Dpp1 5 29996ru full 30 11 2011 – 8 9 – ar


valid until 2018/1/23

Dpp1 5 29996ru full 30 11 2011

Dpp1 5 29996ru full 30 11 2011

Dpp1 5 29996ru full 30 11 2011

Dpp1 5 29996ru full 30 11 2011

Dpp1 5 29996ru full 30 11 2011

14.02.2018 – Provided herein is a recombinant host, such as a microorganism, plant, or plant cell, comprising one or more biosynthesis genes whose expression results in production of steviol glycosides such as rebaudioside A, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, or dulcoside A. Terpenoids can be thought of as terpene derivatives.

Version dpp1 5 29996ru full 30 11 2011 clean app for

Dpp1 5 29996ru full 30 11 2011

What’s New?

1. 6The plasmid vector should be able to be maintained and replicated in bacteria, fungi and yeast. As isolated nucleic acids, these modified sequences can exist as purified molecules and can be incorporated into a vector or a virus for use in constructing modules for recombinant nucleic acid constructs.
2. 10 Optionally, the SUS1 sequence is an A.http://softik.org/zte-max-xl-unlock-code/Thus, said method may further comprise dephosphorylating the farnesyl phosphate to produce farnesol. A recombinant host may contain one or more genes encoding enzymes involved in the methylerythritol 4-phosphate MEP pathway for isoprenoid biosynthesis.

3. 5 Chemical inhibition of squalene synthase, e. Dyes and Pigments http://softik.org/software-house-c-cure-9000-release-date/ http://softik.org/software-house-c-cure-9000-training/X 2 may for example consist of in the range of 4 to 25, such as in the range of 4 to 20, for example of in the range of 4 to 15, such as in the range of 6 to 12, for example in the range of 8 to 12, such as in the range of 9 to 11 nucleotides.

Create an account or sign in to comment

Dpp1 5 29996ru full 30 11 2011

4. 7 Kluyveromyces lactis is a yeast regularly applied to the production of kefir. Such plants can be grown in a manner suitable for the species under consideration, either in a growth chamber, a greenhouse, or in a field.Dpp1 5 29996ru full 30 11 2011A microorganism can be a prokaryote such as Escherichia coli, Rhodobacter sphaeroides, or Rhodobacter capsulatus. Received 2 February

5. 9 A candidate sequence typically has a length that is from 80 percent to percent of the length of the reference sequence, e. The poly-A sequence acts as a buffer to the 3′ exonuclease and thus increases half-life of mRNA.

6. 4 Thus, also disclosed herein is a microbial cell comprising a nucleic acid sequence, said nucleic acid comprising i a promoter sequence operably linked to ii a heterologous insert sequence operably linked to iii an open reading frame operably linked to iv a transcription termination signal, wherein the heterologous insert sequence and the open reading frame are as defined herein above, wherein said microbial cell furthermore comprises a heterologous nucleic acid encoding GGPPS operably linked to a nucleic acid sequence directing expression of GGPPS in said cell. See, the ERG9 section below and Examples

7. 7 The poly-A sequence acts as a buffer to the 3′ exonuclease and thus increases half-life of mRNA.

Dpp1 5 29996ru full 30 11 2011 pro

Kind code of ref document: Ref legal event code: Country of ref document: Year of fee payment: This application claims priority to U. This disclosure relates to the recombinant production of steviol glycosides.

In particular, this disclosure relates to the production of steviol glycosides such as rebaudioside D by recombinant hosts such as recombinant microorganisms, plants, or plant cells.

This disclosure also provides compositions containing steviol glycosides. The disclosure also relates to tools and methods for producing terpenoids by modulating the biosynthesis of terpenoid precursors of the squalene pathway.

Sweeteners are well known as ingredients used most commonly in the food, beverage, or confectionary industries. The sweetener can either be incorporated into a final food product during production or for stand-alone use, when appropriately diluted, as a tabletop sweetener or an at-home replacement for sugars in baking.

Sweeteners include natural sweeteners such as sucrose, high fructose corn syrup, molasses, maple syrup, and honey and artificial sweeteners such as aspartame, saccharine and sucralose.

Stevia extract is a natural sweetener that can be isolated and extracted from a perennial shrub, Stevia rebaudiana. Stevia is commonly grown in South America and Dpp1 for commercial production of stevia extract.

Stevia extract, purified to various degrees, is used commercially as a high intensity sweetener in foods and in blends or alone as a tabletop sweetener. Extracts of the Stevia plant contain rebaudiosides and other steviol glycosides that contribute to the sweet flavor, although the amount of each glycoside often dpp1 among different production batches.

Existing commercial products are predominantly rebaudioside A with lesser amounts of other glycosides such as rebaudioside C, D, and F. Stevia extracts may also contain contaminants such as plant-derived compounds that contribute to off-flavors.

These off-flavors can be more or less problematic depending on the food system or application of choice. ABM Database accession no. AAY Database accession no. US A1 describes a transgenic moss cell that produces or accumulates a terpenoid compound and methods of producing a terpenoid compound through culturing of the transgenic moss.

EP A2 describes nucleotide sequences encoding ent -kaurenoic acid hydroxylase polypeptides and polypeptides having ent- kaurenic acid hydroxylase activity. Provided herein is a recombinant host, such as a microorganism, plant, or plant cell, comprising one or more biosynthesis genes whose expression results in production of steviol glycosides such as rebaudioside A, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, or dulcoside A.

As described herein, EUGT11 has a strong 1,O-glucose glycosylation activity, which is an important step for rebaudioside D production. Typically, stevioside and rebaudioside A are the primary compounds in commercially-produced stevia extracts.

Stevioside is reported to have a more bitter and less sweet taste than rebaudioside A. The composition of stevia extract can vary from lot to lot depending on the soil and climate in which the plants are grown.

Other steviol glycosides are present in varying amounts in stevia extracts. The amount of the minor steviol glycosides affects the flavor profile of a Stevia extract. Full addition, Rebaudioside D and other higher glycosylated steviol glycosides are thought to be higher quality sweeteners than Rebaudioside A.

As such, the recombinant hosts and methods described herein are particularly useful for producing steviol glycoside compositions having an increased amount of Rebaudioside D for use, for example, as a non-caloric sweetener with functional and sensory properties superior to those of many high-potency sweeteners.

In another aspect, the present invention relates to a method of producing a steviol glycoside, comprising: This document features a recombinant host that includes a recombinant gene encoding a polypeptide having the ability to transfer 2011 second sugar moiety to the C-2′ of a O-glucose of rubusoside.

This document also features a recombinant host that includes a recombinant gene encoding a polypeptide having the ability to transfer a second sugar moiety to the C-2′ of a O-glucose of stevioside.

This document also features a recombinant host that includes a recombinant gene encoding a polypeptide having the ability to transfer a second sugar moiety to the C-2′ of the O-glucose of rubusoside and to the C-2′ of the O-glucose of rubusoside.

This document also features a recombinant host that incudes a recombinant gene encoding a polypeptide having the ability to transfer a second sugar moiety to the C-2′ of a O-glucose of rebaudioside A to produce rebaudioside D, wherein the catalysis rate of the polypeptide is at least 20 times faster e.

The UGT85C2 polypeptide can include one or more amino acid substitutions at residues 9, 10, 13, 15, 21, 27, 60, 65, 71, 87, 91,,,,,and of SEQ ID NO: The UGT76G1 polypeptide can have one or more amino acid substitutions at residues 29, 74, 87, 91,,29996ru,,,,,,and of SEQ ID NO: Any of the hosts described herein further can include a gene e.

Any of the hosts described herein further can include one or more of i a gene encoding a geranylgeranyl diphosphate synthase; ii a gene encoding a bifunctional copalyl diphosphate synthase and kaurene synthase, or a gene encoding a copalyl diphosphate synthase 2011 a gene encoding a kaurene synthase; iii a gene encoding a kaurene oxidase; and iv a gene encoding a steviol synthetase.

Each of the genes of iiiiiiand iv can be a recombinant gene. Any of the hosts described herein further can include one or more of v a gene encoding a truncated HMG-CoA; vi a gene encoding a CPR; vii a gene encoding a rhamnose synthetase; viii a gene encoding a UDP-glucose dehydrogenase; and ix a gene encoding a UDP-glucuronic acid decarboxylase.

At least one of the genes of iiiiiiivvviviiviiior ix can be a recombinant gene. Any of the recombinant hosts can produce at least one steviol glycoside when cultured under conditions in which each of the genes is expressed.

The steviol glycoside can be selected from the group consisting of rubusoside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, dulcoside A, stevioside, steviol O -Glucoside, steviol O -glucoside, steviol-1,2-bioside, steviol-1,3-bioside, 1,3-stevioside, as well as other rhamnosylated or xylosylated intermediates.

