FI107342B - Process for producing xylitol from a modified yeast host - Google Patents

Description

107342

A process for the production of xylitol from modified carbon or the like

FIELD OF THE INVENTION The invention is in the field of metabolic manipulation. Specifically, the invention relates to a process for producing xylitol using novel yeast strains having xylitol metabolism modified. The invention further relates to novel yeast strains and to a method for producing such strains.

Background of the Invention

Xylitol is usually prepared by methods of first hydrolyzing a xylan-containing material to produce a xylose-containing monosaccharide mixture. The xylose is then reduced to xylitol, usually in the presence of a nickel catalyst. Numerous methods of this type are described in the literature of the art. U.S. Patent Nos. 3,784,408, 4,066,711, 4,075,406, 4,008,285, and 3,586,537 may be cited as examples.

However, all of these prior art processes are multi-step processes that are relatively expensive and have relatively low efficiency. The major problems relate to the efficient and complete separation of xylose from other hydrolysis byproducts. Purification is: 25 very demanding because the catalysts used in the xylose reduction reaction are very sensitive. The purity of the final product, on the other hand, is highly dependent on how well xylitol can be distinguished from other products formed in the reduction reaction.

The production of xylitol by the biotechnological process is of great interest if such a process is capable of obtaining a very high quality product by a relatively inexpensive process. However, in order to produce xylitol by yeast fermentation for the purposes mentioned above, a yeast strain which is not a requirement of the pathogens must be found. Further, in order to achieve high yields of xylitol by yeast fermentation, it is essential to use a yeast that is capable of reducing xylose to xylitol. Preferably, such a strain would also have a ratio of -5 in its ineffective xylitol metabolic edelleer conversion.

Many yeast strains produce reductase enzymes that catalyze the reduction of sugars to the corresponding sugar alcohols. Many yeasts are also capable of reducing 10 xylose to xylitol as a first step in their metabolism of xylose, and many yeast strains are able to use xyloc as the sole source of carbon and energy. The pathway or pathway used by xylose for the yeast studied is as follows: xylitol is synthesized in the first step where xylose reductase reduces xylose to xylitol. Xylitol is then metabolised (used) in a series of the following n steps. First, xylitol dehydrogenase oxidizes xylitol to xylulose, xylulose kinase (also called xylulokinase) phosphorylates xylulose to xy-20-lulose-5-phosphate, and then part of the xylulose-5-phosphnate is converted to pyruvate and ethanols. Depending on the yeast strain and fermentation conditions, the resulting main and by-products will argue. The reactions are not tightly coupled, which is why some xylitol is always formed in the growth medium.

Research in this field has generally focused on attempts to identify yeast strains that have an enhanced ability to produce ethanol rather than xylitol. 30 However, xylitol production is a relatively common feature of yeast using xylose. For example, 44 of the 5 yeasts of five genera (Candida, Hansenula, Kluyveromyces, Pichia and Pachysolen) produced some xylitol in the medium [Barbosa, M.F.S., et al., J. Industr. Micrc- 107342 3 Biol. 3: 241-251 (1988); and Enzyme Microb. Technol. 10: 66-81 (1988)].

The use of such strains for the industrial production of xylitol has been proposed. For example, the yeasts Candida tro-5 picalis, Candida guillermondii and Candida parapsilosis have been proposed for industrial use (PCT Patent Application Publication Nos. PCT / FI90 / 00015, Ct-tropicalis, PCT / FI91 / 00011, C. tropicalis, and PCT / FI87 / 00162). , Guillermondii, and U.S. Patent Application No. 2,641,545, C. parapsilosis). However, all the above-mentioned Candida strains are potential pathogens that do not meet the requirements of the food industry. Barbosa et al., J. Industr. Microbiol. 3, 241-251 (1988), describe yeasts screened for xylitol production. However, strains that obtained acceptable yields of hy-15 are also all possible pathogens. Therefore, non-pathogenic yeast strains useful for the production of xylitol, which can be used in the food industry and / or for the large-scale production of xylitol, have not been described.

Moreover, profitable industrial production of xylitol by enzymatic conversion of xylose is only possible if the yield is very high. However, no strain of wild yeast can achieve this. Examination of different yeast strains under optimal reaction conditions showed that certain strains of Candida tropicalis produced the best xylitol yield. However, strains of this yeast species, as mentioned above, are potentially pathogenic and therefore cannot be used in the food industry. Acceptable species for the food industry are Saccharomyces cerevisiae, Candida utilis and Kluyveromyces marxianus. Saccharomyces cerevisiae does not normally express the enzymes of the xylose pathway, although Hallborn, J., et al., Bio / Technology 9 (1991) 1090, describes the use of the Pichia stipitic cloned xylose reductase gene 35 to construct strains of Saccharomyces cerevisiae, which have been transformed. 95%.

Mutants have been described for which the use of xylose is not deficient. In Hagedorn, J., et al., Curi.

5 Genet. 16: 27-33 (1989) discloses the identification of yeast stipitis mutants which were unable to use xylose as the sole carbon source and lacking xylose reductase or xylitol dehydrogenase. In James, A. P., et al., Appl. Er -10 Estonian. Microbiol. 55 (1989) 2871 - 2876 discloses H; .J -van Pachysolen tannophilus mutants which are kykere to metabolize D-xylose. Stevis, P.E., et al., Appl. Biochem. Biotechnol. 20: 327-334 (1989) discloses construction of yeast xylulokinase mutants by recombinant DNA techniques.

Candida utilis and K1uyveromyees marxiani s are naturally-inducible enzymes xylose reductase, xylitol dehydrogenase and xylulose kinase, which are essential for the degradation of xylose. However, attempts to regulate the chemical and physical environment of Candle a 20 utilis and Kluyveromyces marxianus to increase the yield of xylitol have failed.

