Field of the invention

The present invention relates to an industrial process for the production of a valuable compound. This process allows high production levels of valuable compounds, such as primary or secondary metabolites, pharmaceutical proteins or peptides, or industrial enzymes, in an economically attractive yield.

Background of the invention

Many valuable compounds are manufactured by fermentative production in large, industrial scale fermentors, i.e. the microorganism which produces the valuable compound of interest is grown under controlled conditions in a fermentor of >10 m ³ . In current industrial scale fermentation processes, the carbon-source (C-source), which is usually also the energy source, is typically selected on the basis of the growth rate of the microorganism, such that a C-source that yields a high biomass-specific growth rate is preferred. A very common C-source is glucose.

In addition to the growth rate, the C-source may also influence the production rate of the valuable compound. At low concentration of the C-source, the production rate depends on the concentration of the C-source, such that an increase of C-source concentration leads to an increase in the production rate. However, depending on the specific combination of microorganism / valuable compound, above a critical concentration the C-source often results in a reduced production rate. This phenomenon is called "repression". A repressing C-source is as defined herein below. It is well known that glucose is a repressing sugar for production of metabolites by many microorganisms under various conditions. For instance Change et al. (J. Industrial Microbiol. (1990), 6, 165-169) describe that excess glucose results in lower penicillin V production in fermentation processes with wild type

Penicillium chrysogenum strains. The fact that glucose is a less favourable carbon source for penicillin production in batch cultures than, for example, lactose, was already discovered in the first years of penicillin production process development (e.g. Moyer and Coghill in J. Bacteriol. (1946), 51 , 57-78).

Later investigations showed that these early observations could be explained by the fact that above a certain threshold concentration, glucose represses the formation of penicillin-synthesizing enzymes in the cell and that this repression does not occur with other C-sources including lactose (e.g. Revilla et al. in J. Antibiotics (1984), 37, 781-789). Such a C-source is therefore designated as a non-repressing C-source as further defined herein below. There are various ways to avoid or circumvent the repressing effect of repressing C-sources in general or glucose in particular. One way is to isolate mutants of the microorganism which are less sensitive to repression. Chang et al. (supre vida) isolated glucose-derepressed mutants of Penicillium chrysogenum which at the same glucose concentration in the fermentation medium gave higher Pen-V titers compared to the wild type strain.

Another way is that to keep repressing C-sources below the critical concentration during the course of the fermentation. This may be achieved by feeding the C-source to the fermentor under such conditions that the concentration of the C-source during fermentation remains below values where the repressing effect occurs. Robin et al. used a glucose feed under limiting conditions for the production of adipyl-7-ADCA by a Penicillium chrysogenum strain transformed with the gene encoding the expandase gene (Metabolic Engineering (2003), 5, 42048 and Biotechnology and Bioengineering

(2001 ), 83, 361-368. However, a disadvantage of a low concentration of C-source is that the biomass-specific growth rate (as defined herein below) of the microorganism is limited by the availability of C-source, resulting in slow formation of biomass and hence a long fermentation time to reach the desired level of valuable compound. Another disadvantage of using a low concentration of C-source is that the biomass-specific production rate (as defined herein below) by the microorganism is also low, again resulting in a low production rate.

A third way is to completely avoid the use of the repressing C-sources which are known to be repressing in fermentation processes using certain microorganisms for the production of selected valuable compounds but instead to use a C-source which is known to be non-repressing. Hence, it would be advantageous to use such a non-repressing C- source instead of a repressing C-source, since non-repressing C-sources have no negative effect on the production rate and can advantageously be used at high concentrations from the onset of the fermentation without the need to limit the concentration.

