FI108300B - Genetic engineering method and host for the production of xylitol - Google Patents

Abstract

Novel methods for the synthesis of xylitol are described.

Description

108300

Genetic engineering method and host for the preparation of xylitol

BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates generally to methods using genetically modified microorganisms for the preparation of useful chemical compounds (metabolic manipulation), and more particularly to the genetic engineering of microbial strains capable of altering readily available carbon sources such as D-glucose, more valuable products such as xylitol.

2. Related Literature 15 Xylitol is a chemical compound of considerable value as a special sweetener. It is about as sweet as sucrose, non-toxic and non-cariogenic.

Xylitol is currently produced by chemically hydrogenating D-xy-20 loses. D-xylose is obtained from hydrolyzates of various plant materials, where it is always present in admixture with other pentoses and hexoses. Purification of xylose and also xylitol is therefore a major problem. A number of methods of this type are known. Examples 25 include U.S. Patent Nos. 3,784,408, 4,066,711, *: **: 4,075,406 and 4,008,285.

Reduction of D-xylose to xylitol can also be achieved by a microbiological process using * · · 1 either strains isolated from nature [Barbosa, M. F. S. et al.

• · I

30, J. Industrial Microbiol. 3: 241-251 (1988)] or gee. #, strain produced technically (Hallborn, J. et al., ... Biotechnology 9: 1090-1095 (1991)). However, the availability of the substrate, D-xy- * t-2 * 108300 loose, in a form suitable for yeast fermentation, is also a major problem because of the inexpensive sources of xylose, such as pulp and paper processes. phyto broth containing yeast growth inhibitory impurities.

One attractive alternative method of making xylitol would be to prepare it by fermenting a cheap and readily available substrate such as D-glucose. However, microorganisms which produce significant amounts of xylitol during single-stage fermentation from common carbon sources other than D-xylose and D-xylulose, both of which are closely related in structure to xylitol, are not known.

On the other hand, many microorganisms, especially osmophilic yeasts, for example, Zygosaccharomyces rouxii, Candida polymorphha and Torulopsis Candida, produce significant amounts of one closely related pentitol, D-arabitol, D-glucose [Lewis, DH and Smith, DC, Phytol. 66: 143-184 (1967)]. Utilizing this property of osmotic yeasts, H. Onishi and T. Suzuki have developed a method for converting D-glucose to xylitol by three successive fermentations [Appl. Micro-biol. 18: 1031-1035 (1969)]. In this process, D-glucose was first converted to D-arabitol by fermentation with an osmophilic *: · * yeast strain. Secondly, D-arabitol was oxidized: * ·,? To D-xylulose by fermentation with Acetobacter suboxydans.

j Finally, D-xylulose was reduced to xylitol by one-third fermentation using as many yeast strain

I | I

30 with the ability to reduce D-xylulose to xylitol.

. One obvious disadvantage of this process is that it involves three different fermentation steps, each of which takes 2-5 days; in addition, additional steps are needed, such as sterilization and cell removal, which increases the manufacturing cost. The yield of the stepwise fermentation process is low and the amount of by-products is high.

«« 108300 3

Thus, there remains a need for methods for economically producing xylitol from readily available substrates in micro-systems.

SUMMARY OF THE INVENTION The present invention provides methods for constructing composite hosts, and thus constructed composite hosts, which have the ability to produce xylitol by growth on carbon sources other than D-xylose or D-xylose and their polymers or oligomers. The carbon sources utilized by the hosts of the invention are cheap and readily available. The microorganisms of the invention also have the ability to secrete synthesized xylitol into the culture medium. This object is achieved by modifying the metabolism of the desired microorganism, preferably a naturally occurring yeast microorganism, by introducing the desired heterologous genes into the body and making them expressed. This object is also achieved by further modifying the metabolism of such desired microorganisms such that certain genes homologous to such microorganism in their natural state are overexpressed and / or their expression activity is inactivated.

It is therefore an object of the present invention to provide ·; a method for producing xylitol, utilizing a novel and novel microbial strain, ·; · *: a recombinant host, also referred to herein as a genetically engineered microorganism, as a producer of such a xylitol, such as xylitol, . ·? -. the treated micro-organism produces such xylitol in either de novo or greater amounts compared to the natural untreated micro-organism.

It is a further object of the present invention to provide a process for the production of xylitol which utilizes a novel metabolic pathway genetically engineered into a microorganism and results in such a microorganism producing xylitol de novo or greater amounts.

It is a further object of the present invention to provide a process for the production of xylitol which utilizes a novel metabolic pathway as described above and which modifies the biosynthesis and / or metabolism pathway of D-arabitol such that the microorganism produces xylitol through the fermentation of carbon sources. , utilized by the unmodified host for the biosynthesis of 10 D-arabitol.

It is a further object of the present invention to provide a process for producing xylitol which utilizes the modified D-arabitol pathway described above and either modifies it by extending 15 existing D-arabitol biosynthetic pathways (by further steps to utilize D-arabitol) or by replacing one or more D-arabitol pathways. steps leading to the formation of xylitol.

It is a further object of the present invention to provide a process for the production of xylitol which utilizes the modified D-arabitol biosynthetic pathway described above and either modifies the existing D-arabitol pathway by introducing D-xylulose ,, 25- arabitol dehydrogenase (EC 1.1.1.11) and * * i ': genes encoding xylitol dehydrogenase (EC 1.1.1.9): * ·, · and rendering them overexpressed.

It is a further object of the present invention to provide a process for the production of xylitol using 30 novel microorganisms such as those described above utilizing the modified D-arabitol biosynthetic pathway and such pathway described above *. · additionally altered by inactivation of chemically induced::: mutagenesis or gene cleavage in said mic. ; In 35 ro-organisms, 5 108300 genes encoding transketolase (EC 2.2.1.1) or D-xylulokinase (EC 2.7.1.17).

It is a further object of the present invention to provide a process for the production of xylitol using 5 novel microorganisms such as those described above employing enzymes of the oxidative branch of the pentose phosphate pathway, more specifically D-glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and / or -phospho-D-gluconate dehydrogenase (EC 1.1.1.44) is the engineered gene overexpression of genes encoding in a microorganism of 10 hours.

It is a further object of the present invention to provide a process for the production of xylitol using a novel microorganism such as the one described above which utilizes genes encoding the enzymes of the pentose-phosphate pathway oxidative branch and the D-ribulose-5-phosphate epimerase gene (EC 5.1.3.1). the resulting altered over-expression.

20 Brief Description of the Drawings

Figure 1 is a restriction map of the insert in plasmid pARL2. This insert is derived from the Kplsiella Terri gene chromosome locus Phpl and contains the D-arabitol dehydrogenase gene of K. terrigena. The open box represents the chromosomal DNA of K. 25 terrigena. The arrow indicates the location: * ·, · and the direction of the gene encoding D-arabitolide *: **: hydrogenase (EC 1.1.1.11) in this DNA.

•. Figure 2 shows the construction of pYARD from pADH and »<.

. *: · From pAAH5. In the plasmid diagrams, a simple line • (· 30 (-) denotes bacterial sequences, a wavy line denotes,. · ". S. cerevisiae 2 pm-DNA; open arrow (=>) denotes the ADCI promoter (ADCI gene). encodes S. cerevi * siae alcohol dehydrogenase (ADCl), formerly known as ADHI); open diamond (O) 35 denotes the RDCI transcription terminator; rectangular-6 108300a denotes the LEi / 2 gene? D arabitolidehydrogenaasigeeniä.

Figure 3 shows construction of plasmid pJDG (AX) -16. XYL2 is a xylitol-5 dehydrogenase gene from Pichia stipitis. dalD is a D-arabitolide dehydrogenase gene. ADCI is the transcriptional regulatory region (promoter) of the ADCJ gene, which precedes and is operably linked to the dalD coding sequence. The symbols are not the same as in Figure 4. In the plasmid diagrams, a single line (-) denotes bacterial sequences and 2 pm-DNA when mentioned; shaded mole ADCJ promoter; a shaded diamond (♦) terminator of ADCI transcription; rectangle LEi72 marker gene; with a shaded arrow the XYL2 gene and a shaded rectangle with 15 dalD genes.

Figure 4 shows the construction of the E. coli-Z. rouxii shuttle vector pSRT (AX) -9. The symbols are as in Figure 3.

Figure 5 shows a restriction map of the cloned T. Candida rDNA fragment-20.

Figure 6 shows construction of plasmid pTC (AX).

Figure 7 shows a restriction map of a cloned T. Candida rDNA fragment.

Figure 7a shows construction of plasmid pCPU (AX).

(Figure 8 shows the cloning of the ZWF1 and gnd genes.

·: **: Figure 8a shows construction of plasmid PAAH (gnd).

Figure 9 shows construction of plasmid pSRT (ZG).

• ·; Figure 10 shows the culture of strain Z. rouxii ATCC 13356 · m [pSRT (AX) -09] in a fermenter.

Figure 11 shows the cultivation of a mutant derived from strain Z. rouxii ATCC 13356 [pSRT (AX) -9] in a fermenter.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. DEFINITIONS 35 The following description extensively uses a number of terms used in recombinant DNA technology. The following definitions are provided in order to permit a clear and uniform understanding of the specification 7 108300 and the claims, including the meaning assigned to such terms.

5 Carbon source other than xylose or xylulose: As used herein, the term "carbon source other than D-xylose and D-xylulose" means a carbon source for the production of xylitol other than D-xylose and D-xylulose or a polymer, oligomer or mixture thereof (such as xylan and hemicellulose). The carbon source preferably supports the growth and fermentation of the genetically engineered microbial hosts of the invention in yeast hosts. Many cheap and readily available compounds can be used as carbon sources to produce xylitol in microbial hosts of this invention, including D-glucose and various D-glucose syrups and D-glucose mixtures with other sugars. Other sugars that can be assimilated by the hosts of the invention, including yeasts and fungi, such as 20 different real and ketohexoses (e.g., D-fructose, D-galactose and D-mannose) and their oligomers and polymers (e.g., sucrose, lactose, starch) , inulin and maltose) are intended to be covered by this term; circuit. Pentoses other than xylose and xylulose and non-; Carbohydrate carbon sources such as glycerol, ethanol, various vegetable oils or hydrocarbons (preferably n-alkanes containing 14 to 16 carbon atoms) are also intended to be included within the scope of this term. The number of carbon sources useful as substrates for producing xylitol by the hosts of this invention varies according to the microbial host. Glucose and glucose-containing syrups are, for example, a preferred source of carbon dioxide for the production of xylitol by the genetically engineered Zygosaccharomyces roux of the invention; . While n-alkanes, preferably containing 14 to 16 carbon atoms, contain 8 108300, being a preferred carbon source for modified Candida tropicalis strains.

Gene: A DNA sequence containing a template for RNA polymerase. The RNA generated from the gene through transcription may or may not encode the protein. The protein encoding RNA is called messenger RNA (mRNA) and is transcribed in eukaryotes by RNA polymerase II. A gene containing an RNA polymerase 10 II template (as a consequence of the RNA polymerase II promoter) can also be constructed to generate an RNA sequence that has a sequence complementary to a particular mRNA, but which is not normally translated. Such a gene construct is referred to herein as an "antisense RNA gene" and such an RNA transcript is referred to as an "antisense RNA". Antisense RNAs are not normally translated because translation stop codons are present in the antisense RNA sequence.

The term "complementary DNA" or "cDNA" gene 20 encompasses recombinant genes that are synthesized, for example, by reverse transcription of mRNA and thus lack intervening sequences (introns). Genes cloned from genomic DNA generally contain introns.

Cloning transporter: Plasmid or phage DNA or other DNA sequence capable of carrying genetic information, particularly DNA, into a host cell. Cloning cul- • *. . the constructor is often characterized by a single endonuclease recognition site, or a small amount of endonuclease recognition site, from which such DNA sequences can be cleaved in an identifiable manner. without destroying the essential biological function of the transporter and to which the desired DNA can be inserted to clone it into the host cell. · **: The cloning carrier may further include a marker suitable for identifying the cells transformed below the cloning site, and replication initiation sites * 9108300 which allow the driver to remain and replicate in one or more prokaryotic or in a euka-ryotic host. Markers include, for example, tetracycline resistance or ampicillin resistance. The word "vector" is sometimes used to refer to "cloning transporter". A "plasmid-midi" is a cloning transporter, usually circular DNA, that is conserved and autonomously replicated in at least one host cell.

Expression transporter: A clone transporter or vector that nevertheless supports self-expression of a cloned gene after host transformation. The cloned gene is usually placed under the control (i.e., operably linked) to certain control sequences, such as promoter sequences, which may be provided by the transporter or the composite structure of the cloned gene. The expression control sequences will vary depending on whether the vector is designed to effect expression of a functionally linked gene in a prokaryotic or eukaryotic host, and may additionally contain transcription-related elements such as promoter elements (above activation sequences) and / or transcriptional sequences. start and end points.

•; v Host: The host is a prokaryotic or eukaryotic (25 cell utilized as a acceptor and carrier of recombinant material *: ···.

The host of the invention: The host of the invention is a microbial host that does not naturally produce significant amounts of xylitol from other * * · 30 common carbon sources during fermentation. than D-xylose or D-xylose or their polymers, oligomers or mixtures, but treated to do so by the methods of the invention. The term "significant amount" means an amount suitable for isolation of xylitol in pure form or an amount that can be readily measured by analytical methods normally used for the analysis of carbohydrates in microbial fermentation broth.

Arabitol Dehydrogenase: There are two types of D-Arabic-5 tolide dehydrogenase, D-xylulose (EC 1.1.11) and D-ribulose. D-ribulose-forming dehydrogenases occur in wild-type yeasts and fungi. D-xylulose-forming arabitolide hydrogenases are known only from bacteria. Unless otherwise indicated, herein is meant D-xylulose forming arabitol dehydrogenase and is referred to herein as arabitol dehydrogenase.

