Antonius J. A. van Maris1 • Aaron A. Winkler2 • Marko Kuyper2 •

Wim T. A. M. de Laat3,4 • Johannes P. van Dijken1,2 • Jack T. Pronk1 (И)

department of Biotechnology, Delft University of Technology, Julianalaan 67,

2628 BC Delft, The Netherlands J. T. Pronk@TUDelft. NL

2Bird Engineering B. V., Westfrankelandsedijk 1, 3115 HG Schiedam, The Netherlands

3DSM Anti-Infectives, A. Fleminglaan 1, 2613 AX Delft, The Netherlands

4Royal Nedalco, Van Konijnenburgweg 100, 4612 PL Bergen op Zoom, The Netherlands

1 Introduction……………………………………………………………………………………………… 180

1.1 Saccharomyces cerevisiae and Fermentation of Lignocellulosic Hydrolysates 180

1.2 Introduction of Heterologous Genes Encoding Xylose Reductase

and Xylitol Dehydrogenase: Redox Restrictions………………………………………. 182

1.3 Native D-Xylose-Metabolising Enzymes in S. cerevisiae………………………………….. 185

1.4 One-Step Conversion of D-Xylose into D-Xylulose via Xylose Isomerase. . 186

2 Xylose Isomerase: Properties and Occurrence………………………………………………. 186

3 Expression of Xylose Isomerases in S. cerevisiae:

a Long and Winding Road…………………………………………………………………….. 187

4 Characterisation of Yeast Strains

with High-Level Functional Expression of a Fungal Xylose Isomerase… 190

5 Metabolic Engineering

for Improved Xylose-Isomerase Based D-Xylose Utilisation……………………… 192

6 Evolutionary Engineering

for Improved Xylose-Isomerase-Based D-Xylose Utilisation……………………… 194

6.1 Evolutionary Engineering of D-Xylose-Consuming S. cerevisiae

for Improved Mixed Substrate Utilisation………………………………………………. 194

6.2 Evolutionary Engineering of S. cerevisiae

only Containing Fungal Xylose Isomerase……………………………………………………. 197

7 Towards Industrial Application:

Fermentation Trials with Xylose-Isomerase-Expressing S. cerevisiae. . . . 198

7.1 From the Laboratory to the Real World: Strains and Media…………………………… 198

7.2 Batch Fermentation of Wheat Straw Hydrolysate…………………………………………. 199

7.3 Fed-Batch Fermentation of Corn Stover Hydrolysate…………………………………….. 200

8 Outlook…………………………………………………………………………………………………… 201

References

Abstract Metabolic engineering of Saccharomyces cerevisiae for ethanol production from D-xylose, an abundant sugar in plant biomass hydrolysates, has been pursued vigorously for the past 15 years. Whereas wild-type S. cerevisiae cannot ferment D-xylose, the keto — isomer D-xylulose can be metabolised slowly. Conversion of D-xylose into D-xylulose is therefore crucial in metabolic engineering of xylose fermentation by S. cerevisiae. Ex­pression of heterologous xylose reductase and xylitol dehydrogenase does enable D-xylose utilisation, but intrinsic redox constraints of this pathway result in undesirable byprod­uct formation in the absence of oxygen. In contrast, expression of xylose isomerase (XI, EC 5.3.1.5), which directly interconverts D-xylose and D-xylulose, does not have these constraints. However, several problems with the functional expression of various bacterial and Archaeal XI genes have precluded successful use of XI in yeast metabolic engineer­ing. This changed with the discovery of a fungal XI gene in Piromyces sp. E2, expression of which led to high XI activities in S. cerevisiae. When combined with over-expression of the genes of the non-oxidative pentose phosphate pathway of S. cerevisiae, the result­ing strain grew anaerobically on D-xylose with a doubling time of ca. 8 h, with the same ethanol yield as on glucose. Additional evolutionary engineering was used to improve the fermentation kinetics of mixed-substrate utilisation, resulting in efficient D-xylose utilisa­tion in synthetic media. Although industrial pilot experiments have already demonstrated high ethanol yields from the D-xylose present in plant biomass hydrolysates, strain ro­bustness, especially with respect to tolerance to inhibitors present in hydrolysates, can still be further improved.

1

Introduction

1.1