ZA200104655B - Thioredoxin and grain processing. - Google Patents

Description

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THIOREDOXIN AND GRAIN PROCESSING

This invention relates to novel methods for grain processing to enhance protein and starch recovery, particularly in corn wet milling and soybean processing, as well as novel transgenic plants useful in such processes.

Thioredoxin (TRX) and thioredoxin reductase (TR) are enzymes that use NADPH to reduce disulfide bonds in proteins. Protein disulfide bonds play an important role in grain processing efficiencies, and in the quality of the products recovered from grain processing.

Development of effective ways to eliminate or decrease the extent of protein disulfide bonding in grain would increase processing efficiencies. Additionally grain performance in livestock feed is also affected by inter- and intramolecular disulfide bonding: grain digestibility, nutrient availability and the neutralization of anti-nutritive factors (e.g., protease, amylase inhibitors etc.) would be increased by reducing the extent of disulfide bonding.

Expression of transgenic thioredoxin and/or thioredoxin reductase in corn and soybeans and the use of thioredoxin in grain processing, e.g., wet milling, is novel and provides an

J alternative method for reducing the disulfide bonds in seed proteins during industrial processing. The invention therefore provides grains with altered storage protein quality as . well as grains that perform qualitatively differently from normal grain during industrial processing or animal digestion (both referred to subsequently as “processing’).

This method of delivery of thioredoxin and/or thioredoxin reductase eliminates the need to develop exogenous sources of thioredoxin and/or thioredoxin reductase for addition during processing. A second advantage to supplying thioredoxin and/or thioredoxin reductase via the grains is that physical disruption of seed integrity is not necessary to bring the enzyme in contact with the storage or matrix proteins of the seed prior to processing or as an extra processing step.

Three modes of thioredoxin utilization in grain processing are provided: 1) Expression and action during seed development to alter the composition and quality of harvested grain; 2) Expression (but no activity) during seed development to alter the quality of the products upon processing; 3) Production of thioredoxin and/or thioredoxin reductase in grain that is used to alter the quality of other grain products by addition during processing.

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The present invention thus provides: — a method to increase efficiency of separation of starch and protein in a grain milling process, comprising steeping the grain at an elevated temperature in the presence of supplemental thioredoxin and/or thioredoxin reductase and separating the starch and protein components of the grain — a method as mentioned hereinbefore wherein the grain includes grain from a transgenic plant wherein the transgene expresses thioredoxin and/or thioredoxin reductase, particularly a thermostable thioredoxin and/or thioredoxin reductase. — a method as mentioned hereinbefore wherein the plant is selected from dicotyledonous or monocotyledonous plants, particularly from cereals and even more particularly from corn (Zea mays) and soybean.

The invention further provides transgenic plants.

In particular, the invention provides: — a plant comprising a heterologous DNA sequence coding for a thioredoxin and/or thioredoxin reductase stably integrated into its nuclear or plastid DNA — a plant as mentioned before wherein the thioredoxin and/or thioredoxin reductase is ] thermostable — a plant as mentioned before wherein the thioredoxin and/or thioredoxin reductase is ] selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQID

NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 — a plant as mentioned herein before wherein the plant is selected from corn and soybean

The invention also provides: — a plant expressible expression cassette comprising a coding region for a thioredoxin and/or thioredoxin reductase operably linked to promoter and terminator sequences which function in a plant — a plant expressible expression cassette as mentioned before wherein the thioredoxin and/or thioredoxin reductase is thermostable — a plant expressible expression cassette wherein the thioredoxin and/or thioredoxin reductase is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID

NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7

The invention further provides

R . — method of producing grain comprising high levels of thioredoxin and/or thioredoxin reductase comprising transforming plants with an expression cassette as mentioned before —a method of producing grain comprising high levels of thioredoxin and/or thioredoxin reductase comprising pollinating a first plant comprising a heterologous expression cassette comprising a transactivator-regulated promoter regulated and operably linked to a DNA sequence coding for a thioredoxin and/or thioredoxin reductase, with pollen from a second plant comprising a heterologous expression cassette comprising a promoter operably linked to a DNA sequence coding for a transactivator capable of regulating said transactivator-regulated promoter; and recovering grain from the plant thus pollinated

In addition the invention provides: — the use of plants or plant material according to the invention as animal feed.

The invention described herein is applicable to all grain crops, in particular corn, soybean, wheat, and barley, most particularly corn and soybean, especially corn. Expression of transgenic thioredoxin and/or thioredoxin reductase in grain is a means of altering the : quality of the material (seeds) going into grain processing, altering the quality of the material derived from grain processing, maximizing yields of specific seed components : during processing (increasing efficiency), changing processing methods, and creating new uses for seed-derived fractions or components from milling streams.

Wet-Milling

Wet milling is a process of separating the starch, protein and oil components of grain, most often cereals, for example corn. It is distinguished herein from dry milling, which is simply pulverizing grain. The first step in wet milling is usually steeping, wherein the grain is soaked in water under carefully controlled conditions to soften the kernels and facilitate separation of the components. The oil-bearing embryos float to the surface of the aqueous solution and are removed, and by a process of watering and dewatering, milling, screening, centrifuging and washing, the starch is separated from the protein and purified. The key difficulty is to loosen starch granules from the complicated matrix of proteins and cell wall material that makes up the endosperm of the grain. One reason for this difficulty is believed to be the presence of inter- or intramolecular disulfide bonds, which render the protein matrix less soluble and less susceptible to proteolytic enzymes and inhibit release of the starch granules from the protein matrix in the grain. At present, the primary means for reducing these bonds is to steep the grain in the presence of sulfur dioxide, but this is costly, environmentally unfriendly, and not optimally effective.

Certain mutations exert beneficial effects on the protein matrix of corn kernel endosperm (floury and opaque), but impair kernel integrity. Transgenic thioredoxin expression provides some of these advantages without creating some of the kernel integrity problems associated with these mutations.

Post-harvest or processing-dependent activity of thioredoxin have equally beneficial effects.