A problem associated with using high levels of C-source at the start of the fermentation, however, is that the resulting high growth rate often results in an undesired, highly viscous fermentation broth. Viscosity is generally determined by two factors, namely by the amount (concentration) of the biomass in the fermentation broth, and by the morphology of the microorganism (called "biomass-specific viscosity"). In industrial fermentation processes where the growth rate is high, filamentous microorganisms, like filamentous bacteria such as Actinomycetes or filamentous fungi such as Penicillium or Aspergillus, typically have a dispersed mycelium with very long and branched hyphae which leads to undesirably high biomass-specific broth viscosity. Hence, even at the start of the fermentation when there is little biomass contributing to the viscosity, but the growth rate is typically high, the biomass-specific viscosity can build up rapidly, leading to poor oxygen transport in the broth. A low (biomass-specific) viscosity of the fermentation broth is therefore advantageous for the production rate.

Of course, one could limit the concentration of the non-repressing C-source in the fermentation medium in order to limit the growth rate and thereby the biomass- specific viscosity, but this scenario is not essentially different from the scenario described herein above, whereby a repressing C-source is fed at low concentration, and which has the same disadvantage, namely a reduced growth rate and consequently a reduced production rate.

An alternative method is to limit the growth rate by the supply of another nutrient than the C-source. For example, Oh et al (Biotechnology and Bioengineering (1988), 32, 569-573) demonstrated that phosphate limitation in a batch fermentation of Penicillium chrysogenum in the presence of a high concentration of a non-repressing sugar (i.e. 3% lactose) results in a 2-fold increase in the biomass-specific production rate of penicillin. However, a great disadvantage of this batch fermentation system is that the volumetric production rate of the phosphate limited fermentation is much lower compared to the control (see Figure 1 in Oh et al.). This may be caused by the fact that the reduction of the amount of active biomass formed in the fermentor caused by the phosphate limitation is stronger than the increase of the biomass-specific production rate of penicillin caused by the same limitation. Oh et al. suggest to apply phosphate limitation in a semi-continuous fermentation system, where the problem of a low amount of active biomass is technically solved by retaining the biomass in the fermentor, by growing biomass as mycelial bioparticles and only harvesting bioparticles-free broth.

Surprisingly, we have found that in a fermentation system without biomass retention, feeding of at least one nutrient at growth rate limiting conditions makes it possible to use high concentrations of a non-repressing C-source and to limit the growth rate and hence the biomass-specific viscosity and increase the biomass-specific production rate resulting in a high volumetric production rate of valuable compounds, such as primary or secondary metabolites, pharmaceutical proteins or peptides, or industrial enzymes.

Detailed description of the invention Definitions

"Biomass-specific production rate", also referred to as "production rate", is defined herein as the amount of valuable compound (based on dry matter) produced per amount of biomass (based on dry matter) per amount of time (in reciprocal time)

"Volumetric production rate" is the amount of valuable compound (based on dry matter) produced per bioreactor volume (in reciprocal cubic meters) per amount of time (in reciprocal time).

"Biomass specific growth rate", also referred to as "growth rate", is defined herein as the amount of biomass (based on dry matter) produced per amount of biomass (based on dry matter) per amount of time (in reciprocal time). "Growth rate limiting nutrient" is defined herein as the nutrient which is the dominant growth rate governing factor.

A "repressing C-source" is a C-source which, at increasing concentration in the fermentor, results in an initial increase of the biomass-specific production rate, but which, above a critical concentration of the C-source, results in a decrease of the biomass-specific production rate.

A "non-repressing C-source" is a C-source which, at increasing concentration, results in an increase of the biomass-specific production rate, until, above a certain concentration, the biomass-specific production rate reaches a plateau value. A non- repressing C-source therefore does not, above a critical concentration, result in a decrease of the biomass-specific production rate. Non-repressing C-sources may therefore advantageously be used in fermentation processes at high concentrations.

In a first aspect, the present invention provides a process for the production of a valuable compound, comprising fermentation of a microbial strain on an industrial scale in a medium comprising a non-repressing C-source and feeding at least one growth rate limiting nutrient, whereby the production of penicillin-G by semi-continuous fermentation of Penicillium chrysogenum with biomass retention is excluded. Preferably, the process for the production of a valuable compound is subject to repression by a repressing C-source as defined.

