BRPI9816369B1 - FERMENTATIVE PRODUCTION OF USEFUL INDUSTRIAL COMPOUNDS USING CHEMICALLY DEFINED MEDIA - Google Patents

Description

FERMENTATIVE PRODUCTION OF INDUSTRIAL SCALE USE COMPOUNDS USING CHEMICALLY DEFINED MEDIA (Split Order PI 9807362-1, filed 02/20/1998) Field of the Invention The present invention relates to the field of fermentation, that is, to the fermentative production of compounds. such as primary or secondary metabolites, pharmaceutical proteins or peptides, or industrial enzymes.

Background of the Invention Many useful compounds are manufactured by fermentative production in large industrial scale fermenters, that is, the microorganism that produces a useful compound of interest is cultivated under controlled conditions in a 10 to 300 m3 fermenter. In today's industrial scale fermentation processes, the production organism is typically fermented in a complex fermentation medium. A complex medium means a medium comprising a source of complex nitrogen and / or carbon, such as soybean meal, cottonseed meal, corn infusion liquor, yeast extract, casein hydrolyzate, molasses and others.

The advantages of complex media are that the constituent complex raw materials are not expensive, readily available and form a complete or nearly complete source of nutrients for the microorganism, containing a source of carbon and nitrogen, as well as vitamins and minerals. In addition, the mixture of biological macromolecules present in complex raw materials, such as proteins, carbohydrates, lipids and others, must be degraded by enzymes excreted by the microorganism before their consumption. As a result, small consumable molecules become uniformly available throughout the fermenter and during the fermentation process, thereby avoiding concentration gradients and mixing problems and keeping the level of these small consumable molecules below repressive concentrations. Moreover, these macromolecules, as well as organic acids also present in complex media, give the medium a buffering capacity, thus facilitating pH control.

In addition to these advantages, complex fermentation media have several important disadvantages. More importantly, complex raw materials have a chemically undefined composition and varying quality, for example due to seasonal variations and different geographical origins. Because the composition of fermentation media has an important influence on fermentation parameters such as viscosity, heat transfer and oxygen transfer, complex raw materials are a major cause of process variability. In addition, it impairs downstream processing and may adversely influence the quality of the final product. For example, fermentation broths, in particular filamentous microorganisms, may have a lower filtration capacity when using complex raw materials.

Complex raw materials may also contain compounds that undesirably accumulate in or are co-isolated with the final product. Heavy metals, pesticides or herbicides are examples of undesirable compounds that may be present in complex raw materials. In addition, complex raw materials may contain or may lead to toxin formation.

Further disadvantages are that complex media generate an unpleasant smell during sterilization and produce undesirable waste streams. Despite the disadvantages identified above in connection with the use of complex media, these media are still preferred for large scale industrial fermentation processes. There are several reasons why media not containing complex raw materials, ie chemically defined media, have not been considered for use in industrial scale fermentation processes. An obvious reason is found in the advantages associated with the use of complex media. More importantly, product yields that would be obtained using chemically defined media on an industrial scale were typically considered to be substantially lower than those obtained using media containing complex raw materials. In addition, highly producing microbial strains that have been developed for industrial processes in complex media may not retain their good performance in chemically defined media. One reason for poor performance in a chemically defined medium may be that current industrial strains have undergone several rounds of mutagenesis and selection without regard to their performance in chemically defined media.

Chemically defined media have so far been applied for research purposes only, ie laboratory cultures in petri dishes and / or shake flasks, or on a relatively small fermentative scale, typically not exceeding a volume of about 20-40 µm. 1. See, for example, the fermentative production of secondary metabolites such as penicillin (Jarvis and Johnson, J. Am. Chem. Soc. 69, 3010-3017 (1947); Stone and Farrell, Science 104, 445-446 (1946). ); White et al., Arch. Biochem. 8, 303-309 (1945)), clavulanic acid (Romero et al., Appl. Env. Microbial. 52, 892 - 897 (1986)) and erythromycin (Bushell et al. Microbial 143, 475-480 (1997)).

However, investigations into the use of chemically defined media in these small research scales provide no teaching for those skilled in the art as to the applicability of these media to large scale industrial fermentation processes for production purposes, typically with a volume scale of about 10 m3 or more.

To avoid the problems associated with using conventional recipes for complex media in current industrial practice, it would be desirable to apply chemically defined recipes to industrial scale fermentations.

Here we describe the use of chemically defined media for industrial scale fermentation processes which allow - in combination with a suitable strain - the production of useful compounds, such as primary or secondary metabolites, pharmaceutical proteins or peptides, or industrial enzymes, in a economically attractive yield.

SUMMARY OF THE INVENTION The present invention provides an industrial process for producing a useful compound comprising the steps of fermentation of a microbial strain in a fermentation medium that is a chemically defined medium consisting essentially of chemically defined constituents and of recovering the same. useful compound of the fermentation broth. The present invention also provides a process for the preparation and / or enhancement of a microbial strain producing a useful compound of interest which is capable of being fermented on an industrial scale in a chemically defined medium, comprising the steps of: submitting a strain suitable for a mutagenic treatment selected from the group of physical and chemical mutagenic media, and / or DNA transformation, - screening of the resulting mutants and / or transformants for their growth performance in a chemically defined medium and their level of production. useful compound of interest, - selection of mutants with similar or better growth performance in a chemically defined medium and / or better level of production of the useful compound of interest as compared to the source strain.

Detailed Description of the Invention The present invention describes the use of chemically defined fermentation media for the industrial scale fermentation of a suitable microbial strain, the appropriate microbial strain being capable of producing a useful compound. Throughout the description of the invention, an industrial scale fermentation process or industrial process should be understood to encompass a volume scale fermentation process that is> 10 m3, preferably> 25 m3, more preferably> 50 m3, more preferably still> 100 m3. The term "chemically defined" is to be understood to be used for fermentation media that are essentially composed of chemically defined constituents. A fermentation medium that is essentially composed of chemically defined constituents includes a medium that does not contain a complex carbon and / or nitrogen source, that is, which does not contain complex raw materials of chemically undefined composition. A fermentation medium that is essentially composed of chemically defined constituents may also include a medium comprising an essentially small amount of a complex nitrogen and / or carbon source, an amount as defined below that is typically not sufficient to maintain growth. microorganism and / or to ensure that sufficient biomass is formed.

In this regard, complex raw materials have a chemically undefined composition because, for example, these raw materials contain many different compounds, including complex heteropolymeric compounds, and have variable composition due to seasonal variations and differences in geographical origin. Typical examples of complex raw materials that function as a source of complex carbon and / or nitrogen in fermentation are soybean meal, cottonseed meal, corn infusion liquor, yeast extract, casein hydrolyzate, molasses and others.