For example, in one embodiment, thioredoxin enzymes are targeted to and accumulated in cell compartments. Protein reduction occurs following physical disruption of the seed. In another embodiment, quiescent endosperm thioredoxin is activated upon steeping. ina preferred embodiment, the invention provides a plant expressing a transgenic thermostable thioredoxin and thioredoxin reductase, e.g. a thioredoxin and thioredoxin reductase derived from a hyperthermophilic organism, such that the thioredoxin and thioredoxin reductase are not significantly active except at high temperatures (e.g. greater than 50°C). In one embodiment, the thermostable thioredoxin and thioredoxin reductase are synergistic with saccharification via expression of other thermostable enzymes in endosperm.

Feed applications

Expression of transgenic thioredoxin and/or thioredoxin reductase in grain is also useful to improve grain characteristics associated with digestibility, particularly in animal feeds.

Susceptibility of feed proteins to proteases is a function of time and of protein conformation.

Kernel cracking is often used in feed formulation as is steam flaking. Both of these processes are designed to aid kernel digestibility. Softer kernels whose integrity can be disrupted more easily in animal stomachs are desirable. Conformational constraints and crosslinks between proteins are major determinants of protease susceptibility. Modifying these bonds by increased thioredoxin expression thereby aids digestion.

Corn dry milling/ masa

Protein content and quality are important determinants in flaking grit production and in masa production. Reduction of disulfide bonds alters the nature of corn flour such that it is suitable for use as a wheat substitute, especially flours made from high-protein white corn varieties.

oo «WO 00/36126 PCT/EP99/09986

Soybean crushing

Over half of the US soybean crop is crushed or milled, and the protein quality in the resulting low-fat soy flour or de-fatted soy flour (or grits) is important for subsequent processing. Protein yield and quality from soybean processing streams are economically important, and are largely dependent upon protein conformation. Increasing thioredoxin activity through expression of transgenic thioredoxin and/or thioredoxin reductase increases protein solubility, and thus increases yield, in the water-soluble protein fractions. Recovery is facilitated by aqueous extraction of de-fatted soybean meal under basic conditions.

Enhancing thioredoxin activity through expression of transgenic thioredoxin and/or thioredoxin reductase also reduces the required pH for efficient extraction and thereby reduces calcium or sodium hydroxide inputs, as well as lowering the acid input for subsequent acid precipitation, allowing efficient recovery of proteins without alkali damage, and reducing water consumption and processing plant waste effluents (that contain substantial biological oxygen demand loads).

Protein redox status affects important functional properties supplied by soy proteins, such : as solubility, water absorption, viscosity, cohesion/adhesion, gelation and elasticity. Fiber removal during soy protein concentrate production and soy protein isolate hydrolysis by : proteases is enhanced by increasing thioredoxin activity as described herein. Similarly, as described for corn above, increasing thioredoxin activity through expression of transgenic thioredoxin and/or thioredoxin reductase enhances the functionality of enzyme-active soy flours and the digestibility of the soybean meal fraction and steam flaking fraction in animal feeds.

Modification of protein quality during seed development and during processing both be provided, although it is preferred that the transgenic thioredoxin and/or thioredoxin reductase be targeted to a cell compartment and be thermostable, as described above, to avoid significant adverse effects on storage protein accumulation will be encountered as a result of thioredoxin activity during seed development. Alternately, the thioredoxin may be added as a processing enzyme, as (in contrast to corn wet milling) breaking the disulfide bonds is not necessary until after grain integrity is destroyed (crushing and oil extraction).

Selection of thioredoxin and thioredoxin reductase for heterologous expression:

Thioredoxin, thioredoxin reductase and protein disulfide isomerase (PDI) genes are found in eukaryotes, eubacteria as well as archea, including hyperthermophilic organisms such as

Methanococcus jannaschii and Archaeoglobus fulgidus. Selection of a particular gene depends in part on the desired application. For the methods of the present invention, preferred thioredoxins have the following characteristics: 1. Heat stability

Thioredoxin and refated proteins from hyperthermophiles are found to have increased stability at high temperatures (>50°C) and relatively low activity at ambient temperatures.

Expression of TRX and/or TR from hyperthermophiles, for example from archea such as

Methanococcus jannaschii and Archaeoglobus fulgidus or other hyperthermophiles is preferred for expression during seed development, so that the thioredoxin activity is not markedly increased until the grain is steeped or processed at elevated temperature. Most grain processing methods involve, or are compatible with, a high temperature step.

Thermostable thioredoxin and thioredoxin reductase is therefore preferred. By thermostable is meant that the enzyme is preferentially active at high temperatures, e.g., temperatures greater than 40°C, most preferably greater than 50°C, e.g. 45-60°C for wet milling, or even higher, e.g., 45-95°C. 2. Substrate specificity

It is also possible to reduce undesirable effects on seed development by selection of a thioredoxin that acts preferentially on certain proteins such as the structural protein in the matrix and has low activity with essential metabolic enzymes. Various TRX's have been shown to differ in reactivity with enzymes that are under redox control. Thus it is possible to select a TRX that will primarily act on the desired targets, minimizing undesirable side- effects of over expression.

Suitable thermostable thioredoxins and thioredoxin reductases include the following:

Sequence of thioredoxin from Methanococcus jannaschii, (SEQ ID NO:1; gi|1591029)

MSKVKIELFT SPMCPHCPAAKRVVEEVANEMPDAVEVEYINVMENPQKAMEYGIMAVPTIVI

NGDVEFIGAPTKEALVEAIKKRL

Sequence of thioredoxin from Archaeoglobus fulgidus (SEQ ID NO:2; gi|2649903)(trx-1)

MPMVRKAAFYAIAVISGVLAAVVGNALYHNFNSDLGAQAKIYFFYSDSCPHCREVKPYVEEF

AKTHNLTWCNVAEMDANCSKIAQEFGIKYVPTLVIMDEEAHVFVGSDEVRTAIEGMK

Sequence of thioredoxin from Archaeoglobus fulgidus (SEQ ID NO:3; gi|2649838) (trx-2)

oo + WO 00/36126 PCT/EP99/09986

MVFTSKYCPYCRAFEKVVERLMGELNGTVEFEVVDVDEKRELAEKYEVLMLPTLVLADGDE

VLGGFMGFADYKTAREAILEQISAFLKPDYKN

Sequence of thioredoxin from Archaeoglobus fulgidus (SEQ ID NO:4; gi|2649295) (trx-3)

