T 1424/18 (Recombinant production of isoprenoids/AMYRIS) 05-09-2022

Main request (claims 1 to 10)

Rule 80 EPC and Article 84 EPC

1. The respondent contended that the amendment "reduced feed" of a carbon source, introduced into claim 1 only intended to restate that the carbon source was maintained at a suboptimal level. This amendment was not occasioned by a ground of opposition as required by Rule 80 EPC, as it materially did not change the scope of granted claim 1.

1.1 If the "reduced feed" introduced a true limitation, in that it defined something else or how maintenance of the carbon source level was effected, then the amendment did not determine with regard to which carbon source reference value it had to be reduced. Thus, the amendment "reduced feed" was either not occasioned by a ground of opposition and contravened Rule 80 EPC or it led to a clarity problem. Either way, the amendment was inadmissible.

2. In the decision under appeal, the introduction of "reduced feed" into claim 1 was considered to be a genuine attempt to overcome a ground of opposition, which was decisive for complying with the requirements of Rule 80 EPC (see item 15.3 of the decision). The amendment clarified and limited the feature "suboptimal" of the subsequent "wherein" clause. The definition of the term "suboptimal" equally applied to the term "reduced feed" and was consistent with paragraph [0194] of the patent application (see item 20.3 of the decision).

3. The board considers that the term "reduced feed" introduced into claim 1 limits and defines the "suboptimal" carbon source level feature against which an objection under Article 83 EPC had been raised (see notice of opposition, item 4.3). For this reason alone, its introduction complies with Rule 80 EPC.

3.1.1 This amendment specifies that the "suboptimal" condition is achieved by applying a "reduced feed" and restricts this condition by excluding excess feed. The amendment therefore also complies with the requirements of conciseness under Article 84 EPC.

3.1.2 The "reduced feed" of a carbon source is defined and limited by the subsequent "wherein" clause which stipulates that the carbon source is maintained at a level that is suboptimal and provides for less than 90% of the maximum specific growth rate as compared to a carbon source level allowing a maximal specific growth rate. Thus, any feed of carbon source relative to the carbon feed which would provide for the maximum specific growth rate is "reduced". Claim 1 is clear.

Article 123(2) EPC

4. The respondent argued that the term "reduced feed" introduced into claim 1 step (b) had no basis in the patent application.

There was no basis in the patent application for a "reduced feed" characterizing the "carbon source" in claim 1 (b) which related to a "level" of the "carbon source" in the subsequent clause of claim 1(b).

The "reduced feed of carbon source" according to claim 1 was only required to improve the amount of isoprenoid produced, but was not mentioned to be reduced "relative to the carbon feed which would provide for the maximum specific growth rate" (see paragraph [0194] of the patent application).

Even if "lowering the carbon source feed rate to a microorganism can improve the amount of isoprenoid produced in the fermentation", said rate was omitted in claim 1(b). The "reduced feed" was in consequence generalized to encompass any carbon source concentration below a certain threshold (see paragraphs [00193] to [00195] of the patent application). The patent application provided no basis for such a method step.

5. The board considers that the patent application relates to a method for producing an improved amount of isoprenoids via optimal redirection of microbial metabolism (see paragraph [0006]). This is achieved by culturing the host cells in a medium, the culturing comprising maintaining the carbon source at a level that is suboptimal and provides for less than 90% of the maximum specific growth rate (see paragraphs [0010],[0186] and [0194] of the patent application and claim 20). The patent application mentions that "[t]he host cells are cultured in a fermentation medium comprises [sic] a carbon source present in an amount that is lower than that which would provide for a maximum specific growth rate" (see patent application paragraph [0186]). It is the amount of carbon source in the medium that has to be lower than the one providing for a maximum specific growth rate not the carbon source feed rate. This view is confirmed in the patent application, which uses the term "reduced feed" and not "reduced feed rate" (see paragraph [00194], first sentence).

