Despite the status of S. cerevisiae as a proven industrial microorganism, con­ferring the ability to rapidly convert pretreated cellulose to ethanol is a daunt­ing proposition. Apart from essential traits, such as high ethanol yield and productivity, industrial strains need to concurrently ferment both hexoses and pentoses under robust industrial conditions that require minimum nutri­ent requirements and high ethanol and inhibitor tolerance. In addition, these strains also have to hydrolyze cellulosics and thus need to produce and se­crete heterologous hydrolases at high enough levels to sustain hydrolysis and fermentation of cellulosics to ethanol (Table 1). Before contemplating these

challenges, it is worth considering the evolutionary development of S. cere­visiae as microorganism of choice for ethanol production.

Through the serendipitous duplication of its entire genome about 100 mil­lion years ago, followed by the further duplication of the alcohol dehydroge­nase (ADH) genes < 80 million years ago, the S. cerevisiae sensu stricto yeast (comprised of 14 Saccharomyces species) adapted the “make-accumulate — consumption strategy” for ethanol production [27,28]. This ability is largely attributed to its overriding glucose repression circuit that suppresses respi­ration of glucose and other C6 sugars above 20-40 mM threshold concentra­tions in the presence of oxygen, a characteristic feature of Crabtree-positive yeasts [29]. This strategy provided the ancestor of S. cerevisiae with an ad­vantage over its competitors because high ethanol levels (concentrations ex­ceeding 4% v/v) are toxic to most other microbes. Once S. cerevisiae has colonized a niche by producing ethanol levels often exceeding 10% v/v from readily available hexoses, the produced ethanol is reconsumed if oxygen is present. These yeasts therefore developed two distinct alcohol dehydrogenase enzymes through the duplication of the ADH genes for the production and

Table 1 Features required from S. cerevisiae as successful CBP microorganism (modified from [2,26])

Suitability of currently available strains of S. cerevisiae

Essential traits:

Only hexoses by native industrial strains. Partial pentose utilization has been engineered in some laboratory and industrial strains Most industrial strains Most industrial strains Most industrial strains

Primarily multicopy expression in laboratory strains

Laboratory and some industrial strains

Manipulated laboratory and some industrial

strains (maltose and glucose utilization)

Most laboratory and industrial strains

Most industrial strains

Some industrial strains, particularly wine

strains

Laboratory and some industrial strains

subsequent utilization of the ethanol: Adh1 that is constitutively produced and is required for ethanol production, and Adh2 that is only induced in the absence of C6 sugars and is necessary for ethanol consumption.

Regardless of the processes used for biomass hydrolysis, CBP-enabling microorganisms may encounter a variety of toxic compounds derived from biomass pretreatment and hydrolysis that could inhibit microbial growth, particularly in the presence of ethanol [30]. However, industrial strains of S. cerevisiae have been adapted to handle stress conditions, such as high ethanol and sugar concentrations (hence osmotolerance), in fermenting sim­ple hexoses (glucose, fructose, galactose, and mannose) or disaccharide (su­crose and maltose) streams. It also has a natural hardiness against inhibitors and has the ability to grow at low oxygen levels. These features confer to S. cerevisiae a general robustness in industrial process conditions [28]. S. cere­visiae has proven itself as a robust ethanol producer in traditional large — scale processes, and therefore presents itself as platform organism for plant biomass conversion to products such as ethanol [2].

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