WO2020231426A1 - Production d'acides aminés de type mycosporine utilisant des souches de production améliorées et de nouvelles enzymes - Google Patents

Production d'acides aminés de type mycosporine utilisant des souches de production améliorées et de nouvelles enzymes Download PDF

Info

Publication number
WO2020231426A1
WO2020231426A1 PCT/US2019/032485 US2019032485W WO2020231426A1 WO 2020231426 A1 WO2020231426 A1 WO 2020231426A1 US 2019032485 W US2019032485 W US 2019032485W WO 2020231426 A1 WO2020231426 A1 WO 2020231426A1
Authority
WO
WIPO (PCT)
Prior art keywords
enzyme
seq
sequence identity
mycosporine
tery
Prior art date
Application number
PCT/US2019/032485
Other languages
English (en)
Inventor
Ulf DÜHRING
Frank ULICZKA-OPITZ
Original Assignee
Algenol Biotech LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Algenol Biotech LLC filed Critical Algenol Biotech LLC
Priority to PCT/US2019/032485 priority Critical patent/WO2020231426A1/fr
Publication of WO2020231426A1 publication Critical patent/WO2020231426A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids

Definitions

  • This application contains a sequence listing submitted by EFS-Web, thereby satisfying the requirements of 37 C.F.R. ⁇ 1.821-1.825.
  • the present invention relates generally to the recombinant production of known as well as novel mycosporine-like amino acids (MAAs) in cyanobacterial host cells and other host cells.
  • MAAs have UV protective ability and can be used as natural skin protectants, such as sunscreens.
  • Cyanobacteria can be utilized as bio-factories to produce compounds of interest from sunlight, CO2, and nutrients.
  • the transformation of the cyanobacterial genus Synechococcus with genes of interest has been described (U.S. Patent Nos. 6,699,696 and 6,306,639, both to Woods et al.).
  • the transformation of the cyanobacterial genus Synechocystis has been described, for example, in PCT/EP2009/000892 and in PCT/EP2009/060526.
  • the transformation of the cyanobacterial genus Cyanobacterium sp. has been described (U.S. Patent No. 8,846,369, U.S. Patent No. 9,315,832, and PCT/US2013/077364).
  • UV-A is regarded as the main cause of skin aging and wrinkling of human skin. Since both UV-A and UV-B are harmful, protection for both kinds of rays are recommended.
  • the FDA has approved a list of active chemical/physical ingredients for use in sunscreens which absorb into the top layers of skin. For some people, however, the ingredients can lead to skin irritation, allergic reactions and even skin damage or aging.
  • sunscreen component oxybenzone has recently been found to contribute to damage to coral reefs by bleaching of corals and disruption of coral reproduction and growth (Downs et al., “Toxicopathological Effects of the Sunscreen UV Filter, Oxybenzone (Benzophenone-3), on Coral Planulae and Cultured Primary Cells and Its Environmental Contamination in Hawaii and the U.S. Virgin Islands”, Arch Environ Contam Toxicol ., 70:265-88 (2016).
  • Mycosporine-like amino acids are natural, water-soluble, carbonaceous, nitrogenous compounds that absorb light in the UV-A/UV-B range between 310 and 362 nm.
  • MAAs are naturally produced by a number of organisms, including certain algae species, cyanobacteria, dinoflagellates and corals. MAAs apparently act as natural sunscreens in these organisms. In addition to their ability to act as a sunscreen, MAAs also have antioxidant activity (Wada et al.“Mycosporine-Like Amino Acids and Their Derivatives as Natural Antioxidants” Antioxidants 4:603-646 (2015)).
  • MAAs such as mycosporine-glycine, shinorine, porphyra-334, mycosporine-2-glycine, palythine, asterina, mycosporine-glutamic acid-glycine, my cosporine-orni thine, usujirene, and palythene (for a review, see Carreto et al.,“Mycosporine- Like Amino Acids: Relevant Secondary Metabolites”, Chemical and Ecological Aspects Mar Drugs , 9:387-446 (2011)).
  • MAA-based bio-sunscreen compounds are exclusively sourced from marine macroalgae, such as Porphyra umbilicalis , containing MAAs such as porphyra-334 and shinorine. Purified MAAs are not currently commercially available, but extracts from the red alga Porphyra umbilicalis are sold and used in cosmetic products (Helioguard® and Helionori®).
  • the Porphyra extracts are reported to contain the MAA porphyra-334 and shinorine with absorption coefficients (e molar) of 42,300 and 44,700 at 334 nm.
  • concentration of the MAAs in the extract is low: in the range of 1 % of total dry weight (Hartmann et al.“Quantitative analysis of mycosporine- like amino acids in marine algae by capillary electrophoresis with diode-array detection”, Jour. Pharm. Biomed. Analysis , 138: 153-157 (2017)).
  • cyanobacteria naturally produce lower amounts of MAAs (0.03 to 0.98 mg MAA/g dry cell weight) compared to P. umbilicalis (10 mg/g dry cell weight), complicating the industrial benefit of cyanobacteria (Garcia-Pichel et al.,“The phylogeny of unicellular, extremely halotolerant cyanobacteria”, Archives of Microbiology, 169:469-482 (1998).
  • modified host cells in particular cyanobacterial host cells, plasmid constructs, and methods to produce various MAAs from cyanobacterial cultures.
  • One embodiment of the present invention is directed to a genetically modified cyanobacterial cell for the production of mycosporine-glycine exhibiting a low pigment phenotype compared to the wildtype with respect to the chlorophyll and phycocyanin content of the cell, comprising
  • the first enzyme having at least 80% sequence identity, preferably at least 90% sequence identity with either the enzyme of SEQ ID NO: 3 or having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 68,
  • a second recombinant gene encoding a second enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 6 or the second enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 70,
  • a third recombinant gene encoding a third enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 9, or the third enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 72 (MysC of HL69).
  • MysC of HL69 Such a genetically modified cyanobacterial cell produces a higher amount of mycosporine glycine due to the fact that it exhibits a low pigment phenotype having a reduced content of chlorophyll and phycocyanin in the cell compared to the respective wildtype cyanobacterial cell.
  • Such a reduced content of the antenna complexes of the cyanobacteria ensures that cells with lower amount of pigment do not shade other cells to a great extent, so that the incident light can penetrate deeper into a cell culture and more light can reach the cells.
  • the second copy of the gene mysA also ensures that higher amount of the first intermediate compound desmethyl-4-deoxygadusol is present in the cyanobacterial cell leading to an increase in the production of the final MAA Mycosporine- glycine, which is produced from desmethyl-4-deoxygadusol by the further enzymatic actions of MysB and MysC.
  • Another embodiment of the present invention is directed to a genetically modified cyanobacterial cell for the production of a mycosporine-like amino acid precursor 4-deoxygadusol, comprising
  • a fusion recombinant gene encoding a fusion protein including first and second enzymes involved in the production of a mycosporine-like amino acid (MAA) in one polypeptide chain, the first enzyme having at least 80% sequence identity, preferably at least 90% sequence identity with either the enzyme of SEQ ID NO: 3 (MysA Ava_3858) or having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 68 (MysA- HL69), the second enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 6, (MysB Ava_3857) or having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 70 (MysB -HL69),
  • the first enzyme having at least 80% sequence identity, preferably at least 90% sequence identity with either the enzyme of SEQ ID NO: 3 or having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 68,
  • a second recombinant gene that encodes a second recombinant enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 6 (Ava_3857) or having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 70 (MysB_HL-69).
  • Such a genetically modified cyanobacterial cell is also able to produce a higher amount of the precursor 4- deoxygadusol due to the“ substrate channeling effect’ of the MysAB fusion protein.
  • genes coding for single proteins MysA and MysB in the genetically modified cyanobacterial cell ensures that the formation of multimeric protein complexes does not lead to the formation of aggregates of multiple MysAB fusion proteins, which either might be less active or completely inactive.
  • the genes coding for single proteins MysA and MysB can form for example dimers with the MysAB fusion protein so that a dimer can be formed between one MysAB fusion protein and one single MysA protein, which as shown in the experimental data section is still enzymatically active.
  • a further embodiment of the present invention is directed to a genetically modified host cell for the production of a mycosporine-like amino acid Tery-347, comprising
  • a first recombinant gene that encodes a first enzyme involved in the production of a mycosporine- like amino acid, the first enzyme having at least 80% sequence identity, preferably at least 90% sequence identity with either the enzyme of SEQ ID NO: 3 or having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 68,
  • a second recombinant gene that encodes the second recombinant enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 6 or the second enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 70,
  • a third recombinant gene encoding a third enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 9, or the third enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 72 (MysC of HL69).
  • a sixth recombinant gene encoding a sixth enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 60 or the sixth recombinant gene encoding a sixth enzyme having at least 80% sequence identity to SEQ ID NO: 92 ( Synechococcus sp. PCC 7335 O-methyltransferase),
  • Such a genetically modified host cell is able to produce a novel MAA compound called Tery-347, which has an absorption maximum at a wavelength of 347 nm.
  • a host cell can produce the different novel MAA compounds Tery-347.1, Tery-347.2 and Tery-347.3 as described herein.
  • Another embodiment of the present invention is directed to a genetically modified cyanobacterial host cell, wherein the O-methyltransferase Tery_2966 can be replaced with other enzymes having low sequence homology of 49% to 60% with Tery_2966. Furthermore, the invention is also directed to a genetically modified cyanobacterial host cell wherein the clavaminic acid synthetase-like enzyme Tery_2972 can be replaced with enzymes having a low sequence homology of 60 to 65 % to Tery_2972.
  • a further variant of the present invention is directed to genetically modified cyanobacterial host cells, wherein the nonribosomal peptide synthetase Tery_2968 is replaced with enzymes having a low sequence homology of 40% to 55% to Tery_2968.
  • Fig. 1 is a general biosynthetic pathway for the synthesis of several MAAs in cyanobacteria.
  • the main enzymes involved in the various conversion steps are indicated above the arrows for each individual step.
  • the enzymes MysA, MysB, and MysC catalyze the conversion of sedoheptulose-7-phosphate to mycosporine-glycine.
  • MysD enzymes attach different amino acids to mycosporine-glycine resulting in different MAAs, in particular shinorine, porphyra-334, mycosporine-2-glycine and mycosporine-glycine-alanine.
  • a first gene encoding for a first enzyme MysA catalyzes the conversion of sedoheptulose-7-phosphate to desmethyl-4-deoxygadusol.
  • This compound is subsequently converted to 4-deoxygadusol catalyzed by the enzyme family MysB, which is a second enzyme encoded by a second gene.
  • 4-deoxygadusol is converted to mycosporine-glycine via the attachment of a glycine catalyzed by the enzyme family MysC, a third enzyme encoded by a third gene.
  • MysA catalyzes the conversion of sedoheptulose-7-phosphate to desmethyl-4-deoxygadusol.
  • MysB is a second enzyme encoded by a second gene.
  • 4-deoxygadusol is converted to mycosporine-glycine via the attachment of a glycine catalyzed by the enzyme family MysC, a third enzyme encoded by a third gene.
  • a fourth gene encoding a fourth enzyme the so-called enzyme family D-alanine-D-alanine ligase“MysD2” can catalyze the formation of shinorine/porphyra-334 through the attachment of either serine or threonine to mycosporine- glycine.
  • Some of the enzymes in this MysD2 enzyme family catalyze the attachment of both serine and threonine to mycosporine-glycine resulting in a mixture of shinorine and porphyra-334 (i.e. D-alanine-D-alanine ligase MysD_HL-69 [ Cyanobacterium stanieri HL-69]).
  • the enzyme marked“Nv mysD” (D-alanine-D-alanine ligase MysD ⁇ Nostoc verrucosum KU005]) catalyzes mainly the attachment of threonine to mycosporine-glycine resulting in porphyra-334 (shinorine/porphyra-334 ratio around 10 to 90 for Nv_MysD) while the enzyme marked “Tery_2970” (D-alanine-D-alanine ligase MysD2 ⁇ Trichodesmium erythraeum IMS101]) catalyzes mainly the attachment of serine to mycosporine-glycine resulting in the formation of shinorine (shinorine/porphyra-334 ratio around 90 to 10 for Tery_2970).
  • Np_MysD NpF5597 of Nostoc punctiforme
  • MysDl Another D-alanine-D-alanine ligase enzyme family called“MysDl” catalyzes the attachment of glycine to mycosporine-glycine resulting in mycosporine-2-glycine.
  • This enzyme family of fifth enzymes is encoded by fifth genes.
  • MysDl Members of this“MysDl” enzyme family are for instance Ap_MysD (also referred to as “Ap3855” from Aphanothece halophytica ) and Tery_2971 (D-alanine-D-alanine ligase MysDl from Trichodesmium erythraeum IMS 101).
  • Ap_MysD also referred to as “Ap3855” from Aphanothece halophytica
  • Tery_2971 D-alanine-D-alanine ligase MysDl from Trichodesmium erythraeum IMS 101.
  • the denomination of the various enzyme families as first, second, third, fourth, fifth etc. enzymes encoded by the respective genes described in Fig. 1, will be used throughout the description of the invention.
  • Table IB also explains the nomenclature employed for the various genes and their respective enzymes for MAA production.
  • FIG. 2A to 2C are graphs representing the growth (FIG. 2A), mycosporine-2-glycine production (FIG. 2B), and the carbon partitioning (FIG. 2C) of low pigment strains AB4102 and AB4111 in comparison to the reference strain AB1334 in GC vial experiments.
  • FIG. 3A to 3D depict the cell growth (FIG. 3A), the mycosporine-2-glycine production (FIG. 3B), the carbon partitioning (FIG. 3C) and the whole cell absorbance spectra (FIG. 3D) of the two low pigment strains AB 4102 and AB4111 in comparison to the reference strain AB1334 in LvPBR cultivations.
  • FIG. 4A to 4C depict the graphs for the growth (FIG. 4A), the shinorine/porphyra-334 production (FIG. 4B) and the carbon partitioning (FIG. 4C) of the low pigment strain AB4101 in comparison to the reference strain AB4094 conducted in GC vial experiments.
  • FIG. 5A to 5D show the cell growth (FIG. 5A), the shinorine production (FIG. 5B), the carbon partitioning (FIG. 5C) and the whole cell absorbance spectra (FIG. 5D) of the low pigment strain AB4101 in comparison to the reference strain AB4094 in LvPBR cultivations.
  • FIG. 6A to 6D show the cell growth (FIG. 6A), the shinorine production (FIG. 6B), the carbon partitioning (FIG. 6C), and the whole cell absorbance spectra (FIG. 6D) for the low pigment strain AB4103 in comparison to the reference strain AB4068 in LvPBR experiments.
  • Fig. 7A and 7B depict the MAA content in the biomass (FIG. 7A) and the cell associated MAA fraction (FIG. 7B) over a time period of 25 days of cultivation for the low pigment strain AB4103 in comparison to the reference strain AB4068.
  • Fig. 7C and 7D depict the MAA content in the biomass (FIG. 7C) and the cell associated MAA fraction over a time period of 25 days of cultivation (FIG. 7D) for the low pigment strain AB4101 in comparison to the reference strain AB4094.
  • FIG. 8A and 8B show the general design of a MysAB fusion protein, and the“peptide linker X” (SEQ ID NO: 82) and“peptide linker Y” (SEQ ID NO: 84) located between the proteins MysA and MysB.
  • FIG. 9A to 9C show the cell growth (FIG. 9A), the shinorine production (FIG. 9B), and the carbon partitioning (FIG. 9C) for the strains AB4179 and AB4181 both expressing a MysAB fusion protein in comparison to the reference strain AB4100.
  • Fig. 10 shows proposed pathways for the synthesis of the novel, not yet known MAA compounds Tery-347.1, Tery-347.2 and Tery-347.3 employing the enzymes Tery_2966, Tery_2971, Tery_2970, Np_MysD, MysD_HL-69 andNv_MysD.
