# Patent

## Title
Soybean cultivar 9505389620962

## Abstract

This is a continuation of International Application No. PCT/SE96/01558, filed Nov. 28, 1996 that designates the United States.

## Background
The present invention relates to a novel starch branching enzyme of potato. More specifically, the present invention relates to an amino acid sequence of a second starch branching enzyme (SBE II) of potato and a fragment thereof as well as their corresponding DNA sequences. Furthermore, the invention relates to vectors comprising such DNA sequences, to processes for production of transgenic potatoes, and to the use of said potatoes for the production of starch.

Starch is a complex mixture of different molecule forms differing in degree of polymerization and branching of the glucose chains. Starch consists of amylose and amylopectin, whereby the amylose consists of an essentially linear .alpha.-1,4-glucan and amylopectin consists of .alpha.-1,4-glucans connected to each other via .alpha.-1,6-linkages and, thus, forming a branched polyglucan. Thus, starch is not a uniform raw material.

Starch is synthesized via at least three enzymatic reactions in which ADP glucose phosphorylase (EC 2.7.7.27), starch synthase (EC 2.4.1.21) and starch branching enzyme (EC 2.4.1.18) are involved. Starch branching enzyme (SBE, also called Q-enzyme) is believed to have two different enzymatic activities. It catalyzes both the hydrolysis of .alpha.-1,4-glucosidic bonds and the formation of .alpha.-1,6-glucosidic bonds during synthesis of the branched component in starch, i.e. amylopectin.

Plant starch is a valuable source of renewable raw material used in, for example, the chemical industry (Visser and Jacobsen, 1993). However, the quality of the starch has to meet the demands of the processing industry wherein uniformity of structure is an important criterion. For industrial application there is a need of plants only containing amylose starch and plants only containing amylopectin starch, respectively.

Processes for altering the amylose/amylopectin ratio in starch have already been proposed. For example, in WO95/04826 there is described DNA sequences encoding debranching enzymes with the ability to reduce or increase the degree of branching of amylopectin in transgenic plants, e.g. potatoes.

In WO92/14827 plasmids are described having DNA sequences that after insertion into the genome of the plants cause changes in the carbohydrate concentration and the carbohydrate composition in regenerated plants. These changes can be obtained from a sequence of a branching enzyme that is located on these plasmids. This branching enzyme is proposed to alter the amylose/amylopectin ratio in starch of the plants, especially in commercially used plants.

WO92/14827 describes the only hitherto known starch branching enzyme in potato and within the art it is not known whether other starch branching enzymes are involved in the synthesis of branched starch of potato.

In Mol Gen Genet (1991) 225:289-296, Visser et al., there is described inhibition of the expression of the gene for granule-bound starch synthase in potato by antisense constructs. Inhibition of the enzyme in potato tuber starch was up to 100% in which case amylose-free starch was provided.

However, the prior known methods for inhibiting amylopectin have not been that successful and, therefore, alternative methods for inhibiting amylopectin are still highly desirable (Muller-Rober and Ko.beta.mann, 1994; Martin and Smith, 1995).

The object of the present invention is to enable altering the degree of amylopectin branching and the amylopectin/amylose ratio in potato starch.

According to the present invention this object is achieved by providing a novel isolated DNA sequence encoding a second starch branching enzyme, SBE II, and fragments thereof, which after insertion into the genome of the plants cause changes in said branching degree and ratio in regenerated plants.

Within the scope of the present invention there is also included the amino acid sequence of SBE II and fragments thereof.

Also variants of the above DNA sequence resulting from the degeneracy of the genetic code are encompassed.

The novel DNA sequence encoding SBEII, comprising 3074 nucleotides, as well as the corresponding amino acid sequence comprising 878 amino acids, are shown in SEQ ID No. 1. One 1393 nucleotides long fragment of the above DNA sequence, corresponding to nucleotides 1007 to 2399 of the DNA sequence in SEQ ID No. 1, as well as the corresponding amino acid sequence comprising 464 amino acids, are shown in SEQ ID No. 2.

