WO2012175574A1

WO2012175574A1 - Increased pantothenate (vitamin b5) production

Info

Publication number: WO2012175574A1
Application number: PCT/EP2012/061889
Authority: WO
Inventors: Ines GRUNER; Zoltan Pragai
Original assignee: Dsm Ip Assets B.V.
Priority date: 2011-06-21
Filing date: 2012-06-20
Publication date: 2012-12-27

Abstract

The present invention relates to an improved production process for pantothenic acid (vitamin B5) using a Bacillus strain in a new medium containing a low amount of sugar such as, e.g., glucose and a higher amount of slowly utilizable carbon source such as, e. g., raffinose. This medium is particularly useful for high throughput (HTP) screening of Bacillus mutants.

Description

INCREASED PANTOTHENATE (VITAMIN B5) PRODUCTION

The present invention relates to an improved production process for pantothenic acid (vitamin B5) using a Bacillus strain in a new medium containing a low amount of sugar such as, e.g. , glucose and a higher amount of slowly utilizable carbon source such as, e.g., raffinose. This medium is particularly useful for high throughput (HTP) screening of Bacillus mutants.

Pantothenate is a member of the B complex of vitamins and is a nutritional requirement for mammals including humans, e.g. , from food sources, as a water-soluble vitamin supplement or as a feed additive. In cells, pantothenate is used primarily for the biosynthesis of coenzyme A and acyl carrier protein.

These essential coenzymes function in the metabolism of acyl moieties, which form thioesters with the sulfhydryl group of the 4'-phosphopantethein portion of these molecules.

Pantothenate has been synthesized conventionally via chemical synthesis from bulk chemicals. However, the substrates required for chemical synthesis are expensive and the racemic intermediates have to be optically resolved.

Accordingly, bacterial or microbial systems have been employed that produce enzymes useful in pantothenate biosynthesis processes. In particular,

bioconversion processes have been evaluated as a means of favoring production of preferred isomer of pantothenic acid. Moreover, methods of direct microbial synthesis have recently been examined as a means of facilitating D- pantothenate production.

The genes involved in biosynthesis of pantothenic acid as well as methods for fermentative production of pantothenic acid are known (see e.g. WO 01 /21772, WO 02/057474, WO 02/061108 or Ullman's Encyclopedia of Industrial Chemistry, 7^th Edition, 2007, Chapter Vitamins).

A method of producing pantothenate or pantoate by culturing genetically modified microorganisms of the genus Bacillus, particularly B. subtilis, in which at least one enzyme selected from the group consisting of PanB (ketopantoate hydroxymethyl transferase), PanC (pantothenate synthetase), PanD (aspartate- a-decarboxylase) and PanE (ketopantoate reductase) is overexpressed is known (WO 01 /21772).

A key enzyme in biosynthesis of pantothenic acid is PanB. This enzyme catalyzes the conversion of a-ketoisovalerate to α-ketopantoate; α-ketopantoate in turn is converted to pantoate via the NADPH-dependent PanE reaction. It has been found that this step forms a serious bottleneck in the biosynthesis of

pantothenate, i. e. is rate-limiting for pantothenate biosynthesis.

Besides genetic engineering of the production strains, there is an ongoing need in optimization of the culture conditions, including but not limited to the use of carbon source, buffer system, pH, oxygen content or the use of growing or resting cells. To achieve higher production rates of the production strains to use them for an industrial production process, this optimization also includes the screening methods, such as e.g. high throughput screening, for more powerful production strains.

Surprisingly, it has now been found out that a specific ratio of glucose to raffinose in the medium such as the use of low amount of glucose combined with higher amount of raffinose in the culture/production medium leads to increased production yield of pantothenate on (consumed) sugar. It turned out that the new medium composition is in particular useful for screening of pantothenate high producing strains, preferably the use of a medium with a concentration of glucose of 10 g/l or less for high throughput screening of pantothenate producing strains of Bacillus, more preferably Bacillus subtilis. Compared to the known culture media used so far (standard medium), the increased production of pantothenate is proportional to the engineering level of the production strain. Using the new medium in a traditional shake flask experiment with cultivation for 48 hours according to the present invention, the pH stayed at a constant level during the whole time and all sugar, e.g. glucose and raffinose, was consumed. ln particular, it is an object of the present invention to provide a pantothenate production process wherein strains of Bacillus, preferably Bacillus subtil is, are cultivated in an (optimized) medium wherein the starting concentration of glucose is 10 g/l or less, preferably 8 g/l, 4 g/l, 2 g/l, 1 g/l or less. In a particular embodiment, the culture medium comprises a mix of glucose

