pine; spruce; and combinations thereof. The softwood feedstocks for the present invention may be selected from loblolly pine (Pinus taeda), radiata pine, jack pine, spruce (e.g., white, interior, black), Douglas fir, Pinus silvestris, Picea abies, and combinations/hybrids thereof. The softwood feedstocks for the present invention may be selected from pine (e.g. Pinus radiata, Pinus taeda); spruce; and combinations/hybrids thereof. [0091] Hardwood feedstocks include, but are not limited to, Acacia; Afzelia; Synsepalum duloificum; Albizia ; Alder (e.g. Alnus glutinosa, Alnus rubra ); Applewood; Arbutus ; Ash (e.g. F. nigra, F. quadrangulata, F. excelsior, F. pennsylvanica lanceolata, F. latifolia, F. profunda, F. americana ); Aspen (e.g. P. grandidentata, P. tremula, P. tremuloides ); Australian Red Cedar ( Toona ciliata ); Ayna ( Distemonanthus benthamianus ); Balsa ( Ochroma pyramidale ); Basswood (e.g. T. americana, T. heterophylla ); Beech (e.g. F. sylvatica, F. grandifolia ); Birch; (e.g. Betula populifolia, B. nigra, B. papyrifera, B. lenta, B. alleghaniensis/B. lutea, B. pendula, B. pubescens ); Blackbean; Blackwood; Bocote; Boxelder; Boxwood; Brazilwood; Bubing a; Buckeye (e.g. Aesculus hippocastanum, Aesculus glabra, Aesculus flava/Aesculus octandra ); Butternut; Catalpa; Chemy (e.g. Prunus serotina, Prunus pennsylvanica, Prunus avium ); Crabwood; Chestnut; Coachwood; Cocobolo; Corkwood; Cottonwood (e.g. Populus balsamifera, Populus deltoides, Populus sargentii, Populus heterophylla ); Cucumbertree; Dogwood (e.g. Cornus florida, 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 ; Greenheart; Grenadilla; Gum (e.g. Nyssa sylvatica, Eucalyptus globulus, Liquidambar styraciflua, Nyssa aquatica ); Hickory (e.g. Carya alba, Carya glabra, Carya ovata, Carya laciniosa ); Hornbeam; Hophornbeam; Ipê; Iroko; Ironwood (e.g. Bangkirai, Carpinus caroliniana, Casuarina equisetifolia, Choricbangarpia subargentea, Copaifera spp., Eusideroxylon zwageri, Guajacum officinale, Guajacum sanctum, Hopea odorata, Ipe, Krugiodendronferreum, Lyonothamnus lyonii ( L. floribundus ), Mesua ferrea, Olea spp., Olneya tesota, Ostrya virginiana, Parrotia persica, Tabebuia serratifolia ); Jacarandá; Jotoba; Lacewood; Laurel; Limba; Lignum vitae; Locust (e.g. Robinia pseudacacia, Gleditsia Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory triacanthos ); Mahogany; Maple (e.g. Acer saccharum, Acer nigrum, Acer negundo, Acer rubrum, Acer saccharinum, Acer pseudoplatanus ); Meranti; Mpingo; Oak (e.g. Quercus macrocarpa, Quercus alba, Quercus stellata, Quercus bicolor, Quercus 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 ); Obeche; Okoumé; Oregon Myrtle; California Bay Laurel; Pear; Poplar (e.g. P. balsamifera, P. nigra , Hybrid Poplar ( Populus×canadensis )); Ramin; Red cedar; Rosewood; Sal; Sandalwood; Sassafras; Satinwood; Silky Oak; Silver Wattle; Snakewood; Sourwood; Spanish cedar; American sycamore; Teak; Walnut (e.g. Juglans nigra, Juglans regia); Willow (e.g. Salix nigra, Salix alba ); Yellow poplar ( Liriodendron tulipifera ); Bamboo; Palmwood; and combinations/hybrids thereof. [0092] For example, hardwood feedstocks for the present invention may be selected from Acacia, Aspen, Beech, Eucalyptus , Maple, Birch, Gum, Oak, Poplar, and combinations/hybrids thereof. The hardwood feedstocks for the present invention may be selected from Populus spp. (e.g. Populus tremuloides ), Eucalyptus spp. (e.g. Eucalyptus globulus ), Acacia spp. (e.g. Acacia dealbata ), and combinations thereof. [0093] Grass feedstocks include, but are not limited to, C4 or C3 grasses, e.g. Switchgrass, Indiangrass, Big Bluestem, Little Bluestem, Canada Wildrye, Virginia Wildrye, and Goldenrod wildflowers, etc, amongst other species known in the art. [0094] Agricultural feedstocks include, but are not limited to, agricultural byproducts such as husks, stovers, foliage, and the like. Such agricultural byproducts can be derived from crops for human consumption, animal consumption, or other non-consumption purposes. 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Methanol-free biosynthesis of fatty acid methyl ester (FAME) in Synechocystis sp. PCC 6803. Metab Eng 57, 217–227. Zhao, D., Zhu, X., Zhou, H., Sun, N., Wang, T., Bi, C., Zhang, X., 2021. CRISPR-based metabolic pathway engineering. Metab Eng 63, 148–159. [0097] Other objects, features, and advantages of the present invention will be apparent to one of skill in the art from the following detailed description and figures. [0098] It is to be understood that, while the invention has been described in conjunction with the preferred specific embodiments thereof, 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 to which the invention pertains. [0099] All patents, patent applications, and publications mentioned herein are hereby incorporated by reference in their entireties. [00100] The invention having been described, the following examples are offered to illustrate the subject invention by way of illustration, not by way of limitation. EXAMPLE 1 Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory Systematic engineering for production of anti-aging sunscreen compound in Pseudomonas putida [00101] Sunscreen has been used for thousands of years to protect skin from ultraviolet radiation. However, the use of modern commercial sunscreen containing oxybenzone, ZnO, and TiO2 has raised concerns due to their negative effects on human health and the environment. In this study, the aim is to establish an efficient microbial platform for production of shinorine, a UV light absorbing compound with anti-aging properties. First, an appropriate host is methodically selected for shinorine production by analyzing central carbon flux distribution data from prior studies alongside predictions from genome-scale metabolic models (GEMs). Shinorine productivity is enhanced through CRISPRi-mediated downregulation and utilized shotgun proteomics to pinpoint potential competing pathways. Simultaneously, the shinorine biosynthetic pathway is improved by refining its design, optimizing promoter usage, and altering the strength of ribosome binding sites. Finally, amino acid feeding experiments is conducted under various conditions to identify the key limiting factors in shinorine production. The study combines meta-analysis of ¹³C-metabolic flux analysis, GEMs, synthetic biology, CRISPRi-mediated gene downregulation, and omics analysis to improve shinorine production, demonstrating the potential of Pseudomonas putida KT2440 as platform for shinorine production. [00102] In this study, an appropriate host is methodically selected for shinorine production by analyzing central carbon flux distribution data from prior studies alongside predictions from genome-scale metabolic models (GEMs). Pseudomonas putida KT2440 is identified as a promising host for shinorine production. Synthetic biology tools are developed for use in this microbe and heterologously expressed shinorine biosynthetic gene cluster (BGC) from Anabaena variabilis ATCC 29413. Shinorine productivity is enhanced through CRISPRi-mediated downregulation of potential competing pathways. Simultaneously, the shinorine biosynthetic pathway is improved by refining its design, optimizing promoter usage, and altering the strength of ribosome binding sites. Finally, amino acid feeding experiments is conducted under various conditions and utilized shotgun proteomics to identify the limiting factors in shinorine production. Altogether, our study provides a comprehensive framework for engineering Pseudomonas putida KT2440 as an efficient chassis for shinorine production. RESULTS AND DISCUSSION Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory Selection of P. putida KT2440 as a suitable host for shinorine production [00103] To select a suitable host for shinorine production, published metabolic flux distribution from different microbes (Table 1) was reviewed alongside predictions from genome-scale metabolic models (GEMs). The primary focus was on analyzing fluxes directed towards 6-phosphogluconate (6PG) as this intermediary metabolite plays important roles in the shinorine biosynthetic pathway.6PG functions as an intermediate molecule in both the pentose-phosphate and the Entner-Doudoroff pathways. In the pentose-phosphate