  • mycosporine-glycine can be converted into shinorine or porphyra-334 via the attachment of either serine or threonine catalyzed by a fourth enzyme encoded by a fourth gene (Tery_2970, Np_MysD, Nv_MysD or MysD_HL-69).
  • a fifth gene encodes a fifth enzyme including among others Tery_2971 or Ap MysD to produce mycosporine-2-glycine via the attachment of glycine.
  • These intermediate compounds shinorine/porphyra-334 or mycosporine-2-glycine can then be converted to the novel compounds Tery-347.1, Tery-347.2 or Tery-347.3 employing the enzyme Tery_2966, which is a sixth enzyme encoded by a sixth gene.
  • a“six gene encoding a sixth enzyme” will in the following be used for the enzyme family including Tery_2966 and OMT PCC7335 which catalyze the addition of a methyl group to shinorine/porphyra-334 or mycosporine-2-glycine.
  • Fig. 11 depicts the HPLC chromatogram of a reference strain AB1333, which produces mycosporine-glycine-alanine, porphyra-334 and shinorine.
  • Fig. 12 shows the HPLC chromatogram of the strain AB4105 producing a mixture of Tery- 347.1 and Tery-347.2.
  • Fig. 13A shows the HPLC chromatogram of the strain AB4104 producing Tery- 347.3.
  • Fig. 13B shows the HPLC profile of the E. coli strain #3186 expressing Ava_MysABC, Tery_2966 and the non-ribosomal peptide synthetase NRPS_NIES2100 from Calothrix sp. NIES-2100, which produces a Tery-347 compound. It is not yet known whether this compound is anyone of the Tery-347.1, Tery-347.2 or Tery-347.3 compounds disclosed herein or a structurally different MAA compound also having an absorption maximum at 347 nm.
  • Fig. 14 shows the absorbance spectra of MAA extracts including Tery-322 for the strains AB4074, AB4075, AB4076 and AB4090.
  • FIG. 15A and 15B show the HPLC spectra of the strains AB4075 (FIG. 15 A) and AB4076 (FIG. 15B) expressing O-methyltransferase-like enzymes having a low sequence homology to Tery_2966, both strains producing Tery-322.
  • Fig. 16 shows the HPLC profile of the ABCyano4 strain AB4140 expressing the low sequence homology enzyme for the enzyme Tery_2972.
  • Fig. 17 shows the gradient between the mobile phase A and mobile phase B employed during a hydrophobic interaction liquid chromatography (HILIC) run.
  • HILIC hydrophobic interaction liquid chromatography
  • Fig. 18 depicts the plasmid map of the plasmid #2848.
  • Fig. 19 depicts the plasmid map of the plasmid #2865.
  • Fig. 20 depicts the plasmid map of the plasmid #2891.
  • Fig. 21 depicts the plasmid map of the plasmid #2892.
  • Fig. 22 shows the plasmid map of the plasmid #2995.
  • Fig. 23 shows the plasmid map of the plasmid #3050.
  • Fig. 24 depicts plasmid map of the plasmid #3075.
  • Fig. 25 depicts the plasmid map of the plasmid #3095.
  • Fig. 26 shows the plasmid map of the plasmid #3096.
  • Fig. 27 shows the plasmid map of the plasmid #3110.
  • Fig. 28 shows the plasmid map of the plasmid #3122.
  • Fig. 29 shows the plasmid map of the plasmid #3123.
  • Fig. 30 shows the plasmid map of the plasmid #3125.
  • Fig. 31 shows the plasmid map of the plasmid #3113.
  • Fig. 32 depicts the plasmid map of the plasmid #3140.
  • Fig. 33 shows the plasmid map of the plasmid #3190.
  • Fig. 34 depicts the plasmid map of the plasmid #3213.
  • Fig. 35 shows the plasmid map of the plasmid #3214.
  • Fig. 36 depicts the plasmid map of the plasmid #3130.
  • Fig. 37 shows the plasmid map of the plasmid #3131.
  • Fig. 38 shows the plasmid map of the plasmid #3186.
  • Fig. 39 shows the general proposed pathways for the production of the MAA compounds shinorine, mycosporine-methylamine-serine, palythine, mycosporine-2-glycine, Tery-322, Tery- 364, Tery-347.2 and Tery-347.3.
  • Fig. 39 shows the general proposed pathways for the production of the MAA compounds shinorine, mycosporine-methylamine-serine, palythine, mycosporine-2-glycine, Tery-322, Tery- 364, Tery-347.2 and Tery-347.3.
  • a sixth enzyme encoded by a sixth gene including Tery_2966 can also catalyze the addition of a methyl group to mycosporine-glycine resulting in another related mycosporine-glycine (Tery-322), which is a methylated mycosporine-glycine variant with a molecular weight of 259 Da and an absorbance maximum at 322nm.
  • An eighth enzyme, encoded by the eighth gene, Tery_2968 further catalyzes the conversion of Tery-322 to the unknown MAA compound Tery-364, a MAA variant with a molecular weight of 356 Da and an absorbance maximum at 364nm.
  • a seventh enzyme, encoded by a seventh gene, Tery_2972 catalyzes the conversion of mycosporine-2-glycine into palythine.
  • a ninth enzyme encoded by a ninth gene, Tery_2969 catalyzes the decarboxylation of shinorine resulting in mycosporine-methylamine- serine or asterina-330.
  • Fig. 40 shows the plasmid map of one endogenous ABCyano4 plasmid, pABCyano4B, having an approximate copy number of 30-50 copies per cell in ABCyano4.
  • the integration site for the plasmids used for transformation of ABCyano4 via homologous recombination is indicated by an arrow.
  • Fig. 41 shows the plasmid map of one endogenous ABCyano4 plasmid, pABCyano4C, having an approximate copy number of 12-20 copies per cell in ABCyano4.
  • the integration site for the plasmids used for transformation of ABcyano4 via homologous recombination is indicated by an arrow.
  • Fig. 42 shows the plasmid map of the plasmid #3094.
  • Fig. 43 shows the plasmid map of the plasmid #2991.
  • Fig. 44 shows the plasmid map of the plasmid #3182.
  • Fig. 45 shows the plasmid map of the plasmid #3183.
  • Fig. 46 shows the plasmid map of the plasmid #3211.
  • Fig. 47 shows the plasmid map of the plasmid #3287.
  • Fig. 48 shows the plasmid map of the plasmid #3289.
  • MAA mycosporine-like amino acid
  • One embodiment of the present invention is directed to a genetically modified cyanobacterial cell for the production of mycosporine-glycine exhibiting a low pigment phenotype compared to the wildtype with respect to the chlorophyll and phycocyanin content of the cell, comprising
  • the first enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with either the enzyme of SEQ ID NO: 3 or having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 68,
  • a second recombinant gene encoding a second enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 6, or the second enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 70 (MysB of HL69),
  • a third recombinant gene encoding a third enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 9, or the third enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 72 (MysC of HL69).
  • MysC of HL69 Such a genetically modified cyanobacterial cell produces a higher amount of mycosporine glycine due to the fact that it exhibits a low pigment phenotype having a reduced content of chlorophyll and phycocyanin in the cell compared to the respective wildtype cyanobacterial cell.
  • the low pigment phenotype with regard to chlorophyll is due to a Aycf37 gene inactivation associated with a low chlorophyll phenotype.
  • the low phycocyanin phenotype is due to a promoter swap in the cpcBA operon.
  • the native PcpcB promoter upstream of the cpcBA operon was replaced by the endogenous PoprB promoter of ABCyano4.
  • the untranslated region of the cpcBA operon was retained in its native form.
  • the photoautotrophic cyanobacterial host cells can accumulate more biomass also leading to enhanced production of mycosporine-glycine and other related MAAs.
  • the second copy of the gene mysA, the first recombinant gene also ensures that higher amount of the first intermediate compound desmethyl-4-deoxygadusol is present in the cyanobacterial cell leading to an increase in the production of the final MAA Mycosporine-glycine, which is produced from desmethyl-4- deoxygadusol by the further enzymatic actions of MysB, the second recombinant protein and MysC, the third recombinant protein.
  • At least one, preferably two, most preferably all recombinant genes are operably linked to a promoter, which is inducible by the addition of an inducer, for example a change in metal-ion concentration.
  • inducers can preferably be promoters being inducible by the addition of zinc-ions, for example the PsmtA promoter or by the addition of copper-ions, for example the PpetE promoter.
  • the cell further is for the production of shinorine and/or porphyra-334, comprising a fourth recombinant gene encoding of fourth enzyme, which is at least 70%, preferably at least 80%, most preferably at least 90% identical to either one of the 12 (NpF5597), 52 (Tery_2970), 74 (MysD [ Cyanobacterium stanieri HL-69]), 76 (MysD ⁇ Nostoc verrucosum KU005]) or 78 (D-alanine-D- alanine ligase ⁇ Actinosynnema mirum DSM 43827]).
  • fourth enzyme which is at least 70%, preferably at least 80%, most preferably at least 90% identical to either one of the 12 (NpF5597), 52 (Tery_2970), 74 (MysD [ Cyanobacterium stanieri HL-69]), 76 (MysD ⁇ Nostoc verrucosum KU005]) or 78
  • This additional fourth recombinant gene encodes a fourth enzyme belonging to a heterogeneous group of so-called MysD2 enzymes, which can catalyze the conversion of mycosporine-glycine into shinorine and/or porphyra-334. In most cases these enzymes produce a mixture of shinorine and porphyra-334, except for the enzyme Nv_MysD (D-alanine-D-alanine ligase MysD ⁇ Nostoc verrucosum KU005]) and Tery_2970 (D- alanine-D-alanine ligase MysD2 ⁇ Trichodesmium erythraeum IMS 101]).
  • Nv_MysD D-alanine-D-alanine ligase MysD ⁇ Nostoc verrucosum KU005]
  • Tery_2970 D- alanine-D-alanine ligase MysD2 ⁇ Tri
  • a further variant of this genetically modified cyanobacterial cell can produce mycosporine- 2-glycine and further comprises a fifth recombinant gene encoding a fifth enzyme D-alanine-D- alanine ligase enzyme family called“MysDl” which is at least 70%, preferably at least 80%, most preferably at least 90% identical to SEQ ID NO. 15 (Ap3855 from Aphanothece halophytica ) and at least 70%, preferably at least 80%, most preferably at least 90% identical to SEQ ID NO. 50 (D-alanine-D-alanine ligase MysDl from Trichodesmium erythraeum IMSIOI).
  • These MAA producing enzymes can catalyze the attachment of glycine to mycosporine-glycine.
  • Another embodiment of the present invention is directed to a genetically modified cyanobacterial cell for the production of mycosporine-like amino acid precursor 4-deoxygadusol, comprising a fusion recombinant gene encoding a fusion protein including first and second enzymes involved in the production of a mycosporine-like amino acid (MAA) in one polypeptide chain, the first enzyme having at least 80 % sequence identity, preferably at least 90 % sequence identity with either the enzyme of SEQ ID NO: 3 or having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 68 (Ava-3858 MysA variant or distant clade MysA variant), the second enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 6 or having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 70 (Ava-3857 MysB variant or
  • a first recombinant gene that encodes the first enzyme involved in the production of a mycosporine-like amino acid (MAA), the first enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with either the enzyme of SEQ ID NO: 3 or having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 68 (Ava-3858 MysA variant or distant clade MysA variant), and
  • a second recombinant gene encoding a second enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 6 or having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 70 (Ava-3857 MysB variant or MysB_HL69 variant).
  • the fusion protein includes a fusion of the two enzymes MysA and MysB. Both enzymes can be separated by a short amino acid sequence in the fusion protein, for example the linker X or the linker Y as described herein.
  • the MysA-MysB fusion protein ensures, that the product of the reaction catalyzed by MysA, desmethyl-4-deoxygadusol is formed in the proximity of the second enzyme MysB, which converts this compound into 4-deoxygadusol, resulting in a so-called“ substrate channeling effect” increasing the amount of 4-deoxygadusol which is available for the synthesis of MAA compounds.
  • the protein MysA is known to form dimers, so that the genetically modified cyanobacterial cell for the production of 4-deoxygadusol also includes a separate single first recombinant gene, coding for MysA and a separate single second recombinant gene, coding for MysB.
  • This approach ensures that one single MysA protein can form a dimer-complex with one MysA-MysB fusion protein. This is supposed to avoid the formation of large aggregates of multimeric MysA-MysB fusion proteins, which might be less active or even inactive due to the large fusion proteins.
  • the genetically modified cyanobacterial cell including the gene for the MysAB fusion protein can furthermore include at least one third recombinant gene encoding a third enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO. 9 (Ava_3856 MysC), or the third enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 72 (MysC of HL69).
  • This third recombinant protein, MysC catalyzes the attachment of glycine to 4-deoxygadusol and the formation of mycosporine-glycine.
  • MysAB fusion protein due to the“ substrate channeling effect” of the MysAB fusion protein, such as genetically modified cyanobacterial cell also produces more mycosporine-glycine in comparison to genetically modified cyanobacterial cells, which do not include a MysAB fusion protein, but which only harbor separate MysA, MysB, and MysC proteins.
  • Another embodiment of the genetically modified cyanobacterial cell including the gene coding for the MysAB fusion protein can additionally include a fourth recombinant gene encoding a fourth enzyme with at least 70%, preferably at least 80%, most preferably at least 90% sequence identity to either one of the SEQ ID NO: 12, 15, 50, 52, 74, 76 or 78.
  • These MysD proteins catalyze the attachment of glycine to mycosporine-glycine resulting in mycosporine-2-glycine or serine to mycosporine-glycine resulting in shinorine or the attachment of threonine to mycosporine-glycine resulting in porphyra-334.
  • Np MysD can also attach alanine to mycosporine-glycine resulting in mycosporine-glycine-alanine.
  • non-ribosomal peptide synthetases such as Ava_3855 (SEQ ID NO. 18) can attach amino acids to mycosporine-glycine. Due to the presence of the MysAB fusion protein, such a genetically modified cyanobacterial cell also produces larger amounts of shinorine/porphyra-334 or mycosporine-glycine-alanine in comparison to other genetically modified cells lacking the MysAB fusion protein.
  • the fusion recombinant gene encodes a linker with between 18 to 24 amino acids between MysA and the MysB.
  • a linker is particularly suited to connect both MysA and MysB sections of the fusion protein, without interfering with the activity of either enzymes.
  • Another embodiment of the present invention is directed to a genetically modified host cell for the production of a mycosporine-like amino acid Tery-347, comprising
  • a first recombinant gene that encodes a first enzyme involved in the production of a mycosporine-like amino acid, the first enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with either the enzyme of SEQ ID NO. 3 or having at least 70%, preferably at least 80 %, most preferably at least 90 % sequence identity with the enzyme of SEQ ID NO. 68,
  • a second recombinant gene encoding a second enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 6, or the second enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 70 (MysB of HL69),
  • a third recombinant gene encoding a third enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO. 9 or the third enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 72 (MysC of HL69)
  • a sixth recombinant gene encoding a sixth enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO. 60 or the sixth recombinant gene encoding a sixth enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity to SEQ ID NO. 92 (O-methyltransferase from Synechococcus PCC 7335),
  • a further recombinant gene encoding a further enzyme wherein the further gene is selected from a group consisting of the following genes:
  • Tery- 347.3 can be produced, which is a methylated mycosporine-2-glycine.
  • the further gene is a fourth gene, encoding for the fourth enzyme as described above, a mixture of either Tery- 347.2/Tery-347.1 can be produced, or in the case of using Nv_MysD as the fourth gene, Tery- 347.1 can be formed.
  • Tery-347.3 is a methylated mycosporine-2-glycine having an absorption maximum at 347 nm.
  • Tery-347.2 is a methylated shinorine derivative also having its absorption maximum at 347 nm.
  • Tery-347.1 is a methylated porphyra-334 with an absorption maximum at 347 nm.
  • the inventors of the present invention also have found, that homologous enzyme with a low degree of homology exist for the sixth enzyme being encoded by the sixth recombinant gene, namely Tery_2966.
