Yeast strains for the production of xylitol

Details Yeast strains for the production of xylitol Yeast strains for the production of xylitol

1994-02-07

2001-08-07

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing oxygen-containing organic compound

C435S155000, C435S254100, C435S255100, C435S255400, C435S440000, C435S441000, C435S443000

Reexamination Certificate

active

06271007

ABSTRACT:

FIELD OF THE INVENTION
The invention is in the field of metabolic engineering. Specifically the invention relates to a process for producing xylitol using novel yeast strains wherein xylitol metabolism is modified. The invention further relates to the novel yeast strains, and a process for producing such strains.
BACKGROUND OF THE INVENTION
Xylitol is usually prepared by methods in which a xylane-containing material is first hydrolyzed to produce a monosaccharide mixture containing xylose. The xylose is then reduced to xylitol, usually in the presence of a nickel catalyst. A number of processes of this type have been described in the literature in this field. U.S. Pat. Nos. 3,784,408, 4,066,711, 4,075,406, 4,008,285 and 3,586,537 may be mentioned as examples.
However, all of these priori methods are multi-step processes which are relatively costly and have a relatively low efficiency. The greatest problems reside in obtaining an effective and complete separation of xylose from other hydrolysis by-products. The purification is very exacting because the catalysts used in the reduction reaction of xylose are very sensitive. The purity of the final product, on the other hand, is greatly dependent on how well xylitol can be separated from the other products produced in the reduction reaction.
The production of xylitol by means of a biotechnological process is highly attractive if such process is able to provide a very high quality product by a comparatively cost effective method. However, in order to produce xylitol by yeast fermentation for the above purposes, a yeast strain must be found which is non-pathogenic and which meets the requirements set by the food industry. Further, in order to achieve high yields of xylitol with the aid of yeast fermentation, it is essential to employ a yeast which is capable of reducing xylose to xylitol. Preferably, such strain would also be relatively inefficient in the further metabolic conversion of xylitol.
Many yeast strains produce reductase enzymes that catalyze the reduction of sugars to corresponding sugar alcohols. Many yeasts are also capable of reducing xylose to xylitol as an initial step in their xylose metabolism and several yeast strains are able to use xylose as a sole source of carbon and energy. The reaction route or pathway of xylose utilization for the yeast studied is the following: xylitol is synthesized in the first step wherein xylose is reduced to xylitol with the aid of xylose reductase. Xylitol is then metabolized (utilized) by a series of successive steps. First, xylitol is oxidized to xylulose with xylitol dehydrogenase, xylulose is phosphorylated to xylulose-5-phosphate with xylulose kinase (also called xylulokinase), and then part of xylulose-5-phosphate is converted to pyruvate and ethanol via several intermediate steps. The resultant main products and by-products vary depending on the yeast strain and the fermentation conditions. The reactions are not tightly coupled, and consequently some xylitol is always produced in the medium.
Generally, research in this area has focused on attempts to identify yeast strains with an enhanced ability to produce ethanol rather than xylitol. Nevertheless, xylitol production is a relative common feature among xylose-utilizing yeasts. For example, of 44 yeasts in five genera (Candida, Hansentla, Kluyveromyces, Pichia and Pachysolen), 42 produced some xylitol in the culture media (Barbosa, M. F. S., et al.,
J. Indust. Microbiol
3:241-251 (1988) and
Enzyme Microb. Technol.
10:66-81 (1988)).
It has been suggested to use such strains for the industrial production of xylitol. For example, the industrial use of
Candida tropicalis, Candida guillermondii
and
Candida parapsilosis
has been suggested (PCT applications PCT/F190/00015 (WO 90/08193),
C. tropicalis
, PCT/FI91/00011 (WO 91/10740),
C. tropicalis, and PCT/FI
87/00162 (WO 88/05467),
C. guillermondii
, and French published application 2641545
, C. parapsilosis
). However, all of the above-mentioned Candida strains are potential pathogens and do not meet the requirements of the food industry. Barbosa et al.,
J. Industr. Microbiol.
3:241-251 (1988) describe yeasts screened for the production of xylitol. However, the strains that gave acceptable yields are also all potential pathogens. Therefore, no non-pathogenic strains of yeast that are useful for the production of xylitol have been described that may be utilized in the food industry and/or for production of xylitol on a large scale.
In addition, profitable industrial production of xylitol by the enzymatic conversion of xylose is possible only if the yield is very high. However, no wild yeast strains can achieve this. When different yeast strains were studied in optimum reaction conditions, it was found that certain
Candida tropicalis
strains gave the best yield of xylitol. However, as stated above, the strains of this yeast species are potentially pathogenic and cannot therefore be utilized in the food industry. Species acceptable to the food industry include
Saccharomyces cerevisiae, Candida utilis
and
Kluyveromyces marxianus. Saccharomyces cerevisiae
does not normally express enzymes of the xylose pathway although Hallborn, J. et al.,
Bio/Technology
9:1090 (1991) describe the use of the cloned xylose reductase gene from
Pichia stipitis
for construction of
Saccharomyces cerevisiae
strains capable of converting xylose into xylitol with a claimed yield of 95%.
Mutants defective in xylose utilization have been described. Hagedorn, J. et al.,
Curr. Genet.
16:27-33 (1989) discloses that mutants of the yeast
P. stipitis
were identified that were unable to utilize xylose as the sole carbon source and which were deficient in either xylose reductase or xylitol dehydrogenase. James, A. P. et al.,
Appl. Environ. Microbiol.
55:2871-2876 (1989) discloses mutants of the yeast
Pachysolen tannophilus
that are unable to metabolize D-xylose. Stevis, P. E. et al.,
Appl. Biochem. Biotechnol.
20:327-334 (1989), discloses the construction of yeast xyulokinase mutants by recombinant DNA techniques.
The yeasts
Candida uitilis
and
Kluyveromyces marxianus
have the naturally inducible enzymes xylose reductase, xylitol dehydrogenase and xylulose kinase necessary for the decomposition of xylose. Attempts to adjust the chemical and physical environment of the
Candida utilis
and
Kluyveromyces marxianus
yeasts to increase the xylitol yield have, however, been unsuccessful.
SUMMARY OF THE INVENTION
It has now been found that the xylitol metabolism of yeasts can be modified, whereby novel yeast strains are developed producing xylitol at high yields.
Thus, the invention is first directed to novel yeast strains, such yeast strains being capable of xylitol synthesis from xylose, but deficient in one or more enzymes of xylitol metabolism, such that xylitol accumulates in the culture medium and may be recovered therefrom.
The invention is further directed to methods of producing xylitol using such yeast strains.
The invention is further directed to a method of constructing modified yeast strains that are capable of reducing xylose, and are incapable or deficient in their expression of xylitol debydrogenase and/or xylulose kinase activity.
DETAILED DESCRIPTION
The modified yeast strains of the invention can synthesize xylitol from xylose but are deficient in one or more enzymes of xylitol utilization, such that xylitol accumulates in the medium when the modified yeast host is grown on xylose. Thus, in the novel strains, xylose reductase activity is present, but xylitol metabolism is markedly decreased. Consequently, the novel strains are not capable of using xylose as their sole carbon source, even though xylose induces the activity of xylose reductase and thereby the conversion of xylose to xylitol.
The reaction route of xylose reduction is shown below.
Xylose reductase catalyzes the reaction:
The first enzyme of xylitol utilization, xylitol dehydrogenase, catalyzes the reaction:
The novel yeast strains produced in accordance with the process of the invention produce xylitol f

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