(mimicking the batch phase of the fermentation cultivation) and raffinose (mimicking the feed phase of the fermentation cultivation), in a ratio range of 1 :2 to 1 :20, such as 1 :4, 1 :5, 1 :6, 1 :8, 1 :9, 1 : 10, 1 : 12, 1 : 15, preferably in a ratio of 1 :9, such as e.g. 1 g/l glucose and 9 g/l raffinose as starting concentration. The glucose concentration varies preferably from 0 to 4 g/l, while the raffinose concentration may be in a range of 5-25 g/l, as long as the amounts fit with the ratios mentioned above. The glucose may be replaced by other quickly metabolized sugars such as e.g. fructose, ribose or sucrose, wherein the ratio of said sugars to raffinose is as mentioned above. The raffinose may be replaced by other oligosaccharides. Thus, the medium according to the present invention can comprise a mixture of glucose with oligosaccharides such as sucrose, maltose, cyclodextrins, maltotriose or lactose, preferably in the ratios mentioned above for glucose to raffinose, in particular in the ratio of 1 :9. A preferred medium is the one described as RSM in WO 2008/ 1 8575 (Example 2), herein further defined as SRG.

Thus, it is an object of the present invention to provide a process for the production of pantothenate, wherein a strain of Bad 11 us is cultivated in a medium comprising a sugar selected from fructose, ribose, sucrose or glucose, preferably glucose, mixed with raffinose and wherein the starting concentration of the sugar selected from fructose, ribose, sucrose or glucose is 10 g/l or less. Optionally, the pantothenate is recovered from the medium. Preferably, the ratio of sugar and raffinose is in the range of 1 :2 to 1 :20 or in any range described above, wherein the sugar is preferably glucose, but might also be selected from fructose, ribose or sucrose. As used herein, the term "pantothenic acid" includes but is not limited to pantothenic acid, precursors and/or derivatives thereof such as salts or esters thereof, i. e. pantothenate, in particular calcium pantothenate, or the alcohol form of pantothenic acid, i. e. pantothenol or panthenol. The terms "pantothenic acid", "pantothenate" and "vitamin B5" are used interchangeably herein.

Precursors/intermediates in the biosynthetic pathway of pantothenic acid which are included may be selected from e.g. pantoate, a-ketopantoate or a- ketoisovalerate. As used herein, the pantothenate production strain may be selected from Bacillus species represented by the Bacillus sensu stricto group, in particular Bacillus subtil is, Bacillus lent i morbus, Bacillus lent us, Bacillus ant hracis, Bacillus firmus, Bacillus pant othenticus, Bacillus cereus, Bacillus circu I ans, Bacillus coagu I ans, Bacillus megateri urn, Bacillus thuringiensis, Bacillus licheniformis, Bacillus amy I oliquef act ens, Bacillus pumi I us, Bacillus halodur ans (Zeigler and Perkins, 2008, Practical Handbook of Microbiology", Second Edition (E. Goldman and L. Green, eds. ), pp 301 -329, CRC Press, Boca Raton, FL). Most preferred, the microorganism is selected from Bacillus subtil is. All microorganisms which can be used for the present invention may be publicly available from different sources, e.g. , Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany, American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, VA 20108 USA or Culture Collection Division, NITE Biological Resource Center, 2-5-8, Kazusakamatari, Kisarazu-shi, Chiba, 292-0818, Japan (formerly: Institute for Fermentation, Osaka (IFO), 17-85, Juso-honmachi 2-chome,Yodogawa-ku, Osaka 532-8686, Japan).

In connection with the present invention it is understood that the above- mentioned microorganisms also include synonyms or basonyms of such species having the same physiological properties, as defined by the International Code of Nomenclature of Prokaryotes. The nomenclature of the microorganisms as used herein is the one officially accepted (at the filing date of the priority application) by the International Committee on Systematics of Prokaryotes and the Bacteriology and Applied Microbiology Division of the International Union of Microbiological Societies, and published by its official publication vehicle International Journal of Systematic and Evolutionary Microbiology (IJSEM).

