JP2020507352A5 - - Google Patents
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- Publication number
- JP2020507352A5 JP2020507352A5 JP2019565596A JP2019565596A JP2020507352A5 JP 2020507352 A5 JP2020507352 A5 JP 2020507352A5 JP 2019565596 A JP2019565596 A JP 2019565596A JP 2019565596 A JP2019565596 A JP 2019565596A JP 2020507352 A5 JP2020507352 A5 JP 2020507352A5
- Authority
- JP
- Japan
- Prior art keywords
- polyketide
- amino acid
- acid residue
- polynucleotide
- dipks
- Prior art date
- Legal status (The legal status 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 status listed.)
- Granted
Links
- 210000005253 yeast cell Anatomy 0.000 claims 18
- 108091033319 polynucleotide Proteins 0.000 claims 16
- 239000002157 polynucleotide Substances 0.000 claims 16
- 102000040430 polynucleotide Human genes 0.000 claims 16
- 238000000034 method Methods 0.000 claims 15
- 108010030975 Polyketide Synthases Proteins 0.000 claims 14
- 125000000539 amino acid group Chemical group 0.000 claims 14
- 229930001119 polyketide Natural products 0.000 claims 11
- 150000003881 polyketide derivatives Chemical class 0.000 claims 10
- 235000018102 proteins Nutrition 0.000 claims 10
- 102000004169 proteins and genes Human genes 0.000 claims 10
- 108090000623 proteins and genes Proteins 0.000 claims 10
- 108091026890 Coding region Proteins 0.000 claims 9
- 241000894007 species Species 0.000 claims 8
- 238000012239 gene modification Methods 0.000 claims 5
- 230000005017 genetic modification Effects 0.000 claims 5
- 235000013617 genetically modified food Nutrition 0.000 claims 5
- 102000004190 Enzymes Human genes 0.000 claims 4
- 108090000790 Enzymes Proteins 0.000 claims 4
- LTYOQGRJFJAKNA-KKIMTKSISA-N Malonyl CoA Natural products S(C(=O)CC(=O)O)CCNC(=O)CCNC(=O)[C@@H](O)C(CO[P@](=O)(O[P@](=O)(OC[C@H]1[C@@H](OP(=O)(O)O)[C@@H](O)[C@@H](n2c3ncnc(N)c3nc2)O1)O)O)(C)C LTYOQGRJFJAKNA-KKIMTKSISA-N 0.000 claims 4
- 102000004357 Transferases Human genes 0.000 claims 4
- 108090000992 Transferases Proteins 0.000 claims 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims 4
- LTYOQGRJFJAKNA-DVVLENMVSA-N malonyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)CC(O)=O)O[C@H]1N1C2=NC=NC(N)=C2N=C1 LTYOQGRJFJAKNA-DVVLENMVSA-N 0.000 claims 4
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims 4
- IRMPFYJSHJGOPE-UHFFFAOYSA-N olivetol Chemical compound CCCCCC1=CC(O)=CC(O)=C1 IRMPFYJSHJGOPE-UHFFFAOYSA-N 0.000 claims 4
- FZOWKKHJBQFDPU-UHFFFAOYSA-N 2-methyl-5-pentylbenzene-1,3-diol Chemical compound CCCCCC1=CC(O)=C(C)C(O)=C1 FZOWKKHJBQFDPU-UHFFFAOYSA-N 0.000 claims 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims 2
- 101150022636 MAFB gene Proteins 0.000 claims 2
- -1 R2 is H Chemical group 0.000 claims 2
- 101100373202 Rattus norvegicus Cx3cl1 gene Proteins 0.000 claims 2
- ZSLZBFCDCINBPY-ZSJPKINUSA-N acetyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZSLZBFCDCINBPY-ZSJPKINUSA-N 0.000 claims 2
- 239000002253 acid Substances 0.000 claims 2
- 229940024606 amino acid Drugs 0.000 claims 2
- 235000001014 amino acid Nutrition 0.000 claims 2
- 150000001413 amino acids Chemical class 0.000 claims 2
- 238000004113 cell culture Methods 0.000 claims 2
- 230000000694 effects Effects 0.000 claims 2
- 229910052739 hydrogen Inorganic materials 0.000 claims 2
- 239000001257 hydrogen Substances 0.000 claims 2
- 101150039067 maf-1 gene Proteins 0.000 claims 2
- 239000002773 nucleotide Substances 0.000 claims 2
- 125000003729 nucleotide group Chemical group 0.000 claims 2