Summary of the Invention

It has now been found that the yeast xylitol metabolite: <a.: 25 can be modified to form new yeast strains.jc that produce xylitol in high yield.

Thus, the invention firstly relates to new yeast strains which are capable of synthesizing xylitol from xylitol but which lack one or more of the xylitol: a-30 bolus enzyme, whereby xylitol is cleaved to a medium from which it can be harvested.

The invention further relates to methods for producing xylito L using such yeast strains.

The invention further relates to a method of constructing modified yeast strains which are capable of xylose-only and which are not capable of expressing xylitol dehydrogenase and / or xylulose kinase activity or have xylitol dehydrogenase and / or xylulose deficiency.

5 Detailed Description

The modified yeast strains of the invention are capable of synthesizing xylitol from xylose, but one or more enzymes of their use in xylitol are deficient, whereby xylitol is accumulated in the medium by culturing the modified yeast host with xylose. Thus, xylose reductase activity is present in the new strains, but xylitol metabolism is significantly reduced. Thus, new strains are unable to use xylose as their sole carbon source, although xylose induces xylose-15 reductase activity and thus the conversion of xylose to xylitol.

The course of the xylose reduction reaction is shown below.

Xylose reductase catalyzes the reaction: 20 xylose

/ NADPH

J. (

NADP

xylitol. The first enzyme in the use of xylitol, xylitol • · dehydrogenase, catalyzes the reaction: xylitol «ΝΑΟ, r (

30 NADH

xylulose • · ·

The novel yeast strains produced by the process of the invention produce xylitol from xylose after addition of 35 other sources of carbon and energy to the growth medium for growth and maintenance of the yeast strain. Under culture conditions, where it is desired to produce xylitol from xylose, the hosts of the invention are genetically unable to use xylose as the sole carbon source because they are genetically unable to completely metabolise xylitol. Since the strains are unable to metabolize xylitol, xylitol accumulates in the culture medium. By "genetically incompetent" is meant that the native yeast DNA has been altered to result in a modified yeast parent with the desired mutation or other genetic modification that is stably inherited.

Thus, the modified yeast strains of the invention have high glucose metabolism and xylose reduction, but at the same time the rate at which they oxidize xylitol to xylulose is significantly reduced. The new strains are thus very effective xylitol producers. Many yeast chambers using xylose are known, including, for example, Cr frareli (such as FT1-20038); guillermondii (such as FTI-20037, IZ-803, iz-1739, IZ-1231, 12: -1322, IZ-1239, IZ-1422, c-138, 17-07 and 20Z-1735); intermedia (such as RJ-245); pseudotropical (such as IZ-431 and 1006); tropicalis (such as 1004, iz-1824, IZ-1958 and 53-51); Cl utilis (such as FTI-20039, '4-64, 1009, EQ2, IZ-1166, IZ-1840 and IZ-1841); anomala (such as IZ-1420, IZ-229, IZ-781, IZ-1033, RJ-510, IZ-271, IZ-1224, IZ-1260 and FTPTAT-106); fragilis (such as FTI-20066); K. marxianus (such as IZ-1821, 145, IZ-1339, 276a-IZ-619); Pach. tannophilus (such as NRRL Y2460); butene (such as 68-1111) and P ^ stipitis (like 79-261). Such yeasts can be obtained from a variety of sources, such as eai-30, such as ATCC, NRRL, The Agronomic Institute (UNICAMP, Campinas, Brazil); the Foundation for Industrial Technology (FTI, Sao Paulo, Brazil); the Microbiology Institute (UFRJ, Rio de Janeiro) and the ZymA-Technic Institute (ESALQ, Piracicaba). All can be maintained on agar slanted surfaces or plates as described in the art, for example, in a medium containing 10 g / liter yeast extract, 20 g / liter peptone and 20 g / liter D-glucose [Barbosa, MFS , et al., J. Industr. Microbiol. 3: 241-251 (1988)].

When non-pathogenic indigenous strains are used to produce new strains, the resulting mutants also meet the specific requirements of the food industry, both in terms of quality and yield. Preferably, species of Candida, Hansenula, Kluyveromyces, Pichia or Pachysolen 10 yeast is used, and especially in yeast Candida utilis, or Kluyveromyces marxianus. In a very preferred embodiment, a strain of Kluyveromyces marxianus var. marxianus (such as CBSC12) or Kluyveromyces marxianus var. Bulgari-cus (such as ATCC 16045) or Kluyveromyces marxianus 15 var. lactis. The methods of the invention are suitable for modifying any yeast to construct or identify a yeast capable of producing xylitol but deficient in one or more xylitol utilization enzymes. The methods, outlined below, in which 20 examples of yeast Kluyveromyces marxianus are used as a model and starting material, can be more extensively applied to modify other yeast species to obtain the modified yeast hosts of the invention.

Kluyveromyces marxianus is a well-known non-pathogenic yeast widely used in the food victualling industry.

Yeast is described, for example, in "The Yeasts, A Taxonomic Study", ed. Kreger-van Rij, N.J., Elsevier Science Publishers B.V., Amsterdam, 1984, where synonyms for this yeast are also mentioned on page 234. This publication 30 and all other references cited herein are hereby incorporated by reference in this application. The wild strain normally uses glucose as its energy source, but it is also able to metabolize xylose. Other yeasts meeting specific requirements of the food industry, such as certain strains of Candida, may be used for the purposes of the invention.