The steviol glycoside e. This document also features a method of producing a steviol glycoside. The method includes growing any of the hosts described herein in a culture medium, under conditions in which the genes are expressed; and recovering the steviol glycoside produced by the host.

The growing step can include inducing expression of one or more of the genes. For example, the steviol glycoside can be rebaudioside D or rebaudioside E.

Other examples of steviol glycosides can include rebaudioside A, rebaudioside Dpp1, rebaudioside C, rebaudioside F, and dulcoside A. This document also features a recombinant host.

The host includes i a gene encoding a UGT74G1; ii a gene encoding a UGT85C2; iii a gene encoding a UGT76G1; iv a gene encoding a glycosyltransferase having the ability dpp1 transfer a second sugar moiety to the C-2′ of a O-glucose of rubusoside or stevioside; and full optionally a gene encoding a UGT91D2e, wherein at least one of the genes is a recombinant gene.

Each of the genes full be a recombinant gene. The host can produce at least one steviol glycoside e. The host further can include a a gene encoding a bifunctional copalyl diphosphate synthase and kaurene synthase, or a gene encoding a copalyl diphosphate synthase and a gene encoding a kaurene synthase; b a gene encoding a kaurene oxidase; c a gene encoding a steviol synthetase; d a gene encoding a geranylgeranyl diphosphate synthase.

This document also features a steviol glycoside composition produced by any of the hosts described herein. The composition has reduced levels of stevia plant-derived contaminants relative to a stevia extract.

In another aspect, this document features a steviol glycoside composition produced by any of the hosts described herein. The composition has a steviol glycoside composition enriched for rebaudioside D relative to the steviol glycoside composition of a wild-type Stevia plant.

In yet another aspect, this document features a method 2011 producing a steviol glycoside composition. The method includes growing a host described herein in a culture medium, under conditions in which each of the genes is expressed; and recovering the steviol glycoside composition produced by the host e.

The composition is enriched for rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F or dulcoside A relative to the steviol glycoside composition of a wild-type Stevia plant.

The steviol glycoside composition produced by the host e. This document also features a method for transferring a second sugar moiety to the C-2′ of a O -glucose or the C-2′ of a O -glucose in a steviol glycoside.

The steviol glycoside can be rubusoside, wherein the second sugar moiety is glucose, and stevioside is produced upon transfer of the second glucose moiety. The steviol glycoside can be stevioside, wherein the second sugar moiety is glucose, and Rebaudioside E is produced upon transfer of the second glucose moiety.

The steviol glycoside can be Rebaudioside A, and Rebaudioside D is produced upon transfer of the second glucose moiety. Also described is an improved downstream steviol glycoside pathway in which materials and methods are provided for the recombinant production of sucrose synthase, and to materials and methods for increasing production of UDP-glucose in a host, specifically for increasing the availability of UDP-glucose in vivo, with the purpose of promoting glycosylation reactions in the cells, and methods for reducing UDP concentrations in the cells are provided.

The document also provides a recombinant host comprising one or more exogenous nucleic acids encoding a sucrose transporter and a sucrose synthase, wherein expression of the one or more exogenous nucleic acids with a glucosyltransferase results in increased levels of UDP-glucose in the host.

Optionally, the one or more exogenous nucleic acids comprise a SUS1 sequence. Optionally, the SUS1 sequence is from Coffea arabica, or encodes a functional homolog of the sucrose synthase encoded 29996ru the Coffea arabica SUS1 sequence, but equally an Arabidopsis thaliana or Stevia rebaudiana SUS may be used as described herein.

In the recombinant host of the invention, the one or more exogenous nucleic acids may comprise a sequence encoding a polypeptide having the sequence set 29996ru in SEQ ID NO: In the recombinant host, the one or more exogenous nucleic acids may comprise a sequence encoding a polypeptide having the sequence set forth in SEQ ID NO: The recombinant host has reduced ability to degrade external sucrose, as compared to a corresponding host that lacks the one or more exogenous nucleic acids.

The recombinant host may be a microorganism, such as a Saccharomycete, for example Saccharomyces cerevisiae. Alternatively, the microorganism is Escherichia coli.

In an alternative embodiment, the recominbant host is a plant or plant cell. Also disclosed is a method for increasing the level of UDP-glucose and reducing the level of UDP in a cell, the method comprising expressing in the cell a recombinant sucrose synthase sequence and a recombinant sucrose transporter sequence, in a medium comprising sucrose, wherein the cell is deficient in sucrose degradation.

Also disclosed is a method for promoting a glycosylation reaction in a cell, comprising expressing in the cell a recombinant sucrose synthase sequence and a recombinant sucrose transporter sequence, in a medium comprising sucrose, wherein the expressing results in a decreased level of UDP in the cell and an increased level of UDP-glucose in the cell, such that glycosylation full the cell is increased.

In either method for increasing the level of UDP-glucose or promoting glycosylation, the cell may produce vanillin glucoside, resulting in increased production of vanillin glucoside by the cell, or may produce steviol glucoside, resulting in increased production of steviol glucoside by the cell.

Optionally, the SUS1 sequence is an A. The recombinant sucrose synthase sequence optionally comprises a nucleic acid encoding a polypeptide having the sequence set forth in SEQ ID NO: In either method, the host is a microorganism, for example a Saccharomycete, optionally such as Saccharomyces cerevisiae.

Or the host may be Escherichia 2011. Or the host may be a plant cell. Also provided herein is a recombinant host, such as a microorganism, comprising one or more biosynthesis 29996ru whose expression results in production of diterpenoids.

At least one of the genes is a recombinant gene. The host can also be a plant cell. Expression of these gene s in a Stevia plant can result in increased steviol glycoside levels in the plant.

In some embodiments the recombinant host further comprises a plurality of copies of a recombinant gene encoding a CDPS polypeptide EC 5. The host can further comprise a plurality of copies of a recombinant gene encoding a KAH polypeptide, e.

The host can further comprise one or more of: The host can further comprise one or more of iv a gene encoding a truncated HMG-CoA; v a gene encoding a CPR; vi a gene encoding a rhamnose synthetase; vii a gene encoding a UDP-glucose dehydrogenase; and viii a gene encoding a UDP-glucuronic acid decarboxylase.

Two or more exogenous CPRs can be present, for example. The expression of one or more of such genes can be inducible. At least one of genes iiiiiiivvviviior viii can be a recombinant gene, and in some cases each of the genes of iiiiiiivvviviiand viii is a recombinant gene.

In one aspect, this document features an isolated nucleic acid encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:

Dpp1 5 29996ru full 30 11 2011 left dead

Furthermore, the concentrations of steviol glycosides produced by recombinant hosts are expected to be higher than the levels of steviol glycosides produced in the Stevia plant, which improves the efficiency of the downstream purification. For example, a useful UGT91D2 homolog can include at least one amino acid substitution at residues, Six isoDPPT-based polymers with optical bandgaps spanning from 1. Research activities in the field of diketopyrrolopyrrole DPP -based polymers are reviewed. An attempt has been made to go through their synthesis, structure and optoelectronic properties in such a way that any future efforts to modify this versatile structure have benefited. Suitable rhamnose synthetases include those made by Arabidopsis thaliana, such as the product of the A.

Xml dpp1 5 29996ru full 30 11 2011 vacuum

Ilias Katsouras for the transistors. Citation data is made available by participants in Crossref’s Cited-by Linking service. For a more comprehensive list of citations to this article, users are encouraged to perform a search in SciFinder.

The authors declare no competing financial interest. Conjugated polymers have attracted an increasing amt. Chemists can design and synthesize a variety of conjugated polymers with different architectures and functional moieties to meet the requirements of these org.

This includes i conjugated polyphenylenes polyfluorenes, polycarbazoles, and various stepladder polymers , ii other polycyclic arom. By summarizing the performances of the different classes of conjugated polymers in devices such as org.

Finally, we summarize the current progress for conjugated polymers and propose future research opportunities to improve their performance in this exciting research field. This progress report summarizes the numerous DPP-contg.

The hole and electron mobilities that are reported in relation to structural properties such as alkyl substitution patterns, polymer mol. The authors also consider important aspects of ambipolar charge transport and highlight fundamental structure-property relations such as the relations between the thin film morphologies and the charge carrier mobilities obsd.

Conjugated polymers containing diketopyrrolopyrrole units in the main chain. Beilstein journal of organic chemistry , 6 , ISSN: Research activities in the field of diketopyrrolopyrrole DPP -based polymers are reviewed.

Synthetic pathways to monomers and polymers, and the characteristic properties of the polymers are described. Potential applications in the field of organic electronic materials such as light emitting diodes, organic solar cells and organic field effect transistors are discussed.

