Field of the invention

The present invention relates to a process for the production of a compound. The invention also relates to a bioreactor. The bioreactor may be used for the production of a compound.

Background to the invention

Disposable bioreactors are used in processes where cleaning validation is difficult and where profit margins are large enough to apply this technology.

Scaling up these disposable bioreactors does, however, lead to several problems. For example, the power input by the stirrer and heat transfer through the plastic wall of the bioreactors is limited. Accordingly, such bioreactor systems are currently restricted in size and by the intensity of growth in the bioreactor. These bioreactor systems are applied only to small-scale bioreactors or limited to slow-growing cells such as mammalian cell cultures.

New bioreactors systems are thus required in which highly intensely growing microorganisms may be grown on a larger scale.

Summary of the invention

The current invention is based on a finding that bubble column technology can be applied to allow heat generated by cells during fermentation to be dissipated. Accordingly, this allows fermentations using cells which grow at high intensity to be carried out at a larger scale where heat generation may otherwise be limiting.

In particular, the invention may be applied to disposable bioreactors, where heat generation is an even greater problem in view of the use of plastics materials which do not transfer heat well.

In particular, application of a gas in the method of the invention allows the temperature of the reaction medium to be controlled. Specifically, cooling may be carried out, potentially completely, by evaporating water from the reaction medium, i.e. the broth. Thus, any adverse effect of increased temperature on the cells in the reaction medium may be controlled and minimized. Accordingly, the intensity of cell growth can be increased because of reduced heat production and better surface to volume ratio. Also, the potential size of a bioreactor, in particular a disposable bioreactor, may be hugely increased, since the stirring and cooling are no longer limitations.

Also, in view of evaporation of reaction medium, the concentration of the end product may advantageously be increased within the reaction medium.

As compared with disposable stirred fermentations, a bubble column may have a number of advantages:

- easier construction in view of the lack of a stirrer shaft; and

- reduced heat production in view of lack of stirring energy and increased evaporation.

The method of the invention will now allow disposable bubble column technology to be applied to fermentations which use rapidly growing organisms, such as bacteria, and to allow such technology to be used at a larger scale.

According to the invention, there is thus provided a process for the production of a compound of interest, which process comprises cultivating cells capable of producing the compound of interest in a reaction medium under conditions which allow for production of the compound of interest, wherein the temperature of the reaction medium is controlled by allowing at least 30% of the heat generated in the reaction medium to be removed by allowing evaporation to take place from the reaction medium, and, optionally, recovering the compound of interest from the reaction medium.

The invention also provides a bioreactor comprising a container for housing cells capable of producing a compound of interest in a reaction medium; and means for maintaining the reaction medium at an approximately constant temperature by allowing at least 30% of the heat generated in the reaction medium to be removed by allowing evaporation to take place from the reaction medium.

Calculations demonstrate that, using the method of the invention, existing E. coli processes may be run in an up to about 2 meter tall bubble column without application of any external cooling. That is to say, cooling of the broth can be carried out in large part or entirely by evaporation. In this way, the method of the invention can be run at large volumes, by increasing the diameter of the bioreactor, and disposable materials may thus be applied to larger fermentations and using cell which grow at high intensity than is currently possible. This will reduce the specific production costs of the product since the batch size (i.e. absolute size of fermentation) will increase. This will especially be beneficial, since the amount of QA work and documentation may be identical as the batch size increases. In addition, increased concentrations of the final product may be achieved for a given batch size so that increased use of the bioreactor may be achieved.

Brief description of the figures

Figure 1 shows oxygen uptake rate, airflow rate, weight development and biomass concentration of a typical E.coli fed batch fermentation process in a stirred and cooled bioreactor.

Figure 2 shows oxygen uptake rate, airflow rate, weight development and biomass concentration in a process similar to that in Figure 1 , but with cooling by evaporation.

Figure 3 shows the same process as in Example 1 but with increased start weight, to make full use of the capacity of the bioreactor.

Figures 4 and 5 show the production of E.coli Penicillin G acylase at small scale.

In all figures the scales have the same units. In these figures, 100 means

OUR: 100 mmol/kg starting weight,

Weight: 100kg,

Airflow: 100Nm ³ /h,

Biomass: 100g/kg,

Temperature: 100 °C.

Detailed description of the invention

Throughout the present specification and the accompanying claims, the words "comprise", "include" and "having" and variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows. The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By way of example, "an element" may mean one element or more than one element.

The invention relates to a method for carrying out fermentations. The fermentations are carried out in such a way that heat generated in the fermentations is dissipated to a large degree, or completely, by evaporation of the reaction medium. That is to say, the method of the invention allows the temperature of the reaction medium to be controlled, so that performance of the organism used is not compromised.

This allows fermentations to be carried out with cells which grow at high intensity and which, consequently, generate a large amount of heat. Typically, fermentations using such cells can only be carried out on small scale or require the application of cooling apparatus.

The application of cooling apparatus to disposable bioreactors is often not possible and, in addition, heat transfer through the plastics wall of such bioreactors is often limited. Accordingly, the size of disposable bioreactors has been limited. The method of the invention, however, allows these limitations to be circumvented, since the temperature of the reaction medium may be controlled in a different way. Thus, the method of the invention allows high oxygen transfer rates to be used in context of larger scale disposable bioreactors.

Previously, this has not been possible since fast growing cells generate large amounts of heat which could not be dissipated and which then adversely affected growth of the cells in question.

