MXPA99007691A - Fermentative production of valuable compounds on an industrial scale using chemically defined media - Google Patents

Abstract

The present invention describes the use of chemically defined media for the fermentative production of valuable compounds on an industrial scale. Microbial strains which are suitable for fermentation on an industrial scale using a chemically defined medium include fungal, yeast and bacterial strains. Suitable strains can be obtained as wild type strains or by screening and selection after mutagenic treatment or DNA transformation.

Description

FERMENTATIVE PRODUCTION TO INDUSTRIAL SCALE OF USEFUL COMPOUNDS USING CHEMICALLY DEFINED MEDIA FIELD OF THE INVENTION The present invention relates to the field of fermentation, that is, to the fermentative production of valuable compounds such as primary or secondary metabolites, pharmaceutical proteins or peptides, or industrial enzymes.

BACKGROUND OF THE INVENTION Many valuable compounds are manufactured by fermentative production in large scale, industrial fermentors, that is, the microorganism which produces a valuable compound of interest is grown under controlled conditions in a fermenter of 10 to 300 m3. In current fermentation processes on an industrial scale, the production organism is typically fermented in a complex fermentation medium.

A complex medium is understood as a medium comprising a complex source of nitrogen and / or carbon, such as soybean syrup, cottonseed syrup, corn cob liquor, yeast extract, casein hydrolyzate, melasas and similar. REF .: 30876 The advantages of complex media is that they are complex constituent raw materials that are not expensive, are easily available and form a complete or almost complete source of nutrients for the microorganism, contain a source of carbon and nitrogen as well as vitamins and minerals. In addition, the mixture of biological macromolecules currently in complex raw materials such as proteins, carbohydrates, lipids and the like, needs to be degraded by enzymes excreted by the microorganism before consumption. As a consequence, consumable small molecules are available uniformly through the fermenter and during the fermentation process, so concentration gradients and mixing problems are avoided and the level of these small consumable molecules is maintained below repression concentrations. In addition, these macromolecules as well as the organic acids that are also present in the complex medium provide the medium with a buffering capacity, and thus control of pH is facilitated. In addition to these advantages, complex fermentation media have several important disadvantages. More importantly, complex raw materials have a chemically undefined composition and variable quality, for example, due to variation by seasons and different geographical origin. Since the composition of the fermentation medium has an important influence on fermentation parameters such as viscosity, heat transfer and oxygen transfer, complex raw materials are a major cause of variability in a process. In addition, they prevent downstream processing and adversely affect the quality of the final product. For example, fermentation broths, in particular filamentous microorganisms, can exhibit a decreased filtration capacity when complex raw materials are used. Complex raw materials may also contain compounds which accumulate unintentionally or are consolidated with the final product. Heavy metals, pesticides or herbicides are examples of undesirable compounds which may be present in complex raw materials. In addition, complex raw materials may contain or may lead to the formation of toxins. The additional disadvantages are that complex media generate an unfavorable odor during sterilization and produce undesirable waste streams. Despite the disadvantages identified above associated with the use of complex media, these media are still preferred for large-scale industrial fermentation processes. There are several reasons why media that do not contain complex raw materials, that is, chemically defined media, have not been considered for use in industrial scale fermentation processes. One obvious reason lies in the advantages associated with the use of complex media. What is more important, product yields which can be obtained using chemically defined media on an industrial scale are typically considered to be substantially lower than those obtained using media containing complex raw materials. In addition, high production microbial strains which have been developed for industrial processes in complex media may not retain their proper functioning in chemically defined media. One reason for unsatisfactory performance in a chemically defined environment is that current industrial strains may undergo various sequences of mutagenesis and selection, without considering their operation in a chemically defined medium. Therefore, the chemically defined media has hitherto been applied only for research purposes, i.e., in laboratory cultures in petri dishes and / or shake flasks or at a relatively small fermentative scale that typically does not exceed a volume of about 20. -40 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. Microbiol., 52, 892-897 (1986) and erythromycin (Bushell et al., Microbiol., 143, 475-480 (1997)). However, research regarding the use of a chemically defined medium at such a small research scale does not provide any teaching to a person familiar with the art regarding the applicability of these media in large-scale industrial fermentation processes for the purposes of production, which typically have a volume scale of approximately 10 m3 or more To avoid the problems associated with the use of conventional recipes for complex media in current industrial practice, it would be desirable to apply chemically defined recipes for industrial-scale fermentation. We describe the use of a chemically defined medium for the industrial scale fermentation process that allows - in combination with a suitable strain - the production of compounds such as primary or secondary metabolites, pharmaceutical proteins or peptides, or industrial enzymes, with an economically attractive performance.

BRIEF DESCRIPTION OF THE INVENTION The present invention describes an industrial process for the production of a useful compound, which comprises the steps of fermenting a microbial strain in a fermentation medium which is a chemically defined medium constituted essentially of chemically defined constituents, and recovery of the useful compound of the invention. fermentation broth. The present invention further discloses a method for preparing and / or improving a microbial strain that produces a useful compound of interest which is capable of being fermented on an industrial scale in a chemically defined medium, which method comprises the steps of: parental or original strain suitable for a mutagenic treatment that is selected from the group of chemical physical and mutagenic media, and / or DNA transformation, * analysis of the mutant results and / or transformants to determine their growth performance in a chemically defined medium and their level of production of the useful compound of interest, * select mutants that have a similar or improved growth performance in a chemically defined medium and / or an improved production level of the useful compound of interest, compared to the parent strain.

DETAILED DESCRIPTION OF THE INVENTION The present invention describes the use of a chemically defined fermentation medium for industrial-scale fermentation of a suitable microbial strain, and the suitable microbial strain is capable of producing a useful compound. Through the description of the invention, it is understood that a process of fermentation on an industrial scale or an industrial process encompasses a fermentation process at a scale in volume which is > 10 m3, preferably > . 25 m3, more preferably > 50 m3, and much more preferably > . 100 m3. The term "chemically defined" is understood to be used for fermentation media which are constituted essentially of chemically defined constituents. A fermentation medium which is composed essentially of chemically defined constituents includes a medium which does not contain a complex source of carbon and / or nitrogen, ie, which does not contain complex raw materials having a chemically undefined composition. A fermentation medium which is composed essentially of chemically defined constituents may further include a medium which comprises an essentially small amount of a complex source of nitrogen and / or carbon, an amount as defined above, which is typically not sufficient to maintain the growth of the microorganism and / or ensure the formation of a sufficient amount of biomass. In this regard, the complex raw materials have a chemically undefined composition due to the fact that, for example, these raw materials contain many different compounds, among which the complex heteropolimeric compounds, and have a variable composition due to the variations between stations and the differences in geographical origin. Typical examples of complex raw materials that function as a complex source of carbon and / or nitrogen in fermentation are soybean syrup, cottonseed syrup, corncob liquor, yeast extract, casein hydrolyzate, melassa and Similar. An essentially small amount of a complex source of carbon and / or nitrogen may be present in the chemically defined medium according to the invention, for example, as an inoculum carrier for the main fermentation. The inoculum for the main fermentation is not necessarily obtained by fermentation in a chemically defined medium. Very often, the transport of the inoculum will be detectable through the presence of a small amount of a complex source of nitrogen in the chemically defined medium for the main fermentation.

