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

Since the selection of spontaneous mutations during metabolic evolution has been a major component of the development of ethanologenic E. coli, many of the underlying changes contributing to ethanologenesis remain unidentified. Identification of these changes will aid in the development of biocatalysts with desired properties for production of other products.

3.1

Physiological Differences Conferring Ethanol Resistance to LY01

LY01 is a derivative of KO11 that was selected in rich medium for increased ethanol tolerance and yield [18]. As described above, LY01 had greater than 80% survival from brief exposure to 100 gL-1 ethanol, compared to only 10% survival for KO11 [18]. The transcriptomes of these two strains were compared in LB with glucose or xylose and with 0, 10, or 20 gL-1 ethanol [72]. Some 205 genes were differentially expressed in LY01 rela­tive to KO11, as determined by the student’s f-test; 49 of these genes were greater than twofold different in each comparison. Functional groups related to amino acid biosynthesis, cell processes, cell structure, central intermediary metabolism, and energy metabolism contained a high percentage of differ­entially expressed genes. Additionally, many stress-related genes, including those related to acid and osmotic stress, were differentially expressed.

Three major physiological differences between LY01 and KO11 were sug­gested by transcriptome data and supported by further analysis: increased glycine degradation, increased expression of genes related to betaine syn­thesis and uptake of protective osmolytes, and lack of FNR regulatory func­tion [72]. Normally, FNR regulates the expression of genes required for fer­mentation and anaerobic respiration (reviewed in [73]). Glycine metabolism and expression of FNR-regulated genes both impact the availability and dis­tribution of pyruvate. It is interesting to note that betaine synthesis genes are affected by FNR via ArcA [74,75]. Thus, the increased ethanol tolerance of LY01 seems to be a combination of several physiological factors, particularly those related to pyruvate partitioning and osmotic protection.

4

Lignin is separated out after glucose fermentation in the Maxifuel concept. Using a filter-type separator, it is possible to obtain the high dry weight lignin necessary to avoid simultaneous removal of xylose and ethanol still present in the liquid phase after initial hydrolysis and fermentation.

4.4

Fermentation

Biomass or agricultural residues consist of the polymers cellulose, hemicel — lulose, pectin, protein, and lignin. Of the carbohydrate monomers, xylose is second-most abundant after glucose in most plant cell walls [21]. Because the raw material cost is > 50% of the overall cost of the ethanol process, fermen­tation of xylose is needed to improve the yield and lower the production cost of ethanol since many biomasses and agricultural wastes contain xylose, in the order of 10-40% of the total carbohydrate mass. Fermentation of both xy­lose and glucose is therefore crucial to reduce the costs of ethanol production from lignocellulosic raw materials.

The baker’s yeast Saccharomyces cerevisiae is a desired process organism for fuel ethanol production due to its extensive use in current large-scale industrial ethanol production processes. Also, the excellent ethanol produc­tivity and tolerance towards ethanol and the inhibitors found in biomass hydrolysates are important reasons for using this organism, even though its natural xylose utilization capability is poor [22].

In the Maxifuel concept, a pentose and hexose fermenting thermophilic microorganism Thermoanaerobacter BG1 is used to ferment the residual sugars in the hydrolysate left after yeast fermentation [23]. Similar to the in­dustrial yeast strains, the thermophilic microorganism is able to grow under the harsh conditions provided by the hydrolysate whilst fermenting sug­ars efficiently. This genetically modified strain has been shown to produce

38.7 g/L or 5.4% v/v of ethanol in a continuous system running directly with non-detoxified lignocellulosic hydrolysate material. The yield from the process is 0.40 g/g total influent sugar or 78% of the theoretical possible value, and productivity is 0.85 g/L/h. The strain is tolerant to 7% of ethanol and higher dry weight in the pretreatment could be used for reaching this concentration.

Furthermore, it grows in temperatures of up to 75 °C, which eases the dis­tillation of ethanol from the reactor. Operation of the fermentation process at thermophilic conditions counteracts contamination by other bacteria, which is generally a problem for mesophilic yeast fermentation. During the residual sugar fermentation, between 0.5 and 1.1 mol of hydrogen/mol of substrate is produced. This is in the same magnitude as hydrogen yields from ded­icated dark fermentation of complex substrates such as sugar beet extract (1.0-1.7 mol hydrogen/mol substrate) [24] and molasses (0.52-1.58 mol hy­drogen/mol substrate) [8]. BG1 and all its mutants are covered by different patent applications.

To optimize the feasibility of the bioethanol production process the ther­mophilic fermentation is conducted in an immobilized reactor system. The immobilization of the fermenting organism in an up-flow reactor brings an array of important traits to the fermentation process like increased ethanol tolerance, high substrate conversions, and decreased sensitivity towards pro­cess imbalances.

