T 2352/09 (Amino acid production/AJINOMOTO) 18-10-2012

Reasons for the Decision

Articles 123(2) and 84 EPC

1. The objections raised under Articles 123(2) and 84 EPC were not admitted into the opposition proceedings for reasons given by the opposition division on page 2, points 9 and 10 of the decision under appeal. This decision has not been contested in appeal and thus, it is not part of the present appeal proceedings.

Article 100(b) EPC; Article 83 EPC

2. Claim 1 as granted is directed to an L-amino acid producing Escherichia bacterium, which has been modified so that its L-amino acid production is increased by enhancing the activities of the proteins as defined in parts (G) or (H) of claim 1. The enhancement is obtained by: i) transformation with a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO:15 (part (G) of claim 1), ii) transformation with a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO:15 which has been mutated as defined in part (H) of claim 1 and has the properties indicated therein (enhanced resistance to L-amino acids and/or its analogs and enhanced sensitivity to S-(2-maminoethyl)cysteine), or iii) alteration of the promoter sequence of the said DNA sequence on the chromosome of the bacterium (cf. Section VII supra).

3. According to the patent-in-suit, the b3434 gene was selected by screening the complete Escherichia coli genome for genes encoding proteins having 4 or more putative transmembrane segments. Thus, the encoded b3434 protein was identified as a putative membrane exporter protein. The nucleic acid sequence of the b3434 gene, also known as yhgN, and the encoded amino acid sequence were shown in SEQ ID NO:15 (cf. page 2, paragraph [0006] and page 4, paragraph [0022] of the patent-in-suit). Transformation of E. coli with plasmid pYHGN, carrying the b3434 gene under the control of a strong lactose promoter (Plac UV5) and prepared as indicated in Example 1 (cf. page 6, paragraph [0042] of the patent-in-suit), resulted in an enhanced production of the L-amino acids Arg and Pro (cf. page 15, Example 10 and page 16, Example 11 of the patent-in-suit). This disclosure has not been contested in appeal proceedings or in the opposition proceedings.

4. Thus, the subject-matter of claim 1 is exemplified by increasing the gene copy number and over-expression of the b3434 gene in order to obtain an E. coli bacterium with an increased production of L-amino acids. No technical problems or difficulties have been encountered when carrying out this embodiment (cf. pages 4 and 5, paragraphs [0026] and [0027] of patent-in-suit).

5. Appellant's first objection is based on the absence of a disclosure in the patent of the actual activity of the encoded b3434 protein (cf. Section IX supra). Claim 1 states that the activity of the protein as defined in part (G) is enhanced by transforming the bacterium with a DNA sequence encoding this protein, i.e. by increasing the expression of a DNA sequence encoding this protein and eventually increasing the amount of this protein in the transformed E. coli bacterium. There is no requirement in claim 1 to enhance the (biological or cellular) activity of the protein as defined in part (G) of this claim other than by increasing its amount. Appellant's objection relates to the clarity of the claim and thus, to Article 84 EPC, rather than to the sufficiency of the disclosure of the patent-in-suit. However, Article 84 EPC is not a ground of opposition.

6. In part (H) of claim 1, the protein is structurally defined as comprising "... an amino acid sequence including deletion, substitution, insertion or addition of 1-20 amino acids in the amino acid sequence shown in SEQ ID NO:15 in Sequence listing ..." (cf. Section VII supra). Since the amino acid sequence shown in SEQ ID NO:15 has 197 residues, the modification of 1-20 amino acids represents a change of no more than about 10% of the whole sequence. Thus, the proteins defined in part (H) of claim 1 must have at least 90% identity to the amino acid sequence shown in SEQ ID NO:15. The production of proteins in accordance with this definition, either by random or site-directed mutagenesis, does not pose any technical problem to a skilled person. This structural definition constitutes a generalization of the specific amino acid sequence SEQ ID NO:15 which is in line with the constant practice, and of general acceptance, in the field.