Oxidative branch of the pentose-phosphate pathway: The term "oxidative branch of the pentose-phosphate pathway" is intended to include that portion of the pentose-phosphate pathway which catalyzes oxidation reactions such as D-glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phospho- D-gluconate dehydrogenase (EC-1.1.1.44) catalysed reactions and utilizes hexose substrates to form pentose phosphate. The "non-oxidative" portion of the pentose-phosphate pathway (which also catalyzes the net formation of ribose from D-glucose) is characterized by non-oxidizing isomers such as ribose-5-phosphate isomerase, D-ribulose-5-phosphate Reactions catalyzed by -3-epimerase and transaldo-25. See H.R. Mahler and E.H. *: ··· Cordes, Biological Chemistry, Harper & Row, Publishers,

New York 1966, pp. 448 - 454.

»· .., Functional derivative: The" functional derivative "of a protein or nucleic acid is a molecule.

30 chemically or biochemically derived. that is to say, of such a protein or nucleic acid, * <> »» · ... with residual (either functional or structural) ♦ »biological activity which is characteristic of nature; : to its protein or nucleic acid. The term "functional derivative" is intended to encompass "fragments", "variants", "analogs" or "chemical derivatives" of the n 108300 molecules which retain the desired activity of the native molecule.

A molecule is referred to herein as a "chemical derivative" of another molecule when it contains additional chemical moieties that are not normally part of that molecule. Such groups may improve the solubility, absorption, biological half-life of the molecule, etc. These groups may reduce, reduce or mitigate the potential toxicity of the molecule, etc. Such groups that are capable of transmitting such effects are described in Remington's Pharmaceutical Sciences 1980. well known in the art.

Fragment: By "fragment" of a molecule, such as a protein or nucleic acid, is meant a portion of the native amino acid or genetic nucleotide sequence, and in particular the functional derivatives of the invention.

Variant or analog: By "variant" or "analog" of a protein or nucleic acid is meant a molecule that is substantially similar in structure and biological activity to a molecule encoded by a functional allele.

:. *. II. Construction of metabolic pathways for xylitol biosynthesis • · · ·

According to the invention, the natural metabolic pathways of the microbial host are manipulated by reducing or eliminating the utilization of carbon 30 for purposes other than xylitol production. All hosts of the invention produce xylitol in a single fermentation step. In one embodiment, the hosts of the invention may have sufficient xylitol dehydrogenase activity *. # 35 (EC 1.1.1.9) for the production of xylitol. However, as described below, 121083300, in hosts where overproduction of xylitol dehydrogenase activity is desired, the host cell can be transformed with recombinant xylitol dehydrogenase encoding genes.

In the practice of the invention, all hosts of the invention are characterized by the ability to synthesize xylitol from carbon sources which are not its structural relatives, such as D-glucose, and not only D-xylose and / or D-xylulose. The hosts of the invention also have the ability to secrete synthesized xylitol into the medium.

In the exemplary and preferred embodiments, hosts according to the invention are characterized more precisely by one or two pathways. The first is the pathway by which arabitol is an intermediate in the formation of xylitol, and the second is the pathway by which xylulose-5-phosphate is directed to the formation of xylitol via dephosphorylation and reduction reactions. Thus, the hosts of the invention are characterized by at least one of the following gene alterations: (1) a gene encoding a protein having D-arabitol dehydrogenase activity (EC 1.1.1.11) constituting? D-xylulose has been cloned into a host and thus obtained Conversion of D-arabitol to D-xylulose (id 25 in pathway I) and / or: (2) the innate gene encoding the host transketolase activity has been inactivated (id in pathway II ·).

In addition, the hosts may undergo various other transformations to improve the xylitol production capacity of such hosts. In addition, as described in (1) and (2): * · *: The hosts may be further modified by cloning a gene encoding a protein having xylitol dehydrogenase activity into the 9 (3) host: 35 (EC 1.1.1.9); 13 1083 (3) the innate gene encoding the host D-xylulokinase (EC 2.7.1.17) has been inactivated; (4) the host has a cloned gene encoding a protein having D-glucose-6-phosphate dehydrogenase activity (EC 1.1.1.49); (4) the host has a cloned gene encoding a protein having 6-phospho-D-gluconate dehydrogenase activity (EC 1.1.1.44); (5) the host has a cloned gene encoding 10 proteins with D-ribucose-5-phosphate-3-epimerase activity (EC 5.1.3.1).

In one preferred embodiment, the hosts of the invention have more than one of the above described gene alterations. In one preferred embodiment, for example, carbon flows "directly" from D-arabitol (i.e.

in one step) to D-xylulose and from D-xylulose to "directly" xylitol. In such an embodiment, the host of the invention is thus modified by cloning a gene encoding a protein having D-xylulose-forming D-arabitol dehydrogenase activity and a gene encoding xylitol dehydrogenase (EC 1.1.1.9).

: · It should be noted that while in many embodiments; In the course of the process, the hosts according to the invention synthesize internally: 25 D-arabitol from other carbon sources, could be D-arabitite. also add externally directly to the substrate.

In another preferred embodiment, the lithol biosynthetic pathway for xy- * · ♦ does not include arabitol as intermediates. Instead, the carbon flow is from D-xylulose-5 »tn» 30 phosphate to D-xylulose and further to xylitol. When D-glucose is used as the «r ··· * carbon source, the carbon flow would be through the oxidative portion of the pentose phosphate pathway, from D-glucose to D-glucose-6-phosphate, to 6-phospho-D- * *« gluconate and further to D- ribulose-5-phosphate. The D-ribulose-5-phosphate would be further epimerized to D-xylulose-10830000 si-5-phosphate, dephosphorylated to D-xylulose and reduced to xylitol. Thus, suitable hosts for use in the present invention would include a host in which the 5 (ai) host has a cloned gene encoding a protein having D-glucose-6-phosphate dehydrogenase activity (EC 1.1.1.49) or an innate host gene. is expressed and / or the (a2) host has a cloned gene encoding 10 proteins with 6-phospho-D-gluconate dehydrogenase activity (EC 1.1.1.44) or the innate host gene is overexpressed and / or the (a3) host has a cloned gene encoding a protein having D-ribulose-5-phosphate-3-epimerase-15 activity or an innate host gene is overexpressed and / or (a4) the host has a cloned gene encoding a protein having xylitol dehydrogenase activity ( EC 1.1.1.9), or the innate gene of the host is overexpressed; (B) the native transketolase gene is inactivated and / or (c) the gene encoding the host's native xylulokinase (EC it'j 2.7.1.17) is inactivated.

•; "The dephosphorylation step (conversion of D-xylulose phosphate to D-xylulose) is the only step catalyzed by an enzyme that has not been characterized in its pure form. the corresponding step • · (from D-ribulose-5-phosphate to D-ribulose) in the natural pathway of D-arabitol formation of the osmophilic yeast has • «. · * (· 30 previously shown to be non-specific and able to catalyze xylulose-5-phosphate dephosphorylation: T; [Ingram, J.M. and Wood, W.A., J. Bacteriol. 89 (1986) 1186-1194] The mutation of the transketolase and the overexpression of these two dehydrogenases in the oxidative pentose phosphate pathway have a dual function. first, the 108300 ίο improves the potency of Road I by increasing the amount of ribulose-5-phosphate in the cell and thus the production of xylitol by arabitol. ttömän ksylu lodge-5-5-phosphate ylikerääntyminen.

Therefore, methods utilizing the naturally occurring pathway leading to the formation of D-arabitol from various carbon sources and extending this pathway by two additional reactions to convert D-arabitol to xylitol) are not always feasible within the scope of the invention. Other pathways leading to xylitol as a final metabolite and not containing D-arabitol as an intermediate can be constructed. Thus, the pathway leading to xylitol from D-ribulose-5-phosphate can be accomplished through one or more multiple reaction chains. D-ribulose-5-phosphate can be efficiently converted to D-xylulose-5-phosphate by D-ribulose-5-phosphate-3-epimerase, and if the conversion of D-xylulose-5-phosphate is further prevented by mutation in the transketolase gene, D-xylulose-5-phosphate accumulated is dephosphorylated by the same non-specific phosphatase as D-ribulose-5-phosphate [Ingram, JM et al., J. Bacteriol. 89, 1186-1194 (1965)] and reduces it to xylitol by means of xylitol dehydrogenase. In addition, realization of this pathway may require the inactivation of the D-xylulokinase gene in order to:. to reduce the energy loss caused by the following unnecessary silyl: D-xylulose-5-phosphate - * D-xylulose - »D-xy- • ♦ · lulose-5-phosphate. Additional genetic alteration - Importation and (over) expression of the D-ribuloki, nase gene (EC 2.7.1.47) - could minimize the simultaneous production of * * D-arabitol in such strains by binding to D-ribulose produced by nonspecific phosphatase. D-ribulose is converted back to D-ribulose-5-phosphate and further to D-xylulose-5-phosphate.

16 1 0 8 300 III. Construction of hosts according to the invention

The method of genetic engineering of hosts according to the invention is facilitated by isolating and sequencing a partially pure protein encoding the enzyme of interest or cloning said protein sequences capable of encoding said protein by polymerase chain reaction methods. As used herein, the term "genetic sequences" refers to a nucleic acid molecule (preferably DNA). Genetic sequences that have the ability to encode proteins are derived from a variety of sources. These sources include genomic DNA, cDNA, synthetic DNA, and combinations thereof. A preferred source of genomic DNA is the cDNA-15 library prepared from yeast mRNA grown under conditions known to induce expression of the desired mRNA or protein.

The cDNA of the invention does not contain naturally occurring introns if the cDNA is prepared using a val-20 mRNA as a template. The genomic DNA of the invention may or may not contain naturally occurring introns. In addition, such genomic DNA may be prepared linked to the 5 'promoter region and / or the 3' end transcription termination region of the gene sequences. In addition: 25 such genomic DNA can be produced associated with genetic sequences encoding the 5 'end of the mRNA, and / or genetic sequences encoding the 5' untranslated region of the mRNA. encode the 3 'untranslated region. If the host cell is able to recognize transcriptional and / or translational signals associated with the expression of mRNA and protein, the 5 'and / or 3' end of the native gene and / or the 5 'and / or 3' ends of the mRNA may be transcribed. The untranslated regions of the 'and / or 3' ends are retained and used to regulate transcription and translation.

Genomic DNA can be isolated and purified from any naturally occurring host cell, particularly a fungal host, of the desired protein (see, for example, Guide to Molecular Cloning Techniques, eds. SL Berger et al., Academic Press). 1987).

Preferably, the mRNA preparation used is enriched for the mRNA encoding the desired protein, either naturally, by isolation from high-yielding cells of the protein, or in vitro, methods commonly used to enrich mRNAs for specific sequences, such as sucrose. -gradient centrifugation, or both.

For cloning into a vector, such suitable DNA preparations (either genomic DNA or cDNA) are cleaved or digested, respectively, and ligated into appropriate vectors (either containing genomic DNA or cDNA) to form a recombinant gene library.

The DNA sequence encoding the desired protein or functional derivatives thereof may be inserted into a DNA-20 vector by conventional techniques, including blunting or stepwise ligation, restriction enzyme cleavage to provide appropriate ends, filling of adhesive ends as needed; ; treatment with alkaline phosphatase to prevent unwanted incorporation and use of appropriate ligases. ligation. Such processing methods are disclosed in Maniatis, T., et al., Molecular Cloning (A Lab- &quot; Oratory Manual), 2 p., Cold Spring Harbor Laboratory 1988, and are well known in the art.

* * 30 Libraries containing the coding sequences for the desired gene can be screened and the desired gene sequence identified: * · *: by any means capable of specifically selecting the coding sequence for such a gene or protein, e.g. hybridization with one or more appropriate nucleic acid probes 18 108300 containing a sequence specific for the DNA corresponding to this protein; or b) hybridization selection translation analysis in which the natural mRNA hybridizing to that clone is read in vitro and further characterized. or c) if the self-cloned genetic sequences are capable of expressing the mRNA, by an immunoprecipitation of the translated protein product produced by the host containing the clone.

Oligonucleotide-10 probes specific for a particular protein, which can be used to identify clones associated with that protein, can be designed by knowing the amino acid sequence of the protein or the nucleic acid sequence of DNA encoding such protein or a related protein. Alternatively, antibodies to purified forms of the protein can be generated and used to detect the presence of unique protein determinants from transformants expressing the desired cloned protein. The amino acid group sequence contained in the peptide is herein designated using either the commonly used three-letter designations or single-letter designations. A list of these three-letter and one-letter entries can be found in textbooks such as Lehninger, A., Biochemistry, Worth] Publishers, New York, NY 1970. When the amino acid sequence: 25 is shown horizontally, the amino terminus is intended to be · ·: unless otherwise stated * · ·. Similarly, unless otherwise stated or revealed

• (I

context, the nucleic acid sequence is shown with the 5 'end to the left.

'* 30 Because the genetic code is degenerate, more than one codon can be used to encode something; * j *. a specific amino acid (Watson, J.D. Molecular

Biology of the Gene, p.3, W. A: Benjamin, Inc., Menlo Park, CA 1977). Peptide fragments are analyzed for such; 35 to identify oligonucleotides with the lowest degree of degeneracy. This is done by identifying sequences containing only one codon-encoded amino acids.

Although the amino acid sequence may be randomly coded for by a single oligonucleotide sequence, the amino acid sequence can often be encoded by any one of a plurality of corresponding oligonucleotides. Importantly, while all members of this set contain oligonucleotide sequences that have the ability to encode the same peptide fragment, and thus possibly contain the same oligonucleotide sequence as the gene encoding that peptide fragment, only one member of this set contains an identical nucleotide sequence. with the exon sequence encoding the gene. Because this member is present in a pool and is capable of hybridizing with DNA even in the presence of a number of other members, it is possible to use an unfractionated set of oligonucleotides in the same way as one oligonucleotide would be used to clone a gene encoding a peptide.

Using the genetic code, one or more different ol, gonucleotides can be identified from the amino acid sequence, each of which would encode the desired protein. The probability that any given oligo-,; nucleotide actually constitutes the actual coding sequence for the protein, it may be appreciated. club abnormal base pairing ratios and frequency, • »9 ·. ···, which actually uses a particular codon •« 4 (to encode a particular amino acid) in eukaryotic cells. Using "codon usage rules", one oligonucleotide sequence or a set of oligonucleotide • · ··· * sequences containing a theoretical “probability: * · *: simpler” nucleotide sequence having the ability to encode protein sequences is identified.