High mobility diketopyrrolopyrrole DPP -based organic semiconductor materials for organic thin film transistors and photovoltaics. Royal Society of Chemistry. In recent years, the electron-accepting diketopyrrolopyrrole DPP moiety has been receiving considerable attention for constructing donor-acceptor D-A type org.

The closely packed mols. Furthermore, the energy levels can be readily adjusted, affording p-type, n-type, or ambipolar org. In the past few years, a no. DPP-based polymer semiconductors have achieved record high mobility values for p-type hole mobility: Power conversion efficiencies PCE of up to 6.

This article provides an overview of the recent exciting progress made in DPP-contg. It focuses on the structural design, optoelectronic properties, mol.

However, the charge carrier mobility of P 3 was reduced because of the steric hindrance between alkyl chains attached to the DPP and BTT units resulting in a twist of the polymer backbone and therefore in decreased order.

Removing the alkyl chains at the BTT excluded the steric interactions between alkyl chains so that P4 exhibited higher order and improved OFET performance.

It was also found that the removal of alkyl chains essentially changed the arrangement of the polymers on the surface due to different interactions between the hydrophobic surface and the macromol.

By this structural optimization, a hole mobility of 0. High-mobility ambipolar near-infrared light-emitting polymer field-effect transistors. The polymer exhibits high hole and electron mobilities of 0.

Both carriers show relatively high activation energies of meVand meV, resp. The authors fabricated inverters and three-stage ring oscillators with record performance based on PSeDPPBT using a selfaligned gate technique.

Inverters exhibited an av. The results establish that the replacement of thiophene with selenophene is a promising synthetic way forward for high-mobility ambipolar polymers. The results demonstrate that with an appropriate polymeric semiconductor, high performance soln.

This implies that ambipolar logic can become a reliable substitute for complementary-based logic in order to realize low-cost polymer-based electronic circuits. Sonar, Prashant; Singh, Samarendra P.

The authors have studied the electronic, phys. Using the electronically neutral benzene B , the weakly accepting benzo-thiadiazole BT , and the strongly accepting benzobisthiadiazole BBT , the accepting strength of the companion unit was systematically modulated.

The BBT moiety also strengthens interchain interactions, which provides higher thermal stability and performance for transistors with BBT-contg. As a characteristic feature of conventional conjugated polymers, it has been generally agreed that conjugated polymers exhibit either high hole transport property p-type or high electron transport property n-type.

Although ambipolar properties have been demonstrated from specially designed conjugated polymers, only a few examples have exhibited ambipolar transport properties under limited conditions.

Furthermore, there is, as yet, no example with equiv. We describe the realization of an equiv. There is a fast-growing demand for polymer-based ambipolar thin-film transistors TFTs , in which both n-type and p-type transistor operations are realized in a single layer, while maintaining simplicity in processing.

Research progress toward this end is essentially fueled by mol. However, ambipolar polymers with even higher performance are still required. By taking into account both the conjugated backbone and side chains of the polymer component, the authors have developed a dithienyl-diketopyrrolopyrrole TDPP and selenophene contg.

A synergistic combination of rational polymer backbone design, side-chain dynamics, and soln. Although the polymers with different length sidechains exhibit almost, same absorption maxima and HOMO energy levels, they show different film-forming ability, intermol.

The longer sidechains 2-decyltetradecyl chains endow PDVT with more uniform thin films and closer n-n stacking distances compared to those of PDVT The mobility higher than 8. These results provide important progress of soln.

Such FETs fabricated from polymer semiconductors can be competitive with small mol. Ambipolar polymer semiconductors are highly suited for use in flexible, printable, and large-area electronics as they exhibit both n-type electron-transporting and p-type hole-transporting operations within a single layer.

This allows for cost-effective fabrication of complementary circuits with high noise immunity and operational stability. Currently, the performance of ambipolar polymer semiconductors lags behind that of their unipolar counterparts.

Here, the authors report on the side-chain engineering of conjugated, alternating electron donor-acceptor D-A polymers using diketopyrrolopyrrole-selenophene copolymers with hybrid siloxane-solubilizing groups PTDPPSe-Si to enhance ambipolar performance.

The alkyl spacer length of the hybrid side chains was systematically tuned to boost ambipolar performance. These results provide guidelines for the mol. A stable solution-processed polymer semiconductor with record high-mobility for printed transistors.

Scientific Reports , 2 , srep, 9 pp. We report herein the processing and optimization of soln. Exceptionally high-gain inverters and functional ring oscillator devices on flexible substrates have been demonstrated.

This optimized polymer semiconductor represents a significant progress in semiconductor development, dispelling prevalent skepticism surrounding practical usability of org. Charge carrier mobility is still the most challenging issue that should be overcome to realize everyday org.

In this Communication, introducing smart side-chain engineering to polymer semiconductors can facilitate intermol. As a result, high hole mobility could be achieved even in devices fabricated using the polymers at room temp.

In this communication, we report the synthesis of a novel diketopyrrolopyrrole-diketopyrrolopyrrole DPP-DPP -based conjugated copolymer and its application in high-mobility org. The synthesis of this novel DPP-DPP copolymer in combination with the demonstration of transistors with extremely high electron mobility makes this work an important step toward a new family of DPP-DPP copolymers for application in the general area of org.

The design, synthesis, and characterization are presented of a series of new low bandgap polymers specifically for tandem polymer solar cells. These structural modifications lead to polymers with different optical, electrochem.

Single junction solar cells were fabricated, and the polymer: PC71BM active layer morphol. New and efficient synthesis of pyrrolo[3,2-b]pyrrole-2,5-diones by double-anion-capture reactions of ester carbanions with bis imidoyl chlorides of oxalic acid.

A variety of pyrrolo[3,2-b]pyrrole-2,5-diones were efficiently prepd. This reaction proceeded by condensation of 2 equiv of the ester with the bis imidoyl chloride and subsequent intramol.

The products, which can be regarded as dilactams of pentalene, represent useful synthetic pigments due to their optical features, their stability, and low soly.

The UV-visible properties of the pyrrolo[3,2-b]pyrrole-2,5-diones could be efficiently controlled by the substituents attached to the heterocyclic core. The scope and the limitations of the new cyclization reaction were investigated.

Lactam analogs of pentalene. A new one-pot synthesis of pyrrolo[3,2-b]pyrrole-2,5-diones deriving from pulvinic acid. Pulvinic acid dilactams I are of interest due to their electronic and optical features and as synthetic pigments.

Cells , , [ Crossref ], [ CAS ] Pyrrolo[3,2-b]pyrrole based small molecules as donor materials for OPVs. A new accepter unit, pyrrolo[3,2-b]pyrrole-2,5-dione, deserves much attention as the electron-deficient unit for the generation of electron donor material for org.

Pyrrolo[3,2-b]pyrrole-2,5-dione unit, regioisomer of the known pyrrolo[3,4-c]pyrrole-1,4-dione, is originated from the structure of stable synthetic pigment. The spectrum of SM-B as the solid thin film shows absorption band with max.

There is no corresponding record for this reference. Synthesis , [ CAS ] Synthesis of unsymmetrical pyrrolo[3,2-b]pyrrole-2,5-diones and bis quinazolinonyls by double-anion-capture reactions of unsymmetrical oxaldi arylimidoyl dichlorides.

N,N’-diaryloxalodiimidoyl dichlorides with arylacetates and 2-aminobenzoates, resp. Synthesis and characteristic properties of polymers P1-P4 contg. P1 and P2 were prepd. Deeply colored polymers with mol.

The polymers were sol. All polymers exhibit broad absorption bands with high extinction coeffs. The biosynthesis of rebaudioside A involves glucosylation of the aglycone steviol.

Specifically, rebaudioside A can be formed by glucosylation of the OH of steviol which forms the O -steviolmonoside, glucosylation of the C-2′ of the O -glucose of steviolmonoside which forms steviol-1,2-bioside, glucosylation of the C carboxyl of steviol-1,2-bioside which forms stevioside, and glucosylation of the C-3′ of the CO-glucose of stevioside.

The order in which each glucosylation reaction occurs can vary. Specifically, rebaudioside E can be formed by glucosylation of the OH of steviol which forms steviol O -glucoside, glucosylation of the C-2′ of the O -glucose of steviol O -glucoside which forms the steviol-1,2-bioside, glucosylation of the C carboxyl of the 1,2-bioside to form 1,2-stevioside, and glucosylation of the C-2′ of the O-glucose of the 1,2-stevioside to form rebaudioside E.

The order in which each glycosylation reaction occurs can vary. For example, the glucosylation of the C-2′ of the O-glucose may be the last step in the pathway, wherein Rebaudioside A is an intermediate in the pathway.

Thus, a recombinant microorganism expressing combinations of these four or five UGTs can make rebaudioside A and rebaudioside D when steviol is used as a feedstock. Typically, one or more of these genes are recombinant genes that have been transformed into a microorganism that does not naturally possess them.