The production strain, e.g. a strain of Bacillus, preferably Bacillus subtil is, used for performing the present invention may be a non-modified or wild-type microorganism or may be a genetically modified/mutated or recombinant one, wherein at least one of the known pantothenate biosynthetic genes has been overexpressed. Preferably, a recombinant Bacillus strain is used. Modifications either on the DNA or protein level include all modification/mutations with a direct impact on the yield, production and/or efficiency of pantothenate production. The person skilled in the art knows how to manipulate a Bacillus strain for said purpose. General techniques are described in Harwood, C.R. and Cutting, S.M. (1990). Molecular Bological Met hods for Bacillus. Chichester: John

J j., Fritsch, E.F. and Maniatis, T. (1989). Molecular Qoning: a Laboratory Manual, 2nd edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. For the scope of the present invention, it is not relevant how the mutant(s) are obtained; such mutants may be obtained, e.g., by site- directed mutagenesis, saturation mutagenesis, random mutagenesis/directed evolution, chemical or UV mutagenesis of entire cells/organisms, and other methods which are known in the art. These mutants may also be generated, e.g., by designing synthetic genes, and/or produced by in vitro (cell-free) translation. For testing of specific activity, these mutants may be (over-) expressed by methods known to those skilled in the art with measurement of the pantothenate production. This and further methods leading to increased pantothenate production by, e.g., the replacement of the natural promoter of the pan genes are described in e.g. WO 2010/018196. Modification also includes manipulation or deregulation of the branch chain amino acid biosynthesis in the pantothenate production strain. The term "overexpressing" or "overexpression" means expression of a gene product at a level higher than that expressed prior to modification of the microorganism or in a comparable microorganism which has not been modified. In one embodiment, the microorganism of the invention overexpresses one or more genes selected from the group consisting of panB, panQ panD, panE, ilvB, ilvC, ilvD, ilvN, glyA, serA, serC, and the gcv genes involved in the glycine cleavage pathway; as well as mutants thereof that result in the synthesis of encoded enzymes of improved catalytic properties. Preferably, the recombinant strain used for the purpose of the present invention carries at least two copies of panB and panD {panBD), more preferably 3 or 4 copies leading to increased pantothenate production. In one particular embodiment, the recombinant strain used for the purpose of the present invention is deregulated in the biosynthesis for branch chain amino acids such as e.g. leucine, valine, isoleucine. Said modification is preferably combined with the overexpression of one or more genes selected from the group consisting of panB, panC, panD, panE, ilvB, ilvC, ilvD, ilvN, glyA, serA, serC, and the gcv genes involved in the glycine cleavage pathway.

Examples of recombinant strains useful for the performance of the present invention are described in WO 2007/131750, i. e. PA12 (reference strain 1 ), PA49 (reference strain 2) or PA73 (reference strain 3), see Figures 1 to 4 or can be generated according to US 5,518,906, US 7,220,561 or WO 2004/005527 (see Table 8 and 9). Besides the overexpression of the whole panBCD operon as in reference strains 1 -3, the strains may be further modified, such as e.g. deregulation of glyA and/or introduction of further copies of panBand panD (i.e. reference strain 4 with glyA deregulated and a second copy of panBD). These further modification may also include deregulation of the branch chain amino acids biosynthesis (see reference strain 5 as further development of reference strain 4) and/or introduction of further copies of panBD, such as e.g. 2, 3 or 4 copies (see reference strain 6 as further development of reference strain 5).

The term "deregulated" or "deregulation" means the alteration or modification of a gene in a microorganism such that the level or activity of the gene product in a microorganism is altered or modified. Preferably, at least one gene is altered or modified such that the gene product is enhanced/increased or

attenuated/decreased. With regards to the genes belonging to the pantothenate operon, this deregulation is meant to be an overexpression of at least one of the genes. The term "genetically engineered" or "genetically altered" means the scientific alteration of the structure of genetic material in a living organism. It involves the production and use of recombinant DNA. More in particular it is used to delineate the genetically engineered or modified organism from the naturally occurring organism. Genetic engineering may be done by a number of

techniques known in the art, such as e.g. gene replacement, gene amplification, gene disruption, transfection, transformation using plasmids, viruses, or other vectors. A genetically modified organism, e.g. genetically modified

microorganism, is also often referred to as a recombinant organism, e.g.

