Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
  • C12N1/16—Yeasts; Culture media therefor

Definitions

• the invention relates to a process for the production of yeast biomass by cultivation of a yeast culture on a commercial scale wherein the yeast culture comprises one or more functionally deleted HXK2 genes or analogues and to yeast biomass obtainable by said process.
• Yeast in particular Saccharomyces cerevisiae, is used in many different biotechnoiogicai processes. These can be broadly divided into two categories depending on the metabolic state of the yeast cell.
• yeast biomass is produced commercially in large scale fermentation processes.
• the yeast biomass thus obtained can be used as the classical ingredient in the baking industry or be further processed to yeast extracts that are used as savoury flavours in the food industry.
• Yet another example comprises the cultivation of yeast for production of proteins or other nutrients. More recently, yeast cultures are increasingly used for the production of recombinant (heterologous) proteins, of secondary metabolites, or other modern biotechnoiogicai substances.
• the production of sufficient amounts of yeast biomass stands also central to, and often initiates or is prerequisite for all fermentative yeast processes and also for making enough yeast available to satisfactorily start a fermentative process.
• Fermentation is the conversion of fermentable substrate into mainly ethanol and CO 2 .
• Respiration is the conversion of substrate with the consumption of oxygen into completely oxidised end products (e.g. CO 2 ).
• Yeast is able to adapt its cellular composition towards each mode of growth, 5 making it a versatile, but sometimes intractable organism. These modes differ considerably in the yield of biomass on substrate.
• respiratory growth is the preferred method.
• metabolic characteristics are required, that are normally obtained during fermentative growth conditions. Both modes need to be dealt with satisfactorily to arrive at optimised 0 production.
• both types of growth are necessary for the baking industry.
• One challenge in baker's yeast production is to optimise the efficiency of conversion of assimilable carbon into biomass during the production phase, while ensuring that the yeast will have good leavening characteristics in the dough. Also important is the maintenance of the leavening characteristics during storage.
• Other respiratory yeast cultivation processes have similar complicated demands for balance between biomass production and optimal end-use characteristics (flavour, heterologous protein production, etc.).
• a fermentative mode of growth is obtained under anaerobic conditions, in which the metabolic balance does not allow the complete oxidation of the substrate without the supply of oxygen. Also, many yeast strains show at aerobic conditions fermentative activity parallel to respiratory metabolism - the so-called respiro-fermentative mode - if fermentable substrates are present in excess. Pure respiratory growth is obtained at aerobic conditions and at non-repressive substrate concentrations. The onset of fermentative activity, in addition to pure respiratory activity, marks the critical growth rate that correlates with a critical concentration of fermentative substrate. This critical growth rate is an expression of the respiratory capacity of yeast.
• Glucose repression is seen with a yeast such as Saccharomyces cerevisiae which uses glucose preferentially to all other sources of carbon and energy. This preference is exercised by repression of genes necessary for the utilisation of other carbon sources when glucose is available (Johnston and Carlson, 1992.). Genes required for utilisation of sucrose, maltose and galactose are glucose-repressed. Moreover, a number of genes required for synthesis of mitochondrial enzymes involved in respiration, hence for utilisation of gluconeogenic carbon sources, are also repressed by glucose (Gancedo, 1998).
• HXK2 hexokinase 2
• Hxk2 Hexokinase 2
• Glucose phosphorylation is the first intracellular step in the glycolytic pathway leading to pyruvate (which is the metabolite that occurs at the branch point between the fermentative and respiratory routes of dissimilation).
• pyruvate which is the metabolite that occurs at the branch point between the fermentative and respiratory routes of dissimilation.
• Two other enzymes, hexokinase 1 and glucokinase are partially redundant to Hxk2; they have different kinetic characteristics and are normally expressed at different phases of growth (for a review see Gancedo, 1998.).
• the production of microbial biomass by fermentation can be carried out in several ways: batch mode, fed-batch mode, continuous culture, a combination of batch and fed-batch mode, repeated fed-batch mode or any other combination.
• Batch mode fermentation processes are characterised by the fact that the growth medium containing the substrates is added to the fermentor followed by inoculation of the medium with a preculture of the micro-organism. Oxygen can be supplied by aeration of the growth medium. After a certain time, one or several nutrients are depleted and a maximal cell density is obtained after which the biomass can be collected. The initial excess of fermentable substrate and possible under-aeration are not favourable for the efficient production of yeast biomass.
• the culture rapidly changes to fermentative growth and starts producing ethanol and CO 2 which decreases the yield and the productivity of the process.
• Fed-batch fermentation processes are characterised by the fact that the complete medium containing the substrates is not added at the onset of the fermentation process, but part of the medium is added as a continuous or intermittent feed.