Preferably, the genes whose expression is desirably inactivated are native genes encoding xylitolicis: -5 hydrogenase and xylulose kinase. These genes are preferred because they are generally the only pathway in xylitol metabolism in the host. Therefore, the final blocking of expression of either (or both) enzymes will ultimately block the use of xylitol and possibly result in a -10 sex accumulation of xylitol in the growth medium in the presence of xylose.

The hosts of the invention may be produced by any method that blocks xylitol metabolism as described above. In one embodiment, the inventive method for producing new yeast strains is characterized in that the parent strain is cultivated on a medium containing assimilable carbon and energy sources, the culture is subjected to mutagenic treatment and, optionally, treatment with a substance that interferes with chromosome 20 the titans are enriched with an antibiotic and the yeast strains deficient in xylitol metabolism are screened;

In this embodiment, for example, genomic mutation-25 is used by chemical or physical treatment. Examples of suitable chemical mutagens include base analogues such as 5'-bromouracil, 2'-aminopuryl and 5'-deoxyuridine, deaminating agents such as nitric acid and hydroxylamine, alkylating agents such as ethyl iris-30-tansulfonate and nitrosoguanidine, , such as acriflavine and ethidium bromide. Loss arrangements for using these agents are known in the art.

Examples of useful physical mutc-35 gene generation methods include, for example, UV and X-ray: 107342 9 irradiation. Procedural arrangements for using these agents are known in the art.

Optionally, the yeast may be treated with a substance that interferes with chromosome division or nuclear succulent formation in the mitotic cycle. The use of benomyl is a preferred embodiment. By this treatment, the chromosome with the desired mutation is isolated from the homologous chromosomes present. This treatment is not required for haploid yeast strains.

Finally, the yeasts modified by the chemical or physical method described above are screened for those having the desired characteristics of the modified yeast hosts of the invention. Enrichment of auxotrophic cells with an antibiotic such as nystatin, which kills only cells with active metabolism, can be used. The treatment is carried out in the presence of xylose. The amount of xylose can be directly determined from the medium according to methods known in the art to determine the best yeast producers.

20 Thus, new strains can be produced by cultivating a starting stock in a suitable medium in the usual manner. The resulting culture may be subjected to mutagenic treatment by chemical or physical methods, for example using insertion or deletion mutagens such as acriflavine or proflavin, point mutation mutagen, ethyl methanesulfonate, UV radiation or the like. After any benomyl treatment, the mutants can be enriched, for example, by treatment with nystatin and finally, the yeast strains are screened by agar on high xylose and low glucose agar plates. Very small colonies deficient in xylose metabolism are harvested and purified by conventional techniques. The advantage of using chemical or physical methods to construct a host according to the invention is that such methods work effortlessly with any parent yeast species, and selection methods (assaying xylitol production from a growth medium) can also be readily employed with any yeast species. As an example, the methods described in the Examples would be expected to yield hosts of the invention not only from the exemplified species, but also from any yeast host which is naturally capable of metabolizing xylose.

An alternative procedure for constructing hosts of the invention involves the use of recombinant DNA techniques. In a first embodiment, the DNA sequence encoding the xylitolmeth i-bolia enzyme is altered to destroy code information in such a gene, and the mutated gene is inserted into a yeast chromosome in place of a native germline gene using homologous DNA fusion or gene cleavage techniques. Examples of genes that can be selected for such targets are xylitol dehydrogenase and xylulose kinase. Xylitol dehydrogenane 20 is preferably selected for inactivation because it is the first enzyme to use xylitol. The inactivation of xylulose cyclase is sufficient to ensure a high yield of xyl L because the equilibrium of the reversible reaction catalyzed by xylitol dehydrogenase shifts very strongly to favor xylitol under the conditions of the yeast cell. If the genes encoding both enzymes are selected for inactivation, any low level of expression of the active form (or fragment) of xylitol dehydrogenase, if any, in the host will be compensated for by the absence of xyllyl-30 lysokinase activity.

Methods for cloning these yeast genes are known in the art (see examples). The genes encoding xylitol dehydrogenase or xylulose kinase can be cloned, for example, by complementing the respective mutations in 35 Escherichia coli or yeast. The cloned genes No. 10734211 are diffused by inserting in the yeast a selectable DNA sequence into the coding region of the cloned gene, preferably at a distance of not less than several hundred base pairs from each end of the cloned gene. The selectable marker (preferably the 5 dominant dominant marker that produces the antibiotic-resistant phenotype) can either be inserted at a suitable restriction site or manipulated to replace the fragment in the disrupted gene. The resulting plasmid construct is amplified in bacteria. Appropriate restriction enzymes are then used to cleave a DNA-10 fragment that has xylitol dehydrogenase (or xylulose kinase) gene sequences at each end and a yeast selection marker in the middle region. This DNA fragment is then used to transform the yeast strain into antibiotic resistant. The ability of individual transformant-15 clones to grow on plates containing xylose as the sole carbon source is tested. Finally, successful gene cleavage can be confirmed by measuring the corresponding enzyme activity in the transformed strain and / or analyzing the structure of the essential region of the yeast chromosome by a method known as South-20 ern hybridization.

A suitable modification is, for example, the replacement of an internal fragment of a cloned gene with a dominant selection marker for yeast. DNAs encoding a bacterial kanamycin resistance gene or a bleomycin resistance 25 gene are examples of useful dominant selection markers for yeast. Such constructs can be operably linked to yeast promoter sequences or use the yeast promoter present at the homologous DNA fusion site. When a promoter of a gene whose coding sequence is cleaved is used, only transformants that insert the coding sequence into the desired translational reading frame will be selected. Thus, it is often more convenient to use a promoter sequence already operatively linked to the construct. Any other gene 107342 12 which enables screening for cells containing transforming DNA can be used in place of the antibiotic resistance gene, for example, the gene encoding the necessary protein.