4.5

The observation that most xylose-utilizing fungi produce considerable amounts of xylitol from xylose, and that only species containing also the

NADH-dependent XR activity are capable of producing ethanol from it, sug­gested that the different cofactor preferences of XR and XDH limit ethanolic xylose fermentation by yeast [21,32]. Since S. cerevisiae ferments xylu­lose [1,2], it was suggested that xylose fermentation could be easily achieved by heterologous expression of an XI [32,33]. Indeed, xylose was fermented to ethanol when extracellular XI was added to the medium [33]. This enzyme, with activity not only for xylose but also for glucose, is industrially used for the production of high-fructose corn syrup (HFCS) [18] to convert starch — derived glucose into the sweeter sugar fructose to reduce the sugar demand in the food industry. Heterologous expression of bacterial XI genes in S. cere­visiae proved to be challenging, and for many years no actively expressed enzyme was reported [34-39]. The first functionally expressed XI in S. cere­visiae [40] originated from the bacterium Thermus thermophilus [41]. It was later shown that the low activity of the bacterial XIs in yeast could be par­tially related to intracellular precipitation [39], and it was suggested that the rigid nature of the thermotolerant T. thermophilus XI aided correct folding of the protein in S. cerevisiae. However, the activity of this enzyme at 30 °C was too low to allow xylose fermentation. Still, when combined with other genetic modifications, aerobic growth on xylose was demonstrated by S. cerevisiae carrying the T. thermophilus XI [42] (strain TMB3050, Table 2).

More recently, an XI from the obligate anaerobe rumen fungus Piro — myces [20] was expressed in S. cerevisiae with an activity of about 1 U/mg protein at 30 °C [43] (strain RWB202, Tables 3 and 4). Later, bacterial XIs with high sequence similarity to the Piromyces XI, such as those from Bac- teroides thetaiotaomicron [44] and Xanthomonas campestris [45], were also expressed in S. cerevisiae, but the activity of these enzymes in S. cerevisiae was lower than that of the Piromyces XI. Despite the relatively high activity of Piromyces XI in S. cerevisiae, the expression of this enzyme alone did only al­low very slow growth on xylose [43], suggesting that the conversion of xylose to xylulose does not alone control the xylose metabolism in S. cerevisiae [42]. This observation may also set in a new light the failures of early trials for het­erologous XI expression where, in many cases, functional XI expression was only assayed as growth on xylose [35,37].

2.3

7.1

From the Laboratory to the Real World: Strains and Media

Successful expression of XI in S. cerevisiae enabled further engineering for high-yield production of ethanol from D-xylose under anaerobic conditions. D-Xylose fermentation rates reported for S. cerevisiae strains based on the

Piromyces sp. E2 XI were, in principle, sufficiently high for industrial imple­mentation. However, the studies on these strains that have hitherto been cited in this review were all performed under “academic” conditions. These in­volved the use of defined synthetic media controlled at pH 5.0 and, perhaps most importantly, the absence of inhibitors that are characteristic for real-life plant biomass hydrolysates [31,37,49,54].

The S. cerevisiae strains expressing the Piromyces sp. E2 XI are based on the S. cerevisiae CEN. PK platform. Interestingly, preliminary tests showed that the parental strain CEN. PK113-7D demonstrated an almost similar performance in industrial-grade molasses compared with industrial bak­ers’ yeast strains. Moreover, deletion of the GRE3 gene (which encodes a non-specific aldose reductase, [66]) was not detrimental for performance in molasses-based industrial fermentations (W. de Laat, unpublished data). Therefore, trials to test the glucose/xylose fermenting strain S. cerevisiae RWB 218 [44] were initiated in both wheat straw and corn stover hydrolysates. Results from these fermentation trials will be briefly discussed below.

7.2

E. coli has the capability of utilizing many different sugar substrates and produce a wide spectrum of fermentation products (Fig. 4). However, redirec­tion of a microorganism’s metabolism for the efficient production of a single compound is often far more complex than anticipated. The expression level of multiple genes, which may not be predictable, must be optimized for performance. Our success in generating microbial biocatalysts capable of pro-

ducing high titers of chemicals has been dependent on an approach that utilizes the organism’s natural ability to evolve. Genetically engineered mi­croorganisms require a period of time to adapt to the growth environment. This was accomplished by growing the microbial biocatalysts in the desired mineral salts medium with high sugar concentrations and allowing them to evolve in the new environment. This method has resulted in microbial bio­catalysts proficient in production of ethanol and other commodity products, demonstrating that this approach can be applied to many different microbial biocatalysts to improve the overall efficiency and titer.

Today, the most commonly used biofuels are bioethanol, generated from sugar — and starch-based processes, and biodiesel, generated from animal fats or vegetable oils. As of 2005, worldwide production capacity for bioethanol fuel was about 45 million Lyear-1 [12]. Global capacity for biodiesel is much lower at about 4 millionL [13-16], although certain countries (notably Ger­many) are investing in expanded capacity for this fuel [14]. The installed capacity for both fuels is rising dramatically in the face of high oil prices; biodiesel production has risen by an average of 50% annually between 2000 and 2005, while about 15% annual growth has been observed in bioethanol production over the same period. While biodiesel is increasing in importance, it is clear that bioethanol will remain the dominant biofuel for some years to come.