MDELELIRQKKLKEMMQKMSGEEKARKVLDSPVKLNSSNFDETLKNNENVVVDFWAEWC

MPCKMIAPVIEELAKEYAGKVVFGKLNTDENPTIAARYGISAIPTLIFFKKGKPVDQLVGAMPK

SELKRWVQRNL

Sequence of thioredoxin from Archaeoglobus fulgidus (SEQ ID NO:5; gi|2648389) (trx-4)

MERLNSERFREVIQSDKLVVVDFYADWCMPCRYISPILEKLSKEYNGEVEFYKLNVDENQD

VAFEYGIASIPTVLFFRNGKVVGGFIGAMPESAVRAEIEKALGA

Sequence of thioredoxin reductase (trxB) from Methanococcus jannaschii (SEQ ID NO:6: 0i|1592167)

MIHDTIIGAGPGGLTAGIYAMRGKLNALCIEKENAGGRIAEAGIVENYPGFEEIRGYELAEKF

KNHAEKFKLPIIYDEVIKIETKERPFKVITKNSEYLTKTIVIATGTKPKKLGLNEDKFIGRGISYC

: TMCDAFFYLNKEVIVIGRDTPAIMSAINLKDIAKKVIVITDKSELKAAESIMLDKLKEANNVEIY

NAKPLEIVGEERAEGVKISVNGKEEIKADGIFISLGHVPNTEFLKDSGIELDKKGFIKTDENCR

. TNIDGIYAVGDVRGGVMQVAKAVGDGCVAMANIIKYLQKL

Sequence of thioredoxin reductase from Archaeoglobus fulgidus (SEQ ID NO:7; gi|2649006) (trxB)

MYDVAIIGGGPAGLTAALYSARYGLKTVFFETVDPVSQLSLAAKIENYPGFEGSGMELLEKM

KEQAVKAGAEWKLEKVERVERNGETFTVIAEGGEYEAKAIIVATGGKHKEAGIEGESAFIGR

GVSYCATCDGNFFRGKKVIVYGSGKEAIEDAIYLHDIGCEVTIVSRTPSFRAEKALVEEVEKR

GIPVHYSTTIRKIIGSGKVEKVVAYNREKKEEFEIEADGIFVAIGMRPATDVVAELGVERDSM

GYIKVDKEQRTNVEGVFAAGDCCDNPLKQVVTACGDGAVAAYSAYKYLTS

The genes which encode these proteins for use in the present invention are preferably designed by back-translation using plant preferred codons, to enhance G-C content and remove detrimental sequences, as more fully described below. The activity of the proteins may be enhanced by DNA shuffling or other means, as described below. The invention therefore comprises proteins derived from these proteins, especially proteins which are substantially similar which retain thioredoxin or thioredoxin reductase activity.

WO CU/36126 PCT/EP99/09986 ,

For engineering thioredoxin expression in seeds for activity during grain development, : promoters which direct seed-specific expression of TRX and TR are preferred, as is targeting to the storage so that the enzyme will have the desired effects on storage proteins, which may be desirable in some applications. In the present invention, however, it is more generally desirable to engineer thioredoxin and/or thioredoxin reductase expression in seeds for accumulation and inactivity during grain development. Several strategies are employed to create seeds that express transgenic thioredoxin and/or thioredoxin reductase without having a significant impact on normal seed development, e.g. (i) To compartmentalize active thioredoxin or thioredoxin reductase such that it does not significantly interact with the target proteins, for example by targeting to or expression in amyloplasts. Plastid targeting sequences are used to direct accumulation in the amyloplast.

Alternatively, the thioredoxin and/or thioredoxin reductase is targeted to an extracellular location in cell walls using secretion signals. Or finally, in the case of monocots, expression in cell types such as aleurone during seed development is used to keep the thioredoxin and/or thioredoxin reductase away from the storage components of the rest of the endosperm. (ii) To engineer the expression of thioredoxin and/or thioredoxin reductase from thermophilic organisms. Enzymes which have little or no activity at ambient temperatures (as high as ) 38-39°C in the field) are less likely to cause problems during development. Preferably, therefore, the enzymes are active primarily at high temperatures, e.g., temperatures greater than 40°C, most preferably 45-60°C for wet milling, or even higher, e.g., 45-95°C. (ili) To place the thioredoxin and/or thioredoxin reductase under control of an inducible promoter, for example a chemically-inducible promoter, a wound-inducible promoter, or a transactivator-regulated promoter which is activated upon pollination by a plant expressing the transactivator. (iv) To utilize thioredoxin having specific requirements for a particular thioredoxin reductase, such that activity of the thioredoxin or thioredoxin reductase is suitably regulated via availability of the appropriate thioredoxin reductase or thioredoxin, respectively. For example, the thioredoxin and thioredoxin reductase are expressed in different plants, so that the active combination is only available in the seed upon pollination by the plant expressing the complimentary enzyme. Alternatively, the thioredoxin or thioredoxin reductase is sequestered in the cell, for example in a plastid, vacuole, or apoplast, as described above, so that it does not become available until the grain is processed.

Ca “© WO 00/36126 PCT/EP99/09986

Methods of grain processing

The invention thus provides a novel method of enhancing separation of the starch from the protein matrix, using thioredoxin and/or thioredoxin reductase. In a first embodiment, thioredoxin activity is found to be useful in a variety of seed processing applications, including wet milling, dry milling, oilseed processing, soybean processing, wheat processing and flour/dough quality, most especially the wet milling of grains, in particular corn.

Accordingly, the invention provides a method to improve milling efficiency or increase milling yield, eo to increase efficiency of separation of starch and protein, to enhance yields of starch and soluble proteins from grain, or « to increase protein solubility in water or other solvents comprising steeping grain in the presence of supplemental thioredoxin and/or thioredoxin and separating the starch and protein components of the grain.