It may be advantageous to use a complex source of carbon and / or nitrogen in the fermentation process of the inoculum for the main fermentation, for example, to accelerate the formation of biomass, that is, to increase the growth rate of the microorganism, and / or to facilitate internal pH control. For the same reason, it may be advantageous to add an essentially small amount of a complex source of carbon and / or nitrogen, for example yeast extract, to the initial stage of the initial fermentation, especially to accelerate the formation of biomass in the early stage of the fermentation process. An essentially small amount of a complex source of carbon and / or nitrogen may be present in the chemically defined medium according to the invention which is defined to be an amount of, at most, about 10% of the total amount of carbon and / or nitrogen (N Kjeldahl) 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, a complex carbon and / or nitrogen source is not present in the chemically defined medium according to the invention. It should be understood that the term "chemically defined medium" as used in the present invention includes a medium in which all necessary components are added to the medium before the start of the fermentation process, and which also includes a medium in which of the necessary components are added before the start, and part is added to the medium during the fermentation process. The present invention further discloses that microbial strains are capable of converting, on an industrial scale, simple raw materials of chemically defined medium into an economically attractive amount of useful product. It has surprisingly been found that the productivity of microbial strains in chemically defined medium, when measured on an industrial scale, can be comparable with, or in some cases even better than, their productivity in complex media. An additional advantage of a chemically defined medium is that the transfer of oxygen from the gas phase to the liquid phase and the transfer of carbon dioxide from the liquid phase to the gas phase is substantially improved as compared to the use of a complex medium. As is known to those familiar in the art, dissolved oxygen and dissolved carbon dioxide concentrations are two important factors in scaling up a fermentation process, and can determine the economic feasibility of an industrial process. The improved mass transfer obtained using a chemically defined medium can be attributed to the absence in that medium of substantial amounts of compounds which promote coalescence of gas bubbles. Compounds that promote coalescence, for example, can be found among certain hydrophobic and / or polymeric compounds present in complex raw materials. The 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 the fermentation process, especially in fermentations of filamentous microorganisms. The improved oxygen transfer capacity that is obtained when fermentation is performed using a chemically defined medium according to the invention provides a much cheaper way of optimizing productivity compared to investments in equipment, such as energy input, enrichment of oxygen from the air inlet or pressure of the fermentor. In industrial fermentation processes, filamentous microorganisms, such as filamentous materials such as Actinomycetes or filamentous fungi such as Penicillium or Aspergillus, are typically grown by having a granule or sediment morphology. In that respect, the proteins and peptides present in the complex fermentation medium have the tendency to produce spongy sediments which are easily separated to the dispersed mycelium with very long and branched hyphae as a consequence of the high growth rates which are typically obtained using a complex medium . Therefore, a spongy sediment morphology can generally cause an undesirably high broth viscosity. The use of a chemically defined medium has a favorable influence on the morphology, for example, by producing a more rigid sediment which does not separate easily during fermentation. In this way, a significant decrease in the viscosity of the filamentous fermentation broths can be obtained by using a chemically defined medium. Since a low viscosity of the fermentation broth is advantageous for product formation, viscosity control is of the utmost importance in commercial scale fermentation processes. Another advantage of using a chemically defined medium is in the downstream or downstream processing of the product. For certain strain-product combinations, especially when filamentous strains are fermented, downstream processing is significantly improved by using a chemically defined medium. A chemically defined medium to be used in the process of the invention typically must contain what are called structural elements and what are called catalytic elements. The structural elements are those elements which are constituents of the microbial macromolecules, that is, hydrogen, oxygen, carbon, nitrogen, phosphorus and sulfur. The structural elements hydrogen, oxygen, carbon and nitrogen are typically contained within the source of carbon and nitrogen. Typically, phosphorus and sulfur are added as phosphate and sulfate and / or thiosulfate ions. The type of carbon and nitrogen source which is used in a chemically defined medium is not critical to the invention, with the proviso that the carbon and nitrogen source have an essentially chemically defined character. Preferably, a carbon source is selected from the group consisting of carbohydrates such as glucose, lactose, fructose, sucrose, maltodextrins, starch inulin, glycerol, vegetable oils, hydrocarbons, alcohols such as methanol and ethanol, organic acids such as acetates and acids. alkanoic higher More preferably, a carbon source of the type consisting of glucose, sucrose and soybean oil is selected. More preferably, the carbon source is glucose. It should be understood that the term "glucose" includes glucose syrups, ie, glucose compositions containing oligomers of glucose in defined amounts. A 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, a nitrogen source is selected from the group consisting of ammonia, ammonium sulfate and ammonium phosphate. More preferably, the nitrogen source is ammonia. The use of ammonia as a source of nitrogen has the advantage that ammonia can additionally function as an agent for pH control. In the case where ammonium sulfate and / or ammonium phosphate is used as a nitrogen source, part or all of the sulfur and / or phosphorus requirement of the microorganism can be met. The catalytic elements are those elements which are constituents of enzymes or cofactors of enzymes. These elements are, for example, magnesium, iron, copper, calcium, manganese, zinc, cobalt, molybdenum, selenium, boron. In addition to these structural and catalytic elements, cations such as potassium and sodium ions must be present to function as a counter ion for intracellular pH and osmorality control. The compounds which may optionally be included in a chemically defined medium or chelating agents such as citric acid, and buffering agents such as monopotassium and dipotassium phosphate, calcium carbonate and the like. Preferably buffering agents are added when working with processes without external pH control. In addition, an antifoaming agent can be dosed before and / or during the fermentation process. Macromolecules and organic acids which are present in a complex medium provide buffering capacity in this medium. Due to the absence of these compounds in a chemically defined medium, pH control is more difficult in a chemically defined medium compared to a complex medium. The present invention shows that a pH control, in which an acid or a base can be dosed, depending on the development of pH in the broth, allows an appropriate pH profile in the process on an industrial scale defined chemically. Vitamins refer to a group of structurally unrelated organic compounds which are necessary for the normal metabolism of microorganisms. A vitamin must be added to the fermentation medium of a microorganism that is not able to synthesize such a vitamin. Typically, chemically defined fermentation media for yeast or bacteria or for certain lower fungi, for example, Mucorales can be supplemented with one or more vitamins. The higher fungi more frequently do not have 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. The structural and catalytic elements and, optionally, the vitamins are necessary for the growth of the microorganism, that is, for the formation of biomass. The amount of necessary compounds, ie compounds comprising structural and catalytic elements and, optionally, vitamins, to be added to a chemically defined medium will depend mainly on the amount of biomass which is going to be formed in the fermentation process. The amount of biomass formed can vary widely, typically from about 10 to about 150 g / 1 of fermentation broth. In general, fermentations that produce a quantity of biomass which is much less than about 10 g / 1 are not industrially important. In addition, the optimal quantity of each constituent of a defined medium, as well as the compounds that are essential and which are not essential, will depend on the type of microorganism which is subjected to fermentation in a defined medium, in the amount of biomass and in the product that is going to be formed. The use of a chemically defined medium, therefore, advantageously allows variation of the concentration of each component of the medium independently of the other components, and in this way the optimization of the composition of the medium is facilitated. For product formation, it may be necessary to supplement the chemically defined medium with additional compounds and / or to increase the concentration of certain co-fractions already present in the chemically defined medium above the level which is necessary for the growth of the microorganism. The function of such compounds may be that they induce and / or improve the production of a compound desired by the microorganism, or that it functions as a precursor for the desired compound. Examples of compounds that are to be supplemented and / or to be added in an increased amount to a chemically defined medium are: sulfate in an increased amount for the production of ß-lactam compounds, nitrogen-containing compounds in an amount increased for the production of amino acids, especially basic amino acids, phenylacetic acid for the production of penicillin G, phenoxyacetic acid for the production of penicillin V, adipic acid for the production of adipyl-7-ADCA and adipyl-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 carbon amount / liter of medium, can vary from 10 to 200 g of C / l, preferably from 20 at 200 g of C / l. The total amount of nitrogen sources added to the chemically defined medium can vary 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 between the carbon and nitrogen source in a fermentation can vary considerably, so a determinant for the optimal ratio between the carbon source and nitrogen is the elemental composition of the product to be formed. Additional compounds necessary for the growth of a microorganism, such as phosphate, sulfate or trace elements, are to be added, using the concentration ranges as indicated in Table 1 as a guide line. The concentration ranges of these additional compounds can vary between different kinds of microorganisms, i.e., between fungi, yeasts and bacteria. Vitamin concentrations are generally within the range of 0.1 (biotin) to 500 (myo-inositol) mg / l. Typically, the amount of medium components needed for the growth of a microorganism can be determined in relation to the amount of carbon source used in the fermentation, since the amount of biomass formed will be determined primarily by the amount of carbon source used. .

