1. Field of the Invention
The present invention relates generally to methods of using genetically modified microorganisms for the manufacture of useful chemical compounds (metabolic engineering) and more specifically to constructing microbial strains by genetic manipulation that are capable of converting readily available carbon sources, such as D-glucose, into a more valuable product, for example, xylitol.
2. Related Art
Xylitol is a chemical compound of a considerable value as a special sweetener. It is approximately as sweet as sucrose, non-toxic, and non-cariogenic.
Currently, xylitol is produced by chemical hydrogenation of D-xylose. D-xylose is obtained from hydrolysates of various plant materials where it is always present in a mixture with other pentoses and hexoses. Purification of xylose and also xylitol presents therefore a significant problem. A number of processes of this type are known. U.S. Pat. Nos. 3,784,408, 4,066,711, 4,075,406, and 4,008,285 can be mentioned as examples.
The reduction of D-xylose into xylitol can also be achieved in a microbiological process using either strains isolated from nature (Barbosa, M. F. S. et al., J. Industrial Microbiol. 3:241-251 (1988)) or genetically engineered strains (Hallborn, J. et al., Biotechnology 9:1090-1095 (1991)). However, obtaining the substrate, D-xylose, in a form suitable for yeast fermentation is also a considerable problem because inexpensive xylose sources such as sulphite liquor from pulp and paper processes contain impurities which inhibit yeast growth.
An attractive alternative method for the manufacture of xylitol would be obtaining it by fermentation of a cheap and readily available substrate, such as D-glucose. However, no microorganisms are known that produce xylitol in significant amounts during one-step fermentation of any common carbon sources other than D-xylose and D-xylulose, both of which are structurally very closely related to xylitol.
On the other hand, many microorganisms, especially osmophilic yeasts, for example Zygosaccharomyces rouxii, Candida polymorpha, and Torulopsis candida, produce significant amounts of a closely related pentitol, D-arabitol, from D-glucose (Lewis D. H. and Smith D. C., New Phytol. 66:143-184 (1967)). Using this property of osmophilic yeasts, H. Onishi and T. Suzuki developed a method for converting D-glucose into xylitol by three consecutive fermentations (Appl. Microbiol. 18:1031-1035 (1969)). In this process, D-glucose was first converted into D-arabitol by fermentation with an osmophilic yeast strain. Second, the D-arabitol was oxidized into D-xylulose in a fermentation with Acetobacter suboxydans. Finally, the D-xylulose was reduced to xylitol in the third fermentation using one of many yeast strains capable of reducing D-xylulose into xylitol.
An obvious disadvantage of this method is that it involves three different fermentation steps, each taking from 2 to 5 days; further additional steps like sterilization and cell removal are also needed, thus increasing processing costs. The yield of the step fermentation process is low and the amount of by-products is high. Thus, a need still exists for methods for the economical production of xylitol in microbial systems from readily available substrates.
The present invention provides methods for constructing recombinant hosts, and the recombinant hosts constructed thereby, such hosts being capable of producing xylitol when grown on carbon sources other than D-xylulose or D-xylose, and other than polymers or oligomers or mixtures thereof. The carbon sources used by the hosts of the invention are inexpensive and readily available. The microorganisms of the invention are also capable of secreting the synthesized xylitol into the culture medium. This goal is achieved through modification of the metabolism of a desired microorganism, preferably a naturally occurring yeast microorganism, by introducing and expressing desired heterologous genes. This goal is also achieved by further modification of the metabolism of such desired microorganism, so as to overexpress and/or inactivate the activity or expression of certain genes homologous to such microorganism in its native state.
Therefore, it is an object of the invention to provide a method for the production of xylitol, such method utilizing a new and novel microbe strain, a recombinant host, also herein termed a genetically engineered microorganism, as the producer of such xylitol, such genetically engineered microorganism producing such xylitol either de novo or in enhanced amounts when compared the native unengineered microorganism.
It is a further object of the invention to provide a method for the production of xylitol, such method utilizing a novel metabolic pathway that has been engineered into a microorganism and which results in the de novo or enhanced production of xylitol by such microorganism.
It is a further object of the invention to provide a method for the production of xylitol, such method utilizing a novel metabolic pathway as above, and such pathway modifying the pathway of D-arabitol biosynthesis and/or metabolism, such pathway being modified so that the microorganism now produces xylitol from fermentation of carbon sources that the unmodified host utilizes for D-arabitol biosynthesis.
It is a further object of the invention to provide a method for the production of xylitol, such method utilizing the altered D-arabitol pathway above, and such pathway being altered either by the extension of the preexisting pathway for D-arabitol biosynthesis (with additional steps for D-arabitol utilization) or by the substitution of one or more steps of the D-arabitol pathway with similar steps leading to the formation of xylitol.
It is a further object of the invention to provide a method for the production of xylitol, such method utilizing the altered D-arabitol biosynthesis pathway above, and such pathway being altered by extending the pre-existing D-arabitol pathway by the introduction and overexpression of the genes coding for D-xylulose-forming D-arabitol dehydrogenase (EC 1.1.1.11) and xylitol dehydrogenase (EC 1.1.1.9) into an D-arabitol-producing microorganism.
It is a further object of the invention to provide a method for the production of xylitol using a novel microorganism as above, such method utilizing the altered D-arabitol biosynthesis pathway above, and such pathway being altered further, by inactivating, using chemically induced mutagenesis or gene disruption, the gene coding for transketolase (EC 2.2.1.1) or the gene coding for D-xylulokinase (EC 2.7.1.17) in such microorganism.
It is a further object of the invention to provide a method for the production of xylitol using a novel microorganism as above, such method utilizing a genetically-engineered altered overexpression of the genes coding for the enzymes of the oxidative branch of the pentose-phosphate pathway, and specifically D-glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and/or 6-phospho-D-gluconate dehydrogenase (EC 1.1.1.44) in such microorganism.
It is a further object of the invention to provide a method for the production of xylitol using a novel microorganism as above, such method utilizing a genetically-engineered altered overexpression of the genes coding for the enzymes of the oxidative branch of the pentose-phosphate pathway, as well as the D-ribulose-5-phosphate epimerase gene (EC 5.1.3.1).