7. All the more so, since these proteins are further defined by functional terms in part (H) of claim 1 by requiring them to have "... an activity of making bacterium having enhanced resistance to L-amino acids and/or its analogs ... and having enhanced sensitivity to S-(2-aminoethyl)cysteine" (cf. Section VII supra). These proteins which, although fulfilling the structural requirements defined in claim 1 do not have similar functional properties as the specific protein having the amino acid sequence SEQ ID NO:15 shown in Table 1 of the patent-in-suit, do not fall under the scope of the claim.

8. Appellant's objection regarding the proteins defined in part (H) of claim 1 is based on two different arguments. As a first argument, it is argued that since the expression "L-amino acids and/or its analogs" is not defined in the patent-in-suit and no correlation is shown in the examples of the patent between the amino acids produced by a transformed bacterium and those to which this bacterium shows enhanced resistance, there is an unlimited number of possible L-amino acids and analogs thereof to which a bacterium transformed with the proteins defined in part (H) could have an enhanced resistance. In the appellant's view, no reliable method would be available to a skilled person for functionally screening these proteins (cf. Section IX supra).

9. The board cannot follow this argument. The results shown in Table 1 of the patent-in-suit have not been contested and, although they do not show the correlation referred to by the appellant, they inform a skilled person of the L-amino acid analogs to which an enhanced resistance may be first expected, namely to those to which an E. coli transformed with the original amino acid sequence SEQ ID NO:15 shows an enhanced resistance. Indeed, this can be expected for a large number of proteins defined in part (H) of claim 1 having only a small number of arbitrary modifications. Even if this expectation is not fulfilled, the patent-in-suit and, in particular Table 1, shows that a large number of L-amino acids and analogs thereof were available to a skilled person for functionally screening the proteins modified in accordance with part (H) of claim 1 (cf. also pages 3 and 4, paragraphs [0016] to [0019] of the patent-in-suit). Again, appellant's objection relates to the clarity of the claim and, thus to Article 84 EPC, rather than to the sufficiency of the disclosure.

10. In the board's view, this also applies for appellant's second argument which presupposes a reading of part (H) of claim 1 so that its subject-matter comprises an embodiment in which the modified protein makes the transformed bacterium to have an enhanced resistance to all L-amino acids and/or analogs thereof (cf. Section IX supra). Whereas the wording of claim 1 may be open to interpretation and thus objectionable under Article 84 EPC, the board does not find any support for appellant's interpretation which, in the light of the whole disclosure of the patent-in-suit and the prior art cited therein as well as that on file, is considered not to be technically sensible.

11. Likewise, the board cannot follow appellant's argument that the exemplified production of the L-amino acids Arg and Pro (Examples 10 and 11) does not allow for a generalization to a method of producing L-amino acids in general (cf. Section IX supra). In absence of any evidence to the contrary, in view of the structure and properties of these two amino acids, and the possible interconnection of their metabolic biosynthetic pathway with that of other (structurally) related amino acids, the board does not see any reason to deviate from the decision of the opposition division on this issue. As a putative membrane exporter, the b3434 protein could well be functionally appropriate for several amino acids other than Arg and Pro. Moreover, the proteins defined in part (H) of claim 1 (at least 90% identical to the amino acid sequence SEQ ID NO:15) may also allow for a certain degree of variation. The appellant's doubts have not been substantiated by verifiable facts as required by the established case law.