Furthermore, there are provided vectors comprising said isolated DNA-sequences and regulatory elements active in potato. The DNA sequences may be inserted in the sense or antisense (reversed) orientation in the vectors in relation to a promoter immediately upstream from the DNA sequence.

Also there is provided a process for the production of transgenic potatoes with a reduced degree of branching of amylopectin starch, comprising the following steps: the genome of a potato cell, and

Finally, the invention provides the use of said transgenic potatoes for the production of starch.

The invention will be described in more detail below in association with an experimental part and the accompanying drawings.

FIG. 1 shows SDS polyacrylamide electrophoresis of proteins extracted from starch of normal potato (lane A) and transgenic potato (lane B). Excised protein bands are marked with arrows. Lane M: Molecular weight marker proteins (kDa).

FIG. 2 shows 4 peptide sequences derived from digested proteins from potato tuber starch.

Isolation of starch from potato tubers

Potato plants (Solanum tuberosum) were grown in the field. Peeled tubers from either cv. Early Puritan or from a transgenic potato line essentially lacking granule-bound starch synthase I (Svalof Weibull AB, international application number PCT/SE91/00892), were homogenized at 4.degree. C. in a fruit juicer. To the "juice fraction", which contained a large fraction of the starch, was immediately added Tris-HCl, pH 7.5, to 50 mM, Na-dithionite to 30 mM and ethylenedinitrilotetraacetic acid (EDTA) to 10 mM. The starch granules were allowed to sediment for 30 min and washed 4.times. with 10 bed volumes of washing buffer (50 mM Tris-HCl, pH 7.5, 10 mM EDTA). The starch, which was left on the bench at +4.degree. C. for 30 min to sediment between every wash, was finally washed with 3.times.3 bed volumes of acetone, air dried over night, and stored at -20.degree. C.

Extraction of proteins from tuber starch

Stored starch (20 g) was continuously mixed with 200 ml extraction buffer (50 mM Tris-HCl, pH 7.5, 2% (w/v) sodium dodecyl sulfate (SDS), 5 mM EDTA) by aspiration with a pipette at 85.degree. C. until the starch was gelatinized. The samples were then frozen at -70.degree. C. for 1 hour. After thawing at 50.degree. C., the samples were centrifuged for 20 min at 12,000.times.g at 10.degree. C. The supernatants were collected and re-centrifuged at 3,000.times.g for 15 min. The final supernatants were filtered through 0.45.mu. filters and 2.25 volumes of ice-cold acetone were added. After 30 min incubation at 4.degree. C., the protein precipitates were collected by centrifugation (3,000.times.g for 30 min at 4.degree. C.), and dissolved in 50 mM Tris-HCl, pH 7.5. An aliquot of each preparation was analyzed by SDS poly-acrylamide gel electrophoresis according to Laemmli (1970) (FIG. 1). The proteins in the remaining portions of the preparations were concentrated by precipitation with trichloroacetic acid (10%) and the proteins were separated on an 8% SDS polyacrylamide gel Laemmli, (1970). The proteins in the gel were stained with Coomassie Brilliant Blue R-250 (0.2% in 20% methanol, 0.5% acetic acid, 79.5% H.sub.2 O).