recombinant microorganism. Using the new cultivation medium according to the present invention with a microorganism described above the biosynthesis of pantothenate can be greatly improved. The amount of pantothenate could be increased by at least 10%, preferably by at least 40, 60, 80, 100, 150, 190, 200, 220, 270, 300% compared to cultivation in a medium known in the art (standard medium), i.e. a medium wherein the only carbon source is glucose and no raffinose is present, in particular a medium comprising at least 20 g/l glucose. Examples of such media used in the art contain e.g. 20 g/l glucose (MMGT), 30 g/l glucose (SVY) or 60 g/l glucose (SVYM and SMG), such as e.g. described in WO 02/057474, WO

02/066665, WO 2004/1 13510 or WO 2007/131750. Compared to cultivation in MMGT minimal medium, the increase was in the range of at least 60 to 190%, compared to SVY medium the increase was in the range of at least 10 to 200%. The increase is dependent on the degree of engineering level of the production strain.

The present invention provides a process for the production of pantothenate by fermentation. In particular, the present invention provides a process for production of pantothenate comprising converting a substrate into

pantothenate. The substrate may be a carbon, nitrogen and phosphate source in which microorganisms are known to utilize to produce biomass, energy, and metabolites, such as pantothenate. A preferred substrate is a-ketoisovalerate. Suitable culture conditions for pantothenate production using e.g. Bacillus subti lis are known in the art and described e.g. in WO 2004/113510 or WO 2007/065602.

The term "culturing a microorganism under suitable culturing conditions" refers to methods of maintaining and/or growing a living microorganism of the present invention which are well known in the art. The microorganisms can be cultured in liquid, solid or semi-solid media. Preferably, the microorganism of the invention is cultured in liquid media comprising nutrients essential or beneficial to the maintenance and/or growth of the microorganism. Such nutrients include, but are not limited to, carbon sources or carbon substrates, such as alcohols, sugars, sugar alcohols, complex carbohydrates such as starches, hydrocarbons, fatty acids, other organic acids, oils, fats; nitrogen sources, e.g. vegetable proteins, yeast extract, peptones, peptides and amino acids derived from grains, beans and tubers, or derived from animal sources such as meat or milk, meat extracts and casein hydrolysates; inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium nitrate and ammonium phosphate; phosphorous sources, e.g. phosphoric acid and sodium or potassium salts thereof; trace elements, e.g. magnesium, iron, manganese, calcium, copper, zinc, boron, molybdenum and/or cobalt salts; as well as growth factors such as vitamins, growth promoters and the like.

The microorganisms are preferably cultured under controlled pH. In one embodiment, microorganisms are cultured at a pH of between 6.0 and 8.5, more preferably at a pH of about 7, wherein the pH is maintained at a constant level during the whole cultivation period, which is e.g. a period of 48 hours. The desired pH may be maintained by any method known to those skilled in the art.

Preferably, the microorganisms are further cultured under controlled aeration and under controlled temperatures. In one embodiment, the controlled temperatures include temperatures between 15 and 70° C, preferably the temperatures are between 20 and 55 ° C, more preferably between 30 and 45 °C or between 30 and 50° C.

The microorganisms may be cultured in liquid media either continuously, semi- continuously or batchwise by conventional culturing methods such as standing culture, test tube culture, shaking culture (shake-flask), aeration spinner culture or fermentation. Preferably, the microorganisms are cultured in a fermenter. Fermentation processes of the invention include batch, fed-batch and continuous methods of fermentation. Varieties of such processes have been developed and are well known in the art. The pantothenate obtained in accordance with the present invention in the fermentation broth can either be used without being recovered or after being recovered. The term "recovering" includes isolating, extracting, harvesting, separating or purifying the compound from the culture medium. Isolating the compound can be performed according to any conventional isolation or purification methodology known in the art including, but not limited to, treatment with a conventional resin, treatment with a conventional adsorbent, alteration of pH, solvent extraction, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, and the like. For example, the panthenol compound can be recovered from the culture medium by first removing the microorganisms from the culture. The solution is then passed through or over a cation exchange resin to remove unwanted cations and then through or over an anion exchange resin to remove unwanted inorganic anions and organic acids.