A suitable oligonucleotide or set of oligonucleotides * '* 35 that has the ability to encode (or is complementary to such an oligonucleotide or set of oligonucleotides) a fragment of a particular gene can be synthesized by methods well known in the art (see Synthesis and Application of DNA and RNA, edited by Narang SA, Aca-5 demic Press, San Diego, CA 1987) and used as a probe for the identification and isolation of a corresponding clone by methods known in the art. Methods for hybridizing nucleic acids and identifying clones are described by Maniatis, T. et al. Molecular 10 Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY 1982; and Hames, B. D. et al. Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC 1985. Those members of the above-described gene library found to be capable of said 15-hour hybridization are then analyzed for the length and nature of the coding sequences they contain.

To facilitate detection of the desired coding DNA sequence, the DNA probe described above is labeled with a detectable moiety. Such a detectable group may be any material having a detectable physical or chemical property. Such materials are highly advanced in the field of nucleic acid hybridization, and generally close to: 25 any of the ft i •. · Useful in such procedures. the marker can be applied to the present invention. Particularly useful are radioactive markers such as 32P, 3H, 14C, 35S, 125I, etc. Any radioactive, radioactive marker which provides a sufficient signal and has a sufficient half-life can be used. If the? 1 ... * oligonucleotide is single-stranded, it can be radiolabeled using kinase reactions. Alternatively, polynucleotides are useful as nucleic acid hybridization probes also labeled with a non-radioactive tag such as biotin, enzyme or fluorescent moiety.

In summary, therefore, elucidation of a portion of a protein sequence provides the opportunity to identify a theoretical "most likely" DNA sequence, or a set of such sequences, capable of encoding such a peptide. By constructing an oligonucleotide complementary to this theoretical sequence (or by constructing a set of oligonucleotides complementary to a set of "most probable" oligonucleotides), a DNA molecule (or set of DNA molecules) is obtained which has the ability to act as a probe (s).

Following one alternative way of cloning the gene, a library is prepared using an expression vector, cloning DNA, or more preferably, a cDNA prepared from a cell capable of expressing the protein in question into the expression vector. The library is then searched for members expressing the desired protein, for example, by examining the library for protein antibodies.

Thus, the methods discussed above are capable of identifying gene sequences capable of encoding a protein or biologically active or antigenic fragments thereof. In order to further characterize such gene sequences and to produce recombinant protein, it is desirable to express the proteins encoded by these sequences. Such expression identifies those clones expressing proteins that have the characteristic features of the desired protein. Such characteristic features may include V, e.g. ability to specifically bind antibody, ability to · · · V * induce production of antibodies capable of binding native non-recombinant protein, ability to give the cell ent-; ·. enzymatic activity, which is a property of the protein in question, and the ability to introduce a non-enzymatic (but specific) function into the recipient cell.

The DNA sequence may be truncated by methods known in the art to isolate a desired gene from a chromosomal region containing more information than is necessary to utilize this gene in the hosts of the invention. For example, a restriction cleavage may be utilized to cleave a full-length sequence at a desired site. Alternatively or additionally, nucleases cleaving manually at the 3 'end of the DNA molecule may be used to cleave a particular sequence into a truncated form, followed by identification and purification by gel electrophoresis and DNA sequencing of the desired length. Such nucleases include, for example, exonuclease III15 and Bal31. Other nucleases are well known in the art.

In practicing the invention, the osmophilic yeast Z. rouxii may be used as a model. Z. rouxii is suitable for food production as it is traditionally used in Japan for making soy sauce. These 20 yeasts are described, for example, in The Yeasts, A Taxonomic Study, ed. Kreger-van Rij, Elsevier Science Publishers BV, Amsterdam 3984, which describes this yeast on pages 462-465. Other D-arabitol producing yeasts such as Candida polymorpha, Torulopsis candida, Can-, 25 did a tropical, Pichia farinosa, Torulaspora hansenii -, etc., as well as D-arabitol producing fungi such as: * · *: Dendryphiella salina or Schizophyllum commune can also be used as host organisms for the purposes of this invention.

·>, 30 D-arabitol Oxidizing enzymes (D-xylulose) (EC 1.1.1.11) are known to occur in bacteria but not in yeast or fungi. In the context of the present invention ,, / Klebsiella terrigena has a D-arabitol dehydrogenase gene: ·. (Gene leading to D-xylulose formation) preferred: 35 source because it is a non-pathogenic terrestrial bacterium and has •> 23 108300 high inducible D-arabitol dehydrogenase activity. The Klebsiella terrigena strain used in the examples, Phpl, was provided by K. Haahtela, University of Helsinki. Isolation of this strain is described by Haahtela et al. in public-5 suspi Help. Env. Microbiol. 45: 563-570 (1983). Cloning of the D-Arabic tolidehydrogenase gene can be conveniently accomplished by constructing a gene library comprising K. terrigena chromosomal DNA in a suitable vector, for example the well-known and commercially available plasmid 10 pUC19. This library transforms one of the many E. coli strains that have the ability to utilize D-xylulose but not D-arabitol as the sole carbon source. The E. coli strain SCSI available from Stratagene is one example of suitable strains. The transformants are then inoculated onto 15 media containing D-arabitol as the sole carbon source and clones capable of growing on this medium are isolated. The D-arabitol dehydrogenase coding region of K. terrigena can be conveniently isolated as a 1.38 kb (kilobase) BclI-Clal fragment and linked to the appropriate promoter and transcription termination sequences. The ADCJ promoter and transcriptional terminator of Saccharomyces cerevisiae are examples of transcriptional regulatory elements which are suitable for the purposes of this invention when used as a host organism: 25 yeast Z. rouxii. The HDCI sequence is available from GenBankis: .f.

Although the majority of yeasts and fungi carry the gene encoding xylitol dehydrogenase (EC 1.1.1.9), overexpression of the mentioned -.... gene is typically necessary for the implementation of the present invention. The XYL2 gene of Pichia stipititis encoding xylitol dehydrogenase (EC *. * * 1.1.1.9) can be conveniently cloned by polymerase chain reaction technology using published data on the XYL2 gene: ·. nucleotide sequence [Kötter et al. Curr. Genet. 18 24 1 0 8 3 0 0 493-500 (1990)]. This gene may be introduced into other yeast species without modification and may be expressed under the control of its own promoter, or the promoter may be changed to another strong yeast promoter.

Genetically stable transformants can be constructed using vector systems or transformation systems, whereby the desired DNA is integrated into the host chromosome. Such integration may take place in a Novo cell, or may be promoted by the transit) formation of a vector functionally located on the host chromosome, for example, a phage, retroviral vectors, transposons or other DNA elements that promote integration of DNA sequences into chromosomes.

Genes encoding D-arabitol dehydrogenase and xylitol dehydrogenase (EC 1.1.1.9), under the control of suitable promoters, can be combined into a single plasmid construct and introduced into host cells of the D-arabitol-producing organism by transformation. The nature of the plasmid vector 20 depends on the host organism. Thus, Z. rouxii ol. . In a preferred embodiment of the present invention, DNA containing vectors of the cryptic plasmid pSR1 are used [Ushio, K. et al. J. Ferment. Technol. 66: 481-488 (1988)]. For other yeasts or fungi for which *. ; Suitable autonomously replicating plasmids are not known, • ·: integration of genes encoding xylitol dehydrogenase (EC 1.1.1.9) and • t1 · · / • D-arabitol dehydrogenase into the host chromosome can be used. Targeting integration into the host ribosome DNA locus (coding for ribosome RNA. ". 30 DNA) is a preferred method for the integration and high-i

• I I

* · * * For recovery; such alignment can be accomplished by • inserting enough recombinant DNA sequences into the recombinant structure to control integration into this locus. Gene markers used in the transformation of D-arabitol producing microorganisms are preferably dominant markers that confer resistance to various antibiotics such as gentamicin or phleomycin, or heavy metals such as copper, etc. The selectable marker can be either. directly expressed DNA sequences or introduced into the same cell by cotransformation.

In addition to introducing genes encoding D-arabitol dehydrogenase and xylitol dehydrogenase (EC 1.1.1.9), other genetic modifications can be used to construct novel xylitol-producing strains. Thus, genes encoding oxidative pentose phosphate pathway enzymes can be over-expressed to improve the synthesis rate of D-arabitol precursor, D-ribulose-5-phosphate. The catabolic enzyme encoding the catabolism of transketolase-pentulose-5-phosphates or pentose-5-phosphates can also be inactivated by conventional mutagenesis or gene cleavage techniques, resulting in an increased accumulation of five-carbon sugar phosphates. Inactivation of the D-xylulokinase gene may, in turn, increase xylitol yield by eliminating D-xylulose loss caused by phosphorylation. The combination of inactivating transketolase simulation with over-expression of D-ribulose-5-epimerase can be used to create a different type of xylitol production pathway that does not use D-Arabic as an intermediate. In order to express the desired protein and / or its active: derivatives, recognizable transcriptional and translational signals from the appropriate host are necessary. The cloned coding sequences obtained by the methods described above, preferably in double-stranded form, can be operably linked to sequences that direct transcriptional expression from the thymium in the expression vector, and introduced into a host cell, either prokaryotic or eukaryote, recombinant protein or. : 35 to produce its functional derivative. According to which strand of the coding sequence is operably linked to sequences directing transcriptional expression, it is also possible to express the antisense RNA or a functional derivative thereof.

Expression of the protein in different hosts can lead to various post-translational modifications that can alter the properties of the protein. The present invention preferably comprises the expression of a protein or a functional derivative thereof in eukaryotic cells, and in particular in yeast.

A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide if it contains expression control sequences containing transcriptional regulatory information, and such sequences are "operably linked" to the nucleotide sequence encoding the polypeptide.

Functional linkage is a linkage in which a sequence is linked to a regulatory sequence (s) in such a way that expression of the sequence is under the control or control of a regulatory sequence. Two DNA sequences (such as the coding sequence and the promoter region linked to the 5 'end of the coding sequence) are said to be operably linked if the induction of the promoter function results in the coding for the desired protein; The nature of the linkage between the 25 mRNAs for transcription and the nature of the linkage between the two DNA sequences (1) does not alter the reading frame of the coding sequence, (2) interferes with the ability of expression control sequences to direct protein expression ..: (antisense RNA) ) and (3) does not interfere with the ability of the DNA template to be copied. Thus, the promoter region would now be operably linked to the DNA sequence if the promoter had the ability to effect the transcription of that DNA sequence.

The exact regulatory regions required for gene expression; The nature of the species may vary between species or cell types, but should generally include, as appropriate, 5'-end untranslated and 5'-end untranslated (unencoded) sequences involved in transcription and translational initiation, respectively. Such as a "TATA box" (TATA sequence), a cap ("Hood") junction region, a CAAT sequence, and the like. In particular, such 5'-end uncovered control sequences include a region that contains a promoter for operably linked gene transcription. Such transcriptional control sequences may also, if desired, include enhancer sequences or Aktiator sequences above.

Protein expression in eukaryotic hosts such as yeast requires the use of regulatory regions, preferably yeast regulatory systems, in such hosts. Many different transcriptional and translational regulatory sequences can be used depending on the nature of the host. These regulatory signals, in their natural state, are preferably associated with a particular gene that has the capacity for strong expression in the host cell.

In eukaryotes in which transcription is not translational coupled, such regulatory regions may or may not provide the initiating methionine codon (AUG), depending on whether the cloned sequence contains such methionine. Such regions generally contain a sufficient promoter region to direct the initiation of RNA synthesis in the host cell. Promoters derived from yeast genes encoding a translationally capable mRNA product are preferred, and particularly strong pro · ·, ···, 30 motors can be used provided they also function as promoters in the host cell. yeast promoters include the GAL1 gene engine, promoters for glycolytic genes such as the phosphoglycerol kinase (PGK) gene promoter, or the constant ligand alcohol dehydrogenase (RDCl) promoter 28 1083300 [Ammerer, G., Met. Enzymol., 101C, 192-201 (1983); Aho, FEBS Lett.

As is well known, translation of eukaryotic mRNA is initiated by a codon encoding first-order methionine. For this reason, it is advantageous to ensure that the linkage region between the eukaryotic promoter and the DNA sequence encoding the desired protein or functional derivative does not contain intermediate codons having the ability to encode methionine. The presence of such codons results in either fusion protein formation (if the AUG codon is in the same reading frame as the protein coding DNA sequence) or mutation in the reading frame (if the AUG codon is not in the same reading frame as the protein coding sequence).

Transcriptional initiation signals that allow repression or activation can be selected so that expression of functionally linked genes can be regulated. Of interest are regulatory signals that are temperature sensitive, so that expression can be suppressed or initiated by temperature change, or subject to chemical regulation, for example, by a metabolic product. Translational signals are not required when expressing antisense RNA sequences.

Untranslated and / or untranslated regions of the 3 'coding sequence of the desired protein may be obtained, if desired, by the cloning methods described above. The non-transcriptional region of the 3 'end can be maintained for its transcription termination control elements; The 3 'end translates • ·, ···. The 30 nucleotide region may be retained for its translational termination *. ** regulatory elements or for the control elements of polyadenylation in the eukaryotic cells.

t * I

When the native expression control sequence signals do not:. function satisfactorily in the host cell, may be replaced; 35 host cell sequences.

The vectors of this invention may further comprise other functionally linked regulatory elements, such as DNA elements, that confer antibiotic resistance, or origin of replication to maintain the vector in one or more host cells.

In another preferred embodiment, for example in the case of Z. roux, the introduced sequence is inserted into a plasmid vector which has the ability to replicate autonomously in the recipient host. Any of the many different vectors can be used for this purpose.

Important factors in selecting a particular plasmid or viral vector include: how easily the recipient cells containing the vector can be identified and selected from the non-vector recipient cells, and whether it is desirable to cause the vector to "shuttle" between host cells of different species.

Preferred yeast plasmids depend on the host. Z.

