US 20210010039A1 IN ( 19 ) United States ( 12 ) Patent Application Publication ( 10 ) Pub . No .: US 2021/0010039 A1 Lim et al . ( 43 ) Pub . Date : Jan. 14 , 2021 ( 54 ) RECOMBINANT CELL AND METHOD OF Publication Classification PRODUCING ITACONIC ACID ( 51 ) Int . Ci . C12P 7/46 ( 2006.01 ) ( 71 ) Applicant: City University of Hong Kong , C12N 15/63 ( 2006.01 ) Kowloon ( HK ) ( 52 ) U.S. CI . CPC C12P 7/46 ( 2013.01 ) ; C12Y 401/01006 ( 72 ) Inventors: Chee Kent Lim , Sabah (MY ); Patrick ( 2013.01 ) ; C12N 15/63 ( 2013.01 ) Kwan Hon Lee , New Territories ( HK ) ( 57 ) ABSTRACT A recombinant cell and a method of producing itaconic acid using such recombinant cell . The recombinant cell is of the ( 21 ) Appl. No .: 16 /509,794 genus Methylorubrum and includes a first polynucleotide sequence as defined in SEQ ID NO : 1 or a homologue thereof operably linked to a regulatory sequence . ( 22 ) Filed : Jul . 12 , 2019 Specification includes a Sequence Listing . PO bosyohusis Disine pretharrat * pynwate . kosustaldehyde meitrijskasse, WAT Acetylcoa fossy #FR Oxaloacetate fost Epiton Qyterest fornate aconic acid dioxide CAD hydroxypynivate Sath ge SERINE CYCLE CYCLE Lexime -ketoglutarata @ -KOOH maate onehundrate succinate que sucony CDA Wide glycose methylene # F glyckyfate malyticon { R } ethykonalossy con ace -CDA Sfatuethydrokartyatak with acetoacetyl SA propiussy COA tyckroxybutyrate PHB CYCLE PATHWAY serdesakakos Dess polyaminyDeoxyixityrate + -3130roxybutyryhos SPExoconyos PARK utstøn giábrits fotonya GOA methylsucÝy CoA PBB Recordation { S } ety stalonyha * {Rlebymolony CoA Patent Application Publication Jan. 14 , 2021 Sheet 1 of 9 US 2021/0010039 A1 biosynthesis Hacwincid late KGO -ethytmaloryl-CoA mesecony.com minnytsucr?Ny-CAR Bcalyplasphata metoshatarata {S} CYCLE ETHYLMALONYL-COA CoRethymolonylCoA-ethylmolonyl($ 1Fig. yruvate 2-phosphor by?roxybutyrangpo-•Tyroxybuty---COM wyberate Blycine toacetite KA methylene4MPT methenyldHMPT formate formaldehyde methenyi methylene yxroxybutyrate3- Carton dioxide Patent Application Publication Jan. 14 , 2021 Sheet 2 of 9 US 2021/0010039 A1 1021 1029 numberTotalregulation-regulationUpDown Numberofgenes 2Fig. WTCADVS101 101WTVsCADAphaR CADWTVSCAD AphaR Patent Application Publication Jan. 14 , 2021 Sheet 3 of 9 US 2021/0010039 A1 CADAphaR 3.Fig Patent Application Publication Jan. 14 , 2021 Sheet 4 of 9 US 2021/0010039 A1 ) L/ mg ( ITA 1 2 prin phar W CM WTAphor 4.Fig IMSeydelMIrendel Swo O Medium Patent Application Publication Jan. 14 , 2021 Sheet 5 of 9 US 2021/0010039 A1 Days 5BFig. OD60010900 6 Days 5AFig. WTCAD CADAphaR ) L/ mg ( ITA Patent Application Publication Jan. 14 , 2021 Sheet 6 of 9 US 2021/0010039 A1 CADAphaR 50Fig. WT101WTCAD o? ( % ) jujuOO SHd Patent Application Publication Jan. 14 , 2021 Sheet 7 of 9 US 2021/0010039 A1 3? Days 6BFig. N ( % ) bujulewes joueyow Days 6AFig. N 00900 Patent Application Publication Jan. 14 , 2021 Sheet 8 of 9 US 2021/0010039 A1 Days 6D.Fig CADAphaR ( 10 ) VOI CADphaR 6CFig. NS ( % ) ju?juoo Hd Patent Application Publication Jan. 14 , 2021 Sheet 9 of 9 US 2021/0010039 A1 2 N 8 7B.Fig 7AFig. US 2021/0010039 A1 Jan. 14 , 2021 1 RECOMBINANT CELL AND METHOD OF [ 0015 ] In one embodiment, the CAD is one derivable from PRODUCING ITACONIC ACID or found in Aspergillus terreus. [ 0016 ] Turning to a second aspect , the present invention SEQUENCE LISTING pertains to a method of producing itaconic acid using a recombinant cell , wherein the recombinant cell comprises a [ 0001 ] The Sequence Listing file entitled " sequencelist first polynucleotide sequence as defined in SEQ ID NO : 1 or ing” having a size of 9,184 bytes and a creation date of Jul . a homologue thereof operably linked to a regulatory 12 , 2019 , that was filed with the patent application is sequence , wherein the recombinant cell is of the genus incorporated herein by reference in its entirety Methylorubrum . TECHNICAL FIELD [ 0017 ] In one embodiment of the second aspect , the fur ther comprises a second polynucleotide sequence as defined [ 0002 ] The present invention relates to a recombinant cell in SEQ ID NO : 2 or a homologue thereof. and a method of producing itaconic acid using such a [ 0018 ] In one embodiment of the second aspect , the first recombinant cell. Particularly , the invention pertains to a polynucleotide sequence or second polynucleotide sequence recombinant cell of the genus Methylorubrum . has at least 80 % sequence identity with the sequence of SEQ ID NO : 1 or SEQ ID NO : 2 , respectively. BACKGROUND OF THE INVENTION [ 0019 ] In one embodiment of the second aspect , the first [ 0003 ] Itaconic acid , a C - 5 dicarboxylic organic acid , is a polynucleotide sequence encodes a protein or fragment high - value polymer building block with broad application