• glucose and other essential nutrients are fed to the culture in a growth-limiting fashion.
• the feed rate is adjusted to a rate that does not exceed the oxygen transfer capacity of the fermentor.
• baker's yeast production usually uses molasses as a substrate, which has a high saccharose concentration.
• the saccharose is hydrolysed by the enzyme invertase into glucose and fructose and, in order to maintain efficient conversion of substrate into biomass, the production process is carried out aerobically by feeding at a rate below the maximum oxygen transfer capacity.
• Irregularities in the mixing may locally cause high concentrations of the sugar substrate with concomitant production of ethanol and repressive effects on the respiratory capacity. Consumption of the ethanol later on at non repressive conditions, still results in a loss of yield due to the properties of the metabolic pathways.
• Production processes under continuous culture conditions resemble the fed- batch processes except that biomass-containing effluent is removed from the fermentation continuously or intermittently.
• Production processes may also be carried out by a combination of different modes of batch and fed-batch and continuous cultivation.
• the costs of yeast biomass production depend heavily on the yield, expressed as amount of biomass per amount of substrate and the productivity of the fermentor. Ethanol production by the yeast decreases the yield and therefore increases the costs.
• the need to keep the growth rate below a critical value in order to avoid ethanol formation reduces the productivity. Therefore, yield and productivity need to be carefully balanced in order to obtain the lowest production costs, whereby the equilibrium point is dependent on the metabolic properties of yeast.
• the sensitivity of yeast strains for glucose repression shifts the aforementioned equilibrium point towards lower productivity. Further demands on the application properties of the biomass product aggravate the establishment of the equilibrium point and the process is still far from being optimised, despite years of biotechnoiogicai and genetic development.
• the present invention provides more cost effective, commercial processes for the production of yeast biomass, by using yeast cultures that are less or not sensitive to glucose repression.
• Saccharomyces cerevisiae mutants provide evidence of hexokinase Pll as a bifunctional enzyme with catalytic and regulatory domains for triggering carbon catabolite repression. J. Bacteriol. 158:29-35 Gancedo, J. M. 1998. Yeast carbon catabolite repression. Microbiol. Mol. Biol. Rev. 62:334-361.
• Figure 1 Influence on growth characteristics of the HXK2 deletion.
• the wildtype strain (o) and the Hxk2 deficient mutant strain (squares) were grown on YNB, 2% glucose. Growth was monitored by measuring the optical density at 600 nm.
• Figure 2 Glucose consumption during growth on glucose.
• the wild type strain (o) and the Hxk2 deficient mutant strain (squares) were grown on YNB, 2% glucose
• Figure 3 Ethanol production during growth on glucose.
• the wild type strain (o) and the Hxk2 deficient mutant strain (squares) were grown on YNB, 2% glucose
• Figure 4 Oxygen consumption capacity during growth on glucose.
• the wildtype strain ( ⁇ , open and closed) and the Hxk2 deficient mutant strain (o, open and closed) were grown on YNB, 2% glucose.
• OD 600 nm, open symbols samples were taken and analysed for oxygen consumption capacity ( ⁇ mole/min/g protein, closed symbols).
• the invention provides a process for the production of yeast biomass characterised by cultivation of a yeast culture on a commercial scale, wherein the yeast culture comprises one or more functionally deleted HXK2 genes or analogues thereof, and by growing said yeast culture aerobically in a growth medium containing one or more fermentable carbon sources
• the invention provides a process for the production of proteins or secondary metabolites using a yeast culture that comprises one or more functionally deleted HXK2 genes or analogues thereof by cultivating the yeast culture according to a process as provided by the invention.
• the invention provides yeast biomass or a substance derived thereof obtainable by a process as provided by the invention.
• the invention provides compressed yeast, cream yeast or dried yeast made from yeast biomass as provided by the invention.
• Yeast biomass is defined as accumulated yeast or yeast cells, or compounds or substances derived from yeast cells, which can subsequently be used for a great many purposes such as ingredient for dough rising (in the form of compressed yeast, cream yeast or dried yeast), as food component in for example processed foods, as a source of (heterologous) proteins or peptides, as a source of nutrients such as amino acids, vitamins, as a source of secondary metabolites, as source of pharmaceutical ingredients.
• a functionally deleted HXK2 gene is defined as a complete deletion of the gene or a gene carrying mutations made by substitution, insertion or deletion that result in a functionally inactive Hxk2.
• Analogues of HXK2-genes are genes encoding proteins that exhibit the same function as Hxk2 with respect to sugar utilisation and regulatory properties.