5.1 Hence, the level of carbon source is equal to the total amount of carbon source added to the medium (feed), whilst the medium in step (b) comprises a reduced feed of carbon source which is further maintained, as introduced by the term "wherein", at a level (i.e. concentration) that is suboptimal and provides for less than 90% of the maximum specific growth rate as compared to a carbon source level that would provide for a maximum specific growth rate for the host cells.

5.2 Since in one aspect of the invention the method of producing an isoprenoid, especially high yield of isoprenoid compounds, involves the steps of culturing the host cells in a medium under conditions that are suboptimal as compared to conditions that would provide for a maximum specific growth rate for the host cells (see page 23, paragraph [00172]), and said culturing the host cells in a medium comprises a reduced feed of a carbon source (i.e. amount) which is explicitly maintained at a level that is suboptimal and provides for less than 90% of the maximum specific growth rate (see paragraphs [00186] and [00194]), the method of claim 1, in particular step (b), complies with the requirements of Article 123(2) EPC.

Article 83 EPC

6. The findings on sufficiency of disclosure in section 21.3 of the decision under appeal were contested by the appellant.

7. The opposition division concluded that establishing the MSGR was not possible without undue burden because the patent was only vague on when and how to measure it. Moreover it considered it implausible that the claimed improvement was achievable across the whole scope of the claim.

8. The respondent argued essentially that

(i) the MSGR could not be reliably determined by the skilled person,

(ii) the technical effect of improved isoprenoid production could not plausibly be achieved over the entire scope of claim 1,

(iii) the skilled person would not be able to reproduce all embodiments falling within the scope of the claim without undue burden, and

(iv) a last method step (c) could not remedy the insufficient disclosure of preceding method steps.

The maximum specific growth rate (MSGR)

9. The respondent contended that (i) the MSGR could not be reliably determined by the skilled person, as several methods of determining MSGR leading to different values were described in the patent.

10. The board considers that the term "maximum specific growth rate" (MSGR) is a standard term known in the art. This term need not be re-interpreted in the light of the description, in a way different from how it would be understood by the person skilled in the art (see e.g. decision T 2221/10, point 33 of the reasons and decision T 197/10, headnote). No general requirement can be derived from Article 69(1) EPC that claims are to be interpreted with the help of the description (see decision T 1646/12, point 2.1), and from the second sentence of Article 69(1) EPC that an unambiguous and generally accepted definition of a term figuring in the claims should be superseded by a different definition found in the description (see decision T 177/08, item 3.3 second paragraph of the reasons).

10.1 Claim 1(b) requires culturing the host cells in a medium comprising

(i) a reduced feed of a carbon source,

(ii) wherein the carbon source is maintained at a level that is suboptimal and provides for less than 90% of the maximum specific growth rate as compared to a carbon source level that would provide for a maximum specific growth rate for the host cells.

10.2 The maximum specific growth rate for cells in a culture medium relates to the effect of substrate concentration on growth rate. The MSGR represents for example the maximal slope obtainable on a cell growth curve on a plot representing the cell biomass concentration or optical density in function of the fermentation time.

10.3 Even if the skilled person turns to the description to understand its technical meaning, it will not come to a different conclusion. A definition for this term is provided in paragraphs [0142] to [0145] of the patent.

Although several time points for determining the MSGR are proposed in paragraph [0145], they are all options introduced by words "often" or "can".

"A theoretical treatment of the relationship between growth rate in culture is well known to those skilled in the art, and is referred to as the Monod equation. [...]. In this theoretical treatment, the maximum specific rate is an asymptotic limit that is never reached until an infinite level of substrate is reached. In practice, however, the maximum specific growth rate can be considered as being obtained when the conditions under investigation (e.g., a substrate level or temperature) support the fastest initial growth rate. For instance, in a fed-batch reactor, the initial condition where the nutrients are supplied in excess is treated as the conditions for the maximum growth rate." (see paragraph [0142], emphasis added).

