Microbial production of natural sweeteners, diterpenoid steviol glycosides
AssigneeMassachusetts Institute of Technology
InventorsGregory Stephanopoulos, Parayil K. Ajikumar
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Abstract
The invention relates to recombinant expression of a steviol or steviol glycosides biosynthetic pathway enzymes in cells and the production of steviol or steviol glycosides.
Claims54
A method comprising:
- recombinantly expressing a copalyl diphosphate synthase (CPS), kaurene synthase (KS) and a geranylgeranyl diphosphate synthase (GGPPS) enzyme in a cell that expresses an endogenous non-mevalonate pathway (MEP) or mevalonic acid pathway (MVA), and which optionally overexpresses one or more components of said pathway.
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A cell that expresses the non-mevalonic acid (MVA) pathway or expresses the non-mevalonate (MEP) pathway, and which optionally overexpresses one or more components of said pathway, and that recombinantly expresses a copalyl diphosphate synthase (CPS) enzyme, a kaurene synthase (KS) enzyme, a geranylgeranyl diphosphate synthase (GGPPS) enzyme, a kaurene oxidase (KO) enzyme or a catalytically active portion of KO fused to a cytochrome P450 reductase enzyme, and a kaurenoic acid 13-hydroxylase (KAH) enzyme or catalytically active portion of KAH fused to a cytochrome P450 reductase enzyme.
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A method for selecting a cell that exhibits enhanced production of kaurene, steviol or steviol glycosides, the method comprising:
- creating or obtaining a cell that overexpresses one or more components of the mevalonic acid pathway (MVA) or non-mevalonate (MEP) pathway,
- producing kaurene, steviol or steviol glycosides from the cell,
- comparing the amount of kaurene, steviol or steviol glycosides produced from the cell to the amount of kaurene, steviol or steviol glycosides produced in a control cell, and
- selecting a first improved cell that produces a higher amount of kaurene, steviol or steviol glycosides than a control cell, wherein a first improved cell that produces a higher amount of kaurene, steviol or steviol glycosides than the control cell is a cell that exhibits enhanced production of kaurene, steviol or steviol glycosides.
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An isolated polypeptide comprising a kaurene oxidase (KO) enzyme, a kaurenoic acid 13-hydroxylase (KAH) enzyme, or a catalytically active portion thereof fused to a cytochrome P450 reductase enzyme, or a catalytically active portion thereof.
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A method for increasing terpenoid production in a cell that produces at least one terpenoid selected from kaurene, steviol or steviol glycosides, comprising controlling the accumulation of indole in the cell or in a culture of the cells, thereby increasing terpenoid production in a cell.
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A method comprising measuring the amount or concentration of indole in a cell that produces at least one terpenoid selected from kaurene, steviol or steviol glycosides, or in a culture of the cells that produce one or more of the terpenoids.
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A method for making a product containing a terpenoid selected from kaurene, a steviol, or a steviol glycoside, the method comprising:
- increasing terpenoid production in a cell that produces one or more terpenoids by controlling the accumulation of indole in the cell or in a culture of the cells,
- recovering the terpenoid from the cell, and optionally conducting one or more chemical or enzymatic steps on the recovered terpenoid;
- incorporating the recovered terpenoid or the terpenoid prepared through one or more chemical or enzymatic steps, into a product to thereby make the product containing a terpenoid.
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Dependencies are inferred from the claim text.