US 2010.0068790A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0068790 A1 Bell et al. (43) Pub. Date: Mar. 18, 2010

(54) NON-RECOMBINANT SACCHAROMYCES (30) Foreign Application Priority Data STRANS THAT GROW ON XYLOSE Jun. 8, 2004 (AU) ...... 2004903.141 (75) Inventors: Philip John Livingstone Bell, New South Wales (AU); Paul Victor O O Attfield, New South Wales (AU) Publication Classification Correspondence Address: (51) Int. Cl. FSH & RICHARDSON PC CI2N L/18 (2006.01) P.O. BOX 1022 MINNEAPOLIS, MN 55440-1022 (US) (52) U.S. Cl...... 435/255.21: 435/255.2 (73) Assignee: MICROBIOGEN PTY LTD, Sydney (AU) (57) ABSTRACT (21) Appl. No.: 12/561,230 The present invention relates to methods for producing Sac (22) Filed: Sep. 16, 2009 charomyces strains that are capable of growth on Xylose as a O O sole carbon source at a desired growth rate (such as at least Related U.S. Application Data one generation per 48 hours), strains made by Such methods, (62) Division of application No. 1 1/570,329, filed on Aug. and Saccharomyces strains that grow at a growth rate of at 8, 2007, filed as application No. PCT/AU2005/000824 least one generation per 48 hours using Xylose as a sole on Jun. 8, 2005. carbon source for growth made by non-recombinant methods.

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FIG. 1 US 2010/006879.0 A1 Mar. 18, 2010

NON-RECOMBINANT SACCHAROMYCES production process, or on glucose- and maltose-rich syrups STRANS THAT GROW ON XYLOSE derived from starch hydrolysis industry.

FIELD OF THE INVENTION SUMMARY OF THE INVENTION 0006. The invention provides methods of producing a Sac 0001. The invention relates to methods for producing non charomyces strain that is capable of growing at a desired recombinant strains of Saccharomyces, strains of Saccharo growth rate (such as at least one generation per 48 hours), myces, and uses thereof. using Xylose as a sole carbon source, and strains of Saccha romyces that grow on Xylose under these growth rates, and the BACKGROUND OF THE INVENTION uses thereof. 0007 Xylose is an example of a naturally occurring, abun 0002 All references, including any patents or patent appli dant and renewable Sugar available from plant biomass that cations, cited in this specification are hereby incorporated by Saccharomyces was previously considered not able to use. reference. No admission is made that any reference consti See for example, Barnett et al. Yeasts Characteristics and tutes prior art. The discussion of the references states what Identification 2nd edition (1990), Cambridge University their authors assert, and the applicants reserve the right to Press, which states that yeast of the species Saccharomyces challenge the accuracy and pertinency of the cited docu cerevisiae are not capable of utilizing Xylose as a sole carbon ments. It will be clearly understood that, although a number of Source for growth. Xylose represents a major potential source prior art publications are referred to herein, this reference for growing yeasts and for manufacturing products, including does not constitute an admission that any of these documents ethanol, from yeasts. The use of xylose as a Sugar Source for yeasts would provide major economic and environmental forms part of the common general knowledge in the art, in advantages. For example, production of fuel ethanol from Australia or in any other country. xylose-rich materials would be a major source of renewable 0003. One of the most important economic groups of energy. yeasts are of the genus Saccharomyces, strains of which are 0008. In a first aspect, the invention provides a method of employed in the brewing, baking, winemaking, distilling and producing a Saccharomyces strain that is capable of growing various other yeast-dependent industries. Species of Saccha at a desired growth rate using Xylose as a sole carbon source romyces areas defined phylogenetically by Kurtzman (2003) for growth, comprising: FEMSYeast Research 3:417-432, and include S. cerevisiae, 0009 (a) providing a population of genetically diverse S. paradoxus, S. mikatae, S. cariocanus, S. kudriavZevi, S. non-recombinant yeast cells of Saccharomyces, pastorianus and S. bayanus. 0.010 (b) culturing the yeast cells under conditions 0004 Saccharomyces spp. are some of the most effective which permits combining of DNA between yeast cells; microorganisms for converting Sugars such as glucose, fruc 0.011 (c) screening or selecting the yeast cells for yeast tose, Sucrose and maltose to biomass, and for fermenting cells which have increased growth rate using Xylose as a these Sugars to ethanol. As a consequence, Saccharomyces Sole carbon Source for growth; spp., and in particular Saccharomyces cerevisiae, is one of the 0012 (d) isolating one or more yeast cells which have most widely used microorganisms in industrial processes. For the desired growth rate. example, in the beer brewing, distilling and wine industries, 0013 Typically, the yeast cells are cultured under condi Saccharomyces are used to ferment Sugars such as glucose, tions which permit combining of DNA between pairs of yeast fructose. Sucrose and/or maltose into ethanol. In the fuel cells. ethanol industry, Saccharomyces cerevisiae strains are cho 0014 Typically, the desired growth rate is an increase in sen for their ability to rapidly convert high concentrations of growth rate relative to that of yeast cells of Saccharomyces Sugars such as glucose, fructose, Sucrose and maltose into strains to which the method has not been applied. For high amounts of ethanol. In the baking industry, Saccharo example, Saccharomyces strains such as strain NL67. More myces cerevisiae strains are used primarily for their ability to typically, the desired growth rate is at least one generation per produce carbon dioxide from Sugars such as glucose, fruc 48 hours, more typically at least one generation per 24 hours. tose, sucrose, and/or maltose in order to leaven bread. Other 0015 Typically, the yeast cells are screened or selected for applications of Saccharomyces cerevisiae include production yeast cells which have the desired growth rate by incubating of yeast extracts and other flavour and aroma products, the yeast cells in or on Xylose-containing medium. Typically, Sources of enzymes such as invertase, production of various the Xylose-containing medium is Xylose-containing culture biochemicals, intermediates, proteins, amino acids, ribo medium. The culture medium may contain Xylose as the only nucleic acid and nucleotides co-factors and vitamins. carbon Source, or xylose may be one of a plurality of carbon 0005 Millions of tonnes of yeast are grown each year in Sources. Typically, the Xylose is the only carbon Source in the industrial processes. Therefore, the ability of Saccharomyces Xylose-containing medium. Typically, the Xylose-containing to grow on abundant and renewable carbon Sources is impor culture medium is Xylose minimal mineral media. The yeast tant in terms of economic production of yeast biomass for cells are typically incubated on or in the Xylose-containing industrial purposes and for economic production of byprod medium for Sufficient time to permit yeast cells having the ucts from yeast metabolism. In particular the ability of Sac desired growth rate using Xylose as a carbon Source to grow. charomyces to grow on waste byproducts of other industrial 0016. In one embodiment, the screened or selected yeast processes is of environmental and economic value. Thus, for cells form the population of genetically diverse non-recom example, baker's yeast biomass is often produced by growing binant yeast cells, and steps (b) and (C) are repeated until yeast on molasses which is a waste product of the Sugar yeast cells having the desired growth rate are obtained. US 2010/006879.0 A1 Mar. 18, 2010

0017. In one embodiment, step (b) is performed prior to medium. In another embodiment, the cells are screened Or step (c). In another embodiment, step (c) is performed before selected in liquid Xylose-containing medium. In yet another step (b). In yet another embodiment, steps (b) and (c) are embodiment, the cells are screened or selected on solid performed simultaneously. Xylose-containing medium, followed by Screening or selec 0018. In a second aspect, the invention provides a method tion in liquid Xylose-containing medium. For example, the of producing a Saccharomyces strain in culture that is capable yeast cells may be screened or selected for a plurality of times of a desired growth rate using Xylose as a sole carbon Source by repeating steps (b) and (c) using Solid Xylose-containing for growth, comprising: medium, followed by screening or selection for a plurality of 0019 (a) providing a population of genetically diverse times by repeating steps (b) and (c) using liquid Xylose non-recombinant yeast cells of Saccharomyces, containing medium. 0020 (b) culturing the yeast cells under conditions which 0029. The solid xylose-containing medium may be any permits combining of DNA between yeast cells; Solid medium that contains Xylose as a carbon Source, and 0021 (c) screening or selecting the yeast cells by incubat which provides a selective advantage to a strain that is capable ing the yeast cells on or in a Xylose-containing medium; of utilising Xylose as a sole carbon Source. For example, the 0022 (d) repeating steps (b) and (c) with the screened or Solid Xylose-containing medium may be a complex solid Selected cells forming the population of genetically diverse medium in which xylose is one of a plurality of carbon non-recombinant yeast cells of Saccharomyces, until one Sources, or the solid Xylose-containing medium may be a or more yeast cells have acquired the ability to grow at a minimal Solid medium in which Xylose is the sole carbon desired growth rate using Xylose as a sole carbon Source for Source. Typically, the Solid Xylose-containing medium will be growth. Solid minimal medium containing Xylose as a sole carbon 0023 Typically, the method comprises the further step (e) SOUC. of isolating one or more yeast cells having the desired growth 0030 The liquid xylose-containing medium may be any rate. In one embodiment, the one or more yeast cells that is liquid medium that contains Xylose as a carbon source. For isolated is a single strain of Saccharomyces. In another example, the liquid Xylose-containing medium may beacom embodiment, the one or more yeast cells that is isolated is a plex liquid medium in which Xylose is one of a plurality of population of genetically diverse yeast cells. carbon Sources, or the liquid Xylose-containing medium may 0024. In one embodiment, the yeast cells are selected in be a minimal liquid medium in which Xylose is the sole step (c). The yeast cells may be selected in step (c) by incu carbon Source. Typically, the liquid Xylose-containing bating the yeast cells on or in Xylose-containing medium for medium will be liquid minimal medium containing Xylose as Sufficient time to permit yeast cells capable of growth on a sole carbon Source. Typically, the liquid minimal medium is Xylose as a sole carbon Source to grow using Xylose as the minimal mineral medium containing Xylose as a sole carbon carbon source, and thereafter collecting the yeast cells that SOUC. grow. The yeast cells may be selected in step (c) by incubating 0031. The desired growth rate is typically at least one the yeast cells on or in Xylose-containing medium for Suffi generation per 48 hours. The desired growth rate may be cient time to permit yeast cells having an increased growth greater than one generation per 24 hours. The desired growth rate using Xylose as a sole carbon Source to grow using Xylose rate may be greater than one generation per 12 hours. The as the carbon Source, and thereafter collecting the yeast cells desired growth rate may be greater than one generation per 10 that grow. hours. The desired growth rate may be greater than one gen 0025. In another embodiment, the yeast cells are screened eration per 8 hours. The desired growth rate may be greater in step (c). The yeast cells may be screened in step (c) by than one generation per 4 hours. The desired growth rate may incubating the yeast cells on or in Xylose-containing medium be greater than one generation per 2 hours. for Sufficient time to permit yeast cells having an increased 0032. The Saccharomyces strain that is produced may be a growth rate using Xylose as a sole carbon Source to grow using strain from any species of the genus Saccharomyces that is Xylose as the carbon Source, and thereafter collecting the capable of growth at a desired growth rate using Xylose as a yeast cells that have an increased growth rate using the Xylose sole carbon source for growth. It will be appreciated by those as a carbon Source. skilled in the art that the strain that is produced will depend on 0026. It is also envisaged that in a portion of the repeats of the species which form the genetically diverse population of steps (b) and (c), the yeast cells will be selected, and in a non-recombinant yeast cells of Saccharomyces. Examples of portion of the repeats of steps (b) and (c), the yeast cells will Suitable species of yeast include S. cerevisiae, S. paradoxus, be screened. S. mikatae, S. cariocanus, S. kudriavZevi, S. pastorianus and 0027 Steps (b) and (c) may be repeated any number of S. bayanus. Typically the strain is of the species Saccharo times that is sufficient to obtain a yeast strain that is capable of myces cerevisiae. Typically the strain will be capable of mat growth at the desired rate using Xylose as a sole carbon Source ing with Saccharomyces of the same species. Typically the for growth. It will be appreciated by persons skilled in the art strain will be capable of mating with Saccharomyces cerevi that the number of times that steps (b) and (c) will be repeated SCé. will depend upon the medium which is used, the starting yeast 0033. The yeast may be cultured under any conditions strains, the culturing conditions, etc. In one embodiment, which permit combining of DNA between yeast cells pro steps (b) and (c) are repeated at least once, typically at least 2 vided the combining of DNA is not by recombinant methods. times, more typically at least 5 times, even more typically at The yeast cells may be cultured under conditions which per least 10 times, still more typically at least 20 times, suitably at mit combining of DNA between yeast cells by, for example, least 30 times. mating or cytoduction, or any other methods known in the art 0028. The yeast cells may be screened or selected on solid for combining of DNA of yeast cells, other than recombinant or in liquid Xylose-containing medium. In one embodiment, methods. In one embodiment, the yeast cells are cultured the cells are screened or selected on Solid Xylose-containing under conditions which permit mating of the yeast cells. US 2010/006879.0 A1 Mar. 18, 2010