Typically, steeping occurs before milling, but may occur afterwards, and there may be more ’ than one milling or steeping step in the process method extraction and increase protein yield from seeds during the steep or points after steeping. Preferably, the supplemental thioredoxin and/or thioredoxin reductase is provided by expression of a transgene in the plant from which the grain is harvested.

The invention further provides: + the use of thioredoxin or thioredoxin reductase in a method to improve milling efficiency or increase milling yield of starches or proteins, for example in any of the methods described above + steepwater comprising an amount of thioredoxin and/or thioredoxin reductase effective to facilitate separation of starch from protein in grain « grain which has been exposed to thioredoxin an amount effective to facilitate separation of starch from protein; and « starch or protein which has been produced by the method described above.

The activity of the thioredoxin in the above method may be enhanced by supplementing the steepwater with thioredoxin reductase and/or NADPH. Other components normally present in steepwater for wet milling may also be present, such as bacteria which produce lactic acid. Preferably, the steeping is carried out at a temperature of about 52°C for a period of 22-50 hours, so it is desirable that the thioredoxin is stable under these conditions.

The grain may be a dicotyledonous seed, for example, an oil seed, e.g., soybean, sunflower or canola, preferably soybean; or may be a monocotyledonous seed, for example a cereal seed, e.g., corn, wheat, oats, barley, rye or rice, most preferably corn.

The thioredoxin may be any protein bearing thiol groups which can be reversibly oxidized to form disulfide bonds and reduced by NAPDH in the presence of a thioredoxin reductase.

Preferably the thioredoxin is derived from a thermophilic organism, as described above.

Thioredoxin and/or thioredoxin reductase for use in the instant invention is suitably produced in an engineered microbe, e.g. a yeast or Aspergillus, or in an engineered plant capable of very high expression, e.g. in barley, e.g., under control of a promoter active during malting, such as a high pl alpha-amylase promoter or other gibberellin-dependent promoters. The thioredoxin (in excreted or extracted form or in combination with the producer organism or parts thereof) is then added to the steepwater.

As an alternative or supplement to adding the thioredoxin to the steepwater, the enzyme can be expressed directly in the seed that is to be milled. Preferably, the enzyme is ' expressed during grain maturation or during a conditioning process.

Accordingly, in a further embodiment, the invention provides ¢ a method of making thioredoxin on an industrial scale in a transgenic organism, e.g., a plant, e.g., a cereal, such as barley or com, or a microorganism, e.g., a yeast or

Aspergillus, for example a method comprising the steps of cultivating a transgenic organism having a chimeric gene which expresses thioredoxin, and optionally isolating or extracting the thioredoxin e a method of using transgenic plants that produce elevated quantities of thioredoxin during seed maturation or germination such that the quality of the proteins in that seed are affected by the endogenously synthesized thioredoxin during seed development, or during the steeping process, thereby eliminating or reducing the need for conditioning with exogenous chemicals or enzymes prior to milling + a method of making transgenic plants that produce elevated quantities of thioredoxin during seed maturation or germination such that the quality of the proteins in that seed are affected by the thioredoxin during seed development or during the steeping process,

SE ? WO 00/36126 ‘ PCT/EP99/09986 : thereby eliminating or reducing the need for conditioning with exogenous chemicals or enzymes prior to milling + a method for milling grain that uses transgenic seed containing thioredoxin, that results in higher starch and soluble protein yields.

Expression of thioredoxin and thioredoxin reductase in transgenic organisms

The invention further comprises a transgenic organism having in its genome a chimeric expression cassette comprising a coding region encoding a thermostable thioredoxin or thioredoxin reductase under operative control of a promoter.

Preferably, the transgenic organism is a plant which expresses a thioredoxin and/or thioredoxin reductase in a form not naturally occurring in plants of that species or which expresses thioredoxin at higher levels than naturally occur in a plant of that species.

Preferably, the thioredoxin is expressed in the seed during seed development, and is therefore preferably under control of a seed specific promoter. Optionally, expression of the thioredoxin is placed under control of an inducible or transactivator-regulated promoter, so that expression is activated by chemical induction or hybridization with a transactivator ’ when desired. The thioredoxin is suitably targeted to the vacuoles of the plant by fusion with a vacuole targeting sequence. : In the present invention, thioredoxin coding sequences are fused to promoters active in plants and transformed into the nuclear genome or the plastid genome. The promoter is preferably a seed specific promoter such as the gamma-zein promoter. The promoter may alternatively be a chemically-inducible promoter such as the tobacco PR-1a promoter; or may be a chemically induced transactivator-regulated promoter wherein the transactivator is under control of a chemically-induced promoter; however, in certain situations, constitutive promoters such as the CaMV 35S or Gelvin promoter may be used. With a chemically inducible promoter, expression of the thioredoxin genes transformed into plants may be activated at an appropriate time by foliar application of a chemical inducer.

Alternatively, the thioredoxin coding sequence is under control of a transactivator-regulated promoter, and expression is achieved by crossing the plant transformed with this sequence with a second plant expressing the transactivator. In a preferred form of this method, the first plant containing the thioredoxin coding sequence is the seed parent and is male sterile, while the second plant expressing the transactivator is the pollinator. Expression of thioredoxin in seeds is achieved by interplanting the first and second plants, e.g., such that the first plant is pollinated by the second and thioredoxin is expressed in the seeds of the first plant by activation of the transactivator-regulated promoter with the transactivator expressed by the transactivator gene from the second parent.

The invention thus provides a plant which expresses a thioredoxin and/or thioredoxin reductase, e.g. a thioredoxin and/or thioredoxin reductase not naturally expressed in plants, for example a plant comprising a heterologous DNA sequence coding for a thioredoxin stably integrated into its nuclear or plastid DNA, preferably under control of an inducible promoter, e.g., a chemically-inducible promoter, for example either operably linked to the inducible promoter or under control of transactivator-regulated promoter wherein the corresponding transactivator is under control of the inducible promoter or is expressed in a second plant such that the promoter is activated by hybridization with the second plant; wherein the thioredoxin or thioredoxin reductase is preferably thermostable; such plant also including seed therefor, which seed is optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a bag with instructions for use, and seed harvested therefrom, e.g., for use in a milling process as described above.