- 102000005369 Aldehyde Dehydrogenase Human genes 0.000 claims 1
- 108020002663 Aldehyde Dehydrogenase Proteins 0.000 claims 1
- 239000004475 Arginine Substances 0.000 claims 1
- 241000168726 Dictyostelium discoideum Species 0.000 claims 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims 1
- 229930182558 Sterol Natural products 0.000 claims 1
- 239000012190 activator Substances 0.000 claims 1
- 235000004279 alanine Nutrition 0.000 claims 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims 1
- 229940009098 aspartate Drugs 0.000 claims 1
- 230000015572 biosynthetic process Effects 0.000 claims 1
- 230000001086 cytosolic effect Effects 0.000 claims 1
- 239000000428 dust Substances 0.000 claims 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 claims 1
- 230000011987 methylation Effects 0.000 claims 1
- 238000007069 methylation reaction Methods 0.000 claims 1
- 230000035772 mutation Effects 0.000 claims 1
- 125000001147 pentyl group Chemical group C(CCCC)* 0.000 claims 1
- 125000000830 polyketide group Chemical group 0.000 claims 1
- 150000003432 sterols Chemical class 0.000 claims 1
- 235000003702 sterols Nutrition 0.000 claims 1
- 230000002194 synthesizing effect Effects 0.000 claims 1
- 230000001131 transforming effect Effects 0.000 claims 1
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201762460526P | 2017-02-17 | 2017-02-17 | |
| US62/460,526 | 2017-02-17 | ||
| PCT/CA2018/050190 WO2018148849A1 (en) | 2017-02-17 | 2018-02-19 | Method and cell line for production of polyketides in yeast |
Publications (4)
| Publication Number | Publication Date |
|---|---|
| JP2020507352A JP2020507352A (ja) | 2020-03-12 |
| JP2020507352A5 true JP2020507352A5 (https=) | 2021-04-01 |
| JP7162018B2 JP7162018B2 (ja) | 2022-10-27 |
| JP7162018B6 JP7162018B6 (ja) | 2024-02-02 |
Family
ID=63169138
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2019565595A Pending JP2020507351A (ja) | 2017-02-17 | 2018-02-19 | 酵母における植物性カンナビノイド及び植物性カンナビノイドアナログの生成のための方法及び細胞株 |
| JP2019565596A Active JP7162018B6 (ja) | 2017-02-17 | 2018-02-19 | 酵母におけるポリケチドの生成のための方法及び細胞株 |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2019565595A Pending JP2020507351A (ja) | 2017-02-17 | 2018-02-19 | 酵母における植物性カンナビノイド及び植物性カンナビノイドアナログの生成のための方法及び細胞株 |
Country Status (11)
| Country | Link |
|---|---|
| US (3) | US11078502B2 (https=) |
| EP (2) | EP3583222A4 (https=) |
| JP (2) | JP2020507351A (https=) |
| KR (2) | KR20200004784A (https=) |
| CN (2) | CN110651047B (https=) |
| AU (2) | AU2018220470B2 (https=) |
| CA (2) | CA3054423A1 (https=) |
| IL (2) | IL268725B2 (https=) |
| MX (2) | MX2019009708A (https=) |
| SG (2) | SG11201907504PA (https=) |
| WO (2) | WO2018148848A1 (https=) |
Families Citing this family (44)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110914416B (zh) | 2017-04-27 | 2023-07-21 | 加州大学董事会 | 产生大麻素和大麻素衍生物的微生物和方法 |
| AU2018346434A1 (en) * | 2017-10-05 | 2020-05-14 | Intrexon Corporation | Microorganisms and methods for the fermentation of cannabinoids |
| WO2019190945A1 (en) * | 2018-03-29 | 2019-10-03 | William Marsh Rice University | Biosynthesis of olivetolic acid |
| AU2019262112A1 (en) | 2018-04-30 | 2020-11-19 | Algae-C Inc. | Engineered microorganism for the production of cannabinoid biosynthetic pathway products |
| WO2020060948A1 (en) | 2018-09-17 | 2020-03-26 | Levadura Biotechnology, Inc. | Production of cannabinoids in yeast using a fatty acid feedstock |
