Genetically modified yeast with improved glycerol catabolism

Abstract

Genetically modified yeast cell of the genus Saccharomyces with improved glycerol catabolism, wherein the yeast cell is genetically modified such that a) the degradation of glycerol to dihydroxyacetone phosphate via the glycerol-3-phosphate pathway is blocked by the deletion of the genes for glycerol kinase (GUT1) and / or mitochondrial glycerol-3-phosphate dehydrogenase (GUT2), and b) a heterologous glycerol uptake facilitator protein is expressed, wherein the heterologous glycerol uptake facilitator protein is Fps1 or Fps2, and c) a heterologous glycerol dehydrogenase catalyzing the oxidation of glycerol to dihydroxyacetone is expressed or a heterologous glycerol dehydrogenase catalyzing the oxidation of glycerol to dihydroxyacetone is overexpressed.or c2) at least one enzyme involved in the degradation of glycerol to glyceraldehyde-3-phosphate via the glyceraldehyde pathway is overexpressed, or at least one enzyme involved in the degradation of glycerol to glyceraldehyde-3-phosphate via the glyceraldehyde pathway is replaced by a heterologous enzyme with the same enzyme activity.

Description

The invention relates to an genetically modified yeast having improved glycerol catabolism.The production of bio-fuels and chemicals from renewable resources is necessary to meet the energy requirements in a world where mineral oil-based fuels are becoming increasingly more knapp and expensive. Biodiesel is an alternative fuel which is currently largely obtained from rapeseed in Germany and, after a survey of the association of the Deutsche Bio-fuel industry e.V., can reduce greenhouse gas emission up to 60% compared with fossil diesel. In principle, used feed fats can also be used. The potential use of fats or oils produced by microalgae or other microorganisms (Gong and Jiang, 2011; Shi et al., 2011) is in the discussion, but so far have not been of any importance commercially. Biodiesel is produced mainly by the transesterification of fats and vegetable oils in the presence of an alcohol (mostly methanol), which leads to the formation of a fatty acid methyl ester (biodiesel) and glycerol (as a byproduct). Biodiesel production and thus also the amount of crude glycerol which is produced in parallel has increased greatly in recent years. 10 weight units of crude glycerol are obtained per 100 weight units of biodiesel produced (Yazdani & Gonzalez, 2007). In Germany, 200,000 t of crude glycerol were produced in 2011 during biodiesel production (market analysis of renewable raw materials, FNR, series of documents of renewable raw materials 34, 2014). The purification of the glycerol fraction from crude glycerol in a manner such that it meets the standards of the food, pharmaceutical or cosmetic industry is not economical, so that other ways must be taken to utilize crude glycerol. One possibility is use as starting material for microbial conversion processes (Almeida et al., 2012).Baker's yeast (Saccharomyces cerevisiae) is a popular object of Metabolism Engineering, as well as an attractive production organism in industrial biotechnology. Numerous approaches have been followed to make this organism usable for the production of industrially relevant products such as bio-fuels, basic and fine chemicals (including medicaments) and protein active ingredients (Borodina & Nielsen, 2014; Hong & Nielsen, 2012; Nevoigt, 2008). However, S. cerevisiae grows only poorly on glycerol as carbon source. Two studies have shown that the S. cerevisiaestrains frequently used in research and industrial use require the addition of supplements (such as amino acids and nucleobases) to grow on glycerol (Merico et al 2011; Swenn et al 2013). Only individual isolates are able to use glycerol as carbon source without such media additives (Swenn et al. 2013).For metabolization, glycerol must first be taken up in the cell. In the case of microorganisms, two types of transport systems have in principle been found (see Fakas et al. 2009 and Lin et al. 1976). The first type is based on facilitated diffusion, does not require energy, and depends on glycerol factors. In contrast, the second type is an energy dependent active transport based on a glycerol / H +- symporter. It has long been moving round how glycerol is transported into the S. cerevisiaecell (Neves et al. 2004). Finally, it has been shown that an active transport system encoded by STL1 is of decisive importance (Ferreira et al. 2005). A glycerol factor encoded by FPS1 is naturally also present in S. cerevisiae, but does not seem to play a decisive role in glycerol uptake in this manner.In general, the initial degradation of glycerol in microorganisms takes place in three ways (see Fakas et al. 2009). The glycerol 3-phosphate pathway (also G3P pathway or phosphorylation pathway) leads via phosphorylation of glycerol by the glycerol kinase to L-glycerol 3-phosphate (G3P), which is oxidized by the glycerol 3-phosphate dehydrogenase to dihydroxyacetone phosphate (DHAP). The DHA pathway (also oxidation pathway) leads via the glycerol dehydrogenase-catalyzed oxidation of glycerol to dihydroxyacetone (DHA), which is phosphorylated by the dihydroxacetone kinase to dihydroxyacetone phosphate (DHAP). The glyceraldehyde pathway leads via glyceraldehyde to glyceraldehyde-3-phosphate, which can be enzymatically converted to DHAP.Experimental data indicate that S. cerevisiaedegradates glycerol exclusively via the phosphorylation pathway, i.e. via L-glycerol-3-phosphate, i.e. uses only the enzymes glycerol kinase and glycerol-3-phosphate dehydrogenase encoded by GUT1 and GUT2. Experimental evidence for the importance of Gut1 and Gut2 for the growth of S. cerevisiae on glycerol was provided for the first time by Sprague and Cronan (1977).The studies by Ferreira et al. (2005) on the importance of the Stl1 transporter or of Sprague and Cronan (1977) on the role of Gut1 and Gut2 for glycerol utilization of S. cerevisiaewere carried out in media which contained complex additives such as several amino acids and