Table 1. Typical ranges of concentration of medium components, in addition to the carbon and nitrogen source, necessary for the growth of various kinds of microorganisms (g / 1) The basal amount of phosphate needed will be 0.5-1% of the dry weight of the biomass. For relatively small-scale batch processes, additional phosphate will be required for pH control. 2Sulfate can also be dosed via the titrant, as K + or Nad 3E1 sulfate can be (partially) substituted by chloride as a counter ion in trace elements, or vice versa. 4 For some elements in traces, the lower limits are difficult to define. Its requirement can be met, for example in the presence of other components in the medium, for example ferrous sulfate, water, small amounts of yeast extract, etc. 5E1 phosphate and sulfate are added as potassium, ammonium and / or sodium salts with a preference of K > NH4 > Na An industrial fermentation process according to the invention using a chemically defined medium can be carried out as a batch fermentation process, repeated batches, repeated or continuous batches of script feeding. In a batch process, all the components of the medium are added directly, in their entirety, to the medium before the start of the fermentation process. In a repeated batch process, a partial collection of the broth takes place accompanied by a partial supplement of the complete medium, optionally repeated several times. In a batch feeding process, either nothing or a part of the compounds comprising one or more of the structural and / or catalytic elements are added to the medium before the start of the fermentation and either all or the remaining part, respectively , of the compounds comprising one or more of the structural and / or catalytic elements is fed during the fermentation process. The compounds which are selected for feeding can be fed together or separated from each other to the fermentation process. Especially in a fermentation process in which the original fermentation medium is diluted approximately twice or more by a feed of compounds comprising one or more of the structural elements, the feed may further comprise catalytic elements and additional components of the medium in addition to the structural elements. In a repeated batch feeding process or a continuous fermentation process, the entire start medium is further fed during fermentation. The starting medium can be fed together with, or separated from, the feeds of structural elements. In a repeated batch feeding process, part of the fermentation broth comprising the biomass is removed at regular time intervals, while in a continuous process, the removal of part of the fermentation broth occurs continuously. In this way, the fermentation process is replenished with a portion of fresh medium corresponding to the amount of fermentation broth extracted.

In a preferred embodiment of the invention, a batch feed or batch feed process is applied, wherein the source of carbon and / or nitrogen and / or phosphate 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 carbon and nitrogen source, as well as the 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 batch feed process typically allows the use of a considerably larger amount of a carbon and nitrogen source than is used in a batch process. Specifically, the amount of carbon and nitrogen source applied in a batch feed process can be at least about twice as high as the highest amount applied in a batch process. This, in turn, leads to a considerably greater amount of biomass that is formed in the batch feeding process. A further aspect of the present invention relates to the processing option downstream of the fermentation broth. After the fermentation process is completed, the useful product can 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 to it depends on the nature and cellular location of the useful compound. First of all, the biomass is separated from the fermentation fluid using, for example, centrifugation or filtration. Then the useful compound of the biomass is recovered, in case the useful product accumulates inside or is associated with microbial cells. Otherwise, when the useful product is excreted from 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 produces a great advantage in downstream processing, since the amount of by-products is substantially less than when using complex media.