12. Although the endogenous promoter sequence of the b3434 gene is not disclosed in the patent-in-suit, there is evidence on file, for instance in document D2, showing that methods for identifying consensus sequences of promoter function and specific recognition sequences of RNA polymerase were standard in the field. Even though this document discloses an extensive compilation of E. coli promoters, it does not disclose a complete list of all E. coli promoters. Document D2 was published in 1993, four years before the publication of the complete E. coli genome sequence in 1997 (document D1) and eight years before the first priority date claimed by the patent-in-suit. The problems identified in document D2 and referred to by the appellant mainly concern deficiencies in the databanks available at that time, such as the presence of duplicated sequences and "inaccuracies in the locations reported in the bank, in comparison to the experimental paper", for which document D2 already indicates possible methods to overcome them (cf. page 1512, left-hand column, first two paragraphs of document D2). In the board's view, the absence of the b3434 promoter sequence in the list of document D2 cannot be taken as evidence that its identification involves real technical difficulties for a skilled person. Taking into account the submissions and evidence on file, the board considers that no undue burden would be required to identify and isolate the b3434 promoter sequence. There is also evidence on file showing that, using random mutagenesis and standard screening methods, altered promoters with the desired properties could also be obtained without undue burden (cf. inter alia, document D4).

13. It is further noted that, for carrying out the claimed invention, it is actually not even necessary to know the endogenous sequence of the b3434 promoter since a replacement of this sequence is also an "alteration" in the sense of claim 1. The patent-in-suit refers to the use of a strong promoter to express the b3434 or yhgN gene (cf. page 5, paragraph [0029] of the patent-in-suit). Indeed, Example 1 describes the construction of plasmid pYHGN carrying the b3434 gene under the control of the lactose promoter (Plac UV5) and Examples 10 and 11 show the enhanced production of Arg and Pro by E. coli strains transformed with this plasmid. As stated in point 3 supra, no technical problems arise from this type of promoter alteration (replacement) which is well-known in the art (cf. page 6, paragraph [0042] of the patent-in-suit).

14. Thus, the board considers the patent-in-suit according to the claims as granted to fulfil the requirements of Article 83 EPC.

Article 100(a) EPC; Article 54 EPC

15. Document D1, the sole document discussed in the decision under appeal in the context of novelty (cf. page 4, point 12 of the decision under appeal), discloses the complete genome of E. coli K-12. The appellant argues that, by determining this sequence, an E. coli bacterium as defined in the claims would have been "inherently" produced (cf. Section IX supra).

16. The genome of E. coli K-12 disclosed in document D1 is obtained by a combination of three different approaches. The first 1.92 Mb (positions 2,686,777 to 4,639,221 in base pairs) are sequenced from an overlapping set of 15 to 20 kb MG1655 lambda clones, a second segment (2,475,719 to 2,690,160) is sequenced using non-overlapping DNA fragments by a popout plasmid approach, and the largest portion of the genome (22,551 to 2,497,976) is sequenced from M13 Janus shotguns with fragments of about 250 kb. The M13 Janus shotgun strategy involves an initial random sequencing at a four to fivefold redundancy and it is the most efficient strategy (cf. page 1453, last paragraph middle column and right-hand column of document D1). There is, however, no information either in document D1 or in any other document on file on the precise location of the b3434 gene within the E. coli K-12 genome and thus, it cannot be excluded with certainty that the b3434 gene is located within the second segment of the E. coli genome in which non-overlapping fragments were used.

17. Similarly, with the evidence at hand, it cannot be said with certainty that the complete, full-length sequence of the b3434 gene is inevitably present in at least one of the clones or subclones resulting from the three cloning strategies used in document D1. There is no evidence on file of the presence of a clone containing and identifying the specific sequence of the b3434 gene (SEQ ID NO:15). There is also no evidence on file that any of the clones or subclones obtained by the strategies used in document D1 results in an E. coli bacterium having an enhanced production of L-amino acids.

18. Moreover, according to the established case law (cf. inter alia T 18/09 21 October 2009, points 10 to 15 of the Reasons), the presence of a cDNA sequence - without any further (indexed) information or identification - in a clone collection, a sequence databank or, as in the present case, in a complete genome sequence of about 4.64 Mb, does not "make available" this cDNA sequence directly and unambiguously to the skilled person. This is in line with the difference made in the case law between an "implicit" and an "inherent" disclosure as laid down in the decision G 2/88 (supra).