Also disclosed herein is that less than five e. For example, a recombinant microorganism expressing a functional EUGT11 can make rebaudioside D when rebaudioside A is used as a feedstock.

A recombinant microorganism expressing two functional UGTs, EUGT11 and 76G 1, and optionally a functional 91D12, can make rebaudioside D when rubusoside or 1,2-stevioside is used as a feedstock.

Similarly, conversion of steviol O -glucoside to rebaudioside D in a recombinant microorganism can be accomplished by the expression of genes encoding UGTs EUGT11, 85C2, 76G1, and optionally 91D2, when fed steviol O -glucoside.

Typically, one or more of these genes are recombinant genes that have been transformed into a host that does not naturally possess them. Thus, UGT76G1 functions, for example, as a uridine 5′-diphospho glucosyl: Functional UGT76G1 polypeptides may also catalyze glucosyl transferase reactions that utilize steviol glycoside substrates that contain sugars other than glucose, e.

The amino acid sequence of a S. EUGT11 polypeptides also can transfer a glucose moiety to the C-2′ of the O -glucose of the acceptor molecule, rubusoside, to produce a O -1,2-diglycosylated rubusoside compound 2 in FIG.

For example, a functional EUGT 11 polypeptide may utilize stevioside as a substrate, transferring a glucose moiety to the C-2′ of the O -glucose residue to produce Rebaudioside E see compound 3 in FIG.

In addition, a functional EUGT11 exhibits significant C-2′ O-diglycosylation activity with rubusoside or stevioside as substrates, whereas UGT91D2e has no detectable diglycosylation activity with these substrates.

Suitable functional UGT91D2 polypeptides include those disclosed herein, e. In addition, a UGT91D2 variant containing substitutions at amino acid residues and can be used. The amino acid sequence encoding the I.

See Osmani et al. In other embodiments the recombinant microorganism expresses one or more genes involved in steviol biosynthesis, e. Also disclosed herein is that the recombinant host may further contain and express a recombinant GGPPS gene in order to provide increased levels of the diterpene precursor geranylgeranyl diphosphate, for increased flux through the steviol biosynthetic pathway.

In some embodiments, the recombinant host further contains a construct to silence the expression of non-steviol pathways consuming geranylgeranyl diphosphate, ent-Kaurenoic acid or farnesyl pyrophosphate, thereby providing increased flux through the steviol and steviol glycosides biosynthetic pathways.

For example, flux to sterol production pathways such as ergosterol may be reduced by downregulation of the ERG9 gene. See, the ERG9 section below and Examples In cells that produce gibberellins, gibberellin synthesis may be downregulated to increase flux of ent-kaurenoic acid to steviol.

In carotenoid-producing organisms, flux to steviol may be increased by downregulation of one or more carotenoid biosynthetic genes. Also disclosed herein is that the recombinant microorganism further can express recombinant genes involved in diterpene biosynthesis or production of terpenoid precursors, e.

One with skill in the art will recognize that by modulating relative expression levels of different UGT genes, a recombinant host can be tailored to specifically produce steviol glycoside products in a desired proportion.

Transcriptional regulation of steviol biosynthesis genes and steviol glycoside biosynthesis genes can be achieved by a combination of transcriptional activation and repression using techniques known to those in the art.

For in vitro reactions, one with skill in the art will recognize that addition of different levels of UGT enzymes in combination or under conditions which impact the relative activities of the different UGTS in combination will direct synthesis towards a desired proportion of each steviol glycoside.

One with skill in the art will recognize that a higher proportion of rebaudioside D or E or more efficient conversion to rebaudioside D or E can be obtained with a diglycosylation enzyme that has a higher activity for the O -glucoside reaction as compared to the O -glucoside reaction substrates rebaudioside A and stevioside.

Other steviol glycosides present may include those depicted in Figure 2 C such as steviol monosides, steviol glucobiosides, rebaudioside A, rebaudioside E, and stevioside. The rebaudioside D-enriched composition produced by the host e.

For instance, a rebaudioside D-enriched composition produced by a recombinant host can be combined with a rebaudioside A, C, or F-enriched composition produced by a different recombinant host, with rebaudioside A, F, or C purified from a Stevia extract, or with rebaudioside A, F, or C produced in vitro.

See, for example, Jewett MC, et al. Molecular Systems Biology, Vol. Sucrose and a sucrose synthase may be provided in the reaction vessel in order to regenerate UDP-glucose from the UDP generated during glycosylation reactions.

The sucrose synthase can be from any suitable organism. For example, a sucrose synthase coding sequence from Arabidopsis thaliana, Stevia rebaudiana, or Coffea arabica can be cloned into an expression plasmid under control of a suitable promoter, and expressed in a host such as a microorganism or a plant.

Conversions requiring multiple reactions may be carried out together, or stepwise. For example, rebaudioside D may be produced from rebaudioside A that is commercially available as an enriched extract or produced via biosynthesis, with the addition of stoichiometric or excess amounts of UDP-glucose and EUGT Phosphatases may be used to remove secondary products and improve the reaction yields.

UGTs and other enzymes for in vitro reactions may be provided in soluble forms or in immobilized forms. The raw materials may be fed during cell growth or after cell growth.

The whole cells may be in suspension or immobilized. The whole cells may be entrapped in beads, for example calcium or sodium alginate beads. The whole cells may be linked to a hollow fiber tube reactor system.

The whole cells may be concentrated and entrapped within a membrane reactor system. The whole cells may be in fermentation broth or in a reaction buffer. A permeabilizing agent may be utilized for efficient transfer of substrate into the cells.

The cells may be permeabilized with a solvent such as toluene, or with a detergent such as Triton-X or Tween. The cells may be permeabilized with a surfactant, for example a cationic surfactant such as cetyltrimethylammonium bromide CTAB.

The cells may be permeabilized with periodic mechanical shock such as electroporation or a slight osmotic shock. The whole cells can be a Gram-negative bacterium such as E.

Tthe whole cell can be a Gram-positive bacterium such as Bacillus. The whole cell can be a fungal species such as Aspergillus, or a yeast such as Saccharomyces.

The term “whole cell biocatalysis” may be used to refer to the process in which the whole cells are grown as described above e. The cells may or may not be viable, and may or may not be growing during the bioconversion reactions.

In contrast, in fermentation, the cells are cultured in a growth medium and fed a carbon and energy source such as glucose and the end product is produced with viable cells. Specifically, dulcoside A can be formed by glucosylation of the OH of steviol which forms steviol O -glucoside, rhamnosylation of the C-2′ of the O-glucose of steviol O -glucoside which forms the 1,2 rhamnobioside, and glucosylation of the C carboxyl of the 1,2 rhamnobioside.

It has been discovered that conversion of steviol to dulcoside A in a recombinant host can be accomplished by the expression of gene s encoding the following functional UGTs: Thus, a recombinant microorganism expressing these three or four UGTs and a rhamnose synthetase can make dulcoside A when fed steviol in the medium.

Alternatively, a recombinant microorganism expressing two UGTs, EUGT11 and 74G1, and rhamnose synthetase can make dulcoside A when fed the monoside, steviol O -glucoside or steviol O -glucoside, in the medium.

Rhamnose synthetase provides increased amounts of the UDP-rhamnose donor for rhamnosylation of the steviol compound acceptor. Suitable rhamnose synthetases include those made by Arabidopsis thaliana, such as the product of the A.

Suitable UGT79B3 polypeptides include those made by Arabidopsis thaliana, which are capable of rhamnosylation of steviol O-monoside in vitro. Sucrose and a sucrose synthase may be provided in the reaction vessel in order to regenerate UDP-glucose from UDP during the glycosylation reactions.

For example, a sucrose synthase coding sequence from Arabidopsis thaliana, Stevia rebaudiana, or Coffea arabica can be cloned into an expression plasmid under control of a suitable promoter, and expressed in a host e.

Reactions may be carried out together, or stepwise. UGTs and other enzymes for in vitro reactions may be provided in soluble forms or immobilized forms. In some embodiments, rebaudioside C, Dulcoside A, or other steviol rhamnosides can be produced using whole cells as discussed above.

In some embodiments, the whole cells are the host cells described in section III A. Here disclosed is that the recombinant host expresses one or more genes involved in steviol biosynthesis, e. In addition, the recombinant host typically expresses an endogenous or a recombinant gene encoding a rhamnose synthetase.

Such a gene is useful in order to provide increased amounts of the UDP-rhamnose donor for rhamnosylation of the steviol compound acceptor. One with skill in the art will recognize that by modulating relative expression levels of different UGT genes as well as modulating the availability of UDP-rhamnose, a recombinant host can be tailored to specifically produce steviol and steviol glycoside products in a desired proportion.

Transcriptional regulation of steviol biosynthesis genes, and steviol glycoside biosynthesis genes can be achieved by a combination of transcriptional activation and repression using techniques known to those in the art.