Transformation of yeast cells with a linear DNA fragment having partial sequences at a target site (such as a xylitol dehydrogenase or xylose kinase gene) at both DNA ends can be performed; a check mark inside the fragment. Such transfection -10 is known to result in a large proportion of transformants carrying a deletion mutation in the corresponding gene.

Alternatively, if desired to maintain the ability of the native yeast to express xylitol dehydrogenase and / or xylcellulose kinase, alternative DNA fusion methods that provide selective inhibition of target enzyme expression may be used. Such methods include the construction of antisense RNA or ribozyme directed against an appropriate target, such as mRNA encoding xylitol dehydrogenase or xylulose kinase. The DNA construct encoding the antisense RNA has a DNA sequence complementary to the target mRNA sequence and of sufficient length to recognize and hybridize to such target mRNA, thereby preventing translation of the target mRNA into the host cell. The DNA construct encoding the appropriate ribozyme also has sufficient DNA sequence to hybridize to the target mRNA; however, upon hybridization to such mRNA, ribozyme activity catalyzes the cleavage of mRNA at a site that destroys the cytoprotein of such mRNA to maintain translation of the active protein encoded therein (see, for example, EP 321,201). The antisense RNA construct or ribozyme sequence can be operably linked to any appropriate yeast promoter sequence using methods known in the art and transformed into yeast hosts by genetically stable method 10734213 using conventional transformation techniques as well known in the art. Expression of such structures is governed by the properties of the operably linked yeast promoter and may be constitutive or inducible or repressible in various media as desired. The advantage of such constructs is that the ability of the native host to express the target enzyme is not destroyed. The advantage of making the expression of such structures adjustable in the on / off mode is that the ability of the host 10 to express the target can be tightly controlled, whereby the expression of the target is only inhibited if xylitol synthesis is desired.

The efficiency of the new yeast strains constructed according to the present invention can be further improved by expressing very strongly xylose reductase cloned from the same yeast or from different yeast strains. A method for cloning the yeast xylose reductase gene has been described by several authors [Hallborn, J., et al., Bio / Technology 9 (1991) 1090-1094; Takuma et al., Appl.

20 Biochem. Biotechnol. 28/29 (1991) 327-340; and Strasser et al., DE 4 009 676 (1991)]. A method for using this cloned gene for xylose-xylitol conversion in yeast has also been described (Hallborn, J., et al., Bio / Technology 9: 1090-1094 (1991)). The cloned xylose reduct-25 gene can be transferred to a suitable vector capable of replication in a selected yeast species. For example, pKD1-based vectors are the preferred vector type for obtaining a high level of expression in K y Marxianus [Bianchi, M. M., et al., Curr. Genet. 12: 185-192 (1987)]. The xyloseire-30 ductase gene can be exchanged for another promoter known to be effective in the selected yeast. The mutant yeast strains obtained according to the present invention can then be used as hosts for transformation with the vector (s) providing very strong expression of the xylose reductase-35 gene.

107342 14

The novel yeast strains obtained in the lime embodiments, regardless of the size they are produced, are characterized by a significantly reduced ability to metabolize xylitol compared to the native h.L: .5 strain. This is because one or more enzymes of xylitol * s-tabolea, such as xylitol * i-hydrogenase activity and / or xylulose kinase activity :., have been modified such that expression of such an enzyme is either lost or decreased (either permanently or by regulation). way) as a result of yeast modification. Any carbon source capable of being used by the host for energy production can be used to maintain the growth of the yeast according to the invention and to provide the only density equivalents for the reduction of xylose. This function (providing the reduction potential) is perhaps more important to the method of the invention than maintaining the growth of the host cell. For example, glucose is preferably added to the substrate as an energy source in amounts known in the art to be necessary for maintaining host growth. Breeding aeration and scouting conditions should be optimal for the host.

When xylose is administered to hosts of the invention, the xylose carbon becomes substantially "captured" as xylitol and is not available for the host's energy requirements.

. Because of this feature, novel strains of the invention

• «. I

are very useful in the production of xylitol. When culturing mutagenized strains of the invention in a high-xylose medium, they convert xylose to xylitol very efficiently. Their conversion efficiency and utility for the desired process can be evaluated using any xylose-containing solutions and hydrolyzates, for example, pure xylose solutions or wood-processing waste liquor, so-called cooking liquor or cooking broth, or a high proportion thereof of xylose. The most efficient xylitol producers 107342 15 are recovered and used for the production of xylitol on an industrial scale.

As a result of the above-described treatment (s), new strains are obtained which yield me-5 to xylose to xylitol in high yield. Because of the deficient xylitol metabolism of the strains, the strains do not degrade (cleave or metabolize or use) the xylitol, but the xylitol is collected in the culture medium and recovered, for example, by chromatography after removal of the yeast cells. Any method known in the art for purifying xylitol from a culture medium may be used (for examples, see US 5,081,026, the disclosure of which is hereby incorporated by reference).

The invention will be described in detail by the following examples, which are not intended to limit the invention. In this context, it is noted that the method is not optimized for xylitol production but for the expression of differences in yeast strains.

