METHODS AND COMPOSITIONS FOR DETECTING MICROBIAL PRODUCTION OF WATER-IMMISCIBLE COMPOUNDS
Provided herein are methods and compositions useful for detecting the production of compounds in a cell, for example, a microbial cell genetically modified to produce one or more such compounds at greater yield and/or with increased persistence compared to a parent microbial cell that is not genetically modified. In some embodiments, the methods comprise contacting a solution with a fluorescent dye that directly binds the recombinantly produced compound, wherein the solution comprises a plurality of cells recombinantly producing the compound; and detecting the fluorescent dye under spectral conditions suitable for the selective detection of the fluorescent dye bound to the recombinantly produced compound.
1 - 60 . (canceled)
61 . A liquid composition comprising:
(a) a cell recombinantly producing and secreting a water-immiscible compound;
(b) water immiscible-compound secreted from said cell;
(c) a fluorescent dye that directly binds to the secreted water-immiscible compound; and
(d) cell culture medium.
62 . The composition of claim 61 , wherein the cell is selected from the group consisting of a yeast cell, a bacterial cell, a mammalian cell, a fungal cell, an insect cell, and a plant cell.
63 . The composition of claim 62 , wherein the yeast is Saccharomyces cerevisiae.
64 . The composition of claim 61 , wherein the recombinantly produced water-immiscible compound is an isoprenoid.
65 . The composition of claim 61 , wherein the fluorescent dye is Nile Red.
66 . The composition of claim 61 , wherein the fluorescent dye is BODIPY 493/503 or BODIPY 505/515.
67 . The composition of any claim 61 , wherein the recombinantly produced water-immiscible compound is a terpene, C5 isoprenoid, C10 isoprenoid or C15 isoprenoid.
68 . The composition of claim 61 , wherein the recombinantly produced water-immiscible compound is farnesene.
69 . The composition of claim 61 , wherein the cell is a recombinant yeast cell comprising one or more heterologous nucleotide sequences encoding one or more enzymes of the mevalonate (MEV) pathway.
70 . The composition of claim 69 , wherein the recombinant yeast cell comprises a nucleic acid encoding farnesene synthase.
71 . The composition of claim 69 , wherein the recombinant yeast cell comprises a heterologous nucleotide sequence that encodes an enzyme that can convert HMG-CoA into mevalonate.
72 . The composition of claim 69 , wherein the recombinant yeast cell comprises a heterologous nucleotide sequence that encodes an enzyme that can convert mevalonate into mevalonate 5-phosphate.
73 . The composition of claim 69 , wherein the one or more heterologous nucleotide sequences encodes more than one enzyme of the mevalonate pathway.
74 . The composition of claim 69 , wherein the cell further comprises a heterologous nucleotide sequence encoding an enzyme that can convert isopentenyl pyrophosphate (IPP) into dimethylallyl pyrophosphate (DMAPP).
75 . The composition of claim 74 , wherein the cell further comprises a heterologous nucleotide sequence encoding an enzyme that can modify IPP or a polyprenyl to form an isoprenoid compound.
76 . The composition of claim 75 , wherein the enzyme that can modify IPP or a polyprenyl to form an isoprenoid compound is selected from the group consisting of carene synthase, geraniol synthase, linalool synthase, limonene synthase, myrcene synthase, ocimene synthase, α-pinene synthase, β-pinene synthase, γ-terpinene synthase, terpinolene synthase, amorphadiene synthase, α-farnesene synthase, β-farnesene synthase, farnesol synthase, nerolidol synthase, patchouliol synthase, nootkatone synthase, and abietadiene synthase.
77 . The composition of claim 75 , wherein the isoprenoid is a C 5 -C 20 isoprenoid.
78 . The composition of claim 77 , wherein the isoprenoid is selected from the group consisting of abietadiene, amorphadiene, carene, α-farnesene, β-farnesene, farnesol, geraniol, geranylgeraniol, isoprene, linalool, limonene, myrcene, nerolidol, ocimene, patchoulol, β-pinene, sabinene, γ-terpinene, terpinolene, and valencene.
79 . A method of detecting, in solution, farnesene produced and secreted from a ce the method comprising:
(a) contacting a solution with Nile Red, wherein the solution comprises a cell recombinantly producing and secreting farnesene; and
(b) detecting Nile Red at an excitation wavelength of about 260 to 290 nm and an emission wavelength of about 530 to 570 nm.
80 . The method of claim 79 , wherein the solution comprising the plurality of cells is contained in a well of a multi-well cell culture plate.
81 . The method of claim 80 , wherein the multi-well cell culture plate is coated with Teflon.
82 . The method of claim 79 , wherein the cells are cultured for a period of at least 12 hours prior to said detecting.
83 . The method of claim 79 , further comprising the step of shaking the multi-well cell culture plate prior to said detecting.
84 . The method of claim 79 , wherein the cell is selected from the group consisting of a yeast cell, a bacterial cell, a mammalian cell, a fungal cell, an insect cell, and a plant cell.
85 . The method of claim 84 , wherein the cell is a yeast cell.
86 . The method of claim 85 , wherein the yeast is Saccharomyces cerevisiae.