10.3.1 Despite the expression "fastest initial growth rate", used in the description in association with the term MSGR, the skilled person would not be motivated to re-interpret the term MSGR, having a well-recognised meaning in the art, in the light of the description.

Even if the skilled person turned nonetheless to the description, the term "initial" could not prima facie refer to the "lag phase", a period of time in which cells are adjusting to their environment. The cell growth rate in this phase is close to zero and increases only during a following acceleration phase.

The fastest (superlative) growth rate is commonly observed during the exponential phase, after which cell growth slows down and stops in a deceleration and a saturation phase respectively. The fastest initial growth rate amounts to the cell growth rate observed when the conditions under investigation (e.g., a substrate level or temperature) support the fastest growth rate, e.g. in a fed-batch reactor where the nutrients are supplied in excess. The term "initial" defines the fastest growth rate when the cell metabolic activity has not yet significantly modified the initial culture conditions e.g. during a multi or bi-phasic cell growth.

10.4 Although other methods for determining the MSGR are suggested in the description, at least one method allows to determine the MSGR of host cells when using a culture medium and conditions supporting a maximum specific growth rate for the host cells. Besides, there is no evidence that the other methods for determining the MSGR mentioned in the description lead to different and inconsistent MSGR values, differences which are so significant that the skilled person is deprived of the promise of the invention due to this ambiguity (see decisions T 608/07, item 2.5.2; T 593/09, catchword; T 61/14; T 1811/13, point 4 of the decision).

The MSGR may be obtained from the experimental data of Example 12 and Figure 12 of the patent. How the MSGR is determined and calculated using methods known to the skilled person is elaborated and detailed in declaration A9, section 7, exhibits A and B (A10 and A11), and declaration A17, section 10, exhibit C (A18).

Standard of proof and burden of proof

11. The respondent argued that decision T 63/06 established that while the initial burden of proof generally lay upon an opponent to establish insufficiency of disclosure, when the patent failed to give any information of how a feature of the invention, i.e. MSGR parameter, could be put into practice, only a weak presumption of validity existed that the invention was sufficiently disclosed.

11.1.1 In such a case, an opponent could discharge their burden of proof by plausibly arguing that common general knowledge would not enable the skilled person to put this feature into practice thereby placing the burden of proof on the patent proprietor for any assertion to the contrary.

11.2 Appellant alleged that no serious doubts substantiated by verifiable facts in accordance with T 19/90 were raised to support the contention that the skilled person would not be able to obtain the technical effect of improved isoprenoid production across the entire claimed scope.

11.3 In the board's view, decision T 63/06 stipulates that after the grant of the patent, i.e. after the end of the examination proceedings, a legal presumption exists that the patent meets the requirements of the EPC. The weight of arguments and evidence required to rebut this presumption depends on its strength. When the patent does not give any information of how a feature of the invention can be put into practice, only a weak presumption exists that the invention is sufficiently disclosed (see Reasons 3.3). In such a case, as also argued by the respondent, the opponent can discharge his burden by plausibly arguing that common general knowledge would not enable the skilled person to put this feature into practice.

11.3.1 This is however not the case here. The method for producing isoprenoids in Example 12 and illustrated in Figures 12 A to D of the patent provides evidence that the invention can be put into practice (see in particular item 10.4 above). Thus, in the absence of verifiable facts, the respondent's comprehensible and plausible arguments are not sufficient to rebut the strong presumption of validity of the patent established by its Example 12.

Technical effect recited in claim not plausibly achieved

12. The opposition division in the decision under appeal and the respondent argued that a technical effect, such as the improved production of isoprenoid, had to be plausibly achievable over the entire ambit of claim 1, i.e. culturing with carbon source concentrations that provide for any growth rate from 0% to just below 90% of the MSGR (see decision G 1/03 point 2.5.2 of the reasons). The carbon source concentration in culture medium should neither be too high nor too low.