The recombinant host may further contain and express a recombinant GGPPS gene in order to provide increased levels of the diterpene precursor geranylgeranyl diphosphate, for increased flux through the rebaudioside A biosynthetic pathway.

The recombinant host may further contain a construct to silence or reduce the expression of non-steviol pathways consuming geranylgeranyl diphosphate, ent-Kaurenoic acid or farnesyl pyrophosphate, thereby providing increased flux through the steviol and steviol glycosides biosynthetic pathways.

The recombinant host may further contain and express recombinant genes involved in diterpene biosynthesis or production of terpenoid precursors, e. Other steviol glycosides present may include those depicted in Figures 2 A and B such as steviol monosides, steviol glucobiosides, steviol rhamnobiosides, rebaudioside A, and Dulcoside A.

The rebaudioside C-enriched composition produced by the host can be further purified and the rebaudioside C or Dulcoside A so purified may then be mixed with other steviol glycosides, flavors, or sweeteners to obtain a desired flavor system or sweetening composition.

For instance, a rebaudioside C-enriched composition produced by a recombinant microorganism can be combined with a rebaudioside A, F, or D-enriched composition produced by a different recombinant microorganism, with rebaudioside A, F, or D purified from a Stevia extract, or with rebaudioside A, F, or D produced in vitro.

The biosynthesis of rebaudioside F involves glucosylation and xylosylation of the aglycone steviol. Specifically, rebaudioside F can be formed by glucosylation of the OH of steviol which forms steviol O -glucoside, xylosylation of the C-2′ of the O-glucose of steviol O -glucoside which forms steviol-1,2-xylobioside, glucosylation of the C carboxyl of the 1,2-xylobioside to form 1,2-stevioxyloside, and glucosylation of the C-3′ of the CO-glucose of 1,2-stevioxyloside to form rebaudioside F.

It has been discovered that conversion of steviol to rebaudioside F in a recombinant host can be accomplished by the expression of genes encoding the following functional UGTs: Thus, a recombinant microorganism expressing these four or five UGTs along with endogenous or recombinant UDP-glucose dehydrogenase and UDP-glucuronic acid decarboxylase can make rebaudioside F when fed steviol in the medium.

As another alternative, a recombinant microorganism expressing a functional UGT 76G1 can make rebaudioside F when fed 1,2 steviorhamnoside. UDP-glucose dehydrogenase and UDP-glucuronic acid decarboxylase provide increased amounts of the UDP-xylose donor for xylosylation of the steviol compound acceptor.

Sucrose and a sucrose synthase can be provided in the reaction vessel in order to regenerate UDP-glucose from UDP during the glycosylation reactions. For example, a sucrose synthase coding sequence from Arabidopsis thaliana, Stevia rebaudiana, or Coffea arabica can be cloned into an expression plasmid under control of a suitable promoter, and expressed in a host, e.

In some embodiments, rebaudioside F or other steviol xylosides can be produced using whole cells as discussed above. In addition, the recombinant host typically expresses an endogenous or a recombinant gene encoding a UDP-glucose dehydrogenase and a UDP-glucuronic acid decarboxylase.

Such genes are useful in order to provide increased amounts of the UDP-xylose donor for xylosylation of the steviol compound acceptor. One with skill in the art will recognize that by modulating relative expression levels of different UGT genes as well as modulating the availability of UDP-xylose, a recombinant microorganism can be tailored to specifically produce steviol and steviol glycoside products in a desired proportion.

Transcriptional regulation of steviol biosynthesis genes can be achieved by a combination of transcriptional activation and repression using techniques known to those in the art. For in vitro reactions, one with skill in the art will recognize that addition of different levels of UGT enzymes in combination or under conditions which impact the relative activities of the different UGTS in combination will direct synthesis towards a desired proportion of each steviol glycosides.

In some embodiments, the recombinant host further contains and expresses a recombinant GGPPS gene in order to provide increased levels of the diterpene precursor geranylgeranyl diphosphate, for increased flux through the steviol biosynthetic pathway.

The recombinant host may further contain a construct to silence the expression of non-steviol pathways consuming geranylgeranyl diphosphate, ent-Kaurenoic acid or farnesyl pyrophosphate, thereby providing increased flux through the steviol and steviol glycosides biosynthetic pathways.

The recombinant host may further contain and express recombinant genes involved in diterpene biosynthesis, e. Other steviol glycosides present may include those depicted in FIGs 2Aand D such as steviol monosides, steviol glucobiosides, steviol xylobiosides, rebaudioside A, stevioxyloside, rubusoside and stevioside.

The rebaudioside F-enriched composition produced by the host can be mixed with other steviol glycosides, flavors, or sweeteners to obtain a desired flavor system or sweetening composition.

For instance, a rebaudioside F-enriched composition produced by a recombinant microorganism can be combined with a rebaudioside A, C, or D-enriched composition produced by a different recombinant microorganism, with rebaudioside A, C, or D purified from a Stevia extract, or with rebaudioside A, C, or D produced in vitro.

Genes for additional polypeptides whose expression facilitates more efficient or larger scale production of steviol or a steviol glycoside can also be introduced into a recombinant host.

As another example, a recombinant host can also contain one or more genes encoding a cytochrome P reductase CPR. In circumstances where NADPH becomes limiting; strains can be further modified to include exogenous transhydrogenase genes.

As another example, the recombinant host can contain one or more genes encoding one or more enzymes in the MEP pathway or the mevalonate pathway. Such genes are useful because they can increase the flux of carbon into the diterpene biosynthesis pathway, producing geranylgeranyl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate generated by the pathway.

The geranylgeranyl diphosphate so produced can be directed towards steviol and steviol glycoside biosynthesis due to expression of steviol biosynthesis polypeptides and steviol glycoside biosynthesis polypeptides.

As another example the recombinant host can contain one or more genes encoding a sucrose synthase, and additionally can contain sucrose uptake genes if desired. The sucrose synthase reaction can be used to increase the UDP-glucose pool in a fermentation host, or in a whole cell bioconversion process.

This regenerates UDP-glucose from UDP produced during glycosylation and sucrose, allowing for efficient glycosylation. In some organisms, disruption of the endogenous invertase is advantageous to prevent degradation of sucrose.

For example, the S. The sucrose synthase SUS can be from any suitable organism. For example, a sucrose synthase coding sequence from, without limitation, Arabidopsis thaliana, Stevia rebaudiana, or Coffea arabica can be cloned into an expression plasmid under control of a suitable promoter, and expressed in a host e.

The sucrose synthase can be expressed in such a strain in combination with a sucrose transporter e. Culturing the host in a medium that contains sucrose can promote production of UDP-glucose, as well as one or more glucosides e.

In addition, a recombinant host can have reduced phosphatase activity as discussed herein. A recombinant host may contain one or more genes encoding enzymes involved in the methylerythritol 4-phosphate MEP pathway for isoprenoid biosynthesis.

Nucleotide sequences encoding DXR polypeptides are described, for example, in U. A recombinant host may contain one or more genes encoding enzymes involved in the mevalonate pathway for isoprenoid biosynthesis.

Suitable genes encoding mevalonate pathway polypeptides are known. For example, suitable polypeptides include those made by E. KO, Nicotiana attenuata, Kitasatospora griseola, Hevea brasiliensis, Enterococcus faecium and Haematococcus pluvialis.

Sucrose synthases can be used to generate UDP-glucose and remove UDP, facilitating efficient glycosylation of compounds in various systems. For example, yeast deficient in the ability to utilize sucrose can be made to grow on sucrose by introducing a sucrose transporter and a SUS.

For example, Saccharomyces cerevisiae does not have an efficient sucrose uptake system, and relies on extracellular SUC2 to utilize sucrose. The combination of disrupting the endogenous S.

The strain was used to isolate sucrose transporters by transformation with a cDNA expression library and selection of transformants that had gained the ability to take up sucrose.

As described herein, the combined expression of recombinant sucrose synthase and a sucrose transporter in vivo can lead to increased UDP-glucose availability and removal of unwanted UDP.

For example, functional expression of a recombinant sucrose synthase, a sucrose transporter, and a glycosyltransferase, in combination with knockout of the natural sucrose degradation system SUC2 in the case of S.

This higher glycosylation capability is due to at least a a higher capacity for producing UDP-glucose in a more energy efficient manner, and b removal of UDP from growth medium, as UDP can inhibit glycosylation reactions.

For example, a sucrose synthase coding sequence from, without limitation, Arabidopsis thaliana, Stevia rebaudiana, or Coffea arabica see, e. It is to be noted that in some cases, a sucrose synthase and a sucrose transporter can be expressed along with a UGT in a host cell that also is recombinant for production of a particular compound e.