  • These enzymes can for example be O-methyltransferases from Synechococcus sp. PCC 7335 (49%) (SEQ ID NO: 92), Chroococcidiopsis sp. TS-821 (58%) (SEQ ID NO: 86), Euhalothece sp.
  • Subject matter of a further variant of the present invention is a mycosporine-like amino acid (Tery-347.1), being a methylated porphyra-334 and having an absorption maximum at 347 nm, being producible by culturing a genetically modified host cell, the genetically modified host cell comprising:
  • a first recombinant gene that encodes a first enzyme involved in the production of a mycosporine-like amino acid, the first enzyme having at least 80% sequence identity, preferably at least 90% sequence identity with either the enzyme of SEQ ID NO: 3 or having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO. 68,
  • a second recombinant gene encoding a second enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 6 or having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO. 70,
  • a third recombinant gene encoding a third enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 9 or having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO. 72,
  • a sixth recombinant gene encoding a sixth enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 60 or the sixth recombinant gene encoding a sixth enzyme having at least 80% sequence identity to SEQ ID NO: 92 (O-methyltransferase from Synechococcus PCC 7335), and
  • a fourth gene encoding a fourth enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 12.
  • NpF5597 or a fourth gene encoding a fourth enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 76
  • Nv_MysD or a fourth gene encoding a fourth enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 74
  • MysD_HL-69 MysD_HL-69
  • the mycosporine-like amino acid exhibits a retention time on a HPLC system with an analytical ZIC-HILIC column (150X, 2.1 mm; 3.5 pm; 200 A). In the area between 8.8 to 9.1 minutes, preferably 8.99 minutes.
  • the inventors have found, that different Tery- 347 MAA molecules can be produced, all of which show the maximum absorption of light at 347 nm, but which exhibit different chemical structures with different hydrophobicities and therefore show different retention times in a liquid hydrophobic interaction liquid chromatography (HILIC) (see also Examples 12 and 16).
  • Mass spectrometry analysis of Tery-347.1 using a Sciex 5600 TripleTOF mass spectrometer revealed the molecular weight of the compound to be 360 Da.
  • Tery- 347.1 has the molecular formula C15H24N2O8.
  • Another embodiment of the present invention is directed to a mycosporine-like amino acid (Tery-347.2), being a methylated shinorine and having an absorption maximum at 347 nm, being producible by culturing a genetically modified host cell, the genetically modified host cell comprising:
  • a first recombinant gene that encodes the first enzyme involved in the production of a mycosporine-like amino acid, the first enzyme having at least 80% sequence identity, preferably at least 90% sequence identity with either the enzyme of SEQ ID NO: 3 or having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 68,
  • a second recombinant gene encoding a second enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 6 or having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 70,
  • a third recombinant gene encoding a third enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 9 or having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 72,
  • a sixth recombinant gene encoding a sixth enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 60 (Tery_2966) or the sixth recombinant gene encoding a sixth enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity to SEQ ID NO: 92 (O- methyltransferase from Synechococcus sp. PCC 7335), and
  • a fourth gene encoding a fourth enzyme wherein the fourth gene is selected from a group consisting of:
  • a fourth gene encoding a fourth enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 52 (Tery_2970), and/or a fourth gene encoding a fourth enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 12 (NpF5597) or a fourth gene encoding a fourth enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 74 (MysD_HL-69).
  • the retention time of this MAA on an HPLC system with an analytical ZIC-HILIC column is in the area between 10.1 to 10.3 minutes, preferably 10.26 minutes (see also Examples 12 and 16).
  • Mass spectrometry analysis of Tery-347.2 using a Sciex 5600 TripleTOF mass spectrometer revealed the molecular weight of the compound to be 346 Da.
  • Tery-347.2 has the molecular formula C14H22N2O8.
  • Another embodiment of the present invention is directed to a mycosporine-like amino acid (Tery-347.3), being a methylated mycosporine-2-glycine and having an absorption maximum at 347 nm, being producible by culturing a genetically modified host cell, the genetically modified host cell comprising:
  • a first recombinant gene that encodes the first enzyme involved in the production of a mycosporine-like amino acid, the first enzyme having at least 80% sequence identity, preferably at least 90% sequence identity with either the enzyme of SEQ ID NO: 3 or having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 68,
  • a second recombinant gene encoding a second enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 6 or having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 70
  • a third recombinant gene encoding a third enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 9 or having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 72,
  • a sixth recombinant gene encoding a sixth enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 60 (Tery_2966) or the sixth recombinant gene encoding a sixth enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity to SEQ ID NO: 92 (O- methyltransferase from Synechococcus sp. PCC 7335), and
  • a fifth gene encoding a fifth enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 50 (Tery_2971) or a fifth gene encoding a fifth enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 15 (Ap3855).
  • a further variant of this mycosporine-like amino acid exhibits a retention time on a HPLC system with an analytical ZIC-HILIC column (150 X 2.1 mm, 3.5 pm, 200 A) in the area between 10.0 to 10.2 minutes, preferably 10.1 minutes (see Examples 12 and 16).
  • Tery- 347.3 has the molecular formula C 13 H 20 N 2 O 7 . Its molecular weight as determined by using a Sciex
  • 5600 TripleTOF mass spectrometer is 316. Based on the different retention times of the MAAs Tery-347.1, Tery-347.2 and Tery-347.3, it is clear that these compounds, although exhibiting the same absorption maximum at 347 nm, have different chemical structures with different hydrophobicities. As shown in Fig. 13B an E. coli strain #3186 expressing Ava_MysABC, Tery_2966 and a non-ribosomal peptide synthetase from Calothrix sp.
  • NIES-2100 having a low sequence homology to the non-ribosomal peptide synthetase Tery_2968 can also produce a MAA compound with an absorption maximum at 347 nm (Tery-347.x). It is not known whether the Tery- 347.x produced by this strain is one of the compounds Tery-347.1, Tery-347.2 or Tery-347.3 disclosed herein or a structurally distinct MAA also having an absorption maximum at 347 nm.
  • a further embodiment of the invention is directed to a mycosporine-like amino acid (Tery- 347.4), being a methylated mycosporine-glycine-alanine and having an absorption maximum at 347 nm, being producible by culturing a genetically modified host cell, the genetically modified host cell comprising:
  • a first recombinant gene that encodes the first enzyme involved in the production of a mycosporine-like amino acid, the first enzyme having at least 80% sequence identity, preferably at least 90% sequence identity with either the enzyme of SEQ ID NO: 3 or having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 68 (MysA),
  • a second recombinant gene encoding a second enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 6 or having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 70 (mysB),
  • a third recombinant gene encoding a third enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 9 or having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 72 (MysC),
  • a sixth recombinant gene encoding a sixth enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 60 (Tery_2966) or the sixth recombinant gene encoding a sixth enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity to SEQ ID NO: 92 (O- methyltransferase from Synechococcus sp. PCC 7335), and
  • a fourth gene encoding a fourth enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 12 (NpF5597 or Np_MysD).
  • This novel MAA compound Tery-347.4 is a methylated mycosporine-glycine-alanine with the chemical formula C 14 H 22 N 2 0 7 . Its molecular weight is 330 Da as determined with a Sciex 5600 TripleTOF mass spectrometer.
  • Another embodiment of the present invention is directed to genetically modified cyanobacterial host cell for the production of a mycosporine-like amino acid Tery-322, comprising
  • a first recombinant gene that encodes a first enzyme involved in the production of a mycosporine- like amino acid (MAA), the first enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity, preferably at least 90 % sequence identity with either the enzyme of SEQ ID NO: 3 or having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 68 (Ava-3858 MysA variant or distant clade MysA variant),
  • a second recombinant gene encoding a second enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 6 or having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 70 (Ava-3857 and MysB_HL-69 variant),
  • a third recombinant gene encoding a third enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 9 or having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 72 (Ava-3856 and MysC_HL-69 variant)
  • PCC 7335 O-methyltransferase (49%) (SEQ ID NO: 92), Chroococcidiopsis sp. TS-821 (58%) (SEQ ID NO: 86), Euhalothece sp. KZN 001 (55%) (SEQ ID NO: 88), and Chondrocystis sp. NIES-4102 (50%), wherein the percentages in parenthesis indicate the percentage of sequence identity to the enzyme Tery_2966 as determined with the NCBI database.
  • Example 17 disclosed herein describes, that the O-methyltransferase-like enzymes from Chroococcidiopsis sp. TS-821, Euhalothece sp. KZN 001 (SEQ ID NO: 88) and Synechococcus sp. PCC 7335 catalyze the same reaction as Tery_2966 leading to the production of Tery-322.
  • Such a genetically modified cyanobacterial cell can furthermore comprise a eighth recombinant gene encoding an eighth enzyme for the production of Tery-364, the eighth enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 56 (Tery_2968) or the eighth recombinant gene encoding an eighth enzyme having at least 80%, preferably at least 90% sequence identity to Euhalothece sp. KZN 001 (low sequence homolog of Tery_2968).
  • homologous proteins with a very low degree of homology to the enzyme Tery_2968 a non-ribosomal peptide synthetase, were found, which potentially catalyze the same reaction.
  • these enzymes are the non-ribosomal peptide synthetases from Oscillatoria sp. PCC 10802 (53%) (SEQ ID NO: 94), Chlorogloeopsis fritschii (51%) (SEQ ID NO: 96), Cyanothece sp. PCC 7424 (49%) (SEQ ID NO: 98), Nostocales cyanobacterium HT-58-2 (49%) (SEQ ID NO. 100), Scytonema cf.
  • Another embodiment of the present invention is directed to a genetically modified cyanobacterial host cell for the production of the mycosporine-like amino acid palythine, comprising:
  • a first recombinant gene that encodes a first enzyme involved in the production of a mycosporine- like amino acid (MAA), the first enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with either the enzyme of SEQ ID NO: 3 or having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 68 (Ava-3858 MysA variant or distant clade MysA variant),
  • a second recombinant gene encoding a second enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 6 or at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 70 (Ava-3857 and MysB_HL-69 variant),
  • a third recombinant gene encoding a third enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 9 or at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 72 (Ava-3856 and MysC_HL-69 variant)
  • a fifth gene encoding a fifth enzyme having at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 50 or at least 70%, preferably at least 80%, most preferably at least 90% sequence identity with the enzyme of SEQ ID NO: 15 (Tery_2971, MysDl and Ap_MysD), and
  • a seventh gene encoding a seventh enzyme having at least 80%, preferably at least 90% sequence identity with SEQ ID NO. 106 ( Calothrix sp. NIES-2100, low sequence homolog of Tery_2972, clavaminic acid synthetase-like).
  • homologous enzymes for the seventh enzyme encoded by a seventh gene, Tery_2972 were found when searching the NCBI database, namely SEQ ID NO. 190 Scytonema tolypothrichoides VB-61278 (63%), and Calothrix sp. NIES-2100 (61%). The percent numbers in parenthesis indicate the percentage of sequence identity to the protein Tery_2972.
  • Example 17 disclosed herein describes that Calothrix sp. NIES-2100 catalyzes the same chemical reaction as Tery_2972.
  • the present invention therefore also discloses alternative enzymes, which can be used for either the sixth enzyme, the seventh enzyme or the eighth enzyme and which still catalyze the same chemical reaction.
  • the invention disclosed herein also describes genetically modified host cells, cyanobacterial cells, wherein each of the recombinant genes is under the transcriptional control of a promoter which is an inducible promoter, and which for example can be induced by the addition of an inducer or which is a constitutive promoter.
  • the promoter can in particular be an inducible promoter which can be inducible by a change of a metal-ion concentration, further wherein the promoter is preferably inducible by an increase in the zinc concentration, such as the PsmtA or by an increase in the copper concentration, such as the PpetE promoter.
  • the mycosporine- like amino acids disclosed herein can all be produced by culturing the host cell, wherein any of the recombinant genes in the host cell are under the transcriptional control of the above described promoters.
  • the genetically modified host cell can be a host cell selected from the following group of host cells or the mycosporine-like amino acids disclosed herein can be produced by the cultivation of the following host cells selected from a group consisting of bacteria and eukaryotic cells, preferably actinobacteria and enterobacteria, such as E. coli , and more preferred cyanobacteria, such as Cyanobacterium sp., further wherein the cyanobacterial host cell is preferably Cyanobacterium sp. ABICyanol or Cyanobacterium sp. ABCyano4. In particular ABICyanol and ABCyano4 can produce high amounts of mycosporine- like amino acids and are also easy to cultivate as described herein.
  • Both of these cyanobacterial species exhibit an extracellular capsular polysaccharide layer (CPS), wherein the CPS of ABCyano4 is particularly thick and can have a thickness of between 1 to 3 pm, preferably between 1.1 to 2 pm.
  • CPS layers are particularly advantageous, because a large fraction of the MAAs produced during the cultivation can stay associated with these CPS layers, so that the MAAs can be particularly easy isolated from a culture, by centrifuging the culture and further isolating the MAAs from the cells as described herein.
  • anyone of the recombinant genes or all recombinant genes are from a cyanobacterium.
  • This variant of the present invention and particularly refers to the first to ninth recombinant genes, coding for the respective enzymes for the production of the MAAs.
  • Such a variant of the present invention is particularly preferred, when the host cell is a cyanobacterial host cell, such as ABICyanol or ABCyano4.
  • anyone of the recombinant genes can be codon optimized for improved expression in the cyanobacterial cell, in order to enhance the number of copies of active enzymes in the cell, leading to a higher production rate of the mycosporine-like amino acids.
  • anyone of the recombinant genes is located on a modified endogenous or a heterologous extrachromosomal plasmid.
  • any one of the recombinant genes is located on an endogenous extrachromosomal high copy plasmid, being present in a cell in at least 10, preferably at least 20, most preferably at least 40 copies per cell.
  • both of the preferred cyanobacterial strains, such as ABICyanol and ABCyano4 include endogenous high copy plasmids, such as endogenous plasmids pABCyano4-B and pABCyano4-C in ABCyano4 or p6.8 in ABICyanol .