Example 1 20 Generation of New Yeast Strains

Kluyyeromyces marxianus strain ATCC 8608 was cultured on YDP, DYM or DYM tartrate medium at pH 6.0 and 30 ° C. YDP (1000 ml) contained 10 g of yeast extract, 20 g of glucose and 20 g of peptone. DYM (1000 mL) contained 5.0 g (NH 2) 2 SO 4, 1.0 g KH 2 PO 4, 0.5 g MgSO 4 · 7H 2 O, 0.1 g NaCl, and 0.1 g. g CaCl2 · 2H2O. The DYM tartrate contained 7.1 g of Na 2 SO 4, 27.6 g of (NH 4) 2-tartrate, 6.8 g of KH 2 PO 4, 0.5 g of MgSO 4 * 7H 2 O, 0.1 g of NaCl and 0.1 g of NaCl. g of CaCl2 * 2H2O. DYM and DYM tartrate growth media were supplemented with micronutrient solution (1: 1000), vitamin solution (1: 100) and carbon source? the last two were added after 'autoclaving'. The sugars and sugar alcohols were autoclaved into Eris as 20% (w / v) solutions and the vitamin mixture was sterilized by filtration. The vitamin solution (1000 ml) contained 35 100 mg of L-histidine * HCl? H 2 O, 0.2 mg of biotin, 40 mg of 107342 16 calcium pantothenate, 0.2 mg of folic acid, 200 mg of inositol, 40 mg of niacin, 20 mg para-aminosulfuric acid, 40 ng pyridoxine * HCl; 20 mg riboflavin and 40 mg plantain HCl. The micronutrient mixture (1000 mL) contained 500 ng of 5 H 3 BO 3, 40 mg of CuSO 4 · 5 H 2 O, 100 mg of K 2, 200 mg of Fed · 6 H 2 O, 400 mg of MnSO 4 * H 2 O, 200 mg of Na 2 Mo 4 · 2H 2 O; a 400 mg ZnSO 4 · 7H 2 O.

A. Mutagenic treatment a) 100 ml of DYM glucose (1% w / v) containing 10, 0.001% (w / v) acriflavin were inoculated with Kluyerveromyces marxianus ATCC 8608 and grown in a nutrient medium for 3 days. The cells were centrifuged, r.e washed with 100 ml of sterile 0.9% NaCl solution; was resuspended in 500 ml of fresh YDP. Cultures over Y4 n15 were further transferred to DYM medium containing 2% glucose for benomyl treatment.

b) Overnight cultures grown on YDP medium for ethylmethanesulfonate (EMS) mutagenesis were centrifuged and concentrated to a cell density of 2 x 10 9 / n].

20 EMS was added to a final concentration of 30 mg / ml and the suspension was incubated for 2 hours 15 minutes. This treatment killed about 70% of the yeast. 2 ml of mutagenized cell suspension was diluted to 100 ml by addition of fresh YDP, needed for 2 days and then further transferred to be-25 DYM medium containing glucose for beomyl treatment.

(c) Similarly, any chemical or physical mutagenization method may be used. Dose s is optimized so that the microbial mortality is 10-30% 100%. After treatment, the microbes are transferred to a growth area or site where growth is optimal. After this, you go to step B.

B. Benomyl Treatment

Cells mutated with acriflavin or EMS and subsequently grown in DYM medium containing 35% glucose were harvested by centrifugation and added to fresh YDP medium at a cell density of 108 / ml. Benomyl (10 mg / ml) was dissolved in dimethyl sulfoxide, the solution was sterilized by filtration and added to 5 cell suspensions to a final concentration of 100 pg / ml benomyl.

The culture was shaken at 30 ° C for 2 days. Cells were then harvested by centrifugation, washed twice with sterile 0.9% NaCl, and resuspended in 5 volumes of fresh YDP. After two 10-day incubations, the culture was transferred to glucose-containing DYM medium for nystatin enrichment. The efficiency of the benomyl treatment was monitored by microscopic estimation of the density of two nucleated cells. Prior to microscopic examination, yeast cells 15 were fixed with acetic acid-formaldehyde alcohol and stained with HCl-Giemsa solution by Streiblova in detail in "Yeast, A Practical Approach", ed. I. Campbell and J. A. Duffus, IRL Press, Oxford, 1988. Similarly, any other compound that interferes with chromosome breakdown or nucleation can be used.

C. Antibiotic Enrichment

The glucose-containing medium, mutagenized and benomyl-treated cells were centrifuged, washed with brine, and resuspended at 10 7 / ml in DYM medium without carbon source. The cells were starved for 8-15 hours, after which 1 g of xylose per 100 ml of cell suspension was added. Nystatin was suspended in ethanol 30 (1 mg / ml) to kill microorganisms (due to its poor solubility it cannot be filtered). The suspension was diluted 1:10 by the addition of sterile water and added to the cultures (10% v / v) 15 hours after the addition of xylose. To enhance the killing effect, another 35 batch of carbon source was added together with nystatin.

• 107342 18

Incubation on a shaker at 30 ° C was continued for 1 hour * 2, after which the cells were harvested and washed with physiological saline. The washed pellet was then resuspended in the original volume of YDI'-5 medium. After the viable yeast cells had recovered (1-3 days), the culture was transferred to DYM medium containing glucose as the sole carbon source. Mortality from nystatin treatment was determined by plate counting (YDP medium) before and after treatment; over 10 99.999% of cells died. In the absence of a carbon source, nystatin killed 0-40% of the cells, respectively. Similarly, another antibiotic may be used whose killing effect is directed exclusively at growing cells.

D. Site-directed Mutagenesis A method for cloning yeast xylitol dehydrogenase has been described [Kötter, P., et al., Curr. Genet. 18: 493-500 (1990)] and a method for cloning a xylulose kinase gene from various yeast species [Ho, N. W. Y., et al., Enzyme Microbiol, Technol. 11: 417-421 (1989); Stev: s, 20 E.P., et al. Appl. Environ. Microbiol. 53: 2975-2977 (1987)]. A suitable source of a dominant selection marker for integrative transformation of yeast is, for example, plasmid pUT332 [Gatignol, A., et al., Gene 91 (1990) 35-41].