For 20 roux, preferred vectors based on native cryptic plasmids pSR1 [Toh, E. et al., J. Bacteriol. 151: 1389-1390 (1982)], pSB1, · pSB2, pSB3 or pSB4 [Toh, E. et al., J. Gen. Microbiol.

130: 2527-2534 (1984)]. Plasmid pSRT303D [Jearnpipatkul, * * 25 A. et al., Mol. Gen. Genet. 206 (1988) 88-84] is an example of a * * * deletion plasmid useful in Zygosaccharomyces yeast.

Once the vector and DNA sequence containing one or more constructs have been prepared for expression, the DNA. ···, 30 constructs or constructs are introduced into a suitable host cell by any of a number of suitable means, including * · · V * transformation. The recipient cells are then grown in a selective medium that is selective for the growth of the cells containing the vector, The expression of one I f <;, 35 or more cloned sequences results in the production of a ha1308300 protein or a fragment thereof. This expression may be continuous or regulated in transformed cells, for example, through induction of expression.

In order to construct hosts of the invention that have been modified so that they can no longer express a particular gene product, directed mutagenesis can be performed using methods known in the art, such as gene cleavage [Rothstein, R. S., Meth. 10 Enzymol. 101C 202-2111 (1983)].

IV. Xylitol production

When used as hosts of the invention, recombinant arabitol yeasts, preferably osmophilic yeasts, can be grown in a medium with high osmotic pressure, for example 10-60%, preferably 25%, in D-glucose. ("Normal" medium usually contains only 2% to 3% glucose.) High osmotic pressure medium induces D-arabitol formation in 20 wild-type strains of osmophilic yeasts such as Z. roux. The medium of the recombinant and (wild type) reference strains is analyzed for the presence of xylitol by methods known in the art at different times of culture. Under culturing conditions that were not optimized to achieve maximal D-arabitol yield, the experimental strain Z. rouxii ATCC13356 [pSRT (AX) - • «;. · · 9] produced and secreted both xylitol and ϊ Γ: D -arabitolia. Only D-arabitol was found in the growth medium of the reference strain. The xylitol yield in the first experiments (see Table 4 in Example 4) was about 7.7 g / L. 30 After 48 hours of rearing.

The xylitol can be purified from the culture medium of the hosts according to the invention by any of the methods known in the art. For example, U.S. Patent 5,081,026, which is incorporated herein by reference, describes a xylitol separator, 4; 35 from yeast cultures. Thus, after the fermentation step, xylitol 311083300 can be purified from the culture medium using chromatographic steps similar to those described in U.S. Patent 5,081,026, followed by crystallization.

Having now generally been described, the invention will be more readily understood by reference to certain specific examples, which are included herein for purposes of illustration only and are not intended to be limiting unless otherwise stated.

10 Examples

Example 1

Cloning of the bacterial strain D-arabitoide dehydrogenase gene

Klebsiella terrigena Phpl [obtained from K. Haahtela, 15 University of Helsinki, see Haahtela et al., Appl. Env. Microbiol. 45, 563-570 (1983)] was grown in 1 L of LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl) at 30 ° C overnight. Bacterial cells (about 5 g) were harvested by centrifugation, washed once in TE (10 20 mmol / L tris-HCl, 1 mmol / L EDTA, pH 7.5) and resuspended in TE which contained 1% sodium dodecyl sulfate and 200 pg / ml proteinase K. The suspension was incubated for 30 minutes at 37 ° C and extracted once with an equal volume of phenol and twice with, 25 chloroform. Sodium acetate solution (3 mol / l, / 1/10 volume) and ethanol (3 times volume) and • · ·: · ί; the precipitated nucleic acids were recovered by centrifugation with a V 'stick and redissolved in 5 ml TE. RNA was removed by centrifugation of the solution in NaCl solution (25 mL, 30 L mol / L) overnight at 30,000 rpm in a "1 Beck- · * '*: man ΤΪ50.2 rotor. j. # K. terrigena chromosomal DNA had an average fragment size of more than 50,000 base pairs (bp). The DNA was then digested with restriction endonuclease Sau3A (Boehringer): 35 in buffer with 5 units (IU) of enzyme / DNA. / 32108300 mg until the average size of the DNA fragments was reduced to about 5-10 kb (as determined by agarose gel electrophoresis) The digestion product was fractionated by electrophoresis through a 0.6% agarose gel (20 x 10 x 0.6 cm) in TBE buffer (0.09 mol / L tris-boric acid, 1 mmol / L EDTAra, pH 8.3) overnight at 5 V / cm, the well was cut into an agarose plate at a position approximately equal to a fragment size of 5 kb, dialyzed. vopal along the recess and continued with electrophoresis-10, until virtually all DNA fragments larger than 5 kb were adsorbed to the membrane. Plasmid DNA of pUC19 (purchased from Pharmacia) was digested with restriction endonuclease BamHI and bacterial alkaline phosphatase using manufacturer's buffer 15 and reaction conditions. The linear form of pUC19 was purified by preparative gel electrophoresis using the membrane electroelution method described above and ligated with the 5-15 kb fraction of Klebsiella terrigena chromosomal DNA. The ligation mixture was transformed into 20 competent E. coli SCSI cells (purchased from Stratagene) to be ampicillin resistant. About 10,000 recombinant clones were obtained in this experiment. Combined cells from Tramform plates were plated on minimal medium plates containing D-arabitol (1%) as the sole carbon source. Fr. , After incubation for 2 days at 37 ° C, / / a few colonies were obtained. Plasmid DNA was isolated from two • · ·: ·; «* (The fastest growing) clone and were used to re-transform E. coli strain HB101 which is unable to catabolize D-arabitol but is capable of using D-xylulose.

*. * ·· 30 All transformants were positive for utilization of D-arabitol in plates containing McConkey agar (Difco) and • · · ^ .j. ^ D-arabitol, while all control 9 * * tail clones (same strain). transformed with pUC19) were; negative. Restriction analysis showed that two isolated 35 clones contained identical plasmids. One of 331083300 of the two isolated plasmids (designated pARL2) was used for further characterization. A restriction map of a (approximately) 9.5 kb K. terrlgena DNA fragment containing the D-arabitol dehydrogenase gene cloned into pARL2 is shown in Figure 1.

Example 2

Expression of the bacterial D-arabitol dehydrogenase gene in yeast

From the original 9.5 kb K. terrlgena DNA clone of the D-arabitol dehydrogenase gene si-10 was subcloned at approximately 1.8 kb using the conventional recombinant DNA techniques and was found to contain the D-arabitol dehydrogenase gene. A plasmid containing this DNA fragment in vector pUC19 (pADH, where 15 ADH stands for D-arabitol dehydrogenase) was isolated in E.

coli strain JM110, digested with restriction endonucleases Bell and Clal and isolated a 1.38 kb DNA fragment by preparative agarose gel electrophoresis. This DNA fragment was treated with the Klenow-20 fragment of DNA polymerase I in the presence of all four deoxynucleotide triphosphates and ligated with the yeast expression vector pAAH5 [Ammerer, Meth. Enzymol. 101: 192-203 (1983)], which had been digested with fllIII and treated with the Klenow fragment (Fig. 2). The resulting expression plasmid, 25 di, pYARD, is an E. coli - Saccharomyces cerevlslae

I 4 I

. · / Vector containing the origin of replication from the bacterium (E. coli) and the ampicillin resistance V 'gene, the origin of replication from the yeast (S. cerevisiae) 2 pm DNA and the yeast ( S. cerevlslae) 30 LE for selection of the [72 gene in yeast. The expression cassette contains the yeast alcohol dehydrogenase I (ADCI) y promoter and the (ADCI) transcription terminator K.

terrigena next to the D-arabitol dehydrogenase gene and; functionally connected to it.

34 108300

Saccharomyces cerevisiae strain GRF18 (ΜΑΤα, leu2-3, 112, his3-II, 15) and S. cerevisiae strain DBY746 (ATCC 44773; ΜΑΤα, leu2-3,112 his3-R11 ura3-52 trpl-289) were each used with the same results. as a host for transforming with the D-arabitol dehydrogenase expression vector pYARD described above. Transformation was performed by the conventional lithium chloride procedure [Ito et al. , Bacteriol. 153: 163-160 (1983)] using the pYARD LEU2 marker for the selection of transformants. Transformants 10 were grown in liquid culture in minimal medium [0.67% Yeast Nitrogen Base ("Difco"), 2% D-glucose, 100 mg / L histidine and tryptophan] overnight with shaking at 30 ° C. Cells were harvested by centrifugation, suspended in a very small volume of potassium phosphate buffer (0.1 mol / L, pH 6.8) containing 1 mM NAD *, and disrupted with 0.5 mm glass beads in a "Biospec products" for six minutes on ice. D-arabitol dehydrogenase activity was measured as described above. The cell extract of K. terrigena grown with D-arabitol was used as a positive control in these experiments and DBY746 transformed with pAAH5 as a negative control. The results presented in Table 2 show that the D-Arabic tolidehydrogenase gene isolated from K. terrigena is efficiently expressed in yeast.

·. ; 25. *. * Table 2 • · ·

Microbial strain D-arabitol dehydrogenase activity • t · * · '' (as optional units) '** 30 K. terrigena Phpl 3.5 DBY746 [pYARD] 1.0. • I ·. DBY746 [pAAH5] 0.00 * «t 35 1 08 300

Example 3

Construction of Yeast Vectors for Overexpression of the Xylitol Dehydrogenase and D-Arabitol Dehydrogenase Genes A known nucleotide sequence of the yeast (Pichia stipitis) xylitol dehydrogenase coding gene, XYL2, was used. Genet. 18 (1990) 493-500] for the synthesis of oligonucleotides by polymerase chain reaction for the cloning of this gene. Two oligonucleotides designed for CGAATTCTAGACCACCCTAAGTCGTCCC (5'-oligonucleotide) [SEQ ID No: 1:] and TTCAAGAATTCAAGAAACTCA-CGTGATGC (3'-oligonucleotide) [SEQ ID No: 2:] are available. and 3 'ends. The 51 oligonucleotide anneals at positions 1 to 24 in XYL2 and the 3 'oligonucleotide anneals at positions 1531 to 1560 following Kötter et al.,

Curr. Genet. 18, 493-500 (1990).

Pichia stipitis CBS6054 (Centraalbureau voor Schimmelcultures, Oosterstraat 1, PO Box 273, 3780 hp)

20 Baarn, The Netherlands) was grown overnight in YEPD medium (1% yeast extract, 2% peptone, 2% D-glucose), cells were harvested by centrifugation, washed once with 1 mM sorbitol solution containing 1 mM EDTA and pH 7.5 was resuspended in the same solution and digested with Lyticase (Sigma). cleavage

«(I

// was controlled by following a 1: 100 l% SDS- · · · ·· ♦; optical density of the cell suspension diluted with the solution at 600 nm. Splitting was stopped when this value dropped to about one-seventh of the original. Sferoplas - "* * 30 The distillate suspension was then washed four times with sorbitol solution (1 mol / L). The spheroplasts were disrupted in 1% SDS solution and treated with a solution containing 200 pg / ml proteinase K for 30 minutes at 37 ° C. After one ·; 'phenol extraction and two chloroform extractions, nucleic acids; 35 were mixed with ethanol and redissolved in a small volume of 36108300 TE buffer. Chromosomal DNA integrity was checked by agarose gel electrophoresis. The average size of DNA fragments was greater than 50 kb. PCR was performed using Taq DNA polymerase (Boehringer) in manufacturer's buffer. The heat cycle was 93 ° C for 30 s; 55 ° C, 30 s; 72 ° C, 60 s. The PCR product was extracted with chloroform, mixed with ethanol and digested with EcoRI and XbaI under standard conditions. After agarose gel purification, the DNA fragment was cloned with Xba I and Eco RI into digested pUC18 [plasmid pUC (XYL2)]. Subsequent restriction analysis confirmed that the restriction map of the cloned fragment corresponds to the nucleotide sequence of the P. stipitis XYL2 gene.

Yeast plasmids for overexpression of D-arabitol dehydrogenase and xy-15 lithol dehydrogenase were constructed as illustrated in Figures 3 and 4. To alter the restriction sites flanking the plasmid pYARD D-arabitol dehydrogenase digestion cassette, the entire cassette was removed by digestion with BamHI and cloned with BamHI into cut-in pUC18. The resulting plasmid pUC (YARD) was digested with SalI and EcoRI and the only 2.0 kb DNA fragment was isolated by preparative gel electrophoresis. This fragment was ligated with the 1.6 kb HindIII-EcoRI DNA fragment isolated from plasmid pUC (XYL2) and E. coli-yeast spliced / vector vector pJDB207 [Beggs, JD , Nature 275 (1978) 104-6: 109] with a 6.6 kb fragment. Plasmid pJDB (AX) -16 · · · * · * 'was isolated after transformation of E. coli with the ligation mixture described above. This plasmid has the ability to replicate in both E. coli and Saccharomyces cerea. In S. cerevisiae it directs the strong synthesis of both D-arabitol dehydrogenase and xylitol dehydrogenase. Plasmid pSRT (AX) -9 was synthesized by ligation of the 4.7 kb SalI fragment from pJDB (AX) -9 and pSRT303D [Jearnpipatkul et al., Mol.

37 108300

Gen. Genet. 206: 88-84 (1987)], a linear form obtained by partial hydrolysis with SalI.