According to the invention, there is thus provided a process for the production of a compound of interest. The process comprises cultivating cells capable of producing the compound of interest in a reaction medium under conditions which allow for production of the compound of interest.

Critically, the cultivation is carried out in such a way that the temperature of the reaction medium is controlled. This is achieved by allowing at least 30% of the heat generated in the reaction medium to be removed by evaporation from the reaction medium. In this way, the temperature of the reaction medium may be controlled, for example the reaction medium may be maintained at an approximately constant temperature. The compound of interest may then be recovered from the reaction medium (or from the cells in the event that the compound of interest is not secreted). Thus, evaporation from the reaction medium is allowed to occur to counterbalance, at least partially, heat generated by growth of the cells in the reaction medium and/or that generated by any agitation from a stirring means, so that the temperature remains approximately constant.

Evaporation from the reaction medium may remove at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or substantially all of the heat generated in the reaction medium.

Removal of heat via evaporation from the reaction medium is defined with reference to the heat generated over a defined cultivation period. Typically, the defined period will be the entire cultivation period. That is to say, at least 30% of all heat generated in the reaction medium over the entire cultivation period is removed via evaporation from the reaction medium. However, removal of at least 30% of all heat generated in the reaction medium may be defined in terms of a period of time shorter than the entire cultivation period, for example over a period of time of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of the entire cultivation period.

Heat production in a fermentation can be measured by the oxygen uptake rate which is proportional to heat produced by the metabolic activity of the cells in the bioreactor. Increase of the intensity of the process will require more heat to be removed. A bioreactor as described in the present invention may allow for heat removal up to 120 W/kg broth. This represents an oxygen uptake rate up to 80 mmol/kg/h. In order to control the temperature of the fermentation the amount of heat to be removed by evaporation can be calculated from the oxygen uptake rate as follows:

Heat Produced = 3.6 ^* Oxygen Uptake Rate ^* Weight Broth ^* Specific Heat Production

, wherein the Factor 3.6 is the conversion factor for hours to seconds and mmol to mol [s/h ^* mmol/mol]; Oxygen Uptake Rate" (in [mmol/kg/h]) is defined by and measured in the process; "Specific Heat Production" relates to the energy content in [J/mol] of the carbon source used in fermentation (for example for glucose 460 ^* 10 ³ ). The calculation then provides "Heat Produced" as [W] (= [J/s]).

If evaporation does not remove all of the heat generated in the reaction medium, then additional cooling means may be used. For example, a heat exchanger may be used. If the method is applied to a disposable system with non-rigid walls, cooling may be provided by means of a closed loop water-jacket, for example.

Evaporation from the reaction medium may be driven by the injection of a gas into the reaction medium. The gas may be, for example, air, oxygen and/or C0 ₂ . That is to say, the degree of evaporation may be controlled via gas flow through the reaction medium. The pressure and rate of flow of such a gas may be such that the appropriate degree of evaporation of the reaction medium takes place so that the temperature of the reaction medium remains approximately constant.

In the method of the invention, the temperature of the reaction medium may be controlled. That is to say, the method of the invention allows heat generated by the fermentation to be dissipated, typically to such an extent that growth of the cells in the fermentation is not impaired. Temperature control according to the invention encompasses temperature rise during the fermentation and fermentations in which the temperature of the reaction medium is held at an approximately constant temperature Typically then, the temperature of the reaction medium will not vary by more than 20°C, more than 15°C, more than 12°C, more than 10°C, more than 8°C, more than 5°C or more than 3°C.

For the purposes of this invention, an approximately constant temperature implies a process in which the temperature of the reaction medium will not vary by more than 2°C or more than 1 °C.

The method of the invention may preferably be carried out using a disposable bioreactor. Herein, "disposable" implies the use of a material or structure (formed from or comprising a material) intended for single usage, or usage for a limited number of times, for example two, three, four or five times, after which it will not be reused and, typically, may be disposed of. This is in contrast to materials or structures intended for multiple usage and which may need to be cleaned and/or sterilized after use and before reuse.

A bioreactor used in the invention may comprise a container for housing cells. Such a container may preferably be in the form of a flexible bag which may be placed in a rigid structure such as a tank shell for support. Such a flexible bag may conveniently be formed from a plastics material. However, the container may comprise a rigid material, for example plastics, metal and/or glass. The support structure may also include/involve a movable dolly, so that the bioreactor may be moved to different locations before, during and after material processing. A bioreactor suitable for use in a method of the invention may be in the form of a bubble column. That is to say, it may be in the form of a column adapted so that introduction of gas takes place at the bottom of the column and causes a turbulent stream to be generated. It may be built in any one of numerous forms of construction. The column may be vertically arranged. It may be cylindrical, but may also be oval or square. Mixing is carried out by liquid flow as a result of the gas sparging. Critically in the method of the invention, introduction of the gas occurs in such a way that evaporation of the reaction medium is allowed to take place to balance heat generation in the reaction medium.

In a bubble column, the liquid can be in parallel flow or counter-current. A bubble column reactor used in the method of the invention may typically have a high liquid content and a moderate phase boundary surface.

The method of the invention may be carried out in the absence of mechanical stirring. The method of the invention may be carried out in the absence of external cooling (in addition to that provided by evaporation of the reaction medium). The method of the invention may be carried out in the absence of mechanical stirring and in the absence of external cooling (in addition to that provided by evaporation of the reaction medium).