nucleobases. Swenn et al. 2013, however, were able to show that the genes STL1, GUT1 and GUT2 also have a decisive and exclusive role for the growth of S. cerevisiae in glycerol-containing medium without additives.The oxidative glycerol degradation pathway via DHA appears to be present in Neurospora crassa parallel to the phosphorylation pathway (Tom et al., 1978). NAD +- dependent glycerol dehydrogenase has been found in some species of the genera and Candida, Torulopsis, and Hansenula(Hofmann and Bebel, 1979); later also in Schizosaccharomyces pombe(May & Sloan, 1981). Beck and Hofmann (1982) were able to measure a particularly high activity of this enzyme in the case of Candida valida. It is unclear, however, whether the oxidative pathway is naturally functional in S. cerevisiae. Indeed, neither a GUT1 nor a GUT2 deletion mutant exhibited significant growth on glycerol (Swenn et al.2013), a result that excludes an important role of the DHA pathway for this function. Moreover, neither Norbeck and Blomberg (1997) nor Nguyen and Nevoigt (2009) were able to measure native glycerol dehydrogenase activity in S. cerevisiae. In contrast, other authors suggest that glycerol is converted to dihydroxyacetone by a NADP +- dependent glycerol dehydrogenase (GDH), where the genes ARA1, GCY1, GRE3 and YPR1 are to be involved (Izawa et al., 2004). Although there is doubt about the existence of a natural glycerol dehydrogenase in S. cerevisiae, a measurable activity of the second enzyme of the DHA pathway, the dihydroxyacetone kinase encoded by the genes and DAK1 and DAK2, is naturally present in S. cerevisiae(Molin et al., 2003). A study by Jung et al. 2011 has recently shown that the expression of a heterologous glycerol dehydrogenase from Ogataea angusta (Syn. Pichia angusta, Hansenula polymorpha) in S. cerevisiae improved glycerol utilization 1.22-fold compared to the wild type strain, i.e. the genetically modified strain consumed 5.18 g / l of 12.62 g / l glycerol after 96 h of culture, whereas the control consumed only 4.24 g / l glycerol in the same time compared thereto.Liu et al. (2013) discloses a study comparing the genomic sequences of S. cerevisiaeand P. tannophilusto examine the genes involved in glycerol transport and glycerol metabolism in these two organisms. The technical article focus exclusively on glycerol transporters and does not address the blocking of metabolic pathways nor the enzymatic optimisation of glycerol catabolism. In particular, Liu et al.,2013 describes heterologous expression of the PtFPS1 transport which leads to improved growth on glycerol. However, Liu et al.,2013 does not disclose blocking the G3P pathway or deletion of genes for glycerol kinase (GUT1) and / or mitochondrial glycerol-3-phosphate dehydrogenase (GUT2).US20130109070A1 discloses a genetically modified yeast strain in which the GPD gene and the glycerol channel FPS1 have been disrupted to block the activity of glycerol 3-phosphate dehydrogenase and to increase bioethanol production. The focus here is on reducing glycerol production in favor of ethanol synthesis, but not on promoting glycerol catabolism via alternative metabolic pathways such as the DHA or glyceraldehyde pathway.WO2011124146A1 describes yeast strains in which FPS1, GPD1 and GPD2 were deleted to block glycerol production and increase ethanol production. The focus is primarily on reducing glycerol synthesis without promoting glycerol degradation or considering the expression or overexpression of enzymes involved in alternative glycerol degradation pathways.The object of the present invention is to improve the ability of yeast to use glycerol as a carbon source.The object is achieved by an genetically modified yeast cell of the genus Saccharomyces with improved glycerol catabolism, wherein the yeast cell is genetically modified in such a way thata) the degradation of glycerol to dihydroxyacetone phosphate via the glycerol 3-phosphate pathway is blocked by the deletion of the genes for glycerol kinase (GUT1) and / or the mitochondrial glycerol 3-phosphate dehydrogenase (GUT2),b) expressing a heterologous glycerol uptake factor protein, wherein the heterologous glycerol uptake factor protein is Fps1 or Fps2, andc1) expresses a heterologous glycerol dehydrogenase catalyzing the oxidation of glycerol to dihydroxyacetone or over-expresses a heterologous glycerol dehydrogenase catalyzing the oxidation of glycerol to dihydroxyacetone, orc2) at least one enzyme involved in the degradation of glycerol to glyceraldehyde-3-phosphate via the glyceraldehyde pathway is overexpressed or at least one enzyme involved in the degradation of glycerol to glyceraldehyde-3-phosphate via the glyceraldehyde pathway is replaced by a heterologous enzyme having the same enzyme activity.The combination of genetic alterations to Saccharomyces cells provided according to the invention brings about efficient uptake and channelization of glycerol by the now established or at least enhanced DHA metabolic pathway. Alternatively, according to the invention, glycerol catabolism is established or optimized via the glyceraldehyde pathway by over-expressing at least one, preferably all of the natural (native) enzymes involved in this pathway up to the glyceraldehyde 3-phosphate or by replacing it with a heterologous enzyme having the same enzyme activity. In this alternative, it is not necessary, nor preferred, to manipulate the DHA pathway. Integration of a heterologous glycerol factor promotes glycerol uptake without requiring energy for this purpose. Blocking of the glycerol-3-phosphate pathway results in glycerol incorporated into the cell being degraded either via the DHA pathway, wherein the introduction of a heterologous glycerol dehydrogenase or the overexpression of a native enzyme having glycerol dehydrogenase activity compensates for the possible deficiency of a natural glycerol dehydrogenase activity, or alternatively via the glycerol aldehyde pathway then optimized by overexpression of the involved enzymes or introduction of heterologous enzyme variants. The FAD