Example 4

Construction of Xylitol Secreting Yeast Strains Zygosaccharomyces rouxii ATCC 13356 was transformed with plasmid pSRT (AX) -9 by slightly modifying the method previously described [Ushio, K. et al. J. Ferment. Technol. 66: 481-488 (1988)]. Briefly, Z. rouxii cells were grown overnight in YEPD medium (resulting in 10 cultures of 400 nm optical density at 3-5), harvested by low speed centrifugation, washed twice with 1 mol / L sorbitol and 1 mmol / l EDTA, pH 7.5, were resuspended in 1/5 of the original culture volume of the same solution containing 1% 2-mercaptoethanol and digested at room temperature with Lyticase (Sigma). . The digestion was monitored by diluting an appropriate dose of cell suspension with 1% SDS and measuring the optical density of the diluted sample at 20,600 nm. When this value dropped to one-seventh of the original, digestion was stopped by cooling the suspension on ice and washing (centrifugation for 10 minutes at 0 ° C at 1000 rpm) with sorbitol solution until no odor of mercaptoethanol was detectable. The sphero plastics were washed once with a cold solution containing * · / 0.3 mol / l calcium chloride and 1 mol / l sorbitol, and resuspended in the same solution used. V 1 about 1/4 of the original volume of the culture. 200 μ1 portions of this suspension were mixed with 10 to 20 pg *: **? 30 plasmid DNA and incubated for 40 minutes at? 0 ° C. 0.8 ml of ice-cold 50% PEG 6000 solution containing 0.3 mol / L total sodium chloride was added to the spheroplast suspension and incubated in the cold for 1 hour. The spheroplasts were concentrated by centrifugation for 10 min: 35 rpm in a new 4000 min'1 table centrifuge, resuspended in 2 no YEPDs containing 1 mol / l sorbitol and left to regenerate overnight at room temperature. Regenerated cells were inoculated into YEPD plates containing 50-100 pg / ml gentamicin and incubated for 4-6 days at 30 ° C.

Transformants were grown in liquid YEPD medium, cell extracts were prepared as described for S. cerevisiae (Example 2), and 10 D-arabitol dehydrogenase and xylitol dehydrogenase activities were measured. The results of these measurements are compared with those of other organisms in Table 3. They show that both genes were efficiently expressed in Z. rouxi.

Z. rouxii ATCC 13356 cells transformed with pSRT (AX) -9 were grown for two days in 50 ml YEPD medium containing 50 pg / ml gentamicin, harvested by centrifugation and inoculated with 100 ml YEPD containing 25 µg / ml gentamicin. by weight glucose and 50 pg / ml 20 gentamicin. This culture was grown in a 1000 ml flask with a rotary shaker (200 min -1) at 30 ° C. In another experiment (Experiment 2), the same medium without yeast extract (low phosphate medium) was used. The culture broth xylitol content was analyzed by conventional HPLC. . By gas chromatography. The results are shown in Table 4.

“Trans- · ·! Grown on the same medium (without gentamicin)! no xylitol was observed in the medium of unformed Z. rouxl. 1 «♦ · · •« «« «« «· • ♦ · •« «39 1 08300

Table 3 D-arabitol dehydrogenase and xylitol dehydrogenase activities in various organisms and strains 5 Organism and strain Plasmid (1) D-arabitol dehydrogenase Xylitol dehydrogenase (IU / mg protein) (IU / g protein)

Klebsiella terri- No plasmid 0.48 ND. (2) Gena Phpl 10 s. Cerevlsiae No plasmid 0.00 <0.01 DBY746 S. eerevlslae pJDB (AX) - 3.0 <0.01 DBY746 S. eerevlslae pJDB (AX) - 16 1.9 1.2 15 DBY746 Z. rouxii No Plasmid 0.00 <0.02 ATCC 13356 Z. rouxi 1 pSRT (AX) -9 1.3 0.7 ATCC 13356 20 (1) Plasmids are designed to express the following enzymes: pJDB (YARD) - D-arabitol dehydrogenase pJDB (AX) -16 - D-arabitol dehydrogenase and xylitol dehydrogenase pSRT (AX) -9 - D-arabitol dehydrogenase and xylitol dehydrogenase 25 (★ 1) ND - not determined

Table 4

Xylitol production (g / l) of Z. rouxii ATCC 13356 containing plasmid pSRT (AX) -9. . 30 Cultivation time (h) Experiment 1 Experiment 2 «· · '· 1 · Medium Medium no • φ ♦ · · ί · 1: yeast extract yeast extract ··· ______ 0 0.0 0.0 *: ··: 35 48 7.7 0.5 144 7.5 6.1 • «· · · 2 • · 1 Using a similar approach, strains that produce xylitol from other: · .. 40 carbon sources can be constructed. For example, Candida tropicalis is capable of converting 40-108300 n-alkanes to D-arabitol in good yield [Hattori, K. and Suzuki, T., Agric. Biol. Chem. 38: 1875-1881 (1974)]. The 4.7 kb Sal I fragment from pJDB (AX) -9 can be inserted into pCU15 [Haas, L. et al., J. Bacteriol. 172: 4571-4577 (1990)] and uses C. tropicalis strain SU-2 (ura3) for transformation. Alternatively, the same expression cassette can be used to transform the prototropic C. tropicalis using a plasmid vector with a dominant selectin marker as the driver.

Example 5

Construction of an Integrating Dominant Selection Vector for Expression of Arabic Tolidehydrogenase and Xylitolide Hydrogenase and Transformation of Toxulopsis Candi-15 dan

Chromosomal DNA was isolated from T. candida (ATCC 20214) by a procedure similar to that used for S. cerevisiae. However, preparation of T. Candida spheroplasts required a high concentration of Lyticase (Sigma), about 50,000 IU / 10 g cells, and a long incubation time, from a few hours to overnight incubation at room temperature, to achieve efficient lysis of the cell walls. The buffer used to prepare the spheroplasts was EDTA buffer (1 mmol / L, pH 8) containing. . 25 mol / l sorbitol and 1% mercaptoethanol. The spheroplasts were washed three times with the same buffer (without mercaptoethanol) and disrupted in 15 ml of a 1% SDS solution. Two phenol extractions were performed immediately after cell lysis and DNA was precipitated by adding twice the volume *! **! 30 ethanol and centrifugation (5 min at rpm; 10,000 rpm). The DNA was washed twice with 70% ethanol and dried under reduced pressure. The DNA was dissolved in 5 ml of Tris. -HCl buffer (10 mmol / L) containing 1 mmol / L EDTA was added at 35 µg / ml to 10 pg / ml RNAase A and incubated for 1 hour at 41103 ° C to 370C. DNA integrity was confirmed by agarose gel electrophoresis using unmodified lambda DNA as a molecular weight reference.

200 µg of T. Candida chromosomal DNA was digested with 5 µl of III and EcoRI, the digestion product was placed in a 6 cm wide well on a 0.7% preparative agarose gel (8 x 15 x 0.8 cm) and electrophoretic separation was performed. A 1 cm wide strip was cut off from the gel and transferred to a positively charged female 10 membrane (Boehringer 1209 299). For the transferred fragments, a 10 kb Zygosaccharomyces bailii rDNA fragment which had been excised with SalI from pAT68 [K. Sugihara et al., Agric. Biol. Chem. 50 (6) 1503-151 (1986)]. The probe was labeled and the spots were welded using a DIG DNA Labeling and Detection Kit (Boehringer 1093 657) according to the manufacturer's instructions. Three hybridization bands corresponding to DNA fragments of about 4.5, 2.7 and 1.1 kb were observed. Using the transferred fragments as refe-20, the band corresponding to the largest (4.5 kb) hybridizing DNA fragment was excised from the preparative gel, electroeluted DNA and ligated with pUC19 digested with Eco RI and Hind III.

The ligation mixture was used to transform E. coli.

. The transformed bacteria were inoculated into a charged female membrane (Bio-Rad 162-0164) on the agar surface of a plate containing ampicillin-1 * * LB medium. When was V; incubated for 24 hours at 37 ° C, the membrane was lifted and the bacterial colonies were in situ, according to the manufacturer's instructions: *: ··: 30. No replica bowls were needed as E.

coli passes through this type of membrane, and after lifting the filter * · · .1. ^, the agar surface shows a trace of each bacterial colony. The membrane was probed with the same Z. bailii «· rDNA fragment probe and using the same DIG-de-35 activators as above. A set of 42108300 positive clones were identified (about 2-5% of all clones). Plasmid minimulates prepared from 8 hybridization positive clones and 4 hybridization negative clones were subjected to restriction enzyme analysis using a mixture of BcoRI, HindIII and EcoRV. All hybridization-positive clones yielded an identical restriction fragment pattern (characteristic fragments of 0.55 and 1.5 kb), whereas the same patterns of hybridization-negative clones were all different. Plasmid DNA of one hybridization positive clone was isolated on a preparative scale and designated pTCrDNA. It was concluded that the cloned fragment was the 27th Candida rDNA fragment because 1) it strongly hybridized with Z. shale rDNA and 2) it was cloned from a partially enriched T. Candida chromosomal DNA cleavage product of large (yeast is known to have about 100 rDNA copies). A partial restriction map of the cloned DNA fragment is shown in Figure 5.

A plasmid in which the T. Candida rDNA fragment binds to the dominant selection marker was constructed as follows. Plasmid pYT332 (Gatignol, A. et al., Gene 91: 35-41 (1990)) was digested with HindIII and Kpn1, the 1.3 kb DNA fragment was isolated by agarose gel electrophoresis, and ligated with EcoRI: and Kpnl digested with the 4.5 kb Hindlll-EcoRl fragment from I * pUC19 (Fig. 6). The ligation mixture was transformed with E. coli and a clone containing plasmid pTC (PHLE) was identified by restriction analysis.

30 pTC (PHLE) was partially digested with SalI and the plasmid in linear ** 'was purified on an agarose gel. by electrophoresis. It was ligated with a 5 kb Sal I fragment isolated from pJDB (AX) -16 (pre-2). Plasmid pTC (AX) was identified from the clones which; 35 was obtained by transformation of this ligation mixture into E. coli 43 108300 (Figure 6). This plasmid contains the following functional elements: (a) a bacterial phleomycin resistance gene under the control of the yeast promoter and transcription terminator, which allows direct selection of the yeast cells transformed with this plasmid; b) a fragment of T. Candida rDNA which provides for homologous recombination of the target with the T. Candida chromosome and enhances the efficiency of transformation; C) an expression cassette for the arabitol dehydrogenase and xylitol dehydrogenase genes, which provides for the synthesis of the enzyme of the two arabitol xylitol pathways. Example 6 Transformation of T. Candida and Analysis of Xylitol Production Plasmid pTC (AX) was used to transform ATCC 20214 of Torulopsis Candida. T. candida was grown for 36 hours in YEPD medium containing 10% glucose. Cells were harvested by centrifugation (10 min at 20 rpm 2000 min -1 at 4 ° C) and washed three times with sterile sorbitol solution (1 mol / L). The cell pellet was suspended in an equal volume of cold. ·, Sorbitol solution (1 mol / L), mixed with 200 µl portions of pUT (AX) DNA (20-100 µg),

* I

,, 25 ice ice-cold electroporation cuvettes (electrode gap • ««. * / 2 mm) and electroporated in Invitrogen ElectroPorator ···: with the following settings: voltage 1,800 V, capacitance V: dance 50 yF, parallel resistance 150 Ω. Cells were transferred to 2 ml YEPD containing 1 mol / l sorbitol and incubated overnight at 30 ° C with a shaker. Transformed cells were harvested by low speed centrifugation and inoculated into plates containing? · * YEPD medium adjusted to pH 7.5 containing 30 µg /

* I

ml of phleomycin. Plates were incubated for 7-10 days at 35 ° C. Most of the yeast colonies developed during this period were background mutants, as an equal number of colonies also appeared on control plates (containing treated cells similarly but without DNA addition). To distinguish the true transformants from the spontaneous mutants, chromosomal DNA was isolated from 72 single yeast colonies by a procedure similar to that described above to isolate T. canadida chromosomal DNA. 10 µg of each of these DNA preparations was digested with a mixture of EcoRI and BamHI. The cleavage products were separated on 1% agarose gels and then transferred to a positively charged nylon film as described in Example 5. The transferred fragments were probed using DNA from plasmid pADH (Example 2) containing arabitol-15 dehydrogenase and pUC sequences but not yeast-derived DNA fragments (to avoid hybridization between homologous yeast sequences). The probe was labeled and the spots were developed using DIG DNA Labeling and Detection Kit (Boehringer 1093 20 657). Only one clone was found [Γ. Candida :: pTC (AX)], whose hybridization signal is consistent with the structure of the transforming plasmid (three bands in the range of 2-3 kb), indicating very low transformation efficiency. For one other clone, a positive hybridization signal of ... was observed; the only hybridizing zone, ·. However, the location of (~ 5-7 kb) indicated that only one pTC (AX) fragment was integrated into the yeast chromosome, or some * t »'rearrangement occurred at the site of integration. We assume that plasmid 30 is integrated into the T. Candida chromosome. This assumption • · · · l '' is consistent with the finding that after culturing and cloning on a non-selective medium, all T. Candida :: pTC (AX) clones retain fleomy-, cin-resistant The phenotype.

1088300 T. Candida :: pTC (AX) transformant was grown for 36 hours in YEPD medium containing 10% glucose and arabitol dehydrogenase and xylitol dehydrogenase activities were measured as described in Example 4. The results are shown in Table 5.

Table 5

Arabitol Dehydrogenase and Xylitol Dehydrogenase Activities in Wild-type T. Candida and 10 pTC (AX) Transformed Strain (IU / mg Protein) T. Candida Arabitol-Xylitol Dehydrogenase Dehydrogenase 15 Wild-type 0.02 0.03

Transformant 0.03 0.05

Xylitol dehydrogenase activity was not significantly elevated above that of endogenous T. Candida xylitol dehydrogenase. The activity of plasmid-encoded arabitol dehydrogenase (EC 1.1.1.11) was difficult to distinguish from the activity of endogenous synonymous but different (ribulose-forming, EC 1.1.1) arabitol dehydrogenase. The only reliable conclusion from the 25 experiments was that the expression of both enzymes was <. ; the level was much lower than in Z. rouxi. Attempts to increase the plasmid copy number and expression level of the integrated plasmid two • · · dehydrogenases by culturing T. candi · · * :: da :: pTC (AX) in media with increased phleomycin levels were not successful. .

'*' T. Candida:: pTC (AX) xylitol production was tested after being grown with YEPD containing 10% glucose for 5-7 days. In three separate experiments

<· I

, ···, the transformant produced 1.1, 1.6 and 0.9 g / L xylitol, whereas no wild-type T. Candida showed 46 x 08300 HPLC at all in xylitol. The limit of detection of the analytical method used is less than 0,1 g / l. Therefore, it is possible to conclude that the plasmid is indeed a dominant factor in the xylitol production of T. Candida :: pTC (AX).

