Disclosed in the present invention is a Zymomonas integrant and derivatives of these integrants that posses the ability to ferment pentose into ethanol. The genetic sequences encoding for the pentose-fermenting enzymes are integrated into the Zymomonas in a two-integration event of homologous recombination and transposition. Each operon includes more than one pentose-reducing enzyme encoding sequence. The integrant in some embodiments includes enzyme sequences encoding xylose isomerase, xylulokinase, transketolase and transketolase. The Zymomonas integrants are highly stable, and retain activity for producing the pentose-fermenting enzyme for between 80 to 160 generations. The integrants are also resistant to acetate inhibition, as the integrants demonstrate efficient ethanol production even in the presence of 8 up to 16 grams acetate per liter media. These stably integrated sequences provide a unique Zymomonas that may then be used for the efficient conversion of pentose sugars (xylose, arabinose) to ethanol. Method of using the Zymomonas integrants and derivatives thereof in production of ethanol from cellulosic feedstock is also disclosed. The invention also provides a method for preparing a Zymomonas integrant as part of the present invention. The host Zymomonas strain found particularly useful in the creation of these compositions and methods is Zymomonas mobilis 31821.
1. A process for using a genetically modified Zymomonas integrant to produce ethanol from a pentose sugar, comprising:
providing a Zymomonas integrant comprising a sequence encoding more than one pentose-sugar fermenting enzymes incorporated by a first integration event of transposition and a second integration event of homologous recombination, wherein said pentose fermenting enzymes comprises xylose isomerase, xylulokinase, transketolase, and transaldolase;
adding the integrant Zymomonas comprising said sequence encoding to a feedstock comprising pentose under fermentation conditions; and
fermenting the pentose sugar to provide a composition comprising ethanol; said Zymomonas integrant being selected from the group consisting of ATCC 31821-5C Penotaltkt/Pgapxyl-AB and ATCC 31821-5C Pgaptaltkt/Pgapxyl AB.
2. The process of claim 1 wherein the first integration event comprises a transposition integration of a first operon comprising a sequence encoding more than one pentose-fermenting enzyme.
3. The process of claim 2 wherein the first operon includes a sequence encoding transaldolase and transketolase.
4. The process of claim 2 wherein the first operon is further defined as comprising Penotaltkt or Pgaptaltkt.
5. The process of claim 1 wherein the second integration event comprises a homologous recombination with a second operon comprising a sequence encoding more than one pentose-fermenting enzyme.
6. The process of claim 5 wherein the second operon is further defined as comprising PgapxylAB.
7. The process of claim 5 wherein the second operon includes a sequence encoding xylose isomerase and xylulokinase.
8. The process of claim 1 wherein the Zymomonas integrant is ATCC31821-5C Penotaltkt/PgapxylAB.
9. The process of claim 1 wherein the Zymomonas integrant is ATCC31821-5C Pgaptaltkt/PgapxylAB.