  • Integrating the recombinant genes in an endogenous high copy plasmid, for example via homologous recombination ensures a high copy number of recombinant genes (high gene dosage) for MAA production in the host cells, thereby leading to a higher production rate of the MAAs.
  • a method for producing an MAA in a cyanobacterial cell or in the host cell comprising:
  • the host cells can be grown with light and CO2 addition, if the genetically modified host cells are cyanobacterial cells, which are photoautotrophic.
  • the method of producing the MAAs at least 60%, more preferred at least 70% of the MAAs produced, is associated with the cyanobacterial cell and wherein in step b) the MAAs are isolated from the cells by separating the cells from the culture medium and isolating the MAAs from the cells. After separation of the cyanobacterial cells from the culture medium for example by centrifugation, the MAAs can be isolated from the cells as disclosed herein, in particular in example 11.
  • the cyanobacterial cell can have a capsular exopolysaccharide layer (CPS) wherein the MAAs produced are associated with the cell via the CPS.
  • CPS capsular exopolysaccharide layer
  • Another embodiment of the present invention is directed to a pharmaceutical composition or a cosmetic composition comprising a UV absorbing compound, wherein the UV absorbing compound is an MAA that has been produced in the genetically modified cyanobacterial cell or host cell as described herein.
  • the term“about” is used herein to mean approximately, in the region of, roughly, or around. When the term“about” is used in conjunction with a numerical value/range, it modifies that value/range by extending the boundaries above and below the numerical value(s) set forth. In general, the term“about” is used herein to modify a numerical value(s) above and below the stated value(s) by a variance of 20%.
  • cyanobacterium refers to a member from the group of photoautotrophic prokaryotic microorganisms which can utilize solar energy and fix carbon dioxide. Cyanobacteria are also referred to as blue-green algae.
  • the terms“host cell” and“recombinant host cell” are intended to include a cell suitable for metabolic manipulation, e.g., which can incorporate heterologous polynucleotide sequences, e.g., which can be transformed.
  • the term is intended to include progeny of the cell originally transformed.
  • the cell is a prokaryotic cell, e.g., a cyanobacterial cell.
  • the term recombinant host cell is intended to include a cell that has already been selected or engineered to have certain desirable properties and to be suitable for further genetic enhancement.
  • “Competent to express” refers to a host cell that provides a sufficient cellular environment for expression of endogenous and/or exogenous polynucleotides.
  • the term“genetically modified” refers to any change in the endogenous genome of a wild type cell or to the addition of non-endogenous genetic code to a wild type cell, e.g., the introduction of a heterologous gene. More specifically, such changes are made by the hand of man through the use of recombinant DNA technology or mutagenesis.
  • the changes can involve protein coding sequences or non-protein coding sequences, including regulatory sequences such as promoters or enhancers.
  • polynucleotide and“nucleic acid” also refer to a polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof.
  • nucleotide polymers in which the nucleotides and the linkages between them include non-naturally occurring synthetic analogs.
  • nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C)
  • this also includes an RNA sequence (i.e., A, U, G, C) in which“U” replaces“T.”
  • nucleic acids of this present invention may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art.
  • modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages, charged linkages, alkylators, intercalators, pendent moieties, modified linkages, and chelators.
  • synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions.
  • nucleic acid also referred to as polynucleotide
  • nucleic acid molecules having an open reading frame encoding a polypeptide, and can further include non-coding regulatory sequences and introns.
  • the terms are intended to include one or more genes that map to a functional locus.
  • the terms are intended to include a specific gene for a selected purpose. The gene can be endogenous to the host cell or can be recombinantly introduced into the host cell.
  • the invention also provides nucleic acids which are at least 60%, 70%, 80% 90%, 95%, 99%, or 99.5% identical to the nucleic acids disclosed herein.
  • the percentage of identity of two nucleic acid sequences or two amino acid sequences can be determined using the algorithm of Thompson et al. (CLUSTALW, 1994, Nucleic Acids Research 22: 4673-4680).
  • a nucleotide sequence or an amino acid sequence can also be used as a so-called“query sequence” to perform a search against public nucleic acid or protein sequence databases in order, for example, to identify further unknown homologous promoters, which can also be used in embodiments of this invention.
  • any nucleic acid sequences or protein sequences disclosed in this patent application can also be used as a“query sequence” in order to identify yet unknown sequences in public databases, which can encode for example new enzymes, which could be useful in this invention.
  • Such searches can be performed using the algorithm of Karlin and Altschul (1990, Proceedings of the National Academy of Sciences U.S.A. 87: 2,264 to 2,268), modified as in Karlin and Altschul (1993, Proceedings of the National Academy of Sciences U.S.A. 90: 5,873 to 5,877).
  • Such an algorithm is incorporated in the NBLAST and XBLAST programs of Altschul et al. (1990, Journal of Molecular Biology 215: 403 to 410).
  • Suitable parameters for these database searches with these programs are, for example, a score of 100 and a word length of 12 for BLAST nucleotide searches as performed with the NBLAST program.
  • BLAST protein searches are performed with the XBLAST program with a score of 50 and a word length of 3. Where gaps exist between two sequences, gapped BLAST is utilized as described in Altschul et al. (1997, Nucleic Acids Research, 25: 3,389 to 3,402).
  • CyanoBase the genome database for cyanobacteria (http://bacteria.kazusa.or.jp/cyanobase/index.html); Nakamura et al. “CyanoBase, the genome database for Synechocystis sp. strain PCC6803 : status for the year 2000”, Nucleic Acid Research, 2000, Vol. 18, page 72.
  • the enzyme commission numbers (EC numbers) cited throughout this patent application are numbers which are a numerical classification scheme for enzymes based on the chemical reactions which are catalyzed by the enzymes.
  • Recombinant refers to polynucleotides synthesized or otherwise manipulated in vitro (“recombinant polynucleotides”) and to methods of using recombinant polynucleotides to produce gene products encoded by those polynucleotides in cells or other biological systems.
  • a cloned polynucleotide may be inserted into a suitable expression vector, such as a bacterial plasmid, and the plasmid can be used to transform a suitable host cell.
  • a host cell that comprises the recombinant polynucleotide is referred to as a“recombinant host cell” or a “recombinant bacterium” or a“recombinant cyanobacterium.”
  • the gene is then expressed in the recombinant host cell to produce, e.g., a“recombinant protein.”
  • a recombinant polynucleotide may serve a non-coding function (e.g., promoter, origin of replication, ribosome-binding site, etc.) as well.
  • the term“homologous recombination” refers to the process of recombination between two nucleic acid molecules based on nucleic acid sequence similarity.
  • the term embraces both reciprocal and nonreciprocal recombination (also referred to as gene conversion).
  • the recombination can be the result of equivalent or non- equivalent cross-over events. Equivalent crossing over occurs between two equivalent sequences or chromosome regions, whereas nonequivalent crossing over occurs between identical (or substantially identical) segments of nonequivalent sequences or chromosome regions. Unequal crossing over typically results in gene duplications and deletions.
  • non-homologous or random integration refers to any process by which DNA is integrated into the genome that does not involve homologous recombination. It appears to be a random process in which incorporation can occur at any of a large number of genomic locations.
  • exogenously refers to polynucleotides that are native to the host cell and are naturally expressed in the host cell.
  • a polynucleotide is“operably linked to a promoter” when there is a functional linkage between a polynucleotide expression control sequence (such as a promoter or other transcription regulation sequences) and a second polynucleotide sequence (e.g., a native or a heterologous polynucleotide), where the expression control sequence directs transcription of the polynucleotide.
  • a polynucleotide expression control sequence such as a promoter or other transcription regulation sequences
  • a second polynucleotide sequence e.g., a native or a heterologous polynucleotide
  • vector as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid generally refers to a circular double stranded DNA molecule into which additional DNA segments may be ligated, but also includes linear double-stranded molecules such as those resulting from amplification by the polymerase chain reaction (PCR) or from treatment of a circular plasmid with a restriction enzyme.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as“recombinant expression vectors” (or simply“expression vectors”).
  • A“promoter” is an array of nucleic acid control sequences that direct transcription of an associated polynucleotide, which may be a heterologous or native polynucleotide.
  • a promoter includes nucleic acid sequences near the start site of transcription, such as a polymerase binding site. The promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription.
  • the term “promoter” is intended to include a polynucleotide segment that can transcriptionally control a gene of interest, e.g., a pyruvate decarboxylase gene that it does or does not transcriptionally control in nature.
  • the transcriptional control of a promoter results in an increase in expression of the gene of interest.
  • a promoter is placed 5' to the gene of interest.
  • a heterologous promoter can be used to replace the natural promoter, or can be used in addition to the natural promoter.
  • a promoter can be endogenous with regard to the host cell in which it is used or it can be a heterologous polynucleotide sequence introduced into the host cell, e.g., exogenous with regard to the host cell in which it is used.
  • Promoters of the invention may also be inducible, meaning that certain exogenous stimuli (e.g., nutrient starvation, heat shock, mechanical stress, light exposure, etc.) will induce the promoter leading to the transcription of the gene.
  • the term“recombinant nucleic acid molecule” includes a nucleic acid molecule (e.g., a DNA molecule) that has been altered, modified or engineered such that it differs in nucleotide sequence from the native or natural nucleic acid molecule from which the recombinant nucleic acid molecule was derived (e.g., by addition, deletion or substitution of one or more nucleotides).
  • the recombinant nucleic acid molecule e.g., a recombinant DNA molecule
  • “gene” refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acids.“Gene” also refers to a nucleic acid fragment that expresses a specific protein or polypeptide, including regulatory sequences preceding (5' non coding sequences) and following (3' non-coding sequences) the coding sequence.
  • the term“endogenous gene” refers to a native gene in its natural location in the genome of an organism.
  • A“foreign” gene or“heterologous” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer.
  • Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes.
  • a “transgene” is a gene that has been introduced into the genome by a transformation procedure.
  • fragment refers to a nucleotide sequence of reduced length relative to the reference nucleic acid and comprising, over the common portion, a nucleotide sequence substantially identical to the reference nucleic acid.
  • a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent.
  • Such fragments comprise, or alternatively consist of, oligonucleotides ranging in length from at least about 6, 50, 100, 200, 500, 1,000, to about 1,500 or more consecutive nucleotides of a polynucleotide according to the invention.
  • ORF open reading frame
  • upstream refers to a nucleotide sequence that is located 5' to reference nucleotide sequence.
  • upstream nucleotide sequences generally relate to sequences that are located on the 5' side of a coding sequence or starting point of transcription. For example, most promoters are located upstream of the start site of transcription.
  • downstream refers to a nucleotide sequence that is located 3' to a reference nucleotide sequence.
  • downstream nucleotide sequences generally relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.
  • homology refers to the percent of identity between two polynucleotide or two polypeptide moieties.
  • the correspondence between the sequence from one moiety to another can be determined by techniques known to the art. For example, homology can be determined by a direct comparison of the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs. Alternatively, homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single- stranded-specific nuclease(s) and size determination of the digested fragments.
  • substantially similar refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence.
  • nucleic acid fragments of the instant invention also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotide bases that do not substantially affect the functional properties of the resulting transcript.
  • the terms“restriction endonuclease” and“restriction enzyme” refer to an enzyme that binds and cuts within a specific nucleotide sequence within double stranded DNA.
  • the term“expression”, as used herein, refers to the transcription and stable accumulation mRNA derived from a nucleic acid or polynucleotide. Expression may also refer to translation of mRNA into a protein or polypeptide.
  • an “expression cassette” or “construct” refers to a series of polynucleotide elements that permit transcription of a gene in a host cell.
  • the expression cassette includes a promoter and a heterologous or native polynucleotide sequence that is transcribed.
  • Expression cassettes or constructs may also include, e.g., transcription termination signals, polyadenylation signals, and enhancer elements.
  • codon refers to a triplet of nucleotides coding for a single amino acid.
  • codon-anticodon recognition refers to the interaction between a codon on an mRNA molecule and the corresponding anticodon on a tRNA molecule.
  • codon optimization refers to the modification of at least some of the codons present in a heterologous gene sequence from a triplet code that is not generally used in the host organism to a triplet code that is more common in the particular host organism. This can result in a higher expression level of the gene of interest.
  • transformation is used herein to mean the insertion of heterologous genetic material into the host cell.
  • the genetic material is DNA on a plasmid vector, but other means can also be employed.
  • General transformation methods and selectable markers for bacteria and cyanobacteria are known in the art (Wirth, Mol Gen Genet. 216: 175-177 (1989); Koksharova, Appl Microbiol Biotechnol 58:123-137 (2002). Additionally, transformation methods and selectable markers for use in bacteria are well known (see, e.g., Sambrook et al, supra).
  • selectable marker means an identifying factor, usually an antibiotic or chemical resistance gene, that is able to be selected for based upon the marker gene's effect, i.e., resistance to an antibiotic, resistance to a herbicide, colorimetric markers, enzymes, fluorescent markers, and the like, wherein the effect is used to track the inheritance of a nucleic acid of interest and/or to identify a cell or organism that has inherited the nucleic acid of interest.
  • selectable marker genes known and used in the art include: genes providing resistance to ampicillin, streptomycin, gentamycin, spectinomycin, kanamycin, hygromycin, zeocin, chloramphenicol, and the like.
  • A“polypeptide” is a polymeric compound comprised of covalently linked amino acid residues.
  • A“protein” is a polypeptide that performs a structural or functional role in a living cell.
  • A“heterologous protein” refers to a protein not naturally produced in the cell.