5 Example 7

Transformation of Candida polymorpha by arabitol dehydrogenase and xylitol dehydrogenase genes

To introduce the arabitol dehydrogenase and xylitol dehydrogenase genes into Candida polymorph, a mutant with a change in the orotidine phosphate decarboxylase gene (hereinafter also designated URA3) was isolated using Boeken et al. a modification of the method [Boeke, J.

D. et al., Mol. Gen. Genet. 1974, 345-346 (1984)]. C. polymorpha strain ATCC 20213 was grown for 24 hours in 15 YEPDs, cells were harvested by centrifugation (2000 min, 10 min), washed twice with water, and suspended in three volumes of sterile sodium phosphate buffer (0.1 mol / l, pH 7.0). Ethyl methanesulfonate was added to a concentration of 1% and the cells were incubated for 2 to 20 hours at room temperature. The reaction was terminated by transferring the cells into sodium thiosulfate solution (0.1 mol / L) and washing three times with sterile water. The mutagenized cells were transferred to 0.5 L of YEPD and cultured at 30 ° C with shaking for two days. The yeast was harvested by centrifugation, washed twice with water, and inoculated with 1 Isa of medium containing 0.7% Yeast Ni · ·: Trogen Base (Difco), 2% glucose (SC medium), and incubated with a rotary shaker. 24 hours at 30 ° C.

• «<

To the culture was added 1 mg of nystatin and incubation was continued for 4 hours. Nystatin-treated cells were separated from the medium by centrifugation, washed twice with water, and then transferred to 1 Isaan SC medium containing 50 mg / l uracil. Cells were incubated on a rotary shaker for 5 days and then inoculated onto SC medium containing 50 mg / l uracil and 1 g / l fluorouric acid. After incubation of 47,108,300 plates for two weeks at 30 ° C, about 400 colonies resistant to fluoro-erotic acid were obtained and all tested for uracil auxotrophy. Five uracil dependent colonies were isolated.

However, three clones grew on a uracil-free medium, albeit at a reduced rate. Two clones (designated C. polymorpha U-2 and C. polymorpha U-5) with a clear uracil-dependent phenotype were used for transformation experiments.

Cloning of the C. polymorpha URA3 gene was accomplished by a conventional strategy. The chromosomal DNA was isolated by the method described in Example 5. The DNA was partially digested with Sau3A and fractionated on agarose gels. A few fractions were recovered and their molar mass distribution was checked by analytical gel electrophoresis.

Fractions in the size range of 5 to 10 kb were used for cloning experiments. The vector used to construct the library, pYEpl3 [Broach et al., Gene 8: 121-123 (1979)], contains the S. cerevisiae LEY2 gene, a 2 μη origin of replication, and a single restriction site for BamHI. The vector was cut with BamHI, purified by agarose gel electrophoresis and dephosphorylated with bacterial alkaline phosphatase. A number of independent vector preparations and ligation conditions were tested, varying the ratio of vector to insert and reaction state .... in small-scale experiments to obtain the highest amount of transformant * · ·, *. * and the highest percentage of recombinant clones * (i) by restriction analysis of mini-preparations from random clones. Large-scale ligations were made using optimized conditions and transformed with E. coli strain XL1-BLUE. The constructed library contained about 15,000 primary transformants, of which about 90% contained the insert.

(··. (Yeast strain DBY746 (MATalpha, leu2-3,112, his3-Al, 35 trpl-289, ura3-52)) was transformed with the C. polymorpha gene-48 108300 libraries. Each library set was transformed separately using approximately 20 pg plasmid. DNA and inoculating the transformation mixture into one plate supplemented with uracil, tryptophan and 5 histidine (i.e., using only leucine assay). (uracil-free plates). Control replicate cultures on histidine-histidine plates (uracil-free plates). however, the number of ids was 2-3 times lower than that of the isolated URA3 clones.

The six uracil independent clones were rescued with plasmids in E. coli, isolated at a prepara-20 level, and re-transformed with DBY746 leucine prototrophic. 10-20 random colonies from each transformation mixture were then checked for uracil dependence. Five of the six rescued plasmids transformed DBY746 into the Leu * Ura * 25 phenotype. Restriction of said five plasmids. analysis yielded very similar restriction · · ♦. *. * patterns. These models were too complex to produce an unambiguous restriction map of the cloned fragment. However, it was found that ffindlll digestion 30 generates in all clones the fragment that covers most of the DNA insert (about 4.5 kb). From one isolate, C. polymorpha URA3 This fragment was purified by agarose gel electrophoresis and partially subcloned in HindIII to 108300 49 pJDB (ΑΧ). The structure of the plasmid pCPU (AX) isolated as a result of this cloning experiment is shown in Figure 7A.

Attempts were made to transform both C. polymorpha U-2 and C. polymorpha U-5 using the conditions described in Example 6 and electroporation. After electroporation-treated cells were shaken overnight in YEPD containing 1 mol / L sorbitol, they were inoculated onto SC medium plates. After incubation for 10 days at 30 ° C, about 100 colonies were observed in a plate containing C. polymorpha U-2 transformed with pCPU (AX), while in a control plate (similarly but without added DNA). cells treated with this strain) had only 14 colonies. There was no significant effect of C. polymorpha U-5 in the transformation assay compared to 15 non-DNA control samples. Three random colonies from a C. polymorpha U-2 transformation plate were first seeded with a fresh SC medium and the inoculated YEPD media containing 15% glucose was inoculated with these stain cultures. Ver-20 lobe culture was inoculated with C. polymorpha U-2. After incubation with a rotary shaker (200 min-1) at 30 ° C for 10 days, the medium was analyzed by HPLC. The results of this:: experiment are shown in Table 6.

25 Table 6. : C. · · · · transformed with C. polymorpha U-2 transformed with pCPU (AX); > · <Xylitol production

• ♦ I

·· ♦ <· ♦ «* · · * Strain Xylitol (mg / ml) 30

• - -M - „I - - - .... I

4 C. polymorpha U-2 0.0 • · · ...: C. polymorpha U-2:: pCPU (AX) -1 0.5 C. polymorpha U-2:: pCPU (AX) -2 0, 0. ·· *. C. polymorpha U-2: pCPU (AX) -3 2.1 50 108300

Significant variations in xylitol production levels were observed between the different clones. This may be due to the integration of plasmid pCPU (AX) into different loci on the C. polymorpha chromosome. The strain C. polymorpha U-2 :: pCPU (AX) -2 5 is probably a revertant and not a true transformant. However, these experiments clearly demonstrated that the arabitol-xylitol pathway can also be introduced into C. polymorpha.

Example 8

Cloning and overexpression of enzymes of the oxidative pentose phosphate pathway in osmophilic yeast

The first enzyme in the oxidative pentose phosphate pathway, D-glucose-6-phosphate dehydrogenase, is encoded by the S. cerevisiae ZWF1 gene. The sequence of this gene is known [Nogae, T. and Johnston, M., Gene 96 (1990) 161-19; Thomas, D. et al., The EMBO J. 10 (1991) 547-553]. The gene, including the entire coding region, 600 bp 5'-end coding region and 450 bp 3'-end coding region, has been cloned by PCR using the following 20 oligonucleotides: CAGGCCGTCGACAAGGATCTCGTCTC (5 'end ol-gonucleotide) [SEQ ID No: : 3:] and AATTAGTCGACCGTTAATTGGGGC-CACTGAGGC (3'-end oligonucleotide) [SEQ ID No: 4:]. The 5'-end oligonucleotide pairs at positions 982-1007 and the 3'-end oligonucleotide at positions 3523-3555 by numbering 25 D-glucose-6-phosphate dehydrogenase in Nogae, T.

. ·. ; and Johnston, M., Gene 96, 161-19 (1990). The chromosomal DNA was isolated from S. cerevislae strain GRF18 as described in Example 3. The PCR parameters were the same as in Example 3. The amplified DNA fragment containing the ZWF1 gene was digested with SalI and cloned into pUC19 digested with the same restriction enzyme, whereby it was digested. .. · resulted in plasmid pUC (ZWF). The identity of the cloned gene; * f. The restriction was checked by restriction analysis.

Another enzyme of the pentose phosphate pathway, 6-phosphoglu-35 conic acid dehydrogenase, is encoded by the gnd gene in E. coli.

1088300 The sequence of this gene is known (Nasoff, M.S. et al., Gene 27: 253-264 (1984)). To clone the gnd gene from E. coli, HB101 chromosomal DNA was isolated from E. coli by a method similar to that used to isolate Klebsiella terrlgena DNA (Example 1). Oligonucleotides for PCR amplification of the gnd gene t GCGAAGCTTAAAAATGTCCAAGCAACAGATCGGCG [SEQ ID No: 5:] and GCGAAGCTTAGATTAATCCAGCCATTCGGTATGG [SEQ ID No: 6:] were designed to amplify the coding region only, and to bring forward the codon fp1. The 5'-end oligonucleotide pairs at positions 56-78 and the 3'-end oligonucleotide at positions 1442-1468 as numbered 6-phosphogluconic acid dehydrogenase as described in Nasof, M. S. et al., Gene 27, 253-264 (1984). The amplified DNA fragment was digested with Hin dIII and ligated with the vector pUC19 digested with fin III. Ten apparently identical clones of the resulting plasmid pUC (gnd) were pooled. This was done to avoid potential problems associated with sequence errors that may have occurred during PCR amplification. The coding region for the gnd gene was inserted into the S. cerevisiae ADCI promoter and. .V: Transferring a 1.4 kb HindIII fragment of pUC (gnd) to the transcription terminator into the expression vector pAAH5 [Ammerer, Meth. Enzymol. 101 (1983). 192-203]. The resultant plasmid pAAH (gnd) was transformed with a few independent clones of S. cerevi-1 ·, * siae GRF18 using the lithium chloride procedure [Ito ♦ · t- 'et al., J. Bacteriol. 153: 163-168 (1983)] and 6-phospho-D-gluconate dehydrogenase activity was measured for 30 transformants. The activity of 6-phospho-D-gluconate dehydrogenase in all transformants was multiplied compared to the untransformed host, indicating that bacterial 6-phospho-D-gluconate dehydrogenase * 35 can be effectively expressed in yeast. The pAAH (gnd) clone, which yielded the highest activity in yeast 52108300, was selected for further constructs. The cloning of the ZWF1 and gnd genes as well as the construction of plasmid pAAH (gnd) are illustrated in Figures 8 and 8a.

A plasmid pSRT (ZG) was constructed for the simultaneous overexpression of both D-glucose-6-phosphate dehydrogenase and 6-phospho-D-gluconate dehydrogenase genes in an osmophilic yeast host. Figure 9 illustrates a method for constructing this plasmid. Briefly, a 6-phospho-D-gluconate tidehydrogenase expression cassette derived from 10 plasmids pAAH (gnd) was transferred to a 3.1 kb BamHI DNA fragment in BamHI digested pUC19. The resulting plasmid pUC (ADHgnd) was digested with SacI and Xjbal and the 3.1 kb DNA fragment was purified by aga-15 rose gel electrophoresis. This fragment was simultaneously ligated with two other DNA fragments, the 2.5 kb fragment of pUC (ZWF), obtained by digestion with SacI and partially PstI, and the plasmid digested with PstI and Xjbal. pSRT (AX) -9 (Example 3) 20 vector fragment. The structure of the resulting plasmid pSRT (ZG) was confirmed by restriction analysis. After Z. rouxii ATCC 13356 was transformed with pSRT (ZG) (as described in Example 4), D-glucose-6-phosphate dehydrogenase and 6-phospho-D-,, gluconate dehydrogenase activity were measured in the transformed strain. The activity of both enzymes in the transformed strain was approximately 20-fold higher than that of the untransformed reference strain (Table 4 ··· · t '· * 7). Similar methods can be used to induce the * * * * 30 genes encoded by the oxidative moieties of the two pentose phosphate pathways in other yeast species. However, since there are no vectors that can be maintained as extra-chromosomal plasmids in most yeast species other than; · Saccharomyces or Zygosaccharorayces, integrative * 35 transformation is the only useful method 53 5383 for such hosts. A preferred type of integrative vectors are vectors that are directed to integrate into the ribosome DNA locus, since these types of vectors provide high copy number integration and thus higher expression levels (Lopes, T.S. et al., Gene 79, 199-206 (1989)).

Table 7 D-glucose-6-phosphate dehydrogenase and 6-phospho-D-10 gluconate activity in pSRT (ZG) transformed Z. rouxii ATCC 13356 and untransformed reference strain [pmol / (min * mg protein)]

Yeast strain D-glucose-6-β-β-β-gluconate-dehydrogenase dehydrogenase Z. rouxii 0.33 0.45 (untransformed) Z. rouxii 7.3 7.7 20 [pSRT (ZG)]

Example 9

Construction of transketolase mutants in yeast

Transketolase mutants are most readily available by the yeast directed gene cleavage method, although conventional chemical mutagenesis methods are also useful. The homologous transketolase gene ♦ · · * · * * cloned from the yeast where the mutation is desired is generally necessary for the use of the gene cut-off * · ** '* 30, although sometimes it may also be used well a heterologous clone from a closely related species. The sequence of the S. cerevisiae transketolase gene is known (Fletcher, T.S. and Kwee, I.

