Microorganisms for Biosynthesis of Limonene on Gaseous Substrates

Abstract

Engineered microorganisms are provided that convert gaseous substrates, such as producer gas, into limonene. In some embodiments, limonene is pumped out of the cell via an efflux pump. In some embodiments, limonene, produced as described herein, is converted through catalytic dimerization into jet fuel. Producer gas used in the processes described herein for production of limonene may be derived from sources that include gasification of waste feedstock and/or biomass residue, waste gas from industrial processes, or natural gas, biogas, or landfill gas.

1 .- 36 . (canceled) 
     
     
         37 . A composition comprising a non-naturally occurring microorganism that is capable of growing on a gaseous substrate as a carbon and energy source, and a culture medium in contact with a gaseous substrate that comprises an inorganic gaseous electron donor and an inorganic gaseous electron acceptor,
 wherein the microorganism comprises at least one exogenous nucleic acid, and   wherein the gaseous substrate is utilized by the microorganism for production of a terpene in the culture medium using a combination of the electron donor and the electron acceptor as an energy source.   
     
     
         38 . The composition of  claim 37 , wherein the gaseous substrate comprises CO 2 , CO, and/or CH 4  as a carbon source. 
     
     
         39 . The composition of  claim 37 , wherein the microorganism is a knallgas microorganism. 
     
     
         40 . The composition of  claim 39 , wherein the gaseous substrate comprises CO 2  as a carbon source, H 2  as an electron donor, and O 2  as an electron acceptor. 
     
     
         41 . The composition of  claim 39 , wherein the gaseous substrate comprises H 2  and O 2  as an energy source. 
     
     
         42 . The composition of  claim 37 , wherein the microorganism comprises at least one exogenous nucleic acid encoding an efflux pump and/or comprises the ability to overexpress a native efflux pump. 
     
     
         43 . The composition of  claim 42 , wherein the exogenous nucleic acid encoding an efflux pump encodes an efflux pump from  A. borkumensis  or  E. coli  AcrB protein. 
     
     
         44 . The composition of  claim 37 , wherein the terpene comprises a monoterpene. 
     
     
         45 . The composition of  claim 44 , wherein the monoterpene comprises limonene and/or pinene. 
     
     
         46 . The composition of  claim 37 , wherein the terpene comprises a triterpene. 
     
     
         47 . The composition of  claim 46 , wherein the triterpene comprises squalene. 
     
     
         48 . The composition of  claim 37 , wherein the microorganism is a  Cupriavidus  sp. or  Ralstonia  sp. or  Hydrogenobacter  sp. 
     
     
         49 . A method for producing a terpene, comprising culturing the non-naturally occurring microorganism of  claim 37  in a bioreactor that comprises the gaseous substrate and the culture medium, wherein the culture medium comprises other nutrients for growth and bioproduct production, under conditions that are suitable for growth of the microorganism and production of the terpene, wherein the microorganism produces the terpene. 
     
     
         50 . The composition of  claim 49 , wherein the gaseous substrate comprises CO 2 , CO, and/or CH 4  as a carbon source. 
     
     
         51 . The method of  claim 49 , wherein the gaseous substrate comprises CO and O 2 ; CO 2 , H 2  and O 2 ; CO, CO 2 , H 2 , and O 2 ; or CO, H 2 , and O 2 . 
     
     
         52 . The method of  claim 49 , wherein the gaseous substrate is producer gas or syngas. 
     
     
         53 . The method of  claim 49 , wherein the microorganism is a knallgas microorganism. 
     
     
         54 . The method of  claim 53 , wherein the gaseous substrate comprises CO 2  as a carbon source, H 2  as an electron donor, and O 2  as an electron acceptor. 
     
     
         55 . The method of  claim 53 , wherein the gaseous substrate comprises H 2  and O 2  as an energy source. 
     
     
         56 . The method of  claim 49 , wherein the gaseous substrate is derived from municipal solid waste, black liquor, agricultural waste, wood waste, stranded natural gas, biogas, sour gas, methane hydrates, tires, pet coke, sewage, manure, straw, lignocellulosic energy crops, lignin, crop residues, bagasse, saw dust, forestry residue, food waste, waste carpet, waste plastic, landfill gas, and/or lignocellulosic biomass. 
     
     
         57 . The method of  claim 49 , wherein the microorganism comprises at least one exogenous nucleic acid encoding an efflux pump and/or comprises the ability to overexpress a native efflux pump, and wherein a greater amount of said terpene is transported out of the microorganism and into the culture medium than an equivalent microorganism that does not comprise the exogenous nucleic acid encoding an efflux pump and/or the ability to overexpress a native efflux pump. 
     
     
         58 . The method of  claim 49 , wherein the terpene is recovered from the surface of the culture medium at the interface between the liquid and gas phases in the bioreactor. 
     
     
         59 . The method of  claim 49 , wherein the culture medium is a biphasic liquid medium that comprises an aqueous phase and an organic phase, and wherein the terpene is recovered in the organic phase. 
     
     
         60 . The method of  claim 49 , wherein the microorganism produces the terpene chemoautotrophically. 
     
     
         61 . The method of  claim 49 , wherein the terpene comprises a monoterpene. 
     
     
         62 . The method of  claim 61 , wherein the monoterpene comprises limonene and/or pinene. 
     
     
         63 . The method of  claim 62 , wherein the monoterpene comprises limonene, and the limonene is dimerized to produce jet fuel and/or is converted to terephthalic acid. 
     
     
         64 . The method of  claim 49 , wherein the terpene comprises a triterpene. 
     
     
         65 . The method of  claim 64 , wherein the triterpene comprises squalene.