pathway, 6PG undergoes decarboxylation to produce ribulose 5-phosphate (Ri5P), which is further metabolized into ribose 5-phosphate (R5P) and xylulose 5-phosphate (X5P) by ribulose-5- phosphate epimerase. While R5P serves as a fundamental building block for nucleic acid synthesis, X5P is converted by a transketolase to glyceraldehyde 3-phosphate (G3P) and sedoheptulose 7-phosphate (S7P), acting as a precursor for shinorine biosynthesis. G3P is converted to 3-phopshoglycerate (3PG) through either glycolysis (the Embden-Meyerhof- Parnas (EMP) pathway) or the Entner-Doudoroff pathway.3PG is used for the biosynthesis of L-serine and glycine, essential amino acids incorporated into the shinorine biosynthetic pathway. [00104] Table 1. Carbon fluxes from G6P to F6P and G6P to 6PG in various organisms. All values are given as relative fluxes normalized to the specific glucose uptake rate. R_{elevant condition} ^{G6P to} G6P to F_{6P 6PG} ^Reference B_{atch cultivation 27.5 56.9} ^{(Panagiotou et al.,} 2₀₀₈₎ Batch cultivation 32.7 56.1 (Lu et al., 2015) ɣ-PGA medium 65.37 33.77 (He et al., 2019) ^{Batch cultivation 47.6 49.2} (Wushensky et al., 2018) B_{acillus subtilis 168} ^{CG medium, batch} (Oliver et al., 2007) c_{ultivation 64.6 33.2 Bacillus subtilis 168} ^{CSEG medium, batch} _54. (Oliver et al., 2007) c_{ultivation 8 43.7} Corynebacterium glutamicum CGX11, 5 g/L glucose, (Zel 0_{C, pH 7, DO >30%} ⁴⁶ le et al., 2021) ATCC 13032 ₃ ^{.2 52.1} Corynebacterium glutamicum ^{Batch culti} (Patrick et al., A_{TCC 21526} ^{vation 36.2 62} 2004) E_{scherichia coli BW25113 Batch cultivation 62 37 Escherichia coli MG1655 Continuous cultivation 75 23 Escherichia coli MG1655 Batch cultivation 73 26} Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory P_{ichia pastoris GS115 Low O2 condition 45 38} ^{(Torres et al.,} 2_{019) Pichia pastoris GS115 Excess O2 condition 44 39} ^{(Torres et al.,} 2₀₁₉₎ Pichia pastoris X33 Chemostat 53 33 (Jordà et al., 2012) P_{seudomonas putida KT2440} ^{M9, 20 mM glucose,} (Nikel et al., 2015) b_{atch cultivation -9 19} P^{seudomonas putida KT2440 M9, batch cultivation -21 52 (Kohlstedt and} W_{ittmann, 2019)} M9, baffled flask, 16.7 (Kukurugya et al., Pseudomonas putida KT2440 mM glucose, batch -6 16 2019) cultivation Rhodosporidium toruloides ^{In silico a} (Bommareddy et D_{SMZ 4444} ^{nalysis 10.3 89.7} S_{accharomyces cerevisiae average from 43 strains 95.29 1.64 2016)} Saccharomyces e_{revisiae ATCC 32167} ^{Purely ox} (Frick and c ^{idative 25.2 54.7} Wittmann, 2005) Saccharomyces i_{siae ATCC 32167} ² (Frick and c_erev ^9.6 Wittmann, 2005) Saccharomyces Mainly fermentative (Fri siae ATCC 32167 _growth ⁷⁷ ck and cerevi ^.9 Wittmann, 2005) Saccharomyces (Yatabe et al., cerevisiae BY4947 (derived SD medium 89.5 4.9 2022) from S288C) Saccharomyces cerevisiae ^{Respiratory (chemostat) 33.6 4} (Karoly et al., C_EN.PK113-7D ^4.2 2001) Saccharomyces cerevisiae Respiro-fermentative ( t_{ch cultivation)} ^{81 1} Karoly et al., CEN.PK113-7D _(ba ⁶ 2001) Saccharomyces (Yatabe et al., cerevisiae Kyokai 7 (sake SD medium 83.2 12.0 2022) yeast) Saccharomyces (Quirós et al., cerevisiae LALVIN EC1118 240 g/L glucose, 28C 95.7 1.88 2013) (wine yeast) Saccharomyces (Quirós et al., cerevisiae LALVIN EC1118 240 g/L glucose, 16C 94.7 2.24 2013) (wine yeast) Saccharomyces (Quirós et al., cerevisiae LALVIN EC1118 280 g/L glucose, 28C 95.5 2.04 2013) (wine yeast) Saccharomyces (Quirós et al., cerevisiae LALVIN EC1118 280 g/L glucose, 16C 92.7 2.67 2013) (wine yeast) Saccharomyces ^S (Yatabe et al., c_{erevisiae QA23 (wine yeast)} ^{D medium 86.8 6.6} 2022) Saccharomyces cerevisiae ^{SD medium 86.8 8.8} (Yatabe et al., R_{edStar (bread yeast)} 2022) Saccharopolyspora erythraea Batch cultivation 81 19 (Chen et al., 2017) Streptomyces coelicolor ^Batc (Coze et al., 2013) M₁₁₄₆ ^{h cultivation 60.2 37.2} Streptomyces coelicolor (Coze et al., 2013) M₁₄₅ ^{Batch cultivation 76.7 21.4} Yarrowia lipolytica Batch cultivation 59.1 35.1 (Sabra et al., 2017) Zymomonas mobilis ZM4 ^{Batch cu} (Jacobson et al., (_{ATCC 31821)} ^{ltivation 0.8 99} 2019) [00105] In most organisms, except for Pseudomonas putida KT2440 and Bacillus Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory megatarium QM B1551, glucose is directly phosphorylated by glucokinase, producing glucose 6-phosphate (G6P) (Fig.1, Figure 7). G6P can be either isomerized to fructose 6- phosphate (F6P) within the glycolytic pathway or undergo oxidation to form 6PG through the oxidative pentose phosphate pathway. The carbon flux distribution from G6P to F6P or G6P to 6PG, as determined through ¹³C metabolic flux analysis (¹³C-MFA), varies among different microorganisms and is influenced by factors such as fermentation conditions and genetic variations (Table 1). Nonetheless, meta-analysis of ¹³C-MFA and predictions from GEMs (Table 2) reveal that only a few microbes can allocate more than 50% of their carbon flux towards 6PG. [00106] Table 2. Carbon flux distribution as predicted by genome-scale metabolic models. Organism Model 6PG Flux-sum (%) Pseudomonas putida iJN1463 100 Zymomonas mobilis iZM4_478 98.9 Synechocystis sp. PCC 6_803. ^{iJN678 89.6} Pichia kudriavzevii iIsor850 53.7 Rhodosporidium toruloides rhto-GEM 43.4 Pichia pastoris iMT1026v3 38.4 Saccharomyces cerevisiae yeast-GEM 36.8 Escherichia coli iML1515 23.6 Aspergillus niger iJB1325 0 Bacillus subtilis iYO844 0 Corynebacterium g_lutamicum ^{iCGB21FR 0} Streptomyces coelicolor Sco-GEM 0 Yarrowia lipolytica iYali4 0 [00107] Zymomonas mobilis converts approximately 99% of it’s glucose supply to 6PG (Table 1). However, the oxidative branch of the pentose phosphate pathway in Z. mobilis appears inactive as Z. mobilis lacks 6PG dehydrogenase enzyme (De Graaf et al., 1999; Jacobson et al., 2019). Instead of using the oxidative branch of the pentose phosphate pathway, Z. mobilis uses the non-oxidative branch and converts F6P to R5P and S7P through a series of enzymatic reactions. Only 0.8% carbon flux is distributed from G6P to F6P, leaving a very small pool of F6P for S7P synthesis. On the other hand, in silico metabolic network analysis of Rhodosporidium toruloides reveals that glucose-fed R. toruloides allocates 89.7% of its carbon flux towards 6PG, creating a significant pool of S7P in the non- Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory oxidative pentose phosphate pathway (Bommareddy et al., 2015). R. toruloides is also an attractive host for industrial scale production of many chemical compounds (Liu et al., 2023, 2020; Schultz et al., 2022; Wehrs et al., 2019). It can readily co-consume C5 and C6 sugars derived from lignocellulosic biomass and grow to very high cell density. However, engineering R. toruloides has been challenging (Wen et al., 2020) with only a few synthetic biology tools available (Brink et al., 2023). [00108] Pseudomonas putida KT2440 exhibits a distinctive central carbon metabolism (Fig.1). In this microbe, glucose can be phosphorylated to G6P in the cytoplasm through direct phosphorylation, or it can undergo oxidation to gluconate catalyzed by glucose dehydrogenase in the periplasm. Subsequently, gluconate can follow one of two routes: it can be phosphorylated to 6PG by gluconokinase in the cytoplasm or oxidized to 2-ketogluconate (2-KG) in the periplasm. The latter compound, 2-KG, is then transported into the cytoplasm and converted to 2-keto-6-phosphogluconate (2K6PG) by 2-KG kinase, which is further reduced to 6PG by 2K6PG reductase. While the majority of glucose is oxidized to gluconate, regardless of the initial step in glucose processing, these pathways converge into the production of 6PG. Furthermore, in contrast to other microorganisms, P. putida KT2440 favors the conversion of F6P to 6PG rather than the reverse reaction. As a result, P. putida KT2440 converts over 90% of its glucose supply into 6PG, creating a substantial pool of 6PG for the biosynthesis of Ri5P and S7P in the non-oxidative pentose-phosphate pathway. In addition, genome editing and synthetic biology tools for P. putida KT2440 have been widely available and well characterized (Nikel and de Lorenzo, 2018). This unique metabolic trait positions P. putida KT2440 as a promising host for shinorine production. Development of synthetic biology tools for gene expression in P. putida KT2440 [00109] To realize the shinorine biosynthetic pathway, it was began by first developing a modular plasmid assembly tool for use in P. putida KT2440 (Fig.2, Panel A). Here, Biopart Assembly Standard for Idempotent Cloning (BASIC) (Storch et al., 2015) plasmid assembly method is used to allow multipart DNA assembly using standard reusable parts in a single pot assembly reaction. The resulting expression plasmids were then used to assess the functionality of genetic elements, such as promoters and ribosome binding sites. The primary objective of this was not an exhaustive characterization of the genetic elements but rather an investigation of the performance of the expression plasmid and individual genetic elements employed in this study. Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory [00110] Six plasmids (pBBR1, pBBR1-B5, pRK2, pRSF1010, pRO1600/p15a, and pVS1/p15a) with distinct backbones, copy numbers, and origin of replications were constructed and characterized (Fig.2, Panel B) by measuring the fluorescence intensity of a red fluorescent protein (RFP). The plasmids selected for construction offer diverse features. First, pBBR1, isolated from Bordetella bronchiseptica S87 (Antoine and Locht, 1992), is a broad host range plasmid origin commonly used for gene expression in metabolic engineering studies of P. putida (Niu et al., 2020; Tiso et al., 2016; X. Wang et al., 2022; Wohlers et al., 2021). Although generally considered a low- to medium- copy plasmid, the copy number of pBBR1 can be altered by mutations in the rep gene. Second, pBBR1-B5, a variant of pBBR1 plasmid with an early stop codon in the rep gene, exhibits a higher copy number than the original pBBR1 plasmid. Third, the plasmid pRK2 is a member of the IncP incompatibility 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 P. putida and has been measured to be a low-copy plasmid in Escherichia coli (De Bernardez and Dhurjati, 1987). Fourth, RSF1010 is a high-copy broad host range plasmid and is a part the IncQ incompatibility group plasmid. It has been routinely used for gene expression in P. putida (Aparicio et al., 2019). Lastly, pRO1600 (Farinha and Kropinski, 1990) and pVS1 (Itoh and Haas, 1985) are plasmids isolated from Pseudomonas species, requiring host-specific origins (e.g., p15a, that replicates in E. coli) to generate shuttle vectors for P. putida. In this study, plasmid characterization reveals that pBBRR1-B5 shows the highest RFP fluorescence level, followed by pBBR1 > pRSF1010 = pRO1600/p15a > pRK2 > pVS1/p15a plasmids (Figure 2, Panel C). [00111] Given its medium-range RFP fluorescence, plasmid pRK2 was then selected as the plasmid backbone for the characterization of different inducible (Figure 2, Panel D) and constitutive (Figure 2, Panel E) promoters. Seven different inducible promoters (Ptet (Cook et al., 2018), P_tet* (Tian et al., 2019), P_A1lacO-1 (Liu et al., 2019), P_BAD (Calero et al., 2016), PNagA (Hüsken et al., 2001), PlacUV5 (Noel Jr. and Reznikoff, 2000), and PXylS/Pm (Calero et al., 2016)), six Anderson promoters (Anderson et al., 2010) with varying strength (PJ23119, P_J23100, P_J23101, P_J23102, P_J23107, P_J23104), and P_trc1-O (Bagdasarian et al., 1983; Calero et al., 2016) (without a LacI repressor) were characterized. P_tet and P_A1lacO-1 promoters exhibited a high basal fluorescence level (Figure 8) compared to other promoters. Among the inducible promoters, P_BAD, the arabinose-inducible promoter, showed the highest RFP fluorescence level. Meanwhile, under the conditions tested, the constitutive promoter P_trc-1O was the Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory strongest promoter among all evaluated promoters. [00112] Lastly, 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), a Shine-Dalgarno E. coli consensus (RBS4; ATCACAAGGAGG) (SEQ ID NO:11), and an anti-Shine-Dalgarno complementary sequence (RBS5: ATTAGTGGAGGT) (SEQ ID NO:12) are characterized based on their varying strengths in E. coli. Fig.2, Panel F shows that the selected ribosome binding sites exhibited varying RFP fluorescence levels. The use of ribosome binding sites with varying strengths has been employed in metabolic engineering studies, proving to be a useful strategy for pathway optimization. Shinorine production in Pseudomonas putida KT2440 [00113] After evaluating the performance and functionality of plasmid backbones, promoters, and ribosome binding sites in Pseudomonas putida KT2440, the shinorine biosynthetic pathway is constructed. The shinorine biosynthetic gene cluster (BGC) has been identified in various microorganisms, including Anabaena variabilis ATCC 29413 (Balskus and Walsh, 2010), Nostoc punctiforme ATCC 29133/PCC 73102 (Qunjie and Ferran, 2011), Actinosynnema mirum DSM 43827 (Miyamoto et al., 2014), Chlorogloeopsis fritschii PCC 6912 (Llewellyn et al., 2020; Portwich and Garcia-Pichel, 2003), and Fischerella sp. PCC 9339 (Yang et al., 2018). In this study, the shinorine biosynthetic pathway is selected from Anabaena variabilis ATCC 29413 as it was one of the earliest characterized routes for shinorine biosynthesis. S7P, an intermediate metabolite of the pentose phosphate pathway, is converted into shinorine through a series of four enzymatic steps. Firstly, S7P undergoes transformation into 4-deoxygadusol (4-DG) by 2-demethyl 4-deoxygadusol synthase (DDGS) and O-methyltransferase (O-MT). Subsequently, glycine is conjugated to 4-DG by ATP- grasp ligase to form mycosporine-glycine (MG). Finally, a serine moiety is attached to MG by a nonribosomal peptide synthetase (NRPS)-like enzyme (Fig.3, Panel A). [00114] To synthesize shinorine in Pseudomonas putida KT2440, it was began by amplifying the whole shinorine BGC (MIBiG Accession Number: BGC0000427) from Anabaena variabilis ATCC 29413, keeping the intergenic regions unaltered. The amplified gene cluster was subsequently cloned into an RK-based vector under the control of a strong constitutive Ptrc1-O promoter (Fig.3, Panel B). The heterologous expression of the shinorine Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory biosynthetic pathway in Pseudomonas putida KT2440 resulted in the production of approximately 60 mg/L shinorine at 48 hr, with over 80% of shinorine accumulating in the supernatant. The chromatographic analysis of the samples (Fig.3, Panel D) showed that the shinorine peak (1) co-eluted with the shinorine peak obtained from the reference Helioguard^TM standard (Fig.3, Panel E). The accuracy of the mass spectra was confirmed through LC-MS/MS (Figure 9) and the results were in agreement with those previously reported in a prior study (Kim et al., 2022). To further validate the chemical structure of shinorine, the extract from a 1-L liquid culture was purified and subjected it to nuclear magnetic resonance (NMR) spectroscopy. Correlations obtained via ¹H-¹H COSY, ¹H-¹H NOESY, ¹H-¹³C HSQC and ¹H-¹³C HMBC enabled the unambiguous assignment of all ¹H and ¹³C resonances (Figure 3, Panel F, G and Table 3). Observed resonances and assignments thereof were highly consistent with previous NMR data reported for shinorine (Miyamoto et al., 2014) (Figures 10-12). To our knowledge, this was the first report of shinorine production in Pseudomonas putida KT2440. [00115] Table 3. Atom assignments for ¹H and ¹³C resonances observed in heterologously produced shinorine. Atom numbering may be found in Figure 4. 3 3. 