L., EMBL DNA sequence library, ID: SCTRANSK, search number; '· 35 M63302). Using this sequence, the S. cere-54 108300 visiae transketolase gene was cloned by PCR. Oligonucleotides AGCTCTAGAAATGACTCAATTCACTGACATTGATAAGCTAGCCG [SEQ ID No: 7:] and GGAGAATTCAGCTTGTCACCCTTATAGAATGCAATGGTCTTTTG [SEQ ID No: 8:] and DNA derived from S. cerevisiae were transfected with the transgenic DNA as described above. The 5'-end oligonucleotide pairs at positions 269-304 and the 3'-end oligonucleotide at positions 2271-2305 as the transketolase T.S. 10 as described by Fletcher and I. L. Kween (EMBL DNA sequence library, ID: SCTRANSK, accession number M63302). The fragment was digested with Xbalc and EcoRI and cloned into pUC19 digested with the same enzymes. Restriction analysis of the resulting plasmid pUC (TKT) confirmed the identity of the cloned DNA fragment. This plasmid was digested with BglII and Clalt and the largest DNA fragment was purified by agarose gel electrophoresis. This fragment was ligated with the following two fragments isolated from plasmid pUT332 (Gatignol, A. et al., Gene 91: 35-41 (1990)): a ClaI-PstI fragment containing the URA3 gene and a 3 'part of the bleomycin resistance gene, and a BamHI-Pst1 DNA fragment containing the yeast TEF1 (transcription extension gene 1) promoter and a 5 'portion of the bleomycin resistance gene encoding sequence. After transformation of E. coli with the above ligation mixture and t <i. 25 plasmid clone restriction analysis, * ll *. * Plasmid pTKT (BLURA) was isolated. This plasmid contains the coding sequence for the S. cerevis transketolase gene, in which the 90 bp fragment between the ClaI and BglII sites has been replaced by a pUT332 fragment containing two S. cerevi-30 the selectable marker, the URA3 gene * and the TEF1 promoter of the bleomycin resistance gene, and CYC1. under the control of a transcription terminator. This plasmid was transformed from S. cerevisiae strain DBY746 (ATCC 44773; *; * measured by his3Al leu2-3,112 ura3-52 trpl-289) to f leomycin? * 35 (10 pg / ml phleomycin in YEPD medium)? 1083300 by the Thium chloride method [I to et al., J. Bacteriol. 153: 163-168 (1983)]. The transformants were tested for uracil prototrophy and ability to grow with D-xylulose. Five URA3 clones, three of which were impaired in growth with D-xylulose-5, were grown in 100 ml cultures, prepared with cellular extracts by shaking with glass beads, and the transketolase activity in the crude extract was measured. The assay was performed in glycylglycine buffer (0.1 mol / L pH 7.6) containing 3 mmol / l magnesium chloride, 0.1 mmol / l thia-10 amine pyrophosphate, 0.25 mmol / l NADH and 0.2 mg / ml bovine serum albumin. Immediately before measurement, 3 μΐ of a solution containing 0.5 mol / L D-xylulose-5-phosphate and 0.5 mol / L ribose-5-phosphate was added to 1 ml of the above buffer followed by 7.5 triose-15 phosphate isomerase; and 1.5 Ia-glycerol phosphate dehydrogenase (both from Sigma). The reaction was initiated by addition of a suitable dose of crude extract and monitored by recording the decrease in optical density at 340 nm. Cell extract of strain DBY746 was used as a ver-20 sample. Transketolase activity was readily measurable from the crude extracts obtained from DBY746 and two transformants growing slowly on D-xylulose; the concentration was about 0.25 IU / mg protein. The retarded growth of trans-,? Formant transketolase activity with three D-xyluloses was our method.

• »I

/] · Below the whitening threshold (at least 20 times lower than the wild type). D-glucose-6-phospho-· * V phate dehydrogenase and 6-phospho-D-gluconate dehydrogenase activities were also measured during the preparation of cell extracts to check for r * 30 inactivation of the enzyme. . The activities of these two enzymes were • 4 1 .1. very similar in all six positions. Therefore, it was concluded that the three clones that grew poorly on the D; xylulose contained a mutation in the transketolase gene; 35 where. The growth of carriers containing the truncated transketolase gene was also somewhat retarded on a synthetic medium lacking the aromatic amino acids phenylalanine and tyrosine, although the effect was less than the effect of this mutation on the utilization of D-xylulose.

Transketolase genes from other yeast species can be conveniently cloned by complementing the transketolase mutation in the S. cerevisiae strains described above. Cloning can preferably be accomplished by constructing a gene library of a non-Saccharomyces yeast strain in an appropriate vector (e.g., the well-known plasmid YEpl3), transforming this library with a S. cerevisiae strain having a transketolase mutation (e.g., mutants generated by gene cleavage described above) transformants in which growth with D-xylulose has been restored. Plasmid DNA can be recovered from such D-xylulose positive transformants and used to transform the same acceptor strain. All strains derived from this transformation-20 should be able to grow well with D-xylulose. Transformants can be measured. transketolase activity and its significant reappearance may serve as an indication of the identity of the cloned gene. Additional and final detection can be obtained by: • venting short sections of the cloned DNA and discovering the fragments of the S. cerevisiae transketolase gene by hologic sequencing or demonstrating the hybridization of the cloned DNA • · · • ♦ t * fragment. With an authentic transketolase clone from S. cerevisiae. Alternatively, the cloning procedure may be based on DNA hybridization as the primary method for selecting clones containing the transketolase gene from a gene library other than Saccharomyces yeast. The S. cerevisiae transketolase gene fragment can be used in a probe colony hybridization assay and the 35 clones that give the strongest hybridization signal “108300 b /

Iin, can be further analyzed by partial sequencing.

Regardless of the method used to clone the transketolase gene 5 from the selected yeast species, the subsequent steps for mutation in the transketolase gene in this yeast are the same. The cloned DNA fragment should be characterized by constructing a partial restriction map and preferably locating the coding region of the transketolase gene. The DNA fragment, which may serve as a selectable marker in the selected yeast, is then inserted into a DNA fragment containing the transketolase gene at least a few hundred base pairs from either end of the fragment. For example, a cassette containing the bacterial phleomycin gene under the control of a strong yeast promoter, such as the pUT332 cassette described above, could be used as a dominant selection marker in many yeast species. It is imperative that the insertion of the DNA fragment containing the selectable marker is accomplished by the way that the inserted DNA either cleaves the coding region of the transketolase gene or, preferably, the inserted DNA fragment. ·; the ment replaces the coding region (part). Such a DNA construct can then be used to cut the chromosomal copy of the transketolase gene in a selected yeast by a method similar to that described above to obtain transketolase mimicry in S. cerevisiae. Any suitable transformation method may be used. Preferred methods include protoplast transformation and electroporation. Selection of <* 1 30 clones containing the truncated transketolase gene can be accomplished by

* M

* ... · similar to those described above for S. cerevisiae.

Analysis of the structure of the transketolase chromosome region *, ··, by Southern hybridization can also be used as an alternative method or in addition to other methods.

»· *« 1 * · 58 1 0 8 300

Example 10 Cloning of D-ribulokinase gene

The preferred method of cloning the D-ribulokinase gene is similar to the method described in Example 1 for cloning D-arabito-5 lidehydrogenase. It is known that the D-ribulokinase gene is present in some bacteria, such as E. coli or Klebsiella aerogenes, as part of the ribitol utilization misoperon [Loviny, T. et al., Biochem. J. 230: 579-585 (1985)]. It is also known that E. coli B strains do not contain this operon and are therefore unable to utilize ribitol as a carbon source. Thus, E. coli strain B (such as common laboratory strains HB101 and JM103 or strains that can be transformed with high efficiency such as Stratagene strain SCSI or XL1-Blue) can be transformed with a ribitol-utilizing bacterial gene library constructed in any suitable vector, preferably in pUC19. Non-pathogenic bacterial species such as Klebsiella terrigena are the preferred source of organisms used to isolate the D-ribulokinase gene. E. coli transformants having the ability to grow on minimal medium containing ribitol as the sole carbon source can then be selected. Plasmid DNA can be isolated from such ribitol-positive clones and used to transform E. coli strain B. For everyone like this. 25 transformants * · ·. *. * Should be able to grow with the only carbon source • · · · ;; · ribitol. A restriction map of the · · · '· * * cloned insert can then be constructed. Using this map, various deletion derivatives of the original clone can be prepared and analyzed for the survival of the ribitol operon by the aforementioned activity test. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · Finally, the (partial) nucleotide sequence of the D-ribu-: 35 loci gene can be determined and used to remove the coding region of this gene, either by using naturally occurring restriction sites or by known PCR techniques to introduce such sites into the molecule. The D-ribulokinase gene can be expressed in other hosts, preferably yeast, by a method that includes conventional procedures such as fusing the coding region of the D-ribulokinase gene to a suitable promoter and transcriptional terminator, transferring the expression cassette to a selected host, and, finally, the efficacy of D-ribulokinase i Imen sea buckthorn.

Example 11 Cloning -15 and Overexpression of D-Ribulose-5-Phosphate-3-Epimerase Gene

A method for isolating homogeneous D-ribulose-5-phosphate-3-epimerase from baker's yeast (industrial Sacc-haromyces cerevisiae) is known [Williamson, W.

T. et al., Meth. Enzymol. 9, 605-608 (1966)]. This enzyme can be isolated and its N-terminal sequence as well as a portion of the internal amino acid sequence determined by commonly known methods. Then the obtained parts-; Such amino acid sequences can be used in reverse translation using a known procedure for oligonucleotide. ; 25, which can then be used as primers in the polymerase chain reaction. PCR generated DNA and DNA fragments can be used as hybridization probes to search for a full-length D-ribulose-5-phosphate-3-epimerase gene in a yeast gene library. Preferred method *: 30 to over-express the D-ribulose-5-phosphate-3-epimerase gene in other yeast hosts is to clone it into a vector with a high copy number in the desired host (e.g. t. · · ·. PSRT303D Z. roux). An alternative and * more efficient way to overexpress the gene is to at least partially determine the nucleotide sequence of the D-ribulose-5-phosphate-3-60-08300 epimerase gene around the translation start codon and use this information in the D-ribulose-codon. Isolation of the coding sequence for the 5-phosphate-3-epimerase gene and its fusion to a promoter known to be effective in the selected host. Example 12 Cloning of the D-xylulokinase gene and construction of D-xylulokinase simulants

Methods for cloning the gene for D-xylulokinase (EC 2.7.1.17) from various yeast species have been described [Ho, N. W. Y. et al., Enzyme Microbiol. Technol. 11: 417-421 (1989); Stevis, P. E. et al., Applied and Environmental Microbiol. 53: 2975-2977 (1987)]. A method for generating a D-xylulokinase mutation through gene cleavage in S. 15 cerevisiae has also been described [Stevis, P. E. et al., Appl. Biochem. Biotechnol. 20: 327-334 (1989)]. Similar methods may be used to provide D-xylulokinase simulants in other yeasts. In the case of hii ova other than S. cerevlslae, the gene markers used in the cleavage of the D-xyluloki-20 nase gene are preferably dominant antibiotic resistance markers (see Example 9). Alternatively, classical mutant formation methods based on chemical (for example treatment with ethylsulfate nisulfonate or acriflavine) or physical * * ·. *. * (Ultraviolet light, X-rays) mutagenesis can be used. Enrichment of the mutant • · ♦ *; · / can be achieved by increasing mutagenized cellular · · · * · * and the only carbon source, D-xylulose, in the presence of a growth-killing antibiotic (such as nystatin) * '”* 30 Inability of D-xylulokinase mutants · · Used to grow D-xylulose as the sole carbon source, ···, can be used to select mutants.

• · · C1 1 08300

dl

Example 13

Strains producing xylitol through D-arabitol in improved yield

Example 4 describes a method for constructing a carbon-5 strain capable of producing xylitol from structurally distant carbon sources, such as D-glucose, via a pathway utilizing D-arabitol as a critical intermediate. In order to improve the yield of xylitol in fermentations using strains utilizing this "D-arabitol pathway", the yield of D-Arabic must be improved. The pathway from D-glucose to D-arabitol in D-arabitol producing yeasts has been described [Ingram, J.M. et al., J. Bacteriol. 89: 1186-1194 (1965)]. D-arabitol is produced from D-ribulose-5-phosphate-15 by dephosphorylation and dephosphorylation by NADPH-linked D-ribulose reductase. The formation of D-ribulose-5-phosphate from D-glucose-6-phosphate through two successive irreversible dehydrogenation steps by D-glucose-6-phosphate dehydrogenase and 6-phospho-D-glucose-20-date dehydrogenase is a ubiquitous pathway known or as the oxidative branch of the hexose monophosphate pathway). In the non-oxidative branch of the pentose phosphate pathway, D-ribulose-5-phosphate is reversibly isomerized to ribose-5-phosphate. 25 si and D-xylulose-5-phosphate. Ribose-5-phosphate and. ' . * D-xylulose-5-phosphate is further metabolized by trans- • · · *, * tolasase. Therefore, a transketo • · · mutation in the D-arabitol-producing microbial strain can be induced and the proportion of D-ribulose-5-phosphate 30 converted to D-arabitol can be increased. Example 9 describes a method for generating transketolase mutants. The D-arabitol yield can be further increased if the rate of D-ribulose-5-phosphate biosynthesis is maximized by overexpressing the two genes encoding the oxidative branch of the pentose phosphate pathway V as described above (Example 8).

62 108300 Strains optimized for D-arabitol yield in this manner can then be further transformed with recombinant DNA constructs containing xylitol dehydrogenase and D-arabol tide dehydrogenase genes (Examples 3 and 4), resulting in strains with xylitol production efficiency improved.

Example 14

Strains Producing Xylitol via Alternative Routes The methods of Examples 4 and 13 are the most straightforward methods for constructing microbial strains capable of converting D-glucose and other carbon sources to xylitol. These methods utilize a naturally occurring pathway leading to the formation of D-arabitol from various carbon sources and extend this pathway by two additional reactions to convert D-arabitol to xylitol. However, this is not the only way. Other pathways leading to xylitol as a final metabolite and not using D-arabitol as an intermediate can be constructed. Thus, the pathway leading to xylitol from the same precursor, D-ribulose-5-phosphate, can be realized through a different reaction chain.