The terms "production" or "productivity" are art-recognized and include the concentration of pantothenate formed within a given time and a given fermentation volume (e.g. , kg product per hour per liter). The term "efficiency of production" includes the time required for a particular level of production to be achieved (for example, how long it takes for the cell to attain a particular rate of output of pantothenate). The term ^"yield^" is art- recognized and includes the efficiency of the conversion of the carbon source into the product (i. e. , pantothenate). This is generally written as, for example, kg product per kg carbon source. By "increasing the yield and/or production /productivity" of the compound it is meant that the quantity of recovered molecules, or of useful recovered molecules of that compound in a given amount of culture over a given amount of time is increased. The terms "biosynthesis" or a "biosynthetic pathway" are art- recognized and include the synthesis of a compound, preferably an organic compound, by a cell from intermediate compounds in what may be a multistep and highly regulated process.

In one particular aspect of the present invention, the new medium as described herein is useful for screening purpose, e.g. for HTP screening of Bacillus strains, preferably Bacillus subtilis, which are in particular useful for high production of pantothenate. HTP screening as well as production of pantothenate using the medium as of the present invention under conditions described herein can be performed according to any method known to the skilled person, including e.g. the use of shake-flask, deep-well assay or micro-fermentation such as e.g. use of a BioLector^® (m2p-l_abs, 52499 Baesweiler, Germany). Useful protocols for shake-flask assay, micro-fermentation using the BioLector^® or deep-well assays are given in the Examples and are furthermore known by the skilled person.

In one aspect, the medium of the present invention is used in a process for production of pantothenate or for screening purposes, wherein the medium comprises glucose and raffinose at a ratio of 1 :2 to 1 :20 and the microorganism is selected from Bacillus strain, preferably B. subtilis.

Advantageous embodiments of the invention become evident from the dependent claims. These and other aspects and embodiments of the present invention should be apparent to those skilled in the art from the teachings herein.

This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patent applications, patents and published patent applications, cited throughout this application are hereby incorporated by reference, in particular WO 01 /21772, WO 02/057474, WO 02/061108, WO 2004/113510 or WO 2007/065602.

The work leading to this invention has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under Grant Agreement No. 244093.

Figures Figure 1. The 6 different strains were submitted to a shake flask assay according to Example 1. Pantothenate production in g/l is shown on the y-axis. Samples were taken after 24 h and 48 h, respectively. The assay was performed using different media, i. e. SVYM (Fig. 1A), SMG (Fig. 1 B) or SRG (Fig. 1 C). Figure 2. The 6 different strains were submitted to a 24 deep-well assay according to Example 1 . Pantothenate production in g/l is shown on the y-axis. Samples were taken after 24 h and 48 h, respectively. The assay was performed using SVYM. Figure 3. 24 Deep-well assay was performed with the 6 different strains according to Example 1 . The x-fold increase of pantothenate production is shown on the y-axis. The production yield of Ref 1 was 1 . Samples were taken after 48 h. The production levels using SMG (Fig. 3A) was tested against production using SRG (Fig. 3B). Figure 4. BioLector^® assay was performed with the 6 different strains using SRG according to Example 1 . The x-fold increase of pantothenate production is shown on the y-axis. The production yield of Ref 1 was 1 . Samples were taken after 48 h.

Examples As otherwise indicated all strains, culture media as well as fermentation conditions are known in the art and described in more detail in WO 2004/ 1 13510 or WO 2007/065602 or WO 2004/ 1 1 1214.

[Example 1 : Strains and Media

Strains and plasmids. Bacillus subtil is strains of the present invention are derived from strain 1A747 (Bacillus Genetic Stock Center, The Ohio State

University, Columbus, Ohio 43210 USA), which is a prototrophic derivative of B. subtil is 168 (trpC2) (GenBank AL009126). The chloramphenicol-resistance gene (cat) cassette was obtained from plasmid pC194 (GenBank M19465, Cat# 1 E17 Bacillus Genetic Stock Center, The Ohio State University, Columbus, Ohio 43210 USA). The S aureus erythromycin resistance gene (GenBank V01278) was amplified from plasmid pDG646 (Guerout-Fleury et al. , 1995). The S aureus spectinomycin resistance gene (X03216) was amplified from plasmid pDG1726 (Guerout-Fleury et al. , 1995). The P_i5 and Ρ promoters of the B. subtil is bacteriophage SP01 (Lee et al. , 1980, Mol. Gen. Genet. 180:57-65) were obtained from derivatives of plasmid pX12 (Hiimbelin et al. , 1999, J. Ind.