6 (s) - 67. Improvement of shinorine production titer by CRISPRi-mediated gene downregulation [00116] To further improve the shinorine titer, CRISPRi-mediated gene downregulation was employed to suppress competing metabolic pathways and redirect carbon flux towards shinorine production. The use of CRISPRi holds the potential to improve Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory the yield of desired metabolites by selectively repressing the expression of specific genes in metabolic pathways. This approach has proven successful in improving the production of various compounds, including isoprenol (Tian et al., 2019; Wang et al., 2022), free fatty acids (Fang et al., 2021), propane (Yunus et al., 2022), fatty alcohols (Kaczmarzyk et al., 2018), and many others (Zhao et al., 2021). In this study, CRISPRi was applied to downregulate a set of twenty-one genes involved in the central carbon metabolism and biosynthesis of L- serine and glycine, two amino acids critical to the shinorine biosynthetic pathway (Fig.4, Panel A). Catalytically inactive Cas9 protein was expressed in RSF1010 plasmid under the control of salicylic acid-inducible promoter (P_nagA) and the single guide RNA (sgRNA) was placed under the constitutive promoter P_J23119 (Fig.4, Panel B). This plasmid was coexpressed with the shinorine-producing RK2 plasmid. [00117] Our findings demonstrated that knocking down the PP_1444 gene led to a significant increase in the shinorine titer. The P. putida KT2440 strain with downregulated PP_1444 produced approximately 400 mg/L shinorine in 72 hr, a 160% increase compared to the control strain (i.e., a strain with a nontarget sgRNA) (Fig.4, Panel C). It is worth noting that the titer of the control strain shown here is approximately 3-fold higher at 48 hr compared to the strain shown in Fig.3, Panel C. The presence of a CRISPRi plasmid appeared to influence the expression levels of DDGS, O-MT, ATP-grasp ligase, and NRPS (Fig.13). These changes might also be attributed to the heightened expression of the aph gene (Fig.14, Panel B), which may suggest that the copy number of the RK2 plasmid was altered in the presence of a second plasmid. [00118] PP_1444 encodes quinoprotein glucose dehydrogenase, the enzyme responsible for the conversion of glucose to gluconate. In previous studies, it was observed that P. putida KT2440 tends to accumulate gluconate and 2-ketogluconate, and the accumulation of these two compounds is effectively eliminated by knocking out PP_1444 (Teresa et al., 2007). Interestingly, the knockout of PP_1444 has been associated with a growth defect in prior studies (Bentley et al., 2020). However, in this study, no growth defects were observed (Fig.15, Panel A). This discrepancy is likely attributable to the partial repression of PP_1444 achieved by CRISPRi (Fig.4, Panel D), resulting in a decrease rather than a complete elimination of gluconate in both the supernatant and cell pellet fractions (Fig. 15). [00119] As the downregulation of PP_1444 appeared to be the only instance resulting Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory in an increase in shinorine titer, it was further investigated to verify the downregulation of other target genes. Among the 21 targeted genes, it was observed that 10 were indeed downregulated, 3 exhibited no downregulation, and 8 could not be verified as the proteins were undetectable in both the sample and control strains (Fig.4, Panel D). Of the three genes that were not downregulated (PP_1010, PP_2930, and PP_4677), with the exception of PP_4677, their respective dCas9 expression levels were low (Fig.14, Panel C) although the expression levels of the gentamicin selection markers were consistently maintained in these strains (Fig.14, Panel A). In the case of PP_4677, the dCas9 expression level was comparable to that of other strains. This leads us to speculate that the lack of downregulation observed in PP_4677 may be attributed to the inefficiency of the designed sgRNA of PP_4677. It underscores the importance of designing more effective sgRNAs for future experiments. [00120] Upon further investigation, in addition to PP_1444, it was found that approximately 83 other genes were unexpectedly significantly downregulated in the PP_1444 strain (Fig.4, Panel E; Table 4). Among them, proteins involved in the metabolism of gluconate into 2-ketogluconate and 6PG (Fig.4, Panel A), such as Q88HH4, Q88HH5, and Q88HH6 (gluconate 2-dehydrogenase, encoded by PP_3384, PP_3383, and PP_3382, respectively), as well as Q88HI1 (ketogluconate-6-P-reductase, encoded by PP_3376) and Q88HH8 (2-ketogluconate epimerase, encoded by PP_3379), were downregulated. Phosphoglucomutase (Q88GY7, encoded by PP_3578), an enzyme responsible for glycogen biosynthesis from G6P, also appeared to be downregulated. This may redirect G6P pool for the synthesis of 6PG (Fig.4, Panel A). Additionally, L-serine dehydratase (Q88P66, encoded by PP_0987), which is involved in the conversion of L-serine to pyruvate, was also downregulated. This might preserve L-serine pool for shinorine biosynthesis. Interestingly, downregulating PP_0987 alone did not improve the shinorine titer (Fig.4, Panel C). In addition to massive, unexpected gene downregulation, it was found 73 genes were significantly upregulated in PP_1444 strain (Fig.4, Panel E, Table 5). For example, 5- methyltetrahydropteroyltriglutamate-homocysteine methyltransferase (Q88JF1, encoded by PP_2698), was upregulated by 28-fold. This enzyme may be responsible for maintaining L- methionine synthesis used for biosynthesis of S-adenosyl-L-methionine (SAM), a methyl donor for O-methyltransferase. [00121] Table 4. List of downregulated proteins. Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory Log2(Fold (-Log10(P- Protein.Group Protein.Names Protein Protein.Description Change) Value)) Category Rank Gluconate - Q88HH5 Q88HH5_PSEPK Pp_3383 dehydrogenase, putative 7.640747266 3.702096017 Downregulated 1 Acetyl-CoA hydrolase/transferase - Q88RH5 Q88RH5_PSEPK Pp_0154 family protein 6.185975706 3.013213077 Downregulated 2 Gluconate dehydrogenase cytochrome c subunit, - Q88HH6 Q88HH6_PSEPK Pp_3382 putative 6.035852694 1.918973926 Downregulated 3 Glucose dehydrogenase (Pyrroloquinoline- - Q88MX4 Q88MX4_PSEPK PP_1444 quinone) 5.847034898 4.650197854 Downregulated 4 Q88HH4 Q88HH4_PSEPK Pp_3384 Uncharacterized protein -5.15429634 4.409810077 Downregulated 5 Protein secretion ABC efflux system, permease and ATP-binding Q88PP4 Q88PP4_PSEPK Pp_0804 protein -4.6194112 3.365265851 Downregulated 6 - Q88F11 Q88F11_PSEPK Pp_4289 Uncharacterized protein 4.576858695 2.337946038 Downregulated 7 Outer membrane efflux - Q88PP3 Q88PP3_PSEPK Pp_0805 protein 3.770988686 1.604855886 Downregulated 8 Phosphoglucomutase, alpha-D-glucose Q88GY7 Q88GY7_PSEPK Pp_3578 phosphate-specific -3.6244109 3.36434885 Downregulated 9 Assimilatory nitrate reductase/sulfite - Q88M71 Q88M71_PSEPK Pp_1703 reductase, putative 3.512007926 2.775954064 Downregulated 10 Ribosomal RNA small subunit - Q88RR3 RSMB_PSEPK Rsmb methyltransferase B 3.294170507 2.996544506 Downregulated 11 Epimerase KguE, - Q88HH8 Q88HH8_PSEPK Pp_3379 putative 3.280290889 3.579416733 Downregulated 12 Chemotaxis protein, - Q88D41 Q88D41_PSEPK Pp_4987 putative 3.254041448 2.524796211 Downregulated 13 Deoxyuridine 5'- triphosphate - Q88C95 DUT_PSEPK Dut nucleotidohydrolase 3.217741206 2.689581495 Downregulated 14 2-ketogluconate 6- - Q88HI1 Q88HI1_PSEPK Kgud phosphate reductase 3.213563148 5.527378824 Downregulated 15 Transcriptional - Q88C83 Q88C83_PSEPK Pp_5300 regulator, LysR family 3.161717961 2.562688919 Downregulated 16 - Q88QY0 Q88QY0_PSEPK Pp_0354 CBS domain protein 3.095890416 4.6249159 Downregulated 17 Biosynthetic arginine - Q88QC7 SPEA_PSEPK Spea decarboxylase 2.934063229 4.007229943 Downregulated 18 ABC transporter, - Q88RL6 Q88RL6_PSEPK Pp_0113 permease protein 2.823397144 1.8784173 Downregulated 19 - Q88LY2 Q88LY2_PSEPK Pp_1794 Uncharacterized protein 2.802247995 4.508039386 Downregulated 20 Chemotaxis protein - Q88PP6 Q88PP6_PSEPK Chev-1 CheV 2.596580353 1.492783501 Downregulated 21 L-serine dehydratase, iron-sulfur-dependent, - Q88P66 Q88P66_PSEPK PP_0987 single chain form 2.591442116 1.518054521 Downregulated 22 - Q88IB0 Q88IB0_PSEPK Pp_3089 Uncharacterized protein 2.500283391 4.487760145 Downregulated 23 - Q88J05 URE2_PSEPK Ureb Urease subunit beta 2.427265402 2.568735002 Downregulated 24 Flagellar hook- - Q88ES7 Q88ES7_PSEPK Flid associated protein 2 2.314479829 3.192912301 Downregulated 25 Q88CF2 Q88CF2_PSEPK Pp_5229 Uncharacterized protein -2.30147798 2.063818138 Downregulated 26 Protein secretion ABC efflux system, membrane fusion - Q88PP5 Q88PP5_PSEPK Pp_0803 protein 2.263604883 2.019710523 Downregulated 27 Dipeptide ABC transporter, permease - Q88PG9 Q88PG9_PSEPK Dppb protein 2.250896966 2.253565262 Downregulated 28 Phosphatidate Q88MH5 Q88MH5_PSEPK Cdsa cytidylyltransferase -2.22756222 1.604498093 Downregulated 29 - Q88EW9 Q88EW9_PSEPK Pp_4333 CheW domain protein 2.213031402 1.593524571 Downregulated 30 Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory - Q88MR9 Q88MR9_PSEPK Pp_1500 Lipoprotein, putative 