Methods of developing terpene synthase variants

Abstract

The present disclosure relates to methods of developing terpene synthase variants through engineered host cells. Particularly, the disclosure provides methods of developing terpene synthase variants with improved in vivo performance that are useful in the commercial production of terpene products. Further encompassed in the present disclosure are superior terpene synthase variants and host cells comprising such terpene synthase variants.

1 .- 30 . (canceled) 
     
     
         31 . A method of testing a terpene synthase variant for improved in vivo performance, comprising the steps of:
 (a) dividing a population of host cells into a control population and a test population;   (b) expressing in the control population a heterologous control terpene synthase that converts a polyprenyl diphosphate to a terpene, wherein the growth rate of the host cells is dependent on having an amount of intracellular polyprenyl diphosphate;   (c) expressing in the test population a heterologous test terpene synthase, wherein the test terpene synthase is a variant of the control terpene synthase that can convert the same polyprenyl diphosphate to the same terpene as the control terpene synthase; and   (d) determining the growth rate of the control population and the test population,   
       wherein the test terpene synthase is identified as having improved in vivo performance compared to the control terpene synthase by a decreased growth rate of the test population compared to the control population. 
     
     
         32 . The method of  claim 31 , wherein the control terpene synthase and the test terpene synthase are both a synthase selected from the group consisting of a monoterpene synthase, a sesquiterpene synthase, a diterpene synthase, a sesterterpene synthase, a triterpene synthase, a tetraterpene synthase, or a polyterpene synthase. 
     
     
         33 . The method of  claim 31 , wherein the control terpene synthase and the test terpene synthase are sesquiterpene synthases, and the polyprenyl diphosphate is farnesyl diphosphate (FPP). 
     
     
         34 . The method of  claim 33 , wherein the sesquiterpene synthase is selected from the group consisting of β-farnesene synthase, α-farnesene synthase, trichodiene synthase, patchoulol synthase, amorphadiene synthase, valencene synthase, farnesol synthase, nerolidol synthase, and nootkatone synthase. 
     
     
         35 . The method of  claim 33 , wherein the control and test terpene synthases are synthases having farnesene synthase activity. 
     
     
         36 . The method of  claim 31 , wherein the control terpene synthase and the test terpene synthase are monoterpene synthases, and the polyprenyl diphosphate is geranyl diphosphate (GPP). 
     
     
         37 . The method of  claim 31 , wherein the control terpene synthase and the test terpene synthase are diterpene synthases, and the polyprenyl diphosphate is geranylgeranyl pyrophosphate (GGPP). 
     
     
         38 . The method of  claim 31 , wherein the test terpene synthase differs from the control terpene synthase by 1 to 10 amino acids. 
     
     
         39 . The method of  claim 31 , wherein the test terpene synthase is expressed at a similar level as the control terpene synthase. 
     
     
         40 . The method of  claim 31 , wherein the population of host cells is a population of yeast cells. 
     
     
         41 . The method of  claim 40 , wherein the yeast cells are  S. cerevisiae  cells. 
     
     
         42 . The method of  claim 31 , wherein the population of host cells is a population of bacterial cells. 
     
     
         43 . The method of  claim 42 , wherein the bacterial cells are  E. coli  cells. 
     
     
         44 . A composition comprising two cell subpopulations derived from a common population of host cells, wherein:
 (a) the first subpopulation comprises a heterologous control terpene synthase that converts a polyprenyl diphosphate to a terpene, wherein the growth rate of the host cells is dependent on having an amount of intracellular polyprenyl diphosphate; and   (b) the second subpopulation comprises a heterologous test terpene synthase that is a variant of the control terpene synthase that can convert the same polyprenyl diphosphate to the same terpene as the control terpene synthase.   
     
     
         45 . The composition of  claim 44 , wherein the growth rate of the second subpopulation is less than the growth rate of the first subpopulation. 
     
     
         46 . The composition of  claim 44 , wherein the control terpene synthase and the test terpene synthase are both a synthase selected from the group consisting of a monoterpene synthase, a sesquiterpene synthase, a diterpene synthase, a sesterterpene synthase, a triterpene synthase, a tetraterpene synthase, or a polyterpene synthase. 
     
     
         47 . The composition of  claim 44 , wherein the control terpene synthase and the test terpene synthase are sesquiterpene synthases, and the polyprenyl diphosphate is farnesyl diphosphate (FPP). 
     
     
         48 . The composition of  claim 47 , wherein the sesquiterpene synthase is selected from the group consisting of β-farnesene synthase, α-farnesene synthase, trichodiene synthase, patchoulol synthase, amorphadiene synthase, valencene synthase, farnesol synthase, nerolidol synthase, and nootkatone synthase. 
     
     
         49 . The composition of  claim 47 , wherein the control and test terpene synthases are synthases having farnesene synthase activity. 
     
     
         50 . The composition of  claim 44 , wherein the control terpene synthase and the test terpene synthase are monoterpene synthases, and the polyprenyl diphosphate is geranyl diphosphate (GPP). 
     
     
         51 . The composition of  claim 44 , wherein the control terpene synthase and the test terpene synthase are diterpene synthases, and the polyprenyl diphosphate is geranylgeranyl pyrophosphate (GGPP). 
     
     
         52 . The composition of  claim 44 , wherein the test terpene synthase differs from the control terpene synthase by 1 to 10 amino acids. 
     
     
         53 . The composition of  claim 44 , wherein the test terpene synthase is expressed at a similar level as the control terpene synthase. 
     
     
         54 . The composition of  claim 44 , wherein the population of host cells is a population of yeast cells. 
     
     
         55 . The composition of  claim 54 , wherein the yeast cells are  S. cerevisiae  cells. 
     
     
         56 . The composition of  claim 44 , wherein the population of host cells is a population of bacterial cells. 
     
     
         57 . The composition of  claim 56 , wherein the bacterial cells are  E. coli  cells.