Typically, mating of yeast comprises sporulating the yeast to for Sufficient time to permit yeast cells having an increased produce spores, germinating the spores, and mating the ger growth rate using Xylose as a sole carbon Source to grow using minated spores. Methods formating yeast are known to those Xylose as the carbon Source, and thereafter collecting the skilled in the art and are described in, for example, Fowell yeast cells that grow fastest using the Xylose as a carbon (1969) “Sporulation and hybridization of yeast, in, The SOUC. Yeasts (A H Rose and J S Harrison, eds.), Academic Press: 0044 Steps (b) and (c) may typically be repeated, whereby European Patent EP 0511 108 B: Attfield and Bell (2003) the derivatives form at least a portion of the population of "Genetics and classical genetic manipulations of industrial genetically diverse Saccharomyces strains, until one or more yeasts in, Topics in Current Genetics, Vol 2. Functional derivatives have acquired an increased growth rate on Xylose Genetics of Industrial Yeasts (J. H. de Winde, ed.), Springer as a sole carbon source for growth. Verlag Berlin Heidelberg or combinations thereof. 0045. In the past, to address the problem of the inability of 0034. The population of genetically diverse non-recombi Saccharomyces cerevisiae to use Xylose, others have used nant yeast cells may be naturally-occurring isolates of Sac recombinant DNA approaches to introduce genes obtained charomyces from any source, spontaneously mutated isolates from yeast which can utilise Xylose as a carbon source to of Saccharomyces, or may be obtained by exposing one or confer on Saccharomyces the ability to utilise Xylose (eg. more Saccharomyces strains to a mutagen. The population of Sonderegger and Sauer (2003) Applied and Environmental genetically diverse non-recombinant yeast cells may be Microbiology 69: 1990-1998). In these approaches, the genes strains from a single species, or may be strains from a plural for Xylose utilisation have been cloned from organisms which ity of species. Species Suitable for use as the genetically are capable of utilising Xylose for growth, and Subsequently diverse population of non-recombinant yeast cells includes S. introduced into Saccharomyces cerevisiae. For example, fun cerevisiae, S. paradoxus, S. mikatae, S. cariocanus, S. kudria gal Xylose isomerase from Piromyces spp. has been integrated vZevi, S. pastorianus and S. bayanus. Typically the strains are into the genome of Saccharomyces cerevisiae to generate of the species Saccharomyces cerevisiae. Typically the strains strains which grow slowly on Xylose as a sole carbon Source are capable of mating with Saccharomyces of the same spe (Kuyper et al. 2003). Xylose reductase from Pichia stipitis cies. Typically the strains are capable of mating with Saccha has also been cloned into Saccharomyces cerevisiae to gen romyces cerevisiae. erate yeast that can utilise xylose as a sole carbon Source for 0035. It will be understood by persons skilled in the art that growth (Wahlbom et al., 2003). in addition to the population of genetically divergent non 0046 Non-recombinant Saccharomyces cerevisiae is recombinant yeast cells, recombinant yeast cells may be “generally regarded as safe” (GRAS) and if recombinant included in steps (b) and (c) as a separate population. techniques are used to develop Saccharomyces cerevisiae that 0036. In a third aspect, the invention provides a method of utilize xylose, they lose the GRAS status. It is therefore not generating a derivative of a Saccharomyces strain with an necessarily industrially or economically useful, desirable, or increased growth rate using Xylose as a sole carbon Source for Suitable to use strains of Saccharomyces cerevisiae that con growth, comprising: tain genes from other genera or species, or that are produced 0037 (a) providing yeast cells of the strain as a portion using recombinant DNA methods. Indeed strains that are of a population of genetically diverse yeast cells of Sac derived through recombinant DNA approaches are not indus charomyces, trially useful where there are socio-, or enviro-political or 0038 (b) culturing the population of genetically diverse other barriers to use of such strains. non-recombinant yeast cells of Saccharomyces under 0047. The use of recombinant DNA techniques reduces conditions which permits combining of DNA between the opportunities and desirability to use yeasts in human the yeast cells of the population; foods etc., whereas non-recombinant strains could be advan 0039 (c) screening or selecting yeast cells for deriva tageously used for human foods etc. The ability to use non tives of the strain that have an increased growth rate on recombinant yeast simultaneously for ethanol and for human Xylose; foods provides opportunities to improve cost-efficiencies of 0040 (d) isolating one or more derivatives of the strain yeast-based processes. For example it is possible to use non which have an increased growth rate using Xylose as a recombinant yeasts to convert Xylose to ethanol and yeast sole carbon source for growth relative to the growth rate biomass. The ethanol may be used for fuel and other applica of the strain using Xylose as a sole carbon Source for tions whereas the yeast produced may be used in other valu growth. able applications such as production of extracts or other by 0041. The yeast cells are typically screened or selected by products. incubating the yeast cells on or in Xylose-containing medium. 0048. The inventors have found that when non-recombi 0042. In one embodiment, the yeast cells are selected in nant wild-type strains of Saccharomyces are incubated on step (c). The yeast cells may be selected in step (c) by incu medium comprising Xylose as a sole carbon Source, very slow bating the yeast cells on or in Xylose-containing medium for growth on thexylose is detectable. This is contrary to the prior Sufficient time to permit yeast cells capable of growth on art, which clearly indicates that Saccharomyces is not capable Xylose as a sole carbon Source to grow using Xylose as the of growth on xylose. The inventors believe that the growth of carbon Source. The yeast cells may be selected in step (c) by Saccharomyces on Xylose is so slow that it has not been incubating the yeast cells on or in Xylose-containing medium previously detected. As described herein, the inventors have for Sufficient time to permit yeast cells having an increased found that when solid minimal mineral medium containing growth rate using Xylose as a sole carbon Source to grow using Xylose as a sole carbon Source was inoculated with wild-type Xylose as the carbon Source. strains of Saccharomyces cerevisiae, growth was detectable 0043. In another embodiment, the yeast cells are screened by microscopic examination of colonies following incubation in step (c). The yeast cells may be screened in step (c) by for 2 months at 30° C. Although the detectable growth incubating the yeast cells on or in Xylose-containing medium allowed application of non-recombinant strategies to be used US 2010/006879.0 A1 Mar. 18, 2010

to derive yeast with improved growth rates, the observed very 0059 (v) at least 1 nanomole of NAD(P)His reduced or slow growth rate does not have industrial utility. oxidised per minute per mg of protein extract at 30°C. 0049. The inventors have extended their finding that Sac under the conditions specified in Test T5; and charomyces is capable of very slow growth on Xylose as a sole 0060 wherein the strain is produced by the method of the first to third aspects. carbon source to develop strains of Saccharomyces that are 0061. In an seventh aspect, the invention provides an iso capable of growth on Xylose as a sole carbon Source at much lated Saccharomyces strain which is capable of a growth rate higher rates than strains of Saccharomyces to which the of at least one generation per 48 hours using Xylose as a sole method of the present invention has not been applied. Prior to carbon Source for growth, wherein: the inventors finding, it was not thought possible that incu 0062 (i) the strain produces at least a 5-fold increase in bating yeast cells of Saccharomyces in or on medium con biomass when grown under the conditions specified in taining Xylose as sole carbon source would result in any Test T1; and selection or enrichment of yeast cells which were capable of 0.063 (ii) the strain produces at least 40 mg dry weight growth on xylose as it was believed that the yeast would not of biomass when grown under the conditions specified grow in the medium because Xylose was seen as a non in Test T2; and useable carbon source for Saccharomyces. Nevertheless, by (0.064 (iii) at least 1 nanomole of NAD(P)H is reduced using their findings and a combination of selection strategies or oxidised perminute per mg of protein extract at 30°C. and methods such as mating of populations of genetically under the conditions specified in Test T4; and diverse Strains of Saccharomyces, Suitably strains of Saccha 0065 (iv) at least 1 nanomole of NAD(P)H is reduced romyces cerevisiae, the inventors have found that Saccharo or oxidised perminute per mg of protein extract at 30°C. myces strains can be produced that are capable of growth on under the conditions specified in Test T5; and Solid medium, and/or in liquid medium, containing Xylose as 0.066 (v) the strain produces at least a 5-fold increase in a sole carbon source at rates that are higher than those of biomass under the conditions specified in Test T7: Saccharomyces strains to which methods of the present 0067 (vi) a concentration of at least 0.04 g/l of ethanol invention have not been applied. Moreover, the inventors have is detected under the conditions specified in Test T8: found that by employing selection Strategies and methods 0068 and wherein the strain is produced by the method Such as mating of genetically diverse strains of Saccharomy of the first to third aspects. ces, Saccharomyces strains can be produced that have a 0069. In one embodiment of the fourth to seventh aspects, growth rate using Xylose as a sole carbon source that is at least the strain produces at least 0.2 grams of ethanol per litre One generation per 48 hours, and in some advantageous within a period of 4 months under the conditions specified in embodiments, may be greater than one generation per 4 Test T9. hours. Thus, by using non-recombinant methods, the inven 0070. In an eighth aspect, the invention provides an iso tors have been able to generate Saccharomyces strains that are lated Saccharomyces strain that is capable of growing at a rate capable of growth in liquid and on Solid media containing of at least one generation per 48 hours using Xylose as a sole Xylose as a sole carbon source at industrially useful rates. carbon source for growth, wherein the capability of the strain 0050. In a fourth aspect, the invention provides a Saccha to utilise Xylose as a sole carbon Source is obtained by non romyces strain produced by the method of the first to third recombinant methods. aspects of the invention. 0071. The rate of growth of the strain may be any rate that 0051. In an fifth aspect, the invention provides an isolated is at least one generation per 48 hours. The rate of growth may Saccharomyces strain which is capable of a growth rate of at be at least one generation per 36 hours. The rate of growth least one generation per 48 hours using Xylose as a sole may be greater than one generation per 24 hours. carbon Source for growth, wherein the strain produces: 0072 The rate of growth may be greater than one genera 0.052 (i) a 5-fold increase in biomass when grown under tion per 12 hours. The rate of growth may be greater than one the conditions specified in Test T1; and generation per 10 hours. The rate of growth may be greater 0053 (ii) at least 10 mg dry weight of biomass when than one generation per 8 hours. The rate of growth may be grown under the conditions specified in Test T2, and greater than one generation per 6 hours. wherein the strain is produced by the method of the first 0073. The rate of growth may be greater than one genera to third aspects. tion per 4 hours. The rate of growth may be greater than one 0054. In a sixth aspect, the invention provides an isolated generation per 2 hours. The rate of growth may be greater than Saccharomyces strain which is capable of a growth rate of at one generation per 80 minutes. least one generation per 48 hours using Xylose as a sole 0074. In one embodiment, the strain is capable of growth carbon Source for growth, wherein: using Xylose as a sole carbon Source at a rate that is Substan tially the same as the rate of growth of the strain using glucose 0055 (i) the strain produces a 10-fold increase in bio as a sole carbon source for growth. mass when grown under the conditions specified in Test 0075. In one embodiment, the strain has a growth rate of at T1; and least one generation per 48 hours on Xylose minimal mineral 0056 (ii) the strain produces at least 50 mg dry weight medium. of biomass when grown under the conditions specified 0076. In one embodiment, the strain produces a 2-fold in Test T2; and increase in biomass when grown under the conditions speci 0057 (iii) at least 0.1 g/l of ethanol is detected under the fied in Test T1. Typically, the strain produces at least a 5 fold conditions specified in Test T3; and increase in biomass when grown under the conditions speci 0058 (iv) at least 1 nanomole of NAD(P)H is reduced fied in Test T1. Suitably, the strain produces at least a 10-fold or oxidised perminute per mg of protein extract at 30°C. increase in biomass when grown under the conditions speci under the conditions specified in Test T4; and fied in Test T1. US 2010/006879.0 A1 Mar. 18, 2010

0077. In one embodiment, the strain produces at least 0.01 charomyces strain may also grow anaerobically or microaero g dry weight of biomass per 50 ml of culture when grown philically using Xylose as a sole carbon Source. under the conditions specified in Test T2. I0088. The Saccharomyces strain may be capable of 0078. In various embodiments: (i) the strain expresses a growth on glucose under anaerobic conditions. non-recombinant enzyme having at least 1 unit of Xylose I0089. The Saccharomyces strain may further be capable of reductase activity under the conditions specified in Test T4: utilising Xylose to produce one or more of a carbon-based (ii) the strain expresses a non-recombinant enzyme having at compound. Examples of Suitable carbon-based compounds least 1 unit of xylitol dehydrogenase activity under the con include alcohols, Xylitol, organic acids such as acetic acid, ditions specified in test T5: glycerol, carbon dioxide, or other yeast components, metabo (iii) the Strain expresses a non-recombinant enzyme having at lites and by-products including yeast extracts, proteins, pep least one unit of xylose reductase activity under the condi tides, amino acids, RNA, nucleotides, glucans, etc. Typically, tions specified in test T4, a non-recombinant enzyme having the alcohol is ethanol. at least one unit of xylitol dehydrogenase activity under the 0090 The strain may produce ethanol when growing on conditions specified in test T5. Xylose. The Strain may use the Xylose to produce ethanol 0079. In a ninth aspect, the invention provides an isolated without growing on the Xylose. The Strain may produce etha Saccharomyces strain which is capable of a growth rate of at nol from Xylose. Typically, the strain ferments Xylose to pro least one generation per 48 hours using Xylose as a sole duce ethanol. carbon source for growth, and which is capable of expressing 0091. The strain may produce ethanolata concentration of non-recombinant enzyme having an activity selected from the at least 0.1 g/L under the conditions specified in Test T3. group consisting of Xylose reductase and Xylitol dehydroge 0092. The strain may produce ethanolata concentration of nase, wherein the Xylose reductase activity is at least 1 unit at least 0.4 g/L under the conditions specified in Test T8. when determined by test T4, and the xylitol dehydrogenase 0093. The strain may produce at least 0.2 g of ethanol per activity is at least 1 unit when determined under the condi litre within a period of 4 months under the conditions speci tions specified in test T5. fied in Test T9. 0080. The strain may be capable of expressing a non recombinant enzyme having Xylose reductase activity and a 0094. The strain may produce at least 0.5g of ethanol per non-recombinant enzyme having Xylitol dehydrogenase L when inoculated at a cell density of at least 5x10 cells per activity. In one embodiment, the strain is capable of express ml into Xylose-containing minimal mineral medium. The ing a non-recombinant enzyme having Xylulose kinase activ Xylose-containing minimal mineral medium may contain ity in addition to one or more non-recombinant enzymes glucose. Typically, the strain produces 0.5 g/L of ethanol having an activity selected from the group consisting of within 5 hours of inoculation of the medium. Xylose reductase and Xylitol dehydrogenase. Typically, the 0095. In one embodiment, the strain ferments xylose to xylulose kinase activity is at least 5 units when determined by produce at least 0.05 grams of ethanol per litre of culture test T6. under the conditions specified in Test T3. 0081 Typically, the strain of the ninth aspect has a rate of 0096. The strain may be capable of utilising xylose as a growth of at least one generation per 48 hours using Xylose as sole carbon Source to grow on Solid medium, and/or in liquid a sole carbon Source. medium. In one embodiment, the strain is capable of growth 0082 In one embodiment, the strain produces at least a in liquid medium containing Xylose as a sole carbon Source. 2-fold increase in biomass when grown under the conditions The liquid medium may be liquid mineral medium containing specified in Test T1. Typically, the strain produces at least a Xylose as a sole carbon source. 5-fold increase in biomass when grown under the conditions 0097. The Saccharomyces strain may be a strain from any specified in Test T1. More typically, the strain produces at species of the genus Saccharomyces. Examples of Suitable least a 5-fold increase in biomass when grown under the species of yeasts include S. cerevisiae, S. paradoxus, S. mika conditions specified in test T1. tae, S. cariocanus, S. kudriavZevi, S. pastorianus and S. 0083. The strain may produce at least 10 mg dry weight of bayanus. Typically the species is Saccharomyces cerevisiae. biomass under the conditions specified in test T2. The strain Typically the strain will be capable of mating with Saccha may produce at least 30 mg dry weight of biomass under the romyces of the same species. Typically the strain will be conditions specified intest T2. The strain may produce at least capable of mating with Saccharomyces cerevisiae. Typically, 40 mg dry weight of biomass under the conditions specified in the strain is Saccharomyces cerevisiae. Test T2. Typically, the strain produces at least 50 mg dry 0098. In one embodiment of the eighth or ninth aspects, weight of biomass under the conditions specified in test T2. the Saccharomyces strain is recombinant. 0084. The strain may produce at least 0.01 g dry weight of 0099. In a tenth aspect, the invention provides an isolated biomass per 50 ml of culture when grown under the condi non-recombinant Saccharomyces strain which produces an tions specified in Test T2. increase in biomass of at least 2-fold under the conditions 0085. The Saccharomyces strain may further be capable of specified in Test T1. using Xylitol as a sole carbon Source for growth. Typically, the 0100. In one embodiment, the increase in biomass is at capability of the strain to utilisexylitolas a sole carbon source least 5-fold, typically at least 10-fold. is obtained through non-recombinant methods. 0101. In a eleventh aspect, the invention provides an iso I0086. In one embodiment, the strain produces at least a lated non-recombinant Saccharomyces strain which has the 5-fold increase in biomass when grown under the conditions following characteristics: specified in Test T7. 0102 (a) Biomass of the strain increases at least 5-fold 0087. The Saccharomyces strain may be capable of aero under the conditions specified in test T1: bic or anaerobic growth using Xylose as a sole carbon Source. 0.103 (b) At least 10 mg dry weight of biomass is pro Typically, the growth is aerobic growth. However, the Sac duced under the conditions specified in test T2. US 2010/006879.0 A1 Mar. 18, 2010