19. Thus, the board considers the claimed subject-matter to fulfil the requirements of Article 54 EPC.

Article 100(a) EPC; Article 56 EPC

20. In the decision under appeal, the document WO 97/23597 cited in paragraph [0004] of the patent-in-suit is identified as the closest prior art, even though this document was not formally introduced into the opposition proceedings (cf. page 5, point 13.1 and page 6, point 13.3 of the decision under appeal). In paragraph [0004] of the patent-in-suit, it is stated that "... increased L-amino acid excretion can enhance the productivity of strain producing L-amino acid. Strain of bacterium belonging to the genus Corynebacterium having increased expression of L-lysine excretion gene (lysE gene) is disclosed (WO 9723597A2)". In the same paragraph, reference is made to other documents stating that "... several Escherichia coli genes coding for putative membrane proteins enhancing L-amino acid production are disclosed", such as the rhtB gene (EP 0 994 190), the rhtC gene (EP 1 013 765), yahN, yeaS, yfiK and yggA genes (EP 1 016 710). Although none of these documents was introduced into the opposition proceedings, in view of the strains used (E. coli), the nature of the isolated genes and the function of the putative membrane proteins (enhancement of L-amino acid production), the board considers the disclosure in these documents to be a whole body of prior art that may represent the closest prior art for the patent-in-suit.

21. Starting from this prior art, the technical problem to be solved is defined as the provision of further E. coli genes encoding membrane proteins that enhance the production of L-amino acids in a transformed E. coli bacterium. Contrary to the appellant's view, the board considers that this technical problem is solved by the claimed subject-matter over the whole scope of the claims without requiring undue burden or the exercise of inventive skills.

22. As in the discussion under Article 83 EPC, the appellant argues that it is not credible that all proteins defined in part (H) of claim 1 and all alterations of the b3434 promoter sequence result in an enhanced production of L-amino acids (cf. Section IX supra). However, claim 1 clearly requires, in an explicit manner, the altered promoters and the proteins defined in part (H) of claim 1 to enhance the L-amino acid production of the transformed E. coli bacterium. In other words, proteins and altered promoters that do not enhance the production of L-amino acids are simply excluded from the scope of the claims. Claim 1 covers solely subject-matter that actually solves the technical problem. The board considers that the achievement of a technical effect stated in a product claim, which effect is a functional feature of the claimed product, is a requirement of Article 83 EPC and not of Article 56 EPC. In view of the conclusion arrived at by the board on Article 83 EPC (cf. points 2 to 14 supra), the board considers the technical problem to be solved by the claimed subject-matter and, indeed, over the whole scope of the claims.

23. Moreover, in view of the prior art on file, the board also considers that the claimed subject-matter is not obvious. Whereas computer-assisted methods for the analysis of the complete E. coli genome and the identification of putative membrane (exporter) proteins were available to the skilled person, the results obtained by these methods could vary significantly depending on the combination of computer programs, parameters and evaluation criteria used. Moreover, as stated in the patent-in-suit (cf. page 2, paragraph [0006], lines 42 and 43 of the patent-in-suit), a further screening was required in order to select those genes which enhanced the production of L-amino acids among all identified genes encoding putative membrane proteins.

24. Although these computer-assisted and screening methods are similar to those methods discussed in the context of Article 83 EPC for the identification of the endogenous b3434 promoter sequence and they were known in the prior art and did not involve any real technical difficulties for a skilled person (cf. point 12 supra), the two situations are not comparable. Whereas for the identification of the b3434 promoter sequence, a skilled person had a starting point in the specific sequence SEQ ID NO:15 disclosed in the patent-in-suit and thus, a clear hint where to look for the promoter sequence (within the 5' upstream region of the b3434 gene), the skilled person had no such starting point in the body of prior art documents identified above that could guide his/her efforts to achieve the specific sequence SEQ ID NO:15 among all other possible sequences in an obvious manner.

25. Thus, the board considers the claimed subject-matter to fulfil the requirements of Article 56 EPC.