The non-repressing C-source may be any assimilable C-source which is non- repressing for the microorganism producing said valuable compound. The non-repressing C-source may be present at high concentrations during any time point in the fermentation process. The total amount of non-repressing C-source required for the entire fermentation process may be added at the beginning of the fermentation process, resulting in high concentrations which, in case a repressing C-source would be used, would result in significant repressing conditions. The non-repressing C-source may also be added at certain time intervals during the fermentation or as a continuous feed. The skilled person is very well capable of designing the fermentation process for a given microorganism and valuable compound in order to get optimal results.

In one embodiment, the non-repressing C-source may be selected from the group consisting of carbohydrates, polyols, oils and triglycerides, alcohols, organic acids, amino acids and proteins. Carbohydrates may include for example monosaccharides such as glucose, fructose and galactose; disaccharides such as sucrose, lactose and maltose; polysaccharides such as starch, dextrin, maltodextrin, inulin, and cellulose. Polyols may include for example glycerol, sorbitol, and mannitol. Oils may include for example soybean oil and rape seed oil. Alcohols may include for example methanol, ethanol, propanol and higher alcohols. Organic acids may include for example formic acid, acetic acid, propionic acid, citric acid and benzoic acid. Proteins may include peptides of any size, for example dipeptides, tripeptides, oligopeptides and polypeptides, including (partially) hydrolyzed proteins (so-called protein hydrolysates).

The growth rate limiting nutrient may be any nutrient provided it is required for growth and may be fed at growth rate limiting conditions. Preferably, the growth rate limiting nutrient is not the C-source. In one embodiment, the growth rate limiting nutrient is selected from the group consisting of a phosphorous source, a nitrogen source, a sulphur source, an oxygen source, one or more vitamins and one or more trace elements. Preferably the growth rate limiting nutrient is the phosphorus source or preferably the growth rate limiting nutrient is the nitrogen source or preferably the growth rate limiting nutrient is the sulphur source. Most preferably, the growth rate limiting nutrient is the phosphorus source.

A suitable phosphorous source that may be added as the growth rate limiting nutrient is well known in the art and may for example be phosphoric acid or a phosphate- containing salt such as ortho-phosphate, hydrogen phosphate, dihydrogen phosphate and/or pyrophosphate. The skilled person is very well capable of selecting the appropriate phosphorus source.

A suitable nitrogen source that may be added as the growth rate limiting nutrient is well known in the art and may for example be urea, ammonia, nitrate, and/or ammonium salts such as ammonium sulphate, ammonium phosphate and/or ammonium nitrate, and amino acids such as glutamate and/or lysine. The skilled person is very well capable of selecting the appropriate nitrogen source.

A suitable sulphur source that may be added as the growth rate limiting nutrient is well known in the art and may for example be sulphuric acid, sulphate and/or thiosulphate. The skilled person is very well capable of selecting the appropriate sulphur source.

In a preferred embodiment, the growth rate limiting nutrient is not the exclusive source of elements (i.e. atoms) which are incorporated into the valuable compound. In this case the growth rate and the production rate are uncoupled. This is advantageous because feeding such nutrient results in limitation of the growth rate (and hence of biomass-specific viscosity) but not of the production rate. The skilled man knows how to select nutrients that are not the exclusive source of elements composing the valuable compound. For example, if the valuable compound contains (in addition to O and H) N, C, and S, the growth rate limiting nutrient in this embodiment should not be the only source of N, C, and S. An example is if such a valuable compound is a β-lactam compound such as a penicillin or a cephalosporin. These compounds contain C, H, N and S-atoms. Therefore, in the fermentation process of the invention, in which the valuable compound is a β-lactam compound, the growth rate limiting nutrient is preferable selected from the group of nutrients that are not the exclusive source of the elements C, H, N and S. A preferred embodiment in this case would be to use a suitable phosphorous source as the growth rate limiting nutrient.

It has been surprisingly found that the production rate in the process of the invention is higher than the production rate in the same process but without feeding at least one growth rate limiting nutrient. Feeding at least one growth rate limiting nutrient advantageously provides a tool to limit the growth rate and hence the biomass-specific viscosity.