0104. In a twelfth aspect, the invention provides an iso ally compatible Saccharomyces, followed by selection for lated non-recombinant Saccharomyces strain which has the strains that are capable of utilising Xylose as a sole carbon following characteristics: source. The strain may be derived from one or more naturally 0105 (a) Biomass of the strain increases at least 10-fold occurring isolates of Saccharomyces, spontaneously mutated under the conditions specified in test T1: isolates, or it May be obtained by exposing one or more 0106 (b) At least 50 mg dry weight of biomass is pro Saccharomyces Strains to a mutagen and Subsequently used in duced under the conditions specified in test T2: mating and selection strategies to screen or select for a mutant 0107 (c) A concentration of at least 0.1 g/L of ethanol is strain that is capable of utilising Xylose as a sole carbon detected under the conditions specified in Test T3; source. The obtained strain may be selected by any of the (0.108 (d) at least 1 nanomole of NAD(P)His reduced or selection methods referred to herein. oxidised per minute per mg of protein extract at 30°C. I0121. In a fourteenth aspect, the invention provides a under the conditions specified in Test T4; and derivative of a strain of the fourth to thirteenth aspects. 0109 (e) at least 1 nanomole of NAD(P)H is reduced or I0122) Methods for the production of derivatives of yeast oxidised per minute per mg of protein extract at 30°C. strains are well known in the art and include matings with under the conditions specified in Test T5. other yeast strains, cell fusions, mutagenesis, and/or recom 0110. In a thirteenth aspect, the invention provides an iso binant methods. lated non-recombinant Saccharomyces strain which has the I0123. In a fifteenth aspect, the invention provides the use following characteristics: ofa Saccharomyces strain of the fourth to fifteenth aspects, or 0111 (a) Biomass of the strain increases at least 5-fold a derivative of the fourteenth aspect, in production of yeast under the conditions specified in test T1: biomass. The yeast biomass may be used in, for example, the 0112 (b) At least 40 mg dry weight of biomass is pro baking industry, for biomass products. duced under the conditions specified in test T2: 0.124. In a sixteenth aspect, the invention provides the use 0113 (c) Biomass of the strain increases at least 5-fold ofa Saccharomyces strain of the fourth to thirteenth aspect, or under the conditions specified in Test T7: a derivative of the fourteenth aspect, for the production of 0114 (d) A concentration of at least 0.04 g/L of ethanol ethanol from Xylose. is detected under the conditions specified in test T8: 0.125. In a seventeenth aspect, the invention provides a 0115 (e) at least 1 nanomole of NAD(P)H is reduced or method for the production of ethanol comprising incubating a oxidised per minute per mg of protein extract at 30°C. Saccharomyces strain of the fourth to thirteenth aspects, or a under the conditions specified in Test T4; and derivative of the fourteenth aspect, with a xylose-containing 0116 (f) at least 1 nanomole of NAD(P)H is reduced or medium, under conditions which permit the Strain to ferment oxidised per minute per mg of protein extract at 30°C. Xylose to produce ethanol. under the conditions specified in Test T5. I0126. In an eighteenth aspect, the invention provides a 0117 Examples of suitable Saccharomyces strains that are method of converting Xylose into yeast biomass, comprising capable of growth at a rate of at least one generation per 48 culturing a Saccharomyces strain of the fourth to thirteenth hours using Xylose as a sole carbon Source for growth are aspects, or a derivative of the fourteenth aspect, with a Xylose those that are deposited under the Budapest Treaty on the containing medium under conditions which permit the strain International Recognition of the Deposit of Microorganisms to grow using the Xylose as a carbon Source for growth. for the Purposes of Patent Procedure at the Australian Gov I0127. In a nineteenth aspect, the invention provides a ernment Analytical Laboratories, 1 Suakin Street, Pymble, method of producing yeast biomass comprising growing a NSW 2073, Australia, under deposit accession no. NM04/ Saccharomyces strain on or in a Xylose-containing medium 41257, NM04/41258, NM05/45177 (ISO 10) and NM05/ wherein at least a portion of the yeast biomass is produced 45178 (ISO 7). Deposit accession numbers NM04/41257 and using Xylose as a carbon Source for growth. NM04/41258 were deposited on 12 May 2004. Deposit I0128. In a twentieth aspect, the invention provides a accession numbers NMO5/45177 and NMO5/45.178 were method of producing a compound from a Saccharomyces deposited on 16 May 2005. strain, comprising culturing the Saccharomyces strain onorin 0118. The Saccharomyces strain may be obtained by any a Xylose-containing medium under conditions which permit method referred to herein in which the ability to utilise xylose production of the compound, wherein the compound is pro as a sole carbon Source for growth is obtained by non-recom duced by the strain using Xylose as a carbon source. binant methods. Methods that may be employed to obtain the I0129. In one embodiment, the compound is produced by strain include combinations of natural selection, mating pro the strain during growth using Xylose as a carbon Source. cedures, mutagenesis, or other so-called classical genetic 0.130. In one embodiment, the method comprises the fur methods known to those skilled in the art and discussed and ther step of recovering the compound. referred to in, for example, Attfield and Bell (2003)"Genetics I0131 The compound may be any compound that can be and classical genetic manipulations of industrial yeasts' in, produced by the Saccharomyces strain. Examples of suitable Topics in Current Genetics, Vol 2. Functional Genetics of compounds include ethanol, CO., enzymes, recombinant Industrial Yeasts (J. H. de Winde, ed.), Springer-Verlag Berlin enzymes, recombinant proteins, yeast by-products, vitamins, Heidelberg or combinations thereof. nucleotides, ribonucleotides, deoxyribonucleotides, lipids, 0119 The ability to grow at a rate of at least one generation yeast proteins, Xylitol. per 48 hours using Xylose as a sole carbon Source is typically 0.132. It will be appreciated by those skilled in the art that obtained by mating between Saccharomyces strains and growth of the yeast cells produces biomass, and therefore any screening or selection for increased growth rate using Xylose method which results in growth will result in production of as a sole carbon source. biomass. Thus, for example, production of ethanol from 0120 For example, the strain may be obtained by conduct growth on Xylose-containing medium will in addition pro ing rare or directed or mass matings between strains of sexu duce biomass. In one embodiment, compounds are contained US 2010/006879.0 A1 Mar. 18, 2010 within the yeast cells in biomass and the compounds may be generation per 48 hours using Xylose as a sole carbon Source. recovered from the yeast cells using methods well known in The inventors have found that it is possible to obtainstrains of the art for extracting compounds from yeast cells. Saccharomyces that are capable of relatively rapid growth 0133. In a twenty-first aspect, the invention provides a using Xylose as a sole carbon source without the need to use compound produced by the method of the eighteenth aspect. recombinant DNA technology. This is an unexpected resultas it was previously believed that strains of Saccharomyces were BRIEF DESCRIPTION OF THE FIGURES not capable of growth on Xylose as a sole carbon Source. Until 0134 FIG. 1 shows a plot of the average doubling time of the present invention, Saccharomyces Strains capable of a population of Saccharomyces cerevisiae strains over time growth at rates of at least one generation per 48 hours using following application of the method of the invention. xylose as a sole carbon source have only be obtained by introducing into Saccharomyces strains cloned Xylose utili DETAILED DESCRIPTION OF THE INVENTION sation genes from other organisms that are capable of growth on Xylose. Alternatively unnaturally occurring combinations 0135 The practice of the present invention employs, of cloned Saccharomyces gene promoters and cloned Saccha unless otherwise indicated, conventional microbiology and romyces DNA sequences have been generated using recom classical genetics. Such techniques are known to the skilled worker, and are explained fully in the literature. See, for binant methods and introduced into Saccharomyces strains. example, Sherman et al. “Methods in Yeast Genetics’ (1981) Thus, prior to the present invention, obtaining strains of Sac Cold Spring Harbor Laboratory Manual, Cold Spring Harbor, charomyces capable of utilising Xylose as a sole carbon N.Y.; European Patent number EP 0511 108 B. Source has not been possible unless either Xylose utilisation 0136. It is also to be understood that the terminology used genes from other organisms that are capable of growth on herein is for the purpose of describing particular embodi xylose have been cloned and introduced into the Saccharo ments only, and is not intended to limit the scope of the myces strain, or strong Saccharomyces gene promoters have present invention which will be limited only by the appended been operably linked to other Saccharomyces DNA claims. It must be noted that as used herein and in the sequences and introduced into the Saccharomyces strain appended claims, the singular forms “a”, “an', and “the using recombinant DNA methods. include plural reference unless the context clearly indicates 0140 Prior to the present invention, no non-recombinant otherwise. Thus, for example, a reference to “a cell includes yeast from the genus Saccharomyces were capable of a a plurality of such cells. Unless defined otherwise, all tech growth rate of at least one generation per 48 hours using nical and Scientific terms used herein have the same meaning xylose as a sole carbon source. U.S. Pat. No. 4.51 1,656 dis as commonly understood by one of ordinary skill in the art to closes yeast strain ATCC No. 20618 which is a non-recom which this invention belongs. Although any materials and binant yeast mutant that is capable of producing ethanol when methods similar or equivalent to those described herein can incubated in medium containing xylose. However, ATCC be used to practice or test the present invention, preferred 20618 is not from the genus Saccharomyces. The morphology materials and methods are now described. of the colonies produced by ATCC No. 20618 is not consis 0.137 All publications mentioned herein are cited for the tent with those produced by Saccharomyces. ATCC No. purpose of describing and disclosing the protocols and 20618 does not sporulate, does not mate with standard strains reagents which are reported in the publications and which of Saccharomyces cerevisiae, and sequencing of the ITS might be used in connection with the invention. Nothing regions and histone H3-H4 intergenic region of ATCC No herein is to be construed as an admission that the invention is 20618 reveals that it is not of the genus Saccharomyces, but is not entitled to antedate such disclosure by virtue of prior closely related to Candida tropicalis. Accordingly, ATCC invention. 20618 is phylogenetically distant from Saccharomyces as 0138 Xylose is a sugar which is inexpensive, can be defined by Kurtzman (2003) FEMS Yeast Research 4:233 obtained from a renewable resource, and is available in large 245 and as defined herein. Candida tropicalis is well known amounts. Xylose can represent a significant portion of hemi to utilise xylose. Additionally, the mutants disclosed in U.S. cellulosic plant biomass. Plant biomass includes agricultural Pat. No. 4,511,656 were only demonstrated to produce etha residues, paper wastes, wood chips and the like, and is renew nol when provided with growth nutrients such as yeast able, and available at low cost in large amounts. Xylose is extract, malt extract and peptone in the growth medium. primarily present in hemicellulosic plant biomass as poly These growth nutrients provide alternative fermentable car mers known as Xylan and hemicellulose. The polymers of bon sources to xylose. Thus, U.S. Pat. No. 4.51 1,656 does not Xylose can be readily broken down into monomeric Sugars disclose Strains of Saccharomyces that are capable of growth either by chemical means Such as acid hydrolysis, or by on, or fermentation of Xylose as a sole carbon source. enzymatic means using enzymes such as Xylanases. In paper 0141 Furthermore U.S. Pat. No. 4,511,656 teaches that manufacturing for example, Xylose is one of the main Sugars yeast capable of producing ethanol are selected which form in the waste stream, where it contributes to biological oxygen colonies on xylose but not xylitol. Without wishing to be demand (BOD) and thereby makes disposal of the waste bound by theory, the inventors believe that contrary to the water environmentally difficult. For every kilogram of paper teaching of U.S. Pat. No. 4.51 1,656, the metabolism of xyli produced from hardwood, typically 100 grams of Sugar is tol, which is an intermediate in Xylose utilisation, might be produced, 35 grams of which is xylose. Because of its abun expected to be advantageous to growth on Xylose and ethanol dance, Xylose presents a major potential carbon Source for production from Xylose. producing yeast biomass and by-products of yeast metabo 0142. The inventors have found contrary to the teaching of lism Such an ethanol. the prior art, that not only are Saccharomyces strains capable 0.139. In one aspect there is provided to a Saccharomyces of very slow growth on Xylose as a sole carbon source, but that strain that is capable of growing at a rate of at least one the growth rate using Xylose as a sole carbon Source of Sac US 2010/006879.0 A1 Mar. 18, 2010