12.1 The respondent contended that an excess of carbon source during fermentation generated poisonous metabolites which prevented an improved production of isoprenoid. The addition of a feed for maintaining a carbon source level providing for 0% of the MSGR included cell cultures that did not proliferate and were close to death. Even if Example 12 of the patent maintained a carbon source level providing for 45% of the MSGR, it provided no examples showing whether upper or lower carbon source concentrations were still capable of enhancing isoprenoid production. Besides, the wording of claim 1 (b) required that the reduced feed of a carbon source was maintained, suggesting that the carbon source concentration in the medium of the method of the invention had to be lower than the carbon source concentration in the MSGR reference culture at all times.

12.2 The board considers that if a technical effect is recited in the claim an alleged lack of reproducibility of the claimed invention needs to be assessed under sufficiency of disclosure (see decision G 01/03, OJ 2004, 413, item 2.5.2 of the reasons). Since the aim of the method of claim 1 is to improve the amount of isoprenoid produced, which is allegedly not reproducible over the whole ambit of the claim, a lack of sufficiency of disclosure may arise. The allegedly non-working embodiments of the method of claim 1 include the culturing of host cells in a medium comprising a reduced feed of a carbon source [...] wherein the carbon source is maintained at a level that is suboptimal and provides for either 0% or just below 90% of the maximum specific growth rate as compared to a carbon source level that would provide for a maximum specific growth rate for the host cells.

12.2.1 The cell growth curve of the fermentation run "050608-1" in Example 12 of the patent, comprising an excess carbon source, is the only fermentation where the cell growth rate may reach a maximum (MSGR) during its exponential phase, as its carbon source is not restricted. Figures 12 A and B of the patent provide evidence that culturing host cells under carbon-restricted conditions, "050629-1", provides for less than 90% of the maximum specific growth rate as compared to a carbon source that would provide for the maximum specific growth rate "050608-1", while Figure 12B of the patent demonstrates that isoprenoid production is higher under the carbon restricted condition "050629-1" compared to the excess carbon condition "050608-1".

There is no evidence that culturing host cells in a medium comprising a carbon source which is maintained at a level that provides for a cell specific growth rate just below 90% of the MSGR will not improve the production of isoprenoid compared to conditions providing for a MSGR for the host cells. Nor is there any evidence that isoprenoids cannot be produced and improved during the stationary phase of cell growth when the specific cell growth amounts to 0% of the maximum specific growth rate (see "050608-1" and "050629-1"; time span from 50 to 70 hours in Figures 12 A and B of the patent). Thus, an improvement in isoprenoid production may still be seen at 0% of the maximum specific growth rate.

12.3 The board considers that claim 1 step (b) requires the step of culturing the host cells in a medium comprising a reduced feed of a carbon source, i.e. a reduced amount of a carbon source in [g], and wherein the carbon source is maintained at a level [g/l] that is suboptimal and provides for less than 90% of the MSGR as compared to a carbon source level that would provide for a maximum specific growth rate for the host cells, which level must be maintained at all times during the cell culture step, regardless of the culture phase.

Even if the carbon source minimum threshold of 5 g/l glucose in "050608-1" is exceeded in "050629-1", which may result in a glucose concentration of over 15 g/l, this is not excluded from the wording of claim 1 as the method only requires that the carbon source level in the medium is maintained at a level at which the host cell specific growth remains below the 90% of the MSGR, while the reduced feed in [g], and not its level [g/l], must be compared to the feed added in a medium which provides for a MSGR at any corresponding time during the fermentation process.

12.4 The respondent contended that the non-working embodiments introduced by steps a) and b) constituted separate limitations of the method according to claim 1, each of which had to be sufficiently disclosed. They were essential to the sufficiency of disclosure and could not be overcome by a subsequent terminal step (c). The facts underlying decision T 292/85 did not align with the present situation. Thus, the standard for sufficiency of T 292/85 was not applicable.

12.5 The board agrees with the decision under appeal, item 21.3, second paragraph, that step c) cannot remedy a lack of insufficiency arising against an independent step of the method claimed, as the disclosure must be sufficient to put into practice essentially all parts of a claim without undue burden.

