Abstract
BackgroundEfficient bioethanol production from hemicellulose feedstocks by Saccharomyces cerevisiae requires xylose utilization. Whereas S. cerevisiae does not metabolize xylose, engineered strains that express xylose isomerase can metabolize xylose by converting it to xylulose. For this, the type II xylose isomerase from Piromyces (PirXI) is used but the in vivo activity is rather low and very high levels of the enzyme are needed for xylose metabolism. In this study, we explore the use of protein engineering and in vivo selection to improve the performance of PirXI. Recently solved crystal structures were used to focus mutagenesis efforts.ResultsWe constructed focused mutant libraries of Piromyces xylose isomerase by substitution of second shell residues around the substrate- and metal-binding sites. Following library transfer to S. cerevisiae and selection for enhanced xylose-supported growth under aerobic and anaerobic conditions, two novel xylose isomerase mutants were obtained, which were purified and subjected to biochemical and structural analysis. Apart from a small difference in response to metal availability, neither the new mutants nor mutants described earlier showed significant changes in catalytic performance under various in vitro assay conditions. Yet, in vivo performance was clearly improved. The enzymes appeared to function suboptimally in vivo due to enzyme loading with calcium, which gives poor xylose conversion kinetics. The results show that better in vivo enzyme performance is poorly reflected in kinetic parameters for xylose isomerization determined in vitro with a single type of added metal.ConclusionThis study shows that in vivo selection can identify xylose isomerase mutants with only minor changes in catalytic properties measured under standard conditions. Metal loading of xylose isomerase expressed in yeast is suboptimal and strongly influences kinetic properties. Metal uptake, distribution and binding to xylose isomerase are highly relevant for rapid xylose conversion and may be an important target for optimizing yeast xylose metabolism.
Highlights
Efficient bioethanol production from hemicellulose feedstocks by Saccharomyces cerevisiae requires xylose utilization
Performance of other xylose isomerase mutants The results described above indicate that both libraries yielded xylose isomerase from Piromyces (PirXI) mutants that caused accelerated growth on xylose
In view of earlier work on the effect of metals on PirXI activity and the observation that mutations influencing manganese homeostasis can improve xylose metabolism, we initially suspected that the modest activity of the enzyme might be due to suboptimal metal loading and that xylose utilization could be improved by engineering PirXI variants carrying mutations surrounding the metal-binding sites
Summary
Efficient bioethanol production from hemicellulose feedstocks by Saccharomyces cerevisiae requires xylose utilization. Efficient xylose conversion is an important property when selecting or engineering yeast strains to be used in second-generation bioethanol production. A xylose isomerase discovered by Kuyper et al [12, 13] in the fungal strain Piromyces E2 through genome mining (PirXI) is an attractive candidate for xylose isomerization in engineered S. cerevisiae strains, and is used in several studies [14]. In vivo performance of the enzyme is modest, as indicated by the high copy number (up to 10) of the chromosomally inserted XI-encoding gene observed in evolved strains that are capable of anaerobic d-xylose fermentation [15]. The engineering of yeast strains showing faster xylose metabolism is an important challenge in the pursuit of strain improvement for second-generation bioethanol production [17,18,19,20]
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