charomyces strains can be increased without introducing 0146 It will be understood by those skilled in the art that cloned Xylose utilisation genes. the genetic information which confers the ability of the Sac 0143. In one aspect, there is provided an isolated Saccha charomyces strain to grow at a rate of at least one generation romyces strain which is capable of growing at a rate of at least per 48 hours utilising Xylose as a sole carbon Source is one generation per 48 hours using Xylose as a sole carbon obtained from the genus Saccharomyces. In other words, all Source. As used herein, a Saccharomyces strain is a strain of the genetic information necessary for growth at a rate of at the genus Saccharomyces as defined in Kurtzman (2003) least one generation per 48 hours is obtained from within the FEMSYeast Research vol. 4 pp. 233-245. gene pool of the genus Saccharomyces. Typically, the genetic 0144. As used herein, the expression “capable of growing information which confers the ability of the Saccharomyces at a rate of at least one generation per 48 hours using Xylose as strain to grow at a rate of at least one generation per 48 hours a sole carbon Source for growth' means that the strain is utilising Xylose as a sole carbon Source for growth is obtained capable of using Xylose as the only source of carbon for from a genetically diverse population of non-recombinant generation of energy, and synthesis of molecules that are Saccharomyces Strains. As discussed above, prior to the necessary for growth of the strain, to attain a growth rate of at present invention, the gene pool of the genus Saccharomyces least one generation per 48 hours. The term “one generation” was thought not to contain the genetic information for con will be clear to those skilled in the art to mean one cycle of cell ferring the ability to utilise xylose as a sole carbon source for division. The strain will preferably be capable of growth in growth. Solid and liquid media in which Xylose is the only carbon 0.147. In another aspect, there is provided isolated Saccha source. It will be appreciated by persons skilled in the art that romyces strains which have the ability to grow on Xylose as a a strain that is capable of growing at a rate of at least one sole carbon Source at a growth rate of at least one generation generation per 48 hours using Xylose as a sole carbon Source per 48 hours, and which are capable of expressing non-re will be capable of growth on solid and/or in liquid minimal combinant enzyme having an activity selected from the group mineral media, in which Xylose is the sole carbon Source. An consisting of Xylose reductase and Xylitol dehydrogenase. example of a Suitable liquid medium is the commercially The enzyme has an activity of at least unit as determined by available DIFCO Laboratories Yeast Nitrogen Base without Test T4 for xylose reductase, and TestT5 for xylitol dehydro amino acids, which contains all essential inorganic salts and genase. Tests T4 and T5 are defined herein. In one embodi Vitamins necessary for cultivation of yeasts excepta source of ment, the Xylose reductase activity is at least 1.5 units, Suit carbohydrate or amino acids, Supplemented with between ably at least 3 units. In one embodiment, the xylitol 0.01% and 50% xylose, typically 1% and 10% xylose, suit dehydrogenase activity is at least 5 units, suitably at least 8 ably 5% xylose. Typically, solid medium is liquid medium units. that has been solidified by the addition of agelling agent Such 0.148. There is also provided a method for producing a as agar. It will also be appreciated by persons skilled in the art Saccharomyces strain that is capable of growth at a desired that a strain that is capable of growth using Xylose as a sole growth rate using Xylose as a sole carbon Source. The “desired carbon Source may also be capable of growth on many other growth rate may be any growth rate which is greater than the Sugars. The capability of the strain to utilise xylose as a sole growth rate of the yeast cells of Saccharomyces prior to carbon Source is obtained by non-recombinant methods. As applying the method of the invention. The desired growth rate used herein, the expression “non-recombinant methods’ refer will be greater than the growth rate of Saccharomyces cerevi to any methods that do not utilise recombinant DNA technol siae strain NL67 on xylose as a sole carbon source. The ogy to produce in the Saccharomyces strain the ability to desired growth rate may be at least one generation per 48 utilise Xylose. In other words, the genes that confer on the hours, Suitably greater than one generation per 24 hours, strain the ability to utilise xylose for growth have not been Suitably greater than one generation per 12 hours, typically introduced into the strain using recombinant methods. As greater than one generation per 10 hours, more typically used herein, a “recombinant method’ is a method in which greater than one generation per 8 hours, and may be as high as one or more genes are introduced into an organism using the rate of growth of Saccharomyces on glucose, which is recombinant DNA techniques. As used herein, recombinant about one generation per 80 minutes. DNA techniques refer to techniques in which genetic infor 014.9 The method comprises providing a population of mation is manipulated in vitro. Such as when genes are iso genetically diverse non-recombinant yeast cells of Saccharo lated and cloned from an organism. Thus, non-recombinant myces. As used herein, the expression "genetically diverse methods are methods which do not involve the manipulation non-recombinant yeast cells' refers to at least two non-re of genetic information in vitro. A non-recombinant strain is a combinant yeast cells that have distinct genotypes. The strain into which recombinant nucleic acid has not been intro genetically diverse non-recombinant yeast cells may be a duced. mixture of yeast cells of different Saccharomyces strains 0145 Non-recombinant methods may include, for obtained from the wild, from wine, distilling and beer fer example, mutagenesis, classical mating, cytoduction, cell mentation, from baking applications, or from any other fusion Such as protoplast fusion, and combinations thereof. Source of Saccharomyces. The genetically diverse population The non-recombinant method may comprise culturing a of non-recombinant yeast cells of Saccharomyces may be population of genetically diverse yeast cells of Saccharomy derived from a single strain that is sporulated to derive geneti ces under conditions which permit combining of the DNA cally diverge progeny. The genetically diverse population of between yeast cells in Vivo, and screening or selecting for non-recombinant yeast cells of Saccharomyces may include yeast cells having an increased growth rate using Xylose as a one or more yeast cells of Saccharomyces strains that have sole carbon Source, typically by incubating a genetically been exposed to a physical or chemical mutagen Such as, for diverse population of yeast cells in or on Xylose-containing example, ultraviolet light, X-ray or Gamma ray, or ethyl medium for Sufficient time to select yeast cells having an methaneSulphonate, nitrosoguanidine, mitomycin C, bleo increased growth rate using Xylose as a sole carbon source. mycin, or any other agents that cause alterations to DNA base US 2010/006879.0 A1 Mar. 18, 2010 sequences. Thus, genetically diverse non-recombinant yeast 0159 (c) hybridisation of compatible mating types of the cells includes within its scope yeast cells of strains that have haploid cells to produce hybrid yeast cells. been generated by mutagenesis. Methods for mutagenesis of 0160 Methods for sporulation, obtaining haploids and yeast, and in particular, mutagenesis of Saccharomyces cer hybridisation of the haploids to produce hybrid yeast cells are evisiae, are known to persons skilled in the art and are known in the art and are described in, for example, Chapter 7, described in, for example. Sherman et al. “Methods in Yeast “Sporulation and Hybridisation of Yeast” by R. R. Powell, in Genetics’ (1981) Cold Spring Harbor Laboratory Manual, “The Yeasts' vol 1 edited by A. H. Rose and J. S. Harrison, Cold Spring Harbor, N.Y. The genetically diverse population 1969, Academic Press: EP 0511 108B. Typically, themating of non-recombinant yeast cells of Saccharomyces may also is mass mating. Mass mating involves culturing at least two include yeast cells with spontaneously occurring mutations. and typically millions of different yeast cells together in a The population of genetically diverse non-recombinant yeast manner which permits mating to occur between compatible cells of Saccharomyces may be all the same species, or may mating types. Methods for mass mating are described in, for be different species of Saccharomyces. Typically, the differ example, Higgins et al. (2001), Applied and Environmental ent species are capable of mating between each other. For Microbiology Vol. 67, pp. 4346-4348; Lindegren (1943), example, the population of genetically diverse non-recombi Journal of Bacteriology Vol. 46 pp. 405-419. nant yeast cells may comprise different strains of Saccharo 0.161. In another embodiment, the yeast are cultured under myces cerevisiae, or any other Saccharomyces species, conditions which permit cell fusion. Methods for the genera obtained from any of the sources mentioned above. Examples tion of intraspecific or interspecific hybrids using cell fusion of Suitable Saccharomyces species include S. cerevisiae, S. techniques are described in, for example, Morgan (1983) paradoxus, S. mikatae, S. cariocanus, S. kudriavZevi, S. pas Experientia suppl. 46: 155-166; Spenceretal. (1990) in, Yeast torianus and S. bayanus. Typically, the population of geneti Technology, Spencer JFT and Spencer D M (eds.), Springer cally diverse yeast cells of Saccharomyces are the same spe Verlag New York. Cytoduction methods are described in, for cies. Typically, the species is Saccharomyces cerevisiae. example, Inge-Vechtomov et al. (1986) Genetika 22: 2625 0150. Examples of yeasts that could be used to provide 2636; Johnston (1990) in, Yeast Technology, Spencer JFT starting inocula for populations of genetically diverse non and Spencer DM (Eds.), Springer Verlag New York; Polaina recombinant yeast cells include Saccharomyces straits avail et al. (1993) Current Genetics 24:369-372. able from well known culture collections such as American 0162 The yeast cells are screened or selected for yeast Type Culture Collection (ATCC), e.g. ATCC 4111, ATCC cells which have an increased growth rate using Xylose as a 26603, ATCC 38559, the National Collection of Yeast Cul sole carbon source for growth. The yeast cells are screened or tures (NCYC) e.g. S. cerevisiae NCYC 995, NCYC 996, the selected to enrich or identify yeast cells which have an Central Bureau voor Schimmel Cultures (CRBS) e.g. S. cer increased rate of growth using Xylose as a sole carbon Source. evisiae CBS 745.95, CBS 755.95, or isolated from commer The yeast cells are screened or selected typically by incubat cially available yeasts sold by any number of companies for ing the yeast cells on or in a Xylose-containing medium. The use in traditional formulations such as baking, brewing, wine, yeast cells are screened or selected for those yeast cells that distilling, etc. have animproved ability to grow using Xylose as a sole carbon 0151. The population of genetically diverse non-recombi source for growth. The improved ability will typically be an nant yeast cells of Saccharomyces are cultured under condi increase in growth rate using Xylose as a sole carbon Source tions which permit combining of DNA between the yeast towards the desired growth rate. An increase in growth rate is cells. Combining of DNA between the yeast cells may be by an increase in the growth rate of a yeast cell relative to the any method suitable for combining DNA from at least two growth rate of cells prior to culturing the yeast cells under yeast cells, provided the method is not a recombinant method. conditions which permit combining of DNA between yeast Examples of suitable methods for combining the DNA of cells. In one embodiment, the yeast cells are selected. The yeast cells include mating and cytoduction. As used herein, terms “selected” or “selecting refers to a process in which the term “mating refers to the process of exchange and the yeast cells that have an improved ability to grow using recombination of DNA between at least two yeast cells, Xylose as a sole carbon source for growth at the desired including by classical genetic mating methods, directed mat growth rate become enriched in the population. The yeast ing, mass mating, rare mating, cell fusion, etc. For example, cells may be selected by incubating the yeast cells on or in classical genetic crosses or cell fusion may be used to gener Xylose-containing medium for Sufficient time to permit yeast ate intraspecific or interspecific hybrids. cells capable of growth on Xylose as a sole carbon Source to 0152. In one embodiment, the yeast cells are cultured grow using Xylose as the carbon Source, and thereafter col under conditions which permit mating between the yeast lecting the yeast cells that grow. The inventors have found that cells. Typically, the yeast cells are cultured under conditions by incubating the yeast cells for sufficient time to allow even which permit mating by sporulating the yeast cells and there slow growing yeast to grow using Xylose as a carbon Source, after mating the sporulated yeast. For example, the yeast cells and thereafter collecting the yeast cells that grow, including may be mated by: the slow growing yeast cells, the faster growing yeast cells 0153 (a) sporulating the yeast cells; will represent a greater proportion of the collected yeast cells 0154 (b) germinating the sporulated yeast cells; and therefore be enriched in the collected yeast cells, but 0155 (c) hybridising compatible mating types of the genetic diversity of the yeast cell population is Substantially sporulated yeast cells. maintained by also including slow growing yeast cells. For 015.6 Typically, the yeast cells are mated by: example, by plating mated yeast cells on Solid minimal media 0157 (a) pooling the population of genetically diverse containing Xylose as a sole carbon Source, cells capable of non-recombinant yeast cells; more rapidly utilising Xylose as a sole carbon Source would 0158 (b) sporulating the pooled cells and germinating the generate larger colonies, and as a consequence, those cells spores to produce haploid cells; would be selected for and the desired genotype(s) enriched in US 2010/006879.0 A1 Mar. 18, 2010 the population. However, by also collecting the Smaller colo able minimal medium may comprise, for example, DIFCO nies, the genetic diversity of the population would be substan Yeast Nitrogen Base Formulae (see Difco manual “Dehy tially maintained for the next repeat of steps (b) and (c). Thus, drated culture media and reagents for microbiology” 10" by incubating the yeast cells on Xylose-containing medium Edition, Difco Labs 1984), together with xylose at a concen for Sufficient time to permit the yeast cells to grow using tration of between 0.1% and 50%, typically 2% and 15%, Xylose as a carbon Source, it is possible to not only select for more typically 5%. In another embodiment, the xylose-con those yeast cells with increased growth rate on Xylose, but to taining medium may be a complex medium. The concentra also maintain the genetic diversity of the population for tion of xylose in the complex medium may be between 0.1% repeated cycles of steps (b) and (c). Enhanced selection may and 50%, typically between 2% and 30%, more typically be applied by reducing the time the yeast cells are incubated between 2% and 15%. In one embodiment, the complex on the Xylose-containing medium Such that only yeast cells medium is complete rich media. An example of complete rich which grow using the Xylose as a carbon source within a medium is medium which comprises yeast extract at between preselected time period are selected. Yeast cells of this type 0.5 and 2%, preferably 0.5%, peptone at between 0.5% and may be selected by incubating the yeast cells, on or in the 2%, preferably 1%, and xylose at a concentration of between Xylose-containing medium for Sufficient time to permit yeast 0.5% and 50%, typically 2% and 15%, more typically 5%. In cells having an increased growth rate using Xylose as a sole another embodiment, the complex medium is selected from carbon source to grow using Xylose as the carbon Source, and the group consisting of molasses, starch hydrolysates, cellu thereafter collecting the yeast cells that grow. Using this losic or hemicellulosic biomass hydrolysate (such as bagasse, approach, greater selective pressure is placed on the yeast stover, wood pulp, Straw, waste paper, etc), and combinations cells to grow at an increased rate, but as there is likely to be thereof, optionally Supplemented with Xylose, and/or nutri insufficient time for a portion of the slow growing yeast cells ents. It will be appreciated by persons skilled in the art that to grow, some genetic diversity may be lost for further repeats Solid medium may be any of the abovementioned media typi of steps (b) and (c). cally supplemented with between 1% and 10% agar, more 0163. In another embodiment, the yeast cells are screened. typically between 1% and 5% agar, still more typically As used herein, the terms "screened' or “screening refers to between 1% and 2% agar. a process in which the yeast cells that have an improved 0.165. The yeast cells may be screened or selected by incu ability to grow using Xylose as a sole carbon Source for growth bating the cells on Solid and/or liquid medium. In one at the desired growth rate are first identified, and subsequently embodiment, the yeast cells are screened or selected by incu isolated. The yeast cells may be screened by incubating the bating the cells on solid medium. In another embodiment, the yeast cells on or in Xylose-containing medium for Sufficient yeast cells are screened or selected by incubating the yeast time to permit yeast cells having an increased growth rate cells in liquid medium. In a preferred embodiment, the yeast using Xylose as a sole carbon Source to grow using Xylose as cells are screened or selected by incubating the yeast cells first the carbon Source, and thereafter collecting the yeast cells that on Solid medium, and Subsequently on liquid medium. For grow fastest using the Xylose as a carbon Source. For example, example, the method may comprise: yeast cells that have an improved ability to grow using Xylose 0166 (a) providing a population of genetically diverse as a sole carbon Source may be identified on rich Xylose non-recombinant yeast cells of Saccharomyces, containing medium as those cells that grow faster and there fore form larger colonies than the yeast cells do not have an 0.167 (b) culturing the yeast cells under conditions to per improved ability to grow using Xylose as a sole carbon Source. mit mating of the yeast cells; The larger colonies that appear on the plate can therefore be 0168 (c) screening or selecting the yeast cells by incubat isolated in preference to the smaller colonies to thereby iso ing the yeast cells on a Solid Xylose-containing medium; late yeast cells that have an improved ability to grow using 0169 (d) repeating steps (b) and (c) with the screened or Xylose as a sole carbon Source for growth at a desired growth selected cells forming the population of genetically diverse rate. non-recombinant yeast cells of Saccharomyces, until one 0164. As discussed above, the yeast cells are screened or or more yeast cells have acquired the ability to grow at the selected typically by incubating the yeast cells on or in a desired growth rate using Xylose as a sole carbon Source for Xylose-containing medium. A "xylose-containing medium’ growth. may be any medium which contains Xylose and provides a 0170 Typically, steps (b) and (c) are repeated until one or selective advantage to those yeast cells which have an ability more yeast cells have acquired a growth rate on Xylose that is to grow using Xylose as a sole carbon source more efficiently sufficient to permit the cells to be conveniently grown in or faster than other strains. As used herein; a “selective advan liquid medium containing Xylose as a sole carbon Source. For tage' is the ability of a cell or strain to undergo a greater example, the growth rate on Solid minimal mineral medium number of cell divisions than other cells or strains because of containing 2% to 5% w/v xylose at which a strain may be some attribute, in this case, the ability to grow efficiently transferred to growth in liquid minimal mineral medium con using Xylose. Thus, a medium that provides a selective advan taining 2% to 5% w/v xylose as a sole carbon source will tage to a strain will permit that strain to grow faster in that typically be reached following at least 2 repeats of steps (b) medium compared to other strains to which the medium does and (c). More typically at least 5 repeats of steps (b) and (c). not provide a selective advantage. It will be appreciated by Even more typically, at least 10 repeats of steps (b) and (c). It those skilled in the art that yeast can be grown on a wide range will be appreciated by those of skill in the art that the time at of media that include at least one source of carbon, a source of which the yeast cells may be selected or screened by incubat nitrogen, a Source of phosphorus, a Source of Sulphate, trace ing in liquid medium will vary depending on the population elements and vitamins. The Xylose-containing medium may and can readily be determined by incubating a small portion be a minimal media or a complex media. In one embodiment, of the population in liquid medium at each repeat of steps (b) the Xylose containing medium is a minimal medium. A Suit and (c). Thereafter, the method may comprise: US 2010/006879.0 A1 Mar. 18, 2010 11