dependent glycerol-3-phosphate dehydrogenase involved in the glycerol-3-phosphate pathway is localized on the outside of the inner mitochondrial membrane (Gancedo et al., 1968; Schneiderberg 1970) and directs electrons from the oxidation of glycerol-3-phosphate directly into the mitochondrial respiratory chain, which is disadvantageous when reducing equivalents are needed, for example, for subsequent production of reduced compounds, for example, 1,2-propanediol. In contrast, the DHA pathway provides valuable reduction equivalents in the form of NADH, the glyceraldehyde pathway in the form of NADPH.The term "glycerol-3-phosphate pathway", "glycerol-3-phosphate pathway", "G3P pathway" or also "phosphorylation pathway" is understood to mean the reaction of glycerol via L-glycerol-3-phosphate (G3P) to give dihydroxacetone phosphate (DHAP). The reactions are catalyzed by glycerol kinase and mitochondrial (FAD-dependent) glycerol-3-phosphate dehydrogenase (GPD-M, mGP, EC 1.1.5.3). In the yeast Saccharomyces cerevisiae, glycerol kinase is encoded by the gene GUT1, the glycerol-3-phosphate dehydrogenase by GUT2.The "DHA pathway", "DHA metabolic pathway" or "oxidation pathway" is understood to mean the reaction of glycerol via dihydroxyacetone (DHA) to give dihydroxacetone phosphate (DHAP). The reactions are catalyzed by glycerol dehydrogenase and dihydroxyacetone kinase.The term "glyceraldehyde pathway" is understood to mean the reaction of glycerol via D-glyceraldehyde to form D-glyceraldehyde-3-phosphate (GAP). The reactions are catalyzed by a NADP-dependent glycerol dehydrogenase (NADP-glycerol dehydrogenase, NADP-GDH) and a glyceraldehyde kinase (triokinase, triose kinase, EC 2.7.1.28).When a "synthetic route for the recovery of glycerol from dihydroxyacetone phosphate" is referred to herein, this means the conversion of dihydroxyacetone phosphate via glycerol-3-phosphate to glycerol. The reduction of DHAP to glycerol-3-phosphate is catalyzed by cytosolic NAD-dependent glycerol-3-phosphate dehydrogenase (GPD-C, cGP, EC 1.1.1.8) occurring in two isoforms. The isoforms are encoded by the genes GPD1 and GPD2, respectively. The dephosphorylation of glycerol 3-phosphate to glycerol is catalyzed by glycerol 3-phosphatase (GPP, EC 3.1.3.21) which also occurs in two isoforms encoded by GPP1 and GPP2.The term "heterologous" is used herein as meaning known to those skilled in the art and refers to the foreign origin of an element, for example an enzyme or other protein. "foreign" means that the element does not so occur in the target cell, and originates from, for example, a cell or organism with diverse genetic equipment, for example an organism of another type.A "glycerol uptake factor protein" or, for short, "glycerol factor" is understood to mean a transmembrane protein which enables the transport of glycerol into a cell, for example a Saccharomycescell, by means of facilitated diffusion.By "glycerol dehydrogenase" (GDH for short) is meant here a cytosolic glycerol:NAD +- oxidoreductase (EC 1.1.1.6) which catalyzes the oxidation of glycerol to dihydroxyacetone by means of NAD + as cofactor. A "heterologous glycerol dehydrogenase" is understood to mean a cytosolic foreign glycerol dehydrogenase, i.e. a glycerol dehydrogenase derived from another organism, e.g. another species, genus etc., but which catalyzes the same reaction, optionally using a different cofactor.The term "NADP glycerol dehydrogenase" (also abbreviated to "NADP-GDH") is understood here to mean a glycerol:NADP+oxidoreductase (EC 1.1.1.72) which oxidizes glycerol to D-glyceraldehyde, wherein NADP+functions as cofactor.The term "dihydroxyacetone kinase" or "DHA kinase" is understood here to mean an ATP:dihydroxyacetone phosphotransferase (EC 2.7.1.29) which catalyzes the phosphorylation of dihydroxacetone by means of ATP.The term "methanol dehydrogenase" (MDH for short) is understood here to mean an enzyme (EC 1.1.1.244, EC 1.1.2.7, EC 1.1.2.8, 1.1.99.37) catalyzing the reduction of formaldehyde to methanol (MeOH), preferably a methanol:NAD +- oxidoreductase (EC 1.1.1.244).A "dihydroxyacetone synthase" or "DHA synthase", also referred to interchangeably as "formaldehyde transketolase" or "transketolase", is understood to mean a D-xylulose-5-phosphate:formaldehyde glycol aldehyde transferase (EC 2.2.1.3) which catalyzes the formation of formaldehyde and D-xylulose-5-phosphate from dihydroxyacetone and glyceraldehyde-3-phosphate.Here, "expression" is understood to mean the conversion of genetic information into a product, for example the formation of a protein or a nucleic acid on the basis of the genetic information. The term encompasses in particular the biosynthesis of proteins on the basis of the genetic information including preceding processes such as transcription, i.e. the formation of mRNA on the basis of a DNA template.The expression that a heterologous protein / enzyme is expressed includes that the protein / enzyme is overexpressed in the target cell under otherwise similar conditions compared to the average level of expression of the protein / enzyme in the parent cell. The term also includes that the protein / enzyme is expressed under the control of any promoter, i.e. also a promoter which may be different from the promoter which is linked to the protein / enzyme in the source cell or which is linked to any orthologous protein / enzyme in the target cell. The skilled person is familiar with how he can bring about an overexpression of a protein / enzyme. One possibility is, for example, the introduction of additional copies of the gene coding for the protein / enzyme into a cell and / or the replacement of the natural promoter under the control of which the expression of the gene takes place with a stronger promoter.The term "blocking" or "blocking" of a metabolic pathway, i.e. a chain of reactions with a starting compound, at least one intermediate product and an end product (which itself may also be the starting compound for another metabolic pathway again) is understood here to mean that the formation of at least