The method of the present invention may be carried out by way of use of a bubble column. In the context of the present invention, the term "bubble column" refers to an apparatus used for gas-liquid reactions consisting of vertically arranged columns that can be built in numerous forms of construction. The columns may be cylindrical, but may also have other shapes such as oval, square to name just a few. The introduction of gas takes place at the lower half of the column and causes a turbulent stream to enable an optimum gas exchange. Mixing occurs through gas sparging and displacement and entrainment of the liquid by rising gas bubbles. Bubble columns are characterized by a high liquid content and a moderate phase boundary surface and are used in various types of fermentations.

When the heat of the bioreactor is dissipated by evaporation, the maximum amount of heat that can be removed is determined by the maximum amount of air that can be supplied to the bioreactor. This air flow is mainly determined by the hydrodynamics of bubble columns. The maximum air velocity is about 30 cm/s; with higher velocities the air flow will result in expulsion of the liquid from the bioreactor. Heat removal is strongly dependent of the temperature of the broth, but typically in a 2 m tall bubble column about 10-15 W/kg can be removed, which corresponds to an oxygen uptake of from 80 to 120 mmol/kg/h.

In an embodiment the bioreactor (e.g. bubble column) has a volume of between 1 L - 100,000L, preferably between 10L - 75,000L, more preferably between 100L - 10,000L.

In an embodiment the bioreactor (e.g. bubble column) has a height (H) of between 0.2 - 5 m, preferably between 0.5 - 4 m, more preferably between 1 - 3 m and in particular between 1 .5 - 2.5 m.

In an embodiment the bioreactor (e.g. bubble column) has a diameter (D) of between 0.4 - 10 m, preferably between 0.5 - 8 m, more preferably between 1 - 5 m and in particular between 1.5 - 2.5 m. When the bioreactor has a square cross-sectional shape, D is defined as the length of one side of the bioreactor. When the bioreactor has a circular cross-sectional shape, D is defined as the diameter.

In an embodiment the bioreactor (e.g. bubble column) comprises an H/D ratio of 1 :2 or higher, for example 1 :1 . Preferably, the bioreactor (e.g. bubble column) comprises a H/D ratio of between 1 :1 .9 and 1 :0.1 , preferably between 1 :1 .8 and 1 :0.2, preferably between 1 :1 .7 and 1 :0.3, preferably between 1 :1.6 and 1 :0.4, preferably between 1 :1 .5 and 1 :0.5, preferably between 1 :1 .4 and 1 :0.6, preferably between 1 :1 .3 and 1 :0.7, preferably between 1 :1 .2 and 1 :0.8, preferably between 1 :1.1 and 1 :0.9.

The method of the invention allows disposable materials to be used in the production of fermentation apparatus at an industrial scale. "Industrial scale" is defined herein as a fermentation process carried out in a bioreactor which has a volume as described above.

Industrial scale may also be defined in terms of concentration of the product at the end of the fermentation in the reaction medium. Accordingly, in a process of the invention, the final product concentration is at least 0.1 g/l, at least 1 g/l, at least 2 g/l, at least 5 g/l, at least 10 g/l at least 50 g/l, at least 100 g/l or more.

The invention also provides a bioreactor adapted for use in the invention. That is to say, the invention relates to a bioreactor comprising:

a container for housing cells capable of producing a compound of interest in a reaction medium; and

means for maintaining the reaction medium at an approximately constant temperature by allowing at least about 30% of the heat generated in the reaction medium to be removed by allowing evaporation to take place from the reaction medium.

In an embodiment the bioreactor may comprise a container. The container then forms the product contact surface for the bioreactor. The container is preferably a flexible bag which may be placed in a rigid structure such as a tank shell for support. The support structure may also include/involve a movable dolly, so that the bioreactor may be moved to different locations before, during and after material processing.

Fittings may added to the container to enable functionality required in a bioreactor such as penetrations and filters to allow for fluid and gas transfer, a mixing interface, sensors and a sparging surface to control bubble size.

For application as a bioreactor, the container plus all attachments, penetrations, sensors, etc. may be sterilized prior to use (for example using gamma-irradiation). After sterilization, the inside of the container, tubing and components may be considered sterile, providing a "sterile envelope" protecting the contents of the vessel from airborne contaminants outside.

Critically, the bioreactor is provided with means for maintaining the reaction medium at an approximately constant temperature by allowing at least about 30% of the heat generated in the reaction medium to be removed by allowing evaporation to take place from the reaction medium.

This means that the bioreactor will typically comprise means for allowing gas to be injected into the reactor which can drive the desired degree of evaporation from the reaction medium. Preferably, then, the bioreactor may be in the form of a bubble column or comprise a bubble column.

Bubble size and distribution can be controlled by passing the inlet gas stream through a porous surface prior to addition to the interior of the bioreactor. Moreover, the sparging surface may be used as a cell separation device by alternating pressurization and depressurization (or application of vacuum) on the exterior surface of the porous surface, for example, or by a Bernoulli effect created by fast flow along one portion of the porous surface causing depressurization along other parts of the surface (e. g. fast flowing air in the center of a tube, exiting at one end of the tube, creating a vacuum along the length of the tube).

A bioreactor of the invention may include one or more of the following: a disposable bioreactor and one or more sensor and/or probes, for example to monitor temperature of the reaction medium. In the event that not all heat generated by the growth of the cells is removed by evaporation, cooling means may be provided. Cooling may be provided by a closed loop water jacket cooled by a control system mounted on the bioreactor or by standard heat exchange through a cover/jacket on the tank. Cooling may also be provided by means of Peltier coolers. For example, a Peltier cooler may be applied to an exhaust line (e.g., to a chamber similar to a small bag, with a large volume to decelerate air and a large surface area) to condense gas in the exhaust air to help prevent an exhaust filter from wetting out.

