Page 48 - Advances in bioenergy (2016)

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1.3+ billion dry tons per year of biomass is potentially available in the United States. Two
issues highlight how metabolic engineering can enable industrial utilization of this feedstock:
xylose utilization and cellulose utilization.

Because hydrolysis of lignocellulosic biomass results in 20–30% carbohydrates in the form of
xylose, utilization of pentose sugars is one of the first steps toward efficiently using cellulosic
materials. Saccharomyces cerevisiae, the most productive of ethanologenic organisms, cannot
ferment xylose; it lacks the ability to convert xylose into xylulose, although xylulose is
metabolized within the pentose phosphate pathway (PPP). Transferring the xylose reductase
(XR) and xylitol dehydrogenase (XDH) enzymes from Scheffersomyces stipitis (formerly

Pichia stipitis) enables the growth of yeast on xylose and production of ethanol. 25

However, growth and production are considerably slower than on glucose, and significant
amounts of xylitol are often produced. Xylitol is the intermediate of the XR/XDH pathway, and
most understand this to result from differences in cofactor specificity between reduced
nicotinamide adenine dinucleotide phosphate (NADPH)-dependent XR and nicotinamide
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adenine dinucleotide (NAD)-dependent XDH. The cofactor imbalance has been addressed in
two different ways: use of (1) xylose isomerase pathway or (2) protein mutagenesis to switch
cofactor specificity.

Additional factors to the limited productivity are the lack of dedicated pentose transporters,

low PPP flux, and inability for the cell to identify xylose as a fermentable sugar. 25-27 However,
more recently, progress has been made in these areas through additional metabolic engineering
strategies: introducing heterologous xylose transporters, overexpressing PPP enzymes,
engineering cofactor specificity, and evolutionary engineering (for a comprehensive review
see Matsushika et al.). 26

Cellulose on the contrary is a polysaccharide composed of β(1→4) linked glucose molecules.
Enzymatic digestion is most commonly used to break these chains down into free glucose
molecules. However, this process is somewhat inefficient, requiring a separate enzymatic unit
operation. To improve the efficiency of this process, recently, cellodextrin transporters from
Neurospora crassa were introduced into S. cerevisiae allowing for utilization of cellobiose,
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cellotriose, and cellotetraose. Upon cellodextrin uptake, β-glucosidase breaks down
cellodextrin into monomeric glucose, allowing for immediate catabolism. When coupled with
enzymatic digestion, this process improves utilization efficiency and allows for the
simultaneous saccharification and fermentation of cellulose.

Because of its preference for glucose, S. cerevisiae will natively repress the utilization of
alternative substrates as well as down-regulate fermentative pathways after glucose is
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depleted. This results in inefficient and delayed utilization of alternative substrates like
xylose, a phenomenon known as diauxic shift. However, the cellodextrin pathway seems to
avoid this by eschewing the generation of extracellular glucose. As a result, strains harboring
both cellodextrin and xylose utilization pathways remarkably exhibit the ability to

simultaneously coferment glucose and xylose. 29
Metabolic engineering has shown how organisms can be adapted to utilize originally foreign