12.6 However, as stated above, the board considers that the skilled person can perform step b) without undue burden. Thus, the board concludes that the requirements of Article 83 EPC are met. The same conclusion applies to claims 2 to 10 dependent thereon.

Priority (Article 87 EPC)

13. In its communication sent in preparation of oral proceedings, the opposition division considered that the auxiliary request, corresponding to the present main request, could not validly claim priority rights from the earliest priority document US 60/808,989 because it did not disclose step (b) of claim 1.

13.1 The finding on priority in the board's communication sent to the parties during appeal proceedings was that the subject-matter of claim 1 does not enjoy priority rights from the earliest claimed priority application for the same reason found by the opposition division (see item 14). This was not contested. Hence, the board sees no reason to deviate from this finding.

Novelty (Article 54 EPC)

14. As a priority right cannot be validly claimed from the first priority application, documents A2 and A4 published before 18 December 2006 represent prior art under Article 54(2) EPC.

14.1 The decision under appeal did not have to address the objections of lack of novelty raised in opposition.

14.2 Respondent did nonetheless not share the provisional finding of the opposition division and of the appellant that the features in the method of claim 1 of "... culturing the host cells in a medium comprising a reduced feed of a carbon source to improve the amount of isoprenoid produced, ...", resulted in a higher production of the isoprenoid for the cell culture showing a lower growth rate/OD-value and vice versa, were neither disclosed in documents A2 and A3.

Document A2 disclosed that cell cultures with a higher OD-value showed a higher production of isoprenoid, whereas the culture with a lower OD-value showed a lower production of isoprenoid (see Figure 3), whereas document A3 disclosed OD-value of E. coli DH10B harboring either the pBBR1MCS-3 (empty plasmid control), pMKPMK, pMevB, pMBI or pMBIS plasmids expressing the various mevalonate operons described in Figure 1 in media supplemented with increasing concentrations of exogenous mevalonate.

14.2.1 Claim 1(b) related to a step of culturing cell under conditions where the carbon source was restricted so that cells did not grow at maximum speed to allow an improved isoprenoid production. Depletion of carbon source during fermentation was a common phenomenon. The cells grown in batch-fed system were known to deplete the nutrients of the fermentation medium, including the carbon source, resulting in a reduction in cell growth rate. In view of this fact, any and all cell culturing systems that utilize a batch fed system for culturing a host cell that comprises the MEV pathway as claimed would fall within the scope of claim 1.

14.3 Documents A2 to A4 relate to the production of Amorphadiene (AMD), an isoprenoid, in E. coli host cells. The bacterial host cells comprised a mevalonate (MEV) pathway for making isopentenyl pyrophosphate (IPP), wherein all of the pathway enzymes were under the control of at least one heterologous transcriptional regulator.

14.3.1 In document A2, the host cells were cultured in TB medium, or in TB medium supplemented with glycerol (TBG) once the cells entered the stationary phase. The TBG medium comprised 1% glycerol (a carbon source).

14.3.2 In document A3 genetically engineered E. coli comprising plasmids pMPKPMX, pMevB, pMBI, or pMBIS were cultured for production of amorphadiene which was recovered (see Figures 1 and 3). The E. coli were cultured in medium supplemented with increasing amounts of mevalonate (a carbon source).

14.3.3 In document A4, the bacteria were cultured for AMD production which was then recovered in an organic layer (see Fig. 2). They contained pMevT, pMBIS and pADS (see Fig. 1) and were grown in C medium containing 3.4% glycerol (a carbon source). When the cell culture had an OD600 of about 0.25, IPTG and varying amounts of mevalonate were added. Fig.2 shows concentrations of AMD produced by the culture cells over time.

15. In the board's view, a host cell which is cultured without addition of fresh media or nutrients (i.e. feed) cannot fall within the scope of the claims, as no feed is added. A feed cannot be reduced if it is absent. For this reason alone document A2 cannot anticipate the method of claim 1.