An essentially small amount of a complex carbon and / or nitrogen source may be present in the chemically defined medium according to the invention, for example as an inoculum remnant for the main fermentation. The inoculum for the main fermentation is not necessarily obtained by fermentation in a chemically defined medium. More often, remnants of the inoculum will be detectable by the presence of a small amount of a complex nitrogen source in the chemically defined medium for the main fermentation.

It may be advantageous to use a complex carbon and / or nitrogen source in the inoculum fermentation process for the main fermentation, for example to accelerate biomass formation, i.e. to increase the growth rate of the microorganism and / or facilitate internal pH control. For the same reason, it may be advantageous to add an essentially small amount of a complex carbon and / or nitrogen source, for example yeast extract, to the early stage of the main fermentation, particularly to accelerate biomass formation at the early stage of the fermentation process. fermentation.

An essentially small amount of a complex carbon and / or nitrogen source that may be present in the chemically defined medium according to the invention and defined as an amount of at most about 10% of the total amount of carbon and / or nitrogen ( Kjeldahl N) which is present in the chemically defined medium, preferably an amount of at most 5% of the total amount of carbon and / or nitrogen, more preferably an amount of at most 1% of the total amount of carbon and / or nitrogen. More preferably, no source of carbon and / or complex nitrogen is present in the chemically defined medium according to the invention.

It is to be understood that the term "chemically defined medium" as used herein includes a medium in which all necessary components are added to the medium prior to the start of the fermentation process, and also includes a medium in which part of the components required are added before the start and part and added to the medium during the fermentation process. The present invention also discloses that microbial strains are capable of converting, on an industrial scale, simple raw materials of chemically defined media into an economically attractive amount of product. Surprisingly, it has been found that the productivity of microbial strains in chemically defined media, when measured on an industrial scale, can be comparable, or in some cases even higher, to their productivity in complex media.

An additional advantage of using chemically defined media is that the oxygen transfer from the gas phase to the liquid phase and the carbon dioxide transfer from the liquid phase to the gas phase are substantially improved compared to the use of complex media. As those skilled in the art are aware, dissolved oxygen and dissolved carbon dioxide concentrations are two important factors for the escalation of a fermentation process, and may determine the economic feasibility of an industrial process. The best mass transfer obtained using chemically defined media can be attributed to the absence in these media of substantial amounts of compounds that promote gas bubble coalescence. Coalescing promoting compounds can be found, for example, among certain hydrophobic and / or polymeric compounds present in complex raw materials. Coalescence of gas bubbles typically results in a lower mass transfer coefficient (van't Riet and Tramper, in: Basic Bioreactor Design, pp. 236 - 273 (1991)). Oxygen transfer is often a limiting factor in fermentation processes, particularly in filamentous microorganism fermentations. The improved oxygen transfer capability obtained when fermentation is performed using a chemically defined medium according to the invention provides a much cheaper way to optimize productivity than equipment investments such as energy ingress, oxygen enrichment. inlet air or fermenter pressure.

In industrial fermentation processes, filamentous microorganisms such as filamentous bacteria such as Actinomycetes or filamentous fungi such as Penicillium or Aspergillus are typically grown with a pellet morphology. In this regard, proteins and peptides present in complex fermentation media have a tendency to produce fuzzy pellets, which easily dissolve into very long, branched hyphae dispersed as a result of the high growth rates that are typically obtained with the pellet. use of complex media. Therefore, a fuzzy pellet morphology can usually cause an undesirable high broth viscosity. The use of chemically defined media has a favorable influence on morphology, for example to produce a stiffer pellet, which does not easily melt during fermentation. Thus, a significant decrease in the viscosity of filamentous fermentation broths can be achieved by the use of chemically defined media. As a low viscosity fermentation broth and advantageous for product formation, viscosity control is of the utmost importance in industrial scale fermentation processes.

Another advantage of using chemically defined media is found in downstream processing of the product. For certain strain-product combinations, particularly when filamentous strains are fermented, downstream processing is significantly improved using chemically defined media.

A chemically defined medium for use in the process of the invention should typically contain so-called structural elements and so-called catalytic elements.

Structural elements are those elements that are constituents of microbial macromolecules, ie hydrogen, oxygen, carbon, nitrogen, phosphorus and sulfur.

The structural elements hydrogen, oxygen, carbon and nitrogen are typically contained in the carbon and nitrogen source. Phosphorus and sulfur are typically added as phosphate and sulfate and / or thiosulfate ions. The type of carbon and nitrogen source that is used in the chemically defined medium is not critical to the invention as long as the carbon source β nitrogen is essentially chemically defined.

Preferably, a carbon source is selected from the group consisting of carbohydrates such as glucose, lactose, fructose, sucrose, maltodextrins, starch and inulin, glycerol, vegetable oils, hydrocarbons, alcohols such as methanol and ethanol, organic acids such as acetate and higher alkanoic acids. More preferably, a carbon source is selected from the group consisting of glucose, sucrose and soybean oil. Most preferably, the carbon source is glucose. It is to be understood that the term "glucose" includes glucose syrups, that is, glucose compositions containing glucose oligomers in defined amounts. The nitrogen source is preferably selected from the group consisting of urea, ammonia, nitrate, ammonium salts such as ammonium sulfate, ammonium phosphate and ammonium nitrate and amino acids such as glutamate and lysine. More preferably, the nitrogen source is selected from the group consisting of ammonia, ammonium sulfate and ammonium phosphate. Most preferably, the nitrogen source is ammonia. The use of ammonia as the nitrogen source has the advantage that ammonia can also act as a pH control agent. If ammonium sulfate and / or ammonium phosphate are used as a source of nitrogen, part or all of the microorganism's sulfur and / or phosphorus requirements can be met.

Catalytic elements are those elements that are enzyme constituents or enzyme cofactors. These elements are, for example, magnesium, iron, copper, calcium, manganese, zinc, cobalt, molybdenum, selenium, boron.

Next to these structural and catalytic elements, cations such as potassium and sodium ions must be present to act as a counterion and to control pH and intracellular osmolarity.

Compounds that may optionally be included in a chemically defined medium are chelating agents, such as citric acid, and buffering agents, such as mono- and dipotassium phosphate, calcium carbonate, and the like. Buffering agents are preferably added when dealing with processes without an external pH control. In addition, a defoaming agent may be dosed prior to and / or during the fermentation process.

Macromolecules and organic acids that are present in complex media confer buffering capacity to these media. Due to the absence of these compounds in chemically defined media, pH control is more difficult in chemically defined media than in complexes. The present invention shows that a pH control wherein: an acid or base may be metered, depending on the pH development in the broth, permits an appropriate pH profile in a chemically defined industrial scale process.