The simplest way to generate bioethanol is to use yeast to ferment hex — ose sugars such as glucose, which can be obtained directly from agricultural crops such as sugarcane or sugar beet. In Brazil, the sugar-based industry currently has the capacity to produce almost half of the world’s bioethanol supply, or about 17 billion Lyear-1 [12]. Another source of the sugars re­quired for fermentation is starch, produced in corn, wheat, and other cereal crops. Starch must be broken down through acid or enzymatic hydrolysis in order to release glucose, which can then be fermented to bioethanol [6]. Both sugar and starch-based processes are employed in Europe, with France (629 millionL) and Spain (520 million L) currently leading production [12]. In North America, corn (or maize) is currently the dominant biomass source for the bioethanol industry, due in part to the high proportion of starch found in its kernels and its high yield per hectare in comparison to other cereal crops. Corn, like sugarcane or switchgrass, is a C4 plant, which can utilize an extra carbon molecule in the photosynthetic process as compared to wheat or trees, which are C3 plants. Warmer growing conditions found across the USA favor C4 plants, while cooler regions (including the Canadian prairie) are well-suited to C3 plant production. Comparatively, C4 plants have relatively high water efficiency, while C3 plants have the ability to increase photosynthetic activity in the presence of elevated CO2 levels. Thus, growing conditions in any given year will determine optimal bioethanol feedstocks for specific regions [17].

The USA has a bioethanol production capacity of over 18 billion L [18], while Canada’s bioethanol production capacity is currently about 245 million L but expected to grow to more than 1 bill L by 2008 [15]. Various other coun­tries around the world have increased bioethanol production significantly since the mid-1990s. The dominant emerging bioethanol producers include China, which is home to Jilin Fuel Alcohol, the world’s largest corn-based bioethanol plant with a current capacity in excess of 350 million L year-1. The development of biofuel capacity over the past quarter century may be seen in Fig. 1.

As bioethanol is the most dominant biofuel found today, it is useful to look at the policies that supported development of this fuel in different jurisdic­tions around the world, and to evaluate the impact that different policies may have on creating increases in production capacity.

2.1

Reducing xylitol formation has been a major challenge in xylose fermentation by recombinant S. cerevisiae carrying the P. stipitis xylose pathway enzymes XR and XDH. Xylitol formation has primarily been ascribed to the differ­ence in cofactor requirements of the two enzymes, so that the intracellular concentration of NAD+ controls the amount of xylitol being converted to xy­lulose [21,32,47,74-76]. However, xylitol formation during ethanolic xylose fermentation also depends on the strain background, i. e., the metabolism of the host cell, since for example some strains of P. stipitis do not produce xyl­itol [47,49,50]. Thus, engineering the redox metabolism of the S. cerevisiae host has been given great attention where the aim primarily has been to ma­nipulate the intracellular concentrations and fluxes of cofactors to minimize xylitol formation.

4.5.1

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.

There has recently been considerable interest in the redirection of metabolism in bacteria such as E. coli for the overproduction of specific metabolites and higher value products. At a commercial level, the large-scale production by Tate & Lyle/Dupont of 1,3-propandiol using a highly engineered strain of E. coli is indicative of an increasing trend towards such bio-based pro­cesses. The fast specific rates of sugar uptake by Z. mobilis, its highly efficient metabolism for a specific product (ethanol), and its relatively small genome size (facilitating genetic manipulation) may make it an ideal candidate for producing other metabolites via its genetic engineering.

As shown in Fig. 7, Z. mobilis has an incomplete TCA cycle and the po­tential exists via “knock out” mutation to redirect metabolism away from end-products such as lactate and ethanol, towards higher value products like succinic acid. As reported recently by Kim et al. [42], succinic acid overproducing Z. mobilis strains have been developed by disruption of the genes for pyruvate decarboxylase (pdc) and lactate dehydrogenase (ldh). Such strains can produce relatively high concentrations of succinic acid at yields of 1.73 mole/mole glucose (86% theoretical). The yield was reported to be more than 30% greater when compared to those of other succinic acid — producing bacteria such as Actinobacillus succinogenes and Mannheimia suc- ciniciproducens (about 1.34 mole/mole glucose). These strains of Z. mobilis were also reported to exhibit higher overall rates of succinic acid production (1.62 gL-1 h-1) under Na bicarbonate supplemented conditions compared to those of other succinic acid producing bacteria (1.35 gL-1 h-1).

Entner-Doudoroff Pathway

f

I

2-keto-3-deoxy-6-P-gluconate

4.3