D-ribulose-5-phosphate can be effectively converted to D-xylulose-5-phosphate by D-ribulose-5-phosphate-3-epimer. 25 (Example 11), and if further conversion of D-xylulose-5-phosphate * * t j * is prevented in the transketolase gene. * ', * Via mutation, the accumulated D-xylulose-5- •' '* * * phosphate can be dephosphorylated by the same non-specific phosphatase as D-ribulose-5-phosphate [Ingram, JM et al. J. Bacteriol. 89, 1186-1194 (1965)] and reduced to xylitol by xylitol dehydrogenase (Example 3). Implementation of this *. ··· pathway may also require inactivation of the D-xylulokinase gene ♦ * * (Example 12) in order to minimize the energy loss caused by the following unnecessary loop: D-xy-: "35 lulose-5 -phosphate -> D-xylulose -> D-xylulose-5-phospho - "103300 63. Phase. Additional genetic change - introduction and (over) expression of the D-ribulokinase gene (EC 2.7.1.47) could minimize the simultaneous production of D-arabitol from such strains. non-binding D-ribulose synthesized by nonspecific phosphatase, converting D-ribulose back to D-ribulose-5-phosphate and further to D-xylulose-5-phosphate.

Example 15 Stability of Z. rouxii combination strain and xylitol) in fermenter culture conditions

The stability of xylitol production during extended culture was checked under both selective conditions (using the following selective medium: YEPD containing 50 mg / L G418 and 30% glucose) and under non-selective conditions (using the same medium without G418). One freshly obtained transformant of Z. rouxii ATCC 13356 [pSRT (AX) - 9] was grown in 200 ml of G418-containing YEP.D. Cells were transferred to 50% glycerol solution and frozen at -70 ° C in 1 ml portions. Ino-20 was cured with four frozen doses of Z. rouxii [pSRT (AX) -9], two on a selective medium and two on non-selective medium. :: 50 ml culture in lectile medium. After the cultures had reached a stationary growth stage of .... (50-60 hours at 30 ° C with a stirring speed of 25 for 200 min -1), a sample was taken for HPLC analysis of pentitol and inoculated with 1 ml: 11a culture others • · · ** !, * 50 ml of the same (selective or non-selective) medium.

• · t ** 'The culture dilution cycle was repeated four more times. The conditions of this experiment approximate the progression of the recombinant strain ··· 4 * ** from a conventional frozen inoculum in large-scale fermentation. The results of this experiment are shown in Table 8. The stability of the recombinant strain is predictably superior to the selective medium.

i

However, even in a non-selective medium, a decrease in xylitol yield was not observed until about 20 generations of ice. Under selective conditions, xylitol production was stable for about 30 generations.

One dose of frozen transformed Z. rouxii stock strain was inoculated with a 2 liter fermenter containing 1 liter of medium of the following composition (per liter): 0.1 g NaCl, 6.8 g potassium phosphate, 0.5 g ammonium sulfate, 20 g yeast extract, 400 g glucose and 50 mg G481, pH 6.0. The culture conditions were as follows: aeration rate of 0.5 V / min; mixing speed 400 min'1; 10 temperature 30 ° C.

Figure 10 shows glucose consumption and xylitol accumulation over time in this fermentation. The dissolved oxygen concentration, which reflects the respiratory activity of the yeast culture, is also shown. A seemingly 15 biphasic increase was observed: the first step reached a level of glucose and xylitol levels in about 40 hours (less than half of the available glucose had elapsed at this step), the second step was observed after about 200 hours of cultivation, when glucose consumption and xylitol production resumed. . The final xylitol content was 15 g / l, almost twice that achieved in bottle cultures. Biphasic growth with a long lag period indicated that a spontaneous mutant (presumably with a better ability to tolerate alcohol than the parent strain) was selected. This hy-:. ·. a single clone at the end of fermentation was isolated from the culture to verify the hypothesis. This isolated · · · clone was grown in a fermentor under the conditions described above. The results of this experiment (Figure 11) confirmed

• · · I I

30 that was a truly isolated mutant capable of fully assimilating 400 g / l glucose in about 60 hours.

: * · *; This experiment also shows that the xylitol-producing strain Z. rouxii ♦ is also able to assimilate xylitol when the glucose in the medium is depleted.

65 1 0 8 3 0 0

Table 8

Stability of Z. rouxii ATCC 13356 [pSRT (ÄX) -9] xylitol production under serial culture conditions (g / l, normalized to total pentitol yield) 5

Culture conditions

Dilution Series Culture Selective- Non-Selective Dilution No. Concentrated 10 1 1 8.3 8.6 2 8.9 8.6 2 1 8.9 8.9 2 8.5 8.4 3 1 8.5 8 , 3 15 2 8.6 8.2 4 1 8.7 8.0 2 8.6 6.9 5 1 8.6 7.6 2 8.1 5.6 20 6 1 7.0 7.0 2 6.9 2.4

All source literature is incorporated herein by reference. Having now fully described the invention, those skilled in the art will recognize that the scope of protection can be achieved by:

Ml ·. *. ··. etc. without affecting the spirit or scope of the invention or any of its embodiments.

I (I

* · • · »i I # •« · • · · * · ·

«I I • I

66 1 08 30 0

Sequence Listing (1) GENERAL INFORMATION: (i) APPLICANT: Harkki, Anu M.

Myasnikov, Andrey N.

Apajalahti, Juha H.A.

Pastinen, Ossi A.

(ii) TITLE OF INVENTION: Manufacture of Xylitol (iii) NUMBER OF SEQUENCES: 8 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: (B) STREET: (C) CITY: (D) STATE: (E) COUNTRY: ( F) ZIP: (v) COMPUTER: READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patent Release # 1.0, Version # 1.25 (VX) CURRENT APPLICATION DATE: (A) APPLICATION NUMBER: PCT (to be assigned) · (B) FILING DATE: herewith (C) CLASSIFICATION: (vi) PRIOR APPLICATION DATE: (A) APPLICATION NUMBER: US 08 / 110,672 (B) FILING DATE: 24-AUG-1993 I-1 (vi) PRIOR APPLICATION DATE :; (A) APPLICATION NUMBER: US 07 / 973,325 (B) FILING DATE: 05-NOV-1992 (viii) ATTORNEY / AGENT INFORMATION: (A) NAME: (B) REGISTRATION NUMBER: • * '\ · (C) REFERENCE / DOCKET NUMBER: * * · (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (B) TELEFAX: 67 1 08 300 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: both (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CGAATTCTAG ACCACCCTAA GTCGTCCC 28 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: both (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: TTCAAGAATT CAAGAAACTC ACGTGATGC 29 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: both • ♦ * · · • · · · · · «(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: • * · CAGGCCGTCG ACAAGGATCT CGTCTC 26. · ♦ . (2) INFORMATION FOR SEQ ID NO: 4: • · ·, .j. #, (I) SEQUENCE CHARACTERISTICS: V: (A) LENGTH: 33 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: both ( D) TOPOLOGY: both 68 10 8 300 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: AATTAGTCGA CCGTTAATTG GGGCCACTGA GGC 33 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: both (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GCGAAGCTTA AAAATGTCCA AGCAACAGAT CGGCG 35 (2) INFORMATION FOR SEQ ID NO: 6: (i) ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: both (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GCGAAGCTTA GATTAATCCA GCCATTCGGT ATGG 34: 2) INFORMATION FOR SEQ ID NO: 7: • ** · (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 44 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: both ... "(D) TOPOLOGY: both «« · • · ·, • · ·

«II

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: AGCTCTAGAA ATGACTCAAT TCACTGACAT TGATAAGCTA GCCG: ·., 44 69 1 08 300 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 44 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: both (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: GGAGAATTCA GCTTGTCACC CTTATAGAAT GCAATGGTCT TTTG 44 * · * «· • · 1 •« · · • · · • · «« • • 1 i • · «« ·.

Claims (30)

A process for the production of xylitol in a recombinant host, characterized in that the process comprises: (a) constructing a novel metabolic pathway in the microbial host which results in the synthesis of xylitol as a final product from a carbon source other than D-xylose, D-xylose; Mixtures of D-xylose and D-xylulose and polymers and oligomers containing D-xylose or D-xylulose as major components, (b) cultivating the combined host of step (a) under conditions that provide synthesis of xylitol using said pathway and - 15 teas other than D-xylose, D-xylulose, mixtures of D-xylose and D-xylulose and polymers and oligomers containing D-xylose or D-xylulose as major components, and (c) recovering the xylitol produced in step (b). li. Process according to Claim 1, characterized in that the arabitol is one intermediate in said path. A method according to claim 2, characterized in that said new metabolic pathway • continues and / or modifies the metabolic pathway of the natural host, leading to the arabitol end product in the natural host. . A method according to claim 3, characterized in that the construction of said novel metabolites involves the transformation of an arabitol-producing microbial host ::: with DNA encoding D-arabitol dehydrogenase (EC 1.1.1.11):. The method of claim 4, *. 35, characterized in that the construction of said novel metabolites 71108300 further comprises transforming the constructed recombinant host with DNA encoding xylitol dehydrogenase (EC 1.1.1.9). Method according to claim 1, characterized in that the natural host is either an arabitol producing yeast or an arabitol producing fungus. The method of claim 6, wherein the host does not express D-xylulokinase (EC 2.7.1.17). A method according to claim 6, characterized in that the host does not express transketo- lase (EC 2.2.1.1). Method according to claim 6, characterized in that the host is further transformed with one or more coding sequences selected from xylitol dehydrogenase, D-glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phospho-D-gluconate decidehydrogenase (EC 1.1.1.44) and DNA encoding D-ribulose-5-phosphate thi-3-epimerase (EC 5.1.3.1). Method according to any one of claims 1 to 9, characterized in that the yeast is selected from the group consisting of Zygosaccharomyces rouxii, Candida polymorpha, Torulopsis Candida, Pichia farinosa and A process according to claim 10, characterized in that said yeast is Zygosac, charomyces rouxii. «« T <4 Process according to claim 1, characterized in that xylitol is formed by converting D-xylulose phosphate to D-xylulose, followed by the reduction of D-xylulose to xylitol. A method according to claim 12, characterized in that the host is transformed with an additional construct encoding one or more enzymes selected from the group consisting of D-glucose-6-phosphate dehydrogenase (EC 1.1. 1.49), 6-phospho-D-Glu-conate dehydrogenase (EC 1.1.1.44), D-ribulose-5-phosph-5-phate-3-epimerase (EC 5.1.3.1), D-ribulokinase (EC 2.7.1.47), and xylitol dehydrogenase (EC 1.1.1.9). A method according to claim 12, characterized in that the host does not express transketo- lase (EC 2.2.1.1). The method of claim 14, wherein the host is further transformed with a construct encoding one or more enzymes selected from the group consisting of D-glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phospho-D-Glu Conate dehydrogenase (EC 1.1.1.44), D-ribulose-5-phosphate-3-epimerase (EC 5.1.3.1), D-ribulokinase (EC 2.7.1.47) and xylitol dehydrogenase (EC 1.1.1.9). 16. The method of claim 12, wherein the host does not express D-xylulokinase (EC 2.7.1.17). 17. The method of claim 16, wherein the host is further transformed with a structure encoding one or more enzymes selected from the group consisting of D-glucose-6-. J phosphate dehydrogenase (EC 1.1.1.49 ), 6-phospho-D-glu-: conate dehydrogenase (EC 1.1.1.44), D-ribulose-5-phosph- * ♦ ·, ···. fatty-3-epimerase (EC 5.1.3.1), D-ribulokinase (EC? · 2.7.1.47) and xylitol dehydrogenase (EC 1.1.1.9). A method according to claim 12, characterized in that the host does not express transketo-lase (EC 2.2.1.1) or D-xylulokinase (EC 2.7.1.17). * The method of claim 18, wherein the host is further transformed with a construct encoding one or more enzymes selected from the group consisting of D-glucose-6-108300 phosphate dehydrogenase (EC 1.1. 1.49), 6-phospho-D-Glu-conate dehydrogenase (EC 1.1.1.44), D-ribulose-5-phosphate-3-epimerase (EC 5.1.3.1), D-ribulokinase (EC 2.7-1.47) and xylitol dehydrogenase (EC 1.1.1.9). A method according to any one of claims 12 to 19, characterized in that the yeast is selected from the group consisting of Zygosaccharomyces rouxii, Candida polymorpha, Torulopsis Candida, Pichia forinosa and Torulaspora hansenii, and the fungus is selected from the group consisting of Dendryphiella Salina and Schizophyllum commune. A method according to claim 20, characterized in that said yeast is Zygosaccharomyces rouxii. 22. A recombinant microbial host, characterized in that it has the ability to synthesize xylitol in a single fermentation from carbon sources other than D-xylose, D-xylulose, mixtures of D-xylose and D-xylulose and D-xylose or D-xylulose as the major constituents. and oligomers, and the synthesis is more abundant than that produced by the corresponding non-recombinant microbial host as a result of transforming the host with DNA encoding D-arabitol dehydrogenase (EC 1.1.1.11) or a gene encoding xylitol dehydrogenase or does not express D-xylulokinase (EC II •. *. 2.7.1.17) or transketolase (EC 2.2.1.1). • · «·. ··, ·. A recombinant host according to claim 22, characterized in that the natural metabolic pathway leading to arabitol as a final product in a non-recombinant host is extended or modified in a manner that enhances; · * Synthesis of xylitol in said recombinant host. A recombinant host according to claim 23, characterized in that said pathway has been left or modified by transformation of a host encoding DNA xylitol dehydrogenase (EC 1.1.1.9). 25. The recombinant host of claim 22, wherein the host does not express D-xylulokinase (EC 2.7.1.17) or transketolase (EC 2.2.1.1). 25 Torulaspora hansenii, and the fungus is selected from the group consisting of Dendryphiella Salina and Schizophyllum commune. • »#« A recombinant host according to claim 22, characterized in that the host is transformed with a gene encoding D-glucose-6-phosphate dehydrogenase (EC 1.1.1.49). The recombinant host of claim 22, wherein the host is transformed with a gene encoding 6-phospho-D-gluconate dehydrogenase (EC 1.1.1.44). The recombinant host of claim 22, wherein the host is transformed with a gene encoding D-ribulose-5-phosphate-3-epimerase (EC 5.1.3.1). A recombinant host according to claim 22, characterized in that the host is transformed with a construct encoding xylitol dehydrogenase. The recombinant host according to any one of claims 22 to 29, characterized in that the non-recombinant microbial host is an arabitol producing yeast or a 25 arabitol producing fungus. «· 108300