Vitamins refer to a group of structurally unrelated organic compounds that are required for normal metabolism of microorganisms. Microorganisms are known to vary widely in their ability to synthesize the vitamins they require. A vitamin should be added to the fermentation medium of a microorganism unable to synthesize this vitamin. Typically, chemically defined fermentation media for yeast or bacteria or certain lower fungi, for example Mucorales, may be supplemented with one or more vitamins. Higher fungi most often have no vitamin requirements.

The vitamins are selected from the group of thiamine, riboflavin, pyridoxal, nicotinic acid or nicotinamide, pantothenic acid, cyanocobalamin, folic acid, biotin, lipoic acid, purines, pyrimidines, inositol, choline and hemines.

Structural and catalytic elements and optionally vitamins are required for the growth of the microorganism, that is, for the formation of biomass. The amount of compounds required, i.e. compounds comprising structural and catalytic elements and, optionally, vitamins to be added to the chemically defined medium will depend mainly on the amount of biomass to be formed in the fermentation process. The amount of biomass formed may vary widely, typically from about 10 to about 150 g / l fermentation broth. In general, fermentations producing less than about 10 g / l biomass are not industrially relevant.

In addition, the optimal amount of each constituent of a defined medium, as well as which compounds are essential and which are non-essential, will depend on the type of microorganism that is fermented in a defined medium, the amount of biomass and the product to be formed. . The use of chemically defined media, therefore, advantageously allows for varying the concentration of each medium component independently of the other components, thereby facilitating the optimization of medium composition.

For product formation, it may be necessary to supplement the chemically defined medium with additional compounds and / or to increase the concentration of certain compounds already present in the chemically defined medium above the level required for growth of the microorganism. The function of such compounds may be to induce and / or enhance the production of a desired compound by the microorganism, or may function as a precursor to a desired compound.

Examples of compounds to be supplemented and / or added in an increased amount to a chemically defined medium are: sulfate in an increased amount for the production of β-lactam compounds, nitrogen-containing compounds in an increased amount for the production of amino acids, particularly amino acids. basic, phenylacetic acid for the production of penicillin G, phenoxyacetic acid for the production of penicillin V, adipic acid for the production of adipil-7-ADCA and adipil-7-ACA, propionic acid for the production of erythromycin.

In an industrial fermentation process according to the invention, the total amount of carbon source added to the chemically defined medium, expressed as amount of carbon / liter of medium, may range from 10 to 200 g of C / l, preferably of 20 to 200 g C / l. The total amount of nitrogen source added to the chemically defined medium may range from 0.5 to 50 g of N / l, preferably from 1 to 25 g of N / l, where N is expressed as Kjeldahl nitrogen. The ratio of carbon to nitrogen source in a fermentation can vary considerably, thus determining the optimal ratio of carbon to nitrogen source to the elemental composition of the product to be formed.

Additional compounds required for the growth of a microorganism, such as phosphate, sulfate or residual elements, should be added using the concentration ranges given in Table 1 as a guideline. The concentration ranges of these additional compounds may vary between different classes of microorganisms, that is, between fungi, yeast and bacteria.

Vitamin concentrations are generally in the range of 0.1 (biotin) to 500 (myoinositol) mg / 1. Typically, the amount of medium components required for the growth of a microorganism can be determined with respect to the amount of source of carbon used in fermentation because the amount of biomass formed will be determined basically by the amount of carbon source used.

Table 1. Typical concentration ranges of media components, besides carbon and nitrogen source, required for growth of various classes of microorganisms (g / 1) · ________________________________________________________ 1 The basal amount of phosphate required will be 0.5 - 1 % of dry weight of biomass. For relatively small scale batch processes, extra phosphate will be required for pH control. 2 Sulphate can also be dosed with the titrator such as K + or Na +. 3 Sulphate may be (partially) substituted by chloride as counterion in the residual elements, or vice versa. 4 For some residual elements, lower boundaries are difficult to define. Their requirement, for example, can be met by the presence in other components of the medium, for example ferrous sulfate, water, small amounts of yeast extract and others. Phosphate and sulfate are added as potassium, ammonium and / or sodium salts, preferably K> NH 4> Na.

An industrial fermentation process according to the invention using a chemically defined medium may be carried out as a batch, repeated batch, fed batch, repeated or continuous batch batch fermentation process.

In a batch process all medium components are added directly as a whole to the medium before the fermentation process begins.

In a repeated batch process, a partial stock collection occurs, accompanied by a partial supplementation of the complete medium, optionally repeated several times.

In a feed batch process, none or part of the compounds comprising one or more of the structural and / or catalytic elements is added to the medium prior to the start of fermentation, and all or the remainder, respectively, of the compounds comprising one or more of the compounds. structural and / or catalytic elements and fed during the fermentation process. Compounds that are selected for feed may be fed together or separated from each other in the fermentation process.

Particularly in a fermentation process wherein the original fermentation medium is diluted about two or more times by a compound feed comprising one or more of the structural elements, the feed may also comprise additional catalytic elements and media components next to the feed elements. structural.

In a preferred continuous or repeated feed batch fermentation process, the starting medium is complete and additionally fed during fermentation. The starting medium may be fed together with or separate from the structural element feed (s).

In a repeated feed batch process, part of the fermentation broth comprising the biomass is removed at regular intervals, whereas in a continuous process, part of the fermentation broth is removed continuously. The fermentation process is thus replenished with a portion of fresh medium corresponding to the amount of fermentation broth removed.

In a preferred embodiment of the invention, a feed batch or repeat feed batch process is applied wherein the carbon and / or nitrogen and / or phosphate source is fed to the fermentation process. In a more preferred embodiment, the carbon and nitrogen source is fed to the fermentation process.

More preferably, the source of carbon and nitrogen as well as phosphate are fed. In this regard, a preferred carbon source is glucose, and a preferred nitrogen source is ammonia and / or ammonium salts. The use of a feed batch process typically allows the use of a considerably greater amount of carbon and nitrogen source than is used in a batch process. Specifically, the amount of carbon and nitrogen source applied to a batch feed process can be at least about twice as large as the largest amount applied to a batch process. This in turn leads to a considerably larger amount of biomass formed in a feed batch process.

A further aspect of the present invention relates to the downstream processing option of the fermentation broth.

Upon completion of the fermentation process, the useful product may optionally be recovered from the fermentation broth using standard technology developed for the useful compound of interest. The relevant downstream processing technology to be applied in this manner depends on the nature and cellular location of the useful compound. Firstly the biomass is separated from the fermentation fluid using, for example, centrifugation or filtration. The useful compound is then recovered from the biomass if the useful product is accumulated within or associated with microbial cells. Otherwise, when the useful product is excreted by the microbial cell, it is recovered from the fermentation fluid. The use of a chemically defined medium in the industrial fermentative production of a useful compound of interest yields a major advantage in downstream processing because the amount of byproducts is substantially less than when using complex media. In addition, product quality is improved as no undesirable by-products are co-isolated with the compound of interest.