In gel digestion and sequencing of peptides

The stained bands marked with arrows in FIG. 1 corresponding to an apparent molecular weight of about 100 kDa were excised and washed twice with 0.2M NH.sub.4 HCO.sub.3 in 50% acetonitrile under continuous stirring at 35.degree. C. for 20 min. After each washing, the liquid was removed and the gel pieces were allowed to dry by evaporation in a fume hood. The completely dried gel pieces were then separately placed on parafilm and 2 .mu.l of 0.2M NH.sub.4 CO.sub.3, 0.02% Tween-20 were added. Modified trypsin (Promega, Madison, Wis., USA) (0.25 .mu.g in 2 .mu.l) was sucked into the gel pieces whereafter 0.2M NH.sub.4 CO.sub.3 was added in 5 .mu.l portions until they had resumed their original sizes. The gel slices were further divided into three pieces and transferred to an Eppendorf tube. 0.2M NH.sub.4 CO.sub.3 (200 .mu.l) was added and the proteins contained in the gel pieces were digested over night at 37.degree. C. (Rosenfeld et al. 1992). After completed digestion, trifluoroacetic acid was added to 1% and the supernatants removed and saved. The gel pieces were further extracted twice with 60% acetonitrile, 0.1% trifluoroacetic acid (200 .mu.l) under continuous shaking at 37.degree. C. for 20 min. The two supernatants from these extractions were combined with the first supernatant. The gel pieces were finally washed with 60% acetonitrile, 0.1% trifluoroacetic acid, 0.02% Tween-20 (200 .mu.l). Also these supernatants were combined with the other supernatants and the volume was reduced to 50 .mu.l by evaporation. The extracted peptides were separated on a SMART.RTM. chromatography system (Pharmacia, Uppsala, Sweden) equipped with a .mu.RPC C2/C18 SC2.1/10 column. Peptides were eluted with a gradient of 0-60% acetonitrile in water/0.1% trifluoroacetic acid over 60 min with a flow rate of 100 .mu.l/min. Peptides were sequenced either on an Applied Biosystems 470A gas phase sequenator with an on line PTH-amino acid analyzer (120A) or on a model 476A according to the instructions of the manufacturer (Applied Biosystems, Foster City, Calif., USA).

Four of the peptides sequenced gave easily interpretable sequences (FIG. 2). A data base search revealed that these four peptides displayed similarity to starch branching enzymes and interestingly, the peptides were more related to starch branching enzyme II from other plant species than to starch branching enzyme I from potato.

Construction of oligonucleotides encoding peptides 1 and 2.

Degenerated oligonucleotides encoding peptide 1 and peptide 2 were synthesized as forward and reverse primers, respectively: to 8 of peptide 1) (Residues 2 to 8 of peptide 2, reversed strand) wherein Y is C or T; bases in lower case were added as tag sequences.

Purification of mRNA from potato tuber, synthesis of cDNA and PCR amplification of a cDNA fragment corresponding to potato starch branching enzyme II.

Total RNA from mature potato tubers (S. tuberosum cv. Amanda) was isolated as described (Logemann et al. 1987). First strand cDNA was synthesized using 2 .mu.g of total RNA and 60 pmol of oligo-dT.sub.30 as downstream primer. The primer was annealed to the polyA of the mRNA at 60.degree. C. for 5 min. The extension of the cDNA was performed according to the technical manual of the manufacturer using the Riboclone.RTM. cDNA Synthesis System M-MLV (H-) (Promega).

cDNA encoding the novel starch branching enzyme II according to the invention was amplified in a Perkin-Elmer GeneAmp.RTM. 9600 PCR thermocycler (Perkin-Elmer Cetus Instruments, Conn., USA) using the two degenerate primers designed from the peptides 1 and 2 (see above) under the following conditions: 1 mM dNTP, 1 .mu.M of each primer and an alicot of the cDNA described above in a total reaction volume of 20 .mu.l with 1.times. AmpliTaq.RTM. buffer and 0.8 U AmpliTaq.RTM. (Perkin-Elmer Cetus). The cycling conditions were: 96.degree. C. for 1', 80.degree. C. while the enzyme was added as a hotstart (approximately 15'), an unintended drop to 25.degree. C., five cycles of 94.degree. C. for 20", 45.degree. C. for 1', ramp to 72.degree. C. for 1' and 72.degree. C. for 2', and 30 cycles of 94.degree. C. for 5", 45.degree. C. for 30", and 72.degree. C. for (2'+2" per cycle) and completed with 72.degree. C. for 10' prior to chilling to 4.degree. C.