• Cultivation on a commercial scale is defined herein as a fermentation process carried out in a fermentor with a volume of more than 0.5 litre.
• the invention provides a process for the production of yeast biomass characterised by cultivation of a yeast culture on a commercial scale, wherein the yeast culture comprises one or more functionally deleted HXK2 genes or analogues thereof, and by growing said yeast culture aerobically in a growth medium containing one or more fermentable carbon sources.
• Carbon sources are preferably sugars and more preferably belong to the group consisting of glucose, fructose, maltose, saccharose, galactose and raffinose.
• the yeast preferably belongs to the genus Saccharomyces or Kluyveromyces. More preferred species are Saccharomyces cerevisiae, Kluyveromyces lactis or Kluyveromyces marxianus.
• yeast cultures in which the glucose repression pathway is functionally deleted have great advantages over classical strains in the production of yeast biomass, specifically under aerobic conditions, because in commercial scale biomass production processes, they improve the productivity of said processes and maintain or improve the yield. Furthermore, it was found that, in contrast to yeast cultures that are traditionally used for biomass production, yeast cultures comprising one or more functionally deleted HXK2 genes or analogues maintain their leavening capacity.
• said yeast culture showing no or diminished glucose repression when grown aerobically in the presence of glucose can advantageously be applied in batch production of inoculation material or in the start up phase of a production culture.
• Said cultures are not hampered by for example the ethanol that is inadvertently produced at those conditions.
• the higher respiratory capacity of said yeast cultures at these conditions allows for higher growth rates in the adaptation time after non repressive conditions are introduced.
• These cultures immediately show respiratory growth when grown aerobically on glucose, not being hampered by glucose induced repression of enzyme synthesis needed for said respiratory growth, as for example characterised by an increased oxygen consumption capacity during initial growth on glucose and exhibit higher growth rates in the period thereafter.
• Reduction of the ethanol production results in improved yield and the application of higher growth rates results in a decreased fermentation time and increased productivity, both resulting in cost price reduction.
• the application of glucose repression resistant strains results also in a reduced ethanol turnover and in apparently higher critical growth rates when irregularities in the mixing lead to locally cause high concentrations of the sugar substrate with concomitant production of ethanol and repressive effects on the respiratory capacity.
• mutants with defects in the glucose repression pathway can be isolated through classical mutagenesis and selection.
• One way of screening for such mutants is by selection for rapid growth on sucrose or ethanol and glycerol of a mutagenised population of cells after preculture in a medium with a high glucose concentration. This procedure is, when so desired, repeated serially in order to optimise the desired phenotype.
• yeast variants can advantageously be used in a culture as provided by the invention.
• Another selection strategy is to use 'gratuitous repressors', i.e. glucose analogues that are not themselves metabolisable (e.g.
• the production process of yeast biomass by fermentation is carried out in batch mode whereby the growth medium initially contains the fermentable carbon source at a non-growth-limiting concentration.
• concentration of said carbon source is more than 1 mmole per litre, more preferably more than 5 mmole per litre, most preferably > 10 mmole per litre.
• the production process of yeast biomass by fermentation is carried out in a fed-batch mode whereby a component of the growth medium is growth limiting and supplied to the fermentation medium in the feed stream.
• This growth limiting component can be a nitrogen source such as ammonia, or a carbon source such as the sugars mentioned above.
• the production process of yeast biomass by fermentation is carried out as a continuous culture and a component of the growth medium is growth limiting.
• This growth limiting component can be a nitrogen source such as ammonia, or a carbon source such as the sugars mentioned above.
• the production process of yeast biomass by fermentation comprises the following steps. First a fermentation in batch mode is carried out as described above. The carbon source at initial non-growth limiting concentration is consumed with concomitant accumulation of yeast biomass. After consumption of the carbon source, the fermentation process is changed to fed-batch mode whereby in this case the carbon source is growth limiting and supplied to the fermentation medium in the feed stream.
• the invention provides yeast biomass or a substance derived thereof obtainable by the production processes of the invention.
• the yeast preferably belongs to the genus Saccharomyces or Kluyveromyces. More preferred species are Saccharomyces cerevisiae, Kluyveromyces lactis or Kluyveromyces marxianus.
• Said biomass in general comprises no or only little ethanol or other byproducts of fermentative growth, despite that it has been produced on a culture medium comprising substantial amounts of glucose.
• the invention provides a process for the production of proteins or secondary metabolites using a yeast culture that comprises one or more functionally deleted HXK2 genes or analogues thereof by cultivating the yeast by the production processes of the invention.
• the yeast comprises a nucleic acid that encodes a desired proteinaceous substance (peptide, polypeptide or protein optionally comprising additional non-protein groups) to be harvested or an enzyme providing for additional enzymatic reaction in said yeast.
