Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
2000-11-01
2003-05-20
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S041000, C435S061000, C435S194000, C435S233000, C435S243000, C435S252300, C435S455000
Reexamination Certificate
active
06566107
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a recombinant
Zymomonas mobilis
strain, bearing xylose and assimilation and pentose-phosphate pathway genes, having improved fermentation performance on xylose or a mixture of glucose and xylose, useful for the fermentation of the pentoses along with glucose in agricultural and cellulosic biomass to ethanol.
This invention also relates to a method which is facile and effective for constructing improved recombinant Zymomonas strains ATCC31821/pZB5. This invention also relates to the process of using ATCC31821/pZB5 strain for rapid and efficient fermentation of the pentose components in agricultural and cellulosic biomass to ethanol. This invention has a significant impact in decreasing the cost of ethanol production from agricultural and cellulosic biomass feedstocks.
BACKGROUND OF THE INVENTION
Cellulosic biomass is a favorable feedstock for fuel ethanol production because it is both readily available and less expensive than either corn or sugarcane. However, substantial hurdles must be overcome before a typical cellulosic feedstock can be utilized effectively as a substrate for the fermentative production of ethanol. The typical feedstock is comprised of approximately 35%-45% cellulose, 30-40% hemicellulose, 15% lignin and 10% of other components. The cellulose fraction is comprised of polymers of the hexose sugar, glucose. The hemicellulose fraction is comprised mostly of pentose sugars, and substantially of xylose.
Whereas microorganisms are known that can efficiently ferment the glucose component in cellulose, conversion of the xylose in the hemicellulose fraction to ethanol has been difficult and this remains to be one of the economical bottlenecks in the biomass to ethanol conversion scheme. The rapid and efficient utilization of the xylose components in cellulosic biomass is desirable in the development of a commercial process.
Zymomonas mobilis
is a bacterium that has been utilized as a natural fermentative agent in the production of alcoholic beverages, such as pulque and palm wines produced from plant saps. Comparative performance trials have suggested that Zymomonas may become an important industrial ethanol-producing microorganism because of its 5-10% higher yield and up to 5-fold higher productivity compared to traditional yeast fermentations. Because of its potential value, several processes based on the use of Zymomonas for production of industrial ethanol from glucose-based feedstocks have been disclosed in U.S. Pat. Nos. 4,731,329, 4,812,410, 4,816,399, and 4,876,196.
While Zymomonas may become an important fuel ethanol-producing microorganism from glucose-based feedstocks, its substrate utilization range is restricted to fermentation of glucose, sucrose and fructose and, as such, it is not naturally suited for fermentation of the xylose and arabinose components in cellulosic feedstocks. Zymomonas contains the Enter-Douderoff pathway that allows it to ferment glucose very efficiently to ethanol as the sole fermentation product. However, Zymomonas is naturally unable to ferment the xylose in cellulosic biomass because it lacks the essential pentose metabolism pathways. Thus, an opportunity exists to genetically engineer this organism for the fermentation of xylose to ethanol.
Genetic engineering attempts have been made to enhance ethanol production by fermentation by transferring genes from one species to another. For example, see U.S. Pat. Nos. 5,000,000 and 5,028,539. Gene cloning and expression of various enzymes including enzymes for creating a new metabolic pathway are also known. For example see U.S. Pat. Nos. 5,272,073, 5,041,378, 5,168,056 and 5,226,475. However, none of these discoveries has successfully broadened the fermentable substrate range of a microorganism which could not previously ferment pentose sugars to ethanol.
Previous attempts to introduce a pentose catabolic pathway from either Xanthomonas or Klebsiella into Zymomonas have been unsuccessful and the recombinant strains were incapable of growth on xylose as the sole carbon source (Feldmann et al., 1992, Appl. Microbiol. Biotechnol. 38:354-361; Liu et al., 1988. J. Biotechnol. 7:61-77).
The wild-type
Z. mobilis
ferments only glucose, sucrose and fructose and is not able to utilize xylose. Enzymatic analysis indicated that
Z. mobilis
lacks functional pentose metabolic pathways necessary to ferment xylose. The xylose-fermenting
Z. mobilis
has been developed by introduction and expression of four genes encoding xylose-assimilating enzymes, xylose isomerase and xylulokinase as well as pentose-phosphate pathway enzymes, transaldolase and transketolase (Picataggio et al., U.S. Pat. No. 5,514,583).
Despite the success of development of xylose-fermenting Zymomonas strains, the xylose utilization rate is much slower as compared to glucose and often residual xylose is left unfermented under fermentation conditions when the pH is not controlled, or at high sugar concentrations, resulting in lower ethanol productivities and process yields.
SUMMARY OF THE INVENTION
One object of the present invention is to provide a new recombinant xylose-fermenting
Zymomonas mobilis
ATCC31821/pZB5 with improved fermentation performance on xylose or on a mixture of glucose and xylose.
Another object of the present invention is to provide an efficient method to transform the strain ATCC31821 at greater than 1000 CFU/&mgr;g DNA through the use of a methylation-deficient host such as
E. coli
JM110.
A further object of the present invention is to provide a process for use of the recombinant strain ATCC31821/pZB5 to ferment 5% xylose without pH control at about 30° C. with a higher xylose utilization rate to obtain about a 12% increase of fermentation performance of xylose utilization compared to the previous strain CP4(pZB5).
A still further object of the present invention is to provide a process for use of the recombinant strain ATCC31821/pZB5 to ferment 1.5% glucose and 3.5% xylose without pH control that utilizes xylose almost completely (99%) in about 69 hours at about 30° C. to obtain about an 18% increase of fermentation performance of xylose utilization compared to the previous strain CP4 (pZB5) which utilizes only about 84% of the xylose under the same conditions, thus resulting in a 12% increase of the ethanol process yield; and to also provide a 20% increase in average volumetric productivity and a 49% increase in xylose sugar up take rate and a 21% increase in total sugar up take rate using Zymomonas strain ATCC31821/pZB5 compared to the previous strain CP4(pZB5).
A further object yet still of the present invention is to provide a process for use of the recombinant strain ATCC31821/pZB5 to ferment 5% xylose without pH control at about 37° C. with a higher xylose utilization rate to obtain about a 34% increase of fermentation performance of xylose utilization compared to the previous strain CP4(pZB5), thus resulting in a 28% increase of the ethanol process yield; and to also provide a 25% increase in average volumetric productivity and a 28% increase in xylose sugar up take using Zymomonas strain ATCC31821/pZB5 compared to the previous strain CP4(pZB5).
Another object of the present invention is to provide a process for use of the recombinant strain ATCC31821/pZB5 to ferment 1.5% glucose and 3.5% xylose without pH control that utilizes xylose nearly completely (96%) in about 69 hours at about 37° C. to obtain about an 18% increase of fermentation performance of xylose compared to the previous strain CP4 (pZB5) under the same conditions, thus resulting in a 13% increase of the ethanol process yield; and to also provide an 18% increase in average volumetric productivity and a 40% increase in xylose sugar up take rate and a 20% increase in total sugar up take rate using Zymomonas strain ATCC31821/pZB5 compared to the previous strain CP4(pZB5).
Another object yet still of the present invention is to provide a process for use of the recombinant strain ATCC31821/pZB5 to ferment 12% glucose and 3% xylose at about pH=5.5 and about 30° C. that utilizes
Fronda Christian L
Midwest Research Institute
Nashed Nashaat T.
White Paul J.
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