A plasmid containing the phleomycin resistance marker inserted into the xy-25 lithol dehydrogenase gene can be constructed by conventional recombinant DNA techniques. Numerous transformation methods can be used to introduce a mutagenized allele of a xylitol-catabolizing gene into a yeast chromosome, for example, by transforming yeast cells treated with lithium chloride or by lysis of the yeast sol-wall with a suitable enzyme preparation (e.g., Lj-ticase). For some strains of yeast, electroporation is not a preferred transformation method. Transformants were selected on 35 plates containing medium rich in nutrients and 5-20 pg / ml of phleomycin and screened for their ability to synthesize xylitol.

E. Screening for Mutants

Potential Mutan-5 titres that had undergone nystatin treatment were plated on DYM agar containing 100 mg glucose and 10 g xylose per liter. Thus, the mutants that were unable to use xylose as their carbon source formed only very small colonies. Xylose-metabolisable yeasts, in turn, received sufficient carbon and therefore formed large colonies. Small colonies were harvested and further cultured on YDP agar. The ability of the isolated mutant strains to grow with glucose, xylose and xylulose was first determined by growing the inoculum overnight in liquid YDP medium. The loop culture was then transferred to 25 ml of DYM tartrate medium containing various carbon sources (glucose, xylose or xylulose). After two or three days, mutant and wild-type growth was measured by determination of A600. Potentially useful mutants grew readily with glucose but were unable to use xylose. Strains with this property were further characterized.

The mutant strains selected for further studies were cultured overnight in YDP medium, then transferred to medium containing 0.5-1.0% glucose as growth medium and 1.0-5.0% xylose to serve as a substrate for xylitol production. Cultures were sampled three and seven days after inoculation for sugar and sugar alcohol analysis. Samples were filtered through 0.2 µm filters to remove cells and precipitates, the filtrates were diluted and analyzed by HPLC. The column used a strong Ca 2+ strong cation exchanger (length 25 cm, diameter 8 mm) loaded with 35 columns of Bio Rad Aminex MicroGuard desalting (Richmond, CA, USA). The column temperature was > 107342 ° C at 85 ° C and was eluted with H 2 O at 0.6 mL / min. The 2r injection volume was 10 μΐ and the elution of the compounds was monitored with an RI detector. External standards were used in the method.

5 Also Kluyveromyces marxianus var. marxianusi, e.g. strain CBSC12, var. bulcraricus, e.g. strain ATCC 16045, and var. 1actis strains using xylose are useful as starting strains. Similarly, any nrx-10 marxianus subspecies or -a3a strain growing on xylose can be used to produce mutants. First, the mutagenization of the yeast strain is optimized with any mutagen. By varying the duration or intensity of the treatment (e.g., chemical mutagen concentration, UV intensity or distance), more than 10% but less than 100% of the organisms are killed. Mutagenized yeasts are grown in the presence of benomyl or some other compound that interferes with nuclear formation (sublethal concentration). Haploic yeast strains do not require benomyl treatment or α-20 response. The culture is transferred to fresh medium using a carbon source as the growth medium, which also allows growth of the desired mutants. When growth begins, the cells are transferred to a medium containing no carbon source for starvation. Xylo or xylitol and growing cells with a lethal antibiotic (for example nystatin) are added. The antibiotic concentration and treatment time are optimized beforehand such that treatment in the presence of xylose kills about 100% of the non-mutated cells but less than 90% without the carbon source. Cells growing with xylose or xylitol die, but the desired mutants survive. Live cells are screened for the correct mutants as described above.

The enzymatic activity of yeast and the productivity of xylitol were determined as described in Examples 2-4., 107342 21

Example 2

New Yeast Strain Enzyme Activity The activities of the major xylose / xylitol metabolism enzymes were determined for parent strain and new yeast strains as follows:

The Kluyveromyces marxianus strain was grown overnight in 50 ml of YDP medium for use as inoculum in one liter of DYM tartrate medium containing 10 g xylose and 20 g glucose as carbon sources and / or inducers. After two days of culture, the culture was centrifuged and the pellet washed with Sörensen's phosphate buffer, pH 7, to remove residual sugar. Cells were resuspended in the same buffer and disrupted by running them 5 times through an X press at -20 ° C. The molten extract li-15 was adjusted to DNase type I (50 mg / ml) and incubated for 1 hour at room temperature. The resulting crude extract was centrifuged at 185,000 x g for 60 minutes. The enzymes to be determined were soluble and remained in the supernatant. The protein content was determined by the Lowry method 20 [J. Biol. Chem. 193: 265-275 (1951)].

Xylose reductase activity was determined by spectrophotometrically monitoring the oxidation of NADPH at 340 nm. The reaction mixture contained 700 μΐ of 50 mM Tris-HCl buffer, pH 7.5, 100 μΐ of 100 mM xylose solution, 25 100 μΐ of diluted cell extract and 100 μΐ of 1 mM NADPH solution. The reaction was initiated by addition of the reduced nucleotide and monitored for 2 minutes. The initial reaction rate was used to determine specific xylose reductase activity.

The xylitol dehydrogenase activity was determined by “following the oxidation of NADH to NAD, a reaction linked to the reduction of xylulose to xylitol. The assay was performed in the same manner as the xylose reductase assay except that xylulose and NADH were used instead of xylose and NADPH.

The results obtained are shown in Table 1.

• 107342 22

Example 3

Formation of xylitoline with new yeast strains

The fermentation steps were carried out as described in Example 1 in a shake flask using an aqueous solution of xylose as the source of xylose. Glucose was added to the fermentation broth as a source of energy and carbon. The vials were shaken and effectively aerated.