0171 (a) providing a population of genetically diverse ditions which select or screen for Xylose-utilising yeast non-recombinant yeast cells of Saccharomyces, strains in populations. By way of example, populations could 0172 (b) culturing the yeast cells under conditions to per be cultured: mit mating of the yeast cells; 0.178 (a) in or on xylose minimal medium under aero bic, microaerophilic, anaerobic conditions; 0173 (c) screening or selecting the yeast cells by incubat 0.179 (b) in or on xylose rich medium under aerobic ing the yeast cells in liquid Xylose-containing medium; conditions, microaerophilic, anaerobic conditions. 0.174 (d) repeating steps (b) and (c) with the screened or 0180. The above medium may include other carbon Selected cells forming the population of genetically diverse Sources (eg. Sugars Such as glucose, galactose, polyols such as non-recombinant yeast cells of Saccharomyces, until one Xylitol, glycerol, organic acids and their salts such as acetic or more yeast cells have acquired the ability to grow at the acid and acetates etc.) in addition to Xylose. For example, the desired growth rate using Xylose as a sole carbon Source for yeast cells may be exposed to stresses such as Sub- or Supra growth. optimal pH, hyper- or hypo-osmotic pressure, ionic stresses 0.175. The yeast cells are typically incubated on or in the from salts, alcohol stress from addition of ethanol or other Xylose-containing medium for Sufficient time to allow the alcohols, stress from other organic inhibitors such as furfurals yeast cells capable of growth using Xylose as a sole carbon and their derivatives, Sub- or Supra-optimal temperatures, Source to grow. As discussed above, the length of time will at presence of absence of xylose and then Subsequently selected or screened for their ability to recover from the stresses whilst least be a sufficient length of time to allow the yeast cells that utilising Xylose as the sole carbon Source. It is anticipated that have an increased rate of growth using Xylose as a sole carbon combinations of different selective conditions could be source to grow. Typically, the length of time will be a suffi applied to the populations. cient length of time to permit growth of yeast cells that do not 0181. It will be appreciated that step (c) may be repeated have an increased rate of growth using Xylose as a sole carbon any number of times before isolating the yeast cells or repeat source. In other words, the length of time will be sufficient to ing step (b). For example, populations selected in liquid permit Substantially all the yeast cells to grow on the Xylose. Xylose-containing medium may be continually Subcultured The length of time may vary depending on the population of into fresh medium to further screen or select for yeast cells genetically diverse non-recombinant yeast cells, and will also having the desired growth rate. vary depending on the types of medium used and how many 0182. It will also be appreciated by those skilled in the art cycles or repeats of steps (b) and (c) have been conducted. As that steps (b) and (c) may be repeated any number of times to each cycle is repeated, it is envisaged that the time will screen or select for non-recombinant yeast cells in which the become less as the method screens or selects for populations rate of growth on Xylose as a sole carbon Source is progres that grow faster using Xylose with each repeat of steps (b) and sively increased with each repeated cycle. This process is thus (c), independent of whether solid or liquid medium is used. It repeated for as many times as is required in order to obtain a will also be appreciated by persons skilled in the art that even strain that exhibits the desired growth rate using Xylose as a in complex medium containing Sugars other than Xylose, it is sole carbon Source. envisaged that some or all of the Sugars other than Xylose will 0183 Thereafter, the method typically comprises the step eventually be depleted through growth, and that the presence of isolating one or more yeast cells which have the desired of xylose will therefore eventually confer a selective advan growth rate. It will be appreciated by persons skilled in the art tage to those strains that grow faster onXylose as a sole carbon that the method typically generates a population of geneti SOUC. cally diverse non-recombinant yeast cells of Saccharomyces 0176 The yeast cells may be incubated in liquid xylose which have the desired growth rate. containing medium for a Sufficient amount of time to permit 0184. Accordingly, in one embodiment, the method may the yeast cells that are most efficient at growing on Xylose as be used to produce a population of Saccharomyces strains a sole carbon source to outgrow those yeast cells that are less which are capable of growth utilising Xylose as a sole carbon efficient at growing on Xylose. Typically the amount of time is Source at a desired growth rate. The population may be iso sufficient to permit growth of eventhose yeast cells that do not lated without separating individual strains. This may be have an increased growth rate using Xylose as a sole carbon achieved, for example, by simply pooling the population of Source. As discussed above, faster growing cells will be Saccharomyces strains that are produced by the method. greater in numbers and therefore selected from. Thus, the use 0185. In another embodiment, individual strains of Sac of liquid Xylose-containing medium provides a convenient charomyces which are capable of growth utilising Xylose as a way to select for those yeast cells that are capable of utilising sole carbon source may be isolated. This may be achieved Xylose at a faster rate than the rest of the yeast cell population. using standard microbiological techniques such as providing The period of growth in liquid Xylose-containing medium is Suitable colonies, streaking the yeast on agar plates for single typically reduced with each cycle as the screened or selected colony isolates, or any other methods for isolation of pure yeast cells become more efficient at utilising xylose. Follow cultures. Such methods are described in Cruickshank et al. ing screening or selecting of the yeast cells by culturing the (1975) “Medical Microbiology”, 12" edition, volume two: cells in liquid Xylose-containing medium, the cells are typi The practice of medical microbiology, published by cally harvested from the liquid medium and mated without Churchill Livingstone. further separation or isolation of the cells. Thus, the screened 0186 Also provided is a method of generating a derivative or selected yeast cells are typically mated as a pool. The of a Saccharomyces strain with increased growth rate on mating methods are the same as those following screening Xylose as a sole carbon Source for growth. The Saccharomy and selection on Solid Xylose-containing medium. ces strain from which the derivative is generated typically has 0177. It will be appreciated that the yeast cells may be a desired property. The method comprises step (a) of provid incubated in or on Xylose-containing medium under any con ing the strain as part of a population of genetically diverse US 2010/006879.0 A1 Mar. 18, 2010 non-recombinant yeast cells of Saccharomyces. In other For example, it is envisaged that the above methods may words, yeast cells of the strain make up a portion of the produce multiple strains of Saccharomyces that are capable of population. The yeast cells of the genetically diverse popula a growth rate of at least one generation per 48 hours utilising tion may be from any of the Sources mentioned above. In step Xylose as a sole carbon source for growth. Thus, a first strain (b), the yeast cells of the population of genetically diverse capable of a growth rate of at least one generation per 48 hours yeast cells are cultured under any of the conditions mentioned utilising Xylose as a sole carbon source for growth may be above which permit mating of the yeast cells of the popula mated with a second strain capable of a growth rate of at least tion. In step (c), the yeast cells are screened or selected for one generation per 48 hours utilising Xylose as a sole carbon derivatives which have an increased growth rate. The source for growth. Matings of this type may be performed, for increased growth rate is an increase in growth rate of the example, to obtain a strain of Saccharomyces with even fur derivative relative to the growth rate of the strain. Typically, ther enhanced or improved ability to utilise xylose as a sole the yeast cells are screened or selected by incubating the yeast carbon Source. For example, it is envisaged that different cell in or on Xylose containing medium as mentioned above. strains of Saccharomyces that are capable of rapid growth The Xylose-containing medium may in addition contain fac utilising Xylose as a sole carbon Source may be mated to tors which select or screen for the derivative strain. For obtain mated Strains that are capable of faster growth on example, the strain may comprise selectable markers such as Xylose, or that are more efficient at producing products Such antibiotic resistance markers or other types of markers which as ethanol or carbon dioxide when using Xylose as a carbon permit derivatives of the strain to be distinguished from other SOUC. yeast cells of the population of genetically diverse yeast cells. 0.190 Strains of Saccharomyces that are capable of growth This allows simultaneous screening or selection for yeast rates of at least one generation per 48 hours using Xylose as a cells having increased growth using Xylose as a sole carbon sole carbon Source may be mated with strains of Saccharo Source, and carrying the selectable marker. This permits dis myces that are less capable of utilising Xylose as a sole carbon tinguishing the derivatives from the rest of the genetically Source. Matings of this type may be performed, for example, diverse population. Suitable selectable markers include, for to transfer the improved ability to utilise xylose as a sole example, ADE2, HIS3, LEU2, URA3, LYS2, MET15, TRP1, carbon Source to a Saccharomyces strain that has one or more URA4, sulfite resistance or p-fluoro-DL-phenylalanine resis desirable properties not found in the strain that has an tance (Cebollero and Gonzalez (2004) Applied and Environ improved ability to utilise xylose as a sole carbon source. For mental Microbiology, Vol 70: 7018-7028). Methods for example, a Saccharomyces cerevisiae strain less capable of screening and selecting for growth using Xylose are as men using xylose as a sole carbon source for growth may have tioned above. Steps (b) and (c) may be repeated any number desirable characteristics for the baking industry, and it may be of times until a derivative is obtained with an increase in advantageous to mate this strain with a strain that is capable of growth rate. Once the growth rate has been increased, the growth, at a rate of at least one generation per 48 hours derivative is isolated. Methods for isolated are as mentioned utilising Xylose as a sole carbon source in order to develop a above. baker's yeast that can be grown more rapidly or efficiently on 0187. Also included within the scope of the present inven Xylose and used Subsequently in baking applications. Simi tion are Saccharomyces strains produced by the method of the larly, yeasts that can be used for other industrial purposes invention. The Saccharomyces strain may be any species of Such as distilling, wine making, yeast extracts, enzymes, het Saccharomyces as defined phylogenetically by Kurtzman erologous proteins, or any other purposes may be mated with (2003) FEMSYeast Research 3:417-432, and include S. cer strains which have an improved ability to use Xylose as a sole evisiae, S. paradoxus, S. mikatae, S. cariocanus, S. kudria carbon Source in order to enable production of biomasses or vZevi, S. pastorianus and S. bayanus. Methods for mating yeast by-products on Xylose media. between strains of Saccharomyces cerevisiae and non-cerevi 0191 By mating a Saccharomyces strain, for example, a siae strains are discussed in, for example, Johnston J R and Saccharomyces cerevisiae strain, in which the capability to Oberman H (1979)Yeast Genetics in Industry, in Bull MJ grow at a rate of at least one generation per 48 hours utilising (ed.) Progress in Industrial Microbiology, Elsevier, Amster Xylose as a sole carbon source is obtained by non-recombi dam vol 15, pp. 151-205; Pretorius IS (2000) Tailoring wine nant methods with a recombinant strain in which one or more yeast for the new millenium: novel approaches to the ancient genes for Xylose utilisation have been introduced by recom art of wine making. Yeast 16: 675-729; P. V. Attfield and P.J. binant methods, it is envisaged that strains with even further L. Bell (2003) Genetics and classical genetic manipulations improvements in xylose utilisation can be produced. It will be of industrial yeasts, in Topics in Current Genetics Vol. 2, J. H. appreciated by those skilled in the art that the genes for xylose de Winde (ed) Functional Genetics of Industrial Yeasts, utilisation that have been introduced recombinantly would Springer-Verlag Berlin Heidelberg. simply Supplement those genes already present in the Strain 0188 It will be appreciated by persons skilled in the art through non-recombinant methods. that once a strain of Saccharomyces is obtained that is capable 0.192 It will also be appreciated by persons skilled in the of utilising Xylose as a sole carbon Source for growth at a art that while the ability to utilise xylose is obtained by non desired growth rate, strains may be derived from that strain recombinant methods, recombinant DNA technology may be using methods known in the art for strain production includ used to supplement the capability of the strain to utilisexylose ing for example, classical genetic crossing methods, as a sole carbon Source. For example, one or more Xylose mutagenesis methods, recombinant methods, or any other utilisation genes from other sources may be introduced into methods for generating strains of Saccharomyces. the strain to supplement Xylose utilisation. For example, 0189 The strain capable of a growth rate of at least one Xylose isomerase from Piromyces sp. may be integrated into generation per 48 hours utilising Xylose as a sole carbon the genome as described in Kuyper et al. merely to Supple Source may be mated with other strains of Saccharomyces, ment xylose utilisation. Xylose reductase (XYL 1), or xylitol preferably with other strains of Saccharomyces cerevisiae. dehydrogenase (XYL2) from Pichia stipitis may be cloned US 2010/006879.0 A1 Mar. 18, 2010

into Saccharomyces as described in Wahlbom et al. (2003) to Yeast Genetics and Molecular Biology'. Methods in Enzy supplement the capability of the strain to utilise xylose. How mology Vol 194, Academic Press). The plasmid can be later ever, it will be understood by persons skilled in the art that the removed from the cell by cultivating the cells in conditions addition of sequences for utilisation of xylose by recombinant that do not select for the plasmid. methods is merely to Supplement the strains existing capabil 0.195 The Saccharomyces spp. strain of the present inven ity of a growth rate of at least one generation per 48 hours tion may be used for any use for which a Saccharomyces spp. utilising Xylose as a sole carbon source. strain of that species would be used. For example, a strain of 0193 In a further example, one or more genes that encode Saccharomyces cerevisiae that is capable of utilising Xylose for metabolic activities that might impinge on efficiency of as a sole carbon source may be used in baking, brewing, Xylose utilisation by Saccharomyces other than through direct biomass production, Sugar fermentation and ethanol produc actions on Xylose, Xylitol or xylulose, could be introduced tion, or any other uses to which standard Saccharomyces into the non-recombinant strains and their expression modi strain are used. fied using recombinant DNA techniques. In some cases it 0196. The ability of the Saccharomyces strain of the might be desirable to increase the expression of certain genes present invention to grow on Xylose as a sole carbon source whereas in other cases it might be desirable to reduce or provides a convenient phenotype that can be used to distin eliminate expression of certain genes. Target genes for altered guish between Saccharomyces strain which are capable of expression could include those encoding cytoplasmic, mito growth rates of at least one generation per 48 hours using chondrial or other organelle metabolic activities. Such genes Xylose as a sole carbon Source, and those that are not for could encode for activities involved in Sugar transport, nutri purposes of “marking yeast strains. ents transport, glycolysis, terminal steps of fermentation, 0.197 In one aspect, there is provided a method of marking pentose phosphate pathway, gluconeogenesis, tricarboxylic a Saccharomyces strain, comprising the steps of: acid pathway, glyoxylate cycle, electron transport chain, 0198 (a) culturing the desired strain with a strain of the intracellular redox balance, interaction between fermentation invention under conditions to permit combining of DNA and respiration, amino acid synthesis and metabolism etc. of the strains; Examples of genes that might be modified by recombinant 0199 (b) selecting or screening for derivatives of the DNA technologies include RPE1, RK11, TAL1, and TKL1 or desired Strain which have an increased growth rate using any other genes that might improve the ability of yeasts to use Xylose as a sole carbon source for growth; Xylose as a sole carbon source. 0200 (c) isolating derivatives of the desired strain (0194 It will be further appreciated by a person skilled in which have an increased growth rate using xylose as a the art that one or more genes for Xylose utilisation may be Sole carbon Source for growth. introduced into the strain using recombinant methods. Meth 0201 In one embodiment, the method comprises: ods for the production of recombinant yeast are well known in 0202 (a) mating the desired strain with a Saccharomy the art and are described in for example, Guthrie and Fink ces strain of the fourth to fourteenth aspect under con (1991) “Guide to Yeast Genetics and Molecular Biology’. ditions which permit combining of DNA of the strains: Methods in Enzymology Vol 194, Academic Press. Genes or and sequences of interest may be cloned into Suitable vectors for 0203 (b) screening or selecting for a derivative of the transformation into the yeast cell. The gene or sequence of desired strain that is capable of growing at a rate of interest is cloned into a suitable expression vector Such as greater than one generation per 48 hours using Xylose as pMA91 Dobson et al. (1984) EMBO Journal 3: 1115), which a sole carbon Source by plating the mated cells on comprises the appropriate regulatory regions for expression Xylose-containing solid medium or by culturing in in the yeast strain. Other vectors include episomal, centro Xylose-containing liquid medium or a combination of meric, integrative, modified expression vectors known to both; those skilled in the art (see for example, Guthrie and Fink 0204 (c) isolating derivatives of the desired strain (1991) “Guide to Yeast Genetics and Molecular Biology’. which have an increased growth rate using Xylose as a Methods in Enzymology Vol 194, Academic Press). Regula Sole carbon Source for growth. tory regions that may be suitable for expression in yeast may 0205 Marked strains may be detected by incubating the include, for example, MAL, PGK1, ADH1, GAL1, GAL10, strain on or in Xylose-containing minimal media and deter CUP1, GAP, CYC1, PHO5. Alternatively, the regulatory mining the growth rate, or comparing the growth rate to that regions of the gene itself may be used to express the gene in of a standard strain with known growth rate on or in Xylose Saccharomyces. The gene or sequence of interest may be containing medium. A Suitable test air detecting a marked integrated into the yeast genome. Such as into the ribosomal strain is Test T1, whereby a marked strain exhibits at least a RNA locus, for instance. For this purpose, the ribosomal 2-fold increase in biomass in Test T1. sequences of a suitable vector (eg. plasmid plRL9) are 0206. In another embodiment, the Saccharomyces spp. released, and cloned appropriately to a BS+ vector. The gene strain of the invention may be used for the production of or sequence of interest is operably linked to Suitable yeast compounds such as ethanol, Xylitol, acetic acid, other yeast promoter and terminator regions to form an expression cas byproducts, enzymes, etc. Methods for the production of sette, and the expression cassette is Subsequently cloned into compounds may comprise the following steps: the cloned ribosomal sequences. From this resulting plasmid 0207 (a) cultivating the Saccharomyces strain in a xylose the expression cassette, flanked by ribosomal sequences can containing medium under conditions which permit the be released as a single fragment using appropriate restriction strain to produce the compound; and enzymes. The released fragment can be cotransformed with 0208 (b) recovering the compound(s) that is produced by an autonomously replicating plasmid carrying a suitable the strain. marker for transformation into a yeast using methods known 0209. The compounds may be one or more compounds or in the art (see for example Guthrie and Fink (1991) “Guide to mixtures thereof that are produced by Saccharomyces when US 2010/006879.0 A1 Mar. 18, 2010