2.183504468 1.762224064 Downregulated 31 Probable malate:quinone - Q88NF9 MQO2_PSEPK Mqo2 oxidoreductase 2 2.085202878 3.60245995 Downregulated 32 - Q88NK1 Q88NK1_PSEPK Oprd Porin D 2.046775562 1.385599889 Downregulated 33 Surface adhesion - Q88PP2 Q88PP2_PSEPK Pp_0806 protein, putative 2.034965288 4.065969666 Downregulated 34 Q88IA6 Q88IA6_PSEPK Pp_3093 Uncharacterized protein -2.02274538 1.369970911 Downregulated 35 - Q88HB5 Q88HB5_PSEPK Pp_3445 Uncharacterized protein 1.959198476 1.568801329 Downregulated 36 Q88MB6 Q88MB6_PSEPK Pp_1658 Uncharacterized protein -1.91997879 2.290526619 Downregulated 37 Quinate dehydrogenase (Pyrroloquinoline- - Q88GZ6 Q88GZ6_PSEPK Pp_3569 quinone), putative 1.914927459 3.250345205 Downregulated 38 NADH-quinone oxidoreductase subunit - Q88FH7 NUOA_PSEPK Nuoa A 1.834759439 2.380011341 Downregulated 39 Dihydrodipicolinate - Q88L99 Q88L99_PSEPK Pp_2036 synthase, putative 1.811189889 1.998070253 Downregulated 40 NADH dehydrogenase - Q88FG7 Q88FG7_PSEPK Nuol I, L subunit 1.753050524 1.830821085 Downregulated 41 - Q88CZ5 Q88CZ5_PSEPK Pp_5034 Uncharacterized protein 1.733300908 3.791293647 Downregulated 42 Non-ribosomal peptide - Q88GD6 Q88GD6_PSEPK Pp_3788 synthetase, putative 1.680066873 1.688551796 Downregulated 43 - Q88M38 Q88M38_PSEPK Pp_1737 Uncharacterized protein 1.676813579 1.738602131 Downregulated 44 Lipoprotein releasing system, permease - Q88KY3 Q88KY3_PSEPK Lole protein 1.673735861 2.161114177 Downregulated 45 Glutathione S- transferase family - Q88QZ9 Q88QZ9_PSEPK Pp_0335 protein 1.667454465 1.970214817 Downregulated 46 - Q88CZ6 HUTU_PSEPK Hutu Urocanate hydratase 1.666394933 1.888125814 Downregulated 47 Hydrolase, haloacid dehalogenase-like Q88CF0 Q88CF0_PSEPK Pp_5231 family -1.66144076 1.772428389 Downregulated 48 GTP - Q88MB8 Q88MB8_PSEPK Rela pyrophosphokinase 1.651854063 3.34991041 Downregulated 49 Flagellar hook- - Q88ES2 Q88ES2_PSEPK Flgk associated protein FlgK 1.621798075 3.476651886 Downregulated 50 Flagellar hook- - Q88ES3 Q88ES3_PSEPK Flgl associated protein FlgL 1.616884997 3.640121665 Downregulated 51 - Q88FP8 Q88FP8_PSEPK Pp_4041 Uncharacterized protein 1.590268818 3.650931945 Downregulated 52 Sodium-solute symporter/sensory box histidine kinase/response - Q88M79 Q88M79_PSEPK Pp_1695 regulator, putative 1.548720877 2.655761364 Downregulated 53 Poly(3- hydroxyalkanoate) - Q88D24 Q88D24_PSEPK Pp_5004 depolymerase 1.544322078 2.865037581 Downregulated 54 - Q88LU9 Q88LU9_PSEPK Pp_1828 Uncharacterized protein 1.539808293 1.376615686 Downregulated 55 Glutamine synthetase, - Q88I53 Q88I53_PSEPK Pp_3148 putative 1.525180346 1.531119671 Downregulated 56 NADH-quinone oxidoreductase subunit - Q88FH1 NUOH_PSEPK Nuoh H 1.520031313 2.384796414 Downregulated 57 Soluble lytic transglycosylase, - Q88L07 Q88L07_PSEPK Pp_2130 putative 1.506900494 1.47322309 Downregulated 58 Acyl-CoA synthetase, - Q88GJ8 Q88GJ8_PSEPK Pp_3724 putative 1.393025235 1.922985872 Downregulated 59 - Q88P67 Q88P67_PSEPK Gcvt-1 Aminomethyltransferase 1.349548022 2.151564892 Downregulated 60 Transcriptional - Q88RG7 Q88RG7_PSEPK Pp_0163 regulator, GntR family 1.347213776 1.727705936 Downregulated 61 Methyl-accepting Q88LV8 Q88LV8_PSEPK Pp_1819 chemotaxis transducer -1.31136696 1.77084019 Downregulated 62 Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory Q88KQ0 Q88KQ0_PSEPK Pp_2240 protein, putative 1.025339541 2.558982374 Downregulated 84 [00122] Table 5. List of upregulated proteins. Protein.Group Protein.Names Protein Protein.Description Fold Change Change) Value)) Category Rank Q88FX3 Q88FX3_PSEPK Pp_3954 Periplasmic binding protein, putative 52.408004 5.711715259 1.950163191 Upregulated 1 5- methyltetrahydropteroyltriglutamate- Q88JF1 Q88JF1_PSEPK PP_2698 homocysteine methyltransferase 28.7842473 4.84720758 3.33197127 Upregulated 2 Ketoglutarate semialdehyde Q88GW5 Q88GW5_PSEPK Pp_3602 dehydrogenase 18.6346344 4.219914611 3.494649745 Upregulated 3 Q88I79 Q88I79_PSEPK Pp_3122 CoA-transferase, subunit A, putative 17.5120065 4.130272494 1.642963727 Upregulated 4 Indole-3-glycerol phosphate Q88QR6 TRPC_PSEPK Trpc synthase 15.7210765 3.974628107 4.691773936 Upregulated 5 Protocatechuate 3,4-dioxygenase, Q88E12 Q88E12_PSEPK Pcah beta subunit 14.8336964 3.890806238 3.079505614 Upregulated 6 Q88H28 Q88H28_PSEPK Poba 4-hydroxybenzoate hydroxylase 13.81804 3.788481087 1.778984498 Upregulated 7 Q88GK8 Q88GK8_PSEPK Cata Catechol 1,2-dioxygenase 13.5424966 3.759421823 1.542704137 Upregulated 8 Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory Q88K44 Q88K44_PSEPK Pp_2448 Uncharacterized protein 10.9505495 3.452931366 3.20922295 Upregulated 9 Q88HZ0 Q88HZ0_PSEPK Pp_3212 Rieske 2Fe-2S family protein 10.5533072 3.399623275 2.798760365 Upregulated 10 Q88CW0 Q88CW0_PSEPK Pp_5070 ParA family protein 8.97797975 3.166390843 2.424439163 Upregulated 11 Leucyl/phenylalanyl-tRNA--protein Q88FS7 LFTR_PSEPK Aat transferase 7.74071887 2.952467554 2.94202538 Upregulated 12 Q88HF7 Q88HF7_PSEPK Pp_3402 Uncharacterized protein 7.45150928 2.897532668 2.466424863 Upregulated 13 Dipeptide ABC transporter, Q88PG5 Q88PG5_PSEPK Pp_0885 periplasmic peptide-binding protein 7.40256831 2.888025899 3.743808768 Upregulated 14 Sugar ABC transporter, periplasmic Q88P38 Q88P38_PSEPK Pp_1015 sugar-binding protein 6.60925726 2.724488152 4.976240495 Upregulated 15 Q88BZ4 Q88BZ4_PSEPK Pp_5389 Uncharacterized protein 6.15933588 2.622774802 1.409824999 Upregulated 16 Sugar ABC transporter, permease Q88P36 Q88P36_PSEPK Pp_1017 protein 5.65699991 2.500037149 3.359690681 Upregulated 17 Dipeptide ABC transporter, ATP- Q88PH2 Q88PH2_PSEPK Dppf binding protein 5.6050645 2.486730974 1.847332029 Upregulated 18 Sigma-54 dependent transcriptional Q88HE1 Q88HE1_PSEPK Pp_3419 regulator/response regulator 5.49247745 2.457457043 1.359659088 Upregulated 19 Q88PF7 Q88PF7_PSEPK Pp_0893 ThiJ/PfpI family protein 4.67093 2.223709825 1.63618281 Upregulated 20 Q88PD7 Q88PD7_PSEPK Pp_0913 Uncharacterized protein 4.40314924 2.138535743 4.474518109 Upregulated 21 Q88QE0 Q88QE0_PSEPK Acob Acetoin dehydrogenase, beta subunit 4.2913686 2.101437825 2.46049567 Upregulated 22 Peptide ABC transporter, periplamic Q88FF0 Q88FF0_PSEPK Pp_4147 peptide-binding protein 4.2130399 2.074861579 1.322236917 Upregulated 23 Nucleoside diphosphate kinase Q88CF8 Q88CF8_PSEPK Rnk regulator 4.19603 2.069024991 2.527092515 Upregulated 24 Q88KQ7 Q88KQ7_PSEPK Pp_2233 Hydrolase, isochorismatase family 4.07442933 2.026598009 3.324474398 Upregulated 25 Q88NR1 Q88NR1_PSEPK Pp_1144 GGDEF domain protein 3.80081028 1.926307013 2.229544057 Upregulated 26 Sugar ABC transporter, permease Q88P37 Q88P37_PSEPK Pp_1016 protein 3.74460043 1.904811783 3.182483042 Upregulated 27 Q88KZ8 Q88KZ8_PSEPK Pp_2140 Uncharacterized protein 3.69210587 1.884443923 1.869762527 Upregulated 28 Oxygen-insensitive NAD(P)H Q88K59 Q88K59_PSEPK Pp_2432 nitroreductase 3.68919518 1.883306118 1.529412168 Upregulated 29 Aromatic-amino-acid Q88GX7 Q88GX7_PSEPK Tyrb-2 aminotransferase 3.67277511 1.876870559 3.984589652 Upregulated 30 Q88GX6 Q88GX6_PSEPK Pp_3591 Malate dehydrogenase, putative 3.54458253 1.82561572 1.945207737 Upregulated 31 Q88PQ1 Q88PQ1_PSEPK Pp_0797 Uncharacterized protein 3.34610991 1.742484835 1.704728555 Upregulated 32 Q88E32 Q88E32_PSEPK Pp_4636 Beta-ketothiolase 3.2463164 1.698803617 1.906473734 Upregulated 33 Multidrug efflux MFS membrane Q88NE0 Q88NE0_PSEPK Pp_1272 fusion protein, putative 3.05768322 1.612438948 3.035254919 Upregulated 34 Dipeptide ABC transporter, ATP- Q88PH1 Q88PH1_PSEPK Dppd binding protein 3.05759318 1.612396463 2.757440835 Upregulated 35 Sugar ABC transporter, ATP- Q88P35 Q88P35_PSEPK Pp_1018 binding subunit 3.0487297 1.608208247 4.146921516 Upregulated 36 Glyceraldehyde-3-phosphate Q88P44 Q88P44_PSEPK Gap-1 dehydrogenase 3.03667706 1.602493492 5.098100656 Upregulated 37 DNA-binding response regulator Q88P41 Q88P41_PSEPK Gltr-2 GltR 3.00203594 1.58594125 3.223831446 Upregulated 38 Multidrug efflux MFS outer Q88ND9 Q88ND9_PSEPK Pp_1273 membrane protein, putative 2.88686625 1.52950427 1.498491976 Upregulated 39 Branched-chain amino acid ABC transporter, periplasmic amino acid- Q88NR4 Q88NR4_PSEPK Brac binding protein 2.70014594 1.433037386 3.901875029 Upregulated 40 Anthranilate Q88QR7 TRPD_PSEPK Trpd phosphoribosyltransferase 2.68238302 