In addition, the quality of the product is improved, since unwanted by-products are not co-isolated with the compound of interest. In a further aspect of the present invention, a strain suitable for an industrial fermentation process is identified using a chemically defined medium. A suitable microbial strain for an industrial fermentation process using a chemically defined medium can be any wild type strain that produces a useful compound of interest, with the proviso that the wild-type strain has a good growth performance in a medium chemically defined. In addition, a suitable microbial strain for an industrial fermentation process using a chemically defined medium can be a strain which is obtained and / or improved by subjecting a parent strain of interest to a classical mutagenic treatment or a recombinant DNA transformation , also with the proviso that the resulting mutant or the transformed microbial strain has a good growth performance in a chemically defined medium. Therefore, it will depend both on the growth performance of the parental strain in a chemically defined medium if the resulting mutant or transformed strains should have improved or similar growth performance in a chemically defined medium, as compared to the parental 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 which is > . 0.05 hd, preferably > 0.1 h "1, more preferably 0.2 h" 1, and more preferably > , 0.4 h "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 in a culture in a shake flask and / or in a cabinet fermentation of 10 1. It is preferred to include a cabinet fermentation of 10 1, with a control of pH, temperature and oxygen concentration, in the growth performance analysis In one embodiment of the invention, the microbial strains which are capable of being fermented in a chemically defined medium are obtained and / or improved by subjecting a parent strain of interest to a classical mutagenic treatment using a physical medium, such as UV irradiation or a suitable chemical mutagen, such as N-methyl -N'-nitro-N-nitrosoguanidine or ethyl methane sulfonate In another embodiment of the invention, the microbial strains which are capable of being fermented in a medium Chemically defined proteins are obtained and / or improved by subjecting a parental strain of interest to recombinant DNA technology, whereby the parental strain is transformed with one or more functional genes of interest. In general, the present invention considers two groups of parental strains of interest to be subjected to classical mutagenesis and / or DNA transformation. In one embodiment of the invention, a parental strain of interest is selected from the group of strains which have good growth performance in a chemically defined medium, but which need to be improved with respect to their level of production of a compound of interest. In another embodiment of the invention, a parental strain of interest is selected from the group of strains which have a high production level of a compound of interest, but which have a relatively poor growth performance in a chemically defined medium. Microbial strains with a specific growth rate which is less than about 0.05 h "1 are understood to have a relatively poor growth performance in a chemically defined medium, both processes, the classical mutagenic treatment as well as the transformation process with DNA , are followed by an analysis of the resulting mutants or transformants both in their growth performance in a chemically defined medium as well as in their level of production of a compound of interest.S mutant or transformant strains are selected which have a good growth performance in a chemically defined medium and / or an improved production level of a compound of interest, compared to the parental strain. It should be noted that some microbial strains, in particular industrial strains which have already undergone extensive mutagenic treatment to improve production levels, may function poorly or may not grow in any way in a chemically defined medium. Such 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, it is possible that a mutagenized strain possesses a mutation that causes an unknown growth requirement (unknown auxotrophic mutation). Microbial strains which are suitable for industrial fermentation using a chemically defined medium include filamentous and non-filamentous strains. For example, microbial strains which are suitable for fermentation in a chemically defined medium include fungal strains such as strains of Aspergillus, Penicillium or Mucorales, yeast strains such as Saccharomyces, Pichia, Phaffia or Kluyveromyces and bacterial strains such as Actinomycetes. The use of a chemically defined medium according to the invention is especially advantageous for the industrial fermentation of filamentous microorganisms. The process according to the invention using a chemically defined medium is suitable for the industrial scale fermentative production of any useful compound of interest, which includes primary or secondary metabolites, pharmaceutical proteins or peptides, or industrial enzymes. Preferred useful compounds are secondary metabolites, such as antibiotics or β-lactam compounds, especially β-lactam antibiotics. Examples of strain-product combinations include A. niger, for example A. niger CBS 513.88 for amyloglucosidase, A. oryzae for α-amylase, A. terreus, for example A. terreus CBS 456.95 for lovastatin, Mortierella alpina for archidonic acid or lipids containing arachidonic acid, Mucor 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 tetraacetyl phytosphingosine, Phaffia rhodozyma, for example P. rhodozyma CBS 6938 for astaxanthin, Saccharopolyspora erythraea for erythromycin, L. lactis for lactase, Streptomyces natalensis for natamycin. The present invention also considers the use of microbial strains which are transformed with one or more functional genes of interest, which result in a transformed strain which can overexpress a product which is already produced by the strain, or which results in a strain transformed which can express a product that is not produced naturally by the strain. In this way, the type of strain that will be selected for transformation is left to the choice of a person familiar with the art, provided that the selected strain has a good growth performance in a chemically defined medium. For example, a strain can be selected for transformation which has already been subjected to one or more mutagenic treatments. Alternatively, a non-mutagenized or wild type strain can be selected.

After analyzing the level of expression of a desired compound, the transformants that are obtained after the transformation of a selected strain with one or more functional genes of interest must be analyzed to determine its growth performance in a chemically defined medium. Examples of recombinant strains that make a product that is not naturally produced by the strain are: * Streptomyces lividans, for example S. lividans TK21, which contains a cassette (sequence) of expression that allows the expression of glucose isomerase, the gene that encodes glucose isomerase originating, for example, from Actinoplanes missouriensis, * Penicillium chrysogenum, for example P. chrysogenum CBS 455.95, which contains one or more expression cassettes that allow the expression of an expandase, and optionally, a hydroxylase and / or an acetyltransferase, the genes encoding expandase, hydroxylase and acetyltransferase originate, for example, from Acremonium chrysogenum or Streptomyces clavuligerus, which allows the production of cephalosporin compounds such as 7-ADCA or 7-ACA using adipic acid (see EP 532341) or 3-carboxymethylthiopropionic acid (see WO 95/04148) as a chain precursor lateral, * Aspergillus niger, for example Aspergillus niger CBS 513.88, which contains an expression cassette allowing the expression of human lactoferrin (see WO 93/22348) or bovine chymosin. * Kluyveromyces lactis containing a cassette expressing bovine chymosin or phospholipase A2, insulin or recombinant human albumin. Examples of recombinant strains that overproduce an enzyme that is already produced by the strains are: * A. niger for example A. niger CBS 513.88, which contains 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 strain of Streptomyces, and by the advantageous use of a chemically defined medium for large scale Penicilium fermentation, in comparison with a complex medium. Additional examples are directed to chemically defined media which can be used to measure the growth performance and productivity of a strain of interest when grown on such a small-scale medium, in order to identify microbial strains which are suitable for fermentative production of a compound useful on an industrial scale in a chemically defined medium.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Contour of pWGx.GIT. Figure 2. Total glucose isomerase development produced during fermentation.