utilising Xylose as a carbon source. For example, the com 0221) The inoculated media may be incubated for at least pound may be ethanol. Xylitol, acetic acid, carbon dioxide, or 2 hours, typically at least 5 hours. any component, metabolites and byproducts of yeast cells and 0222. In another aspect, there is provided a method of their metabolism. Components of yeast cells may include producing ethanol comprising incubating a non-recombinant enzymes, co-factors, vitamins, amino acids, peptides, pro Saccharomyces strain in Xylose-containing media under con teins, nucleosides, nucleotides, oligonucleotides, DNA, ditions sufficient to produce ethanol and thereafter recovering RNA, cell wall components, glucans, mannoproteins, mem the ethanol. brane components, lipids, sterols, storage carbohydrates, tre halose, glycogen, organic acids, succinic acid, acetic acid, Definition of Tests T1 to T9. lactic acid, polyols such as glycerol, Xylitol. 0223 T 1: Growth Using Xylose as Sole Carbon Source 0210. It will be appreciated by persons skilled in the art Yeast strains are streaked onto on Glucose Yeast extract Bac that the type of compounds produced by the Saccharomyces teriological Peptone medium solidified with 2% Agar using spp. Strain may depend on the growth conditions to which the standard microbiological techniques. After incubation for 72 strain is subjected. For example, temperature, aerobic or hours at 30 deg Celsius, yeast cells are taken from plates using anaerobic growth, pH of the medium, nitrogen Source, pres a sterile microbiological loop and inoculated to an ODoo ence of carbon Sources other than Xylose, other compounds in (Optical Density at 600 nm) of between 0.1 and 0.2 units the medium on their own or in combination may impact on the (ODoo at To) in 50 ml of broth. The broth contains xylose types of compounds that are produced by the Saccharomyces (5% w/v), Difco Yeast Nitrogen Base w/o amino acids cerevisiae Strain. However, precise conditions for production (0.67%) in distilled water in a 250 ml Erlenmeyer flask. of any one or more compounds can be readily determined by Cultures are incubated at 30 deg Celsius with shaking at 220 a person skilled in the art through standard procedures. rpm (10 cm orbital diameter) for 48 hours prior to measuring 0211. It will also be appreciated by persons skilled in the ODoo (OD at Ts). The fold increase in biomass is deter art that the Saccharomyces strains of the invention will be mined by the equation: capable of utilising Sugars such as glucose, fructose, man nose, galactose, maltotriose, maltose, Sucrose, melibiose, raffinose, Xylose, melizitose, alpha-methyl-glucoside, treha OD600 at T48 hrs lose, isomaltose. The strains may also be capable of using OD600 at To non-Sugar carbon Sources such as ethanol, acetate, glycerol, xylitol. 0212. The strain of the invention will typically be capable of fermenting Sugars Such as glucose, fructose, mannose, T2: Cell Biomass Yield Using Xylose as Sole Carbon Source. galactose, maltotriose, maltose, Sucrose, melibiose, raffinose, 0224 Yeast strains are streaked onto on Glucose Yeast Xylose, melizitose, alpha-methyl-glucoside, trehalose or iso extract Bacteriological Peptone medium solidified with 2% maltose in addition to xylose for production of carbon dioxide Agar using standard microbiological techniques. After incu and ethanol. bation for 72 hours at 30 deg Celsius, yeast cells are taken 0213. Thexylose in the xylose-containing medium may be from plates using a sterile microbiological loop and inocu in any form that can be utilised by the yeast. For example, the lated to an ODoo (Optical Density at 600 nm) of between 0.1 Xylose may be purified, or it may be in a crude form Such as a and 0.2 units in 50 ml of broth. The broth contains xylose (5% hemicellulose hydrolysate obtained from acid hydrolysis of w/v), Difco Yeast Nitrogen Base w/o amino acids (0.67%) in biomass such as Sugar cane, Straw, corn Stover, timber prod distilled water in a 250 ml Erlenmeyer flask. Cultures are uctS etc. incubated at 30 deg Celsius with shaking at 220 rpm (10 cm 0214. In the production of biomass and compounds, the orbital diameter) for 72 hours prior to measuring the yield of inoculated Xylose-containing media is typically cultivated at dry yeast matter. The dry weight content of the yeast is mea a temperature range of between 22°C. and 40°C. for between Sured by transferring 5 mls of yeast culture to a pre-weighed 2 hours and 4 days. glass test tube (W1), followed by centrifugation at 3000 g for 0215. The inventors have further found that in order to 10 minutes at 22 deg C. The supernatant is removed without produce ethanol from Xylose using the strains of the inven disturbing the yeast pellet, and the cells are resuspended in 5 tion, it is not necessary that the strains exhibit Substantial ml of distilled water, prior to re-centrifugation at 3000 g for 10 growth on the xylose. The inventors have found that heavy minutes at 22 deg C. The Supernatant is again removed with inocula of Xylose-containing medium with strains of the out disturbing the yeast pellet, and the cells are resuspended invention results in ethanol production within 2 hours, typi in 5 ml of distilled water, prior to re-centrifugation at 3000 g cally 4 hours, more typically 5 hours. Under Such conditions, for 10 minutes at 22 deg C. The glass test tube containing the substantial growth is not detectable. Thus, there is also pro yeast cells is baked at 105 deg C. for 24 hours and weighed vided a method of producing ethanol comprising: (W2). Dry yeast matter is calculated by subtracting W1 from 0216 (a) inoculating Xylose-containing media with the W2 and multiplying the obtained value by 10. Assays are strain of the first to eighth, twelfth or thirteenth aspects performed in duplicate and the average is calculated. to a density of at least 1x10 yeast cells per ml of media; 0217 (b) incubating the inoculated media for sufficient T3: Production of Ethanol Using Xylose as Sole Carbon time to permit ethanol production; Source. 0218 (c) recovering the ethanol. 0225 Inoculum of yeast was prepared by growing 1 stan 0219. The xylose containing media may be any of the dard microbial loopful of pure cells of the strain for 16 hr at Xylose-containing media mentioned above. 30°C. with shaking at 200 rpm in a 250 mL Erlenmeyer flask 0220. The density of yeast cells may be at least 5x10, containing in 50 mL distilled water: 2.5g Xylose, 0.5g yeast typically at least 6x10 yeast cells per ml of media. extract, 0.5g bacteriological peptone. Sevenx50 mL cultures US 2010/006879.0 A1 Mar. 18, 2010

were grown simultaneously. The cultures were harvested by 3 and 5 units. If cells did not reach the required density after centrifugation at 22°C. and 3,000xg for 5 min. Supernatant 24h incubation they were nevertheless harvested. Cells were was discarded and cell pellets were resuspend in sterile dis harvested by centrifugation at 3,000xg and 4°C. for 5 min. tilled water and re-centrifuged. Supernatant was discarded The Supernatant was discarded and the cell pellet was resus and cell pellets resuspended and pooled in 20 mL of xylose pended in chilled distilled water and re-centrifuged. Super minimal medium which contained per L of distilled water: natant was discarded and the process repeated so as to remove 0226 50 gxylose, 13.4 g Difco Yeast Nitrogen Base with all traces of medium. outamino acids, supplemented with 0.4 mg of CuSO4.5H2O, 0231. Cells were re-suspended in disintegration buffer and 1 mg ZnSO.7H2O, 2 mg of MnSO4HO, 1 mg NaMoO. cell extracts prepared as described in Eliasson A. etal. (2000) 2H2O, 1 mg of NaBO7.10H2O, 2 mg Ca-pantothenate, 2 mg Applied and Environmental Microbiology, Volume 66 pages thiamine HCl, 2 mg pyridoxine HCl, 4 mg inositol. 1 mg 3381-3386. The cell extracts were then assayed for xylitol nicotinic acid, and 0.4 mg biotin. dehydrogenase activities according to the methods described 0227. The cells were then inoculated into 980 mL of the and referred to in Eliasson A., et al. (2000) Applied and same Xylose minimal medium which had been prewarmed to Environmental Microbiology, volume 66 pages 3381-3386. 30° C. and aerated to 20% oxygen is a Braun Biostat B2 L One unit of activity is defined as 1 nanomole of NAD(P)H fermentation vessel. Yeast was grown with air pumped at 10L reduced or oxidised per minute per mg of protein at 30° C. permin, stirring at 1200 rpm and pH maintained at pH5 using Protein was assayed by the method described by Lowry OH, additions of KOH or phosphoric acid as required. After 24 h. Rosebrough N.J. Farr AL, and Randall RJ (1951) Journal of 300 mL of the culture volume was removed and replaced with Biological Chemistry 193:265-275, using a bovine serum 300 ml fresh xylose minimal medium comprising 50 gxylose albumin standard. plus 10 g. Difco Yeast Nitrogen Base without amino acids and 0232 T6: Assay of Xylulose kinase. Strains of yeast were trace salts and vitamins in the amounts described above. After grown on glucose, yeast extract and bacteriological peptone a further 7h 30 g of fresh xylose was added to the culture and agar as described previously, were inoculated to an optical air Supply was reduced to 4L/min and stirrer speed reduced to density at 600 nm of between 0.1 and 0.2 units in a 250 mL 200 rpm. Ethanol was assayed after a further 20h by use of a shake flask in 50 mL medium that contained 5% w/v xylose, YSI 2700 Select Biochemistry Analyzer fitted with a YSI 0.5% w/v yeast extract and 1% w/v bacteriological peptone membrane 2786 for ethanol detection (YSI Inc. Yellow (XYP). Cultures were incubated at 30° C. and 180 rpm until Springs, Ohio, USA). they reached optical density at 600 nm of between 3 and 5 0228 T4: Assay of Xylose reductase: Strains of yeast were units. If cells did not reach the required density after 24 h. grown on glucose, yeast extract and bacteriological peptone incubation they were nevertheless harvested. Cells were har agar as described previously, were inoculated to an optical vested by centrifugation at 3,000xg and 4°C. for 5 min. The density at 600 nm of between 0.1 and 0.2 units in a 250 mL Supernatant was discarded and the cell pellet was resuspended shake flask in 50 mL medium that contained 5% w/v xylose, in chilled distilled water and re-centrifuged. Supernatant was 0.5% w/v yeast extract and 1% w/v bacteriological peptone discarded and the process repeated so as to remove all traces (XYP). Cultures were incubated at 30° C. and 180 rpm until of medium. they reached optical density at 600 nm of between 3 and 5 0233 Cells were re-suspended in disintegration buffer and units. If cells did not reach the required density after 24h cell extracts prepared as described in Eliasson A. etal. (2000) incubation they were nevertheless harvested. Cells were har Applied and Environmental Microbiology, Volume 66 pages vested by centrifugation at 3,000xg and 4°C. for 5 min. The 3381-3386. The cell extracts were then assayed for xylulose Supernatant was discarded and the cell pellet was resuspended kinase activities according to the methods described and in chilled distilled water and re-centrifuged. Supernatant was referred to in Eliasson A., et al. (2000) Applied and Environ discarded and the process repeated so as to remove all traces mental Microbiology, volume 66 pages 3381-3386. One unit of medium. of activity is defined as 1 nanomole of NAD(P)H reduced or 0229 Cells were re-suspended indisintegration buffer and oxidised per minute per mg of protein at 30° C. Protein was cell extracts prepared as described in Eliasson A. etal. (2000) assayed by the method described by Lowry OH. Rosebrough Applied and Environmental Microbiology, Volume 66 pages NJ, Farr AL, and Randall RJ (1951) Journal of Biological 3381-3386. The cell extracts were then assayed for xylose Chemistry 193:265-275, using a bovine serum albumin stan reductase activities according to the methods described and dard. referred to in Eliasson A., et al. (2000) Applied and Environ mental Microbiology, volume 66 pages 3381-3386. One unit of activity is defined as 1 nanomole of NAD(P)H reduced or T7: Growth Using Xylitol as Sole Carbon Source oxidised per minute per mg of protein at 30° C. Protein was 0234 Yeast strains are streaked onto on Glucose Yeast assayed by the method described by Lowry OH. Rosebrough extract Bacteriological Peptone medium solidified with 2% NJ, Farr AL, and Randall RJ (1951) Journal of Biological Agar using standard microbiological techniques. After incu Chemistry 193:265-275, using a bovine serum albumin stan bation for 72 hours at 30 deg Celsius, yeast cells are taken dard. from plates using a sterile microbiological loop and inocu 0230 T5: Assay of Xylitol dehydrogenase. Strains of lated to an ODoo (Optical Density at 600 nm) of between 0.1 yeast were grown on glucose, yeast extract and bacteriologi and 0.2 units (ODoo at T) in 50 ml of broth. The broth cal peptone agar as described previously, were inoculated to contains xylitol (5% w/v), Difco Yeast Nitrogen Base w/o an optical density at 600 nm of between 0.1 and 0.2 units in a amino acids (0.67%) in distilled water in a 250 ml Erlenmeyer 250 mL shake flask in 50 mL medium that contained 5% w/v. flask. Cultures are incubated at 30 deg Celsius with shaking at xylose, 0.5% w/v yeast extract and 1% w/v bacteriological 220 rpm (10 cm orbital diameter) for 48 hours prior to mea peptone (XYP). Cultures were incubated at 30° C. and 180 Suring ODoo (ODoo at Tass). The fold increase in biomass rpm until they reached optical density at 600 nm of between is determined by the equation: US 2010/006879.0 A1 Mar. 18, 2010