1.423515253 3.382573721 Upregulated 41 Q88FI0 Q88FI0_PSEPK Acea Isocitrate lyase 2.64979586 1.405881219 2.501772997 Upregulated 42 Q88IC0 Q88IC0_PSEPK Ppic-2 Peptidyl-prolyl cis-trans isomerase C 2.64261318 1.40196526 2.448624944 Upregulated 43 Glycine betaine/L-proline ABC transporter, periplasmic binding Q88R38 Q88R38_PSEPK Pp_0296 protein 2.63026412 1.395207678 2.308483368 Upregulated 44 Exodeoxyribonuclease 7 small Q88QG5 EX7S_PSEPK Xseb subunit 2.6102214 1.384172182 1.441557258 Upregulated 45 Acetoin dehydrogenase, alpha Q88QD9 Q88QD9_PSEPK Acoa subunit 2.59347856 1.374888445 2.780810052 Upregulated 46 Q88QE2 Q88QE2_PSEPK Adh 2,3-butanediol dehydrogenase 2.5551391 1.353401831 2.005234109 Upregulated 47 Outer membrane lipoprotein, Q88NS3 Q88NS3_PSEPK Pp_1131 putative 2.55314799 1.352277163 1.983545328 Upregulated 48 4-hydroxyphenylpyruvate Q88JU3 Q88JU3_PSEPK Pp_2554 dioxygenase, putative 2.53711225 1.34318735 2.122322209 Upregulated 49 Cobalt/cadmium/zinc transporter, Q88RV3 Q88RV3_PSEPK Pp_0026 CDF family 2.51871358 1.332687073 1.546213133 Upregulated 50 Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory Alcohol dehydrogenase, zinc- Q88J22 Q88J22_PSEPK Pp_2827 containing 2.4690491 1.303955526 2.288416378 Upregulated 51 Q88KC6 Q88KC6_PSEPK Pp_2364 Uncharacterized protein 2.39780997 1.261717326 1.94909243 Upregulated 52 Q88QT3 Q88QT3_PSEPK Pp_0405 Uncharacterized protein 2.39029813 1.257190571 1.542770761 Upregulated 53 Q88DV4 TPIS_PSEPK Tpia Triosephosphate isomerase 2.38055357 1.251297093 3.178106687 Upregulated 54 Branched-chain amino acid ABC transporter, periplasmic amino acid- Q88DF8 Q88DF8_PSEPK Pp_4867 binding protein 2.34826837 1.231597294 3.227340331 Upregulated 55 Q88BY7 Q88BY7_PSEPK Pp_5400 Uncharacterized protein 2.32915409 1.219806087 2.470731355 Upregulated 56 General amino acid ABC transporter, periplasmic binding Q88NB5 Q88NB5_PSEPK Aapj protein 2.28207702 1.190347482 1.852186821 Upregulated 57 Transporter, NCS1 nucleoside Q88FQ2 Q88FQ2_PSEPK Pp_4035 transporter family 2.25503219 1.173148028 2.588023663 Upregulated 58 Q88I78 Q88I78_PSEPK Pp_3123 CoA-transferase, subunit B, putative 2.22252364 1.152198764 3.324176565 Upregulated 59 Q88QV5 PQQB_PSEPK Pqqb Coenzyme PQQ synthesis protein B 2.21264185 1.145769947 1.511128585 Upregulated 60 Q88QR8 Q88QR8_PSEPK Trpg Anthranilate synthase, component II 2.20708905 1.142144837 3.655438686 Upregulated 61 ABC transporter, periplasmic Q88RL7 Q88RL7_PSEPK Pp_0112 binding protein, putative 2.20173361 1.138639923 2.021039004 Upregulated 62 N-carbamoyl-beta-alanine Q88FQ3 Q88FQ3_PSEPK Pp_4034 amidohydrolase, putative 2.18522774 1.127783645 3.963963296 Upregulated 63 Alginate biosynthesis transcriptional Q88RJ6 ALGB_PSEPK Algb regulatory protein AlgB 2.16930256 1.117231286 2.585652472 Upregulated 64 Q88DJ8 Q88DJ8_PSEPK Cobi Precorrin-2 C20-methyltransferase 2.15793659 1.109652475 1.900859624 Upregulated 65 Q88I10 Q88I10_PSEPK Pp_3192 Uncharacterized protein 2.14176644 1.098801164 2.772069671 Upregulated 66 Betaine aldehyde dehydrogenase, Q88MT7 Q88MT7_PSEPK Pp_1481 putative 2.13156264 1.091911453 2.329629217 Upregulated 67 Q88GA2 Q88GA2_PSEPK Pp_3823 Cytochrome c-type protein 2.08949239 1.063152506 2.508671486 Upregulated 68 ABC transporter, periplasmic Q88M48 Q88M48_PSEPK Pp_1726 binding protein 2.08682424 1.061309095 1.626200399 Upregulated 69 Branched-chain amino acid ABC Q88NR8 Q88NR8_PSEPK Brag transporter, ATP-binding protein 2.06228614 1.04424452 1.738825142 Upregulated 70 Uncharacterized 2.0417519 1.029807572 1.597299489 71 Q88QZ8 MSRA_PSEPK Msra reductase MsrA 2.00321069 1.002314163 1.73982064 Upregulated 73 [00123] These results may demonstrate that the downregulation of PP_1444 not only reduces the accumulation of gluconate but also has far-reaching effects on the expression of multiple genes in Pseudomonas putida KT2440. Many of the listed proteins appear to be involved in various metabolic pathways, including amino acid metabolism and central carbon metabolism. Their direct roles in shinorine production are not clear. While some of these gene expression changes align with our goal of increasing shinorine production by redirecting carbon flux and preserving critical metabolites, others 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, offering promising avenues for further optimization and enhancing the yield of shinorine and other valuable metabolites in biotechnological applications. Pathway optimization through refining promoter usage and altering the strength of ribosome binding sites Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory [00124] In tandem with CRISPRi-mediated gene downregulation approach, it was sought to improve the shinorine titer by refining the design of the genetic constructs, including optimizing promoter usage, and altering the strength of ribosome binding sites. Previous studies have demonstrated the effectiveness of adjusting 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 for biosynthetic pathways involving multiple genes can be labor-intensive. To streamline this process, a modular linker- based plasmid construction method (Storch et al., 2015) was adopted to facilitate the creation of plasmid constructs with various RBSs, thereby tuning the expression levels of proteins involved in the shinorine pathway (Fig.5, Panel A). [00125] By employing two distinct promoters (PBAD and Ptrc1-O) and restructuring the shinorine BGC into either one or two transcriptional units, coupled with variations in RBSs, twenty-one different plasmids were successfully assembled (Fig.5, Panel B). This combinatorial approach led to a significant increase in shinorine production, elevating it from 100 mg/L (Fig.5, Panel C; JBx_250483, represented by the white bar) to approximately 467 mg/L shinorine (Fig.5, Panel C; JBx_250497, represented by the black bar) within a 72-hr post inoculation. [00126] Our shotgun proteomics analysis revealed that manipulating promoters and RBSs proved to be a viable strategy for enhancing the expression levels of shinorine pathway proteins (Fig.5, Panel D). The arabinose-inducible promoter P_BAD appeared to outperform the constitutive Ptrc1-O promoter. Substituting the native RBS with foreign RBS sequences, except for the native O-MT RBS, resulted in a significant increase in protein expression levels. By employing a foreign RBS, substantial improvements in the expression levels of DDGS, O- MT, ATP-grasp ligase, and NRPS in JBx_250497, were achieved with improvements of 2.1- fold, 2.6-fold, 40.5-fold, and 6.7-fold, respectively, compared to the original strain (JBx_250483) (Fig.5, Panels E-H). [00127] To discern the enzyme(s) with the most significant impact on shinorine production, a multiple linear regression analysis was performed. The Ordinary Least Squares (OLS) regression results indicate that the expression levels of O-MT and ATP-grasp ligase carry considerable statistical significance in predicting shinorine titer, whereas DDGS and NRPS do not appear to have a significant effect in this model. This may imply that further improvements in the expression levels of O-MT and ATP-grasp ligase could lead to a more Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory substantial increase in the shinorine titer. However, having exhaustively explored the available permutations of RBS variations, it was found that optimizing these two enzyme's expression levels beyond a certain point did not yield significant improvements in shinorine production (Fig.16). Therefore, while O-MT and ATP-grasp ligase play a crucial role in predicting shinorine titer, there may be other factors or pathways that need to be considered for further enhancement. Glycine and L-serine supplementation further improves shinorine production [00128] Following the successful implementation of CRISPRi-mediated gene downregulation and the refinement of genetic constructs, the two strategies were then combined to further improve the shinorine production. The highest shinorine producing strain (JBx_250497) was used as the base strain and co-transformed this strain with a CRISPRi plasmid harboring PP_1444 sgRNA or nontarget sgRNA (Fig.6, Panel A). The PP_1444 strain produced approximately 524 mg/L shinorine at 66 hr while the control nontarget strain produced approximately 365 mg/L shinorine (Fig.17). The glucose consumption profiles indicate that both samples completely consumed glucose after 42 hr. No significant growth and shinorine production were observed after 66 hr. These results might imply that the glucose supply was limiting the shinorine production under the tested condition. [00129] To increase the supply of glucose, 20 g/L of glucose was added at 18 hr. Upon the addition of extra glucose, the PP_1444 strain produced approximately 723 mg/L of shinorine at 66 hr (Fig.6, Panel B). The shinorine titer peaked at 902 mg/L at 90 hr. To explore if another set of glucose supplementation would further improve the titer, an extra 20 g/L of glucose was added at 42 hr (Fig.6, Panel C). While the shinorine titer has not peaked over 114 hr of fermentation, the highest shinorine titer was 900 mg/L achieved at 114 hr. These results indicate that another factor was potentially limiting the shinorine production. [00130] Shinorine biosynthetic pathway 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 (Fig.4, Panel A). The ATP-grasp ligase, a key enzyme in the shinorine biosynthetic pathway, facilitates the incorporation of glycine into 4- deoxygadusol to form mycosporine-glycine, which is a precursor of shinorine. Subsequently, an NRPS-like enzyme attaches a serine moiety to mycosporine-glycine, resulting in the formation of shinorine. Considering the role of these amino acids in shinorine biosynthesis, it Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory is hypothesized that their external supplementation could potentially alleviate any metabolic bottleneck due to limited intracellular availability, thereby enhancing the overall production of shinorine. [00131] To test this hypothesis, a series of fermentation experiments of PP_1444 and nontarget strains was conducted (Fig.6, Panel D) with supplementation of glycine and L- serine. The supplementation of glycine and L-serine resulted in a marked increase in shinorine production, both in the PP_1444 and nontarget strains (compared Fig.6, Panels E and B). For the PP_1444 strain, the shinorine titer increased sharply within the first 66 hr and then continued to steadily increase up to 1,134 mg/L at 114 hr (Fig.6, Panel E). Approximately 1.7 g/L of glucose remained in the liquid cultures at 66 hr. To further improve the shinorine titer, an extra 20 g/L of glucose was added at 42 hr (Fig.6, Panel F). Here, a sharp increase of shinorine titer was observed over 114 hr. The highest shinorine titer of 1,601 mg/L was achieved at 114 hr from the PP_1444 strain, where the strain completely consumed the glucose. [00132] Shinorine was found exclusively in the supernatant whereas for the PP_1444 strain, 91% and 9% shinorine was found in the supernatant and cell pellet, respectively (Fig. 7, Fig.18). From a biotechnological downstream processing perspective, these findings added significance to the use of P. putida KT2440 as a microbial platform for shinorine production. When the shinorine accumulates in the liquid medium rather than within the cells, it has several significant advantages for industrial biotechnology. Products in the supernatant can be harvested more easily as there is no need for cell disruption which simplifies the downstream processing and reduces costs (Ying Wang et al., 2019). It might also require less rigorous purification steps, which can be advantageous from a cost perspective. Additionally, if the product is secreted into the medium, the process could be adapted to a continuous production system where the product is continuously harvested while the culture is maintained. Accumulation of products within the cells can often lead to stress and eventual cell death. Secretion avoids this issue, maintaining cell viability and potentially increasing the overall yield of shinorine. MATERIALS AND METHODS Strains, plasmids, media, and growth conditions [00133] E. coli XL 1-Blue strain (Thermo Fisher Scientific) was used to propagate all Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory the plasmids used in the study. The strain was routinely cultivated in lysogeny broth (LB) medium (LB Broth, Sigma Aldrich), 37 °C, 180 rpm, and supplemented with appropriate antibiotic(s) (final concentration: gentamicin 10 μg/mL, and kanamycin 50 μg/mL). Plasmids were constructed using a modular plasmid assembly method, namely Biopart Assembly Standard Idempotent Cloning (BASIC) (Storch et al., 2015) with modification. [00134] Self replicating plasmids (RK2 and RSF1010-based plasmids) were transformed into Pseudomonas putida KT2440 by electroporation. The electroporation procedure was modified from (Choi et al., 2006). Briefly, one fresh colony of Pseudomonas putida KT2440 was inoculated into 5 mL of LB and incubated for overnight at 30 °C, 200 rpm. Overnight culture was centrifuged for 1 min at 13,000 x g, washed three times with 1 mL 10% glycerol, and resuspended in 500 μL of 10% glycerol at room temperature. Electroporation was performed by adding approximately 100 ng DNA into 100 μL cell aliquot and shocked with Bio-Rad GenePulser II (USA) using 1 mm cuvette (1.8k kV, 200 ^). After electroporation, a volume of 1 mL LB media was added into the cuvette and the cell mixtures were transferred into a fresh 1.5 mL microtube. For cell recovery, the cell mixtures were allowed to grow at 30 °C, 200 rpm, for 1 hr. After incubation, 20 μL of cell mixtures was plated onto a selective agar plate containing appropriate antibiotic and incubated at 30 °C overnight. Fluorescence measurement [00135] For fluorescence measurement taken by plate reader, overnight P. putida KT2440 liquid cultures grown in LB media containing appropriate antibiotic(s) (final concentration: gentamicin 10 μg/mL, and kanamycin 50 μg/mL) or otherwise stated were diluted 1,000 times in LB media. A volume of 200 μL liquid culture was grown in a 96-well plate and RFP fluorescence was measured using a Tecan Infinite F200 PRO instrument with excitation wavelength at 575 ^ 10 nm and emission wavelength at 620 ^ 10 nm for 24 hr with continuous shaking except when taking measurement. For measurement using the flow cytometer, 2 mL of LB media was inoculated with overnight culture (0.1% v/v) and supplemented with appropriate antibiotics. After 24 hr of incubation, 1-3 μL of samples was added to 150 μL 1X phosphate buffered saline. Single-cell RFP and GFP fluorescence from at least 30,000 cells was immediately recorded using a BD C6 Accuri flow cytometer (BD Bioscience). GFP and RFP fluorescence was measured using FL1 and FL4 detector, respectively. Protein fluorescent level was determined by taking the average of the Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory fluorescence distribution. Shinorine standard preparation [00136] Shinorine-producing strain was cultivated in 250 mL M9 medium in a 1-L flask with a starting OD6000.2. At 48 h post inoculation, sample was centrifuged at 4 °C, 4,500 x g for 30 min. 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% aceteonitrile. The eluent was evaporated to dryness on a LabConco SpeedVac. The dried SPE elution was then reconstituted in MilliQ water and semi-purified using an Agilent 1260 HPLC system equipped with a Machery-Nagel Nucleosil 100-10 SB strong anion exchange column (250 mm x 4.6 mm, 10 μm particle size) operating at a flow rate of 1 mL/min using an isocratic mobile phase composed 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 retention time for shinorine was approximately 12.4 minutes. Collected fractions were then evaporated to dryness on a LabConco SpeedVac. [00137] Dried fractions from anion exchange semi-purification were reconstituted in water and injected onto an Agilent 1260 HPLC system equipped with a Thermo HyperCarb column (150 mm x 4.6 mm, 5 μm particle size) operating at 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. Fractions were manually collected by monitoring the absorbance of the shinorine chromophore (λ = 334 nm). The observed retention time for shinorine was approximately 9.2 minutes. Fractions containing shinorine were then flash frozen in liquid nitrogen and placed on a LabConco lyophilizer. The dried fractions were reconstituted in water, frozen, and lyophilized three times to volatilize residual ammonium formate. The resulting dried solid of purified shinorine