the first intermediate product of the pathway is suppressed or substantially reduced. "blocking" or "blocking" here means in particular that, compared to a yeast cell in which the metabolic pathway is not blocked, the formation of the first intermediate product is reduced by at least 75%, preferably at least 80%, at least 85%, at least 90%, at least 95% or at least 98%. With respect to the glycerol-3-phosphate pathway, blockade is therefore understood to mean that at least the degradation of glycerol to glycerol-3-phosphate is blocked by glycerol kinase. In relation to the synthetic route for the regression of glycerol from dihydroxyacetone phosphate (DHAP), blockage is understood to mean that at least the reduction of DHAP to glycerol-3-phosphate is blocked by glycerol-3-phosphate dehydrogenase or at least the reduction of glycerol-3-phosphate to glycerol is blocked by glycerol-3-phosphatase. The term also includes that all intermediate steps are blocked on the route to the end product, for example by deleting or mutating the genes of all enzymes involved in the route, so that no functional gene product or only one gene product, i.e. enzyme, which is substantially restricted in its function results. The term can also mean that the formation of functional enzyme is reduced by the percentages mentioned above, for example by replacing the natural promoters with correspondingly weaker promoters, or that only such an amount of an enzyme whose functionality is restricted is formed that the formation of the intermediate product is reduced as defined above. Means for modifying a metabolic pathway by means of "Metabolic Engineering" are well known to the skilled person (see, for example, Nevoigt 2008; Mapelli 2014). For example, a metabolic pathway can be blocked by deleting or deactivating a gene or a DNA section relevant for the regulation of a gene, for example by means of targeted mutation. Modification to express a heterologous enzyme can be accomplished by genetic engineering techniques known to those skilled in the art, for example, by introducing a suitable expression vector having a gene encoding the enzyme (along with a suitable promoter and terminator) into the cell.When an enzyme having a certain enzyme activity is referred to here, e.g. a dihydroxyacetone kinase (DHA kinase), the term also covers an enzyme which, in addition to at least one other enzyme activity, also has at least the corresponding enzyme activity. A "DHA kinase", for example, is therefore also understood to mean an enzyme having DHA kinase activity that in addition has at least one other enzyme activity, for example a triose phosphate isomerase activity. A specific example of this is Saccharomyces cerevisiae2,3-butanediol dehydrogenase (Bdh1) which has been shown to also have glycerol dehydrogenase activity (Gonzalez et al., 2000). When the expression of a native glycerol dehydrogenase is referred to here, this therefore also comprises, for example, the overexpression of Bdh1or of another native enzyme having corresponding activity.The genetically modified yeast cell according to the present invention is preferably a yeast cell of the species Saccharomyces cerevisiae.In a particularly preferred embodiment, the genetically modified yeast cell according to the invention is further genetically modified in that c1a) the degradation of glycerol to glycerol aldehyde-3-phosphate is blocked via the glycerol aldehyde pathway, and / or d) the expression of the natural active glycerol transporter encoded by STL1 is blocked, and / or e) the synthetic pathway for the regression of glycerol from dihydroxyacetone phosphate is blocked, and / or f) a heterologous dihydroxyacetone kinase is expressed or the native dihydroxyacetone kinase is overexpressed.The features d), e) and f) can be realized individually or in any combination or else all together with the features a), b) and c1 or the mermalens a), b) and c2. Feature c1a) is preferably realized only together with features a), b) and c1), wherein features d), e) and f) can additionally be realized individually, in any desired combination or in cumulative form. A preferred combination of features is, for example, a combination of features a), b), c1) and f). In this embodiment, the DHA path is activated or optimized in a particularly advantageous manner.In the genetically modified yeast cell according to the invention, in one embodiment all features cla) to f) or all features d) to f) are preferably realized together with features a) to c1). In the case of the alternative embodiment of the invention having the features a) to c2), it is preferred if the features d) to f) are likewise all realized.The additional blocking of the glyceraldehyde pathway is advantageous, for example, for the production of fermentation products for which reduction equivalents are required only in the form of NADH, e.g. succinate.Blocking the expression of the natural active transporter encoded by STL1 avoids unnecessary consumption of ATP, especially under conditions where the cells may not deliver sufficient ATP, for example under oxygen limitation. Expression of the heterologous glycerol factor still allows glycerol to be incorporated into the yeast cell. The production of an STL1 deletion mutant is described, for example, in Liu et al. 2013 and Swenn et al. 2013.Blocking the synthetic route for the regression of glycerol from dihydroxyacetone phosphate additionally prevents dihydroxyacetone phosphate formed via the DHA route from being converted back into glycerol-3-phosphate or even glycerol, as a result of which, in particular, unnecessary outflow of reduction equivalents can be avoided. Blockade can be achieved, for example, by deleting the genes (GPD1 / 2 and / or GPP1 / 2) which code for the enzymes catalyzing regression. Deletions of GPD1 and GPD2 are described in Hubmann et al. (2011). The deletion of GPP1 and GPP2, respectively, is described in Påhlmann et al. (2001). Alternatively, the natural promoters can be replaced by much weaker promoters, as has already been described for GPD1 or GPD2 (Hubmann et al., 2011). Instead of shutting down GPD1 / 2, it is also possible, as a further alternative, to shut down GUT2and thus block the expression of the FAD-dependent mitochondrial glycerol 3-phosphate dehydrogenase (mGDP). Thus, in particular in aerobic culture, the loss of electrons via the glycerol-3-phosphate / DHAP shuttle system would be avoided.Expression of a heterologous DHA kinase can be carried out instead of or in addition to the native DHA kinase, i.e. that is naturally present in the cell. Preferably, a heterologous DHA kinase having a higher activity (higher turnover rate) compared to the native DHA kinase is used or the DHA kinase is overexpressed. In a preferred embodiment of the present invention, the yeast cell is genetically engineered such that degradation of glycerol to dihydroxyacetone phosphate via the glycerol-3-phosphate pathway is blocked by blocking expression of glycerol kinase and / or mitochondrial glycerol-3-phosphate dehydrogenase (mGDP). It is preferred that both the expression of glycerol kinase and the mitochondrial glycerol-3-phosphate dehydrogenase is blocked. This can be achieved using various methods known to the skilled worker, for example by deleting the genes encoding the enzymes (see, for example, Swenn et al. 2013).Preferably, the heterologous glycerol uptake factor protein is Fps1or Fps2, preferably Fps2from Pachysolen tannophilus(see Liu et al. 2013; PtFPS1: GenBank: JQ481631.1 GI:394916125, AFN43530.1 GI:394996126; PtFPS2: JQ481632.1 GI:39496127, AFN43531.1 GI:394996128). Other proteins are also suitable, however, for example Fps1 from Pichia pastoris, Pichia jadiniior Yarrowia lipolytica. The heterologous glycerol dehydrogenase is preferably a glycerol dehydrogenase from Ogataea angusta (see Jung et al. 2011, GenBank: AB185335.1 GI:50582511, BAD32688.1 GI:50582512). Instead of introducing a heterologous glycerol dehydrogenase, the Bdh1 naturally occurring in Saccharomyces cerevisiae and having glycerol dehydrogenase activity can also be overexpressed. Heterologous DHA kinases are preferably derived from organisms which have been shown to naturally use the DHA pathway, e.g. Dak1 from Schizosaccharomyces pombe(see Itoh et al., 1999, UniProtTM / Swiss-Prot 013902, Ver 98, 1106.2014.). Examples of suitable heterologous substitutes for the NADP +- dependent glycerol dehydrogenase of the glyceraldehyde pathway are the product of the gene gldB from Aspergillus(Emericella) nidulans(de Vries et al. 2003, UniProtTM / Swiss-Prot Q7Z8L1, Ver 64, 11.06.2014). A heterologous DHA kinase is also suitable as a substitute for the triose kinase. Alternatively or additionally, the genes DAK1 and / or DAK2 coding for the triose kinases Dak1 or Dak2 in S. cerevisiae can be overexpressed.The yeast cell of the invention is modified to be particularly suitable for the production of reduced fermentation products. In the alternative embodiment, in which the DHA pathway is activated and / or enhanced, the preparation of succinate, for example, can be realized in a particularly advantageous manner, because the two NADH required in the redox balance are produced here. Depending on the desired fermentation product, the yeast cell can be correspondingly manipulated in such a way that the necessary number and type of reduction equivalents are produced. This also includes, for example, activating and / or enhancing the DHA pathway in a manner that provides NADPH. For this purpose, it may be advantageous to make further changes to the yeast cell.Thus, in a further embodiment of the present invention, the yeast cell is genetically modified to the effect that a heterologous formate dehydrogenase (FDH) is expressed in the yeast cell or the native formate dehydrogenase is overexpressed. Examples of suitable heterologous formate dehydrogenases are bacterial formate dehydrogenases, for example from E. coli. However, formate dehydrogenases of eukaryotic origin are preferred, for example from Candida (e.g. FDH1 from Candida boidinii, UniProtTM / Swiss-Prot 013437 (FDH_CANBO), Ver 72, 16.04.14). The overexpressed formate dehydrogenase can also be the formate dehydrogenase from Saccharomyces cerevisiaeencoded by the FDHI gene. When culturing a yeast cell modified in this way with formate as cosubstrate, additional reduction equivalents can be obtained, which can be advantageous, for example, when using the yeast for the production of, in particular, 1,2-propanediol or other greatly reduced products. Formate (used as cosubstrate) can function in this context as an additional electron donor. In the case where the glyceraldehyde pathway is optimized by over-expressing the relevant enzymes or introducing heterologous variants and therefore NADPH is obtained as reduction equivalent, the 1,2-PDO formation can also be operated with NADPH, and alternatively to formate dehydrogenase, a native glucose dehydrogenase could be overexpressed or heterologous glucose dehydrogenase could be introduced and glucose could be used as co-substrate.In a further preferred embodiment, the yeast cell according to the invention is additionally modified by genetic engineering in such a way that a native formaldehyde transketolase catalyzing the formation of formaldehyde from dihydroxyacetone is overexpressed in the yeast cell or a heterologous formaldehyde transketolase catalyzing the formation of formaldehyde from dihydroxyacetone is expressed or overexpressed in the yeast cell, and a heterologous methanol dehydrogenase (MDH), preferably a NAD +- variant, is expressed or overexpressed in the yeast cell. This embodiment of a yeast cell according to the invention is particularly suitable for the production of methanol from glycerol. Optionally, instead of or in addition to the native formaldehyde transketolase(s), a heterologous formaldehyde transketolase may be expressed.As already mentioned in the introduction to the specification, glycerol is obtained in the production of biodiesel. At the same time, methanol is generally used for transesterification of the fats and oils. It would therefore be