In yet another aspect of the present invention, a suitable strain is identified for an industrial fermentation process using a chemically defined medium.

A suitable microbial strain for an industrial fermentation process using a chemically defined medium may be any wild type strain producing a useful compound of interest, provided that the wild type strain has good growth performance in a chemically defined medium. In addition, a microbial strain suitable for an industrial fermentation process using a chemically defined medium may be a strain that is obtained and / or enhanced by subjecting a strain of original interest to classical mutagen treatment or recombinant DNA transformation, also provided that the resulting mutated or transformed microbial strain has good growth performance in a chemically defined medium. Thus, it will depend on the growth performance of the original strain in a chemically defined medium whether the resulting mutant or transformed strains should have similar growth performance in a chemically defined medium compared to the source strain. It is understood that a microbial strain has a good growth performance in a chemically defined medium when the strain has a specific growth rate (μ) in a chemically defined medium that is> 0.05 h'1, preferably> 0, 1h "1, more preferably> 0.2h" 1, more preferably still> 0.4h'1. The growth performance of a microbial strain in a chemically defined medium is conveniently analyzed by fermentation of the strain in a chemically defined medium on a relatively small scale, for example a shaking balloon culture and / or a 10 1 laboratory fermentation. It is preferable to include a 10 1 laboratory fermentation with pH, temperature and oxygen concentration control in the growth performance analysis.

In one embodiment of the invention, microbial strains that can be fermented in a chemically defined medium are obtained and / or perfected by subjecting a strain of original interest to classical mutagen treatment using physical means such as UV irradiation or a chemical mutagen. suitable, such as N-methyl-N'-nitro-N-nitrosoguanidine or ethyl methane sulfonate. In another embodiment of the invention, microbial strains that can be fermented in a chemically defined medium are obtained and / or enhanced by subjecting a strain of original interest to recombinant DNA technology, whereby the original strain is transformed with one or more genes. of interest.

In general, the present invention contemplates two groups of original strains of interest for submission to classical mutagenesis and / or DNA transformation. In one embodiment of the invention, an original strain is selected from the group of strains that has good growth performance in a chemically defined medium but needs to be refined with respect to its production level of the compound of interest. In another embodiment of the invention, an original strain is selected from the group of strains that has a high level of production of a compound of interest, but has relatively poor growth performance in a chemically defined medium. Microbial strains with a specific growth rate that is less than about 0.05 h -1 are understood to have relatively poor growth performance in a chemically defined medium.

Both processes, the classical mutagenic treatment and the DNA transformation process, are followed by screening of the resulting mutants or transformants for their growth performance in a chemically defined medium, as well as their level of production of a compound of interest. Mutant or transformant strains that have good growth performance in a chemically defined medium and / or a better level of production of a compound of interest are selected compared to the source strain.

It should be noted that some microbial strains, in particular industrial strains that have already undergone extensive mutagenic treatment to improve production levels, may perform poorly or may not grow in a chemically defined medium. This poor performance or lack of growth of a mutagenized strain may be caused by the fact that growth in a chemically defined medium has never been applied as a criterion for the selection of appropriate mutants. For example, the mutagenized strain may have a mutation that causes an unknown growth requirement (unknown auxotrophic mutation).

Microbial strains that are suitable for industrial fermentation using a chemically defined medium include filamentous and non-filamentous strains. For example, microbial strains that are suitable for fermentation in a chemically defined medium include fungal strains such as Aspergillus, Penicillium or Mucorales, yeast strains such as Saccharomyces, Pichia, Phaffia or Kluyveromyces, and bacterial strains such as Actinomycetes. The use of chemically defined media according to the invention is particularly advantageous for the industrial fermentation of filamentous microorganisms. The process according to the invention using a chemically defined medium suitable for the industrial-scale fermentative production of any useful compound of interest, including primary or secondary metabolites, pharmaceutical proteins or peptides, or industrial enzymes. Preferred useful compounds are secondary metabolites such as antibiotics or β-lactam compounds, particularly β-lactam antibiotics.

Examples of strain-product combinations include A. niger, for example, A. niger CBS 513.88, for amyloglycosidase, A. oryzae for α-amylase, A. terreus, for example, A. terreus CBS 456.95, for lovastatin, Mortierella alpina for arachidonic acid or lipid containing arachidonic acid, Mucro miehei for protease, P. chrysogenum, for example, P. chrysogenum CBS 455.95 or other suitable strains, for β-lactam compounds (penicillin G or V), Streptomyces clavuligerus, for example S. clavuligerus ATCC 27064, for clavulanic acid, Pichia ciferrii, for example, P. ciferrii NRRL Y-1031 F-60-10, for tetraacetylphyphosphingosine, Phaffia rhodozyma, for example, P. rhodozyma CBS 6938, for astaxanthin, Saccharopolraea erythra erythromycin, K. lactis for lactase, Streptomyces natalensis for natamycin. The present invention also contemplates the use of microbial strains that are transformed with one or more functional genes of interest, resulting in a transformed strain that may overexpress a product that is already produced by the strain, or resulting in a transformed strain may express non-naturally produced. by the strain.

Therefore, it is left to the choice of those skilled in the art which strain will be selected for transformation as long as the selected strain has good growth performance in a chemically defined medium. For example, you can select a strain for transformation that has already undergone one or more mutagenic treatments. Alternatively, a mutagenized or wild type strain may be selected. Following analysis of the expression level of a desired compound, transformants obtained after transformation of a selected strain with one or more functional genes of interest should be analyzed for their growth performance in a chemically defined medium.

Examples of recombinant strains that produce a product not naturally produced by the strain are: Streptomyces lividans, for example, S. lividans TK21, containing an expression cassette that allows expression of glucose isomerase, the gene that encodes glucose isomerase, e.g. from Actinoplanes missouriensis, * Penicillium chrysogenum, e.g. P. chrysogenum CBS 455.95, containing one or more expansionase expression cassettes and optionally a hydroxylase and / or an acetyltransferase, the genes encoding the expandase, hydroxylase and acetyltransferase originating, for example, from Acremonium chrysogenum or Streptomyces clavuligerus, which allows the production of cephalosporin compounds such as 7-ADCA or 7ACA using adipic acid (see EP 532341) or 3-carboxymethylthiopropionic acid (see WO 95 / 04148) as a side chain precursor, * Aspergillus niger, for example Aspergillus niger CBS 513.88, containing an expression cassette allowing and the expression of human lactoferrin (see WO 93/22348) or bovine chymosin. * Kluyveromyces lactis, containing an expression cassette allowing expression of bovine chymosin or phospholipase A2, insulin or recombinant human albumin.