| IL326299A (en) * | 2018-10-31 | 2026-04-01 | Baymedica Inc | Cannabinoid analogs and methods for their preparation |
| CN113227353A (zh) * | 2018-11-14 | 2021-08-06 | 马努斯生物合成股份有限公司 | 用于产生大麻素的微生物细胞及方法 |
| MY205459A (en) * | 2018-12-06 | 2024-10-22 | Cytomx Therapeutics Inc | Matrix metalloprotease-cleavable and serine or cysteine protease-cleavable substrates and methods of use thereof |
| US20220127649A1 (en) * | 2019-01-30 | 2022-04-28 | Genomatica, Inc. | Engineered cells for improved production of cannabinoids |
| EP3917642A4 (en) | 2019-01-30 | 2023-04-05 | Genomatica, Inc. | RECOVERY, DECARBOXYLATION AND PURIFICATION OF CANNABINOIDS FROM GMO CULTURES |
| CA3130763A1 (en) | 2019-02-25 | 2020-09-03 | Ginkgo Bioworks, Inc. | Biosynthesis of cannabinoids and cannabinoid precursors |
| US20220186267A1 (en) * | 2019-03-27 | 2022-06-16 | Rynetech Bio, Inc. | Biosynthetic Cannabidiol Production In Engineered Microorganisms |
| SG11202112690YA (en) * | 2019-05-22 | 2021-12-30 | Hyasynth Biologicals Inc | Methods and cells for production of phytocannabinoids and phytocannabinoid precursors |
| ES3037766T3 (en) | 2019-06-11 | 2025-10-07 | Symrise Ag | Synthesis and purification of cannabigerol and its application |
| CA3151799A1 (en) * | 2019-08-18 | 2021-02-25 | Ginkgo Bioworks, Inc. | Biosynthesis of cannabinoids and cannabinoid precursors |
| EP4025705A4 (en) * | 2019-09-06 | 2024-01-17 | Genomatica, Inc. | PRENYL TRANSFERASE VARIANTS AND METHOD FOR PRODUCING PRENYLATED AROMATIC COMPOUNDS |
| CN120555521A (zh) * | 2019-10-01 | 2025-08-29 | 杭州恩和生物科技有限公司 | 用于大麻素合成的酶和制备大麻素的方法 |
| US20240084337A1 (en) * | 2019-10-11 | 2024-03-14 | National University Of Singapore | Biosynthesis of cannabinoid precursors using novel aromatic prenyl transferases |
| KR20220079578A (ko) | 2019-10-11 | 2022-06-13 | 내셔널 유니버시티 오브 싱가포르 | 신규 칸나비노이드 신타아제를 사용한 칸나비게롤산으로부터 칸나비노이드의 생합성 |
| MX2022008463A (es) | 2020-01-10 | 2022-10-18 | Univ California | Plataforma biosintética para la producción de ácido olivetólico y análogos del ácido olivetólico. |
| US20250346871A1 (en) * | 2020-03-06 | 2025-11-13 | Cellibre, Inc. | Prenyltransferases and methods of making and use thereof |
| EP4126916A2 (en) | 2020-03-27 | 2023-02-08 | Willow Biosciences Inc. | Compositions and methods for recombinant biosynthesis of cannabinoids |
| US12529077B2 (en) * | 2020-04-08 | 2026-01-20 | Københavns Universitet | Production of geranyl diphosphate-derived compounds |
| EP3901256A1 (en) * | 2020-04-21 | 2021-10-27 | Synbionik GmbH | Optimized production of cbga from olivetol acid and geranyl pyrophosphate via synnphb |
| CA3175683A1 (en) | 2020-04-29 | 2021-11-04 | Xueyang FENG | Compositions and methods for enhancing recombinant biosynthesis of cannabinoids |
| WO2021225952A1 (en) * | 2020-05-08 | 2021-11-11 | Lygos, Inc. | Large scale production of olivetol, olivetolic acid and other alkyl resorcinols by fermentation |
| EP4176054A1 (en) * | 2020-07-03 | 2023-05-10 | Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie e.V. -Hans-Knöll-Institut- (HKI) | Method for producing olivetolic acid in an amoebozoa host species |
| KR20230042073A (ko) * | 2020-07-24 | 2023-03-27 | 히야신스 바이오로지컬스 아이엔씨. | 치환된 칸나비노이드 및 전구체를 제조하기 위해 변형 효소를 이용하는 방법 및 이를 함유하는 세포 |
| CN111979134B (zh) | 2020-08-28 | 2022-02-15 | 江南大学 | 一种合成胭脂红酸的重组酿酒酵母的构建及其应用 |
| CN112042677B (zh) * | 2020-09-21 | 2021-09-14 | 中国农业科学院麻类研究所 | 一种大麻叶提取物及其制备方法、用途 |
| US11427840B2 (en) | 2020-11-20 | 2022-08-30 | Hyasynth Biologicals Inc. | Olivetolic acid cyclase variants with improved activity for use in production of phytocannabinoids |