15.1 Even if the MSGR can be calculated based on the growth curve shown in Figure 3 of document A2, resulting in a MSGR on the cell growth curve (o) and (white square) in low carbon and high carbon medium of 0.217 [s**(-1)] and 0,385 [s**(-1)] respectively, the cell culture in low carbon did not receive any feed. The specific cell growth rate during the exponential growth phase of the cell culture in low carbon was 100% (MSGR) and thus was not maintained at a level of less than 90% of the MSGR. In spite of the relative ratio of the growth rates between the carbon medium conditions of 0.2171/0.3856 = 56%, demonstrating that the low carbon source in the culture was maintained at a level less than 90% of the MSGR, no feed was added during the whole cell culture carried out in low carbon source. Under these circumstances, it is of no relevance to determine whether the production of amorphadiene (AMD) is improved or not.

15.2 As the MSGR and maintenance of a carbon source at a level providing for a specific cell growth less than 90% of the MSGR value is not deemed to be an unusual parameter, the burden of proof cannot be reversed (see decisions T 388/15 and T 1406/14 and item 11.3 above).

15.3 Documents A3 and A4 disclose batch culture methods. For this reason alone, respondent's novelty objection cannot succeed.

15.3.1 In addition, in document A4, Figure 2 provides no information with respect to the growth rate of the single culture with cells comprising all of the MEV pathway enzymes, let alone the MSGR determined under unrestricted carbon source. The growth curve shown in Figure 3 was obtained with cells not comprising a complete mevalonate pathway (i.e. pMevT, pMBIS and pADS) and in consequence cannot be mapped onto the different culture conditions of Figure 2.

15.4 Hence, in the board's view, there is no convincing evidence, going beyond reasonable doubt, that the methods disclosed in documents A2 to A4 include all the method steps of claim 1. Thus, none of the documents A2 to A4 is considered to anticipate the subject-matter of claim 1. The main request fulfils the requirements of Article 54 EPC.

Inventive step (Article 56 EPC)

16. It is common ground between the parties that document A2 represents the closest prior art for claim 1.

16.1 Document A2 relates to the production of Amorpha-4,11-diene (AMD), an isoprenoid, in E. coli host cells. The bacterial host cells comprised a mevalonate (MEV) pathway, wherein all of the pathway enzymes were under the control of at least one heterologous transcriptional regulator (see Table I, page 685, col.2, second paragraph). The host cells were cultured in TB medium, or in TB medium supplemented with glycerol (TBG) once the cells entered the stationary phase (see page 688, col.1, Figure 3).

16.2 The method step (b) of claim 1, culturing the host cells in a medium comprising a reduced feed of a carbon source, is the distinguishing feature resulting in an improved production of isoprenoids.

16.3 Based on the experimental data provided in Example 12 of the patent and the arguments submitted under Article 83 EPC above, the technical effect is considered to be achieved.

16.4 Starting from document A2, the technical problem underlying the method of claim 1 may be formulated as the provision of a method for improved isoprenoid production.

Obviousness

16.5 The question that remains to be answered is whether the skilled person starting from the teaching of document A2 and faced with the technical problem formulated above was motivated to solve the technical problem by modifying the culture conditions of the host cell as proposed by claim 1.

16.5.1 Document A2 mentions that further optimizing bioreactor media and conditions to use a medium with balanced nutrients and optimal carbon delivery was planned. "A future, detailed study of medium development should further increase product concentrations" (document A2, page 690, right column, middle of the page).

16.5.2 Even if the skilled person reading these lines may have been motivated to optimize the composition of the medium for improving the isoprenoid production yield, there is no indication whatsoever how this optimization may be achieved.