Examples of recombinant strains that overproduce an enzyme already produced by the strain are: A. niger, for example, A. niger CBS 513.88, containing an expression cassette that allows overexpression of phytase (see EP 420358) or endoxylanase I (see EP 463706). The present invention is exemplified by an industrial scale fermentation process using a chemically defined medium for the production of glucose isomerase by a recombinant Streptomyces strain, and by the advantageous use of chemically defined medium for large scale Penicillium fermentation in comparison. with complex media.

Additional examples refer to chemically defined media that can be used to measure the growth performance and productivity of a strain of interest when grown on this medium on a small scale to identify microbial strains that are suitable for the fermentative production of a useful compound. on an industrial scale in a chemically defined medium.

Brief Description of the Figures Figure 1. pWGx.GIT Scheme.

Figure 2. Development of total glucose isomerase produced during fermentation.

Example 1 Industrial Glucose Isomerase Production Using Streptomyces lividans Construction of a Glucose Isomerase-producing Streptomyces Strain The Actinoplanes missouriensis glucose isomerase gene was originally cloned as a 5.0 kb DNA fragment into E. coli K12 strain JM101.

A 1.7 kb fragment internal to the 5.0 kb fragment was found to represent the complete coding sequence for A. missouriensis glucose isomerase and its upstream regulatory region (see also Amore and Hollenberg (1989), Nucl. Acids Res. 17, 7515).

A glucose isomerase mutant exhibiting enhanced thermostability was obtained by altering within the glucose isomerase gene the triplet AAG encoding lysine at position 253 of the glucose isomerase protein for CGG encoding arginine (Quax et al. (1991)). , Bio / Technology 9, 738-742).

For Streptomyces cloning, plasmid pi 1486 (Ward et al. (1986), Mol. Gen. Genet. 203, 468-478) was used as a vector. The 1737 base pair A. missouriensis DNA fragment encoding glucose isomerase was combined with the large piJ486 Psti DNA fragment. The resulting plasmid, called pWGx.GIT, essentially contained the replicon region of plasmid piJIO1, the thioestreptone resistance gene, and the GIT-encoding A. missouriensis DNA fragment. A schematic map of pWGx.GIT is given in Figure 1. The glucose isomerase producing strain was constructed by transformation of Streptomyces lividans strain TK21 (Hopwood et al. (1985), Genetic Manipulation of Streptomyces: A Laboratory Manual. The John Innes Foundation , Norwich, England) with plasmid pWGx.GIT.

Industrial Glucose Isomerase Production A functional cell bank of a production strain constructed as mentioned in Example 1 was prepared by choosing a thioestreptone resistant colony and growing it in 20 ml of thiostreptone-containing Sodium Tryptone Broth (SO mg / 1). in a 100 ml stirred flask at 30 ° C for 40 - 48 hours and stirring at 280 rpm.

A mycelium equivalent to 1 ml of the functional cell bank (fresh or stored as frozen mycelium at -80 ° C) was inoculated into 100 ml of inoculum growth medium in a 500 ml shake flask containing 16 g / 1 of Bactopepton (Difco 0123/01), 4 g / 1 soytone Bacto (Difco 0436/01), 20 g / 1 casein hydrolyzate (Merck 2239), 10 g / 1 dipotassium phosphate.3 aq (Merck, Reagent Anal.), 16.5 g / l aq glucose, 0.6 g / l soybean oil and 50 mg / l thioestreptone. The pH of the medium is adjusted to 7.0 with sodium hydroxide and / or sulfuric acid prior to sterilization. Glucose and thioestreptone are added separately after sterilization. Thioestreptone is dissolved in DMSO at a concentration of 50 g / l and sterilized by filtration on a 0.2 m Nalgene filter. The culture is grown for 24 hours at 30 ° C on an incubator shaker at 280 rpm. 50 ml of the fully grown culture is transferred to 6 1 of the second-stage inoculum growth medium, with a composition similar to the aforementioned medium except for a double glucose concentration (33 g / 1 glucose aq), defoamer extra (SAG5693, 0.6 g / 1; a Basildon Company silicic defoamer) and without thioestreptone. The glucose is separately sterilized separately in a 50% solution and added after sterilization of the medium (60 minutes, 121 ° C). The culture is grown for 36 hours in a sterile 840 l aerated sterile air column / hr, with a nozzle containing 4 holes with a diameter of 2 mm, and the temperature maintained at 22 ° C. Alternatively, this step may be performed in shaking buckets (eg 12 x 500 ml medium in Erlenmeyer buckets with 2 L bulkheads) with similar inoculation ratios and shaken at 280 rpm on an orbital incubator shaker. The complete growth culture is aseptically transferred to an inoculum fermenter containing 4.5 m3 inoculum medium containing 16.3 kg citric acid. 1 aq, 70.8 g of iron sulfate. 7 aq, 109 g of zinc sulfate. 7 aq, 109 g of manganese sulfate. 1 aq, 32.7 g cobalt dichloride.6 aq, 5.45 g disodium molybdate.2 aq, 5.45 g boric acid, 5.45 g copper sulfate.5 aq, 10.9 kg diammonium sulfate, 10.9 magnesium sulfate. 7 aq, 463 g of calcium chloride aq, 1,090 g of soybean oil, 21.8 kg of monopotassium phosphate and 139 kg of glucose. 1 aq and 5.9 kg of yeast extract (brewer's yeast) with 10% Kjeldahl nitrogen based on dry weight). The medium is prepared as follows: All components except glucose are loaded in the sequence indicated in approximately 2,700 l of tap water. The pH is adjusted to 4.5 with sodium hydroxide and / or phosphoric acid, and the medium is sterilized at 122 ° C for 60 minutes. Glucose is sterilized in 1,000 l of water at pH 4.5 for 60 minutes at 122 ° C in a separate container. Upon cooling of both parts, glucose is aseptically transferred to the inoculum container. After mixing both parts, the pH is adjusted to 7.0 3 with ammonia, and the volume is adjusted with 4.5 m3 sterile water. The fermentation temperature is controlled at 30 ° C, and the fermenter is aerated at 0.5 - 1.0 vvm, while the pH is maintained at 7.0 ± 0.1 with gaseous ammonia, and excess pressure is maintained. at 1.3 - 1.4 bar. Foaming is controlled, if necessary, with a sterile mixture of soybean oil and a silicon defoamer such as SAG5693 at a ratio of 3: 1. Oxygen concentration is maintained above 25% of air saturation by agitator speed adjustment (0.5 to 3 Kw / m3). The culture is transferred to the main fermentation before all glucose is consumed (as in all previous growth phases) and before the oxygen uptake rate exceeds a level of 30 mmol / l broth volume.h. The main fermentation medium contains 245.1 kg of citric acid. 1 aq, 1.062 g of iron sulfate. 7 aq, 1.634 g zinc sulfate.7 aq, 1.634 g manganese sulphate.1 aq, 490 g cobalt dichloride. 6 aq 1 82 g of disodium molybdate.2 aq 1 82 g of boric acid, 82 g of copper sulphate.5 aql 16314 kg of diammonium sulphate, 16314 of magnesium sulphate.7 aq, 6194 g of calcium chloride 2 aql 16.3 g of soybean oil, 327 kg of monopotassium phosphate, 880 kg of brewer's yeast extract (10% Kj-N based on dry weight) and 556 kg of glucose. 1 aq. The medium is prepared as described for inoculum fermentation (glucose is sterilized separately). For glucose, a DE-95 sugar syrup may alternatively be used. Medium volume before inoculation and 65 m3 after pH is corrected to 710 with ammonia.

