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Methods of developing terpene synthase variants

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Methods of developing terpene synthase variants


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.

Browse recent Amyris, Inc. patents - Emeryville, CA, US
Inventors: Lishan Zhao, Lan Xu, Patrick Westfall, Andrew Main
USPTO Applicaton #: #20120270260 - Class: 435 29 (USPTO) - 10/25/12 - Class 435 
Chemistry: Molecular Biology And Microbiology > Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip >Involving Viable Micro-organism

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The Patent Description & Claims data below is from USPTO Patent Application 20120270260, Methods of developing terpene synthase variants.

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1. CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No. 13/363,588, filed Feb. 2, 2012, which claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61/438,948, filed Feb. 2, 2011. Each of these priority documents are incorporated herein by reference, in their entirety.

2.

FIELD OF THE INVENTION

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.

3. BACKGROUND

Terpenes are a large class of hydrocarbons that are produced in many organisms. They are derived by linking units of isoprene (C5H8), and are classified by the number of isoprene units present. Hemiterpenes consist of a single isoprene unit. Isoprene itself is considered the only hemiterpene. Monoterpenes are made of two isoprene units, and have the molecular formula C10H16. Examples of monoterpenes are geraniol, limonene, and terpineol. Sesquiterpenes are composed of three isoprene units, and have the molecular formula C15H24. Examples of sesquiterpenes are farnesenes, farnesol and patchoulol. Diterpenes are made of four isoprene units, and have the molecular formula C20H32. Examples of diterpenes are cafestol, kahweol, cembrene, and taxadiene. Sesterterpenes are made of five isoprene units, and have the molecular formula C25H40. An example of a sesterterpenes is geranylfarnesol. Triterpenes consist of six isoprene units, and have the molecular formula C30H48. Tetraterpenes contain eight isoprene units, and have the molecular formula C40H64. Biologically important tetraterpenes include the acyclic lycopene, the monocyclic gamma-carotene, and the bicyclic alpha- and beta-carotenes. Polyterpenes consist of long chains of many isoprene units. Natural rubber consists of polyisoprene in which the double bonds are cis.

When terpenes are chemically modified (e.g., via oxidation or rearrangement of the carbon skeleton) the resulting compounds are generally referred to as terpenoids, which are also known as isoprenoids. Isoprenoids play many important biological roles, for example, as quinones in electron transport chains, as components of membranes, in subcellular targeting and regulation via protein prenylation, as photosynthetic pigments including carotenoids, chlorophyll, as hormones and cofactors, and as plant defense compounds with various monoterpenes, sesquiterpenes, and diterpenes. They are industrially useful as antibiotics, hormones, anticancer drugs, insecticides, and chemicals.

Terpenes are biosynthesized through condensations of isopentenyl pyrophosphate (isopentenyl diphosphate or IPP) and its isomer dimethylallyl pyrophosphate (dimethylallyl diphosphate or DMAPP). Two pathways are known to generate IPP and DMAPP, namely the mevalonate-dependent (MEV) pathway of eukaryotes, and the mevalonate-independent or deoxyxylulose-5-phosphate (DXP) pathway of prokaryotes. Plants use both the MEV pathway and the DXP pathway. IPP and DMAPP in turn are condensed to polyprenyl diphosphates (e.g., geranyl disphosphate or GPP, farnesyl diphosphate or FPP, and geranylgeranyl diphosphate or GGPP) through the action of prenyl disphosphate synthases (e.g., GPP synthase, FPP synthase, and GGPP synthase, respectively).

The polyprenyl diphosphate intermediates are converted to more complex isoprenoid structures by terpene synthases. Terpene synthases are organized into large gene families that form multiple products. Examples of terpene synthases include sesquiterpene synthases, which convert FPP into sesquiterpenes. An example of a sesquiterpene synthase is farnesene synthase, which converts FPP to farnesene. The reaction mechanism of terpene synthases has been extensively investigated and is well understood. Overall, three steps are required to convert a diphosphate substrate such as FPP to its isoprenoid product: a) formation of enzyme-substrate complex (ES), b) formation of an enzyme-bound reactive carbocation intermediate, subsequent rearrangements, and the formation of product (EP), and c) release of product from the enzyme-product complex. In vitro kinetic and pre-steady state kinetic studies on terpene synthase catalyzed reactions have shown that the overall rate-limiting step for the reactions is the release of product (Cane et al. (1997) Biochemistry, 36(27):8332-9, and Mathis et al. (1997) Biochemistry 36(27):8340-8). The turnover rates of terpene synthases are low, generally measured at less than 0.5 per second (Cane, D. C. (1990) Chem. Rev. 90:1089-1103).