16.5.3 It is true that the skilled person knew that the volumetric yield of recombinant products could be improved by controlling the specific growth rate and the substrate concentration while the formation of inhibitory by-products could be minimized in fed-batch cultures (see document A13, abstract; document A13, page 1550, col.2, last paragraph; page 1551, col.2 last full paragraph; document A22, page 1004, col.1). In yeast production, ethanol could be produced from excess glucose even if the dissolved oxygen concentration was sufficient. This phenomenon was known as "Crabtree effect" and arises in E. coli as well (see document A13, page 1550, col.2 2nd full paragraph; document A16, abstract). It consists of the repression of the consumption of an energy source (respiration) by another energy source (fermentation) and was explained by the repression of the respiratory enzymes synthesis in exponentially growing yeast or bacteria at high glucose concentrations (see documents A16 and A22). A straightforward approach to prevent acetate formation was to force glucose limited cells to grow below the threshold specific growth rate. Cells could be constrained in this way merely by restricting the supply of glucose; therefore, a fed-batch process is superior to a batch process for protein production (see document A26).

16.5.4 There is no teaching in document A4 using a batch culture fermentation - not a fed-batch fermentation - that the Crabtree effect was actually a problem in the production of isoprenoids - not of proteins - and that any potentially supplied feed medium is important and must be controlled.

16.5.5 Although the Crabtree effect was known in the art (see documents A13, A15, A16, A22 and A26) to have an impact on the production of recombinant proteins or enzymes, on the Baker's yeast's fermentative capacity (i.e. ethanol production), on E. coli's growth rate and biomass yield, none of these documents teaches or suggests that the Crabtree effect is or has a limiting and/or detrimental effect on the host cells defined in claim 1 for the production of isoprenoids.

16.5.6 Even if document A13 discloses that the Crabtree effect affects the production of proteins and that the host cells in claim 1 express recombinant enzymes, document A2 fails to teach or suggest that the expression of one or more of the heterologous enzymes of the mevalonate pathway was compromised and had to be increased in order to achieve an improved production of isoprenoids. There is even less of an indication as to why a suboptimal feed of carbon source would have found its way only partially to the production of proteins while isoprenoid production was improved.

16.6 Starting from the closest prior art document A2, there is equally no indication and motivation why the skilled person would turn to document A1 to provide for a method for improved production of isoprenoid. Indeed, document A1 relates to a method for cultivating yeast or bacteria in a culture medium, wherein the carbon source concentration in the culture medium is maintained at a constant low level. The compound to be produced was L-lysine and other amino acids, such as glutamic acid (see Example 1).

16.6.1 The board finds no evidence in documents A2 or A1 that the production of isoprenoids in host cells should be identical or similar to the production of glutamic acid or other amino acids. There is no evidence either why the method for producing isoprenoids using host cells in document A2 should be improved, if the skilled person followed the teaching of documents A3 or A4, using a batch fermentation process - lacking any feed of carbon - in which the basal medium is supplemented with mevalonate, to balance the flow of carbon through the mevalonate pathway, leading to improved growth and production of isoprenoids in E. coli (see document A4, bridging sentence col 1 and 2 on page 194).

16.6.2 Although the skilled person knew that the balancing of nutrients was important when recombinant host cells were fermented using a fed-batch method for producing isoprenoids and that document A5 relates to fed-batch techniques in microbial processes, there is nevertheless no indication why the skilled person should turn to document A5 if the method for producing isoprenoids has to be improved. Document A5 does neither address the production of isoprenoids using recombinant host cells nor does it provide any indication that isoprenoid production could be improved by decreasing the feed of carbon source with a reasonable expectation of success.

16.6.3 Finally, there is no apparent motivation for the skilled person to combine the teaching of document A2 with that of A6, which concerns the cultivation of algae -which are neither bacteria nor fungi- and which do not comprise one or more heterologous enzymes of the mevalonate pathway under the control of at least one heterologous transcriptional regulator.

16.7 Thus, the subject-matter of claim 1 satisfies the requirements of Article 56 EPC. This applies, mutatis mutandis, also to the subject-matter of dependent claims 2 to 10.

17. The board concludes that the claims according to the present main request and the invention to which they relate, and the description adapted thereto, which had not been objected to, comply with the requirements of the EPC.