A glucose feed is prepared at 275 to 550 g glucose / l feed solution, either as glucose.1 aq or as glucose equivalents of a DE> 90 syrup. PH adjusted to 4.5 - 5.0 with a solution of phosphoric acid. Sterilization is done by batching (122 ° C, 45 minutes) or continuously by a thermal shock or filter set. The main fermentation is controlled at 30 ° C ± 015 and the pH at 710 ± 01 1 (by a pH control using ammonia and phosphoric acid). The air flow is adjusted to 0.5-1.5 vvm, preferably 0.7 vvm, the excess pressure is 0.5-1.5 bar, and the fermenter is stirred with Rushton turbines at an intensity of 01 5 to 3 Kw / m3 1 to prevent oxygen concentration from falling below 0.2 mmoles / 1 as measured at the bottom stirrer height. Glucose feeding begins when there is a sudden drop in oxygen uptake rate, and dissolved oxygen concentration increases, as does the pH, which goes from 6.9 to 7.1. The glucose concentration in the broth should be << 0.2 g / 1 at this time. The glucose feed rate is equivalent to 93 kg of glucose / h, initially rising linearly to 186 kg / h 64 hours after the start of feeding. After 100 hours of feeding at 186 kg / hr, the feeding rate is increased to 298 kg of glucose / hr until approximately 200 hours of feeding. Foaming is controlled by dosing sterile soybean oil at 5.5 kg / hr or alternatively in 45 kg injections every 8 hours during the first 100 hours of fermentation. If necessary, an additional foam control is made with a mixture of soybean oil and a silicon defoamer such as SAG471 (Basildon silicon defoamer) at a ratio of 3: 1. Ammonia concentration is maintained between 750 and 1,500 mg / 1, measured every 12 hours and sterile ammonium sulfate added in portions equivalent to 500 mg of ammonia / 1 as soon as the level is below 1 000. mg / 1. The phosphate concentration in the culture filtrate should be maintained above 500 mg P04 / 1 by the addition of sterile monopotassium phosphate in 500 mg / 1 equivalent portions. Glucose isomerase production can be measured as harvested and purified broth protein, followed by protein determination procedures known in the art or assayed in an enzyme assay applied to a stabilized broth sample. The broth samples are stabilized by weighing 2 g of broth and adding 5 ml of stabilizer solution containing 12 g / l trishydroxymethylaminomethane and 2.4 g / l CoCl2.6 aq, which is subsequently heated for 30 minutes. minutes at 73 ° C. After cooling, 42 ml of stabilized sample is mixed with 0.8 ml of glucose solution (containing 27.25 g / 1 Tris / HCl buffer, pH 81 2, glucose 67 56 g / 1 MgC12.6 aq, 22 33 g / l Na2-EDTA.2 aq and 5 mg / l Triton X-100) and incubated at 60 ° C. Activity is determined by measuring glucose to fructose conversion rates, expressed as GU / g. (1 GU is the amount of enzyme required to form 1 mol of fructose / min.) Using the specific activity of 12 Units per mg of protein, the amount of protein per kg of broth can be determined.

In Figure 2, the total amount of enzyme produced is indicated.

As shown in this example, 850 kg of the enzyme can be manufactured in a feed batch production operation.

Example 2 Production of penicillin v Conidiospores of a P. chrysogenum CBS 455.95 (or other suitable Wisconsin-derived strain 54.1255 by mutation and selection for higher productivity, preferably in the recipe presented below) are inoculated at 105 - 106 conidios / ml in one. production medium containing: glucose.H20.5; lactose. H2 O, 80; (NH 2) 2co, 415; (NH 4) 2 SO 4, 11 1; Na2 SO4, 219; KH2 P04.2; residual element solution (HP04.3H201 41 8; citric acid.H201 150; Fe804 .7H201 155 MgSOl.7H20 I 150Ϊ H3B03 1010075; Cu804 .5H201 0124; CO804 .7H201 01 375; ZnS04.7H201 11 5; Mn804 · H201 21 28; CaCl2 · 2H2 01 0.9), 10 (ml / l); 10% potassium phenoxyacetate solution 1 pH 7.75 (ml / l) (pH before sterilization 6.5). The culture is incubated at 25 ° C on an orbital shaker at 280 rpm for 144 - 168 hours. At the end of the fermentation, the mycelium is removed by centrifugation or filtration, and the amount determined, and the medium is assayed for penicillin formed by HPLC procedures well known in the art.

Example 3 Large-Scale Penicillium Fermentation with Complex and Defined Media Penicillium chrysogenum Wisconsin 54.1255 was optimized for growth and production of penicillin in a chemically defined medium by mutation and selection in defined media as described in Example 2. Feed batch batch fermentations were performed. on a 60 m3 scale with a complex medium as described by Hersbach et al. (Biotechnology of Industrial Antibiotics, pp. 45-1401 Marcei Dekker Inc.1 19841 Table 4, Medium B, including salts, as mentioned in Medium A) I containing 50 kg / m3 Corn Infusion solids. At the same time, fermentation was performed in a defined medium, as shown in Example 2, where dosages were doubled because of the high cell density character of these feed-fed batches, while lactose and urea were omitted. Glucose was fed to the fermenter, maintaining glucose concentration below 2 g / 1 to prevent repression by glucose. Ammonium, diammonium sulfate and phenylacetic acid were fed to the fermenter to control pH and ammonium sulfate and phenylacetic acid concentrations in the desired ranges (Hersbach, 1984). As oxygen transfer is an important parameter in determining the economic feasibility of an industrial fermentation process, the performance of the above fermentation processes was analyzed by determining the extent of oxygen transfer in each process. A good measure of the oxygen transfer obtained in a fermentation process is the relative kLa value as determined in a system. KLa is defined as the oxygen transfer coefficient and is calculated according to Van't Riet and Tramper (Basic Biorector Design, 20 Marcei Dekker Inc. (1991), pp. 236 - 273).