advantageous to be able to at least partially recover methanol from the glycerol obtained in a biotechnological process. This is made possible with the aid of this embodiment of a yeast cell modified by genetic engineering according to the invention. The yeast cell is modified by genetic engineering in such a way that it carries out or can carry out the following reaction of dihydroxyacetone (DHA) to methanol (GAP=glyceraldehyde-3-phosphate, XP5=xylulose-5-phosphate): While the first step, i.e. the formation of formaldehyde from DHA, naturally also occurs in wild type in Saccharomyces cerevisiae(Molin and Blomberg 2006) and it is therefore preferred to over-express the enzyme or enzymes involved therein, the second step, in the absence of a native methanol dehydrogenase, only occurs when a heterologous methanol dehydrogenase is introduced into the yeast cell. In particular, bacterial methanol dehydrogenases are suitable for this purpose. Methanol dehydrogenases are known, for example, from Gram-negative and Gram-positive methylotrophic bacteria (Hanson & Hanson 1996; Duine et al. 1986). An example of a suitable methanol dehydrogenase is NAD +- dependent methanol dehydrogenase from the gram-positive bacterium Bacillus methanolicus(Arfman et al. 1997).NAD +- dependent methanol dehydrogenase is preferred because in this manner the degradation of glycerol to methanol is balanced with respect to the consumed and generated reduction equivalents. The NAD + reduced in the oxidation of glycerol to DHA is recovered in the reduction of formaldehyde.Baker's yeast has two homologous formaldehyde transketolases, transketolase 1 (Tkl1, YPR074C, s. http: / / www.yeast genome.org / locus / S0000006278 / overview) and transketolase 2 (Tkl2, YBR117C, s. http: / / www.yeast genome.org / locus / S0000321 / overview), to catalyze the first step in the above reaction equation (Molin and Blomberg 2006). Overexpression of one or both transketolases can be effected in a manner known to the skilled worker, for example by introducing further gene copies or a stronger promoter.The glyceraldehyde-3-phosphate (GAP) required for the transketolase reaction is available in yeast via the central carbon metabolism. GAP can also be obtained, for example, if DHA is converted to DHAP by means of the DHA kinase naturally present in yeast and encoded by DAK1 and DAK2, and is further converted to GAP by means of the triose phosphate isomerase encoded by TPI1 (EC 5.3.1.1). This pathway is particularly preferred when degradation of glycerol to glyceraldehyde-3-phosphate is blocked via the glyceraldehyde pathway.In this embodiment, it is particularly preferred if the yeast cell is further genetically modified in such a way that the native DHA kinase is inhibited or the expression of the native DHA kinase is reduced. A reduction in gene expression of the genes DAK1 and / or DAK2 can be effected, for example, by introducing a weaker promoter (Nevoigt et al. 2006). A reduction in the activity of the DHA kinase in the yeast cell according to the invention in a suitable manner and to a suitable extent makes it possible to control the reaction of DHA in such a way that GAP formation and methanol formation are in a ratio suitable for methanol production.In this connection, it should be noted that both DHA and formaldehyde are cytotoxic. Formaldehyde is therefore dissimulated by S. cerevisiaeto carbon dioxide via formate for detoxifying (Molin and Blomberg 2006; Molin et al 2003), the corresponding steps being catalyzed by a glutathione-dependent formaldehyde dehydrogenase (EC 1.1.1.284) encoded by SFA1 and a formate dehydrogenase (EC 1.2.1.2) encoded by FDH1. In a further preferred embodiment, the yeast cell of the invention is therefore genetically engineered to either block this path of detoxification or, preferably, to be genetically engineered to reduce or eliminate expression of the SFA1 gene when the formaldehyde content in the cell is low enough to produce undesirable toxic effects in the cell or, alternatively, to switch on or enhance expression of the SFA1 gene when the formaldehyde content in the cell threatens to exceed a level capable of producing undesirable toxic effects in the cell. Genetic engineering methods for carrying out these modifications are known to the person skilled in the art (see, for example, Borodina and Nielsen 2014). Alternatively or optionally additionally, it is also possible to implement so-called "metabolic channeling". In this case, enzymes which catalyze successive reactions are arranged in a common complex, so that intermediates are prevented from entering the cytosol (see Dueber, J. E. et al. 2009).In a further aspect, the present invention also relates to a method for producing a fermentation product from glycerol, wherein a yeast cell according to the aspect described above, which has been modified by genetic engineering according to the invention, is cultivated in a glycerol-containing culture medium under suitable fermentation conditions and the fermentation product is separated from the culture medium. Examples of fermentation products are succinate, 1,2-propanediol and methanol. In the case of 1,2-propanediol (1,2-PDO), it is particularly preferred if the yeast cell used contains a heterologous formate dehydrogenase and / or a native formate dehydrogenase, which is overexpressed. Depending on the desired fermentation product, a yeast cell can be used which is optimized for the respective purpose, in particular with regard to the redox balance.The invention is explained in greater detail below by way of example with reference to the attached figure.FIG. 1 shows a schematic illustration of the degradation of glycerol in an embodiment of an genetically modified yeast cell according to the invention.Figure 1 schematically shows the degradation of glycerol in a preferred embodiment of an genetically engineered yeast cell according to the invention. The figure shows the glycerol-3-phosphate pathway (left), the DHA pathway (middle) and the glyceraldehyde pathway (right), i.e. those three metabolic