A sample of this reaction (0.1 .mu.l) was reamplified using the cycling conditions: 96.degree. C. for 1', 80.degree. C. while the enzyme was added as a hotstart (approximately 5'), five cycles of 94.degree. C. for 20", 45.degree. C. for 1', and 72.degree. C. for 2', and 25 cycles of 94.degree. C. for 5", 45.degree. C. for 30", and 72.degree. C. for (2'+2" per cycle) and completed with 72.degree. C. for 10' prior to chilling to 4.degree. C. After completion of the PCR amplification, the reaction was loaded on a 1.5% Seakem.RTM. agarose gel (FMC Bioproducts, Rockland, Me., USA). After electrophoresis and staining with ethidium bromide a major band with an apparent size of 1500 bp was excised and the fragment was eluted by shaking in water (200 .mu.l) for 1 h. This fragment was used as template in sequencing reactions after reamplification using primers corresponding to the tag sequences (in oligonucleotides 1 and 2), purification by agarose gel electrophoresis as above and extraction from the gel using the Qiaex.RTM. gel extraction kit according to the manufacturer's instructions (DIAGEN GmbH, Hilden, Germany). The sequencing reactions were done using the DyeDeoxy.RTM. Terminator Cycle Sequencing kits (Perkin-Elmer Cetus Instruments) using tag sequences and internal primers. The sequencing reaction were analyzed on an Applied Biosystems 373A DNA sequencer according to the manufacturer's protocol. The sequence was edited and comprised 1393 bp.

To complete the determination of the sequence of starch branching enzyme II, the 5' and 3' ends of the full length cDNA were amplified from the same total RNA as above using rapid amplification of cDNA ends, RACE methodology with specific primers from the 1393 bp sequence. In the 3' end amplification, an oligo T.sub.29 G primer was used against the poly A tail and in the 5' end, the 5'/3' RACE kit from Boehringer Mannheim (Cat. No. 1734792) was used. The fragments from these amplifications were sequenced in the same way as above using internal and end primers. The sequences from the two ends were aligned together with the 1393 base pairs to give a composite full length cDNA sequence. Primers were designed from this sequence to amplify the whole coding region in one part. Partial sequencing of the amplified coding cDNA confirmed the presence of a cDNA corresponding to the composite sequence. The full length cDNA is 3074 bp and the translated sequence comprises 878 amino acids. The mature protein comprises 830 amino acids.

Comparisons of the consensus sequence with the EMBL and GenBank databases showed 68% identity to potato starch branching enzyme I and about 80% identity to starch branching enzyme II from other plant species. The present inventors therefore denote the enzyme encoded by the new branching enzyme sequence potato starch branching enzyme II.

Transformation of potato plants

The isolated full length cDNA of potato starch branching enzyme II and other functionally active fragments in the range of 50-3 074 bp are cloned in reverse orientation behind promoters active in potato tubers. By the term "functionally active" is meant fragments that will affect the amylose/amylopectin ratio in potato starch. The DNA and amino acid sequence of SBE II according to the invention as well as one fragment of the DNA and corresponding amino acid sequence are shown in SEQ ID No. 1 and 2, respectively.

The promoters are selected from, for example, the patatin promoter, the promoter from the potato granule-bound starch synthase I gene or promoters isolated from potato starch branching enzymes I and II genes.

The constructs are cloned by techniques known in the art either in a binary Ti-plasmid vector suitable for transformation of potato mediated by Agrobacterium tumefaciens, or in a vector suitable for direct transformation using ballistic techniques or electroporation. It is realized that the sense (see below) and antisense constructs must contain all necessary regulatory elements.

Transgenic potato plants transcribe the inverse starch branching enzyme II construct specifically in tubers, leading to antisense inhibition of the enzyme. A reduction and changed pattern of the branching of amylopectin as well as a changed amylose/amylopectin ratio thereby occur in tuber starch.