A disposable bioreactor may typically comprise a plastic, flexible container, but may also comprise a rigid material for example, plastics, metal or glass). The sensors and/or probes typically are connected to sensor electronics, the output of which may be to a controller system, such as a PC.

A mixing system may be provided, in addition to any mixing provided by the gas injection, which may generally include a motor for driving an impeller positioned in the bioreactor.

However, a bioreactor of the invention may be provided which does not comprise external cooling means and/or mechanical stirring means.

Since gas, such as air, oxygen and/or C0 ₂ gas (compressed or pumped) will typically be so as to allow evaporation to occur from the contents of the bioreactor, a filter, a flow meter, and/or a valve (for example a pneumatic valve) may be provided inline, the latter of which may be controlled by a controller system, which, may be a PC.

Such a controller system may include a combination of electronic, mechanical or pneumatic systems to control gas flow to the disposable bioreactor.

A disposable bioreactor preferably is supported by a support structure, which may be a tank (e. g., stainless steel). The tank may be designed to include a height and diameter similar to standard stainless steel bioreactors. Baffles may be built into the interior of the hard tank shell to improve mixing by causing the container for housing cells to conform a shape that protrudes into the bioreactor, which preferably breaks up circular flow and/or prevents vortexing (for example). At a top portion of the tank, one or more connections (e.g., tubes, valves, openings) for fluids, gases, and the like, to be added or withdrawn (e.g., intakes/exhausts) from the bioreactor, each of which may include a flow sensor and/or filter may be provided. A utility tower may be provided on or adjacent to the tank, which may be used to house one or more pumps, controllers and electronics (e.g., the sensor electronics, electronics interfaces, pressurized gas controller, etc.).

Sensors/probes and controls for monitor and controlling important process parameters include any one or more, and combinations of: temperature, pressure, pH, dissolved oxygen (DO), dissolved carbon dioxide (pC0 ₂ ), mixing rate, and gas flow rate (for example).

Preferably process control may be achieved in ways which do not compromise the sterile barrier established by the bioreactor. In particular, gas flow may be monitored and/or controlled by a rotameter or a mass flow meter upstream of an inlet air filter.

The rising gas bubble and the lower density of gas-saturated liquid rise, displacing gas-poor liquid which falls, may provide top-to-bottom circulation. The path of rising liquid can be guided by means of dividers inside the chamber of the container for housing cells. For example, using a sheet of plastic which bisects the interior of the bioreactor, preferably vertically, with a gap at the top and the bottom. Gas may be added on one side of the divider, causing the gas and gas-rich liquid to rise on one side, cross over the top of the barrier sheet, and descend on the other side, passing under the divider to return to the gas-addition point.

In this way, the bioreactor may allow the mixing of fluids or solids of any type. In particular, fluids inside the bioreactor may be mixed to provide distribution of nutrients and dissolved gasses for cell growth. The same bioreactor may be used for mixing buffers and media or other solutions in which a disposable product contact surface is desirable. This may also include applications in which the vessel is not required to be sterile or maintain sterility. Moreover, the container for housing cells and holding the fluids/mixtures/gases may be removed and discarded such that any tank supporting the container, for example, is not soiled by the fluids that are mixed in the bioreactor. Thus, the tank need not be cleaned or sterilized after every use.

The compound of interest in the method according to the invention can be any biological compound.

The compound of interest may be the cells themselves, i.e. biomass, in which case cells may be recovered from the medium following cultivation according to the method of the invention.

The biological compound may be biomass (i.e. the cells in the fermentation themselves) or any biopolymer or metabolite. The biological compound may be encoded by a single polynucleotide or a series of polynucleotides composing a biosynthetic or metabolic pathway or may be the direct result of the product of a single polynucleotide or products of a series of polynucleotides. The biological compound may be native to the host cell or heterologous.

The term "heterologous biological compound" is defined herein as a biological compound which is not native to the cell; or a native biological compound in which structural modifications have been made to alter the native biological compound.

The term "biopolymer" is defined herein as a chain (or polymer) of identical, similar, or dissimilar subunits (monomers). The biopolymer may be any biopolymer. The biopolymer may for example be, but is not limited to, a nucleic acid, polyamine, polyol, polypeptide (or polyamide), or polysaccharide.