The transgenic plant of the invention may optionally further comprise genes for enhanced production of thioredoxin reductase and/or NADPH.

The invention further provides: : » a method for producing thioredoxin comprising cultivating a thioredoxin-expressing plant as described above » a method for producing starch and/or protein comprising extracting starch or protein from seed harvested from a plant as described above; and a method for wet milling comprising steeping seed from a thioredoxin-expressing plant as described above and extracting starch and/or protein therefrom.

The invention further provides: ¢ a plant expressible expression cassette comprising a coding region for a thioredoxin or thioredoxin reductase, preferably a thioredoxin derived from a thermophilic organism, e.g., from an archea, for example from M. jannaschii or A. fulgidus, e.g., as described above, wherein the coding region is preferably optimized to contain plant preferred codons, said coding region being operably linked to promoter and terminator sequences which function in a plant, wherein the promoter is preferably a seed specific promoter or an inducible promoter, e.g., a chemically inducible or transactivator-regulated promoter:

© + WO 00/36126 PCT/EP99/09986 for example a plastid or nuclear expressible expression cassette comprising a promoter, e.g., a transactivator-regulated promoter regulated by a nuclear transactivator (e.g., the

T7 promoter when the transactivator is T7 RNA polymerase the expression of which is optionally under control of an inducible promoter) e a vector comprising such a plant expressible expression cassette a plant transformed with such a vector; or * a transgenic plant which comprises in its genome, e.g., its nuclear or plastid genome, such a plant expressible expression cassette.

The invention also comprises a method of producing grain comprising high levels of thioredoxin or thioredoxin reductase comprising pollinating a first plant comprising a heterologous expression cassette comprising a transactivator-regulated promoter regulated and operably linked to a DNA sequence coding for a thioredoxin or thioredoxin reductase, the first plant preferably being emasculated or male sterile: with pollen from a second plant comprising a heterologous expression cassette comprising a promoter operably linked to a

DNA sequence coding for a transactivator capable of regulating said transactivator- : regulated promoter; recovering grain from the plant thus pollinated. : DEFINITIONS

In order to ensure a clear and consistent understanding of the specification and the claims, the following definitions are provided: “Expression cassette” as used herein means a DNA sequence capable of directing expression of a gene in plant cells, comprising a promoter operably linked to a coding region of interest which is operably linked to a termination region. The coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a nontranslated RNA that, in the sense or antisense direction, inhibits expression of a particular gene, e.g., antisense RNA. The gene may be chimeric, meaning that at least one component of the gene is heterologous with respect to at least one other component of the gene. The gene may also be one which is naturally occurring but has been obtained in a recombinant form useful for genetic transformation of a plant.

Typically, however, the expression cassette is heterologous with respect to the host, i.e., the particular DNA sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event.

A "nuclear expression cassette" is an expression cassette which is integrated into the nuclear DNA of the host.

A “plastid expression cassette" is an expression cassette which is integrated into the plastid

DNA of the host. A plastid expression cassette as described herein may optionally comprise a polycistronic operon containing two or more cistronic coding sequences of interest under control of a single promoter, e.g., a transactivator-regulated promoter, e.qg., wherein one of the coding sequences of interest encodes an antisense mRNA which inhibits expression of cIpP or other plastid protease, thereby enhancing accumulation of protein expressed the other coding sequence or sequences of interest. “Heterologous” as used herein means "of different natural origin®. For example, if a plant is transformed with a gene derived from another organism, particularly from another species, that gene is heterologous with respect to that plant and also with respect to descendants of the plant which carry that gene. "Homoplastidic" refers to a plant, plant tissue or plant cell wherein all of the plastids are genetically identical. This is the normal state in a plant when the plastids have not been transformed, mutated, or otherwise genetically altered. In different tissues or stages of development, the plastids may take different forms, e.g., chloroplasts, proplastids, ) etioplasts, amyloplasts, chromoplasts, and so forth.

An "inducible promoter" is a promoter which initiates transcription only when the plant is exposed to some particular external stimulus, as distinguished from constitutive promoters or promoters specific to a specific tissue or organ or stage of development. Particularly preferred inducible promoters for the present invention are chemically-inducible or transactivator-regulated promoters. Chemically inducible promoters include plant-derived promoters, such as the promoters in the systemic acquired resistance pathway, for example the PR promoters, e.g, the PR-1, PR-2, PR-3, PR-4, and PR-5 promoters, especially the tobacco PR-1a promoter and the Arabidopsis PR-1 promoter, which initiate transcription when the plant is exposed to BTH and related chemicals. See US Patent 5,614,395, incorporated herein by reference, and WO 98/03536, incorporated herein by reference.

Chemically-inducible promoters also include receptor-mediated systems, e.g., those derived from other organisms, such as steroid-dependent gene expression, copper-dependent gene expression, tetracycline-dependent gene expression, and particularly the expression system utilizing the USP receptor from Drosophila mediated by juvenile growth hormone and its

Co £ WO 00/36126 PCT/EP99/09986 agonists, described in PCT/EP 96/04224, incorporated herein by reference, as well as systems utilizing combinations of receptors, e.g., as described in PCT/EP 96/00686, incorporated herein by reference. Chemically inducible promoters may be directly linked to the thioredoxin gene or the thioredoxin gene may be under control of a transactivator- regulated promoter while the gene for the transactivator is under control of a chemically inducible promoter. See generally, C. Gatz, "Chemical Control of Gene Expression", Annu.

Rev. Plant Physiol. Plant Mol. Biol. (1997) 48: 89-108, the contents of which are incorporated herein by reference. Transactivator regulated promoters are described more fully below, and may also be induced by hybridization of a plant comprising the thioredoxin gene under control of a transactivator-regulated promoter with a second plant expressing the transactivator.