The results obtained with the parent cinnamon and the new mutagenized hii '/ a strains are shown in Table 1.

10

table 1

Xylitol Dehydrogenase Activity of Kluyveromyces marxiani is Strains Cultured in Shake Flasks Strain Xylitol-Xylitol Dehydro Yield% Genase (XDH) nmol / min / mg wild 42 330 II-7 64 76 II-8 51 39 20 II-1 60 255 II-1 II-3 54 289 II-4 57 329 II-5 55 315 ·: 25 II-6 57 352

The results obtained with mutants II-1 to II-6 are consistent with the mutation in the xylulose kinase gene Ls, since these hosts still retain xylitol deh 30 d rogenase activity.

Example 4

Xylitol formation on a larger scale

The ability of the parent strain and new yeast strains to produce xylitol on a larger scale process has also been investigated. All chemicals used in fermentation oL

107342 23 technical quality. The xylose source was a soup from two sulphite processes using different hardwood species.

The inoculum was prepared by inoculating the yeast colony into YM-5 broth (100 mL) and growing the culture for one day at 30 ° C on a shaker (200 rpm). The culture was then transferred to 900 ml NH4-tartrate buffered substrate pH 6.0 containing 30 g / liter glucose. The inoculum was grown for another day at 30 ° C in 10 shakers (200 rpm) and then aseptically transferred to the fermenter.

Xylose, xylitol, ethanol and glucose were analyzed by HPLC as described in Example 1. Cell mass was determined by dry weight measurement. The fermentation broth was centrifuged, the cell mass was washed with water and dried overnight at 105 ° C.

The volumetric rate of production of xylitol was determined without taking into account the time required to initiate xylose consumption. The xylitol yield was calculated as the ratio of xylitol produced to consumed xylose.

The fermentation parameters were as follows: pH 6.0 (25% NaOH), stirring 500 rpm, aeration 4.0 Nl / min (vmm) and temperature 30 ° C.

The composition of the medium was as follows: *: 240 g glucose 250 g CSL (corn soak water; 45.5% ka) 20 g (NH4) 2 SO4 20 g (NH4) 2HPO4 8 g KH2PO4 30 8 g MgSO4 ♦ 7H2O 5 liters H2O approximately 600g xylose / 5 liters (12-13%)

The results are shown in Table 2.

«10734:! 24

Table 2

Ability of Kluyveromyces marxianus strains to produce xylitol on a larger scale

Strain Injected Glucose Yield Xylitol 5 g / liter / hour% yield rate g / liter / hour [g / (g / liter / hour /] wild - 35.0 1.6 [0.060] 10 wild 0.80 36.7 1, 9 [0.076] II-7 0.65 68.5 2.8 [0.130] II-7 to 55.0 1.2 [0.094] II-8 0.70 60.0 1.6 [0.09] II -1 0.77 57.0 2.8 [0.09] 15 II-4 0.80 46.0 1.7 [0.07] II-6 0.65 40.1 2.0 [0.10 ]

The specific rate of production of xylitol (g xylitol / g yeast) is shown in parentheses.

The results show that the new yeast strains are excellent xylitol producers as compared to the parent strain.

Thus, using the method of the invention, several mutant strains of Kluyveromyces marxianus were obtained which have significantly lower specific activity of xylitol dehydrogenase (XDH) or xylulose kinase * than both of the previous yeast strains and are thus capable of producing more xylitol. Example 5

Construction of Antisense RNA Yeast Strains by Compound L-30 RNA DNA Methods The yeast xylitol dehydrogenase gene or xylulose kinase gene from a desired yeast host can be cloned according to methods known in the art for cloning these genes [Kötter, P., et al., Curr. Genet. 18: 35493-500 (1993); Ho, N. W. Y., et al., Enzyme MicrobioL.

«I 107342 25

Technol. 11: 417-421 (1989); Stevis, P.E., et al., Appl. Environ. Microbiol. 53: 2975-2977 (1987)]. Alternatively, a xylitol dehydrogenase or xylulose kinase gene cloned from the first yeast species may be used to identify and clone the 5 genes from the second yeast species, provided that these genes are sufficiently complementary to allow for the first hybridization DNA as a hybridization probe was digested with restriction enzymes of another desired yeast host using Southern blot analysis.

From the cloned DNA, a complementary sequence which hybridizes to the coding sequence of the desired enzyme is predicted. This sequence can be synthesized in vitro by chemical means as described in "Oligonucleotide Synthesis, A Practical Approach", by M.J. Gait, ed., IRL Press, Oxford, 1984. The antisense sequence can be obtained even without knowing the exact coding sequence of any of the 20 cloned DNAs targeted to the desired gene sequence. Preferably, such cloned DNA is in double stranded form where one strand is a "sense" (or coding) strand and one strand is an "antisense" (or non-coding) strand. Antisense constructs can be prepared using, ·; By employing techniques described above and known in the art, operably linking such double-stranded DNA to a desired yeast promoter in a manner that results in transcription of an RNA having an antisense (non-coding) sequence. Preferably, the antisense DNA is operably linked to a sequence comprising the yeast xylitol dehydrogenase promoter or the yeast xylulose kinase promoter, wherein the expression of the antisense RNA occurs under the conditions where the target enzyme mRNA is expressed.

107342 26 Such antisense constructs can be introduced into vectors, transformed into yeast hosts, for example yeasts of Candida species. Hansenula, Kluyveromyces, Pichia tel Pachysolen, and preferably Kluyveromyces marxianus and 5 dida utilis and most preferably Kluyveromyces marxianus. marxianus, Kluyveromyces marxianus var. bulgaricus And Kluyveromyces marxianus var. Laet is grown and grown using an antibiotic, for example, as described above for nystates. Yeasts are grown as described above and for further studies mutant strains are selected which are unable to use xylose for growth or which grow very poorly under xylose but which are capable of growing using glucose.