It is an object of the disclosure to produce terpenoids based on the concept of increasing the accumulation of terpenoid precursors of the squalene pathway. Non-limiting examples of terpenoids include Hemiterpenoids, 1 isoprene unit 5 carbons ; Monoterpenoids, 2 isoprene units 10C ; Sesquiterpenoids, 3 isoprene units 15C ; Diterpenoids, 4 isoprene units 20C e.

Hemiterpenoids include isoprene, prenol and isovaleric acid. Monoterpenoids include Geranyl pyrophosphate, Eucalyptol, Limonene and Pinene. Sesquiterpenoids include Farnesyl pyrophosphate, Artemisinin and Bisabolol.

Triterpenoids include Squalene and Lanosterol. Tetraterpenoids include Lycopene and Carotene. Terpenes are hydrocarbons resulting from the combination of several isoprene units. Terpenoids can be thought of as terpene derivatives.

The term terpene is sometimes used broadly to include the terpenoids. Just like terpenes, the terpenoids can be classified according to the number of isoprene units used. By terpenoids is understood terpenoids of the Hemiterpenoid class such as but not limited to isoprene, prenol and isovaleric acid; terpenoids of the Monoterpenoid class such as but not limited to geranyl pyrophosphate, eucalyptol, limonene and pinene; terpenoids of the Sesquiterpenoids class such as but not limited to farnesyl pyrophosphate, artemisinin and bisabolol; terpenoids of the diterpenoid class such as but not limited to geranylgeranyl pyrophosphate, steviol, retinol, retinal, phytol, taxol , forskolin and aphidicolin; terpenoids of the Triterpenoid class such as but not limited to lanosterol; terpenoids of the Tetraterpenoid class such as but not limited to lycopene and carotene.

The disclosure provides for production of terpenoids, which are biosynthesized from Geranylgeranyl-pyrophosphate GGPP. In particular such terpenoids may be steviol. The present disclosure provides a cell, such as any of the hosts described in section III, modified to comprise the construct depicted in FIG.

Accordingly, the present disclosure provides a cell comprising a nucleic acid, said nucleic acid comprising i a promoter sequence operably linked to ii a heterologous insert sequence operably linked to iii an open reading frame operably linked to iv a transcription termination signal, wherein the heterologous insert sequence has the general formula I: In addition to above mentioned nucleic acid comprising a heterologous insert sequence, the cell may also comprise one or more additional heterologous nucleic acid sequences e.

The heterologous insert sequence can adapt the secondary structure element of a hairpin with a hairpin loop. The hairpin part comprises sections X 2 and X 4 which are complementary and hybridize to one another.

Sections X 2 and X 4 flank section X 3 , which comprises nucleotides that form a loop – the hairpin loop. The term complementary is understood by the person skilled in the art as meaning two sequences compared to each other, nucleotide by nucleotide counting from the 5′ end to the 3′ end, or vice versa.

The heterologous insert sequence is long enough to allow a hairpin to be completed, but short enough to allow limited translation of an ORF that is present in-frame and immediately 3′ to the heterologous insert sequence.

Thus, the heterologous insert sequence may comprise nucleotides, preferably nucleotides, more preferably nucleotides, more preferably nucleotides, more preferably nucleotides, more preferably nucleotides, more preferably 19 nucleotides.

X 2 and X 4 may individually consist of any suitable number of nucleotides, so long as a consecutive sequence of at least 4 nucleotides of X 2 is complementary to a consecutive sequence of at least 4 nucleotides of X 4.

X 2 and X 4 may consist of the same number of nucleotides. X 2 may for example consist of in the range of 4 to 25, such as in the range of 4 to 20, for example of in the range of 4 to 15, such as in the range of 6 to 12, for example in the range of 8 to 12, such as in the range of 9 to 11 nucleotides.

X 4 may for example consist of in the range of 4 to 25, such as in the range of 4 to 20, for example of in the range of 4 to 15, such as in the range of 6 to 12, for example in the range of 8 to 12, such as in the range of 9 to 11 nucleotides.

X 2 may consist of a nucleotide sequence, which is complementary to the nucleotide sequence of X 4 , i. X 4 may consiss of a nucleotide sequence, which is complementary to the nucleotide sequence of X 2 , i.

Very preferably, X 2 and X 4 consists of the same number of nucleotides, wherein X 2 is complementary to X 4 over the entire length of X 2 and X 4. X 3 may be absent, i. It is also possible that X 3 consists of in the range of 1 to 5, such as in the range of 1 to 3 nucleotides.

X 1 may be absent, i. It is also possible that X 1 consists of in the range of 1 to 25, such as in the range of 1 to 20, for example in the range of 1 to 15, such as in the range of 1 to 10, for example in the range of 1 to 5, such as in the range of 1 to 3 nucleotides.

X 5 may be absent, i. It is also possible that X 5 may consist of in the range 1 to 5, such as in the range of 1 to 3 nucleotides. The sequence may be any suitable sequence fulfilling the requirements defined herein above.

Squalene synthase SQS is the first committed enzyme of the biosynthesis pathway that leads to the production of sterols. It catalyzes the synthesis of squalene from farnesyl pyrophosphate via the intermediate presqualene pyrophosphate.

The enzyme is sometimes referred to as farnesyl-diphosphate farnesyltransferase FDFT1. The mechanism of SQS is to convert two units of farnesyl pyrophosphate into squalene.

SQS is considered to be an enzyme of eukaryotes or advanced organisms, although at least one prokaryote has been shown to possess a functionally similar enzyme. In terms of structure and mechanics, squalene synthase most closely resembles phytoene syntase, which serves a similar role in many plants in the elaboration of phytoene, a precursor of many carotenoid compounds.

A high level of sequence identity indicates likelihood that the first sequence is derived from the second sequence. Amino acid sequence identity requires identical amino acid sequences between two aligned sequences.

Identity may be determined by aid of computer analysis, such as, without limitations, the ClustalW computer alignment program as described in section D. Using this program with its default settings, the mature bioactive part of a query and a reference polypeptide are aligned.

The number of fully conserved residues are counted and divided by the length of the reference polypeptide. The ClustalW algorithm may similarly be used to align nucleotide sequences.

Sequence identities may be calculated in a similar way as indicated for amino acid sequences. A promoter is a region of DNA that facilitates the transcription of a particular gene.

Promoters are located near the genes they regulate, on the same strand and typically upstream towards the 5′ region of the sense strand. Promoters contain specific DNA sequences and response elements which provide a secure initial binding site for RNA polymerase and for proteins called transcription factors that recruit RNA polymerase.

These transcription factors have specific activator or repressor sequences of corresponding nucleotides that attach to specific promoters and regulate gene expressions. In bacteria, the promoter is recognized by RNA polymerase and an associated sigma factor, which in turn are often brought to the promoter DNA by an activator protein binding to its own DNA binding site nearby.

In eukaryotes the process is more complicated, and at least seven different factors are necessary for the binding of an RNA polymerase II to the promoter. As promoters are normally immediately adjacent to the open reading frame ORF in question, positions in the promoter are designated relative to the transcriptional start site, where transcription of RNA begins for a particular gene i.

In prokaryotes, the promoter consists of two short sequences at and positions upstream from the transcription start site. The Pribnow box is essential to start transcription in prokaryotes.

Its presence allows a very high transcription rate. Both of the above consensus sequences, while conserved on average, are not found intact in most promoters. On average only 3 of the 6 base pairs in each consensus sequence is found in any given promoter.

Eukaryotic promoters are typically located upstream of the gene ORF and can have regulatory elements several kilobases kb away from the transcriptional start site.

In eukaryotes, the transcriptional complex can cause the DNA to fold back on itself, which allows for placement of regulatory sequences far from the actual site of transcription.

The TATA box typically lies very close to the transcriptional start site often within 50 bases. The cell of the present disclsoure comprises a nucleic acid sequence which comprises a promoter sequence.

The promoter sequence is not limiting for the invention and can be any promoter suitable for the host cell of choice. The promoter may be a constitutive or inducible promoter.

Post-transcriptional regulation is the control of gene expression at the RNA level, therefore between the transcription and the translation of the gene. The first instance of regulation is at transcription transcriptional regulation where due to the chromatin arrangement and due to the activity of transcription factors, genes are differentially transcribed.

After being produced, the stability and distribution of the different transcripts is regulated post-transcriptional regulation by means of RNA binding protein RBP that control the various steps and rates of the transcripts: These proteins achieve these events thanks to a RNA recognition motif RRM that binds a specific sequence or secondary structure of the transcripts, typically at the 5′ and 3′ UTR of the transcript.

Modulating the capping, splicing, addition of a Poly A tail, the sequence-specific nuclear export rates and in several contexts sequestration of the RNA transcript occurs in eukaryotes but not in prokaryotes.