The oxygen transfer capacity, calculated as the KLa value, was found to be between 10 and 20% higher in the chemically defined medium than in the complex medium during the main part of the fermentation.

Example 4 Production of 7-ADCA The process described in Example 2 and modified using a P. chrysogenum CBS 455.95 (or other suitable Wisconsin-derived strain 54.1255 by r: mt3. <; preferably in the recipe shown below) which is transformed with an expandase expression cassette, wherein the expandase coding region has the IPNS promoter as described in EP 532341, and using a 10% potassium adipate solution, instead of phenoxyacetate, and using a modification of the above medium containing 400 ml of a 10% potassium adipate solution 1 pH 5.5 instead of phenoxyacetate (medium pH prior to sterilization of 5.5, instead of 6.5). The resulting adipoyl-7-ADCA is subsequently converted to 7-ADCA using the enzymatic deacylation procedure substantially as described in Example 4 or 5 of WO 95/04148.

Example 5 Production of lova.sta.tina Aspergillus terreus conidiospores strain CBS 456.95 (or strains derived therefrom by mutation and selection for highest yield, preferably in any of the recipes shown below) are inoculated at 105 - 106 conidios / ml, in a production medium containing (g / 1): dextrose, 40; CH3COONH4 1 212; Na.S04 14; KH2 P04 1316; K2HP04 .3H2 01 3511; residual element solution (see above, Ex. 2) I 10 (ml / 1). The culture is incubated at 28 ° C on an orbital shaker at 220 rpm for 144 - 168 hours. At the end of the fermentation, the mycelium is removed by centrifugation or filtration and the amount calculated, and medium is assayed for lovastatin formed by HPLC procedures well known in the art.

Example 6 Production of clavulanic acid Streptomyces clavuligerus strain ATCC 27064 or a mutant thereof and inoculated into a preculture medium consisting of (g / 1): (NH4) 2804 1 2175; KH2 P04 1 01 5; Mg804.7H20, 0.5; CaCl2 .2H2 O, 01.01; 3- (N-morpholino) propanesulfonic acid, 21; glycerol, 19.9; sodium succinate, 5.5; solution of residual elements (ZnSO4 .7H201 218; ferric ammonium citrate 217; CuSO.SH 201 01125; MnSO4 .H2 01 1; CoC12.6H20.011; Na2 B4 07 .10H201 0116; Na2MoO. 2H 201 01 054) 1 0106 (ml / l). The culture is incubated on an orbital shaker at 220 rpm at 28 ° C for 48 - 72 hours and used to inoculate 20 volumes of production medium containing (g / 1): (NH4) 2 804 1 2; asparagine, 4; KH2 P04, 115; MgSO4.7H2 01 01 5; CaCl2 · 2H201 0 1 01; 3- (N-morpholino) propanesulfonic acid, 21; glycerol, 1919; sodium succinate, 525; residual element solution (see above) 1 0 1 06 (ml / l); FeSO 4 .7H 2 O, 0.5; MnCl 2 · 4H 2 O, 0.1; 20 ZnSO4 • 7H2 0.01 1. Incubation is continued for 144 hours, preferably in a 500 ml Erlenmeyer bulkhead flask containing 50 ml culture volume. At the end of fermentation, the mycelium is removed by centrifugation or filtration, and the amount calculated, and the filtrate is assayed by HPLC procedures well known in the art.

Example 7 Production of Amyloglycosidase Aspergillus niger strain CBS 513.88 or a mutant thereof and inoculated at 105 - 106 conidiospores / ml in a germination medium consisting of (g / 1): K2HP04.3H20, 0.75; KH 2 PO, 6.6; Na 3 · 3H 2 O citrate, 5.3; citric acid.H 2 O, 0.45; glucose, H2 O, 25; (NHq) 264.8; NaCl, 0.5; MgSO4. 7H2 O, 0.5; Fe-SO,. 7H2 O, 0.1; ZnSO4. 7H2 O, 0.05; CaCl2, 0.005; CuS04. 5H 2 O, 0.0025; Mn804.4H20, 0.0005; H3B03, 0.0005; Na2Mo04. 2H20, 0.0005; EDTA, 0.13; Tween80,3.

If necessary, 50 g / ml arginine and / or proline may be added to improve germination. The culture is incubated on an orbital shaker for 48 - 72 hours at 33 ° C, 295 rpm, and then used to inoculate 10 - 20 volumes of a production medium consisting of (g / 1): KH2PO4, 1-5 ; NaH2 P04. H2 O, 0.5; Na 3 · 3H 2 O citrate, 53; citric acid, H2 O, 4.05; dextrose polymers, 70; (NH 4) 2804.8; (NaCl, MgSO4 · 7H2O, FeSO4 · 7H2O, ZnSO4 · 7H2O, CaCl2, CUSO4.5H2O MnSO4.4H2O H3B03, Na2MoO4.2H2O EDTA): equal to the germination medium. Incubation is continued for 96 hours, preferably in a 500 ml Erlenmeyer flask containing 100 ml medium. At the end of fermentation, centrifugation or filtration, the mycelium is removed by calculated amount, and the filtrate is assayed for amylolytic activity.

Example 8 Production of Astaxanthin Phaffia rhodozyma strain CBS 693 8 or a mutant thereof and inoculated into 25 ml of a preculture medium containing 5 (g / 1): yeast extract 10; peptone, 20; glucose, 20. The culture is incubated for 72 hours at 20Â ° C in a 100 ml Erlenmeyer bulkhead flask on an orbital shaker at 25 rpm. 1 ml of the preculture is then used to inoculate 100 ml of a production medium containing (g / 1): glucose, 30; NH4 Cl1 4183; MgSO4 .7H201 0188; NaCl 11106; solution of residual elements (acid (NH) 2 Fe (S04) 2. 6H2 01 90; ZnS04.7H2 01 1617; CaCl2 · 6H201 01 15; citric.H20I 50; CuS04 · 5H2 01 215; MnS04 · 4H2 01 2; 01 4 N) 10 13 (ml / 1); vitamin solution (myoinositoll 40; nicotinic acid, 2; Cal 2 D-pantothenate; vitamin B11 2; p.aminobenzoic acid, 11 2; vitamin B61 012; biotin, 01 008; in H 2 SO 4 to 01 07 N) 1 5 ( ml / 1); Pluronicl 0.04; KH2P01.1; potassium hydrogen phthalate, 20 (pH before sterilization of 5.4). Incubation is continued for 96 hours, preferably in an Erlenmeyer flask and 500 ml with shields. At the end of the fermentation, the astaxanthin content of the biomass (calculated amount) is determined by solvent extraction and measurement of the extract's optical density at 470 - 490.