pathways which can in principle lead to glycerol degradation. In the yeast cell, the expression of the natural transport protein Stl1 was blocked by deletion of the corresponding gene. In addition, the cell was genetically engineered to express a heterologous glycerol factor, here Fps2from Pachysolen tannophilus. The glycerol-3-phosphate pathway is blocked by deletion of the genes for glycerol kinase (GUT1) and the mitochondrial glycerol-3-phosphate dehydrogenase (GUT2). In addition, the reverse reaction via the NAD-dependent glycerol 3-phosphate dehydrogenase and the glycerol 3-phosphatase is also blocked by deletion of the genes coding for this (GPD 1, GPD 2, GPP 1, GPP 2). Glycerol transported into the cell via the glycerol factorilator is thus converted via the DHA pathway shown in the middle via dihydroxyacetone to dihydroxyacetone phosphate (DHAP). In the yeast cell, a heterologous glycerol dehydrogenase, here a glycerol dehydrogenase from Ogataea angusta, is expressed. Furthermore, the glyceraldehyde pathway is preferably also completely blocked by deletion of the coding enzymes in this embodiment. This embodiment is particularly advantageous for the case where reduction equivalents are required exclusively in the form of NADH, for example in the preparation of succinate. The formation or consumption of reduction equivalents or ATP is shown only for the DHA pathway and the glyceraldehyde pathway.ReferencesAlmeida, J. R., Favaro, L. C., Quirino, B. F. 2012. Biodiesel biorefinery: opportunistics and challenges for microbial production of fuels and chemicals from glycerol waste. Biotechnol Biofuels, 5(1), 48.Arfman, N., Hector, HJ., Bystrykh, L.V., Govorukhina, NI., Dijkhuizen, L., Frank, J. (1997. Properties of an NAD(H)-containing methanol dehydrogenase and its activator protein from Bacillus methanolicus. Eur. J. Biochem. 244:426-433.Bebel, W., Hofmann, K. H. 1982. The relation between the association of methanol and glycerol in yeast. Arch Microbiol, 132, 179-184.Borodina, I., Nielsen, Advances in Metabolic Engineering of Yeast Saccharomyces cerevisiae for Production of Chemicals. J., Biotechnol J. 2014 May; 9(5):609-620. doi: 10.1002 / biot.201300445.De Vries RP, Flitter SJ, van de Vondervoort PJ, Chaveroche MK, Fontaine T, Fillinger S, Ruijter GJ, d'Enfert C, Visser J. 2003. Glycerol dehydrogenase encoded by gldB is essential for osmotolerance in Aspergillus nidulans. Mol Microbiol. 49(1):131-41. Dueer J. E., Wu G. C., Malmirchegini G. R., Moon T. S., Petzold C. J., Ullal A. V., Prather K. L., Keasling J. D. 2009. Synthetic protein scaffolds provide modular control over metabolic flux. Nat. Biotechnol. 27, 753-759Duine, J. A., Frank Jzn., J., Jongejan J. A. 1986. PQQ and quinoprotein enzymes in microbial oxidation. FEMS Microbiol. Lett. 32:165-178.Fakas, S., Makri, A., Bellou, S., Aggelis, G. 2009. Pathways to aerobic glycerol catabolism and their regulation. in: Microbial conversions of raw glycerol, (Ed.) G. Aggelis, Nova Science Publishers, Inc. New York, pp. 9-18.Ferreira, C., van Voorst, F., Martins, A., Neves, L., Oliveira, R., Kielland-Brandt, M.C., Lucas, C., Brandt, A. 2005. A member of the sugar transporter family, Stllp is the glycerol / H+ symporter in Saccharomyces cerevisiae. Mol Biol Cell, 16(4), 2068-76. Gancedo, C., Gancedo, J. M., Sols, A. 1968. Glycerol metabolism in yeast. Pathways of utilization and production. Eur J Biochem, 5(2), 165-72.Gonzalez, E., Fernandez, M.R., Larroy, C., Sola, L., Pericas, M.A., Pares, X., Biosca, J.A. 2000. Characterization of a (2R,3R)-2,3-butanediol dehydrogenase as the Saccharomyces cerevisiae YAL060W gene product. Disruption and induction of the gene. J Biol Chem., 275(46), 35876-85.Hanson, RS., Hanson, TE. 1996. Methanotrophic bacteria. Microbiol. Rev. 60: 439-471. Hofmann KH, Bebel W (1979) Synthesis and accumulation of glycerol dehydrogenase in Candida valida H 122 as a function of the C source. VII. Reinhardsbrunner Symposium of the Microbiology Section of the Biological Society of DDR, from May 6 to 12. In: Abendl Akad Wiss DDR (N3):229-234. Berlin, Akademia-Verlag, 1981, Hong, K. K., Nielsen, J. 2012. Metabolism engineering of Saccharomyces cerevisiae: a key cell factory platform for future biorefines. Cell Mol Life Sci, 69(16), 2671-90. Hubmann, G, Guilloutet, S., Nevoigt, E. 2011. Gpd1 and Gpd2 Fine-Tuning for Sustained Reduction of Glycerol Formation in Saccharomyces cerevisiae. Appl. Env. Microbiol. 77, 5857-5867.Itoh N, Tupibata Y, Liu JQ 1999. Cloning and overexpression in Escherichia coli of the gene encoding dihydroxyacetone kinase isoenzyme I from Schizosaccharomyces pombe and its application to dihydroxyacetone phosphate production. Appl Microbiol Biotechnol. 51(2):193-200.Izawa, S., Sato, M., Yokogawa, K., Inoue, Y. 2004. Intracellular glycerol influence to freeze stress in Saccharomyces cerevisiae: analysis of a quadruple mutant in glycerol dehydrogenase genes and glycerol-enriched cells. Appl Microbiol Biotechnol, 66(1), 108-14.Jung, J. Y., Yun, H. S., Lee, J., Oh, M. K. 2011. Production of 1,2-propanediol from glycerol in Saccharomyces cerevisiae. J Microbiol Biotechnol, 21(8), 846-53.Schneiderberg, M. 1970. Localization of the glycerol phosphate dehydrogenase in the outer phase of the mitochondrial inner membrane. Eur J Biochem, 13(2), 247-52.Lin, E. C. 1976. Glycerol dissimilation and its regulation in bacteria. Annu Rev Microbiol, 30, 535-78.Liu, X., Mortensen, U.H., Workman, M. 2013. Expression and functional studies of genes involved in transport and metabolism of glycerol in Pachysolen tannophilus. Microb Cell Fact, 12, 27.Market analysis of renewable raw materials 2014, series of writing of renewable raw materials 34, technical agency of renewable raw materials e.V. (FNR)May, JW, Sloan J., 1981. Glycerol utilization by Schizosaccharomyces pompe dehydrogenation as the initial step. J Gen Microbiol 123:182-186Merico, A., Ragni, E., Galagragi, S., Poplo, L., Compagno, C. 2011. Generation of an evolved Saccharomyces cerevisiae strain with a high freeze tolerance and an improved abliability to grow on glycerol. J Ind Microbiol Biotechnol, 38(8), 1037-44.Molin, M., Norbeck, J., Blomberg, A. 2003. Dihydroxyacetone kinases in Saccharomyces cerevisiae are involved in detoxication of dihydroxyacetones. J Biol Chem., 278(3), 1415-23.Molin, M., Blomberg, A. 2006. Dihydroxyacetone detoxication in Saccharomyces cerevisiae involves formaldehyde dissimilation. Mol. Microbiol. 