Abstract
BackgroundDecarboxylation of α-ketoisovalerate to isobutyraldehyde is a key reaction in metabolic engineering of Saccharomyces cerevisiae for isobutanol production with published studies relying on overexpression of either the native ARO10 gene or of the Lactococcus lactis kivD decarboxylase gene resulting in low enzymatic activities. Here, we compare relevant properties for isobutanol production of Aro10, KivD and an additional, less studied, L. lactis decarboxylase KdcA.ResultsTo eliminate interference by native decarboxylases, each 2-oxo acid decarboxylase was overexpressed in a ‘decarboxylase-negative’ (pdc1Δ pdc5Δ pdc6Δ aro10Δ) S. cerevisiae background. Kinetic analyses in cell extracts revealed a superior Vmax/Km ratio of KdcA for α-ketoisovalerate and a wide range of linear and branched-chain 2-oxo acids. However, KdcA also showed the highest activity with pyruvate which, in engineered strains, can contribute to formation of ethanol as a by-product. Removal of native decarboxylase genes eliminated growth on valine as sole nitrogen source and subsequent complementation of this growth impairment by expression of each decarboxylase indicated that based on the increased growth rate, the in vivo activity of KdcA with α-ketoisovalerate was higher than that of KivD and Aro10. Moreover, during oxygen-limited incubation in the presence of glucose, strains expressing kdcA or kivD showed a ca. twofold higher in vivo rate of conversion of α-ketoisovalerate into isobutanol than an ARO10-expressing strain. Finally, cell extracts from cultures grown on different nitrogen sources revealed increased activity of constitutively expressed KdcA after growth on both valine and phenylalanine, while KivD and Aro10 activity was only increased after growth on phenylalanine suggesting a difference in the regulation of these enzymes.ConclusionsThis study illustrates important differences in substrate specificity, enzyme kinetics and functional expression between different decarboxylases in the context of isobutanol production and identifies KdcA as a promising alternative decarboxylase not only for isobutanol production but also for other branched-chain and linear alcohols.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0374-0) contains supplementary material, which is available to authorized users.
Highlights
Decarboxylation of α-ketoisovalerate to isobutyraldehyde is a key reaction in metabolic engineering of Saccharomyces cerevisiae for isobutanol production with published studies relying on overexpression of either the native ARO10 gene or of the Lactococcus lactis kivD decarboxylase gene resulting in low enzymatic activities
In view of the industrial relevance of yeast-based isobutanol production and the essential role of decarboxylation in this process, the goal of the present study is to evaluate the suitability of the ‘novel’ 2-oxo acid decarboxylase KdcA from L. lactis B1157 [20], the frequently used KivD from L. lactis IFPL730 [18], and the native S. cerevisiae 2-oxo acid decarboxylase Aro10 [14] for metabolic engineering strategies aimed at constructing efficient isobutanol-producing S. cerevisiae strains
In vitro enzymatic analysis of 2‐oxo acid decarboxylase overexpression The substrate specificity of the decarboxylases encoded by kivD and kdcA has been previously analysed using purified enzyme isolated from L. lactis IFPL730 and using cell extracts via overexpression in E. coli [18, 20]
Summary
Decarboxylation of α-ketoisovalerate to isobutyraldehyde is a key reaction in metabolic engineering of Saccharomyces cerevisiae for isobutanol production with published studies relying on overexpression of either the native ARO10 gene or of the Lactococcus lactis kivD decarboxylase gene resulting in low enzymatic activities. In S. cerevisiae, isobutanol is a natural product of valine catabolism via the Ehrlich pathway [3, 4] In this pathway, valine is first transaminated to yield 3-methyl-2-oxobutanoate (α-ketoisovalerate, KIV), which is subsequently decarboxylated to isobutyraldehyde, whose NAD(P) H-dependent reduction by a yeast alcohol dehydrogenase yields isobutanol. While the three Pdc isoenzymes exhibit a low activity with KIV, their much higher activity and affinity for pyruvate [13] renders them unsuitable for high-efficiency production of isobutanol and other fusel alcohols. When ammonium sulphate is the nitrogen source, the wild-type ARO10 gene is not transcribed and even the expression of ARO10 from a constitutive promoter yields minimal enzyme activity indicating an as yet unknown mechanism of post-transcriptional regulation [13, 14]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