Example 6 Construction of Ribozyme Yeast Strains with Compounds * - DNA Losses

The yeast xylitol dehydrogenase gene or the xyluloc quinase gene from a desired yeast host can be cloned according to methods known in the art [Kötter, P., et al. , 20 Curr. Genet. 18: 493-500 (1990); Ho, N. W. Y., et al., Enzyme Microbiol, Technol. 11: 417-421 (1989); Stevis, P.E., et al., Appl. Environ. Microbiol. 53: 297-2977 (1987)].

Alternatively, the yeast chloro ·; 25 xylitol dehydrogenase or xylulose kinase genes for the identification and cloning of that gene from another yeast species, if these genes are sufficiently complementary to allow for cross-hybridization, for example, as demonstrated in the first 30 from this cloned DNA as a hybridization probe to ge: p-mi DNA digested with restriction enzymes of another target yeast using Southern blot analysis.

The complementary sequence is predicted from the sequence of the cloned 35 DNA which hybridizes to the 2773 coding sequence of the desired enzyme, such as in the context of an antisense RNA construct. This sequence can be synthesized in vitro by chemical means as described in "Oligonucleotide Synthesis, A Practical Approach", by M.J. Gait, 5 eds., IRL Press, Oxford, 1984. Such DNA is preferably obtained from cloned DNA in double-stranded form, with one strand being a "sense" (or coding) strand and one strand being "antisense" (or non- coding) thread.

The ribozyme construct of the invention contains the catalytic activity of a ri-10 bozyme, such as Tetrahymena ribozyme, as disclosed in EP 321,201, and a sufficient length of the antisense RNA sequence directed against the mRNA of the desired target mRNA so that the antisense The RNA structure hybridizes to the target mRNA and causes the ribozyme to cleave the mRNA into too small a form to produce the active enzyme.

Ribozyme constructs can be prepared using techniques described above and known in the art by operably linking such double-stranded DNA to a desired yeast promoter in a manner that results in transcription of the ribozyme antisense RNA. Preferably, the ribozyme anti-sense DNA is operably linked to a sequence comprising the yeast xylitol dehydrogenase promoter or the yeast xylulose kinase promoter such that expression of the ribo *: 25 enzyme antisense RNA occurs under the conditions whereby the target enzyme: .

Such ribozyme constructs can be introduced into vectors, transformed into yeast hosts, for example yeast 30 from Candida, Hansenula, Kluyveromyces, Pichia or 'Pachysolen, and preferably Kluyveromyces marxianus and Cani t dida utilis, and most preferably Kluyveromyces marx. marxianus, Kluyveromyces marxianus var. bulgaricus and Kluyveromyces marxianus var. lactis, and cultured using 35 antibiotics, for example nystatin, as described above. Yeasts are grown as described above and mutant strains that are not capable of using xylose for growth or which grow very poorly with xylose, but which are capable of using growth-glucose, are selected for further studies.

Having now fully described the invention, those skilled in the art will appreciate that the scope of the invention can be accomplished using a wide variety of equivalent conditions, parameters, and the like without affecting the spirit or scope of the invention or any of its embodiments.

• I

Claims (12)

107342 A process for producing xylitol from a yeast host, characterized in that a yeast host capable of synthesizing and metabolizing xylitol is selected, wherein said yeast host is selected from the group consisting of Candida species. Hansenula. Kluvveromvces and Pichia: b. Modifying the expression of one or more enzymes involved in xylitol metabolism in the yeast of step (a) by reducing or eliminating xylitol metabolism; c. cultivating the yeast of step (b) under conditions which produce xylitol synthesis; and 15 d. recovering the xylitol produced in step (c). 2. The method of claim 1, wherein the modification in step (b) deletes or reduces expression of a gene encoding xylitol dehydrogenase or xylulose kinase 20, or a gene encoding both xylitol dehydrogenase and xylulose kinase. 3. A method according to claim 1, characterized in that the modification in step (b) involves exposing the yeast to a mutagen or an agent that interferes with chromosome breakdown or nucleation. 4. A method according to claim 3, characterized in that the agent that interferes with chromosome division or nuclear spindle formation is Benomyl. '· · The process according to claim 2, characterized in that the modification in step (b) is carried out in step (b). involves exposure to a substance that causes insertion, deletion, or point mutation in a yeast gene. 107342 Process according to Claim 5, characterized in that the substance is selected from the group consisting of acriflavine, ethyl methanesulfonate, t-radiation and X-ray radiation. The method of claim 1, characterized in that the modification in step (b) comprises modifying the ability of the yeast host to express one to several enzymes involved in xylitol metabolism by gene manipulation methods, preferably by gene-10 cleavage. A method according to claim 7, characterized in that the gene manipulation method comprises the step (a) of transfecting the yeast with a recombinant DNA construct encoding ribozyme or antisense RNA for xylitol metabolism enzymes. Process according to Claim 1, characterized in that the yeast is selected from the group consisting of Kluvveromvces marxianus and Candida utilis. Method according to claim 9, characterized in that Kluvveromvces marxianus is selected from the group consisting of Kluvveromvces marxianus var. marxianus. Kluvveromvces marxianus var. bulgaricus j s Kluvveromvces marxianus var. lactis. The process according to claim 1, characterized in that the method further comprises the step of growing the yeast in step (b) in the presence of an antibiotic, preferably nystatin. The method according to claim 1, characterized in that step (b) further comprises cultivation and selection in a medium rich in xylose and low glucose. • * 107342