| CA3199891A1 (en) * | 2020-11-24 | 2022-06-02 | Ruth Gallily | Cannabinoid derivatives and their use for the treatment of inflammation and/or pain and/or obesity |
| US20240026314A1 (en) * | 2020-11-25 | 2024-01-25 | Steven Rothstein | Polypeptides for use in the synthesis of bioactive phenolic compounds |
| WO2022125960A1 (en) | 2020-12-11 | 2022-06-16 | Willow Biosciences, Inc. | Recombinant acyl activating enzyme (aae) genes for enhanced biosynthesis of cannabinoids and cannabinoid precursors |
| EP4347800A1 (en) | 2021-05-26 | 2024-04-10 | Invizyne Technologies, Inc. | Prenyltransferase variants with increased thermostability |
| WO2023010083A2 (en) | 2021-07-30 | 2023-02-02 | Willow Biosciences, Inc. | Recombinant prenyltransferase polypeptides engineered for enhanced biosynthesis of cannabinoids |
| CA3227236A1 (en) | 2021-08-19 | 2023-02-23 | Trish Choudhary | Recombinant olivetolic acid cyclase polypeptides engineered for enhanced biosynthesis of cannabinoids |
| CN113881584B (zh) * | 2021-09-16 | 2023-06-27 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | 一株经航天育种技术选育的酿酒酵母及其突变位点应用 |
| WO2023056350A1 (en) * | 2021-09-29 | 2023-04-06 | Ginkgo Bioworks, Inc. | Biosynthesis of cannabinoids and cannabinoid precursors |
| WO2023069921A1 (en) | 2021-10-19 | 2023-04-27 | Epimeron Usa, Inc. | Recombinant thca synthase polypeptides engineered for enhanced biosynthesis of cannabinoids |
| CN114621982B (zh) * | 2022-03-16 | 2023-11-07 | 嘉兴欣贝莱生物科技有限公司 | 香叶基二磷酸酯的生物合成方法及其在制备大麻类化合物中的应用 |
| CN116024111B (zh) * | 2022-12-03 | 2025-09-05 | 中国科学院深圳先进技术研究院 | 一种产大麻萜酚的微生物细胞及其构建方法与应用 |
| CN119685467A (zh) * | 2023-12-01 | 2025-03-25 | 上海市东方医院(同济大学附属东方医院) | 诊断和治疗阿尔茨海默病的医药制剂 |
| CN119753053A (zh) * | 2025-03-07 | 2025-04-04 | 中国农业科学院植物保护研究所 | 磷酸泛酰巯基乙胺基转移酶3x32、其编码基因、含有该基因的重组载体和重组菌的应用 |
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| ATE455785T1 (de) | 2000-11-02 | 2010-02-15 | Ajinomoto Kk | Neue pyrazolderivate und diese enthaltende mittel gegen diabetes |
| US7238514B2 (en) | 2001-01-05 | 2007-07-03 | William Marsh Rice University | Diterpene-producing unicellular organism |
| EP1338608A3 (en) * | 2001-12-20 | 2004-05-06 | Cellzome Ag | Protein complexes and methods for their use |
| AU2002367554A1 (en) | 2001-12-20 | 2003-09-09 | Cellzome Ag | Protein complexes and methods for their use |
| US7186519B2 (en) | 2002-11-13 | 2007-03-06 | Trustees Of Dartmouth College | Method for detecting intracellular cholesterol |
| WO2006060839A2 (en) | 2004-12-10 | 2006-06-15 | Universität Für Bodenkultur Wien | Method for identifying ppt inhibitors |
| US7361483B2 (en) | 2005-01-28 | 2008-04-22 | The Salk Institute For Biological Studies | Aromatic prenyltransferases, nucleic acids encoding same and uses therefor |
| US20080233628A1 (en) * | 2006-09-14 | 2008-09-25 | The Salk Institute For Biological Studies | Incorporation of type III polyketide synthases into multidomain proteins of the type I and III polyketide synthase and fatty acid synthase families |
| CA2770774C (en) | 2009-08-12 | 2020-07-14 | National Research Council Of Canada | Aromatic prenyltransferase from cannabis |
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| CN103562398A (zh) | 2011-03-22 | 2014-02-05 | Opx生物工艺学公司 | 化学产品的微生物生产以及相关的组合物、方法和系统 |
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| AU2012283708B2 (en) * | 2011-07-13 | 2017-02-23 | National Research Council Of Canada | Genes and proteins for alkanoyl-CoA synthesis |