Throughout the description of the invention, an industrial scale fermentation process or an industrial process may be understood to encompass a fermentation process on a volume scale which is > 10 m ³ , preferably > 25 m ³ , more preferably > 50 m ³ , most preferably > 100 m ³ , preferably less than 5000 m ³ .

The process of the invention may be performed as a fed-batch, a repeated fed- batch, a semi-continuous fed batch or a continuous fermentation process.

In a repeated batch process, a partial harvest of the broth accompanied by a partial supplementation of complete media occurs, optionally repeated several times.

In a fed-batch process, either none or part of the fermentation media compounds are added to the media before the start of the fermentation and either all or the remaining part, respectively, of the compounds is fed during the fermentation process. The compounds which are selected for feeding can be fed together or separate from each other to the fermentation process.

In a semi-continuous fed batch or a continuous fermentation process, the complete starting medium is additionally fed during fermentation. In a repeated fed-batch process, part of the fermentation broth comprising the biomass is removed at regular time intervals, whereas in a continuous process, the removal of part of the fermentation broth occurs continuously. The fermentation process is thereby replenished with a portion of fresh medium corresponding to the amount of withdrawn fermentation broth.

The process of the invention is suitable for the fermentative production of any valuable compound of interest, including primary or secondary metabolites, pharmaceutical proteins or peptides, or industrial enzymes. Primary metabolites are biomolecules that are essential to the growth, development or reproduction, and are shared by many species. Primary metabolites are for example intermediates of the main metabolic pathways such as the glycolytic pathway or the TCA cycle. Examples of primary metabolites are amino acids and nucleic acids.

Secondary metabolites are not essential for growth, development, or reproduction, but instead have an ecological function. Examples of secondary metabolites are antibiotics or β-lactam compounds, especially β-lactam antibiotics. A preferred valuable compound is a β-lactam compound. Examples of β-lactam compounds are clavulanic acid, penicillin (e.g. penicillin-G, penicillin-V or 6-aminopenicillinic acid) and semisynthetic penicillins such as amoxicillin and cephalosporins such as cephalosporin C.

In a highly preferred embodiment the β-lactam compound is an N-acylated derivative of β-lactam intermediates such as 7-ADCA, 7-ACA, 7-ADAC, 7-ACCA, 7-PACA or 7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid. The acyl-group at the 7- amino position is preferably adipic acid yielding the corresponding adipoyl-derivate as disclosed in WO93/05158, WO93/08287 or WO 2004/106347. Alternative suitable side chains have been disclosed in WO95/04148, WO95/04149, WO96/38580, WO98/48034 and WO98/48035

A suitable microbial strain for the process of the invention may be any wild type strain producing a valuable compound of interest. Furthermore, a suitable microbial strain of the invention may be a strain which has been obtained and/or improved by subjecting a parent or wild type strain of interest to a classical mutagenic treatment or to recombinant

DNA transformation. In a preferred embodiment, the microbial strain which is suitable for the process of the invention is a yeast, a fungus, a protozoa or a bacterium. The microbial strain may include filamentous and non-filamentous strains. Preferably, the microbial strain is a filamentous strain, preferably a bacterium or a fungus. A preferred filamentous bacterium is an Actinomycete. Preferably, the Actinomycete is Streptomyces clavurigerus. Which preferably produces clavulanic acid as the valuable compound.

A filamentous fungus is preferably selected from the group consisting of Aspergillus, Trichoderma, Penicillium and Acremonium. Preferred examples are Penicillium chrysogenum for the production of PenG or PenV, Acremonium chrysogenum for the production of cephalosporin C and Aspergilli such as Aspergillus niger or Aspergillus oryzae either as wild type or classically improved strains producing, or genetically modified to overexpress genes encoding, enzymes such as amylases, lipases, phospholipases, galactolipases, hemicellulases, xylanases, cellulases, proteases and other enzymes known to be used in industry.