was then used for characterization by NMR spectroscopy. NMR spectroscopy of purified shinorine [00138] Approximately 4.6 mg of purified shinorine can be dissolved in 400 μL methanol-d₄ with 0.03% trimethylsilane (>99.8% atom % D; Sigma-Aldrich). NMR spectra were obtained on a Bruker Avance NEO 500 MHz equipped with a 5 mm ¹H/BB iProbe. Samples were held at 298 K during acquisition. Standard Bruker pulse sequences were used Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory for each of the following experiments: ¹H, ¹³C, ¹H-¹H COSY, ¹H-¹H NOESY (750 ms mixing time), ¹H-¹³C HSQC, and ¹H-¹³C HMBC. Spectra were recorded using the Bruker TopSpin 4.0.6 software and analyzed using MestReNova 14.3.2. Chemical shifts (δ, ppm) were referenced internally to trimethylsilane. Routine shinorine extraction and analysis [00139] For routine shinorine analysis from a whole liquid culture, 100 μL of liquid culture was mixed with 250 μL of methanol and 125 μL of chloroform. The resulting mixture was vortexed for 5 min at 3,000 rpm. Next, 125 μL of ultrapure water and 100 μL of chloroform were added and the samples were re-vortexed for 5 min at 3000 rpm followed by centrifugation for 1 min at 13,000 x g. Shinorine was then sampled from the top aqueous layer and measured using a NanoDrop™ 2000/2000c Spectrophotometers at 334 nm. To determine the concentration of shinorine in the sample, serially diluted purified shinorine standards were prepared and the concentration was determined using the Beer-lambert law with ε = extinction coefficient of shinorine (ε = 44,700 M⁻¹ cm⁻¹). (Llewellyn et al., 2020; Wada et al., 2015). [00140] For the analysis of extracellular shinorine, 100 μL of liquid culture was centrifuged for 5 min at 13,000 x g, and shinorine was measured from the supernatant without extraction. For the analysis of intracellular shinorine, 100 μL of liquid culture was centrifuged for 5 min at 13,000 x g. The supernatant was removed, and the cell pellet was washed three times with 500 μL of ultrapure water. Finally, the cell pellet was resuspended in 100 μL of ultrapure water and mixed with 250 μL of methanol and 125 μL of chloroform, following the extraction method described above for the whole liquid culture. CONCLUSION [00141] Growing interest in sourcing a sustainable and environmentally friendly sunscreen has motivated scientists to produce shinorine, a naturally occurring compound with UV-absorbing properties, in microbes. Our study provides a comprehensive approach for engineering Pseudomonas putida KT2440 as an efficient chassis for shinorine production. Comprehensive review of metabolic flux distribution from different microbes pinpoint Pseudomonas putida KT2440 as a potential host for shinorine production. By leveraging synthetic biology approaches and metabolic engineering strategies, shinorine yield have significantly increased and productivity CRISPRi-mediated gene downregulation, particularly Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory targeting the PP_1444 gene, was significantly improved shinorine production compared to the first-engineered strain. Proteomics analysis shows downregulation of PP_1444 has far- reaching effects on the expression of multiple genes in Pseudomonas putida KT2440. Refinement of genetic design, promoter usage, and ribosome-binding sites also contributed to the titer improvement. Feeding studies indicate that the supply of two critical amino acids, L- serine and glycine, might be the limiting factor in shinorine biosynthesis. The final titer, productivity, and yield of 900 mg/L or 10 mg/L/h or 22.5 mg/g glucose (without glycine and L-serine supplementation) and 1,601 mg/L or 14 mg/L/h or 26 – 28.35 mg/g glucose (with glycine and L-serine supplementation), respectively, represent a substantial improvement over the initial production levels and surpass achievements of earlier studies. Additionally, the exclusive secretion of shinorine into the culture medium offers advantages for downstream processing in industrial applications. These findings underscore the potential of P. putida KT2440 as a microbial platform for the production of valuable natural compounds. Through continued optimization and scale-up efforts, our work paves the way for the commercialization of shinorine as a bio-based alternative in the sunscreen industry. [00142] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory What is claimed is: 1. A genetically modified host cell capable of producing porphyra-334 and/or shinorine comprising: (a) (i) 2-demethyl 4-deoxygadusol synthase (DDGS), (ii) O- methyltransferase (O-MT or OMT), (iii) ATP-grasp ligase, and (iv) nonribosomal peptides 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 preceding enzymes is a homologous enzyme thereof. 2. The genetically modified host cell of claim 1, wherein the 2-demethyl 4-deoxygadusol synthase (DDGS) is a cyanobacteria DDGS, or homologous enzyme thereof. 3. The genetically modified host cell of claim 1, wherein the O-methyltransferase (O- MT or OMT) is a cyanobacteria OMT, or homologous enzyme thereof. 4. The genetically modified host cell of claim 1, wherein the ATP-grasp ligase is a cyanobacteria ATP-grasp ligase, or homologous enzyme thereof. 5. The genetically modified host cell of claim 1, wherein the nonribosomal peptides synthetase (NRPS) or D-ala-D-ala ligase is a cyanobacteria nonribosomal peptides synthetase (NRPS) or D-ala-D-ala ligase, or homologous enzyme thereof. I 6. The genetically modified host cell of claim 1, wherein the MysA, MysB, MysC, MysD, and/or MysE are independently derived or obtained from Anabaena ATCC 29413, Nostoc ATCC 29133, Porphyra umbilicalis, and/or Chondrus crispus. Cyanobacterial MysD or D-ala-ala ligase have a relaxed substrate specificity, with condensation of threonine instead of serine onto mycosporine-glycine to yield porphyra-334. 7. The genetically modified host cell of claim 1, wherein the DDGS, OMT, ATP-grasp ligase, and/or nonribosomal peptides synthetase (NRPS) or D-ala-D-ala ligase are each independently obtained or derived from Porphyra umbilicalis, Nostoc punctiforme, 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 incapable of metabolizing in nature. Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory 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 is an aromatic compound 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 for producing porphyra-334 and/or shinorine comprising: (a) providing a genetically modified host cell comprising: (1)(i) 2-demethyl 4-deoxygadusol synthase (DDGS), (ii) O-methyltransferase (O-MT or OMT), (iii) ATP-grasp ligase, and (iv) nonribosomal peptides 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 such that porphyra- 334 and/or shinorine is produced. 17. The method of claim 16, comprising (c) extracting or separating the porphyra- 334 and/or shinorine from the host cells, and/or culture or medium to form an isolated or purified porphyra-334 and/or shinorine. 18. The method of claim 16, comprising (d) mixing the porphyra-334 and/or shinorine with a lotion, oil or water to form a ultra-violet light (UV) blocking or filtering composition. Attorney Docket: 2021-119-02 Lawrence Berkeley National Laboratory 19. The method of claim 18, wherein the mixing step further comprises mixing the porphyra-334 and/or shinorine or the UV blocking or filtering composition with another active UV filtering agent. 20. The method of claim 18, wherein the active UV filtering agent is oxybenzone, octinoxate, octisalate and avobenzone, zinc oxide, and/or titanium dioxide, or a mixture thereof. 21. The method of claim 18, wherein the UV blocking or filtering composition is suitable for application on human skin. 22. The method of claim 18, wherein the UV blocking or filtering composition does not contain an ingredient or component that causes coral bleaching. 23. The method of claim 18, wherein the culturing or growing step (b) takes place 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 hydrolysate thereof. 26. The method of claim 25, wherein the biomass is obtained from softwood feedstock (such as poplar), hardwood feedstock, grass feedstock, and/or agricultural feedstock, or mixture thereof. 27. The method of claim 18, wherein the culture or medium comprises one or more aromatic compounds.