The biopolymer may be a polypeptide. The polypeptide may be any polypeptide having a biological activity of interest. The term "polypeptide" is not meant herein to refer to a specific length of the encoded product and, therefore, encompasses peptides, oligopeptides, and proteins. The polypeptide may be a pharmaceutical polypeptide and/or a bioactive. Polypeptides further include naturally occurring allelic and engineered variations of the above- mentioned polypeptides and hybrid polypeptides. The polypeptide may be native or may be heterologous to the host cell. The polypeptide may be a collagen or gelatin, or a variant or hybrid thereof. The polypeptide may be an antibody or parts thereof, an antigen, a clotting factor, an enzyme, a hormone or a hormone variant, a receptor or parts thereof, a regulatory protein, a structural protein, a reporter, or a transport protein, protein involved in secretion process, protein involved in folding process, chaperone, peptide amino acid transporter, glycosylation factor, transcription factor, synthetic peptide or oligopeptide or intracellular protein. The intracellular protein may be an enzyme such as, a protease, ceramidases, epoxide hydrolase, aminopeptidase, acylases, aldolase, hydroxylase, aminopeptidase, lipase. The polypeptide may be an enzyme secreted extracellularly. Such enzymes may belong to the groups of oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, catalase, cellulase, chitinase, cutinase, deoxyribonuclease, dextranase, esterase. The enzyme may be a carbohydrase, e.g. cellulases such as endoglucanases, β-glucanases, cellobiohydrolases or β-glucosidases, hemicellulases or pectinolytic enzymes such as xylanases, xylosidases, mannanases, galactanases, galactosidases, pectin methyl esterases, pectin lyases, pectate lyases, endo polygalacturonases, exopolygalacturonases rhamnogalacturonases, arabanases, arabinofuranosidases, arabinoxylan hydrolases, galacturonases, lyases, or amylolytic enzymes; hydrolase, isomerase, or ligase, phosphatases such as phytases, esterases such as lipases, proteolytic enzymes, oxidoreductases such as oxidases, transferases, or isomerases. The enzyme may be a phytase. The enzyme may be an aminopeptidase, asparaginase, amylase, a maltogenic amylase, carbohydrase, carboxypeptidase, endo- protease, metallo-protease, serine-protease, catalase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase, protein deaminase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phospholipase, galactolipase, polyphenoloxidase, ribonuclease, transglutaminase, or glucose oxidase, hexose oxidase, monooxygenase. The enzyme may catalyse a non-naturally occurring conversion, for example an enzyme suitable for use in a biocatalysis reaction.

The enzyme may be involved in the synthesis of a primary or secondary metabolite

According to the present invention, a polypeptide or enzyme also can be a product as described in WO2010/102982. According to the present invention, a polypeptide can also be a fused or hybrid polypeptide to which another polypeptide is fused at the N- terminus or the C-terminus of the polypeptide or fragment thereof. A fused polypeptide is produced by fusing a nucleic acid sequence (or a portion thereof) encoding one polypeptide to a nucleic acid sequence (or a portion thereof) encoding another polypeptide.

Techniques for producing fusion polypeptides are known in the art, and include, ligating the coding sequences encoding the polypeptides so that they are in frame and expression of the fused polypeptide is under control of the same promoter (s) and terminator. The hybrid polypeptides may comprise a combination of partial or complete polypeptide sequences obtained from at least two different polypeptides wherein one or more may be heterologous to the host cell. Example of fusion polypeptides and signal sequence fusions are for example as described in WO2010/121933.

The biopolymer may be a polysaccharide. The polysaccharide may be any polysaccharide, including, but not limited to, a mucopolysaccharide^. g., heparin and hyaluronic acid) and nitrogen-containing polysaccharide (e.g., chitin). In a more preferred option, the polysaccharide is hyaluronic acid.

The polynucleotide of interest according to the invention may encode an enzyme involved in the synthesis of a primary or secondary metabolite, such as organic acids, carotenoids, (beta-lactam) antibiotics, and vitamins. Such metabolite may be considered as a biological compound according to the present invention. The term "metabolite" encompasses both primary and secondary metabolites; the metabolite may be any metabolite, such as organic acids, carotenoids, (beta-lactam) antibiotics, and vitamins. Such metabolites may be considered as a biological compound according to the present invention.

Metabolites include citric acid, gluconic acid, itaconic acid, a dicarboxylic acid such as fumaric acid, succinic acid or adipic acid, lactic acid or a keto acid such as levulinic acid.

The metabolite may be encoded by one or more genes, such as in a biosynthetic or metabolic pathway. Primary metabolites are products of primary or general metabolism of a cell, which are concerned with energy metabolism, growth, and structure. Secondary metabolites are products of secondary metabolism (see, for example, R. B. Herbert, The Biosynthesis of Secondary Metabolites, Chapman and Hall, New York, 1981 ).

Primary metabolites are for example intermediates of the main metabolic pathways such as the glycolytic pathway or the TCA cycle. The primary metabolite may be, but is not limited to, an amino acid, fatty acid, such as a polyunsaturated fatty acid, nucleoside, nucleotide, sugar, triglyceride, or vitamin.

The secondary metabolite may be, but is not limited to, an alkaloid, coumarin, flavonoid, polyketide, quinine, steroid, peptide, or terpene. The secondary metabolite may be an antibiotic, antifeedant, attractant, bacteriocide, fungicide, hormone, insecticide, or rodenticide. Antibiotics include cephalosporins, beta-lactams and macrolides, such as erythromycin.

Examples of β-lactams are clavulanic acid, penicillin (e.g. penicillin G, penicillin V or 6-aminopenicillinic acid) and semi synthetic penicillins such as amoxicillin and cephalosporins such as cephalosporin C. The β-lactam may be an N-acylated derivative of β-lactam intermediates such as 7-amino-3-carbamoyloxymethyl-3-cephem-4- carboxylic acid (7ACCCA), 7-aminocephalosporanic acid (7-ACA), 7-amino-3-chloro-3- cephem-4-carboxylic acid (7-ACCA), 7-aminodesacetoxycephalosporanic acid (7-ADCA), 7-aminodesacetylcephalosporanic acid (7-ADAC), 7-amino-3-[(Z/£)-1 -propen- 1 -yl]-3-cephem-4-carboxylic acid (7-PACA) and the like. The acyl group at the 7-amino position is preferably adipic acid yielding the corresponding adipoyl derivate as disclosed in WO 93/05158, WO 93/08287 or WO 2004/106347. Alternative suitable side chains have been disclosed in WO 95/04148, WO 95/04149, WO 96/38580, WO 98/48034 and WO 98/48035. Further examples of secondary metabolites are adipoyl-7-ADCA, adipoyl-7-ACA, adipoyl-7-ADAC, adipoyl-7-ACCA, adipoyl-7-PACA or adipoyl-7-ACCCA, most preferred is adipoyl-7-ADCA.