  • An“isolated polypeptide” or“isolated protein” is a polypeptide or protein that is substantially free of those compounds that are normally associated therewith in its natural state (e.g., other proteins or polypeptides, nucleic acids, carbohydrates, lipids).
  • fragment of a polypeptide refers to a polypeptide whose amino acid sequence is shorter than that of the reference polypeptide. Such fragments of a polypeptide according to the invention may have a length of at least about 2, 50, 100, 200, or 300 or more amino acids.
  • A“variant” of a polypeptide or protein is any analogue, fragment, derivative, or mutant which is derived from a polypeptide or protein and which retains at least one biological property of the polypeptide or protein.
  • Different variants of the polypeptide or protein may exist in nature. These variants may be allelic variations characterized by differences in the nucleotide sequences of the structural gene coding for the protein, or may involve differential splicing or post- translational modification. The skilled artisan can produce variants having single or multiple amino acid substitutions, deletions, additions, or replacements.
  • the invention also provides polypeptides or proteins that have amino acid sequences that are at least about 50%, 60%, 70%, 80% 90%, 95%, 97%, 99%, 99.5% or more identical to the amino acid sequences disclosed herein.
  • primer is an oligonucleotide that hybridizes to a target nucleic acid sequence to create a double stranded nucleic acid region that can serve as an initiation point for DNA synthesis under suitable conditions. Such primers may be used in a polymerase chain reaction.
  • PCR polymerase chain reaction
  • PCR refers to an in vitro method for enzymatically amplifying specific nucleic acid sequences. PCR involves a repetitive series of temperature cycles with each cycle comprising three stages: denaturation of the template nucleic acid to separate the strands of the target molecule, annealing a single stranded PCR oligonucleotide primer to the template nucleic acid, and extension of the annealed primer(s) by DNA polymerase. PCR provides a means to detect the presence of the target molecule and, under quantitative or semi-quantitative conditions, to determine the relative amount of that target molecule within the starting pool of nucleic acids.
  • MAA mycosporine-like amino acid
  • exemplary MAAs include, for example, mycosporine-glycine, shinorine, and mycosporine-2-glycine. These molecules absorb UV light, and exhibit photoprotection functions.
  • UV refers to light between the wavelengths of 280 - 400. This is divided between“UV-A” and“UV-B”.
  • UV-A refers to light between the wavelengths of 315 - 400 nm.
  • UV-B refers to light between the wavelengths of 280 - 315 nm.
  • selection-free refers to a growth medium that does not include a selection agent, such as an antibiotic, that would allow only cells having a functional selectable marker gene to survive.
  • heterologous gene in the context of genes coding for enzymes involved in the production of MAA and the term “MAA gene” will be used interchangeably. If more than one heterologous gene coding for enzymes involved in the production of MAA is present in a genetically modified cyanobacterial cell, then these genes will be referred to as the first heterologous gene, the second heterologous gene and so on.
  • extracellular polysaccharides EPS
  • capsulear polysaccharides CPS
  • EPS extracellular polysaccharides
  • CPS capsulear polysaccharides
  • FIG. 1 shows the general pathways for synthesizing MAAs, also naming enzymes, which were not yet implicated in the synthesis of MAAs.
  • MysA a family of homologous enzymes, which among others include the homologous enzymes Ava_3858, MysA_HL-69, and Tery_2977 convert this compound into desmethyl-4-deoxygadusol.
  • This intermediate compound is then subsequently converted into 4- deoxygadusol by the enzyme family MysB, which among others include the homologous enzymes Ava_3857, MysB_HL-69, and Tery_2976.
  • glycine can be attached to 4- deoxygadusol resulting in Mycosporine-glycine catalyzed by the enzyme family called MysC, which among others include the enzymes Ava_3856, MysC_HL-69, and Tery_2975.
  • MysD the enzyme family all named MysD.
  • Glycine can be attached to mycosporine-glycine resulting in mycosporine-2-glycine catalyzed by either one of the enzymes Ap3855 (Ap_MysD) or Tery_2971.
  • Ap3855 Ap_MysD
  • Tery_2971 The attachment of alanine to mycosporine-glycine leads to mycosporine-glycine-alanine catalyzed by the enzyme Np_MysD (NpF5597 of Nostoc punctiforme).
  • Threonine can be attached to mycosporine-glycine via the catalytic action of any of the enzymes MysD_HL-69, Np MysD (D- ala-D-ala ligase NpF5597 from [.
  • Fig. 39 depicts further proposed pathways for the formation of mycosporine-like amino acids, which are partly known from the literature.
  • Some of the MAAs, such as Tery-322, are a methylated mycosporine-glycine, Tery-364, Tery-347.3, which is a methylated mycosporine- 2-glycine, and Tery-347.2 which is a methylated shinorine are not yet known from the literature.
  • Table 1A includes a description of the genes used to genetically modify the cyanobacterial host cells for MAA production and their respective SEQ ID Nos in the sequence listing:
  • Table 1A Recombinant Genes and enzymes involved in MAA production encoded by the recombinant genes
  • Table IB includes a nomenclature for the recombinant genes and the respective enzymes encoded by these genes as used throughout this invention. Table IB also lists the substrates of the various enzymes and the products into which the substrates are converted by the various enzymes. Some of the enzymes can recognize more than one substrate or are able to convert the same substrate into different products. For example the genes and respective enzymes of the MysD2 family, referred to as the fourth genes encoding fourth enzymes can convert mycosporine-glycine into either one of shinorine, porphyra-334, mycosporine-glycine-alanine: Table IB: Nomenclature of Genes and enzymes involved in MAA production
  • At least some of the recombinant MAA genes to be expressed in cyanobacterial host cells can be codon improved for optimal expression in the target cyanobacterial strain.
  • the underlying rationale is that the codon usage frequency of highly expressed genes is generally correlated to the host cognate tRNA abundance. (Bulmer, Nature 325:728-730; 1987). Codon improvement (sometimes referred to as codon optimization or codon adaptation) can be performed to increase the expression level of foreign genes.
  • the codon usage for ABIcyanol is the same as for ABCyano4. Therefore, we refer to Table 3, the codon usage table of ABICyanol, which is disclosed in the PCT application WO 2014/100799A2 and which is incorporated in its entirety.
  • the inserted genes can be controlled by one promoter, or they can be controlled by different individual promoters.
  • the promoters can be constitutive or regulatable.
  • the promoters can be, for example, inducible.
  • the promoter sequences can be derived, for example, from the host cell, from another organism, or can be synthetically derived.
  • Any desired promoter can be used to regulate the expression of the inserted MAA biosynthesis genes.
  • Exemplary promoter types include but are not limited to, for example, constitutive promoters, regulatable promoters such as inducible promoters (e.g., by nutrient source, nutrient starvation, heat shock, mechanical stress, environmental stress, metal concentration, specific metabolites, light exposure, etc.), endogenous promoters, heterologous promoters, and the like.
  • Suitable promoter sequences are also disclosed, for example, in U.S. Patent No. 9,315, 820, U.S. Patent No. 9,551,014, PCT/EP2012/067534, U.S. Patent No. 9,476,067, U.S. Patent No. 9, 157,101, PCT/US2013/077364, U.S. Patent No. 9,493,794, and PCT/US2015/000210, all of which are hereby incorporated by reference in their entireties.
  • the recombinant MAA biosynthesis gene(s) can be under the transcriptional control of a constitutive promoter.
  • a constitutive promoter can be endogenous to the cyanobacterial cell. This has the advantage that no recombinant transcription factor has to be present in the host cell.
  • the endogenous promoter is usually well-recognized by the metabolically enhanced cyanobacterial cell without the need to introduce further genetic modifications.
  • Suitable constitutive promoters include, without limitation, the PrpsL promoter (Gene ID: ABICyanol_orfl758), PpsaA promoter (ABICyanol_orf 243), PpsbB (ABICyanol_orf2107), PcpcB promoter (ABICyanol_orf2472), PatpG (ABICyanol_orfl814), PrbcL promoter (ABICyanol_orfl369), and variations thereof.
  • Suitable endogenous constitutive promoters from genes with unknown function exhibiting appropriate transcriptional activity include, without limitation, the promoters of Gene IDs ABICyano_orfl924, ABICyano_orfl997, ABICyano orf 3446, ABICyano_orf0865, ABICyano_orfl919,
  • the promoters can be derived from the cyanobacterial strain Cyanobacterium sp. PTA-13311, or they can be derived from another cyanobacterium or from another organism. In an embodiment, the promoters can be about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100 % identical to the promoter sequences described herein.
  • the promoters can be regulatable promoters, such as inducible promoters.
  • certain promoters are up-regulated by the presence of a compound, which is called the inducer, while other promoters can be up-regulated by the absence of a compound (also termed “repressible”).
  • promoters that can be used include promoters that are regulatable by the presence (or in other promoters, by the absence) of inductors such as different metal ions, different nutrient sources, different metabolites, different external stimuli such as heat, cold, salinity or light.
  • the regulatable or inducible promoters are induced under conditions such as nutrient starvation or nutrient source, stationary growth phase, heat shock, cold shock, oxidative stress, salt stress, light, darkness, metal ions, organic chemical compounds, and combinations thereof.
  • a particularly tight control of the expression of gene can be achieved if a gene is under the transcriptional control of a Zn-, Ni-, Cu-, or Co-inducible promoter.
  • Exemplary Zn-regulatable promoters and their variants are described, for example, in International Application No. PCT/EP2013/077496.
  • Exemplary Zn, Ni, and Co-regulatable promoters are described, for example, in International Application No. PCT/2012/076790, both of which are incorporated by reference herein in their entireties.
  • the regulatable or inducible promoter is inducible by a change of a metal-ion concentration.
  • a change of metal-ion concentration includes for instance the addition or depletion of certain metal ions.
  • Suitable inducible promoters include, without limitation, the PziaA promoter, the PsmtA promoter, PaztA promoter, the PcorT promoter, the PnrsB promoter, the PpetJ promoter, the PpetE promoter, the Porf03 16, the Porf01460 promoter, the Porf0221 promoter, the Porf ' 0223 promoter, the Porf 126 promoter, the PmntC promoter, and variations thereof.
  • the promoter being inducible by a change of a metal-ion concentration can be inducible by Zinc-ions and/or Cu-ions, such as the PsmtA promoter or the PpetE promoter.
  • the regulatable or inducible promoter is endogenous to the cyanobacterial cell.
  • An endogenous inducible promoter is usually well-recognized by the metabolically enhanced cyanobacterial cell without the need to introduce further genetic modifications.
  • the choice of regulatable or inducible promoters can include, but are not limited to, PntcA, PnblA, PisiA, PpetJ, PpetE, PggpS, PpsbA2, PsigB, PlrtA, PhtpG, PnirA, PnarB, PnrtA, PhspA, PclpB l, PhliB, PcrhC, PziaA, PsmtA, PcorT, PnrsB, PnrsB916, PaztA, PbmtA, Pbxal, PzntA, PczrB, PnmtA, PpstS, and the like.
  • the inducible promoter can, for instance, also be a nitrate inducible promoter.
  • Suitable nitrate inducible promoters include, without limitation, the PnirA promoter, the PnrtA promoter, the PnarB promoter, and variations thereof.
  • truncated or partially truncated versions of these promoters including only a small portion of the native promoters upstream of the transcription start point, such as the region ranging from -35 to the transcription start can often be used.
  • introducing nucleotide changes into the promoter sequence e.g. into the TATA box, the operator sequence, 5’ -untranslated region and/or the ribosomal binding site (RBS) can be used to tailor or optimize the promoter strength and/or its induction conditions, e.g. the concentration of inductor required for induction.
  • the different inducible promoters are inducible by different metal ions.
  • the promoters PhspA, PclpBl, and PhliB can be induced by heat shock (raising the growth temperature of the host cell culture from 30°C to 40°C), cold shock (such as, for example, reducing the growth temperature of the cell culture from 30°C to 20°C), oxidative stress (for example by adding oxidants such as hydrogen peroxide to the culture), or osmotic stress (for example by increasing the salinity).
  • the promoter PsigB can be induced by stationary growth, heat shock, and osmotic stress.
  • the promoters PntcA and PnblA can be induced by decreasing the concentration of nitrogen in the growth medium and the promoters PpsaA and PpsbA2 can be induced by low light or high light conditions.
  • the promoter PhtpG can be induced by osmotic stress and heat shock.
  • the promoter PcrhC can be induced by cold shock.
  • An increase in copper concentration can be used in order to induce the promoter PpetE, whereas the promoter PpetJ is induced by decreasing the copper concentration. Additional details of these promoters can be found, for example, in PCT/EP2009/060526, which is incorporated by reference herein in its entirety.
  • the promoters of any of the above embodiments may be selected from the endogenous inducible promoters identified in Cyanobacterium sp. with the ATCC accession number PTA-13311 (“ABICyanol”) as listed in table 2A below, and variants thereof.
  • the promoters of any of the above embodiments may be selected from the endogenous inducible promoters identified in Cyanobacterium sp. with the ATCC accession number PTA-125253 (“ABCyano4”) as listed in table 2B below, and variants thereof.
  • Table 2B Cyanobacterium sp. ABCyano4 endogenous promoter sequences
  • truncated or partially truncated versions of these promoters including only a small portion of the native promoters upstream of the transcription start point, such as the region ranging from -35 to the transcription start can often be used.
  • introducing nucleotide changes into the promoter sequence e.g. into the TATA box, the operator sequence and/or the ribosomal binding site (RBS) can be used to tailor or optimize the promoter strength and/or its induction conditions, e.g. the concentration of inductor required for induction.
  • Table 2C shows the nucleotide sequences of two intergenic regions IScpcBA and IScpcBA* 1 employed to enhance the expression of the enzymes involved in the production of MAAs as described herein.
  • the cyanobacterial host cell is Cyanobacterium sp. ATCC accession number PTA-13311 (“ABICyanol”, also termed“ABl”). This strain has been found to grow well under various indoor and outdoor conditions, and can be genetically modified to produce a compound of interest. The strain, as well as its endogenous plasmid p6.8, has been described, for example, in U.S. Patent NO: 8,846,369, U.S. 9,315,832, and U.S. Patent NO: 9,157, 101, all of which are hereby incorporated by reference in their entireties. [00180] In another embodiment of the invention, the cyanobacterial host cell is Cyanobacterium sp.
  • ABCyano4 can produce more biomass than AB 1 in batch cultivation and exhibits a much thicker capsular exopolysaccharide layer (CPS layer) compared to AB1, so that MAAs produced with genetically modified ABCyano4 cells are mainly associated with this CPS layer or are produced intracellularly, enabling an easier purification of the MAAs.
  • CPS layer capsular exopolysaccharide layer
  • the strain as well as its endogenous plasmids pABCyano4-B and pABCyano4-C (FIG. 40 and 41), has been applied for recombinant MAA production by insertion of MAA biosynthesis genes of interest from different cyanobacterial origin.
  • the 16S rDNA of Cyanobacterium sp. ABCyano4 shows a high sequence identity of around 99% to the 16S rDNA sequences of different cyanobacterial species of the genus Cyanobacterium , including Cyanobacterium IHB-410, Cyanobacterium aponinum ETS-03, and Cyanobacterium sp. MBIC10216.