generations, the cells on medium containing Xylose had pro gressed through 9 to 10 generations. OD600 at T48 hrs OD600 at To Example 2 Generation of Populations Comprising Diverse Strains of Saccharomyces Capable of Rapid Growth T8: Ethanol Production Using Xylose in a Rich Medium. on Xylose as Sole Carbon Source 0235 Yeast strains are streaked onto on Glucose, Yeast 0239. By obtaining yeast strains from a variety of sources, Extract, Bacteriological Peptone, medium solidified with 2% which included strains obtained from the wild, from wine, Agar using standard microbiological techniques. After incu distiller's and beer fermentations, and baking applications, bation for 96 hours at 30 deg Celsius, yeast cells are taken inducing them to sporulate and using mass mating techniques from plates using a sterile microbiological loop and inocu to generate a genetically diverse population it was possible to lated to an ODoo (Optical Density at 600 nm) of between 1 apply selection pressure to enrich for yeast strains capable of and 2 units in 50 ml of broth. The broth contains xylose (5% more vigorous growth using Xylose as a sole carbon Source. w/v), Yeast extract (0.5% w/v) and Bacteriological Peptone By spreading the genetically diverse populations onto Xylose (1% w/v) in distilled water in a 250 ml Erlenmeyer flask. minimal mineral medium (and simultaneously onto the same minimal mineral medium without a carbon Source) and incu Cultures are incubated at 30 deg Celsius with shaking at 220 bating for two months, it was observed that there was hetero rpm (10 cm orbital diameter). Samples of the culture super geneity in the colony size. By comparing the plates with natant are assayed hourly for a period of 24 hours for ethanol Xylose and without Xylose, the observation was made that by use of aYSI2700Select Biochemistry Analyzer fitted with contrary to accepted dogma, growth had been due to the aYSI membrane 2786 for ethanol detection (YSI Inc. Yellow addition of Xylose to the medium; implying that there was Springs, Ohio, USA). Levels of ethanol are expressed as heterogeneity in the ability of the yeast strains within the grams per litre. population to grow on Xylose. By harvesting the entire Xylose grown population, and sporulating them, it was possible to T9: Ethanol Production in Xylose Minimal Mineral Medium generate new populations of yeast that were enriched for Under Anaerobic Conditions. genetic information conferring ability to grow more effec tively on Xylose. By using large population sizes (at least 0236 Yeast strains were streaked onto on 5% w/v xylose, 100,000) and pooling cells including those that did not grow 0.67% w/v. Difco Yeast Nitrogen Base without amino acids optimally, the genetic diversity of the population was main medium Solidified with 2% agar using standard microbiologi tained in each generation. As the number of cycles of mating cal techniques. After incubation for 96 hours at 30 deg Cel and selection increased, the heterogeneity of colony sizes was sius, yeast cells are taken from plates and inoculated directly maintained but the final size of the colonies increased. into 10 mL of sterile 5% w/v xylose plus 0.67% w/v. Difco 0240. After between 5 and twenty cycles of selection on Yeast Nitrogen Base without amino acids contained in a 15 agar plates, the populations were introduced into liquid cul mL volume sterile PP-Test Tubes (Cellstar, Greiner bio-one) ture containing minimal mineral medium with Xylose as a to an optical density at 600 nm of 0.1 to 0.4. Medium was sole carbon source, and continually Sub-cultured for approxi overlayed with 2 mL sterile mineral oil to inhibit oxygen mately 50 generations. The Subsequent populations were transfer, screw caps fully tightened and tubes were incubated sporulated and new populations were constructed by mass without shaking at 30° C. Samples for ethanol assay we mating. The new populations of heterogeneous Saccharomy removed using sterile Pasteur pipettes, without disturbing cell ces cells were grown in liquid culture in Xylose minimal pellets that had formed through growth of original inoculum. medium for approximately 50 generations. After this time Ethanol was assayed using a YSI 2700 Select Biochemistry Some of the sample was stored and some of the sample was Analyzer fitted with aYSI membrane 2786 for ethanol detec sporulated. Germinated spores derived from the selected tion (YSI Inc. Yellow Springs, Ohio, USA). population were mated en masse to generate a new population 0237. The invention will now be described in detail by way of heterogeneous Saccharomyces cerevisiae cells to grow of reference only to the following non-limiting examples. under selection pressure in liquid Xylose minimal medium for approx. 50 generations. This process can be reiterated until Example 1 desired growth rates are achieved. 0241. To determine whether growth rates of populations Saccharomyces is Capable of Slow Growth Using on Xylose minimal medium were increasing, samples taken Xylose as the Sole Carbon Source after 365,569, 1013, 1059, 1170, and 1377 days respectively of selection were inoculated into Xylose minimal medium at 0238 Baker's yeast Saccharomyces cerevisiae strain an optical density of 600 nm (OD) between 0.1 and 0.2 with NL67 (Higgins et al. (1999) Applied and Environmental shaking at 220 rpm and 30 deg C. OD was re-assayed after 24 Microbiology 65:680-685) was inoculated onto solidified h and used to calculate average doubling time over the 24h minimal mineral medium with or without Xylose as a sole period according to standard microbiological method. The carbon source and incubated at 30°C. for two months. It was results were plotted graphically and are shown in FIG. 1. observed using a light microscope that microscopic colonies 0242 FIG. 1 shows the improvement in growth rates of the occurred on both types of plates, but that the colonies on the populations en masse is exponential as they progress through medium containing Xylose were detectably larger than those rounds of mating and selection for growth on Xylose as the provided with no carbon source. Whereas the cells on sole carbon source. This improvement in growth rate on medium containing no Xylose had progressed through 5 to 6 Xylose as sole carbon Source is predicted to continue until US 2010/006879.0 A1 Mar. 18, 2010 growth rate on Xylose is equivalent to the growth rate on lation and mating of Subsequently derived haploids with glucose as sole carbon Source. known laboratory strains of Saccharomyces cerevisiae with genetic markers. Example 3 Ethanol Production in Xylose Rich Medium by Het TABLE 2 erogeneous Populations of Non-Recombinant Sac Growth of pure strains of Saccharomyces cerevisiae charomyces Strains Correlates with Growth Rate on on Xylose minimal medium as described in Test T1. Xylose Minimal Mineral Medium Initial Optical Final Optical Fold 0243 Doubling times of the populations in xylose mini STRAIN Density Density Increase mal medium were determined as described in Example 2, CEN.PK O.128 O.132 1.03 FIG. 1. Ethanol production was determined by inoculating NL67 O.154 O.198 1.29 samples of populations at an optical density at 600 nm of NMO4,41257 O-111 2.57 23.15 between 1 and 2 into 250 mL shake flasks at in 50 mL medium NMO4,41258 O-110 2.9 26.36 ISO 10 O.127 6.3 49.61 that contained 5% w/v xylose, 0.5% w/v yeast extract and 1% NM 05.45.177 w/v bacteriological peptone (XYP). Cultures were incubated ISO 7 O.170 6.8 40 at 30°C. and 220 rpm and ethanol production monitored over NMOS,451.78 a 2 day period. 0244. The data obtained (Table 1) indicate that the ability 0246 Biomass yields of pure strains of Saccharomyces to produce ethanol in Xylose rich medium increased as dou cerevisiae on Xylose minimal medium were assayed as bling time of heterogeneous populations on Xylose minimal described in Test T2. Strains CEN.PK and NL67 were again medium was reduced. Both characteristics improved in con included as representing strain types that have not been gen cert with the number of reiterations and length of time that the erated using the methods described herein. CEN.PKyield=1 mating and selection process was applied. mg dry yeast matter per 50 mL culture; NL67 yield=2 mg dry yeast matter per 50 mL culture: NM04/41257 yield=50 mg TABLE 1. dry yeast matter per 50 mL culture; NM04/41258 yield=55 Ethanolic fermentation of xylose in rich medium improves as mg dry yeast matter per 50 mL culture; ISO 10 yield=145 mg growth rate of heterogeneous populations of non-recombinant dry yeast matter per 50 mL culture, ISO 7 yield=114 mg dry Saccharomyces in xylose minimal mineral medium increases yeast matter per 50 mL culture. 0247 The above data indicate that the protocol generates Doubling time Minutes incubation in Xylose rich medium strains that possess the ability to rapidly grow and yield well (h) in xylose 1035 1155 1275 1395 1455 2465 using Xylose as a sole carbon Source. Taken with the popula Population minimal medium min min min min min min tions data in Example 2 and FIG. 1 the data demonstrate that 1 142 O O O O O O the protocol can be applied reiteratively to derive strains that 2 76 O O O O O O have a maximum possible growth rate and yield on Xylose, 3 17 O O O O O O2 which could be equivalent to growth rate and yield on glu 4 14 O O O O.O1 O.O1 O.O2 COSC. 5 10 O O O.06 0.09 O.12 O.69 6 7 O.O1 O.OS 014 O.16 O.21 11 Example 5 Numbers for ethanol production represent gethanol/L produced at the time (min) indicated. Non-Recombinant Saccharomyces Strains Capable of Rapid Growth and of Producing Ethanol Using Example 4 Xylose as the Sole Carbon Source 0248 Inoculum of strain ISO10 was prepared by growing Characterization of Pure Non-Recombinant Strains 1 standard microbial loopful of pure cells of the strain for 16 of Saccharomyces Capable of Growth on Xylose as hr at 30°C. with shaking at 200 rpm in a 250 mL Erlenmeyer Sole Carbon Source According to Tests T1 and T2 flask containing in 50 mL distilled water: 2.5 g xylose, 0.5g 0245 Saccharomyces strain isolates were purified from yeast extract, 0.5 g bacteriological peptone. Sevenx50 mL Xylose-utilising populations of Example 2 by standard micro cultures were grown simultaneously. The cultures were har biological procedure and tested for growth and yields on vested by centrifugation at 22°C. and 3,000xg for 5 min. Xylose as sole carbon Source according to Test T1 and T2. Supernatant was discarded and cell pellets were re-suspended Strains CEN.PK (Karhumaa et al. (2005)Yeast 22:259-368) in sterile distilled water and re-centrifuged. Supernatant was and NL67 (Higgins et al. (1999) Applied and Environmental discarded and cell pellets re-suspended and pooled in 20 mL Microbiology 65:680-685) were included as representing of xylose minimal medium which contained per L of distilled strain types existing prior to the application of the methods water: 50 gxylose, 13.4 g Difco Yeast Nitrogen Base without described herein. Strains NMO4/41257 and NMO4/41258 amino acids, supplemented with 0.4 mg of CuSO4.5H2O, 1 (deposited) are derived from populations that had gone mg ZnSO.7H2O, 2 mg of MnSO.4H2O, 1 mg NaMoO. through 1059 days of running of the protocol described in 2H2O, 1 mg of NaBO7.10H2O, 2 mg Ca-pantothenate, 2 mg Example 2 and FIG. 1. Strains ISO 10 (NM 05/45.177) and thiamine HCl, 2 mg pyridoxine HCl, 4 mg inositol. 1 mg ISO 7 (NM 05/45178) were obtained from populations that nicotinic acid, and 0.4 mg biotin. had gone through 1377 days, and 1431 days, respectively, of 0249. The cells were then inoculated into 980 mL of the running of the protocol described in Example 2 and FIG. 1. same Xylose minimal medium which had been pre-warmed to The individual isolated yeast strains were confirmed to be 30° C. and aerated to 20% oxygen is a Braun Biostat B2 L members of the species Saccharomyces cerevisiae by sporu fermentation vessel. Yeast was grown with air pumped at 10 L US 2010/006879.0 A1 Mar. 18, 2010

permin, stirring at 1200 rpm and pH maintained at pH5 using 0253) These data suggest that the reiterative mating and additions of KOH or phosphoric acid as required. After 24 h. selection methodology applied to obtain the yeasts that grow 300 mL of the culture volume was removed and replaced with on Xylose as the Sole carbon Source has resulted in improved 300 ml fresh xylose minimal medium comprising 50 gxylose XR and XDH activities that are critical for metabolism of plus 10 g. Difco Yeast Nitrogen Base without amino acids and Xylose and Xylitol. trace salts and vitamins in the amounts described above. This gave a culture of cell mass of 4 g dry yeast equivalents per L. 0254. It is likely that other enzymes and activities such as Aeration and stirring was maintained at 10 L/min and 1200 those involved in pentose phosphate pathway have also been rpm, respectively and pH maintained at pH5. Under these naturally improved in our strains due to the selective pres conditions yeast biomass doubled in 4 hours (based on dry Sures applied in the breeding and selection procedures. Those yeast matter) with a yield of 4 g dry yeast matter from 10 g skilled in the art would realize that our strains could obviously consumed Xylose. be used as a basis for further improvement using further 0250 When yeast grown by the procedure described selections and combinations of breeding, mutagenesis, pro above reached a density of about 12 g (dry yeast equivalent) toplast fusion, cytoduction and/or recombinant DNA tech per L, 30 g of fresh xylose was added to the culture and air niques that optimize activities of XR, XDH, XK, or introduce Supply was reduced to 4 L/min and stirrer speed reduced to and optimize Xylose isomerase, or other genetic changes 200 rpm. Under these conditions where dissolved oxygen was required to improve xylose, Xylitol and Xylulose metabolism. undetectable, 17 g of xylose was consumed and a further 1 g dry yeast matter was produced along with 1.75 g ethanol/L and 1 g xylitol/L in 20 h. Example 7 Example 6 Characterization of Pure Non-Recombinant Strains of Saccharomyces Capable of Growth on Xylitol as Activities of Xylose Reductase (XR), Xylitol Dehy Sole Carbon Source According to Test T7 drogenase (XDH) and Xylulokinase (XK) in Expo nentially Growing Non-Recombinant Yeast Strains 0255 Pure strains were grown on glucose, yeast extract 0251 Strains of yeast were grown on glucose, yeast and bacteriological peptone agar as described in test T1. extract and bacteriological peptone agar as described in test Colonies of the strain were inoculated into 50 mL of 5% w/v. T1, were inoculated to an optical density at 600 nm of xylitol plus 0.67% Difco Yeast Nitrogen Base without Amino between 0.1 and 0.2 units in a 250 mL shake flask in 50 mL, Acids and tested for growth as described in Test T7. medium that contained 5% w/v xylose, 0.5% w/v yeast extract and 1% w/v bacteriological peptone (XYP). Cultures TABLE 4 were incubated at 30° C. and 180 rpm until they reached Growth of pure strains of Saccharomyces cerevisiae optical density at 600 nm of between 3 and 5 units. If, as in the on xylitol minimal medium as described in Test T7. case of strains such as NL67 and CEN.PK, cells did not reach the required density after 24 h incubation they were never Initial Optical Final Optical Fold theless harvested and assayed. Cells were harvested by cen STRAIN Density Density Increase trifugation at 3,000xg and 4°C. for 5 min. The supernatant CEN.PK O.146 O.130 O.89 NL67 O.156 O.164 1.OS was discarded and the cell pellet was resuspended in chilled NMO4,41257 O.130 O.171 1.32 distilled water and re-centrifuged. Supernatant was discarded NMO4,41258 O.1OS 3.03 28.86 and the process repeated so as to remove all traces of medium. ISO 10 O.108 O. 115 1.06S 0252 Cells were re-suspended indisintegration buffer and ISO 7 O.198 6.96 35.15 cell extracts prepared as described in Eliasson A. etal. (2000) Applied and Environmental Microbiology, Volume 66 pages 3381-3386. The cell extracts were then assayed for XR, XDH 0256 These data suggest that the reiterative mating and and XK activities according to the methods described and selection methodology applied to obtain the yeasts that grow referred to in Eliasson A., et al. (2000) Applied and Environ on Xylose as the sole carbon source has resulted in some mental Microbiology, volume 66 pages 3381-3386. The fol strains that can also utilize Xylitol as the sole carbon Source. lowing activities were found where one unit of activity is defined as 1 nanomole of NAD(P)H reduced or oxidised per Example 8 minute per mg of protein at 30° C. Production of Ethanol Using Xylose Rich Medium TABLE 3 by Pure Non-Recombinant Strains of Saccharomyces Capable of Growth on Xylose as Sole Carbon Source Units of enzyme activities of yeast strains According to Test T8 Strain XR activity XDH Activity XK Activity CEN.PK O.74 O.O2S 23 0257 Pure isolates of yeasts grown on glucose, yeast NL67 O.74 O.12 2O2 extract and bacteriological peptone agar as described in test NMO4,41257 1.16 1.09 20.9 T1, were inoculated to an optical density at 600 nm of NMO4,41258 1.61 1.13 22.5 between 1 and 2 units in a 250 mL shake flask in 50 mL ISO 10 3.81 8.09 22.5 ISO 7 5.39 13.35 29.75 medium that contained 5% w/v xylose, 0.5% w/v yeast extract and 1% w/v bacteriological peptone (XYP) and assayed as described in Test T8. US 2010/006879.0 A1 Mar. 18, 2010 19