This modulation is a result of a protein or transcript which in turn is regulated and may have an affinity for certain sequences. The cap also helps in ribosomal binding.

Splicing removes the introns, noncoding regions that are transcribed into RNA, in order to make the mRNA able to create proteins. Cells do this by spliceosomes binding on either side of an intron, looping the intron into a circle and then cleaving it off.

The two ends of the exons are then joined together. Polyadenylation is the addition of a poly A tail to the 3′ end, i. The poly-A sequence acts as a buffer to the 3′ exonuclease and thus increases half-life of mRNA.

In addition, a long poly A tail can increase translation. Thus the poly-A tail may be used to further modulate translation of the construct of the present invention, in order to arrive at the optimal translation rate.

The poly A tail is also important for the nuclear export, translation, and stability of mRNA. RNA editing is a process which results in sequence variation in the RNA molecule, and is catalyzed by enzymes.

These enzymes are termed ‘APOBEC’ and have genetic loci at 22q13, a region close to the chromosomal deletion which occurs in velocardiofacial syndrome 22q11 and which is linked to psychosis. RNA editing is extensively studied in relation to infectious diseases, because the editing process alters viral function.

Use of a post-transcriptional regulatory elements PRE is often necessary to obtain vectors with sufficient performance for certain applications. Schambach et al in Gene Ther. The enhancing activity of the PRE depends on the precise configuration of its sequence and the context of the vector and cell into which it is introduced.

Thus use of a PRE such as a woodchuck hepatitis virus post-transcriptional regulatory elements WPRE may be useful in the preparation of the cell of the present invention when using a gene therapeutic approach.

Accordingly, the nucleic acid sequence of the cell defined herein may further comprises a post-transcriptional regulatory element. Further, the post-transcriptional regulatory element may be a Woodchuck hepatitis virus post-transcriptional regulatory element WPRE.

To insert genetic sequences into host DNA, viruses often use sequences of DNA that repeats up to thousands of times, so called repeats, or terminal repeats including long terminal repeats LTR and inverted terminal repeats ITR , wherein said repeat sequences may be both 5′ and 3′ terminal repeats.

The nucleic acid sequence or the vector of the cell defined herein may comprise a 5′ terminal repeat and a 3′ terminal repeat. The microbial cells of the present invention may in preferred embodiments contain a heterologous nucleic acid sequence encoding Geranylgeranyl Pyrophosphate Synthase GGPPS.

In particular, the GGPPS to be used with the present invention may be any enzyme capable of catalysing the following reaction: However, it is frequently the case that the heterologous nucleic acid is nucleic acid sequence encoding any particular GGPPS, where said nucleic acid has been codon optimised for the particular microbial cell.

Thus, by way of example, if the microbial cell is S. Methods for determining sequence identity are described herein above in the section “Squalene synthase” and in section D.

The nucleic acid sequence directing expression of GGPPS in the microbial cell may be a promoter sequence, and preferably said promoter sequence is selected according the particular microbial cell.

The promoter may for example be any of the promoters described herein above in the section “Promoter”. A vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell.

The major types of vectors are plasmids, viruses, cosmids, and artificial chromosomes. Common to all engineered vectors is an origin of replication, a multicloning site, and a selectable marker.

The vector itself is generally a DNA sequence that consists of an insert transgene and a larger sequence that serves as the “backbone” of the vector. The purpose of a vector which transfers genetic information to another cell is typically to isolate, multiply, or express the insert in the target cell.

Vectors called expression vectors expression constructs specifically are for the expression of the transgene in the target cell, and generally have a promoter sequence that drives expression of the transgene.

Simpler vectors called transcription vectors are only capable of being transcribed but not translated: Transcription vectors are used to amplify their insert. Insertion of a vector into the target cell is usually called transformation for bacterial cells, transfection for eukaryotic cells, although insertion of a viral vector is often called transduction.

Plasmid vectors are double-stranded generally circular DNA sequences that are capable of automatically replicating in a host cell. Plasmid vectors minimalistically consist of an origin of replication that allows for semi-independent replication of the plasmid in the host and also the transgene insert.

Modem plasmids generally have many more features, notably including a “multiple cloning site” which includes nucleotide overhangs for insertion of an insert, and multiple restriction enzyme consensus sites to either side of the insert.

In the case of plasmids utilized as transcription vectors, incubating bacteria with plasmids generates hundreds or thousands of copies of the vector within the bacteria in hours, and the vectors can be extracted from the bacteria, and the multiple cloning site can be cut by restriction enzymes to excise the hundredfold or thousandfold amplified insert.

These plasmid transcription vectors characteristically lack crucial sequences that code for polyadenylation sequences and translation termination sequences in translated mRNAs, making protein expression from transcription vectors impossible.

Viral vectors are generally genetically-engineered viruses carrying modified viral DNA or RNA that has been rendered noninfectious, but still contain viral promoters and also the transgene, thus allowing for translation of the transgene through a viral promoter.

However, because viral vectors frequently are lacking infectious sequences, they require helper viruses or packaging lines for large-scale transfection. Viral vectors are often designed for permanent incorporation of the insert into the host genome, and thus leave distinct genetic markers in the host genome after incorporating the transgene.

For example, retroviruses leave a characteristic retroviral integration pattern after insertion that is detectable and indicates that the viral vector has incorporated into the host genome. Here disclosed is a viral vector capable of transfecting a host cell, such as a cell that can be cultured, e.

The vector is then capable of transfecting said cell with a nucleic acid that includes the heterologous insert sequence as described herein. The viral vector may also be selected from the group consisting of alphavirus, adenovirus, adeno associated virus, baculovirus, HSV, coronavirus, Bovine papilloma virus, Mo-MLV and adeno associated virus.

In embodiments of the invention wherein the microbial cell comprises a heterologous nucleic acid encoding GGPPS, then said heterologous nucleic acid may be positioned on the vector also containing the nucleic acid encoding squalene synthase, or the heterologous nucleic acid encoding GGPPS may be positioned on a different vector.

Said heterologous nucleic acid encoding GGPPS may be contained in any of the vectors described herein above. Also disclosed is a microbial cell that comprises a heterologous nucleic acid encoding HMCR, then said heterologous nucleic acid may be positioned on the vector also containing the nucleic acid encoding squalene synthase, or the heterologous nucleic acid encoding HMCR may be positioned on a different vector.

Said heterologous nucleic acid encoding HMCR may be contained in any of the vectors described herein above. It is also contained within the invention that the heterologous nucleic acid encoding GGPPS and the heterologous nucleci acid encoding HMCR may be positioned on the same or on individual vectors.

Transcription is a necessary component in all vectors: Thus, even stable expression is determined by stable transcription, which generally depends on promoters in the vector.

However, expression vectors have a variety of expression patterns: This expression is based on different promoter activities, not post-transcriptional activities. Thus, these two different types of expression vectors depend on different types of promoters.

Viral promoters are often used for constitutive expression in plasmids and in viral vectors because they normally reliably force constant transcription in many cell lines and types.

Inducible expression depends on promoters that respond to the induction conditions: Expression vectors require sequences that encode for e. Creates a polyadenylation tail at the end of the transcribed pre-mRNA that protects the mRNA from exonucleases and ensures transcriptional and translational termination: UTRs contain specific characteristics that may impede transcription or translation, and thus the shortest UTRs or none at all are encoded for in optimal expression vectors.

Above conditions are necessary for expression vectors in eukaryotes but not in prokaryotes. Modem vectors may encompass additional features besides the transgene insert and a backbone such as a promoter discussed above , genetic markers to e.

The cell of the present disclosure may comprise a nucleic acid sequence integrated in a vector such as an expression vector. The the vector may be selected from the group consisting of plasmid vectors, cosmids, artificial chromosomes and viral vectors.

The plasmid vector should be able to be maintained and replicated in bacteria, fungi and yeast. The present disclosure also concerns cells comprising plasmid and cosmid vectors as well as artificial chromosome vectors.

The important factor is that the vector is functional and that the vector comprises at least the nucleic acid sequence comprising the heterologous insert sequence as described herein.

The vector can be functional in fungi and in mammalian cells. Here disclosed is a cell transformed or transduced with the vector as defined herein above. As mentioned herein above, the cell of the present invention i.

The cell of the invention may therefore be used in various set-ups in order to increase accumulation of terpenoid precursors and thus to increase yield of terpenoid products resulting from enzymatic conversion of said upstream terpenoid precursors.

Accordingly, here disclosed is a method for producing a terpenoid compound synthesized through the squalene pathway, in a cell culture, said method comprising the steps of a providing the cell as defined herein above, b culturing the cell of a.

By providing the cell comprising the genetically modified construct defined herein above, the accumulation of terpenoid precursors is enhanced FIG. The terpenoid product of the method of the present invention as defined herein above, may be selected from the group consisting of hemiterpenoids, monoterpenes, sesquiterpenoids, diterpenoids, sesterpenes, triterpenoids, tetraterpenoids and polyterpenoids.