• a desired proteinaceous substance peptide, polypeptide or protein optionally comprising additional non-protein groups
• Such an enzyme is particularly useful to provide for additional modifications of a desired product, such as a protein or secondary metabolite to be harvested from said yeast.
• yeast in addition to comprising one or more functionally deleted HXK2 genes also comprise a heterologous nucleic acid, is used to produce any product, such as a heterologous protein or a secondary metabolite with a greatly enhanced conversion efficiency as a result of the fact that the sugar substrate is oxidised without wasteful production of ethanol.
• the invention provides compressed yeast, cream yeast or dried yeast made from said yeast biomass that is obtainable by the production processes of the invention.
• the yeast preferably belongs to the genus Saccharomyces or Kluyveromyces. More preferred species are Saccharomyces cerevisiae,
• the Saccharomyces cerevisiae wildtype strain CEN.PK 113-7D (MATa, MAL2-8c SUC2) which was kindly provided by Dr. P. K ⁇ tter (Frankfurt, Germany) was used for the construction of a mutant strain which lacks the HXK2 gene.
• Genetic modification of yeast i.e. deleting or mutating one or more parts of the nucleic acid of a yeast genome, or providing a yeast genome with additional heterologous nucleic acid is in itself a skill known in the art.
• the HXK2 gene was functionally deleted by PCR-based gene disruption and replaced by a kanMX-marker.
• PCR primers AK53 (SEQ ID 1) and AK54 (SEQ ID 2) were used in a polymerase chain reaction with plasmid pFA6A- kanMX4 (Wach et al., 1994), deoxynucleotides, and ExpandTM DNA polymerase (Boehringer Mannheim) to produce an HXK2-specific kanMX gene replacement module.
• This DNA module was transformed into the yeast strain, and transformants were selected for resistance to the antibiotic G418. Resistant isolates were verified to have replacements of HXK2 with kanMX by diagnostic PCR.
• a wildtype yeast strain and a Hxk2 functional deletion strain were grown in batch cultures in a fermentor with a volume of 1 litre on minimal medium containing 2% glucose. The growth of both strains was monitored by measuring the optical density of the cultures at 600nm. During growth, samples were taken for glucose and ethanol determination. Samples were taken for determination of oxygen consumption rates as an indicator of respiratory activity. The characteristics of glucose transport were determined at certain key points during growth.
• the molasses feed started at a constant rate, that was slightly in excess of the respiratory capacity in the initial hours of the fermentation, resulting in some ethanol formation, the rate of which decreased with the increase in biomass.
• the feed rate of molasses was exponentially increased but was kept below the critical rate during the remaining part of the fermentation.
• the aeration rate is high and no ethanol formation is obtained except occasionally a small amount at the end of the fermentation, when the oxygen consumption rate is close to the oxygen transfer capacity.
• Part of the biomass of this fermentation is used as inoculum for the next production fermentation after washing and concentration and storage in the refrigerator for a limited amount of time.
• the main (production) fermentation follows basically the same scheme, except for the application of a higher amount of inoculum material and by consequences higher feed rates of molasses and NH 4 + .
• the feed rate of the molasses was designed in this experiment with a maximum that did not exceed the respiratory capacity of either the parent or the mutant strain. No ethanol formation occurred under these conditions. Due to limitations in the oxygen transfer capacity the feed rate was kept at a maximum value in the last phase of the fermentation, resulting in a decrease of the growth rate with increasing biomass concentration.
• a maturation step was included at the end of the fermentation before washing and concentration by centrifugation and final concentration in a filter press.
• the resultant wet yeast product has a dry matter content of about 30 %.
• the dough is mixed in a normal way to get a properly developed dough and then put in a gas- production measurement device essentially as described by Burrows and Harrison (1959) at 28°C and incubated for up to 3 hours.
• the amount of gas produced is recalculated to the amount of gas produced by a quantity of yeast containing 1 mg of nitrogen determined according to Kjeldahl.
• the amount of gas produced by the parent strain under these conditions is about 7 ml.
• the test is repeated on samples that have been stored during 1 to 7 days in an incubator at 30 °C, resulting in a figure for the keepability of the gassing power.
• Table 1 the relative gassing power values of both strains are compared (the values for the parent strain are set at 100%).
• the critical growth rate of the parent strain and the Hxk2-mutant were determined according to this method. Values of respectively 0.3 h "1 and 0.35 h '1 were found. This indicates an increase of 16% in the respiratory capacity. This allows a significant increase in growth rate in the first phase of the fermentation and allows an optimisation of the process towards a higher productivity.

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