L. R. Jarboe1,2 (И) • T. B. Grabar1 • L. P. Yomano1 • K. T. Shanmugan1 •

L. O. Ingram1

department of Microbiology and Cell Science, University of Florida,

Gainesville, FL 32611, USA Jarboe@UFL. edu

department of Chemical and Biological Engineering, Iowa State University,

Ames, IA 50011, USA

1 Introduction……………………………………………………………………………………………… 238

2 Engineering and Performance of Ethanologenic E. coli……………………………………. 240

2.1 Ethanologenic Biocatalysts KO11 and LY01………………………………………………… 240

2.1.1 Engineering Scheme………………………………………………………………………………….. 240

2.1.2 Utilized Substrates…………………………………………………………………………………… 242

2.1.3 Limitations and Challenges………………………………………………………………………. 243

2.2 Ethanologenic Biocatalyst, Strain LY168 …………………………………………………….. 243

2.2.1 Conversion of SZ110 to LY168 ………………………………………………………………….. 244

2.2.2 Ethanol Production by LY168 …………………………………………………………………… 244

2.3 Other Recombinant Ethanologenic E. coli Strains…………………………………………… 245

2.4 Non-recombinant Ethanologenic E. coli…………………………………………………………. 246

2.5 Ethanol Production in Organisms Other than E. coli………………………………………. 246

3 Metabolic and Transcriptomic Changes Accompanying Ethanologenicity. 247

3.1 Physiological Differences Conferring Ethanol Resistance to LY01…………………… 248

4 Challenges for Ethanol Production……………………………………………………………… 248

4.1 Cost Effective Growth Media……………………………………………………………………… 248

4.2 Osmolyte Stress Limits Performance in Mineral Salts Media………………………… 249

4.3 Hemicellulose Hydrolysate Contains Inhibitors……………………………………………. 250

4.4 Reducing the Requirement for Fungal Cellulases………………………………………….. 251

5 Application of Ethanol Design Scheme to Other Commodity Products. . 252

5.1 Optically Pure d(-)-and L(+)-Lactic Acid……………………………………………………. 252

5.2 Acetate and Pyruvate………………………………………………………………………………… 253

5.3 Xylitol……………………………………………………………………………………………………… 254

5.4 Succinate………………………………………………………………………………………………….. 255

5.5 L-Alanine…………………………………………………………………………………………………. 255

6 Summary…………………………………………………………………………………………………. 256

References……………………………………………………………………………………………………. 257

Abstract The utilization of lignocellulosic biomass as a petroleum alternative faces many challenges. This work reviews recent progress in the engineering of Escherichia coli and Klebsiella oxytoca to produce ethanol from biomass with minimal nutritional supplemen-

tation. A combination of directed engineering and metabolic evolution has resulted in microbial biocatalysts that produce up to 45 g L-1 ethanol in 48 h in a simple mineral salts medium, and convert various lignocellulosic materials to ethanol. Mutations contributing to ethanologenesis are discussed. The ethanologenic biocatalyst design approach was ap­plied to other commodity chemicals, including optically pure d(-)- and L(+)-lactic acid, succinate and L-alanine with similar success. This review also describes recent progress in growth medium development, the reduction of hemicellulose hydrolysate toxicity and reduction of the demand for fungal cellulases.

Keywords Escherichia coli ■ Ethanol ■ Hemicellulose hydrolysate ■ Lactic acid

1

Introduction

Increasing petroleum costs, together with our increasing dependency on crude oil imports, have provided an opportunity for bio-based fuels and chemicals to become economically competitive. With the development of new technologies, replacement of the current petroleum-based automotive fuels and petrochemicals and supplementation of the national energy supply with sustainable resources, such as plants and plant-derived materials, is now feas­ible. Currently, 65% of the oil consumed in the USA is imported. More than 211 billion gallons, or roughly half of the total US energy consumption, were burned as automotive fuel in 2005 [1]. Therefore, development of an alterna­tive renewable transportation fuel, such as ethanol, will significantly reduce US imported oil dependency, contribute to preservation of finite natural re­sources, and improve the environment.

The use of sugar-derived ethanol as the chief component of automotive fuel was successfully implemented in Brazil nearly three decades ago. While the USA already has automobiles capable of utilizing ethanol blended with gasoline and the infrastructure required to distribute ethanol across the na­tion, ethanol production lags significantly behind the 168 billion gallon do­mestic fuel demand. In 2006, the USA produced approximately 4.9 billion gallons of fuel ethanol [2]. Lignocellulosic materials provide the opportunity to further expand ethanol production.

Lignocellulose is a complex substance that accounts for approximately 90% of the dry weight of plant material. It represents the most abundant renewable energy source in the world and is comprised of cell wall structural polymers (cellulose, hemicellulose, pectin, and lignin) (Fig. 1). Due to the complexity of lignocellulose and the biological limitations of existing biocatalysts, the cur­rent conceptual process designs for lignocellulose-based ethanol production are more complex than starch-based processes. The development of a micro­bial biocatalyst that is capable of metabolizing lignocellulose and all of the constitutive sugars will simplify the process and reduce the cost of ethanol production

The common bacterial ethanol production pathway, shown in Eq. 1 and Fig. 2, does not allow complete, balanced conversion of glucose to ethanol. In contrast, the homoethanol pathway, comprised of pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH), allows balanced production of two ethanol molecules per glucose. The homoethanol pathway is present in yeast, plants, and fungi, but is rare in prokaryotes and animals. Bacterial PDCs have a low pyruvate Km relative to other pyruvate-utilizing enzymes, resulting in effective competition for the pyruvate pool [3]; Km values are indicated for pyruvate-consuming reactions in Fig. 2.

Native E. coli Glucose ^ Ethanol + Acetate + 2 Formate (1)

Homoethanol Glucose ^ 2 Ethanol + 2CO2 (2)

Recombinant expression of the Zymomonas mobilis homoethanol pathway in E. coli was first described nearly 20 years ago and has been previously re­viewed [4-8]; this review will focus on progress made in the past 10 years. Additionally, this review will discuss advances in hemicellulose utilization and the application of the ethanologenic microbial biocatalyst design scheme to successful production of other commodity chemicals.