in thereof having CAD activity for producing itaconic acid . various industries . Polymers derived from itaconic acid have [ 0020 ] In one embodiment, the method further comprises wide - ranging industrial uses , including as ingredients for a step of incubating the recombinant cell in a medium making superabsorbent polymers , as co - builders in deter containing a carbon substrate for producing itaconic acid . gents , and anti - scaling agents in water treatment processes [ 0021 ] In one embodiment of the second aspect , the car to name a few . In view of its role as an important pre - cursor bon substrate is selected from a group comprising of metha to various products , maximizing itaconic acid production is nol , methylamine, formate, pyruvate , succinate , lactate and of commercial value . acetate . [ 0004 ] Conventionally, itaconic acid production is by fer [ 0022 ] In one embodiment of the second aspect , the car mentation with Aspergillus terreus is expensive and with bon substrate is selected from a group consisting of acetate , undesirable characteristics in cultivation such as spore for succinate and methanol. mation , susceptibility to damage by shear stress and fila [ 0023 ] In one embodiment of the second aspect , the car mentous growth . Other deficiencies when relying on the bon substrate is methanol . conventional method of producing itaconic acid is that [ 0024 ] In one embodiment of the second aspect , the Aspergillus terreus grows slowly and does not produce recombinant cell is Methylorubrum extorquens. itaconic acid in its spore - forming stage . [ 0025 ] In one embodiment of the second aspect , the [ 0005 ] Accordingly, there is a strong need for a method recombinant cell is Methylorubrum extorquens AM1 . that produces itaconic acid inexpensively and in high yield . [ 0026 ] In one embodiment of the second aspect , the regu latory sequence is a constitutive or strong promoter as SUMMARY OF THE INVENTION defined in SEQ ID NO : 3 or a homologue thereof. [ 0006 ] In a first aspect , the present invention pertains to a [ 0027 ] In one embodiment of the second aspect , the CAD recombinant cell that comprises a first polynucleotide is one derivable from or found in Aspergillus terreus and the sequence as defined in SEQ ID NO : 1 or a homologue method further comprises fermenting the recombinant cell in thereof operably linked to a regulatory sequence , wherein a suitable fermentation medium to produce itaconic acid . the recombinant cell is of the Methylorubrum genus . [ 0028 ] Those skilled in the art will appreciate that the [ 0007 ] In one embodiment, the recombinant cell com invention described herein is susceptible to variations and prises a second polynucleotide sequence as defined in SEQ modifications other than those specifically described . The ID NO : 2 or a homologue thereof. invention includes all such variations and modifications. The [ 0008 ] In one embodiment, the first polynucleotide invention also includes all steps and features referred to or sequence encodes a protein or fragment thereof having indicated in the specification , individually or collectively, cis -aconitic decarboxylase ( CAD ) activity for producing and any and all combinations of the steps or features. itaconic acid from a carbon substrate . [ 0029 ] Other features and aspects of the invention will [ 0009 ] In one embodiment, the carbon substrate is selected become apparent by consideration of the following detailed from a group comprising of methanol, methylamine, for description and accompanying drawings . mate , pyruvate , succinate , lactate and acetate . [ 0010 ] In one embodiment, the carbon substrate is selected from a group consisting of acetate , succinate and methanol. BRIEF DESCRIPTION OF DRAWINGS [ 0011 ] In one embodiment, the carbon substrate is metha [ 0030 ] FIG . 1 is a schematic diagram illustrating carbon nol . metabolic pathways in the formation of itaconic acid . [ 0012 ] In one embodiment, the recombinant cell is Meth [ 0031 ] FIG . 2 is a comparison of the number of up ylorubrum extorquens . regulated and down - regulated genes between wildtype [ 0013 ] In one embodiment, the recombinant cell is Meth Methylorubrum extorquens AM1 carrying a PTE 101 plas ylorubrum extorquens AM1 . mid ( WT_101 ) , wildtype Methylorubrum extorquens AMI [ 0014 ] In one embodiment, the regulatory sequence is a expressing a heterologous codon -optimized gene encoding constitutive or strong promoter as defined in SEQ ID NO : 3 cis - aconitic decarboxylase ( CAD ) (WT_CAD ) and mutant or a homologue thereof. Methylorubrum extorquens AM1 with a truncated phaR US 2021/0010039 A1 Jan. 14 , 2021 2 gene expressing a heterologous codon - optimized gene [ 0041 ] FIG . 7A is a line graph showing optical density encoding