An “isolated DNA molecule” is a nucleotide sequence that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated nucleotide sequence may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell.

A “protein” as defined herein is the entire protein encoded by the corresponding nucleotide . sequence, or is a portion of the protein encoded by the corresponding portion of the nucleotide sequence.

An ‘isolated protein” is a protein that is encoded by an isolated nucleotide sequence and is therefore not a product of nature. An isolated protein may exist in a purified form or may exist in a non-native environment, such as a transgenic host cell, wherein the protein would not normally expressed or would be expressed in a different form or different amount in an isogenic non-transgenic host cell.

A "plant" refers to any plant or part of a plant at any stage of development, and is specifically intended to encompass plants and plant material which have been damaged, crushed or killed, as well as viable plants, cuttings, cell or tissue cultures, and seeds. ‘DNA shuffling” is a method to introduce mutations or rearrangements, preferably randomly, in a DNA molecule or to generate exchanges of DNA sequences between two or more DNA molecules, preferably randomly. The DNA molecule resulting from DNA shuffling is a shuffled DNA molecule that is a non-naturally occurring DNA molecule derived from at least one template DNA molecule. The shuffled DNA encodes an enzyme modified with respect to the enzyme encoded by the template DNA, and preferably has an altered biological activity with respect to the enzyme encoded by the template DNA.

In its broadest sense, the term "substantially similar’, when used herein with respect to a nucleotide sequence, means a nucleotide sequence corresponding to a reference nucleotide sequence, wherein the corresponding sequence encodes a polypeptide having substantially the same structure and function as the polypeptide encoded by the reference nucleotide sequence, e.g. where only changes in amino acids not affecting the polypeptide function occur. Desirably the substantially similar nucleotide sequence encodes the polypeptide encoded by the reference nucleotide sequence. The percentage of identity between the substantially similar nucleotide sequence and the reference nucleotide sequence desirably is at least 80%, more desirably at least 85%, preferably at least 90%, more preferably at least 95%, still more preferably at least 99%. Sequence comparisons are carried out using a Smith-Waterman sequence alignment algorithm (see e.g. Waterman,

M.S. Introduction to Computational Biology: Maps, sequences and genomes. Chapman &

Hall. London: 1995. ISBN 0-412-99391-0, or at http:/www- : hto.usc.edu/software/seqaln/index.html). The localS program, version 1.16, is used with following parameters: match: 1, mismatch penalty: 0.33, open-gap penalty: 2, extended-gap : penalty: 2.

A nucleotide sequence "substantially similar" to reference nucleotide sequence hybridizes ) to the reference nucleotide sequence in 7% sodium dodecyl! sulfate (SDS), 0.56 M NaPO,, 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C, more desirably in 7% : sodium dodecy! sulfate (SDS), 0.5 M NaPO,, 1 mM EDTA at 50°C with washing in 1X SSC, 0.1% SDS at 50°C, more desirably still in 7% sodium dodecyl! sulfate (SDS), 0.5 M NaPOQ,, 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C, preferably in 7% sodium dodecyl! sulfate (SDS), 0.5 M NaPO,, 1 mM EDTA at 50°C with washing in 0.1X

SSC, 0.1% SDS at 50°C, more preferably in 7% sodium dodecy) sulfate (SDS), 0.5 M

NaPO,, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 65°C.

The term "substantially similar”, when used herein with respect to a protein, means a protein corresponding to a reference protein, wherein the protein has substantially the same structure and function as the reference protein, e.g. where only changes in amino acids not affecting the polypeptide function occur. When used for a protein or an amino acid sequence the percentage of identity between the substantially similar and the reference protein or amino acid sequence desirably is at least 80%, more desirably 85%, preferably at least 90%, more preferably at least 95%, still more preferably at least 99%.

A “transactivator” is a protein which, by itself or in combination with one or more additional proteins, is capable of causing transcription of a coding region under control of a

Cs © WO 00/36126 PCT/EP99/09986 corresponding transactivator-regulated promoter. Examples of transactivator systems include phage T7 gene 10 promoter, the transcriptional activation of which is dependent upon a specific RNA polymerase such as the phage T7 RNA polymerase. The transactivator is typically an RNA polymerase or DNA binding protein capable of interacting with a particular promoter to initiate transcription, either by activating the promoter directly or by inactivating a repressor gene, €.g., by suppressing expression or accumulation of a repressor protein. The DNA binding protein may be a chimeric protein comprising a binding region (e.g., the GAL4 binding region) linked to an appropriate transcriptional activator domain. Some transactivator systems may have multiple transactivators, for example promoters which require not only a polymerase but also a specific subunit (sigma factor) for promoter recognition, DNA binding, or transcriptional activation. The transactivator is preferably heterologous with respect to the plant.

Moditication of Microbial Genes to Optimize Nuclear Expression in Plants

If desired, the cloned thioredoxin genes described in this application can be modified for expression in transgenic plant hosts. For example, the transgenic expression in plants of genes derived from microbial sources may require the modification of those genes to achieve and optimize their expression in plants. In particular, bacterial ORFs that encode : separate enzymes but which are encoded by the same transcript in the native microbe are best expressed in plants on separate transcripts. To achieve this, each microbial ORF is isolated individually and cloned within a cassette which provides a plant promoter sequence at the 5’ end of the ORF and a plant transcriptional terminator at the 3’ end of the ORF.

The isolated ORF sequence preferably includes the initiating ATG codon and the terminating STOP codon but may include additional sequence beyond the initiating ATG and the STOP codon. In addition, the ORF may be truncated, but still retain the required activity; for particularly long ORFs, truncated versions which retain activity may be preferable for expression in transgenic organisms.

By “plant promoter” and “plant transcriptional terminator” it is intended to mean promoters and transcriptional terminators which operate within plant cells. This includes promoters and transcription terminators which may be derived from non-plant sources such as viruses (examples include promoters and terminators derived from the Cauliflower Mosaic Virus or from opine synthase genes in Agrobacterium Ti or Ri plasmids).