Example 1 Industrial production of glucose isomerase using Strevtomvces lividans Construction of a strain of Streptomyces that produces glucose isomerase The gene for glucose isomerase of Actinoplanes missouriensis was originally cloned as a 5.0 kb DNA fragment in E. coli K12 strain JM101. It is found that a 1.7 kb fragment, internal to the 5.0 kb fragment, represents the complete coding sequence of glucose isomerase from A. missouriensis and its regulatory region towards the 5 'end (see also Amore and Hollenberg (1989), Nuci. Acids, Res. 17., 7515). A glucose isomerase mutant is obtained which shows improved thermostability by shifting the AAG triplet encoding lysine at position 253 of the glucose protein isomerase into CGG, which codes for arginine, into the gene for glucose isomerase (Quax et al. ), Bio / Technology 9_, 738-742). For cloning in Streptomyces, plasmid pIJ486 (Ward et al (1986), Mol.Gen.Genet, 203, 468-478) is used as vector. The 1737 base pair DNA fragment of A. missouriensis coding for glucose isomerase is combined with the large PstI DNA fragment of pIJ486. The resulting plasmid, designated pWGx.GIT contains essentially the replication region of the pIJIOI plasmid, the thiostrepton resistance gene, and the DNA fragment of A. missouriensis that codes for GIT. A schematic map of pWGx.GIT is provided in Figure 1. The glucose isomerase producing strain is 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 the plasmid pWGx.GIT.

Industrial production of glucose isomerase By working a cell bank of a production strain constructed as mentioned in Example 1 which is prepared by taking a thiostrepton resistant colony and growing it in Tryptone Soytone broth 20 ml containing thiostrepton (50 mg / l) in a shake flask of 100 ml at 30 ° C for 40-48 hours and shaking at 280 rpm. Mycelium equivalent to 1 ml of the working cell bank (fresh or stored as mycelium frozen at -80 ° C) is inoculated in 100 ml of inoculum growth medium in a 500 ml shake flask containing 16 g / 1 of Bactopeptone (Difco 0123/01), 4 g / 1 Bacto soytone (Difco 0436/01), 20 g / 1 casein hydrolyzate (Merck 2239), 10 g / 1 phosphate dipotassium.3aq (Merck, Analytical Reagent ), 16.5 g / 1 glucose. laq, 0.6 g / 1 of soybean oil and 50 mg / l of thiostrepton. The pH of the medium is adjusted to 7.0 with sodium hydroxide and / or sulfuric acid before sterilization. Glucose and thiostrepton are added separately after sterilization. The thiostrepton is dissolved in DMSO at a concentration of 50 g / 1 and sterilized by filtration on a Nalgene 0.2 μm filter. The culture is grown for 24 hours at 30 ° C in an incubator shaker at 280 rpm. 50 ml of the full growth culture is transferred to 6 1 of second phase inoculum growth medium having a composition similar to the medium mentioned above, except for a double glucose concentration (33 g / 1 glucose, lac), with additional antifoam (SAG5693, 0.6 g / 1, a silicon antifoam from Basildon Company) and without thiostrepton. The glucose is again sterilized separately in 50% solution and added after sterilization of the medium (60 minutes, 121 ° C). The culture is grown for 36 hours in a sterilized bubble column, aerated with 840 1 sterile air / h with a nozzle containing 4 holes with a diameter of 2 mm and the temperature is maintained at 22 ° C. Alternatively, this phase can be carried out in shake flasks (for example, 500 ml of 12x medium in Erlenmeyer flasks of 2 1 with baffles) with similar inoculation ratios and which are shaken at 280 rpm in an orbital shaking incubator. The full growth culture is transferred aseptically to an inoculum fermenter containing 4.5 m3 of inoculum medium containing 16.3 kg of citric acid. laq, 70.8 g of ferrous sulfate.7aq, 109 g of zinc sulfate.7aq, 109 g of manganese sulfate, laq, 32.7 g of cobalt dichloride.6aq, 5.45 g of disodium molybdate.2aq, 5.45 g of boric acid , 5.45 g of copper sulfate.5aq, 10.9 kg of diammonium sulfate, 10.9 kg of magnesium sulphate.7aq, 763 g of calcium chloride.2aq, 1090 hg of soybean oil, 21.8 kg of monopotassium phosphate and 139 kg of glucose, laq, and 5.9 kg of yeast extract (brewer's yeast with Kjeldahl nitrogen 10% on a dry weight basis). The medium is prepared as follows: all the components, except glucose, are charged in the indicated sequence in approximately 2700 1 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. The glucose is sterilized in 1000 1 of water at pH 4.5 for 60 minutes at 122 ° C in a separate vessel. After cooling both portions, the glucose is transferred aseptically to the inoculum vessel. After mixing both portions, the pH 7.0 is adjusted with ammonia and the volume is adjusted with sterile water to 4.5 m3. The fermentation temperature is controlled at 30 ° C and the fermenter is aerated at 0.5-1.0 vvm while maintaining the pH at 7.0 +/- 0.1 with gaseous ammonia and maintaining the excess pressure at 1.3-1.4 bar. The foaming is controlled if necessary with a sterilized mixture of soybean oil and silicon antifoam, such as SAG 5693, in a ratio of 3: 1. The oxygen concentration above 25% air saturation is maintained by adjusting the agitator speed (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 per volume of broth. h.

The main fermentation medium contains 245.1 kg of citric acid. laq, 1062 g of ferrous sulfate.7 aq, 1634 g of zinc sulfate-7aq, 1634 g of manganese sulfate. laq, 490 g of cobalt dichloride.6aq, 82 g of disodium molybdate .2aq, 0.82 g of boric acid, 82 g of copper sulfate.5aq, 163.4 kg of diammonium sulfate, 163.4 kg of magnesium sulfate.7aq, 6.94 kg of calcium dichloride.2aq, 16.3 kg of soybean oil, 327 kg of monopotassium phosphate, 880 kg of brewer's yeast extract (10% of Kj -N on a dry weight basis) and 556 kg of glucose. laq The medium is prepared as described for inoculum fermentation (the glucose is sterilized separately). For the glucose, a sugar syrup DE-95 can alternatively be used. The volume of the medium before inoculation is 65 m3 after the pH is corrected to 7.0 with ammonia. A glucose feed is prepared at 275 to 550 g of glucose / 1 of feed solution, either as glucose. laq or as glucose equivalents from the syrup > 90-DE The pH is adjusted to 4.5-5.0 with a phosphoric acid solution. Sterilization is carried out either batchwise (122 ° C, 45 minutes) or continuously via thermal shock or filter placement. The main fermentation is controlled at 30 ° C +/- 0.5 and pH 7.0 +/- 0.1 (by means of a pH control using ammonia and phosphoric acid). The air flow is adjusted to 0.5-1.5 vvm, preferably 0.7 vvm, the overpressure is 0.5-1.5 bar and the fermenter is agitated with Rushton turbines at an intensity of 0.5 to 3 Kw / m3 in order to prevent the concentration of oxygen decrease below 0.2 mmol / 1, measured at the height of the lower agitator. The glucose feed is started when there is a sudden decrease in oxygen uptake, and the concentration of dissolved oxygen is increased, in the same way as the pH which becomes 6.9 to 7.1. The concentration of glucose in the broth should be < < 0.2 g / 1 at this point in time. The glucose feed rate is equivalent to 93 kg of glucose / h that initially increases linearly to 186 kg / h at 64 hours after the start of feeding. After 100 hours of feeding at 186 kg / h, the feed rate is increased to 298 kg of glucose / hr until about 200 feeding hours. Foaming is controlled by dosing sterile soybean oil at 5.5 kg / h or alternatively in 45 kg loads every 8 hours during the first 100 hours of fermentation. If necessary, additional foam control is carried out with a mixture of soybean oil and a silicon antifoam such as SAG471 (Basildon Silicon Defoamer) in a 3: 1 ratio. The ammonia concentration is maintained between 750 and 1500 mg / l measured every 12 hours and by adding sterile ammonium sulfate in portions equivalent to 500 mg of ammonia / 1 as soon as the level has dropped below 1000 mg / l. The phosphate concentration in the culture filtrate should be maintained above 500 mg P04 / 1 by adding sterile monopotassium phosphate in portions equivalent to 500 mg / l. The portion of glucose isomerase can be measured as protein collected and purified from the broth followed by protein determination methods known in the art or assayed in an enzyme assay applied to a sample of stabilized broth. The broth samples are stabilized by weighing 2 g of broth and adding 5 ml of stabilizing solution containing 12 g / 1 of Tris-hydroxymethylaminomethane and 2.4 g / 1 of CoCl2.6aq which is subsequently heated for 30 minutes at 73 ° C . After cooling, 0.42 ml of stabilized sample is mixed with 0.8 ml of glucose solution (containing 27.25 g / 1 of Tris / HCl buffer, pH 8.2, 67.56 g / 1 of glucose, MgCl2-6aq, 22.33 g / 1 of Na2-EDTA.2aq and 5 mg / l of Triton X-100) and incubate at 60 ° C. Activity is determined by measuring the conversion rates from glucose to fructose and expressed as GU / g. (1 GU is the amount of enzyme necessary for the formation of 1 μmol 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. Figure 2 shows the total amount of poducide enzyme.