  • the 16S ribosomal RNA (rRNA) gene sequences (16S rDNA) of ABICyanol was predicted from the genome sequence with the RNAmmer program (Lagesen K, et al. (2007) RNAmmer: consistent and rapid annotation of ribosomal RNA genes.
  • a culture of the cyanobacterial cell is grown in an outdoor photobioreactor system, and the MAA that is produced can be isolated, for example, from the culture medium or from the cells.
  • a culture of the cyanobacterial cells is grown in an indoor photobioreactor system.
  • the method for producing a genetically enhanced Cyanobacterium sp. host cell uses an extrachromosomal plasmid derived from an endogenous plasmid of the host cell to introduce a recombinant nucleic acid sequence into the host cell.
  • This endogenous plasmid can be, for example, an extrachromosomal plasmid derived from the 6.8 kb endogenous plasmid of ABICyanol.
  • the cyanobacterial strain ABICyanol is transformed with at least one recombinant gene in order to produce the desired MAA. It has been found that the use of modified endogenous plasmids improves the stability of the plasmid in the host cell.
  • the cyanobacterial strain ABICyanol contains three endogenous plasmids. In combination with other genotypic and phenotypic attributes, these endogenous plasmids differentiate ABICyanol from other cyanobacterial species.
  • One plasmid is 6,826 base pairs, another is 39,702 base pairs, and a third plasmid is 28,554 base pairs.
  • the 6,826 bp endogenous plasmid is alternatively referred to herein as pABICyanol, p6.8 or 6.8.
  • plasmid 6.8 has been modified in vivo and in vitro for use as a plasmid vector containing genes of interest for the production of compounds of interest.
  • a modified endogenous vector derived from p6.8 from ABICyanol was developed.
  • the modified endogenous vector from ABICyanol can be used to transform cyanobacteria from a broad range of genera, including ABICyanol itself, as described in the PCT application WO 2014/100799 A2 which is hereby incorporated by reference.
  • the cyanobacterial strain ABICyano4 is transformed with the recombinant genes in order to produce the desired MAA. It has been found that the integration of recombinant genes into the endogenous plasmids improves expression of the respective enzymes.
  • the cyanobacterial strain ABCyano4 comprises three endogenous plasmids, which, in combination with other genotypic and phenotypic attributes, differentiate ABICyano4 from other Cyanobacterium species.
  • One plasmid herein termed“pABCyano4B” is 37990 base pairs.
  • a second, smaller plasmid“pABCyano4C” is 31678 base pairs.
  • both plasmids can be used for integration of recombinant genes via homologous recombination.
  • Table 3A Integrative plasmids for production of a MAAs in ABCyano4
  • Table 3B Plasmids for production of a MAAs in Escherichia coli
  • E. coli strains FIB 101 (Promega), NEB 10 and NEB Turbo (New England Biolabs), andEClOOD (Epicentre)) were grown in Luria-Bertani (LB) medium at 37 °C. carbencillin (100 pg/mL), kanamycin (50 pg/mL), and chloramphenicol (34 pg/mL), zeocin (25 pg/ml), streptomycin (25 pg/ml) were used when appropriate. Cultures were continuously shaken overnight at 200 rpm and at 100 rpm when used for conjugation.
  • ABICyanol and ABCyano4 were cultured at from 28 °C to 37 °C in liquid BG11 fresh water medium in bubbled bottles under continuous illumination or 12h/12h day/night cycles of approximately 30 - 40 pmol photons*m 2 *sec 1 .
  • Plasmid DNA from E. coli strains was isolated using a GeneJet Plasmid Miniprep Kit (Fermentas) according to the manufacture’s protocol.
  • total DNA was prepared according to Saha et al. (2005), World Jour. Microbiol Biotechnol 21 :877-881.
  • BG-11 stock solution was purchased from Sigma Aldrich (Sigma Aldrich, St. Louis, MO). Marine BG-11 (MBG-11) was prepared by dissolving 35 g Instant Ocean (United Pet Group, Inc, Cincinnati, OH) in 1 L water and supplementing with BG-11 stock solution. Vitamin B12 (Sigma Aldrich) was supplemented to MBG-11 to achieve a final concentration of 1 pg/L, as needed. Stock solutions of the antibiotics were purchased from Sigma Aldrich or Carl Roth GmbH).
  • the ABICyanol and ABCyano4 transformants were selected on solid BG11 medium containing 10 - 20 pg/mL of the appropriate antibiotic.
  • the choice of culture medium can depend on the cyanobacterial species.
  • the following BG11 medium for growing cyanobacteria can be used.
  • artificial seawater recipe Table 4 is used to prepare the culture medium to yield artificial seawater medium BG11, denoted “ASW BG11”.
  • the endogenous 6.8 kb plasmid of ABICyanol (p6.8) was used as a means of shuttling exogenous DNA to cyanobacterial host cells.
  • an origin of replication that is effective in E. coli such as R6Kori or oriVT
  • the p6.8 kb plasmid DNA was manipulated in bacteria, such as E. coli , to incorporate genes and sequences of interest into a recombinant p6.8 kb.
  • modifications to decrease the effectiveness of endogenous restriction systems that are present in ABICyanol, such as methylation can be performed.
  • the following method was used to treat cells of the strain Cyanobacterium sp. ABICyanol prior to conjugation.
  • the method involves several steps: treatment of cells with N-acetylcysteine (NAC), washing steps that utilize NaCl, treatment with lysozyme, and subsequent washing followed by a conjugation procedure.
  • NAC N-acetylcysteine
  • Two hundred mL of an exponentially growing culture (OD750nm greater than about 0.5 and less than about 1.0) was incubated with NAC for 2 days at 16 °C (end concentration of NAC is about 0.1 mg/mL) without shaking. This pretreatment was followed by several steps to weaken the cell wall.
  • the pretreated culture was pelleted at 4400 rpm and washed with 0.9% NaCl containing 8 mM EDTA.
  • the cell pellet was resuspended in 0.5 M sucrose and incubated 60 minutes at room temperature (RT) with slow shaking (85 rpm). Then, cells were centrifuged and resuspended in 40 mL of a solution containing 50mM Tris (pH 8.0), 10 mM EDTA (pH 8.0), 4% sucrose, and 20-40 pg/mL lysozyme.
  • Triparental mating was performed as follows: E. coli strain J53 bearing a conjugative RP4 plasmid and E. coli strain HBIOI bearing the cargo to be introduced into ABICyanol and the pRL528 helper plasmid (for in vivo methylation) were used. E. coli strains were grown in LB broth supplemented with the appropriate antibiotics overnight at 37 °C with shaking at 100 rpm. An aliquot of 3 - 5 mL of each culture was centrifuged, washed twice with LB medium and resuspended in 200 pL LB medium. Subsequently, the E.
  • coli strains were mixed, centrifuged and resuspended in 100 pL BG11 medium.
  • Two hundred mL of exponentially growing cyanobacterial culture (OD750nm of greater than 0.5 and less than 1.0) was centrifuged (3000 rpm, 10 minutes), pretreated as described in Example 4, and subsequently washed and resuspended in 400 pL BG11 culture medium containing Tris/sucrose buffer (Example 4).
  • a 100 pL aliquot of resuspended cyanobacterial and E. coli cultures was mixed and applied onto a membrane filter (Millipore GVWP, 0.22 pm pore size) placed on the surface of solid BG11 medium supplemented with 5% LB.
  • Petri dishes were incubated under dim light (5 pE/m 2 -sec ) for 2 days. Cells were then resuspended in fresh BG11 medium and plated onto selective medium containing 10 and 15 pg/mL kanamycin, respectively. The following selection conditions were used: light intensity of approximately 20 - 40 pE/m 2 -sec at a temperature of approximately 28 °C. Transformants were visible after approximately 7-10 days. The transformant colonies were then plated on BG11 media containing 15 pg/mL kanamycin and then transferred stepwise to higher kanamycin concentrations (up to kanamycin 60 pg/mL) to aid in the selection process.
  • Electroporation can also be used for successful transformation of Cyanobacterium sp. ABICyanol and ABCyano4, or other strains such as Arthrospira, Synechococcus , and Synechocystis , using, for example, the same plasmids as for conjugation, but with lower efficiency.
  • strain-specific adaptations of standard electroporation protocols can be made to avoid DNA digestion by endogenous restriction enzymes and to allow DNA entry through the CPS layer.
  • DNA is protected against endogenous restriction enzymes by methylation.
  • ABICyanol and ABCyano4 cells are pretreated with positively charged polyaminoacids such as poly-L-lysine hydrobromide or poly-L-ornithine hydrochloride or combinations thereof (in particular poly-L-lysine hydrobromide) in order to increase the DNA uptake efficiency.
  • the cells were washed sequentially once more with 1 mM HEPES and ETMT buffer containing 0.1 mM HEPES, 0.2 mM K2HPO4 and 0.2 mM MgCb.
  • the cells were harvested by centrifugation at 15000 x g for 5 minutes. All of the washes and centrifugations were carried out on ice or in a pre-chilled centrifuge (4 °C). For each electroporation procedure, 3 pg methylated DNA is added to 100 pL of concentrated cells.
  • E. coli strains were generally grown in LB broth supplemented with the appropriate antibiotics overnight at 37 °C with shaking at 100 rpm. An aliquot of 3-5 ml of each culture was centrifuged, washed twice with LB medium and resuspended in 200 pi LB medium.
  • the E. coli strains were mixed, centrifuged and resuspended in 100 m ⁇ BG11 medium.
  • a 100 m ⁇ aliquot of the resuspended cyanobacterial cells and the E. coli cultures was mixed and applied onto a membrane filter (Millipore GVWP, 0.22 pm pore size) placed on the surface of solid BG11 medium supplemented with 5% LB.
  • Petri dishes were incubated under dim light of 5 pmol photons m 2 s 1 for two days.
  • Cells were then resuspended in fresh BG11 medium and plated onto selective medium containing > 10 pg/ml of the respective antibiotic.
  • the following selection conditions were used: light intensity approximately 20 - 40 pmol photons m 2 sec 1 at a temperature of approximately 30 °C. Transformants were visible after approximately 3-4 days. The transformant colonies were then stepwise transferred to higher antibiotic concentrations.
  • Cultivations were performed in 1.2L LvPBRs in BG11 artificial seawater (35 psu).
  • the cultures were cultivated at a 12h / 12h dark / light cycle illuminated with 350 pE m 2 s 1 from one side of the LvPBRs. During the night the temperature was 25 °C with a ramping to 37 °C during the day.
  • the strains were cultivated at pH 7.3 ⁇ 0.01, controlled by CO2 (15 % CO2 in air) injection into the liquid phase. Mixing of the culture was ensured by continuous aeration at 38 mL / minutes.
  • the carbon-partitioning was determined by calculation from the OD measured at 750nm using an experimentally determined DW/OD ratio (1L ABCyano4 culture with an OD of 1 corresponds to 0.2g/L dry biomass).
  • MAA concentration was determined spectrophotometrically using Lambert-Beer’s law and known absorbance properties of the various MAAs (molar extinction coefficient and molecular weight) as described earlier.
  • a culture of modified cyanobacteria is scaled-up to flasks, then 1 liter containers, then 5 liter containers, then to outdoor or indoor photobioreactors, in a suitable culture medium, with the pH set at 7.3 by use of air bubbling with CO2 addition on demand. After 2 days, the host cells are induced to produce the MAA and production allowed to proceed for a specified period of time, usually not more than 30 days of continuous growth under production conditions.
  • a culture of MAA-producing cyanobacterial cells is grown and the production of the MAA of interest is induced in an indoor or outdoor photobioreactor. After a selected time period of growth, the culture is harvested by centrifugation or other means, and the MAA is collected from the cell-free medium using crossflow ultrafiltration combined with bulk ion exchange chromatography, or alternatively through absorption/de-absorption cycling using an organic substrate such as activated charcoal as an isolation matrix. The concentrated MAA is quantified and further purified, if needed. If desired, other products of interest are isolated from the culture in addition to the MAA, such as biomass, pigments, proteins, lipids, etc.
  • MAAs can be isolated from the concentrated culture biomass.
  • the cell biomass is harvested by centrifugation or other means of harvesting, with the result that the cells are separated from the culture medium.
  • the concentrated cell slurry is dried and the cells disrupted by grinding or other means.
  • the cellular material is resuspended in water or 30 %(v/v) ethanol in water, mixed for 30 minutes at room temperature, and centrifuged to separate the insoluble solid biomass from the soluble MAA.
  • the MAA-enriched cell extract can then be used without further purification, or it can be further purified, such as, for example, using crossflow ultrafiltration, centrifugation, and ion exchange, as needed.
  • the purity of the MAA is determined using mass spectrometry or by other means (i.e. photometrically in combination with a TOC analyzer).
  • the amount (and type) of MAA produced is quantified by Capillary Electrophoresis, following the method described in Hartmann et al. “Quantitative analysis of mycosporine-like amino acids in marine algae by capillary electrophoresis with diode-array detection”, Jour. Pharm. Biomed. Analysis, 138: 153-157 (2017).
  • Another suitable method for quantitation and analysis of MAAs is the use of HPLC, as described, for example, in Rastogi et al., Appl. Microbiol. 119:753-762 (2015). Hydrophilic interaction liquid chromatography (HILIC) is also utilized (Hartmann et al., Mar. Drugs 13 :6291-6305 (2015).
  • the flow rate was 0.3 mL/minutes and the diode array detector (DAD) was set to an absorption maximum of 320 nm.
  • the injection volume for each HILIC run was 50 pi.
  • the sample was prepared by diluting the sample (culture medium ASW or BG11) to 1 : 10 or 1 :5 with mobile phase A (Acetonitrile/ 5mM ammonium acetate in water 9: 1). The mixture is then centrifuged to allow the separation of eventual precipitate (not always visible) and further filtered through a PTFE filter (0.45 pm) and finally applied on the HILIC column.
  • the gradient elution mode was employed, starting with a mixture of 80 vol % mobile phase A and 20 vol % mobile phase B.
  • Fig. 17 shows the composition of the buffer during the gradient elution starting with 80 vol % mobile phase A and 20 vol % mobile phase B and then gradually increasing the fraction of mobile phase B until 100 vol % B are reached after 30 minutes. For 8 minutes 100 vol % B are maintained and subsequently within 1 minute the composition is gradually changed to of 80 vol % mobile phase A and 20 vol % mobile phase B.
  • An MAA is produced in an indoor or outdoor photobioreactor containing modified cyanobacterial strain according to the above examples.
  • the MAA is obtained from the culture, and is further purified using either a tangential cross-flow filtration unit or a batch addition of an ion exchange medium.
  • the MAA obtained via ion exchange method has a purity of at least 95% by HPLC analysis.
  • the MAA is further purified to about 99% using additional chromatography methods.
  • the purified MAA is mixed with one or more different MAAs (of different absorbance maxima) that have also been prepared from cyanobacteria. This mixture of various MAAs increases the breadth of the UV protection over a wider absorbance range.
  • the MAA mixture is then mixed with a naturally obtained, suitable carrier for application to human skin (such as an oil or a lotion) to result in a spreadable material that protects the skin from sun damage.
  • ABCyano4 cells AB4102 exhibiting a low content of both chlorophyll and phycocyanin in comparison to the wildtype cell were generated by transforming ABCyano4 cells AB1322 with the plasmid #2848 conferring a lower chlorophyll content, the plasmid #2995 conferring a lower phycocyanin content and plasmid #3113 encoding D-ala-D-ala ligase (Ap3855) from Aphanothece halophytica for mycosporine- 2-glycine production.
  • ABCyano4 cells AB4111 which in comparison to AB4102 additionally include two mysA (Ava_3858) copies instead of one copy were generated by transforming AB 1322 with the plasmids #2848, #2995 and plasmid #3140 encoding D-ala-D-ala ligase (Ap3855) from Aphanothece halophytica for mycosporine-2-glycine production and a further copy of the gene Ava_3858 (mysA).
  • GC vial data were generated as described above.
  • Fig. 2 A shows that the strain AB4111 exhibits a lower growth (OD750nm) compared to the reference strain AB1334 and the AB4102 cells only containing one mysA copy.
  • Fig. 3A to Fig. 3C show the corresponding data for cell growth, mycosporine-2-glycine production and carbon partitioning for cells grown in LvPBRs.