60:925-938.Neves, L., Leits, F., Lucas, C. 2004. New insights on glycerol transport in Saccharomyces cerevisiae. FEBS Lett, 565(1-3), 160-2.Nevogt, E., Kohnke, J., Fischer, CR., Alper, H., Stahl, U., Stephanopoulos, G. 2006. Engineering of promoter replacement cassettes for fine-tuning of gene expression in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 72: 5266-5273Nevoigt, E. 2008. Progress in Metabolic Engineering of Saccharomyces cerevisiae. Microbiol Mol Biol Rev, 72(3), 379-412.Nguyen, H.T., Nevoigt, E. 2009. Engineering of Saccharomyces cerevisiae for the production of dihydroxyacetone (DHA) from sugar: a proof of concept. Metab Eng, 11(6), 335-46.Norbeck, J., Blomberg, A. 1997. Metabolism and regulatory changes associated with growth of Saccharomyces cerevisiae in 1.4 M NaCl. Evidence for osmotic induction of glycerol dissimilation via the dihydroxyacetone pathway. J Biol Chem., 272(9), 5544-54.Pahlman AK, Garneth K, Ansell R, Hohmann S, Adler L. 2001. The yeast glycerol 3-phosphatases Gppplp and Gppp2p are required for glycerol biosynthesis and differentially involved in the cellular responses to osmotic, anaerobic, and oxidative stress. J Biol Chem 276(5):3555-563.Shi, S., Valle-Rodriguez, J. O., Siewers, V., Nielson, J. 2011. Products for microbial biodiesel production. Biotechnol. J. 6:277-285.Sprague, G. F., Chonan, J. E. 1977. Isolation and characterization of Saccharomyces cerevisiae mutants defective in glycerol catabolism. J Bacteriol, 129(3), 1335-42. Swenn, S., Klein, M., Carrillo, M., McInnes, J., Nguyen, H.T., Nevoigt, E. 2013. Recovery of glycerol utilization in Saccharomyces cerevisiae: characterization of an isolate that growths on glycerol with support supplements. Biotechnol Biofuels, 6(1), 157.Tom GD, Viswanath-Reddy M, Howe Jr, Branch H (1978) Effect of carbon source on enzymes involved in glycerol metabolism in Neurospora crassa. Arch Microbiol 117:259-263Yazdani, S. S., Gonzalez, R. 2007. Anaerobic fermentation of glycerol: a path to economic viability for the biofuel industry. Curr Opin Biotechnol, 18(3), 213-9.

Claims

An genetically engineered yeast cell of the genus Saccharomyces having enhanced glycerol catabolism, wherein the yeast cell is genetically engineered such that a) degradation of glycerol to dihydroxyacetone phosphate via the glycerol 3-phosphate pathway is blocked by deletion of the genes for glycerol kinase (GUT1) and / or mitochondrial glycerol 3-phosphate dehydrogenase (GUT2), and b) a heterologous glycerol uptake factor protein is expressed, wherein the heterologous glycerol uptake factor protein is Fps1 or Fps2, and c1) a heterologous glycerol dehydrogenase catalyzing the oxidation of glycerol to dihydroxyacetone is expressed or a heterologous glycerol dehydrogenase catalyzing the oxidation of glycerol to dihydroxyacetone is overexpressed, or c2) over-expressing at least one enzyme involved in the degradation of glycerol to glyceraldehyde-3-phosphate via the glyceraldehyde pathway, or replacing at least one enzyme involved in the degradation of glycerol to glyceraldehyde-3-phosphate via the glyceraldehyde pathway with a heterologous enzyme having the same enzyme activity.The genetically modified yeast cell of claim 1, wherein the yeast cell is a yeast cell of the species Saccharomyces cerevisiae.The genetically modified yeast cell of claim 1 or 2, wherein additionally c1a) the degradation of glycerol to glyceraldehyde-3-phosphate is blocked via the glyceraldehyde pathway, and / or d) the expression of the natural active glycerol transporter encoded by STL1 is blocked, and / or e) the synthetic pathway for recovering glycerol from dihydroxyacetone phosphate is blocked, and / or f) a heterologous dihydroxyacetone kinase is expressed or the native dihydroxyacetone kinase is overexpressed.The genetically modified yeast cell of any one of the preceding claims, wherein the heterologous glycerol uptake factor protein is Fps1 or Fps2 from Pachysolen tannophilus, or Fps1 from Pichia pastoris, Pichia jadini or Yarrowia lipolytica, and the heterologous glycerol dehydrogenase is a glycerol dehydrogenase from Ogataea angusta.The genetically modified yeast cell of any preceding claim, wherein the yeast cell is further genetically modified to express a heterologous formate dehydrogenase in the yeast cell or to overexpressed the native formate dehydrogenase.The genetically modified yeast cell of any one of claims 1 to 4, wherein the yeast cell is further genetically modified such that a native formaldehyde transketolase catalyzing the formation of formaldehyde from dihydroxyacetone is overexpressed in the yeast cell or a heterologous formaldehyde transketolase catalyzing the formation of formaldehyde from dihydroxyacetone is expressed or overexpressed in the yeast cell, and a heterologous methanol dehydrogenase, preferably a NAD +- is expressed or overexpressed in the yeast cell.The genetically modified yeast cell of claim 6, wherein the yeast cell is further genetically modified to inhibit or reduce expression of the native dihydroxyacetone kinase.A method for producing a fermentation product from glycerol, wherein an genetically modified yeast cell according to any one of the preceding claims is cultured in a glycerol-containing culture medium under suitable fermentation conditions and the fermentation product is separated from the culture medium.The method of claim 8, wherein the yeast cell is an genetically engineered yeast cell of claim 5, and the yeast cell is cultured with formate as a cosubstrate.The process of claim 8 or 9, wherein the fermentation product is succinate or 1,2-propanediol.The process of claim 6 or 7, wherein the fermentation product is methanol.

Images