Terpene synthases are important in the regulation of pathway flux to an isoprenoid because they operate at metabolic branch points and often compete with other metabolic enzymes for a prenyl diphosphate pool. For example, FPP is the precursor to many cellular molecules including squalene, dolichols, and the cofactor heme. In engineered microbes where the production of sesquiterpenes such as farnesene is desired, the terpene synthases hold the key to high yield production of such terpenes. However, because they are slow enzymes, terpene synthases are often the bottlenecks in the metabolic pathways. In addition, they can suffer from other shortcomings such as substrate inhibition that limit the kinetic capacity required for efficient production of terpenes in engineered microbial hosts (Crock et al. (1997) Proc. Natl. Acad. Sci. USA 94:12833-12838).

Hence, there are potentially enormous benefits to improving the catalytic efficiency of terpene synthases so that these enzymes would no longer limit the overall metabolic flux to an isoprenoid. Attempts to engineer terpene synthases for altered product specificity as well as the use of rational approaches such as those based on structural guidance or adaptive evolution have been described previously (Greenhagen et al. (2006) Proc. Natl. Acad. Sci. USA 103:9826-9831; O\'Maille et al. (2008) Nat. Chem. Biol. 4:617-623; Yoshikuni et al. (2006) Nature 440:1078-1082; Yoshikuni et al. (2008) Chem. Biol. 15:607-618). However, these studies have fallen short of improving the kinetic capacity of terpene synthases while also maintaining their product specificity. In addition, the application of conventional protein engineering strategies, such as directed evolution, has been devoid for terpene synthases primarily because of the lack of available and effective high throughput screening methods (Yoshikuni et al. (2008) (supra)). There thus remains a need for reliable and high throughput methods for improving the catalytic efficiency of terpene synthases, and for terpene synthase variants that have such improved catalytic efficiency.

4.

SUMMARY

OF THE INVENTION

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. The methods also allow for the continued improvement of the in vivo performance of these enzymes.

In one aspect, the present invention provides a screening method for a sesquiterpene synthase variant with improved in vivo performance, comprising the steps of: a) engineering a host cell expressing a control sesquiterpene synthase to comprise an elevated level of FPP, wherein the elevated level of FPP reduces the viability of the host cell compared to a parent cell not comprising the elevated level of FPP; b) expressing in the host cell a test sesquiterpene synthase instead of the control sesquiterpene synthase, wherein the test sesquiterpene synthase is a variant of the control sesquiterpene synthase; and c) identifying the test sesquiterpene synthase as having improved in vivo performance compared to the control sesquiterpene synthase by an increase in viability of the host cell expressing the test sesquiterpene synthase compared to the host cell expressing the control terpene synthase.

In some embodiments, the host cell is plated on an agar plate, and a host cell comprising a test terpene synthase variant with improved in vivo performance is identified by colony growth. In some embodiments, the method further comprises selecting and/or isolating the test sesquiterpene synthase having improved in vivo performance.

In some embodiments, a collection of sesquiterpene synthase variants is expressed in a collection of host cells. In some embodiments, the collection of sesquiterpene synthase variants comprises from 2 to 5, from 5 to 10, from 10 to 50, from 50 to 100, from 100 to 500, from 500 to 1,000, from 1,000 to 10,000, from 10,000 to 100,000, from 100,000 to 1,000,000, and more, sesquiterpene synthase variants.

In some embodiments, the screening method is used in an iterative fashion, wherein the test sesquiterpene synthase identified in an iteration is used as the control sesquiterpene synthase of the next iteration, and wherein the host cell in an iteration comprises such elevated level of FPP that it has reduced viability in the presence of the test sesquiterpene synthase identified in the previous iteration compared to a parent cell not comprising the elevated level of FPP.