The terpenoid may be selected from the group consisting of farnesyl phosphate, farnesol, geranylgeranyl, geranylgeraniol, isoprene, prenol, isovaleric acid, geranyl pyrophosphate, eucalyptol, limonene, pinene, farnesyl pyrophosphate, artemisinin, bisabolol, geranylgeranyl pyrophosphate, retinol, retinal, phytol, taxol , forskolin, aphidicolin, lanosterol, lycopene and carotene.

The terpenoid prioduct can be used as starting point in an additional refining process. Thus, said method may further comprise dephosphorylating the farnesyl phosphate to produce farnesol.

The enzyme or enzymes used in the process of preparing the target product terpenoid compound is preferably an enzyme “located downstream” of the terpenoid precursors Farnesyl-pyrophosphate, Isopentenyl-pyrophosphate, Dimethylallyl-pyrophosphate, Geranyl-pyrophosphate and Geranylgeranyl-pyrophosphate such as an enzyme located downstream of the terpenoid precursors Farnesyl-pyrophosphate, Isopentenyl-pyrophosphate, Dimethylallyl-pyrophosphate, Geranyl-pyrophosphate and Geranylgeranyl-pyrophosphate depicted in the squalene pathway of FIG.

The enzyme used in the process of preparing the target product terpenoid, based on the accumulation of precursors achieved through the presentdisclosure, may thus be selected from the group consisting of Dimethylallyltransferase EC 2.

The present disclosure may operate by at least partly, sterically hindering binding of the ribosome to the RNA thus reducing the translation of squalene synthase.

The method of the disclosure as defined herein above may further comprise recovering the Farnesyl-pyrophosphate, Isopentenyl-pyrophosphate, Dimethylallyl-pyrophosphate, Geranyl-pyrophosphate or Geranylgeranyl-pyrophosphate compound.

The recovered compound may be used in further processes for producing the desired terpenoid product compound. The further process may take place in the same cell culture as the process performed and defined herein above, such as the accumulation of the terpenoid precursors by the cell of the present disclosure.

Alternatively, the recovered precursors may be added to another cell culture, or a cell free system, to produce the desired products. As the precursors are intermediates, however mainly stable intermediates, a certain endogenous production of terpenoid products may occur based on the terpenoid precursor substrates.

Also, the cells of the disclosure may have additional genetic modifications such that they are capable of performing both the accumulation of the terpenoid precursors construct of the cell of the disclosure and whole or substantially the whole subsequent biosynthesis process to the desired terpenoid product.

Occasionally it may be advantageous to include a squalene synthase inhibitor when culturing the cell of the present disclosure. Chemical inhibition of squalene synthase, e. It has also been suggested that variants in this enzyme may be part of a genetic association with hypercholesterolemia.

Other squalene synthase inhibitors include Zaragozic acid and RPR Thus, the culturing step of the method s defined herein above may be performed in the presence of a squalene synthase inhibitor.

The cell of the disclosure may furthermore be genetically modified to further enhance production of certain key terpenoid precursors. As described herein above the microbial cell may comprise both a nucleic acid encoding a sqalene synthase as described herein above as well as a heterologous nucleic acid encoding a GGPPS.

Accordingly, here disclosed is a method for preparing GGPP, wherein the method comprises the steps of a. Cultivating the microbial cell of a. Also disclosed herein is a method for preparing a terpenoid of which GGPP is an intermediate in the biosynthesis pathway, wherein the method comprises the steps of a.

Cultivating the microbial cell of a; and c. Recovering the terpenoid, wherein said terpenoid may be any terpenoid described herein above in the section “Terpenoids” having GGPP as intermediate in its biosynthesises; and said microbial cell may be any of the microbial cells described herein above in the section “The cell”; and said promoter may be any promoter, such as any of the promoters described herein above in the section “Promoter”; and said heterologous insert sequence may be any of the heterologous insert sequences described herein above in the section “Heterologous insert sequence”; and said open reading frame encodes a squalene synthase, which may be any of the squalene synthases described herein above in the section “Squalene synthase”; and said GGPPS may be any of the GGPPS described herein above in the section “Geranylgeranyl Pyrophosphate Synthase”.

Said microbial cell may also optionally contain one or more additional heterologous nucleic acids encoding one or more enzymes involved in the biosynthesis pathway of said terpenoid.

Also disclosed herein is a method for preparing steviol, wherein the method comprises the steps of a. Recovering steviol, wherein said microbial cell may be any of the microbial cells described herein above in the section “The cell”; and said promoter may be any promoter, such as any of the promoters described herein above in the section “Promoter”; and said heterologous insert sequence may be any of the heterologous insert sequences described herein above in the section “Heterologous insert sequence”; and said open reading frame encodes a squalene synthase, which may be any of the squalene synthases described herein above in the section “Squalene synthase”; and said GGPPS may be any of the GGPPS described herein above in the section “Geranylgeranyl Pyrophosphate Synthase”.

Said microbial cell may also optionally contain one or more additional heterologous nucleic acids encoding one or more enzymes involved in the biosynthesis pathway of steviol. Recovering the terpenoid, wherein said terpenoid may be any terpenoid described herein above in the section “Terpenoids” having GGPP as intermediate in its biosynthesises; and said microbial cell may be any of the microbial cells described herein above in the section “The cell”; and said promoter may be any promoter, such as any of the promoters described herein above in the section “Promoter”; and said heterologous insert sequence may be any of the heterologous insert sequences described herein above in the section “Heterologous insert sequence”; and said open reading frame encodes a squalene synthase, which may be any of the squalene synthases described herein above in the section “Squalene synthase”; and said GGPPS may be any of the GGPPS described herein above in the section “Geranylgeranyl Pyrophosphate Synthase”; and said HMCR may be any of the HMCR described herein above in the section “HMCR”.

Said microbial cell may also contain one or more additional heterologous nucleic acids encoding one or more enzymes involved in the biosynthesis pathway of said terpenoid. Recovering steviol, wherein said microbial cell may be any of the microbial cells described herein above in the section “The cell”; and said promoter may be any promoter, such as any of the promoters described herein above in the section “Promoter”; and said heterologous insert sequence may be any of the heterologous insert sequences described herein above in the section “Heterologous insert sequence”; and said open reading frame encodes a squalene synthase, which may be any of the squalene synthases described herein above in the section “Squalene synthase”; and said GGPPS may be any of the GGPPS described herein above in the section “Geranylgeranyl Pyrophosphate Synthase” and said HMCR may be any of the HMCR described herein above in the section “HMCR”.

Said microbial cell may also contain one or more additional heterologous nucleic acids encoding one or more enzymes involved in the biosynthesis pathway of steviol. The cell may comprise a mutation in the ERG9 open reading frame.

The method of the present invention the cell may comprise an ERG9[Delta]:: The step of recovering the compound in the method of the present disclosure may further comprise purification of said compound from the cell culture media.

Functional homologs of the polypeptides described above are also suitable for use in producing steviol or steviol glycosides in a recombinant host. A functional homolog is a polypeptide that has sequence similarity to a reference polypeptide, and that carries out one or more of the biochemical or physiological function s of the reference polypeptide.

A functional homolog and the reference polypeptide may be natural occurring polypeptides, and the sequence similarity may be due to convergent or divergent evolutionary events.

As such, functional homologs are sometimes designated in the literature as homologs, or orthologs, or paralogs. Variants of a naturally occurring functional homolog, such as polypeptides encoded by mutants of a wild type coding sequence, may themselves be functional homologs.

Functional homologs can also be created via site-directed mutagenesis of the coding sequence for a polypeptide, or by combining domains from the coding sequences for different naturally-occurring polypeptides “domain swapping”.

Techniques for modifying genes encoding functional UGT polypeptides described herein are known and include, inter alia, directed evolution techniques, site-directed mutagenesis techniques and random mutagenesis techniques, and can be useful to increase specific activity of a polypeptide, alter substrate specificity, alter expression levels, alter subcellular location, or modify polypeptide: Such modified polypeptides are considered functional homologs.

The term “functional homolog” is sometimes applied to the nucleic acid that encodes a functionally homologous polypeptide. Functional homologs can be identified by analysis of nucleotide and polypeptide sequence alignments.

For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologs of steviol or steviol glycoside biosynthesis polypeptides. Amino acid sequence is, in some instances, deduced from the nucleotide sequence.

Amino acid sequence similarity allows for conservative amino acid substitutions, such as substitution of one hydrophobic residue for another or substitution of one polar residue for another. If desired, manual inspection of such candidates can be carried out in order to narrow the number of candidates to be further evaluated.

Manual inspection can be performed by selecting those candidates that appear to have domains present in steviol biosynthesis polypeptides, e.