In a preferred embodiment the fungus is Penicillium chrysogenum and the secondary metabolites are adipoyl-7-ADCA, adipoyl-7-ADCA, adipoyl-7-ACA, adipoyl-7- ADAC, adipoyl-7-ACCA, V7-PACA or adipoyl^-amino-S-carbamoyloxymethyl-S-cephem^- carboxylic acid, most preferred is adipoyl-7-ADCA. As disclosed in WO93/05158, the

Penicillium chrysogenum strain is transformed with and expressing a gene encoding an expandase. This engineered Penicillium chrysogenum strain, when grown in the presence of adipic acid as the side chain precursor in the fermentation vessel, produces and excretes adipoyl-7-ADCA.

Figures legends

Figure 1. Phosphate concentration during fermentation. Solid line, fermentation A; bold solid line, fermentation B; dashed line, fermentation C. Fermentation A and

C, left-hand side scale; Fermentation B, right hand side. Figure 2. Relative stirrer speed. Solid line, fermentation A; bold solid line, fermentation

B; dashed line, fermentation C.

Figure 3. Adipoyl^-aminodeacetoxycephalosporinic acid production. Solid line, fermentation A; bold solid line, fermentation B; dashed line, fermentation C.

Figure 4. Phosphate concentration during fermentation. Solid line, fermentation D; dashed line, fermentation E. Figure 5. Adipoyl-7-ADCA concentration during fermentation. Solid line, fermentation D; dashed line, fermentation E.

MATERIALS AND METHODS Determination of adipoyl-7-ADCA

The adipoyl-7-ADCA concentration was determined via HPLC as described in US 6,410,259. EXAMPLES

Example 1

Penicillium chrysogenum CBS 455.95 which has been transformed with an expandase expression cassette wherein the expandase coding region is provided with the IPNS promoter, as described in WO93/05158, was fermented in media according to Table 1. Three separate fermentations (A-C) were carried out. Trace element solution is according to Table 2. The fermentation conditions are listed in Table 3. The fermentations were inoculated at an inoculation strength of 5 vol% with inoculum grown in the same medium as given for fermentation A in Table 1 , except that the concentration of glucose was 50 g/kg and no potassium adipate was present.

Table 2. Trace element solution:

In fermentations A and C no phosphate is present in the initial medium. Instead, in these fermentations phosphate is fed to the medium as a solution containing 2.76 g/kg KH ₂ PO ₄ and 1.96 g/kg K ₂ HPO ₄ according to the feed profile as listed in Table 4.

Table 3. Fermentation conditions

Table 4. Phos hate feed rofile

t = fermentation time (hours)

Figure 1 shows the phosphate concentration during the fermentation. Figure 2 shows the relative stirrer speed during the fermentation. Higher stirring speeds correspond to higher viscosity. Figure 3 shows the adipoyl-7-ADCA production.

Example 2

Two fermentations D and E were carried out as described in Example 1 except that the composition of the media was according to Table 5. Phosphate is not present in the initial media but is instead fed as a solution containing 2.76 g/kg KH ₂ PO ₄ and 1.96 g/kg K ₂ HPO ₄ according to the feed profile as listed in Table 4.

Table 5. Com osition media /k

In fermentation D and E the phosphate is fed as described in Example 1.

Figure 4 shows the phosphate concentration during the fermentation. Figure 5 shows the adipoyl-7-ADCA acid production. Example 3

Penicillium chrysogenum CBS 455.95 was fermented in media according to Table 1. Three fermentations F, G and H were carried out as described in Example 1 for A, B and C respectively, except that the media (Table 1) did not contain potassium adipate, but instead contained 1 g/kg of potassium phenylacetic acid. Another difference with Example 1 was that the untransformed Penicillium chrysogenum CBS 455.95 was used. The fermentation conditions were identical to Example 1 (Table 6) except that the concentration of phenylacetic acid was kept between 0.6 and 2.0 g/kg by analysing the concentration in the broth every 4 hours and dosing an adequate amount of a concentrated solution of potassium phenylacetic acid to the broth.

The results of the three fermentations were essentially the same as in Example 1 with respect to the fact that the titer of PenicillinG at the end of fermentation H was higher than that of fermentations F and G.

Table 6. Fermentation conditions