Serious development of yeasts other than S. cerevisiae has been muted; this is partly because of the ease of genetic transformation of S. cerevisiae and closely related strains with bacterial yeast shuttle vectors. Among nonconventional yeasts, C. she — hatae has, however, properties highly desirable for bioethanol production from lig — nocellulosic substrates:133

• Ethanol production is more efficient from a mixture of glucose and xylose than from either sugar alone.

• Ethanol production can be demonstrated at elevated temperatures (up to 45°C).

• Ethanol formation from xylose is not affected by wide variation in the xylose concentration in the medium.

• Ethanol can be produced from rice straw hemicellulose hydrolysates.

With a straightforward liquid hot water pretreatment of alfalfa fibers, C. shehatae could produce ethanol at a concentration of 9.6 g/l in a batch fermentation with a conversion efficiency of 0.47 g/g sugar consumed; hemicellulose utilization was, however, poor because of the presence of inhibitors.134 The methylotrophic yeast Hansenula poly — morpha can, on the other hand, ferment xylose as well as glucose and cellobiose; this species is thermotolerant, actively fermenting sugars at up to 45°C and with a higher ethanol tolerance than P. stipitis (although less than S. cerevisiae); a vitamin B2 (ribo — flavin)-deficient mutant exhibited increased ethanol productivity from both glucose and xylose under suboptimal riboflavin supply and the consequent growth restriction.135

P. stipitis, the host organism for genes of a xylose metabolism pathway success­fully expressed in S. cerevisiae, has been developed as an ethanologen by a research group at the University of Wisconsin, Madison, since the early 1990s.136 Part of this work was the development of a genetic system for P. stipitis, which was used to endow the yeast with the ability to grow and produce ethanol anaerobically.

P. stipitis is Crabtree-negative and is poorly productive for ethanol. S. cerevisiae derives its ability to function anaerobically from the presence of a unique enzyme, dihydroorotate dehydrogenase (DHOdehase), converting dihydroorotic acid to orotic acid in the pyrimidine biosynthetic pathway for nucleic acids; in S. cerevisiae, DHO — dehase is a cytosolic enzyme catalyzing the reduction of fumaric acid to succinic acid,[25] and the enzyme may constitute half of a bifunctional protein with a fuma — rate reductase or be physically associated with the latter enzyme inside the cell.137 Expression of the S. cerevisiae gene for DHOdehase (ScURAl) in P. stipitis enabled rapid anaerobic growth in a chemically defined medium with glucose as sole carbon source when essential lipids were supplied; 32 g/l of ethanol was produced from 78 g/l glucose in a batch fermentation.138

In mixtures of hexoses and pentoses, xylose metabolism by P. stipitis is repressed, whereas glucose, mannose, and galactose are all used preferentially, and this may limit the potential of the yeast for the fermentation of lignocellulosic hydrolysates; neither cellobiose nor L-arabinose inhibits induction of the xylose catabolic path­way by D-xylose.139 Ethanol production from xylose is also inhibited by the CaSO4 formed by the neutralization of sulfuric acid hydrolysates of lignocellulosic mate­rials with Ca(OH)2, whereas Na2SO4 (from NaOH) had no effect on either xylose consumption or ethanol production and (NH4)2SO4 (from NH4OH) reduced growth but enhanced the xylose utilization rate, the rate of ethanol production, and the final ethanol concentration.140 P. stipitis has been shown to produce ethanol on an array of lignocellulosic substrates: sugarcane bagasse, red oak acid hydrolysate, wheat straw, hardwood hemicellulose hydrolysate, and corn cob fractions.141145

Strains of the thermotolerant Kluyveromyces yeasts are well known to modern biotechnology as vehicles for enzyme and heterologous protein secretion.146 K. marx — ianus is one of the extraordinary biodiversity of microbial flora known to be present in fermentations for the spirit cachaga in Brazil (chapter 1, section 1.2); more than 700 different yeast species were identified in one distillery during a season, although S. cerevisiae was the usual major ethanologen except in a small number of cases where Rhodotorula glutinis and Candida maltosa predominated.147 Strains of K. marxia — nus isolated from sugar mills could ferment glucose and cane sugar at temperatures up to 47°C and to ethanol concentrations of 60 g/l, although long fermentation times (24-30 hr) and low cell viability were operational drawbacks.148 In another study, all eight strains that were screened for D-xylose use were found to be active and one K. marxianus strain was capable of forming ethanol at 55% of the theoretical maximum yield from xylose.149 A medium based on sugarcane molasses was fermented to a final ethanol level of 74 g/l at 45°C, but osmotic stress was evident at high concentra­tions of either molasses or mixtures of sucrose and molasses.150 Brazilian work has shown that K. marxianus is strictly Crabtree-negative, requiring (at least in labora­tory chemostat experiments) the O2 supply to be shut down for ethanol to be formed; a high tendency to divert sugars via the oxidative pentose phosphate pathway may be the major obstacle to this yeast as an ethanologen, but metabolic engineering could be applied to redirect carbon flow for fermentative efficiency.151

Some other yeast species are considered in the next chapter when microbes natu­rally capable of hydrolyzing polysaccharides (or engineered to do so) as well as fer­menting the resulting sugars are considered.