  • the cells were cultured in LvPBRs as mentioned above.
  • the AB4111 cells with two mysA (Ava_3858) copies produce the highest amount of mycosporine-2-glycine.
  • Fig. 3D shows that in the strain AB4102 exhibiting a lower content of phycocyanin than strain AB1334 shows a reduced absorption at 615 nm, the absorption maximum of phycocyanin. Absorption at 680 nm for chlorophyll a is also reduced.
  • AB4094 cells served as a reference strain.
  • ABCyano4 strain AB4101 with a low content of antenna complexes was transformed with all the plasmids of strain AB4094.
  • AB4101 furthermore exhibits a lower chlorophyll content owing to transformation with plasmid #2848, and a lower phycocyanin content due to transformation with plasmid #2995.
  • Fig. 4A, 4B and 4C show that AB4101 with two copies of mysA and a low content of chlorophyll and phycocyanin antenna complexes in comparison to the wildtype strain produces more shinorine/porphyra-334 than AB4094 (Fig. 4B), but exhibits a similar growth as assessed by the OD750 nm (Fig. 4A).
  • the carbon-partitioning towards the production of shinorine/porphyra-334 is also higher for AB4101 (Fig. 4C).
  • Fig. 5 A to 5D The corresponding experimental data for LvPBR cultivations for both strains AB4094 and AB4101 are shown in Fig. 5 A to 5D. These Fig. indicate again that the production rates for shinorine/porphyra-334 are higher for the AB4101 strain exhibiting a low chlorophyll and phycocyanin content compared to AB4094.
  • Fig. 5D shows that the absorption intensity at 615 nm (phycocyanin) and 680 nm (chlorophyll) is reduced for AB4101 compared to AB4094 due to the lower content of both antenna complexes.
  • Fig. 6A to Fig. 6D present the cell growth, the shinorine/porphyra-334 production, carbon partitioning and the whole cell absorbance spectra for the ABCyano4 strains AB4068 and AB4103 obtained by LvPBR cultivations.
  • AB4068 exhibits a wildtype level of antenna complexes and is able to produce shinorine/porphyra-334 due to integration of plasmid #2865 containing Anabaena variabilis mysA, mysB and mysC genes (Ava_3858, Ava_3857 and Ava_3856) and the integration of plasmid #3075 including a second copy of Ava_3858 and the gene mysD of HL 69.
  • the strain AB4103 was transformed with plasmids #2848 and #2995 conferring a lower chlorophyll and phycocyanin content.
  • FIG. 6A shows that the cell growth for both strains is comparable until day 12 of the cultivation with AB4103 performing better from that point on to day 25 of the cultivation.
  • Fig. 6B and 6C demonstrate that the shinorine/porphyra-334 production and the carbon partitioning towards shinorine/porphyra-334 is better for the low pigment strain AB4103.
  • Fig. 6D demonstrates the reduced absorption at 615 nm for phycocyanin and at 680 nm for chlorophyll for the strain AB4103.
  • Fig. 7A evidences that a higher amount of MAA of the low pigment strain AB4103 stays associated with the biomass (MAA content in dry weight) compared to the strain AB4068 exhibiting a wildtype level of the antenna complexes chlorophyll and phycocyanin. Consequently, Fig. 7B shows that initially all the MAAs produced in the cell remain in or at the cell during day 1 (100% cell-associated MAA) and subsequently the amount of cell associated MAA decreases over the course of the cultivation until day 25, because a fraction of the MAAs is released into the cultivation medium.
  • FIG. 7C evidences that a higher amount of MAA of the low pigment strain AB4101 stays associated with the biomass (MAA content in dry weight) compared to the strain AB4094 exhibiting a wildtype level of the antenna complexes chlorophyll and phycocyanin.
  • Fig. 7D illustrates that after day 20 of the cultivation more than 60% of the MAAs were cell-associated for the low pigment strain AB4101, whereas less than 50% were found to be cell-associated for the strain AB4094 with wild-type pigmentation.
  • Fig. 8A and Fig. 8B depict the general principle of increasing the production of MAAs by introducing a MysAB fusion protein in order to allow a “ substrate channeling effect’ taking place, leading to a higher production of MAAs.
  • This“ substrate channeling effect’ due to the presence of a MysAB fusion protein allows the rapid conversion of the intermediate desmethyl-4-deoxygadusol produced by MysA into 4-deoxygadusol by the enzyme MysB, because both enzymes are in close proximity in the fusion protein.
  • the ABCyano4 strain AB4100 serves as a reference strain and was transformed with the plasmids #2991 including the genes Ava_3858 (mysA) and NpF5597 (mysD) and the plasmid #3123 including the genes mysA, mysB and mysC (AvA_3858, AvA_3857, and AvA_3856) without including MysAB gene fusion.
  • the ABCyano4 strain AB4179 was transformed with the same plasmid #3123 as reference strain AB4100, but not the plasmid #2991.
  • AB4179 was transformed with the plasmid #3213 encoding a MysAB fusion protein, wherein MysA and MysB are connected via the so-called “ linker A” and additionally containing NpF5597 (NP_mysD).
  • a second ABCyano4 strain AB4181 was generated by transforming ABCyano4 with the plasmids #3123 and #3214.
  • Plasmid #3214 includes the genes coding for a MysAB fusion protein, wherein MysA and MysB are connected via the so called“ linker F” and the plasmid additionally contains a gene coding for NpF5597 (MysD).
  • FIG. 8A also shows the amino acid sequences of both peptide linkers,“ linker A” and“ linker T Fig. 8B represents the differences in the expression of the various proteins between the reference strain AB4100 and the two strains AB4179 and AB4181 both expressing the recombinant MysAB fusion protein.
  • Both strains AB4179 and AB4181 also include genes coding for the single proteins MysA and MysB. This approach ensures, that the formation of large aggregates of MysAB fusion protein multimers, which might be enzymatically inactive, can be avoided.
  • the protein encoded by the gene mysA is known to form a dimer (ACS Chem Biol. 2017 Apr 21;12(4):979-988).
  • both the MysAB fusion protein, as well as the single MysA proteins enables the formation of dimers of for example the MysAB fusion protein together with single MysA monomers, which are enzymatically active as evidenced by the above discussed experimental data.
  • Fig. 9A, 9B and 9C depict the cell growth (OD750nm), the formation of shinorine/porphyra-334 (Fig. 9B) and the carbon partitioning (Fig. 9C) for the reference strain and for both strains overexpressing the recombinant MysAB fusion protein.
  • the strains overexpressing the recombinant MysAB fusion protein, AB4179 and AB4181 exhibit improved properties with regard to the growth and up to 30 % improved production rate for shinorine/porphyra-334 compared to the reference strain.
  • the carbon partitioning towards shinorine/porphyra-334 is also improved for the strains expressing the recombinant MysAB fusion proteins.
  • Fig. 10 depicts different proposed pathways for producing the novel mycosporine-like amino acids Tery-347.1, Tery-347.2 and Tery-347.3 employing different enzymes. It is possible that the steps denoted“7” and“2” in Fig. 10 take place in reverse order, because the O-methyltransferase is also able to convert mycosporine glycine into Tery-322. According to the proposed pathways labeled with “7”, mycosporine-glycine serves as a precursor for all 3 different novel MAAs Tery-347.1, Tery-347.2 and Tery-347.3.
  • This compound can be converted into mycosporine-2- glycine by the enzyme Tery_2971 (MysDl) or Ap_MysD.
  • the enzyme Tery_2970 (MysD2) converts mycosporine-glycine into shinorine (>90%).
  • NpF5597 of Nostoc punctiforme (Np MysD) or the enzyme MysD_HL-69 [ Cyanobacterium stanieri HL-69] can convert mycosporine-glycine into a mixture of shinorine/porphyra- 334 by using either serine or threonine (Np_MysD with a ratio of about 60%:40% shinorine/porphyra-334 and MysD_HL-69 with a ratio of about 40%:60% shinorine/porphyra-334.
  • the gene Nv mysD encoding a D-alanine-D-alanine ligase MysD from Nostoc verrucosum KU005 can convert mycosporine-glycine into porphyra-334 by using threonine (>90%).
  • the O-methyltransferase Tery_2966 can convert these intermediate compounds mycosporine-2-glycine, or shinorine/porphyra- 334 into the novel mycosporine like amino acids Tery-347.1, Tery-347.2 and Tery- 347.3 (step“2”), which all show an absorption maximum at 347 nm.
  • These novel compounds all exhibit different retention characteristics in a Hydrophobic Interaction Liquid Chromatography (HILIC) indicating different chemical structures for these MAAs as shown in the following.
  • HILIC Hydrophobic Interaction Liquid Chromatography
  • Fig. 11 depicts a hydrophobic interaction liquid chromatogram of the MAAs isolated from the ABCyano4 strain AB 1333, which lacks the O-methyltransferase Tery_2966 and which was transformed with the plasmids #2865 and #2891, which were already described in more detail above.
  • This ABCyano4 strain produces a mixture of shinorine/porphyra-334 and serves as a reference strain for the ABCyano4 strain AB4105 producing the novel mycosporine like amino acids Tery-347.1 and Tery-347.2.
  • Fig. 12 shows a hydrophobic interaction liquid chromatogram of the MAAs isolated from the ABCyano4 strain AB4105, which produces a mixture of Tery-347.1 and Tery-347.2.
  • This ABCyano4 strain was transformed with the plasmids #3094, including a gene coding for Tery_2966 and the plasmid #3123, which encodes the enzymes for MAA production Ava_3858, Ava_3857, and Ava_3856 (MysA to MysC) and additionally the NpF5597 of Nostoc punctiforme (Np_MysD).
  • the main peaks eluted from the HILIC column are denoted as “ 347 nm Tery 347.1”, “ 347 nm Tery 347.2”,“ 334 nm Shinorine” ,“ 334 nm j porphyra-334” and“ 334 nmJvi-Gly- Ala” , indicating the absorption maxima of the respective components and their chemical structure, if known.
  • the peak being assigned to porphyra-334 is dramatically reduced in comparison to the chromatogram shown in Fig. 11 and a new peak, which could be assigned to the novel MAA compound Tery- 347.1 appears in the chromatogram.
  • Tery-347.2 is produced from porphyra- 334 and that the novel compound MAA Tery-347.2 is produced from shinorine.
  • the fact that Tery-347.2 is produced from shinorine is known from another ABcyano4 strain AB4169, including a fourth gene encoding for the fourth enzyme Tery_2970, catalyzing the formation of shinorine (>90%), this strain additionally being transformed with a sixth gene encoding the sixth enzyme Tery_2966.
  • This AB4169 strain produces mainly Tery-347.2 so that it is clear that shinorine is further converted to Tery-347.2 by the enzyme Tery_2966.
  • Tery-347.1 exhibits a retention time of 8.994 minutes, whereas the other MAA compound Tery-347.2 has a retention time of 10.256 minutes, indicating different hydrophobicities and therefore different chemical structures for both compounds.
  • the compound Tery-347.1 is supposed to be a methylated porphyra-334 compound, whereas the compound Tery-347.2 is a methylated shinorine compound.
  • the ABCyano4 strain AB4105 was shown to also produce another novel MAA compound Tery-347.4, which is a methylated mycosporine-glycine-alanine with the molecular formula C 14H22N2O7 as described herein.
  • Fig. 13 A shows the hydrophobic interaction chromatogram of the MAA compounds produced by the ABCyano4 strain AB4104.
  • This ABCyano4 strain was transformed with the plasmids #3122 including the genes mysA, mysB and mysC (Ava_3858, Ava_3857, and Ava_3856), as well as Ap_mysD (ap3855) and which additionally was transformed with the plasmid #3094 including the gene coding for the O-methyltransf erase Tery_2966.
  • the HILIC chromatogram shows that the compound Tery-347.3, which is a methylated mycosporine-2-glycine was formed from mycosporine-2-glycine.
  • the compound Tery-322 with an absorption maximum at 322 nm was also formed.
  • This novel compound Tery-347.3 exhibits a retention time of 10.110 minutes, which is different to the novel MAA compounds described above, Tery-347.1 and Tery-347.2.
  • These novel MAA compounds exhibit different absorption characteristics in comparison to the other MAA compounds disclosed in this application or MAA compounds known from the literature and therefore can be used as UV-absorbing compounds with characteristics different to known MAAs.
  • Fig. 13B depicts an HPLC spectrum of the main MAA compound produced by the E. coli strain #3186, which is an E. coli BL21 strain transformed with the plasmid #3186 containing the genes for mysA, mysB, and mysC (Ava_3858, Ava_3857, and Ava_3856), the gene encoding Tery 2966 and a further gene encoding a nonribosomal peptide synthetase from Calothrix sp. NIES-2100 (SEQ ID NO. 193).
  • This E. coli strain produces a new MAA compound having an absorption maximum at 347 nm, called Tery-347.x. It is currently not known whether this compound is Tery-347.1, Tery-347.2 or Tery-347.3 or a different MAA compound, structurally distinct from any of these MAA compounds with an absorbance maximum at 347nm.
  • New O-methyltransferases-like enzymes were found employing a BLAST search with the NCBI database, which have a low sequence homology of less than 70% to the known O-methyltransf erase Tery_2966, a Hydroxyneurosporene-O- methyltransferase from Trichodesmium erythraeum IMS101 (GenBank: ABG52124.1), which can be used to produce the novel MAA compounds Tery-347.1, Tery-347.2, Tery-347.3, Tery.347.x and Tery-322, which also is a novel MAA compound having an absorption maximum at 322 nm and which is a methylated mycosporine-glycine.
  • clavaminic acid synthetase-like enzymes could be identified, having a low sequence homology of less than 70% to the enzyme Tery 2972 from Trichodesmium erythraeum IMSIOI (GenBank: ABG52130.1). These low sequence homology enzymes can for example be used for the conversion of mycosporine-2-glycine into palythine.
  • NRPS non-ribosomal peptide synthetases
  • Fig. 14 depicts the UV-absorbance spectra of the main MAA compound Tery-322 produced by the ABCyano4 strain AB4075, expressing the O- methyltransferase from Chroococcidiopsis sp. TS-821 (SEQ ID NO: 86), the strain AB4076, including the O-methyltransferase from Synechococcus sp. PCC 7335 (SEQ ID NO: 92), and the strain AB4090, including the O-methyltransferase from Euhalothece sp. KZN 001 (SEQ ID NO: 88).
  • FIG. 15A shows the HPLC profile of the main MAA compound produced by the ABCyano4 strain AB4075.
  • This ABCyano4 strain was transformed with the plasmid #2865 containing the genes mysA, mysB, and mysC (Ava_3858, Ava_3857, and Ava_3856) for the production of mycosporine-like-glycine.
  • this strain was also transformed with the integrative plasmid #3095 containing the gene encoding the O-methyltransferase from Chroococcidiopsis sp. TS-821 (SEQ ID NO: 86), a low sequence homolog having only 58% sequence identity with Tery_2966.
  • Fig. 15B shows the chromatogram of the main MAA compound produced by the ABCyano4 strain AB4076. This strain contains the O- methyltransferase from Synechococcus sp. PCC 7335 instead of the O- methyltransferase from Chroococcidiopsis sp. TS-821.
  • the O-methyltransferase from Synechococcus sp. PCC 7335 has only 49% sequence identity with the O- methyltransf erase Tery_2966.
  • the same compound Tery-322 is also produced by the O-methyltransferase from Synechococcus sp. PCC 7335, thereby evidencing that the O- methyltransferase from Synechococcus sp. PCC 7335 can catalyze the same chemical reaction as Tery_2966.
  • Fig. 16 shows the HPLC profile of palythine, the MAA compound produced from mycosporine-2-glycine by the ABCyano4 strain AB4140.
  • This ABCyano4 strain was transformed with the plasmid #2892, already described above and additionally was transformed with the plasmid #3190, containing the genes mysA, mysB, and mysC (Ava_3858, Ava_3857, and Ava_3856) and the gene encoding the clavaminic acid synthetase from Calothrix sp.
  • NIES-2100 an enzyme having only 61% sequence identity with the enzyme Tery_2972. Despite the low sequence identity, both enzymes Tery_2972 and the clavaminic acid synthetase from Calothrix sp. NIES-2100 catalyze the same reaction.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne des cellules hôtes modifiées capables de produire divers acides aminés de type mycosporine (MAA), utiles en tant que composés absorbant des UV naturels.
PCT/US2019/032485 2019-05-15 2019-05-15 Production d'acides aminés de type mycosporine utilisant des souches de production améliorées et de nouvelles enzymes WO2020231426A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/US2019/032485 WO2020231426A1 (fr) 2019-05-15 2019-05-15 Production d'acides aminés de type mycosporine utilisant des souches de production améliorées et de nouvelles enzymes