As demonstrated in this example, 850 kg of enzyme can be manufactured in a batch feed production run.

Example 2 Production of penicillin V Conidiospores of P. chrysogenum CBS 455.95 (or another suitable strain derived from Wisconsin 54.1255 are inoculated by mutation and selection for higher productivity, preferably in the recipe as set forth below) at 105-106 conidia / ml in a production medium containing (g / 1): glucose.H20, 5; lactose. H20, 80; (NH2) 2C0, 4.5; (NH4) 2S04, 1.1; Na2SO4, 2.9; KH2P04, 5.2; K2HP04.3H20, 4.8; Trace element solution (citric acid, H20, 150, FeS04.7H20, 15; MgSO4.7H20, 150; H3B03, 0.0075; CuS04.5H20, 0.24; CoS04.7H20, 0. 375; ZnS04.7H20, 1.5; MnS04.H20, 2.28; CaCl2.2H20, 0.99), (ml / 1); 10% potassium flushoxide solution, pH 7, 7.5 (ml / 1). (pH before sterilization, 6.5). The culture is incubated at 25 ° C in an orbital shaker at 280 rpm for 144-168 hours. At the end of the fermentation, the mycelia are removed by centrifugation or filtration and the amount quantified, and tests are performed on the medium to determine penicillin formed, by CLAP methods, well known in the art.

Example 3 Large scale fermentation of Penicillium with complex medium and defined medium Penicillium chrysogenum Wisconsin 54.1255 is optimized for growth and production of penicillin in a chemically defined medium by mutation and selection in defined media, as described in example 2. Batch feed fermentations are carried out at a scale of 60 m3 with a medium complex as described by Hersbach et al. (Biotechnology of Industrial Antibiotics pp 45-140, Marcel Dekker Inc. 1984, table 4, medium B, which includes the salts as mentioned in medium A) containing 50 kg / m3 of corn syrup solids. Parallel to this, a fermentation is carried out in a defined medium as indicated in example 2, where dosages are doubled due to the high cell density character of these batch feed fermentations, while lactose and urea are omitted. Glucose is supplied to the fermenter, maintaining the glucose concentration below 2 g / 1 to avoid suppression by glucose. Ammonium, diammonium sulfate and phenylacetic acid are fed to the fermenter in order to control the pH and concentrations of ammonium, sulfate and phenylacetic acid at the desired intervals (Hersbach 1984). Since oxygen transfer is an important parameter to determine the economic feasibility of an industrial fermentation process, the performance of the previous fermentation process was analyzed by determining the amount of oxygen transfer in each process. A good measurement for the transfer of oxygen obtained in a fermentation process is relative KLa value determined within a system. KLa is defined as the oxygen transfer coefficient and is calculated according to van 't Riet and Tramper (Basic Biorector Design, Marcel Dekker Inc. (1991), pp. 236-273). It is found that the oxygen transfer capacity calculated as the KLa value is between 10 and 20% higher in a chemically defined medium compared to a complex medium, during the main part of the fermentation.

Example 4 Production of 7-ADCA The process is modified as described in the example 2 when using P. chrysogenum CBS 455.95 (or another suitable strain derived from Winsonsin 54.1255 by mutation and selection for greater productivity, preferably in the recipe as set forth below, which is transformed with an expanse expression cassette wherein the region that encode expandasa is provided with the IPNS promoter, as described in EP 532341, and using a solution of 10% potassium adipate instead of phenoxyacetate, and using a modification of the above medium containing 400 ml of an adipate solution. %, pH 5.5, instead of phenoxyacetate (pH of the medium before sterilization 5.5 instead of 6.5) The resulting adipoyl-7-ADCA is subsequently converted to 7-ADCA using the enzymatic deacylation process substantially as described in example 4 or 5 of WO95 / 04148.

Example 5 Production of lovastatin Conidiospores from Aspergill us terreus strain are inoculated CBS 456.95 (or strains derived therefrom by mutation and selection for superior productivity, preferably in any of the recipes as set forth below), at 105-105 conidia / ml in a production medium containing (g / 1): dextrose , 40; CH3COONH4, 2.-2; Na2SO4 / 4; KH2P04, 3.6; K2HP04.3H20, 35.1; solution of trace elements (vide supra, example 2), 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 mycelia are removed by centrifugation or filtration and the amount quantified, and a test of the medium is performed to determine lovastatin formed, by CLAP methods well known in the art.