| BR112014010448A2 (pt) * | 2011-11-02 | 2017-04-18 | Genomatica Inc | microorganismos e métodos para a produção de caprolactona |
| US9670494B2 (en) | 2012-01-06 | 2017-06-06 | Firmenich Sa | Genetically engineered yeast cells |
| WO2014018982A1 (en) * | 2012-07-27 | 2014-01-30 | The Regents Of The University Of California | Systems and methods for enhancing gene expression |
| WO2014134281A1 (en) | 2013-02-28 | 2014-09-04 | Full Spectrum Laboratories Limited | Biosynthesis of cannabinoids |
| EP3008178A1 (en) | 2013-06-14 | 2016-04-20 | Technical University of Denmark | Microbial production of 3-hydroxypropionic acid |
| WO2014207113A1 (en) | 2013-06-26 | 2014-12-31 | Abengoa Bioenergia Nuevas Tecnologias S.A. | Yeasts engineered for the production of valuable chemicals from sugars |
| US20160230200A1 (en) | 2013-09-05 | 2016-08-11 | Danmarks Tekniske Universitet | Microorganisms for efficient production of melatonin and related compounds |
| US9822384B2 (en) | 2014-07-14 | 2017-11-21 | Librede Inc. | Production of cannabinoids in yeast |
| US9394510B2 (en) | 2014-08-25 | 2016-07-19 | Full Spectrum Laboratories Limited | Apparatus and methods for the simultaneous production of compounds |
| DK3277827T3 (da) | 2015-04-02 | 2021-06-14 | Biopetrolia Ab | Svampeceller og fremgangsmåder til fremstilling af produkter afledt af meget langkædede fedtsyrer |
| WO2017139496A1 (en) | 2016-02-09 | 2017-08-17 | Cevolva Biotech, Inc. | Microbial engineering for the production of cannabinoids and cannabinoid precursors |
| EP3430151A4 (en) * | 2016-03-16 | 2020-09-02 | William Marsh Rice University | MICROBIAL SYNTHESIS OF ISOPRENOID, ISOPRENOID AND DERIVATIVE PRECURSORS INCLUDING PRENYLATED AROMATIC COMPOUNDS |
| CN110914416B (zh) | 2017-04-27 | 2023-07-21 | 加州大学董事会 | 产生大麻素和大麻素衍生物的微生物和方法 |
-
2018
- 2018-02-19 EP EP18754525.6A patent/EP3583222A4/en not_active Withdrawn
- 2018-02-19 US US16/486,618 patent/US11078502B2/en active Active
- 2018-02-19 KR KR1020197027008A patent/KR20200004784A/ko not_active Withdrawn
- 2018-02-19 US US16/486,610 patent/US11312979B2/en active Active
- 2018-02-19 SG SG11201907504PA patent/SG11201907504PA/en unknown
- 2018-02-19 JP JP2019565595A patent/JP2020507351A/ja active Pending
- 2018-02-19 CA CA3054423A patent/CA3054423A1/en active Pending
- 2018-02-19 EP EP18754640.3A patent/EP3583217A4/en not_active Withdrawn
- 2018-02-19 WO PCT/CA2018/050189 patent/WO2018148848A1/en not_active Ceased
- 2018-02-19 AU AU2018220470A patent/AU2018220470B2/en active Active
- 2018-02-19 AU AU2018220469A patent/AU2018220469B2/en active Active
- 2018-02-19 CA CA3054152A patent/CA3054152A1/en active Pending
- 2018-02-19 IL IL268725A patent/IL268725B2/en unknown
- 2018-02-19 CN CN201880025281.2A patent/CN110651047B/zh not_active Expired - Fee Related
- 2018-02-19 WO PCT/CA2018/050190 patent/WO2018148849A1/en not_active Ceased
- 2018-02-19 CN CN201880025692.1A patent/CN110637088B/zh not_active Expired - Fee Related
- 2018-02-19 SG SG11201907469YA patent/SG11201907469YA/en unknown
- 2018-02-19 JP JP2019565596A patent/JP7162018B6/ja active Active
- 2018-02-19 IL IL268723A patent/IL268723B2/en unknown
- 2018-02-19 MX MX2019009708A patent/MX2019009708A/es unknown
- 2018-02-19 MX MX2019009712A patent/MX2019009712A/es unknown
- 2018-02-19 KR KR1020197027012A patent/KR102589523B1/ko active Active
-
2020
- 2020-03-24 US US16/828,696 patent/US10975395B2/en active Active
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