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US2019/032485 WO2020231426A1 (fr) 2019-05-15 2019-05-15 Production d'acides aminés de type mycosporine utilisant des souches de production améliorées et de nouvelles enzymes

Publications (1)

Publication Number Publication Date
WO2020231426A1 true WO2020231426A1 (fr) 2020-11-19

Family

ID=66867770

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2019/032485 WO2020231426A1 (fr) 2019-05-15 2019-05-15 Production d'acides aminés de type mycosporine utilisant des souches de production améliorées et de nouvelles enzymes

Country Status (1)

Country Link
WO (1) WO2020231426A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2752609C1 (ru) * 2021-04-30 2021-07-29 Федеральное государственное бюджетное образовательное учреждение высшего образования "Московский государственный университет имени М.В. Ломоносова" (МГУ) ШТАММ ЦИАНОБАКТЕРИИ Synechococcus sp. ПРОДУЦЕНТ МИКОСПОРИН-ПОДОБНЫХ АМИНОКИСЛОТ
CN113980918A (zh) * 2021-10-18 2022-01-28 中国海洋大学 一种南极冰藻MAAs合成酶及其编码基因和应用
KR102603304B1 (ko) * 2023-06-28 2023-11-16 전남대학교산학협력단 포피라-334 생산용 조성물 및 생산방법

Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6306639B1 (en) 1997-02-19 2001-10-23 Enol Energy Inc. Genetically modified cyanobacteria for the production of ethanol, the constructs and method thereof
WO2011158041A2 (fr) * 2010-06-18 2011-12-22 The Boots Company Plc Composition topique
US20140044677A1 (en) * 2012-08-07 2014-02-13 TopGeniX, Inc. Topical composition comprising transformed bacteria expressing a compound of interest
WO2014100799A2 (fr) 2012-12-21 2014-06-26 Algenol Biofuels Inc. Cyanobacterium sp. pour la production de composés
US8846369B2 (en) 2012-12-21 2014-09-30 Algenol Biofuels Inc. Cyanobacterium sp. host cell and vector for production of chemical compounds in cyanobacterial cultures
US9315820B2 (en) 2011-12-30 2016-04-19 Algenol Biotech LLC Metabolically enhanced cyanobacterium with sequentially inducible production genes for the production of a first chemical compound
US9353400B2 (en) 2013-12-30 2016-05-31 Algenol Biotech LLC Methods for analyses of cyanobacterial restriction endonucleases
US9476067B2 (en) 2012-12-21 2016-10-25 Algenol Biotech LLC Shuttle vector capable of transforming multiple genera of cyanobacteria
US9493794B2 (en) 2013-06-14 2016-11-15 Algenol Biotech LLC Metabolically enhanced cyanobacterial cell for the production of ethanol
US9551014B2 (en) 2011-12-30 2017-01-24 Algenol Biotech LLC Genetically enhanced cyanobacteria for the production of a first chemical compound harbouring Zn2+, Co2+ or Ni2+ -inducible promoters
US20170202762A1 (en) * 2014-05-13 2017-07-20 The Kitasato Institute Method for producing mycosporine-like amino acid using microbes
WO2017185018A1 (fr) * 2016-04-21 2017-10-26 Naked Biome, Inc. Bactéries synthétiques et leurs méthodes d'utilisation
US9965364B2 (en) 2014-10-31 2018-05-08 Red Hat, Inc. Fault tolerant listener registration in the presence of node crashes in a data grid
WO2019094447A2 (fr) * 2017-11-07 2019-05-16 Algenol Biotech LLC Production d'acides aminés de type mycosporine dans des cyanobactéries

Patent Citations (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6306639B1 (en) 1997-02-19 2001-10-23 Enol Energy Inc. Genetically modified cyanobacteria for the production of ethanol, the constructs and method thereof
US6699696B2 (en) 1997-02-19 2004-03-02 Enol Energy Inc. Genetically modified cyanobacteria for the production of ethanol, the constructs and method thereof
WO2011158041A2 (fr) * 2010-06-18 2011-12-22 The Boots Company Plc Composition topique
US9551014B2 (en) 2011-12-30 2017-01-24 Algenol Biotech LLC Genetically enhanced cyanobacteria for the production of a first chemical compound harbouring Zn2+, Co2+ or Ni2+ -inducible promoters
US9315820B2 (en) 2011-12-30 2016-04-19 Algenol Biotech LLC Metabolically enhanced cyanobacterium with sequentially inducible production genes for the production of a first chemical compound
US20140044677A1 (en) * 2012-08-07 2014-02-13 TopGeniX, Inc. Topical composition comprising transformed bacteria expressing a compound of interest
US9476067B2 (en) 2012-12-21 2016-10-25 Algenol Biotech LLC Shuttle vector capable of transforming multiple genera of cyanobacteria
US9315832B2 (en) 2012-12-21 2016-04-19 Algenol Biotech LLC Cyanobacterium sp. host cell and vector for production of chemical compounds in Cyanobacterial cultures
US9157101B2 (en) 2012-12-21 2015-10-13 Algenol Biotech LLC Cyanobacterium sp. for production of compounds
WO2014100799A2 (fr) 2012-12-21 2014-06-26 Algenol Biofuels Inc. Cyanobacterium sp. pour la production de composés
US8846369B2 (en) 2012-12-21 2014-09-30 Algenol Biofuels Inc. Cyanobacterium sp. host cell and vector for production of chemical compounds in cyanobacterial cultures
US9493794B2 (en) 2013-06-14 2016-11-15 Algenol Biotech LLC Metabolically enhanced cyanobacterial cell for the production of ethanol
US9353400B2 (en) 2013-12-30 2016-05-31 Algenol Biotech LLC Methods for analyses of cyanobacterial restriction endonucleases
US20170202762A1 (en) * 2014-05-13 2017-07-20 The Kitasato Institute Method for producing mycosporine-like amino acid using microbes
US9965364B2 (en) 2014-10-31 2018-05-08 Red Hat, Inc. Fault tolerant listener registration in the presence of node crashes in a data grid
WO2017185018A1 (fr) * 2016-04-21 2017-10-26 Naked Biome, Inc. Bactéries synthétiques et leurs méthodes d'utilisation
WO2019094447A2 (fr) * 2017-11-07 2019-05-16 Algenol Biotech LLC Production d'acides aminés de type mycosporine dans des cyanobactéries

Non-Patent Citations (30)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. ABG52126.1
"Handbook Of Microalgal Culture Biotechnology And Applied Phycology", 2003, BLACKWELL PUBLISHING
"The cyanobacteria, molecular Biology, Genomics and Evolution", 2008, CAISTER ACADEMIC PRESS
ACS CHEM BIOL, vol. 12, no. 4, 21 April 2017 (2017-04-21), pages 979 - 988
ALTSCHUL ET AL., JOURNAL OF MOLECULAR BIOLOGY, vol. 215, 1990, pages 403 - 410
ALTSCHUL ET AL., NUCLEIC ACIDS RESEARCH, vol. 25, 1997, pages 3,389 - 3,402
ALTSCHUL SF ET AL.: "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", NUCL. ACIDS. RES., vol. 25, no. 17, 1997, pages 3389 - 3402, XP002905950, DOI: doi:10.1093/nar/25.17.3389
BULMER, NATURE, vol. 325, 1987, pages 728 - 730
CARRETO ET AL.: "Mycosporine-Like Amino Acids: Relevant Secondary Metabolites", CHEMICAL AND ECOLOGICAL ASPECTS MAR DRUGS, vol. 9, 2011, pages 387 - 446
COURT ET AL.: "Genetic engineering using homologous recombination", ANNUAL REVIEW OF GENETICS, vol. 36, 2002, pages 361 - 388
DOWNS ET AL.: "Toxicopathological Effects of the Sunscreen UV Filter, Oxybenzone (Benzophenone-3), on Coral Planulae and Cultured Primary Cells and Its Environmental Contamination in Hawaii and the U.S. Virgin Islands", ARCH ENVIRON CONTAM TOXICOL., vol. 70, 2016, pages 265 - 88, XP035869668, DOI: doi:10.1007/s00244-015-0227-7
E. P. BALSKUS ET AL: "The Genetic and Molecular Basis for Sunscreen Biosynthesis in Cyanobacteria", SCIENCE, vol. 329, no. 5999, 24 September 2010 (2010-09-24), pages 1653 - 1656, XP055083282, ISSN: 0036-8075, DOI: 10.1126/science.1193637 *
GARCIA-PICHEL ET AL.: "The phylogeny of unicellular, extremely halotolerant cyanobacteria", ARCHIVES OF MICROBIOLOGY, vol. 169, 1998, pages 469 - 482
HARTMANN ET AL., MAR. DRUGS, vol. 13, 2015, pages 6291 - 6305
HARTMANN ET AL.: "Quantitative analysis of mycosporine-like amino acids in marine algae by capillary electrophoresis with diode-array detection", JOUR. PHARM. BIOMED. ANALYSIS, vol. 138, 2017, pages 153 - 157, XP029949129, DOI: doi:10.1016/j.jpba.2017.01.053
HARTMANN ET AL.: "Quantitative analysis of mycosporine-like amino acids in marine algae by capillary electrophoresis with diode-array detection", JOUR. PHARM. BIOMED. ANALYSIS,, vol. 138, 2017, pages 153 - 157, XP029949129, DOI: doi:10.1016/j.jpba.2017.01.053
KARLINALTSCHUL, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES U.S.A., vol. 87, 1990, pages 2,264 - 2,268
KARLINALTSCHUL, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES U.S.A., vol. 90, 1993, pages 5,873 - 5,877
KIM ET AL.: "Occurrences, toxicities, and ecological risks of benzophenone-3, a common component of organic sunscreen products: a mini-review", ENVIRON INT., vol. 70, 2014, pages 143 - 157, XP028860664, DOI: doi:10.1016/j.envint.2014.05.015
KOKSHAROVA, APPL MICROBIOL BIOTECHNOL, vol. 58, 2002, pages 123 - 137
LAGESEN K ET AL.: "RNAmmer: consistent and rapid annotation of ribosomal RNA genes", NUCLEIC ACIDS RESEARCH, vol. 35, no. 9, 2007, pages 3100 - 3108, XP055005462, DOI: doi:10.1093/nar/gkm160
MARIO O. CARIGNAN ET AL: "Characterization of mycosporine-serine-glycine methyl ester, a major mycosporine-like amino acid from dinoflagellates: a mass spectrometry study", JOURNAL OF PHYCOLOGY., vol. 49, no. 4, 1 August 2013 (2013-08-01), US, pages 680 - 688, XP055381724, ISSN: 0022-3646, DOI: 10.1111/jpy.12076 *
NAKAMURA ET AL.: "CyanoBase, the genome database for Synechocystis sp. strain PCC6803: status for the year 2000", NUCLEIC ACID RESEARCH, vol. 18, 2000, pages 72
RASTOGI ET AL., APPL. MICROBIOL., vol. 119, 2015, pages 753 - 762
SAHA ET AL., WORLD JOUR. MICROBIOL BIOTECHNOL, vol. 21, 2005, pages 877 - 881
SAMBROOK, J. ET AL.: "Molecular Cloning: A Laboratory Manual", 2001, COLD SPRING HARBOR LABORATORY PRESS
SINGH ET AL.: "Mycosporine-like amino acids (MAAs): chemical structure, biosynthesis and significance as UV-absorbing/screening compounds", INDIAN J EXP BIOL, vol. 46, 2008, pages 7 - 17
THOMPSON ET AL.: "CLUSTALW", NUCLEIC ACIDS RESEARCH, vol. 22, 1994, pages 4673 - 4680
WADA ET AL.: "Mycosporine-Like Amino Acids and Their Derivatives as Natural Antioxidants", ANTIOXIDANTS, vol. 4, 2015, pages 603 - 646
WIRTH, MOL GEN GENET, vol. 216, 1989, pages 175 - 177

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2752609C1 (ru) * 2021-04-30 2021-07-29 Федеральное государственное бюджетное образовательное учреждение высшего образования "Московский государственный университет имени М.В. Ломоносова" (МГУ) ШТАММ ЦИАНОБАКТЕРИИ Synechococcus sp. ПРОДУЦЕНТ МИКОСПОРИН-ПОДОБНЫХ АМИНОКИСЛОТ
CN113980918A (zh) * 2021-10-18 2022-01-28 中国海洋大学 一种南极冰藻MAAs合成酶及其编码基因和应用
CN113980918B (zh) * 2021-10-18 2023-11-24 自然资源部第一海洋研究所 一种南极冰藻MAAs合成酶及其编码基因和应用
KR102603304B1 (ko) * 2023-06-28 2023-11-16 전남대학교산학협력단 포피라-334 생산용 조성물 및 생산방법

Similar Documents

Publication Publication Date Title
WO2019094447A2 (fr) Production d'acides aminés de type mycosporine dans des cyanobactéries
EP2935566B1 (fr) Cyanobacterium sp. pour la production de composés
CN110106209B (zh) 一种利用解脂耶氏酵母途径定位合成萜类化合物的方法
Cunningham Jr et al. Carotenoid biosynthesis in the primitive red alga Cyanidioschyzon merolae
CN112301013B (zh) 复合酶及其在制备麦角硫因中的应用
Song et al. The generation of metabolic changes for the production of high-purity zeaxanthin mediated by CRISPR-Cas9 in Chlamydomonas reinhardtii
WO2020231426A1 (fr) Production d'acides aminés de type mycosporine utilisant des souches de production améliorées et de nouvelles enzymes
Huang et al. ISOLATION AND CHARACTERIZATION OF THE PHYTOENE DESATURASE GENE AS A POTENTIAL SELECTIVE MARKER FOR GENETIC ENGINEERING OF THE ASTAXANTHIN‐PRODUCING GREEN ALGA CHLORELLA ZOFINGIENSIS (CHLOROPHYTA) 1
Scaife et al. Characterization of cyanobacterial β‐carotene ketolase and hydroxylase genes in Escherichia coli, and their application for astaxanthin biosynthesis
WO2002085293A2 (fr) Production d'acide alpha-lipoique
KR20170121051A (ko) Ｒｇｅｎ ｒｎｐ를 이용한 미세조류의 교정 방법
WO2019079135A1 (fr) Production de protéines contenant de l'hème dans des cyanobactéries
US11352601B2 (en) Cyanobacterial hosts and methods for producing chemicals
US20170175148A1 (en) Recombinant Cyanobacterial Cell For Contamination Control In A Cyanobacterial Culture Producing A Chemical Compound Of Interest
KR102473375B1 (ko) 재조합 미생물, 그 제조방법 및 보효소 q10의 생산에 있어서 그의 사용
US10138489B2 (en) Cyanobacterial strains capable of utilizing phosphite
He et al. Genetic deletion of proteins resembling Type IV pilins in Synechocystis sp. PCC 6803: their role in binding or transfer of newly synthesized chlorophyll
Zheng et al. Overexpression of OHPs in Neopyropia yezoensis (Rhodophyta) reveals their possible physiological roles
Yu et al. Gene cloning, sequence analysis, and expression profiles of a novel β-ring carotenoid hydroxylase gene from the photoheterotrophic green alga Chlorella kessleri
JP2003525626A (ja) 微生物におけるルテインの生産
KR20210047992A (ko) 알파-휴물렌 생산용 형질전환 메탄자화균 및 이의 용도
Chen et al. Enhancing astaxanthin accumulation through the expression of the plant-derived astaxanthin biosynthetic pathway in Dunaliella salina
CN113817752B (zh) slr0681基因在合成集胞藻类胡萝卜素中的应用
WO2022220263A1 (fr) Procédé de production d'ergothionéine
KR102277385B1 (ko) Famt 유전자를 이용한 남조류 세포 내 바이오디젤 생산방법

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19731008

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19731008

Country of ref document: EP

Kind code of ref document: A1