WILLIAM HOYT HOWE
Pilots at Ogden St, Denver, CO

2003016, Aug 28, 2003

Apr 27, 2002

10/134655

Min Zhang - Lakewood CO, US
Yat-Chen Chou - Golden CO, US
William Howe - Golden CO, US
Christine Eddy - Golden CO, US
Kent Evans - Littleton CO, US
Ali Mohagheghi - Northglenn CO, US

C12P007/06, C12N001/18, C12N015/74

435/161000, 435/483000, 435/254200

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.