Microbiol. Biotech. 22: 1 -7) containing the SP01 -15 and SP01 -26 promoters from RB50: : [pRF69] : : [pRF93] (Perkins et al. , 1999, J. Ind. Microbiol. Biotech. 22:8- 18). Strains PA12 (Ref 1 ), PA49 (Ref 2) and PA73 (Ref 3) are described in detail in WO 2007/ 131750. Strains Ref 4, Ref 5 and Ref 6 are further deregulated

n_ig^ p_rocj_uctj_on of pantothenate. This includes deregulation of glyA and a second copy of panBD for Ref 4, deregulation of glyA plus a second copy of panBD plus deregulation of the branch chain amino acid synthesis for Ref 5, and deregulation of glyA plus 3 or 4 copies of panBD plus deregulation of the branch chain amino acid synthesis for Ref 6. Production media. To test the B. subtil is reference strains (Ref 1 to Ref 6) for their B5 production capability 5 different media were used. The main characteristics of the media (carbon source, buffer, pH and YE and protein supplementation) are described in Table 1. The composition of MMGT minimal medium is described in WO 2004/1 13510 (as VFB MMGT), the SVY medium is described in WO 2002/066665, and the composition of SMG is described in WO 2007/065602.

The composition of SRG is as follows: 100 ml 10x SMS [20 g (NH₄)₂S0₄, 140 g K₂HP0₄, 60 g KH₂P0₄, 10 g Na₃citrate-H₂0, 2 g MgS0₄-7H₂0, add water to a final volume of 1 I, autoclave (20 min at 121 °C)], 10 ml 100x Trace Elements SMS medium [12.5 g MgCl₂-6H₂0, 0.55 g CaCl₂, 1 .35 g FeCl₂-6H₂0, 0.1 MnCl₂-4H₂0, 0.17 g ZnCl₂, 0.043 g CuCl₂-2H₂0, 0.06 g CoCl₂-6H₂0, 0.06 g Na₂Mo0₄-2H₂0, add water to a final volume of 1 I, autoclave (20 min at 121 °C)], 2 ml 50% glucose [50 g glucose add water to a final volume of 100 ml, filter sterilized], 36 ml 25% raffinose [25 g raffinose (D(+)-raffinose), add water to a final volume of 100 ml, filter sterilize], 10 ml 10% yeast extract and 842 ml sterile distilled water. This medium has been also described as RSM in WO2008/ 148575 (Example 2).

The composition of SVYM is as follows: Dissolve 62.78 g MOPS in 600 ml dist. water adjust pH to 7.2. Add 25 g Difco Veal Infusion Broth (Becton Dickinson, Catalog # 0344), 5 g Difco Yeast Extract (Merck, Catalog # 103753), 5 g Na- glutamate, 2.7 g (NH₄)₂S0₄, 1 ml PSTE-1000x solution [0.2 g MnCl₂-4H₂0, 0.15 g ZnS0₄-7H₂0, 0.2 g CoCl₂-6H₂0, 0.025 g CuS0₄-5H₂0, 0.075 g Na₂Mo0₄-2H₂0, add water to a final volume of 1 I, filter sterilized], add water to 770 ml, autoclave (20 min at 121 °C), after autoclaving add 100 ml 1 M K-phosphate buffer (pH 7.2), 120 ml 50% glucose and 10 ml 1 M MgS0₄-7H₂0.

Table 1 : Main characteristics of media tested

MMGT SRG SVY SVYM SMG

Carbon 22 g/l 1 g/l 30 g/l 60 g/l 60 g/l source glucose glucose glucose glucose glucose

9 g/l raffinose

E3uffer 0.12 M 0.2 M 0.3 M 0.3 M

phosphate phosphate MOPS MOPS

0.1 M 0.1 M phosphate phosphate

PH 6.8 7.0 7.0 7.2 7.2

YE and No 1 g/l YE 5 g/l YE 5 g/l YE 20 g/l Soy protein 10 g/l 10 g/L flour content peptone peptone

10 g/l lean 10 g/l lean

veal veal

Shake flask assay. Single colonies grown on selective TBAB plates or frozen glycerol working cell banks were used to inoculate 10 ml of VY medium (WO 2004/1 13510) in baffled Erlenmeyer flasks with urethane closures. The culture was grown overnight (about 17 h) at 37°C with an agitation of 300 rpm. 1 ml of overnight cultures were added to 40 ml of the different prewarmed production media and the cultures were grown at 37° C with an agitation of 300 rpm

(Kiichner shaker with humidity) in a 250-ml baffled Erlenmeyer flasks with urethane closures. After 24 and 48 h growth, (i) pH was determined, (ii) cell turbidity was measured at 600 nm (OD600nm), and (iii) for determination of sugar, pantothenate and 3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (HMBPA) side-product content using HPLC assay 2 ml of cultures were

centrifuged and 1 ml of supernatants were carefully transferred into Eppendorf tubes. 2 x 150 μΐ of supernatant per sample was used for HPLC analysis of sugar, B5 and HMBPA.