The biological compound may also be the product of a selectable marker. A selectable marker is a product of a polynucleotide of interest which product provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Selectable markers include, but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricinacetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), trpC (anthranilate synthase), ble (phleomycin resistance protein), hyg (hygromycin), NAT or NTC (Nourseothricin) as well as equivalents thereof.

A suitable cell for use in the method of the invention may be any wild-type strain producing a 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 nucleic acid transformation. Thus, a cell suitable for use in the method of the invention may already be capable of producing the compound of interest. However, the cell may also be provided with a homologous or heterologous expression construct that encodes a polypeptide involved in the production of the compound of interest.

The person skilled in the art will be aware of methods for modification of a cell, such as a microbial cell, such that it is capable of production of the polypeptide involved in the production of the compound of interest.

Typically, this may involve the use of an expression construct (or nucleic acid construct) which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature. Such an expression cassette/construct typically may contain all the control sequences required for expression of a coding sequence, wherein said control sequences are operably linked to said coding sequence.

The term "operably linked" is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the coding sequence of the DNA sequence such that the control sequence directs the production of a polypeptide.

The term "control sequences" is defined herein to include all components, which are necessary or advantageous for the expression of mRNA and / or a polypeptide, either in vitro or in a host cell. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, Shine-Delgarno sequence, optimal translation initiation sequences (as described in Kozak, 1991 , J. Biol. Chem. 266:19867-19870), a polyadenylation sequence, a pro-peptide sequence, a pre-pro-peptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. Control sequences may be optimized to their specific purpose.

The method of the invention is particularly suited to the use of rapidly growing cells, i.e. it can be applied to high intensity fermentation. Significant amounts of heat are produced in such fermentations, such that the performance of the cells used, in terms of production of the product of interest, would be impaired in the absence of removal of that heat. A high intensity fermentation, for the purposes of this invention may be defined as one which has an oxygen uptake rate of at least about 10mmol/kg/h, least about 30mmol/kg/h, at least about 50mmol/kg/h or at least about 100mmol/kg/h or higher.

A cell suitable for use in the method according to the invention may be a prokaryotic cell. Preferably, the prokaryotic cell is a bacterial cell. The term "bacterial cell" includes both Gram-negative and Gram-positive microorganisms. The bacterium may be an Actinomycete.

Suitable bacteria may be selected from e.g. Escherichia, Anabaena, Caulobactert, Gluconobacter, Rhodobacter, Pseudomonas, Paracoccus, Bacillus, Brevibacterium, Corynebacterium, Rhizobium (Sinorhizobium), Flavobacterium, Klebsiella, Enterobacter, Lactobacillus, Lactococcus, Methylobacterium, Staphylococcus or Acintomycetes such as Streptomyces and Actinoplanes species. Preferably, the bacterial cell is selected from the group consisting of B. subtilis, B. amyloliquefaciens, B. licheniformis, B. puntis, B. megaterium, B. halodurans, B. pumilus, G. oxydans, Caulobacter crescentus CB 15, Methylobacterium extorquens, Rhodobacter sphaeroides, Pseudomonas zeaxanthinifaciens, Pseudomonas putida, Pseudomonas fluorescens, Paracoccus denitrificans, E. coli, C. glutamicum, Staphylococcus carnosus, Streptomyces lividans, Streptomyces clavuligerus, Sinorhizobium melioti and Rhizobium radiobacter.

The host cell according to the invention may be a eukaryotic host cell. Preferably, the eukaryotic cell is a fungal or an algal cell. The eukaryotic cell may be a fungal cell, for example a yeast cell, such as Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia strain. More preferably from Kluyveromyces lactis, S. cerevisiae, Hansenula polymorpha, Yarrowia lipolytica and Pichia pastoris.

The eukaryotic cell may be a filamentous fungal cell. Filamentous fungi include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK). The filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. Filamentous fungal strains include, but are not limited to, strains of Acremonium, Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Phanerochaete, Pleurotus, Schizophyllum, Talaromyces/Rasamsonia, Thermoascus, Thielavia, Tolypocladium, and Trichoderma.

Preferred filamentous fungal cells belong to a species of an Acremonium, Aspergillus, Chrysosporium, Myceliophthora, Penicillium, Talaromyces/Rasamsonia, Thielavia, Fusarium or Trichoderma genus, and most preferably a species of Aspergillus niger, Acremonium chrysogenum, Acremonium alabamense, Aspergillus awamori, Aspergillus sojae, Aspergillus fumigatus, Rasamsonia emersonii, Aspergillus oryzae, Chrysosporium lucknowense, Fusarium oxysporum, Myceliophthora thermophila, Trichoderma reesei, Thielavia terrestris, Penicillium chrysogenum or Penicillium citrium. A more preferred host cell belongs to the genus Aspergillus, more preferably the host cell belongs to the species Aspergillus niger. When the host cell according to the invention is an Aspergillus niger host cell, the host cell preferably is CBS 513.88, CBS124.903 or a derivative thereof.