described herein results in increased efficiency of anaerobic TABLE 5 ethanol production by non-recombinant strains. Ethanol production using xylose rich medium Example 10 Rapid Ethanol Production by Non-Recombinant Sac Initial Optical Optical Density Ethanol (gL) charomyces Inoculated at High Density into Minimal STRAIN Density after 24h after 24h Media Under Aerobic Conditions CEN.PK 1.16 1.9 O 0261 Strain ISO 10 grown on glucose, yeast extract, bac NL67 1.29 1.8 O NMO4,41257 1.06 14.5 O.04 teriological peptone agar (as described above) was inoculated NMO4,41258 1.17 17.2 O.O7 into 50 mL of 5% w/v xylose, 0.5% w/v yeast extract and 1% ISO 10 1.18 2O.O O.66 w/v bacteriological peptone (XYP) contained in 250 mL ISO 7 1.15 21.2 1.32 Erlenmeyer flasks and incubated for 72 hat 30° C. and 220 rpm. Twenty flasks were incubated simultaneously. Cells were harvested by centrifugation at 3,000xg and 22°C. for 10 0258. These data show that the selection strategy based on min. Supernatant was discarded and cell pellet re-suspended growth of yeasts onXylose as the sole carbon Source generates in 10 mL sterile water before re-centrifugation. The superna yeast strains that are capable of producing ethanol from tant was again discarded and the Cells re-suspended in dis xylose. These data coupled with data of Example 3 (Table 1), tilled sterile water before a further centrifugation. Finally, show that the ability to grow and yield cell biomass using cells were re-suspended in 20 mL sterile distilled water. 0262. Fermentation medium was prepared, filter-steril Xylose as the sole carbon Sugar increases in concert with ized as follows:YNB Contained 0.67% Difco Yeast Nitrogen increasing ability to produce ethanol from Xylose. Moreover Base without amino acids: XYNB was YNB plus 5% xylose; strains such as NM04/41258 and ISO 7that can utilize xylitol GXYNB was YNB plus 0.5% glucose and 4.5% xylose. and xylose are able to produce ethanol. The finding that Media were contained in 125 mL conical glass flasks. They strains NM04/41258 and ISO 7 (NM 05/45178) which can were inoculated with 3 mL of cell Suspension and placed on a utilize Xylitol and make ethanol from Xylose goes against the 220 rpm orbital shaker at 30° C. and ethanol production read teaching of U.S. Pat. No. 4.51 1,656, which states strains at hourly intervals as described previously. Immediately after should be screened to isolate the specific colony or colonies inoculation optical densities at 600 nm of cultures were mea which will utilize D-xylose but not xylitol. sured and it was determined that cell densities for cultures were at 5.7 to 6.1x10es per mL. Example 9 TABLE 7 Isolation and Characterisation of Pure Non-Recom binant Strains of Saccharomyces Capable of Ethanol Production of ethanol by high, density inoculum Production Using Xylose as Sole Carbon Source of Non-recombinant Saccharomyces cells. Under Anaerobic Conditions Medium 1 h 2h 3h 4h Sh YNB O41 0.44 O.36 O.29 O.09 0259 Isolates were purified from Xylose-utilising popula XYKB 0.75 1.1 1.67 2.1 2.9 tions that had undergone 1106 days of selection and were GXYNB 1.69 2.2 2.41 3.2 3.8 subjected to Test T9. Fifty individual isolates were tested and ethanol production assayed after three weeks to 4 months 0263. Numbers refer to gethanol per L in culture super ranged from 0.24 g/L to 0.75 g/L. Strains NM04/41257 and natant at the indicated time after inoculation. The small NM04/41258 were also included in these assays. amount of ethanol produced by cells incubated in YNB with out a carbon source was derived most likely from carry over TABLE 6 of endogenous storage Sugars synthesized and accumulated Production of ethanol by pure yeast strains in xylose by the yeast during the 72 h inoculum preparation phase. minimal medium under anaerobic conditions. Decline in the ethanol concentration of YNB cultures after 3 h indicates that storage Sugars were exhausted or becoming g/L Ethanol exhausted and ethanol was being consumed by cells and/or Strain Incubation Period Produced ethanol was evaporating. NMO4,41257 4 months O.82 0264. These data show that it is possible for non-recom NMO4,41258 4 months O.48 Isolate no. 2 1 month O.70 binant strains of Saccharomyces to be obtained that can fer Isolate no. 6 1 month 0.75 ment Xylose rapidly to produce ethanol in minimal medium Isolate no. 21 1 month O.70 where xylose is the sole carbon source. Moreover, these Isolate no. 23 1 month 0.73 strains are able to produce ethanol from Xylose in medium Isolate no. 31 3 weeks O.64 where fermentable hexose Sugar Such as glucose was also Isolate no. 36 3 weeks O.63 added since the ethanol produced by 4h in GXYNB was in Isolate no. 37 3 weeks O.65 excess of that expected solely from the glucose present. Example 11 0260 These data indicate that it is possible to obtain non recombinant Saccharomyces yeasts that are capable offer Non-Recombinant Saccharomyces Biomass Grown menting Xylose under anaerobic conditions to produce etha on Xylose as the Sole Carbon Source Exhibits Broad nol. The NM04/41257 and NM04/41258 strains were purified Industrially Useful Features from earlier selected populations relative to the other strains 0265 Those skilled in the art will know that yeasts have in the Table. This data shows that the reiterative protocol proven industrial utility in various fermentation applications US 2010/006879.0 A1 Mar. 18, 2010 20

(e.g. bread, and ethanol (potable and non-potable) produc Wilkinson J F (1971) “Chemical Extraction Methods of tion) and also as a Source of amino acids and protein, nucle Microbial Cells”, in Methods in Microbiology Vol 5B (Eds. J otides (e.g. DNA and RNA), enzymes (e.g. invertase and R Norris and D W Ribbons), Chapter IV pp. 345-383, Aca phytase), antioxidants (e.g. glutathione) and other cellular demic Press London and New York. According to Sutherland components such as cell wall components (e.g. glucans). The et al. this method produces “cell wall glucan which is free aforementioned properties are by way of example and are not from contaminating material”. Using the described procedure meant to be limiting. an amount of 8 mg of glucan was obtained from the dry yeast 0266. It would be useful if yeasts could be grown on media starting material. that contain Xylose and then Subsequently used for the various 0272 Amino Acid/Protein content. An amount of yeast industrial purposes. Therefore, by way of example strain material equivalent to 24.6 mg dry yeast matter was Sus ISO10 was tested for various features following growth on pended in a glass test tube in 2.5 mL of 1 MNaOH and placed Xylose. in a boiling water bath for 15 min. The boiled sample was 0267 Strain ISO 10 was grown in per Litre: 50 g xylose, cooled to 22°C. and volume made to 10 mL using distilled 13.4 g Difco Yeast Nitrogen Base without amino acids, water. Amino acid/protein was assayed according to Lowry O supplemented with 0.4 mg of CuSO4.5H2O, 1 mg ZnSO. H. Rosebrough N.J. Farr AL, and Randall RJ (1951) Journal 7H2O, 2 mg of MnSO.4H2O, 1 mg NaMoO.2H2O, 1 mg of of Biological Chemistry 193:265-275, using a bovine serum NaBO7.10H2O, 2 mg Ca-pantothenate, 2 mg thiamine HCl, albumin standard. This assay indicated the yeast contained 2 mg pyridoxine HCl, 4 mginositol. 1 mg nicotinic acid, and equivalent of 39% amino acid/protein material per wt dry 0.4 mg biotin. Cells were stirred at 1200 rpm with air supply matter. at 12 L permin and temperature at 30°C. and pH maintained 0273 Nucleotide content. An amount of yeast material at pH5 using 1M KOH and 1M phosphoric acid. When cell equivalent 8.3 mg of dry yeast was resuspended in 1 mL of4% density was at Asoo 14, cells were harvested by centrifugation w/v NaCl and autoclaved at 121°C. for 15 min. Upon cooling at 3,000xg and 22°C. for 10 min. Cell pellet was resuspended to 22°C. the yeast suspension was centrifuged at 3,000xg for in distilled water and re-centrifuged. This washing procedure 10 min at 22°C. and the supernatant assayed for nucleotide was carried out three-times and the biomass placed on What content by the spectrophotometric UV absorbance method man filter paper no. 1 to achieve a wet biomass of 22 to 25% described by Herbert D, Phipps PJ, and Strange RE (1971) Solids. “Chemical Analysis of Microbial Cells’, in Methods in 0268. The biomass was assayed for the following features Microbiology Vol 5B (Eds. J R Norris and D W Ribbons), by way of demonstrating that it is possible to grow non Chapter III pp. 209-344, Academic Press London and New recombinant Saccharomyces on Xylose as Sole carbon Source York. The yeast contained 1.9% nucleotide material per wt to obtain yeast biomass with properties generally relevant to dry matter. industrial applications. 0274) Glutathione content. An amount of yeast material 0269. Ethanolic fermentation power. Yeast was inoculated equivalent 6.82 mg of dry yeast was resuspended in 0.8 mL of into a molasses-based medium to test for ethanolic fermen 80:20 ethanol:distilled water, vortex mixed and centrifuged at tation power, defined as the ability to produce ethanol from 10,000xg for 2 min at 22°C. The supernatant was assayed as fermentable Sugars such as Sucrose, glucose and fructose. follows: Phosphate buffer contained 3.99g Na HPO, 0.43g Sugar cane molasses was diluted in water and sterilized by NaH2POHO, 0.59 g disodium-EDTA.2H2O per 250 mL at heating to 121° C. for 5 min. When cooled to 22°C., the pH7.5. NADPH solution contained 26.6 mg NADPH tetra molasses was centrifuged at 4,000xg at 22°C. for 10 minto sodium salt in 100 mL of phosphate buffer, 5.5"-dithio-bis(2- remove Solids. The Supernatant was diluted to a final concen nitrobenzoic acid) (DTNB) was prepared by weighing 23.8 tration of 18% w/w sucrose equivalents and supplemented mg DTNB in 10 mL of phosphate buffer. Glutathione stan with filter-sterilised Difco Yeast Nitrogen Base at 0.67% w/v. dard was prepared in distilled water to a stock concentration Forty mL of this medium was inoculated with an equivalent of of 0.1 mM. Glutathione reductase stock solution from Fluka. 6.8 mg dry yeast matter and incubated at 30° C. without Chemie AG contained 162.72 units of activity per mL. shaking. Ethanol was assayed at 24h intervals by use of aYSI 0275 Assay was carried out in a 3 mL spectrophotometer 2700 Select Biochemistry Analyzer fitted with a YSI mem cuvette which contained 1.4 mL of NADPH solution+200 brane 2786 for ethanol detection (YSI Inc. Yellow Springs, microlitre DTNB solution+10 microlitre of sample, standard Ohio, USA). After 24 h the yeast produced 16.8g ethanol per GSH solution, or distilled water and 390 microlitre of dis L., after 1 week the yeast had produced 59 g ethanol per L. tilled water. Sample was prewarmed to 30° C. and reaction 0270 Leavening of bread dough. To test for leavening started by addition of three microlitre of glutathione reduc power, defined as the ability to ferment Sugars such as glu tase. Cuvettes were incubated at 30° C. for 30 min then the cose, fructose and maltose and thereby produce carbon diox absorbance read against the distilled water blank at 412 nm ide which raises a flour dough, the yeast was added to a bread wavelength using a Shimadzu UV-1201 spectrophotometer. dough mixture and tested for its ability to produce leavened This assay indicated the yeast contained 0.36% total glu bread. A dough was prepared that contained 500 g wholemeal tathione per wt dry matter. flour soy and linseed bread mix (Kitchen Collection, 0276 Phytase activity. An amount of yeast material Christchurch), 300 mL tap water and 10 g yeast (at 24% equivalent to 13.5 mg dry yeast was resuspended in 0.5 mL solids). Dough was leavened and baked using a Breville Bak 0.2M sodium acetate buffer at pH4.9. Phosphatase substrate ers Oven on setting 3B (HWI Electrical, Sydney Australia). from Sigma-Aldrich Chemie GmbH was made to 1 mg per The yeast raised (leavened) the dough mixture to produce a mL in 0.2M sodium acetate buffer at pH4.9, and Phytase loaf of bread with height of 14 cm. enzyme from Sigma-Aldrich Chemie GmbH (defined by the 0271 Glucan content. An amount of yeast biomass Supplier as having 1.1 units of phytase activity per mg Solid equivalent to 59 mg dry matter was extracted for glucans material) was made to 0.909 units per mL in 0.2M sodium according to the method described by Sutherland I W. and acetate buffer at pH4.9. Assay was performed in a cuvette US 2010/006879.0 A1 Mar. 18, 2010

containing 500 microlitre of sodium acetate buffer+250 48 hours using Xylose as a sole carbon Source, wherein microlitre of phosphatase substrate solution and 250 microli steps (b) and (c) are optionally repeated until one or tre of either yeast Suspension, phytase enzyme or distilled more yeast cells having a growth rate of at least one water. Cuvettes were incubated for 20 min at 30° C. at which generation per 48 hours can be isolated. time 300 microlitre of 1 OMNaOH was added. Absorbance at 77. The method of claim 76, wherein the growth rate is at 405 nm was read against the distilled water blank. The least one generation per 24 hours using Xylose as a sole phytase activity of the yeast was 0.068 units per mg dry yeast carbon Source. matter. 78. The method of claim 76, wherein the yeast cells are 0277 Invertase activity. An amount of yeast material incubated on Xylose-containing medium. equivalent to 14.48 mg dry yeast was resuspended in 1 mL of 79. The method of claim 76, wherein the xylose-containing distilled water. The suspension was further diluted one hun medium contains Xylose as the Sole carbon Source. dred-fold in distilled water. Invertase activity was assayed 80. The method of claim 76, wherein the yeast cells are according to the colorimetric method described in described selected by incubating the yeast cells on the Xylose-contain in test T3 of U.S. Pat. No. 4,396,632 and U.S. Pat. No. ing medium. 5,741,695. The yeast produced 0.93 units of invertase activity 81. The method of claim 76, wherein the xylose-containing where a unit of activity is defined as 1 micromole of glucose medium is Xylose minimal medium. released from sucrose per minute at 30° C. and pH4.9 per mg 82. The method of claim 76, wherein the xylose-containing of dry yeast. medium is solid Xylose-containing medium. 0278. These data indicate the potential for non-recombi 83. The method of claim 76, wherein the combining of nant Saccharomyces cerevisiae to be grown on Xylose as the DNA between the yeast cells occurs by mating. sole carbon Source to produce biomass, metabolism, cellular 84. The method of claim 83, wherein the mating occurs by components, and/or enzyme activities that are relevant to sporulation of the yeast cells and hybridizing of sexually broad types of industrial applications. Those skilled in the art competent offspring. will know that the exact levels or amounts of these features can be manipulated not only through classical and recombi 85. The method of claim 76, wherein the population of nant genetical methods but also through means of altering genetically diverse yeast cells comprises the species Saccha culturing conditions, such that the results given above are romyces cerevisiae. merely indicative. 86. The method of claim 76, wherein the population of 1-75. (canceled) yeast cells comprises naturally occurring Saccharomyces 76. A method of producing a strain of Saccharomyces that Strains. is capable of growing at a desired growth rate using Xylose as 87. The method of claim 85, wherein the xylose-containing a sole carbon Source, the method comprising: medium is one in which Xylose is the sole carbon source. (a) providing a population of genetically diverse nonre 88. The method of claim 85, wherein the combining of combinant yeast cells of the genus Saccharomyces, DNA between the yeast cells occurs by mating. (b) culturing the yeast cells under conditions that permit 89. A Saccharomyces cerevisiae strain produced by the combining of DNA between the yeast cells; method of claim 76. (c) screening or selecting the yeast cells by incubating the 90. A Saccharomyces cerevisiae strain produced by the yeast cells on or in Xylose-containing medium for a time method of claim 77. Sufficient to permit yeast cells capable of growing on 91. A Saccharomyces cerevisiae strain produced by the Xylose-containing medium to grow; and method of claim 83. (d) isolating one or more of the screened or selected yeast cells having a growth rate of at least one generation per