Deep well assay. Single colonies grown on selective TBAB plates or frozen glycerol working cell banks were used to inoculate in 24-DW plate containing 3 ml of VY medium per well. The bacteria were grown overnight (about 17 h) at 39°C with an agitation of 550 rpm and humidity of 85 % (Infors HT microtron shaker). Next day a new 24-DW plate was prepared containing 3 ml of the different production media per well. For inoculation 30 μΐ of overnight cultures were used. Plates were covered with a gas-permeable sealing film that supports the oxygen exchange. The cultures grew for 48 h in Infors HT microtron shaker as described above. After 24 h and 48 h growth, samples were taken for determining the sugar, B5 and HMBPA content by HPLC and the cell turbidity at 600 nm (OD600nm). In addition, the pH was determined after 48 h growth. BioLector^® assay. BioLector^® (m2p-Labs, 52499 Baesweiler, Germany) is a micro fermentation system allowing HTP screening for optimal fermentation parameters using 48 or 96 well plates with a volume of 100-2000 μΐ. It performs real-time monitoring of the biomass formation, pH and dissolved oxygen (p0₂). Using these data the optimum B5 production conditions can be determined.

Single colonies grown on selective TBAB plates were used to inoculate VY medium at 37° C with an agitation of 250 rpm (Innova 4000 shaker). After 8 h growth the cell turbidity was measured to determine the inoculation volume. The 48-well Flowerplate™ (m2p-Labs, 52499 Baesweiler, Germany) was filled with 1 ml of the different production media per well and inoculated with the pre-cultures to a starting

in triplicates. The plate was covered with a gas-permeable sealing film that supports the oxygen exchange and a second foil with a perforated sealing film on the top to reduce evaporation. The cultures were grown in the BioLector^® at 39 ° C for 48 h with an agitation of 900 rpm and humidity of 92%, the parameters biomass, pH and p0₂ were measured every 12 min. In addition, after 48 h sugar, B5 and HMBPA contents were determined by HPLC. Samples were prepared as described in herein.

HPLC analytics for vitamin B5 and HMBPA Chromatography of samples was performed on a Phenomenex LUNA C8 column, using an Agilent 1 100 HPLC system equipped with a thermostatted autosampler and a diode array detector. The column dimensions are 150 x 4.6 mm, particle size 5 micron. The column temperature was kept constant at 20° C. The mobile phase is a mixture of 0.1 % acetic acid (A) and methanol (B). Gradient elution is applied, ranging from 1 % B to 45% B in 15 minutes. The flow rate is 1 ml/ min. Pantothenate was monitored using UV absorption at 220 nm, and is eluted at approximately 9.6 min. The calibration range of the method is from 1 to 100 mg/l pantothenate. Other pantothenate related metabolites were also separated and the concentration was determined using this method.

HPLC assay for the analysis of sugars The concentration of glucose and raffinose in the culture broth was analyzed by an Agilent 1 100 series HPLC system using a quaternary pump, an autosampler a UV- and a refractive index detector. The separation was achieved on a CAPCELL PAK NH2 UG80 column (4.6 mm x 250 mm, 5μ) (Shiseido). The optimal column temperature was 35 °C. The mobile phase was a mixture of acetonitrile and Dl water at a 65/35 ratio. The flow rate was 1 .0 ml/min and the injection volume set to 5 μΐ. The refractive index signal was monitored and used for detection. The calibration range for — , , *— ! Q 5 _mg/_mi o 30 mg/ml. Example 2: Production of vitamin E35 in shake flask experiments

First the six reference strains (Ref 1 to Ref 6) and the five media described above were tested in traditional shake flask assays to determine the benchmarks and select the best media for HTP 24- DW plate assay. The best results were achieved with SRG medium and SMG medium (see Figure 1 B and 1 C). At 48 h the B5 production of the strain Ref 6 was 3.6-fold higher in SRG and 2-fold higher in SMG medium than for strain Ref 1 . The B5 yields on sugar were the highest for all reference strains in both SMG and SRG medium (not shown). All the sugar was consumed using both media compared to the other media. The pH was constant in SMG and dropped only slightly (from pH 7 to pH 6.7) in SRG. In contrast thereto, using MMGT minimal medium, the B5 yield on the consumed sugar was much less, the pH dropped dramatically (pH was -4.5 at 24 h) and only half of the glucose was consumed. All reference strains showed similar B5 production levels in SVY (not shown) and SVYM media (Figure 1A) despite of different genetic engineering levels of these strains, constant pH during the production and all the sugar was consumed.