The antisense construct for potato starch branching enzyme II is also used in combination with antisense constructs for potato starch branching enzyme I, for potato granule-bound starch synthase II, for potato soluble starch synthases II and III, for potato starch disproportionating enzyme (D-enzyme) or for potato starch debranching enzyme to transform potato to change the degree of branching of amylopectin and the amylose/amylopectin ratio. This gives new and valuable raw material to the starch processing industry.

The full-length cDNA sequence encoding the enzyme is, in different constructs, cloned in sense orientation behind one or more of the promoters mentioned above, and the constructs are transferred into suitable transformation of potato. Regenerated transformed potato plants will produce an excess of starch branching enzyme II in the tubers leading to an increased degree and changed pattern of branching of amylopectin or to inhibition of transcription of endogenous starch branching enzyme II transcription due to co-suppression, resulting in a decreased branching or amylopectin.

References quantity and starch quality in transgenic plants. Plant Cell Environm. 17, 601-613. the head of bacteriophage T4. Nature 227, 680-685. isolation of RNA from plant tissues. Anal. Biochem. 163, 16-20. digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal. Biochem. 203, 173-179. starch content and composition. TibTech 11, 63-68.

## Claims

1. A vector which contains an isolated DNA sequence encoding starch branching enzyme II (SBE II) of potato, wherein the DNA sequence is in the antisense orientation in relation to a promoter operable in a plant which is operably linked to said DNA sequence, wherein said DNA affects the amylose/amylopectin ratio in potato starch and the pattern of amylopectin branching when expressed in a potato plant.

2. A process for the production of transgenic potatoes with a reduced degree of branching of amylopectin starch, which comprises the following steps: genome of a potato cell, and

3. A process according to claim 2, wherein the vector also comprises an antisense construct of starch branching enzyme I (SBE I).

4. A process according to claim 2, wherein the vector also comprises an antisense construct of potato granule bound starch synthase II.

5. A process according to claim 3, wherein the vector also comprises an antisense construct of potato granule bound starch synthase II.

6. A process according to claim 2, wherein the vector also comprises an antisense construct of potato starch disproportionating enzyme (D-enzyme).

7. A process according to claim 3, wherein the vector also comprises an antisense construct of potato starch disproportionating enzyme (D-enzyme).

8. A process according to claim 4, wherein the vector also comprises an antisense construct of potato starch disproportionating enzyme (D-enzyme).

9. A process according to claim 2, wherein the vector also comprises an antisense construct of potato starch debranching enzyme.

10. A process according to claim 3, wherein the vector also comprises an antisense construct of potato debranching enzyme.

11. A process according to claim 4, wherein the vector also comprises an antisense construct of potato debranching enzyme.

12. A process according to claim 6, wherein the vector also comprises an antisense construct of potato debranching enzyme.

13. A transgenic potato obtainable by the process according to claim 2.

14. A transgenic potato obtainable by the process according to claim 3.

15. A transgenic potato obtainable by the process according to claim 4.

16. A transgenic potato obtainable by the process according to claim 6.

17. A transgenic potato obtainable by the process according to claim 9.

18. A process for isolating starch or starch proteins from the transgenic potato of claim 13, comprising:

19. A process for isolating starch or starch proteins from the transgenic potato of claim 14, comprising:

20. A process for isolating starch or starch proteins from the transgenic potato of claim 15, comprising:

21. A process for isolating starch or starch proteins from the transgenic potato of claim 16, comprising:

22. A process for isolating starch or starch proteins from the transgenic potato of claim 17, comprising:

23. The vector of claim 1, which comprises a DNA having SEQ ID NO:1 in antisense orientation.

24. The vector of claim 1, wherein said promoter is selected from the group consisting of potato granule-bound starch synthase I, or a promoter derived from the potato starch branching enzyme I or II gene.