Methods and systems for methylotrophic production of organic compounds

Abstract

The present disclosure identifies pathways, mechanisms, systems and methods to confer production of carbon-based products of interest, such as sugars, alcohols, chemicals, amino acids, polymers, fatty acids and their derivatives, hydrocarbons, isoprenoids, and intermediates thereof, in engineered and/or evolved methylotrophs such that these organisms efficiently convert C1 compounds, such as formate, formic acid, formaldehyde or methanol, to organic carbon-based products of interest, and in particular the use of organisms for the commercial production of various carbon-based products of interest.

1 .- 51 . (canceled) 
     
     
         52 . A methylotrophic host cell comprising:
 a heterologous polynucleotide encoding a 1-deoxy-D-xylulose-5-phosphate synthase protein having a sequence that is at least 90% identical to the  E. coli  1-deoxy-D-xylulose-5-phosphate synthase protein encoded by SEQ ID NO: 1, wherein the 1-deoxy-D-xylulose-5-phosphate synthase protein encoded by SEQ ID NO: 1 corresponds to NCBI Reference Sequence: AAC46162;   a heterologous polynucleotide encoding an isoprene synthase protein having a sequence that is at least 90% identical to the  Populus nigra  isoprene synthase protein encoded by SEQ ID NO: 3, wherein the isoprene synthase protein encoded by SEQ ID NO: 3 corresponds to NCBI Reference Sequence: CAL69918;   a heterologous polynucleotide encoding an E-alpha-bisabolene synthase protein having a sequence that is at least 90% identical to the  Picea abies  E-alpha-bisabolene synthase protein encoded by SEQ ID NO: 4, wherein the E-alpha-bisabolene synthase encoded by SEQ ID NO: 4 corresponds to NCBI Reference Sequence: AAS47689;   a heterologous polynucleotide encoding a propionyl-CoA synthase having a sequence that is at least 90% identical to SEQ ID NO:5; or   a combination thereof.   
     
     
         53 . The methylotrophic host cell of  claim 52 , wherein the methylotrophic host cell comprises a heterologous polynucleotide encoding a 1-deoxy-D-xylulose-5-phosphate synthase protein having a sequence that is at least 90% identical to the  E. coli  1-deoxy-D-xylulose-5-phosphate synthase protein encoded by SEQ ID NO: 1, wherein the 1-deoxy-D-xylulose-5-phosphate synthase protein encoded by SEQ ID NO: 1 corresponds to NCBI Reference Sequence: AAC46162. 
     
     
         54 . The methylotrophic host cell of  claim 52 , wherein the methylotrophic host cell comprises a heterologous polynucleotide encoding an isoprene synthase protein having a sequence that is at least 90% identical to the  Populus nigra  isoprene synthase protein encoded by SEQ ID NO: 3, wherein the isoprene synthase protein encoded by SEQ ID NO: 3 corresponds to NCBI Reference Sequence: CAL69918. 
     
     
         55 . The methylotrophic host cell of  claim 52 , wherein the methylotrophic host cell comprises a heterologous polynucleotide encoding an E-alpha-bisabolene synthase protein having a sequence that is at least 90% identical to the  Picea abies  E-alpha-bisabolene synthase protein encoded by SEQ ID NO: 4, wherein the E-alpha-bisabolene synthase encoded by SEQ ID NO: 4 corresponds to NCBI Reference Sequence: AAS47689. 
     
     
         56 . The methylotrophic host cell of  claim 52 , wherein the methylotrophic host cell comprises a heterologous polynucleotide encoding a propionyl-CoA synthase having a sequence that is at least 90% identical to SEQ ID NO:5. 
     
     
         57 . The methylotrophic host cell of  claim 52 , wherein the methylotrophic host cell comprises a genomic integration of the heterologous polynucleotide encoding the 1-deoxy-D-xylulose-5-phosphate synthase protein, a genomic integration of the heterologous polynucleotide encoding an isoprene synthase, a genomic integration of the heterologous polynucleotide encoding an E-alpha-bisabolene synthase protein, a genetic integration of the heterologous polynucleotide encoding a propionyl-CoA synthase, or a combination thereof. 
     
     
         58 . The methylotrophic host cell of  claim 52 , wherein the methylotrophic host cell is from the genus  Paracoccus.    
     
     
         59 . The methylotrophic host cell of  claim 58 , wherein the host cell is a  Paracoccus versutus  cell. 
     
     
         60 . The methylotrophic host cell of  claim 58 , wherein the host cell is a  Paracoccus denitrificans  cell. 
     
     
         61 . The methylotrophic host cell of  claim 58 , wherein the host cell is a  Paracoccus zeaxanthinifaciens  cell. 
     
     
         62 . The methylotrophic host cell of  claim 52 , wherein the methylotrophic host cell further comprises one or more genetic modifications that enhance the ability of the methylotrophic host cell to grow on an electrolytically generated C1 compound relative to a corresponding methylotrophic host cell lacking the one or more genetic modifications. 
     
     
         63 . The methylotrophic host cell of  claim 62 , wherein the C1 compound is formate, formic acid, formaldehyde, or methanol. 
     
     
         64 . The methylotrophic host cell of  claim 63 , wherein the C1 compound is formate, and the methylotrophic host cell further comprises a formate transporter. 
     
     
         65 . The methylotrophic host cell of  claim 52 , wherein the methylotrophic host cell is capable of converting a C1 compound into an isoprenoid. 
     
     
         66 . A high-cell density bioreactor comprising a methylotrophic host cell according to  claim 52 . 
     
     
         67 . A method comprising culturing a methylotrophic host cell according to  claim 52 , under conditions such that it produces a product of interest. 
     
     
         68 . A method of producing an isoprenoid, comprising culturing a methylotrophic host cell according to  claim 65 , under conditions such that it produces the isoprenoid. 
     
     
         69 . The method of  claim 68 , wherein the isoprenoid is a triterprene. 
     
     
         70 . The method of  claim 68 , wherein the isoprenoid is isoprene. 
     
     
         71 . The method of  claim 68 , wherein the conditions comprise formate.