In some cases, modification to the ORF coding sequences and adjacent sequence will not be required, in which case it is sufficient to isolate a fragment containing the ORF of interest

I

-18 - and to insert it downstream of a plant promoter. Preferably, however, adjacent microbial sequences left attached upstream of the ATG and downstream of the STOP codon should be minimized or eliminated. In practice, such construction may depend on the availability of restriction sites.

In other cases, the expression of genes derived from microbial sources may provide problems in expression. These problems have been well characterized in the art and are particularly common with genes derived from certain sources such as Bacillus. The modification of such genes can be undertaken using techniques now well known in the art.

The following problems are typical of those that may be encountered: 1. Codon Usage

The preferred codon usage in plants differs from the preferred codon usage in certain microorganisms. Comparison of the usage of codons within a cloned microbial ORF to usage in plant genes (and in particular genes from the target plant) will enable an identification of the codons within the ORF which should preferably be changed. Typically plant evolution has tended towards a strong preference of the nucleotides C and G in the third base position of monocotyledons, whereas dicotyledons often use the nucleotides Aor

T at this position. By modifying a gene to incorporate preferred codon usage for a particular target transgenic species, many of the problems described below for GC/AT content and illegitimate splicing will be overcome. 2. GC/AT Content

Plant genes typically have a GC content of more than 35%. ORF sequences which are rich in A and T nucleotides can cause several problems in plants. Firstly, motifs of ATTTA are believed to cause destabilization of messages and are found at the 3' end of many short- lived mRNAs. Secondly, the occurrence of polyadenylation signals such as AATAAA at inappropriate positions within the message is believed to cause premature truncation of transcription. In addition, monocotyledons may recognize AT-rich sequences as splice sites (see below). 3. Sequences Adjacent to the Initiating Methionine

Plants differ from microorganisms in that their messages do not possess a defined ribosome binding site. Rather, it is believed that ribosomes attach to the 5’ end of the message and scan for the first available ATG at which to start translation. Nevertheless, it is believed that there is a preference for certain nucleotides adjacent to the ATG and that expression of microbial genes can be enhanced by the inclusion of a eukaryotic consensus

J + WO 00/36126 PCT/EP99/09986 translation initiator at the ATG. Clontech (1993/1994 catalog, page 210) have suggested the sequence GTCGACCATGGTC (SEQ ID NO: 8) as a consensus translation initiator for the expression of the E. coli uidA gene in plants. Further, Joshi (NAR 15: 6643-6653 (1987)) has compared many plant sequences adjacent to the ATG and suggests the consensus TAAACAATGGCT (SEQ ID NO: 9). In situations where difficulties are encountered in the expression of microbial ORFs in plants, inclusion of one of these sequences at the initiating ATG may improve translation. In such cases the last three nucleotides of the consensus may not be appropriate for inclusion in the modified sequence due to their modification of the second AA residue. Preferred sequences adjacent to the initiating methionine may differ between different plant species. A survey of 14 maize genes located in the GenBank database provided the following results:

Position Before the Initiating ATG in 14 Maize Genes: -10 9 8 7 6 5 4 3 2 A ’ C 3 8 4 6 2 5 6 0 10 7

T 3 0 3 4 3 2 1 1 1 0 : A 2 3 1 4 3 2 3 7 2 3

G 6 3 6 0 6 5 4 6 1 5

This analysis can be done for the desired plant species into which the thioredoxin or thioredoxin reductase genes are being incorporated, and the sequence adjacent to the ATG modified to incorporate the preferred nucleotides. 4. Removal of lllegitimate Splice Sites

Genes cloned from non-plant sources and not optimized for expression in plants may aiso contain motifs which may be recognized in plants as 5’ or 3’ splice sites, and be cleaved, thus generating truncated or deleted messages. These sites can be removed using the techniques described in patent application WO 97/02352, hereby incorporated by reference.

Techniques for the modification of coding sequences and adjacent sequences are well known in the art. In cases where the initial expression of a microbial ORF is low and it is deemed appropriate to make alterations to the sequence as described above, then the construction of synthetic genes can be accomplished according to methods well known in the art. These are, for example, described in the published patent disclosures EP 0 385 962, EP 0 359 472 and WO 93/07278. In most cases it is preferable to assay the expression of gene constructions using transient assay protocols (which are well known in the art) prior to their transfer to transgenic plants.

A major advantage of plastid transformation is that plastids are generally capable of expressing bacterial genes without substantial modification. Codon adaptation, etc. as described above is not required, and plastids are capable of expressing multiple open reading frames under control of a single promoter.

Construction of Plant Transformation Vectors and Selectable Markers

Numerous transformation vectors are available for plant transformation, and the genes of this invention can be used in conjunction with any such vectors. The selection of vector for use will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers used routinely in transformation include the npt/l gene : which confers resistance to kanamycin and related antibiotics (Vieira & Messing, Gene 19: } 259-268 (1982); Bevan et al., Nature 304:184-187 (1983)), the bar gene which confers resistance to the herbicide phoSph/nothricin (White et al., Nucl Acids Res 18: 1062 (1990), -

Spencer et al. Theor Appl Genet 79: 625-631(1990)), the hpt gene which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann, Mol Cell Biol 4: 2929-2931), the dhfr gene, which confers resistance to methotrexate (Bourouis et al, EMBO J. 2(7): 1099-1104 (1983)); and the mannose-6-phosphate isomerase gene which confers the ability to metabolize mannose, as described in US 5767378.