Several strains of filamentous fungi are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL), and All-Russian Collection of Microorganisms of Russian Academy of Sciences, (abbreviation in Russian - VKM, abbreviation in English - RCM), Moscow, Russia. Useful strains in the context of the present invention may be Aspergillus niger CBS 513.88, CBS124.903, Aspergillus oryzae ATCC 20423, IFO 4177, ATCC 101 1 , CBS205.89, ATCC 9576, ATCC14488-14491 , ATCC 1 1601 , ATCC12892, P. chrysogenum CBS 455.95, P. chrysogenum Wisconsin54-1255(ATCC28089), Penicillium citrinum ATCC 38065, Penicillium chrysogenum P2, Thielavia terrestris NRRL8126, Rasamsonia emersonii CBS 124.902, Acremonium chrysogenum ATCC 36225 or ATCC 48272, Trichoderma reesei ATCC 26921 or ATCC 56765 or ATCC 26921 , Aspergillus sojae ATCC1 1906, Myceliophthora thermophila C1, Garg 27K, VKM-F 3500 D, Chrysosporium lucknowense C1 , Garg 27K, VKM-F 3500 D, ATCC44006 and derivatives thereof.

In the method of the invention, the cells are cultivated in a reaction medium and under conditions suitable for production of the compound of interest. Typically, in the method of the invention the cells may be cultivated at a large-scale (including, for example, in the form of a continuous, batch or fed-batch cultivations) in a suitable reaction medium and under conditions allowing the compound of interest to be produced and/or isolated. The amount of reaction medium may be maintained at an approximately constant volume in the method of the invention. That is to say, evaporated reaction medium may be replenished so that the volume of reaction medium remains approximately constant.

The cultivation takes place in a suitable reaction medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art (see, e. g., Bennett, J. W. and LaSure, L, eds., More Gene Manipulations in Fungi, Academic Press, CA, 1991). Suitable media are available from commercial suppliers or may be prepared using published compositions (e. g., in catalogues of the American Type Culture Collection). If the compound of interest is secreted into the reaction medium, the compound can be isolated directly from the reaction medium. If the compound of interest is not secreted, it can be isolated from, for example, cell lysates.

The compound of interest may be isolated by methods known in the art. For example, the compound of interest may be isolated from the reaction medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. The isolated compound of interest may then be further purified by a variety of procedures known in the art including, but not limited to, chromatography (e. g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e. g., ammonium sulphate precipitation), or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989). In some applications the compound of interest may be used without substantial isolation from the culture broth; separation of the reaction medium from the biomass may be adequate.

A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that that document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.

The disclosure of each reference set forth herein is incorporated herein by reference in its entirety.

The present invention is further illustrated by the following Examples:

EXAMPLES

Example 1 : Production of E.coli Penicillin G acylase in a classical stirred

fermentor, with temperature control by external cooling.

This Example describes the cultivation of recombinant E. coli Penicillin G acylase producing strain (WO00/66751 ) in a standardized fermentation set-up.

The following is prepared:

Yeast Extract (total Nitrogen content of 2.1 g/l) 1 14 g

Na ₂ HP0 ₄ .2H ₂ 0 53.4 g

KH ₂ P0 ₄ 40.8 g

NH ₄ CI 14.4 g

Distilled water to 6 kg final reaction medium.

When all components are completely dissolved, the pH is adjusted to 6.8, and the reaction medium is brought into a steel inoculum bioreactor and sterilized in an autoclave at 121 °C for 30 minutes.

After sterilization, glucose and neomycin are added aseptically to a final concentration of 2 g/l and 10 mg/l respectively.

The inoculum bioreactor is placed in a water bath at 27°C. Air is supplied to the bioreactor for aeration and mixing.

This inoculum can is inoculated with 1 ml vegetative suspension of the E. coli production strain and incubated at 27°C until late log-phase culture is obtained. This inoculum culture is used to inoculate 380 I of reaction medium in a 1 .3 meter wide and 2 meter tall bioreactor. The reaction medium has the following composition per kg reaction medium:

Yeast extract 30.0 g

Bacto Peptone 20 mmol/kg (e.g. Difco) 12.5 g

(NH ₄ ) ₂ )S0 ₄ 5.0 g

Citric acid 9.0 g

CaCI ₂ .2H ₂ 0 1 .25 g

FeS0 ₄ .7H ₂ 0 0.63 g

MnS0 ₄ .H ₂ 0 0.025 g

Antifoam agent (e.g. Basildon) 1.0 g

pH is adjusted to 5.5 with NaOH.

This reaction medium is sterilized and brought into the pre-sterilised bioreactor aseptically. Then, other components are added from sterile stock solutions to a final amount per kg reaction medium of:

Glucose 10.0 g

K ₂ HP0 ₄ 7.5 g

MgS0 ₄ .7H ₂ 0 2.0 g

Neomycin 0.01 g

Necessary nutritional supplements (e.g. Vit B1 , amino acids like leucine and proline) are added aseptically to batch reaction medium and/or together with the carbon feed in such amounts that, during fermentation, no shortages of these components occur. Heat labile components are filter sterilised.

As carbon feed, a sterile 500 g/kg glucose solution or an equivalent concentration of another carbon source suitable to the host as DE glucose syrup, glycerol etc. is applied.

