Stable recombinant yeasts for fermenting xylose to ethanol

• US 7,527,927 B1
• Filed: 05/06/1997
• Issued: 05/05/2009
• Est. Priority Date: 05/06/1996
• Status: Expired due to Fees

• Current Assignee: Purdue Research Foundation (The Trustees of Purdue University (d/b/a Purdue University))
• Original Assignee: Purdue Research Foundation (The Trustees of Purdue University (d/b/a Purdue University))
• Inventors: Chen, Zheng-Dao, Ho, Nancy W. Y.
• Primary Examiner(s): Robinson; Hope A
• Application Number:
  US09/180,340
• Time in Patent Office: 4,382 Days
• Field of Search: 435/158, 435/252.3, 435/139, 435/169, 435/105, 435/161, 435/320.1, 435/163, 435/172.3, 435/183, 435/325, 435/254.21, 435/69.1, 530/380, 530/350, 536/23.5, 536/23.7, 536/23.1
• US Class Current: 435/6.18
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 15/905 in yeast
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12P 7/06 Ethanol, i.e. non-beverage
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

Described are recombinant yeast which ferment xylose to ethanol and which maintain their ability to do so when cultured for numerous generations in non-selective media. The preferred yeast contain multiple copies of integrated genes encoding xylose reductase, xylitol dehydrogenase, and xylulokinase fused to promoters which are non-glucose inhibited and which do not require xylose for induction. Also described are preferred methods for integrating multiple copies of exogenous DNA into host cells by transforming cells with replicative/integrative vectors, and then replicating the cells a number of times under selective pressure to promote retention of the vector in subsequent generations. The replicated vectors thus serve to integrate multiple copies of the exogenous DNA into the host cells throughout the replication/selection phase. Thereafter the selective pressure can be removed to promote loss of the vector in subsequent generations, leaving stable integrants of the exogenous DNA.

Claims

1. A method of integrating multiple copies of exogenous DNA into reiterated chromosomal DNA of cells, comprising:

(a) transforming the cells with a replicative and integrative plasmid comprising an autonomous replicating sequence, exogenous DNA, and a first selection marker; and

(b) repeatedly replicating the cells from step (a) to produce a number of generations of progeny cells while selecting for cells which include the selection marker, promoting the retention of the replicative and integrative plasmid in subsequent generations of the progeny cells to produce progeny cells having multiple integrated copies of the exogenous DNA, and wherein the method further includes the step of repeatedly replicating the progeny cells to produce a number of generations of progeny cells in the absence of selection for cells which include the selection marker, so as to promote the loss of the plasmid in subsequent generations of progeny cells and recovering the cells each containing multiple copies of the exogenous DNA integrated into its chromosomal DNA, and wherein the cells are yeast cells and the exogenous DNA includes genes encoding xylose reductase, xylitol dehydrogenase, and xylulokinase, which also serve as the first selection marker.

3. The method of claim 1, wherein the plasmid also includes a second selection marker for selecting cells which include the plasmid.

2. A plasmid vector comprising a functional yeast autonomous replicating sequence and exogenous DNA including genes encoding xylose reductase, xylitol homologous to a reiterated DNA sequence of a target yeast cell, the plasmid vector for use in integrating the exogenous DNA sequence into chromosomal DNA of the target yeast to form stable integrants which ferment xylose to ethanol.