In another aspect, provided herein is a composition comprising two cell subpopulations derived from a common population of host cells comprising an elevated level of FPP, wherein: a) the first subpopulation comprises a control sesquiterpene synthase, wherein the elevated level of FPP reduces the viability of cells of the first subpopulation compared to the viability of a parent cell not comprising the elevated level of FPP; and b) the second subpopulation comprises a test sesquiterpene synthase, wherein the test sesquiterpene synthase is a variant of the control sesquiterpene synthase.

In some embodiments, the viability of the cells of the second subpopulation is greater than the viability of the cells of the first subpopulation.

In another aspect, the present invention provides a second screening method for identifying terpene synthase variants with improved in vivo performance, comprising the steps of: a) providing a host cell expressing a control terpene synthase and having a growth rate; b) expressing in the host cell a test terpene synthase instead of the control terpene synthase, wherein the test terpene synthase is a variant of the control terpene synthase; and d) identifying the test terpene synthase as having improved in vivo performance compared to the control terpene synthase by a decreased growth rate of the host cell expressing the test terpene synthase compared to the growth rate of the host cell expressing the control terpene synthase.

In yet another aspect, the present invention provides a competition method for identifying and/or ranking the in vivo performance of terpene synthase variants, 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 control terpene synthase and a comparison terpene synthase, wherein the control terpene synthase can convert a polyprenyl diphosphate to a first terpene, and wherein the comparison terpene synthase can convert a polyprenyl diphosphate to a second terpene; c) expressing in the test population the comparison terpene synthase and a test terpene synthase, wherein the test terpene synthase is a variant of the control terpene synthase, and wherein the comparison terpene synthase is expressed at similar levels in the test population and in the control population; and d) measuring a ratio of the first terpene over the second terpene in the test population and in the control population.

In separate embodiments, the competition method is applied to identify and/or to rank terpene synthases selected from the group consisting of monoterpene synthases, diterpene synthases, sesquiterpene synthases, sesterterpene synthases, triterpene synthases, tetraterpene synthases, and polyterpene synthases.

In some embodiments, the competition method is used to screen a library of mutant terpene synthases on the basis that compared to the control terpene synthase, a terpene synthase variant with improved in vivo performance is capable of diverting more flux from a polyprenyl diphosphate substrate to its terpene product, thus, giving a higher ratio of terpene of interest/comparison terpene (i.e., first terpene/second terpene). In such embodiments, it is important that the test terpene synthase is expressed at a similar level in the test population as the control terpene synthase is expressed in the control population.

In other embodiments, the competition method is used to identify a promoter of a desired strength. In such embodiments, the control terpene synthase and the test terpene synthase are identical, and the control population and test population differ in the expression level of the control terpene synthase.

In another aspect, provided herein is a composition comprising two cell subpopulations derived from a common population of host cells, wherein: a) the first subpopulation comprises a control terpene synthase and a comparison terpene synthase, wherein the control terpene synthase converts a polyprenyl diphosphate to a first terpene, and wherein the comparison sesquiterpene synthase converts the polyprenyl diphosphate to a second terpene; and b) the second subpopulation comprises a test terpene synthase and the comparison terpene synthase, wherein the control terpene synthase converts the polyprenyl diphosphate to the first terpene, and wherein the test terpene synthase is a variant of the control terpene synthase.

In some embodiments, the ratio of the first terpene over the second terpene is greater in the second subpopulation compared to that in the first subpopulation.

In yet another aspect, provided herein are isolated β-farnesene synthase variants, and isolated nucleic acids comprising a nucleotide sequence encoding such β-farnesene synthase variants, having an amino acid sequence as given in SEQ ID NO: 111 but comprising one or more amino acid substitutions at positions selected from the group consisting of positions 2, 3, 4, 6, 9, 11, 18, 20, 24, 35, 38, 50, 61, 72, 80, 89, 105, 115, 144, 196, 211, 251, 280, 288, 319, 348, 357, 359, 369, 371, 385, 398, 423, 433, 434, 442, 444, 446, 460, 467, 488, 495, 505, 526, 531, 556, 572, and 575 of SEQ ID NO: 111.



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stats Patent Info
Application #
US 20120270260 A1
Publish Date
10/25/2012
Document #
File Date
09/02/2014
USPTO Class
Other USPTO Classes
International Class
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