11.08.2015   Advances in Biochemical Engineering/Biotechnology

Zymomonas mobilis for Fuel Ethanol and Higher Value Products

P. L. Rogers1 (И) • Y. J. Jeon1 • K. J. Lee2 • H. G. Lawford3

School of Biotechnology and Biomolecular Sciences, UNSW, 2052 Sydney, Australia p. rogers@unsw. edu. au

2School of Biological Sciences, Seoul National University, 151-742 Seoul, Korea 3Department of Biochemistry, University of Toronto, Toronto Ont., M5S 1A8, Canada

1 Introduction……………………………………………………………………………………………… 264

2 Development of Recombinant Strains of Z. Mobilis…………………………………………. 265

2.1 Increased Substrate Range Through Expression

of a Single Heterologous Gene……………………………………………………………….. 265

2.2 Strain Construction for Utilization of C5 Sugars………………………………………….. 266

2.3 NMR Analysis of Metabolic Characteristics of Recombinant Strains…. 269

2.4 Kinetic Characteristics of Recombinant Strains…………………………………………….. 269

2.5 Kinetic Model Development……………………………………………………………………….. 273

2.6 Effect of Inhibitors in Lignocellulosic Hydrolysates………………………………………. 274

2.7 Application to Industrial Raw Materials……………………………………………………… 275

3 Genome Sequence of Z. Mobilis…………………………………………………………………….. 278

4 Applications for Higher Value Products……………………………………………………… 278

4.1 Metabolites and Related Products………………………………………………………………. 278

4.2 Metabolic Engineering for Organic Acids and TCA Cycle Intermediates. . 279

4.3 Enzyme Based Biotransformations……………………………………………………………… 281

4.3.1 Sorbitol/Gluconate Production…………………………………………………………………… 281

4.3.2 Pharmaceutical Intermediates and Fine Chemicals………………………………………. 282

5 Discussion and Conclusions……………………………………………………………………….. 283

References…………………………………………………………………………………………………….. 286

Abstract High oil prices, increasing focus on renewable carbohydrate-based feedstocks for fuels and chemicals, and the recent publication of its genome sequence, have provided continuing stimulus for studies on Zymomonas mobilis. However, despite its apparent advantages of higher yields and faster specific rates when compared to yeasts, no com­mercial scale fermentations currently exist which use Z. mobilis for the manufacture of fuel ethanol. This may change with the recent announcement of a Dupont/Broin part­nership to develop a process for conversion of lignocellulosic residues, such as corn stover, to fuel ethanol using recombinant strains of Z. mobilis. The research leading to the construction of these strains, and their fermentation characteristics, are described in the present review. The review also addresses opportunities offered by Z. mobilis for higher value products through its metabolic engineering and use of specific high activity enzymes.

Keywords Ethanol production • Glycose/Xylose fermentations • Higher value products • Lignocellulosics • Metabolic engineering • Zymomonas mobilis

1

Introduction

Zymomonas mobilis has attracted considerable interest over the past decades as a result of its unique metabolism and ability to rapidly and efficiently pro­duce ethanol from simple sugars. An early paper by Millis [1] characterized the role which Zymomonas sp. play in causing cider sickness and a compre­hensive review by Swings and DeLey [2] provided much of the background for the subsequent stimulus in research activity in the early 1980s which fol­lowed the first of the “oil price shocks”. Further reviews over the ensuing decades [3-9] included extensive data on genetic and kinetic characteriza­tion of strains of Zymomonas mobilis capable of growing on an increasingly wide range of sugars. In a fine example of metabolic (pathway) engineering, recombinant strains of Z. mobilis were reported in 1995/6 from the National Renewable Energy Laboratory (NREL) Golden, CO, USA, that were capable of the efficient conversion to ethanol of the C5 sugars, xylose and arabinose present in lignocellulosic hydrolysates [10,11]. Most recently, the reporting of the complete genome sequence of Z. mobilis ZM4 (ATCC 31821) [12] has opened up further potential for strain enhancement and for its use for higher value products.

Table 1 provides an outline of the key research milestones which have oc­curred for Z. mobilis over the past three decades with the present review focusing particularly on those developments which have been reported over the past 5-10 years.

Table 1 Zymomonas research milestones

Activity

Period

Refs.

Review of ethanologenic potential of Z. mobilis

Late 1970s

Swings & DeLey [2]

Kinetic confirmation of high rate, high ethanol yields

Early 1980s

Rogers et al. [13] Lee et al. [14]

Batch, continuous and cell recycle evaluations of various strains

Early 1980s

Lavers et al. [15] Lawford et al. [16] Doelle et al. [17]

Development of genetic engineering techniques for Z. mobilis

Early 1980s

Skotnicki et al. [18] Dally et al. [19] Drainas et al. [20]

Table 1 (continued)

Activity

Period

Refs.

Cloning of individual heterologous genes to extend substrate range beyond glucose, fructose and sucrose

Mid 1980s

Carey et al. [21] Goodman et al. [22] Strzelecki et al. [23] Su et al. [24]

Characterization of enzymes in the Entner-Doudoroff Pathway

Mid 1980s

Scopes et al. [25] Neale etal. [26,27]

Cloning of genes to complete pathways for xylose/arabinose utilization

Mid 1990s

Zhang et al. [10] Deanda et al. [11]

Kinetic evaluation of rec strains

Late 1990s/

Joachimsthal et al. [28]

using glucose/xylose/arabinose media

early 2000s

Joachimsthal & Rogers [29] Lawford et al. [30-38] Mohagheghi et al. [39]

Evaluation of industrial lignocellulosic hydrolysates

Early 2000s

Lawford et al. [38,40] Mohagheghi et al. [41]

Publication of complete genome sequence of Z. mobilis ZM4

2005

Seo et al. [12]

Metabolic engineering for efficient succinate production

2006

Kim et al. [42]

Dupont/Broin Partnership announced to develop Zymomonas-based process for ethanol from corn stover

October 2006

Industry report [43]

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