Based on these results SRG and SMG media were selected for further analysis in 24- DW plate assay.

[Example 3: Production of vitamin B5 in 24 deep-well experiments Elevating the B5 production assay to high-throughput screening level, the shake flask assay was down -scaled to 24 deep- well plate assay.

A 24-well plate assay using SVYM showed a reduced pantothenate production after 48 h in the high engineered strains Ref 4 to Ref 6 (Fig. 2) in comparison to the shake flask assay (see Example 2). A 24-well plate assay was performed using SMG and the different reference strains (Fig. 2). Compared to shake flask assay (see Example 2) the amount of pantothenate production was 2-3 fold increased. Growth, ph and glucose consumption were comparable to the results of the shake flask assay.

Based on these results, a more detailed analysis of the induction of B5 was performed in 24- DW plate assay with the six reference strains tested in SRG and SMG media (Figure 3).

Surprisingly, using the SMG production medium, the B5 production levels of the more engineered strains decreased approximately 2-fold compared to the shake flask results. In addition, the B5 production performance of the strains was not reflecting the engineering levels (Fig. 3A). The data indicated that SMG is not appropriate for HTP B5 production assay.

Using the SRG medium in 24-DW plate similar results were obtained as in the shake flask experiments (Fig. 3B). The B5 production of strain Ref 6 was 4.3-fold higher than of strain Ref 1 (increasing B5 production with increasing strain engineering levels of the reference strains). The B5 yields on sugar were the comparable with the shake flask results (not shown).

[Example 4: Production of vitamin B5 in BioLector^® experiments The BioLector^® micro-fermentation system was applied to define optimal B5 production conditions such as pH and p0₂ content. BioLector^® performs realtime monitoring of the biomass formation, pH and p0₂. Using the SRG medium in BioLector^®, the reference strains showed a continuous increase of B5 production correlating to the engineering levels (Fig. 4). Similarly to the 24-DW plate assay the B5 production of strain Ref 6 was 4.1 -fold higher than that of strain Ref 1 . There was no oxygen limitation during the 48 h growth and the pH decreased only slightly. The BioLector^® results indicated no need for further optimization of the 24-DW plate assay in the context of p0₂.

Claims

1. A process for the production of pantothenate, wherein a strain of Bacillus is cultivated in a medium comprising a sugar selected from fructose, ribose, sucrose or glucose mixed with raffinose and wherein the starting concentration of the sugar selected from fructose, ribose, sucrose or glucose is 10 g/l or less.

2. A process according to claim 1 wherein the pantothenate is recovered from the medium.

3. A process according to claim 1 or 2, wherein the sugar is glucose.

4. A process according to any one of claims 1 to 3, wherein the ratio of sugar mixed with raffinose is in the range of 1 :2 to 1 :20.

5. A process according to claim 4, wherein the ratio is 1 :9.

6. A process according to any one of claims 1 to 5, wherein at least one of the genes encoding enzymes PanB, PanC, PanD, PanE and IlvD is overexpressed.

7. A process according to any one of claims 1 to 6, wherein the microorganism has a deregulated branch chain amino acid biosynthesis.

8. A process according to any one of claims 1 to 7, wherein the microorganism comprises at least 2 copies of panBD.

9. A process according to any one of claims 1 to 8, wherein the production of pantothenate is at least 10% higher compared to a process using standard medium.

10. A process according to any one of claims 1 to 9, wherein the pH is maintained at a constant level during cultivation period of 48 hours.

11. A process according to any one of claims 1 to 10, wherein all sugar is consumed after cultivation period of 48 hours.

12. Use of a medium comprising a mixture of glucose of 10 g/l or less and raffinose for production of pantothenate in a Bacill us strain, preferably Bacillus subtilis.