Example 9 Arachidonic Acid Production A 1 ml vial of a suspension of stored aseptically opened Mortierella alpina strain ATCC 16266 and the contents is used to inoculate 500 ml of a production medium containing (g / 1): glucose, 70 ° C; yeast extract, 01 5; NH4 NO3 3 (ml / 1); (pH before sterilization of 71 0). The culture is incubated in a 2 liter bulkhead shake flask at 25 ° C for 72 hours on an orbital shaker at 250 rpm. At the end of the fermentation, the biomass amount and the arachidonic acid content of the biomass are determined after centrifugation, freeze drying and solvent extraction by GC methods well known in the art.

Example 10 Production of Erythromycin Saccharopolyspora erythraea strain NRRL2338 or a mutant thereof (selected for highest yield, preferably in the recipe shown below) and inoculated into 25 ml of a preculture medium containing (g / 1): soluble starch, 15 ; NaCl, 5; soybean meal, 15; CaC031 10; yeast extract, 5; millol infusion solids 5; CoC12.6H201 670 1 of a 1 g / 1 solution. The culture is incubated in a 100 ml unblocked shake flask at 32-34 ° C for 3 days at 250 rpm in a shaker-incubator. 0.4 ml of the culture is inoculated into 25 ml of a sterile production medium containing (g / 1): citric acid. H2 O, 2 (NH4) 2 SO4.2; MgSO 4 .7H 2 O 2; CaCl 2 .2H 2 O, 0.085; KH2 P04, 0.25; HEPES (N- (2-hydroxyethyl) piperazine-N '- (2-ethanesulfonic acid)), 5; glucose, 1.5; soluble starch, 20; soybean oil, 0.4; solution of residual elements (grams in 250 10 ml of distilled water: citric acid. H2 O, 621.5i FeSO4.7H2 O, 0.8215; CuSO4 .5H2 O, 0.0625; COC12.H2 O, 0.375; H3B03, 0.0625; ZnSO 4 .7H 2 O, 1.25; MnSO 0, 1 H 2 O, 1.25;

Na 2 MoO 4 (2H 2 O, 0.0625), 3.6 ml / l. pH = 7.0. To each flask, 0.25 ml of n-propanol is added. The culture is incubated in a 100 ml bulkhead shake flask at 32-34 ° C for 5 days on a shaker-incubator at 300 rpm. At the end of the fermentation, the broth is centrifuged and the amount of biomass calculated. The erythromycin content of the supernatant is measured by HPLC procedures known in the art.

Example 11 Production of β-Carotene A suspension of Blakeslea trispora CBS 130.59 spores is used to inoculate 114 ml of preculture medium (yeast extract, 10 g / 1; peptone, 20 g / 1; glucose, 20 g / 1) in a 500 ml agitation flask without bulkheads. The preculture is incubated for 42 h on a rotary shaker at 250 rpm at 26 ° C. The biomass and collected by filtration is washed 3 times with 100 ml of sterile demineralized water to remove components from the preculture medium. Subsequently, the biomass is homogenized by mixing until only small fragments are left, and resuspended in 40 ml of demineralized water. The production medium is prepared in 100 ml portions in bulkhead shake flasks. The composition of the production medium is as follows (in g / 1): glucose, 40; asparagine monohydrate, 2; KH 2 P04, 0.5; MgSO4. 7H2 01 0.25. In addition, a solution of residual elements (0.3 ml / l) having the following composition (in g / l) (NH4) 2 Fe (SO4) 2 • 6H2 0.90 is added; ZnSO 4 · 7H 2 O, citric acid H 2 O, 50; 161 7;

CuSO4 · 5H2 O, 2.5; MnSO4 · 4H20.2; H3B03.2; Na 2 Mo 4 · 2H 2 0.2; Kl, 01-5; in 0.4 N H2 SO4. Prior to sterilization, the pH of the medium is adjusted to 6.2. The flasks are sterilized for 20 minutes at 120 ° C and after sterilization 0.05 ml of a 1 mg / ml solution of thiamine hydrochloride in demineralized (filter sterilized) water are added. Production cultures are inoculated 0.5 to 10 ml of the fragmented mycelium suspension prepared above.

The cultures are incubated for 139 h on a rotary shaker (250 rpm; 26 ° C). The fungal biomass is collected by filtration, washed with demineralized water to remove medium components and quantified. The amount of β-carotene produced is determined by acetone extraction and measuring extinction at 450 nm of the acetone fraction on a spectrophotometer.

Claims (10)

1. Process for the production of arachidonic acid characterized by comprising the steps of: fermentation of a filamentous microbial strain, which is a Mortierella strain, on an industrial scale in a fermentation medium, which is a chemically medium. defined, essentially composed of chemically defined constituents, and recovery of arachidonic acid from the fermentation broth. Process according to claim 1, characterized in that the chemically defined medium contains an amount of a complex carbon source expressed as the amount of carbon / liter of the medium from 10 to 200 g of C / L and / or complex nitrogen source of 0,5 to 50 g of N / L, where N is expressed as Kjeldahl nitrogen. Process according to either of Claims 1 and 2, characterized in that the source of complex carbon and / or nitrogen is used in the inoculum fermentation process for the main fermentation. Process according to any one of claims 1, 2 and 3, characterized in that the chemically defined constituents of the chemically defined medium comprise a carbon source selected from the group consisting of carbohydrates such as glucose, lactose, fructose. , sucrose, maltodextrins, starch and inulin, glycerol, vegetable oils, hydrocarbons, alcohols such as methanol and ethanol, organic acids such as acetate and higher alkanoic acids and a nitrogen source selected from the group consisting of urea, ammonia, nitrate , ammonium salts such as ammonium sulfate, ammonium phosphate and ammonium nitrate and amino acids such as glutamate and lysine. Process according to Claim 4, characterized in that the carbon source is glucose and the nitrogen source is ammonia and / or an ammonium salt. Process according to any one of Claims 1, 2, 3, 4 and 5, characterized in that the fermentation takes place by a process of batch fermentation, repeated batching, fed batching, fed batching. repeated or continuous fermentation process. Process according to Claim 6, characterized in that the fermentation takes place by a batch feeding process. Process according to Claim 7, characterized in that the source of carbon and / or nitrogen is fed to the process. Process according to any one of claims 1, 2, 3, 4, 5, 6, 7 and 8, characterized in that the strain is Mortierella alpina. Process according to Claim 9, characterized in that the lipid comprising arachidonic acid is a triglyceride.