Example 6 Production of clavulanic acid Streptomyces clavuligerus strain ATCC 27064 or a mutant thereof is inoculated in preculture medium consisting of (g / 1): (NH4) 2S04, 2.75; KH2P04, 0.5; MgSO4.7H20, 0.5; CaCl2.2H20, 0.01; 3- (N-morpholino), propanesulfonic acid, 21; glycerol; 19.9; sodium succinate, 5.5; trace elements solution (ZnS04.7H20, 2.8; ferric ammonium citrate, 2.7; CuSO4.5H20, 0.125; MnSO4H20, 1; C? Cl2.6H20, 0.1; Na2B407.10H2O, 0.16; Na2Mo04.2H20, 0.054 ), 0.06 (ml / 1). 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) 2S04, 2; asparagine, 4; KH2P04, 1.5; MgSO4.7H20, 0.5; CaCl2.2H20, 0.01; 3- (N-morpholino), propanesulfonic acid, 21; glycerol, 19.9; sodium succinate, 5.5; solution of trace elements (vide supra), 0.06 (ml / 1), FeS04.7H20, 0.5; MnCl2.4H20, 0.1; ZnS04.7H20, 0.1. The incubation is continued for 144 hours, preferably in a 500 ml Erlenmeyer flask with baffles, containing 50 ml of culture volume. At the end of the fermentation, the mycelium is removed by centrifugation or filtration and the amount quantified, and the filtrate is assayed by CLAP methods well known in the art.

Example 7 Production of amylogogyucosidase Aspergillus niger strain CBS 513.88 or a mutant thereof is inoculated at 105-10s conidiospores / ml in a germination medium consisting of (g / 1): K2HP04.3H20, 0.75; KH2P04, 6.6; Na3 citrate .3H20, 5.3; citric acid. H20 0.45; glucose. H20, 25; (NH4) 2S04 / 8; NaCl, 0.5; MgSO4.7H20, 0.5; FeS04.7H20, 0.1; ZnS04.7H20, 0.05; CaCl2, 0.005; CuS04.5H20, 0.0025; MnS04.4H20, 0. 0005; H3B03, 0.0005; Na2Mo04.2H20, 0.00005; EDTA, 0.13; Tween 80, 3. If necessary, 50 μg / ml of arginine and / or proline can be added to improve germination. The culture is incubated in an orbital shaker for 48-72 hours at 33 ° C, 295 rpm and then used to inoculate -20 volumes of production medium consisting of g / 1: KH2P04, 1-5; NaH2P04.H20, 0.5; Na3citrate .3H20, 53; citric acid. H20, 4.05; Dextrose 70 polymers; (NH4) 2S04, 8; (NaCl, MgSO4.7H20, FeSO4H20, ZnSO4.7H20, CaCl2, CuSO4.5H20, MnSO4.4H20, H3B03, Na2Mo04.2H20, EDTA): same as in the germination medium. Incubation is continued for 96 hours, preferably in a 500 ml Erlenmeyer flask containing 100 ml of medium. At the end of the fermentation, the mycelia are removed by centrifugation or filtration and the amount is quantified, and the filtrate is assayed for amylolytic activity.

Example 8 Production of astaxanthin Phaffia rhodozyma strain CBS 6938 or a mutant thereof is inoculated in 25 ml of preculture medium containing (g / 1): yeast extract, 10; peptone 20; glucose, 20. The culture is incubated for 72 hours at 20 ° C in a 100 ml Erlenmeyer flask with baffles, on an orbital shaker at 275 rpm. Then 1 ml of preculture is used to inoculate 100 ml of production medium containing (g / 1): glucose, 30; NH4C1, 4.83; MgSO4.7H20, 0.88; NaCl, 0.06; CaCl2.6H20, 0.15, trace element solution (citric acid, H20, 50; (NH4) 2Fe (S04) 2.6.H20, 90; ZnS04.7H20, 16.7; CuS04.5H20, 2.5; MnS04, 4H20, 2; H3B03, 2; Na2Mo04.2H20, 2; Kl, 0.5; in 0.4N H2SO4), 0.3 (ml / 1); solution of vitamins (myo-inositol, 40; nicotinic acid, 2; Ca-D-pantothenate, 2; vitamin Bl, 2: p-aminobenzoic acid, 1.2; vitamin B6, 0.2; biotin 0.008; in 0.07N H2S04) 1- 5 (ml / 1); Pluronic, 0.04; KH2P04, 1; phthalate acid potassium, 20 (pH before sterilization, 5.4).

Incubation is continued for 96 hours, preferably in a 500 ml Erlenmeyer flask with baffles. At the end of the fermentation the astaxanthin content of the biomass (quantity quantified) is determined by extraction with solvents and measurement of the optical density of the extract at 470-490 nm.

Example 9 Production of arachidonic acid A 1 ml bottle of a suspension of Mortierella alpina strain ATCC 16266 stored at -80 ° C is aseptically opened, and the content is used to inoculate 500 ml of a production medium containing (g / 1): glucose, 70; Yeast extract 0.5; NH4N03, 3.0; KH2P047.2; MgS04.7H20, 1.5 trace element solution (citric acid, H20 50 (NH4) 2Fe (S04) 2.6H20, 90; ZnS04.7H20, 16.7; CuS04.5H20, 2,5 MnS04.4H20, 2; H3B03, 2; Na2M? 04.2H20; 2; Kl, 0.5; in 0.4NH2SO4), 0.3 (ml / 1); (pH before sterilization, 7.0). The culture is incubated in a 2 liter shake flask with baffles, at 25 ° C using 72 hours in an orbital shaker at 250 rpm. At the end of the fermentation, the amount of biomass and the arachidonic acid content of the biomass is determined, after centrifugation, lyophilization and solvent extraction, by GC methods well known in the art.

Example 10 Production of erythromycin Saccharopolyspora erythraea strain NRRL2338 or a mutant thereof (which is selected for increased productivity, preferably in the recipe as set forth below) is inoculated in 25 ml of a preculture medium containing (g / 1): Soluble starch, fifteen; NaCl, 5; Soybean meal, 15; CaCO3, 10; Yeast extract, 5, macerated corn solids 5; CoCl2.6H20, 670 μl of a 1 g / 1 solution. The culture is incubated in a shake flask of 100 ml without deflectors at 32-34 ° C for 3 days at 250 rpm in an agitator-incubator. 0.4 ml of culture are inoculated into 25 ml of a sterile production medium containing (g / 1): citric acid. H20, 2; (NH4) 2S04, 2; MgSO4.7H20, 2; CaCl2.2H20, 0.085; KH2P04, 0.25; HEPES (= N- (2-hydroxyethyl) piperazine-N '- (2-ethanesulfonic acid)), 5; glucose 1.5, soluble starch, 20; soybean oil, 0.4; trace elements solution (grams in 250 ml of distilled water: citric acid H20, 62.5, FeS04.7H20, 0.8215, CuS04.5H20, 0.0625, CoCl2.H20 * 0.375, H3B03, 0.0625, ZnS04.7H20, 1.25, MnS04 .H20, 1.25; Na2Mo04.2H20, 0.0625), 3.6 ml / 1. pH = 7.0. to each flask, 0.25 ml of n-propanol are added. The culture is incubated in a 100 ml shake flask with baffles at 32-34 ° C for 5 days in an agitator-incubator at 300 rpm. At the end of the fermentation, the broth is centrifuged and the amount of biomass is quantified. The erythromycin content of the supernatant is measured by CLAP methods known in the art.