Requirements for Construction of Plant Expression Cassettes

Gene sequences intended for expression in transgenic plants are firstly assembled in expression cassettes behind a suitable promoter and upstream of a suitable transcription terminator. These expression cassettes can then be easily transferred to the plant transformation vectors described above. 1. Promoter Selection

The selection of promoter used in expression cassettes will determine the spatial and temporal expression pattern of the transgene in the transgenic plant. Selected promoters will express transgenes in specific cell types (such as leaf epidermal cells, mesophyll cells,

Co + WO 00/36126 IK PCT/EP99/09986 iE root cortex cells) or in specific tissues or organs (roots, seeds, leaves or flowers, for example) and this selection will reflect the desired location of biosynthesis of the thioredoxin. In the present invention, seed specific promoters are preferred. Alternatively, the selected promoter may drive expression of the gene under a light-induced or other temporally regulated promoter. A further alternative is that the selected promoter be inducible by an external stimulus, e.g., application of a specific chemical inducer, or by hybridization with a second plant line, providing the possibility of inducing thioredoxin transcription only when desired. 2. Transcriptional Terminators

A variety of transcriptional terminators are available for use in expression cassettes. These are responsible for the termination of transcription beyond the transgene and its correct polyadenylation. Appropriate transcriptional terminators and those which are known to function in plants and include the CaMV 35S terminator, the tm/ terminator, the nopaline synthase terminator, the pea rbcS E9 terminator. These can be used in both monocotyledons and dicotyledons. 3. Sequences for the Enhancement or Regulation of Expression

Numerous sequences have been found to enhance gene expression from within the transcriptional unit and these sequences can be used in conjunction with the genes of this : invention to increase their expression in transgenic plants.

Various intron sequences have been shown to enhance expression, particularly in monocotyledonous cells. For example, the introns of the maize Adht gene have been found to significantly enhance the expression of the wild-type gene under its cognate promoter when introduced into maize cells. Intron 1 was found to be particularly effective and enhanced expression in fusion constructs with the chloramphenicol acetyltransferase gene (Callis et al,, Genes Develop 1: 1183-1200 (1987)). In the same experimental system, the intron from the maize bronze1 gene had a similar effect in enhancing expression. Intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader.

A number of non-translated leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous cells.

Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the “Q-sequence”), Maize

Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (e.g. Gallie et al. Nucl. Acids Res. 15: 8693-8711 (1987);

Skuzeski et al. Plant Molec. Biol. 15; 65-79 (1990)). 4. Targeting of the Gene Product Within the Cell

Various mechanisms for targeting gene products are known to exist in plants and the sequences controlling the functioning of these mechanisms have been characterized in some detail. Aminoterminal sequences are responsible for targeting to the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler & Ho, Plant Cell 2: 769- 783 (1990)). Additionally, aminoterminal sequences in conjunction with carboxyterminal sequences are responsible for vacuolar targeting of gene products (Shinshi et al. Plant

Molec. Biol. 14: 357-368 (1990)). By the fusion of the appropriate targeting sequences described above to transgene sequences of interest it is possible to direct the transgene product to any organelle or cell compartment. The signal sequence selected should include the known cleavage site and the fusion constructed should take into account any amino acids after the cleavage site which are required for cleavage. In some cases this requirement may be fulfilled by the addition of a small number of amino acids between the cleavage site and the transgene ATG or alternatively replacement of some amino acids within the transgene sequence.

The above-described mechanisms for cellular targeting can be utilized not only in conjunction with their cognate promoters, but also in conjunction with heterologous } promoters so as to effect a specific cell targeting goal under the transcriptional regulation of a promoter which has an expression pattern different to that of the promoter from which the targeting signal derives.

Examples of Expression Cassette Construction

The present invention encompasses the expression of thioredoxin genes under the regulation of any promoter that is expressible in plants, regardless of the origin of the promoter.

Furthermore, the invention encompasses the use of any plant-expressible promoter in conjunction with any further sequences required or selected for the expression of the thioredoxin or thioredoxin reductase gene. Such sequences include, but are not restricted to, transcriptional terminators, extraneous sequences to enhance expression (such as introns [e.g. Adh intron 1], viral sequences [e. g. TMV-Q]), and sequences intended for the targeting of the gene product to specific organelles and cell compartments.

Claims (17)

What is claimed is:

1. A method to increase efficiency of separation of starch and protein in a grain milling process, comprising steeping the grain at an elevated temperature in the presence of supplemental thioredoxin and/or thioredoxin reductase and separating the starch and protein components of the grain.

2. The method of claim 1 wherein the grain includes grain from a transgenic plant wherein the transgene expresses thioredoxin and/or thioredoxin reductase.

3. The method of claim 2 wherein the plant is selected from corn (Zea mays) and soybean.

4. A plant comprising a heterologous DNA sequence coding for a thioredoxin and/or thioredoxin reductase stably integrated into its nuclear or plastid DNA.

5. The plant according to claim 4, wherein the thioredoxin and/or thioredoxin reductase is thermostable.

6. The plant of claim 5 wherein the thioredoxin and/or thioredoxin reductase is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.

7. The plant of any one of claims 4 to 6 wherein the plant is selected from com and soybean.

8. A plant expressible expression cassette comprising a coding region for a thioredoxin and/or thioredoxin reductase operably linked to promoter and terminator sequences which function in a plant.

9. The plant expressible expression cassette according to claim 8, wherein the thioredoxin and/or thioredoxin reductase is thermostable.

oo Case S-30758A

10. The plant expressible expression cassette of claim 9, wherein the thioredoxin and/or thioredoxin reductase is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.

11. A method of producing grain comprising high levels of thioredoxin and/or thioredoxin reductase comprising transforming plants with an expression cassette of claims 8 to 10.

12. A method of producing grain comprising high levels of thioredoxin and/or thioredoxin . reductase comprising pollinating a first plant comprising a heterologous expression cassette comprising a transactivator-regulated promoter regulated and operably linked to a DNA sequence coding for a thioredoxin and/or thioredoxin reductase, with pollen from a second plant comprising a heterologous expression cassette comprising a promoter operably linked to a DNA sequence coding for a transactivator capable of regulating said transactivator-regulated promoter; and ’ . recovering grain from the plant thus pollinated. :

13. Use of plants or plant material according to any one of claims 4 to 7 as animal feed.

14. A method to increase efficiency of separation of starch and protein in a grain milling process substantially as herein described and exemplified.

15. A plant substantially as herein described and exemplified.

16. A plant expressible expression cassette substantially as herein described and exemplified.

17. A method of producing grain comprising high levels of thioredoxin and/or thioredoxin reductase substantially as herein described and exemplified. AMENDED SHEET