After all glucose in the batch reaction medium is consumed, a carbon feed is started according to an exponential feed profile. The feed rate is increased to maintain exponential growth of the cells until the maximum oxygen uptake rate is reached. Then the feed rate is maintained constant. Due to the feeding, the weight of the fermentation increases during the process. In Figure 1 , the air flow rate, feed rate, weight, and biomass concentration are presented. The increase of the biomass concentration reduces during the process due to dilution by the feed solution. The bioreactor is filled completely only at the end of the process at 120 hrs. In this process, heat is produced by stirring and biological activity. At an Oxygen Uptake Rate of 80 mmol/kg/h, about 10.5 J/s is produced per kg of broth by biological activity, and about 2-3 J/s is added by the stirrer. For the complete process this amounts to 4200kJ/kg. In a standard stirred fermentation with external cooling, about 15% of that heat is removed by evaporation. The remainder of the heat, i.e. 3570kJ/kg needs to be removed by active cooling using cooling water so as to maintain a constant temperature of 27°C.

Example 2: Production of E.coli Penicillin G acylase in a bubble column fermentor

The process basics in the bubble column bioreactor are the same as in the process of Example 1 and composition of media is identical in all phases of the process. The concentration of the feed is reduced to compensate for the evaporation in the bubble column process, but the total feed on a carbon-basis is the same.

Critically, however, the air flow rate is used to control the temperature of the reaction medium. Consequently, the airflow rate is increased as compared with the stirred process of Example 1 . In this example the airflow is adjusted so as to maintain a constant temperature in the bioreactor by means of evaporation. Since the airflow is much higher, stirring is no longer required for mixing and oxygen transfer.

Dependent on the fermentation temperature, the amount of heat that can be removed can be determined by the saturated water vapour pressure at that temperature, and the evaporative heat of water. For example, at 27°C, the vapour in the outlet air is about 19 g/m ³ . The evaporative heat of water is 2257 J/g. If dried air is used to aerate the bioreactor, more than 40 kJ can be removed by 1 m ³ of air so as to maintain the constant temperature of 27°C. Since, in this example, all the heat produced is removed by evaporation, airflow needs to dissipate 4200kJ/kg, and external cooling is not required. Accordingly, 105 m ³ of air is required per kg of broth to carry out cooling that would, in the fermentation of Example 1 , be carried out by external cooling.

The result of this process is given in Figure 2. In Figure 2, the air flow rate, feed rate, weight and biomass concentration are given at the same scales as Figure 1 .

Because of evaporation the weight of the broth does not increase, resulting in a partial filling of the bioreactor during the process. Since the total carbon fed is the same as in the process of Example 1 , the amounts of biomass (and product) are the same as compared with Example 1 , but the concentrations of biomass (and product) are increased. Since the concentration of biomass is increased, the size of the down-stream processing equipment may be reduced.

Example 3: Production of E.coli Penicillin G acylase in a bubble column fermentor with increased starting weight of broth

In Example 3 the same process is described as in Example 2, except that the starting weight is increased to make full use of the available bioreactor volume. Since the feeds are proportional to the starting weight of the fermentation, more feed can be added to the process, allowing for increased production of biomass (and product). All concentrations are identical to Example 2. The airflow rate is increased proportionally to the feed rates and starting weight.

In Figure 3 the air flow rate, feed rate, weight and biomass concentration are given at the same scales as figure 1 . In this process design, also 4200kJ/kg of needs to be removed so as to maintain a constant temperature of 27°C, but since the starting weight is increased, the airflow needs to be increased proportionally.

The biomass and product concentrations will develop identically to Example 2, but due to the increased overall volume more biomass/product is made in this process design, i.e. higher productivity may be achieved and larger batch sizes generated. This may, inter alia, reduce costs for quality control.

Example 4: Production of E.coli Penicillin G acylase at small scale

A small scale evaluation of the process as described in Example 3 was performed in a thermally insulated laboratory bioreactor of about 10 litre volume. Conditions such as pH, temperature, running time of the process etc. are identical as in Example 3.

The diameter of the bioreactor is about 30 cm and the H/D ratio is between 1 :1 .5 and 1 :0.5. The bubble column bioreactor is fitted with a ring sparger for aeration. Since the height of the bioreactor is lower than examples 2 in and 3 and the amount of heat produced is proportional to the height, there is excess cooling capacity compared to a higher bioreactor; consequently a lower airflow of 5-15 WM is required.

The airflow rate is controlled by the fermentation temperature. The airflow rate is kept at the level where temperature is below the maximum allowed set-point.

To compensate for the loss of water due to evaporation two strategies can be employed either separately or in combination: 1. sterilized water is added to maintain the weight of the broth (including the regular feed additions, if any) during the fermentation, or 2. The feed concentration is prepared in such a way that the calculated or experimentally determined amount of water from option 1 is included in the feed stream.

Actual performance of the bubble column in the small scale bioreactor confirmed that this approach to remove heat via evaporation is feasible.

In Figure 4 the temperature control and the Oxygen Uptake Rate are given for a late stage of the process. This figure shows that the temperature can be controlled properly at 27 °C in a situation where the E.coli cells in the broth are biologically active and therefore produce heat.

In the set-up available, the requirements to generate high biomass was limited by insufficient oxygen transfer, most likely due to the sparger design. Engineering solutions (more and/or smaller openings, pure oxygen suppletion) are known in the art to improve this aspect for smaller scale applications.

In Figure 5 the weight of the fermentation and the cumulative feed is presented. The weight increase at the 25 hour time-point is caused by the addition of water to compensate for the reduced broth weight.

In Table 1 the physiological fermentation parameters of the evaporation controlled bubble column are compared with the those of traditional stirred tank fermentations. Within the experimental error these data show that the performance of the bubble column fermentation is similar to the stirred tank process.

Table 1 : Comparison between physiological fermentation performance parameters of a bubble column fermentation cooled by evaporation only and a classical stirred tank fermentation cooled by external means.