Example 11 Production of ß-carotene A suspension of spores of Blakeslea trispora CBS 130.59 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 shake flask without baffles. The preculture is incubated for 42 h on a rotary shaker a 250 rpm at 26 ° C. The biomass is collected by filtration, and washed 3 times with 100 ml of sterile demineralized water to remove the components of the preculture medium.

Subsequently, the biomass is homogenized by combination, until only small fragments remain, and is resuspended in 40 ml of demineralized water.

The production medium is prepared in 100 ml portions, in 500 ml shake flasks with baffles. The composition of the production medium is as follows (in g / 1): glucose, 40; Asparagine monohydrate, 2; KH2P04, 0.5; MgS04.7H20, 0.25. In addition, a trace element solution (0.3 ml / 1) is added with the following composition (in g / 1): citric acid.H20, 50; (NH4) 2Fe (S04) 2.6H20, 90; ZnS04.7H20, 16.7; CuS04.5H20, 2.5; MnS04.4H20, 2; H3B03, 2; Na2M? 04.2H20, 2; Kl, 0.5; in 0.4N H2S04. Before 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 water (sterilized by filtration) is added. The production cultures are inoculated with 0.5 to 10 ml of the suspension of fragmented mycelium prepared before. 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 the components of the medium and quantified. The amount of β-carotene produced is determined by extraction with acetone and the extinction at 450 nm of the acetone fraction in a spectrophotometer is measured.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (35)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A process for the production of a useful or valuable compound, characterized in that it comprises the steps of: * fermentation of a microbial strain on an industrial scale in a fermentation medium which is a chemically defined medium, composed essentially of chemically defined constituents, and * recovery of the useful compound from the fermentation broth.

2. The process according to claim 1, characterized in that the chemically defined medium contains an essentially small amount of a complex source of carbon and / or nitrogen.

3. The process according to claim 1 or 2, characterized in that the chemically defined constituents of the chemically defined medium comprise a carbon source which 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 acetates and higher alkanoic acids, and a nitrogen source which is selected from the group consisting of urea, ammonia, nitrate, ammonium salts such such as ammonium sulfate, ammonium phosphate and ammonium nitrate, and amino acids such as glutamate and lysine.

4. The process according to claim 3, characterized in that the carbon source is glucose and the nitrogen source is ammonia and / or an ammonium salt.

5. The process according to any of claims 1 to 4, characterized in that the fermentation takes place via a batch fermentation process, by repeated batches, by batch feeding, by repeated or continuous batch feeding.

6. The process according to claim 5, characterized in that the fermentation takes place via a batch feed process.

7. The process according to claim 6, characterized in that the source of carbon and / or nitrogen is fed into the process.

8. The process according to claim 7, characterized in that the carbon source is glucose and the nitrogen source is ammonia and / or an ammonium salt.

9. The process according to any of claims 1 to 8, characterized in that the useful compound is a protein or pharmaceutical peptide, a primary or secondary metabolite, or an industrial enzyme.

10. The process according to claim 9, characterized in that the useful compound is a secondary metabolite.

11. The process according to claim 10, characterized in that the secondary metabolite is a β-lactam compound.

12. The process according to claim 9, characterized in that the useful compound is an enzyme.

13. The process according to any of claims 1 to 9, characterized in that the microbial strain is a yeast.

14. The process according to claim 13, characterized in that the yeast is Phaffia rhodozyma and the useful compound is astaxanthin.

15. The process according to any of claims 1 to 9, characterized in that the microbial strain is a filamentous microbial strain.

16. The process according to claim 15, characterized in that the filamentous strain is a fungus.

17. The process according to claim 16, characterized in that the fungus is a strain of Aspergillus.

18. The process according to claim 17, characterized in that the fungus is Aspergillus terreus and the useful compound is lovastatin.

19. The process according to claim 16, characterized in that the fungus is a Penicillium strain.

The process according to claim 19, characterized in that the fungus is Penicillium chrysogenum and the useful compound is a β-lactam compound.

21. The process according to claim 16, characterized in that the fungus is a Mucorales strain.

22. The process according to claim 21, characterized in that the Mucorales strain is a Mortierella strain.

23. The process according to claim 22, characterized in that the Mucorales strain is Mortierella alpina and the useful compound is a lipid comprising arachidonic acid.

24. The process according to claim 23, characterized in that the lipid comprising arachidonic acid is a triglyceride.

25. The process according to claim 21, characterized in that the Mucorales strain is a Blakeslea strain.

26. The process according to claim 25, characterized in that the Mucorales strain is Blakeslea trispora and the useful compound is β-carotene.

27. The process according to claim 15, characterized in that the filamentous strain is a bacterium.

28. The process according to claim 27, characterized in that the bacterium is an Actinomycete.

29. The process according to claim 28, characterized in that the Actinomycete is a Streptomyces strain and the useful compound is glucose isomerase.

30. The process according to claim 28, characterized in that the Actinomycete is Streptomyces clavuligerus and the useful product is clavulanic acid.

31. The process according to claim 28, characterized in that the Actinomycete is Saccharopolyspora erythraea and the useful compound is erythromycin.

32. A method for preparing and / or improving a microbial strain that produces a useful compound of interest which is capable of being fermented on an industrial scale in a chemically defined medium, the method is characterized in that it comprises the steps of: * submitting a strain suitable for mutagenic treatment that is selected from the group of chemical physical and mutagenic media, and / or to DNA transformation, * analyze the resulting mutants and / or transformants to determine their growth performance in a chemically defined medium and their production level. valuable compound of interest, * select mutants and / or transformants which have good growth performance in a chemically defined medium and / or an improved production level of the useful compound of interest, as compared to the parent strain.

33. The method according to claim 32, characterized in that the parental strain is selected from the group consisting of strains which have good growth performance in a chemically defined medium, but which need to be improved with respect to the level of production.

34. The method according to claim 32, characterized in that the parental strain is selected from the group consisting of strains which have a high production level of a desired compound, but a relatively poor growth performance in a chemically defined medium.

35. The use of a chemically defined fermentation medium, characterized in that it is used for the production of a useful compound by fermentation of a microbial strain on an industrial scale.