Tryptophan plays an important role in yeast\u2019s tolerance to isobutanol

Hsien-Lin Liu,Wen-Hsiung Li,Chieh-Chen Huang,En-Pei Isabel Chiang,Christine H.-T Wang

doi:10.1186/s13068-021-02048-z

Abstract

BackgroundIsobutanol is considered a potential biofuel, thanks to its high-energy content and octane value, limited water solubility, and compatibility with gasoline. As its biosynthesis pathway is known, a microorganism, such as Saccharomyces cerevisiae, that inherently produces isobutanol, can serve as a good engineering host. Isobutanol’s toxicity, however, is a major obstacle for bioproduction. This study is to understand how yeast tolerates isobutanol.ResultsA S. cerevisiae gene-deletion library with 5006 mutants was used to screen genes related to isobutanol tolerance. Image recognition was efficiently used for high-throughput screening via colony size on solid media. In enrichment analysis of the 161 isobutanol-sensitive clones identified, more genes than expected were mapped to tryptophan biosynthesis, ubiquitination, and the pentose phosphate pathway (PPP). Interestingly, adding exogenous tryptophan enabled both tryptophan biosynthesis and PPP mutant strains to overcome the stress. In transcriptomic analysis, cluster analysis of differentially expressed genes revealed the relationship between tryptophan and isobutanol stress through some specific cellular functions, such as biosynthesis and transportation of amino acids, PPP, tryptophan metabolism, nicotinate/nicotinamide metabolism (e.g., nicotinamide adenine dinucleotide biosynthesis), and fatty acid metabolism.ConclusionsThe importance of tryptophan in yeast’s tolerance to isobutanol was confirmed by the recovery of isobutanol tolerance in defective strains by adding exogenous tryptophan to the growth medium. Transcriptomic analysis showed that amino acid biosynthesis- and transportation-related genes in a tryptophan biosynthesis-defective host were up-regulated under conditions similar to nitrogen starvation. This may explain why ubiquitination was required for the protein turnover. PPP metabolites may serve as precursors and cofactors in tryptophan biosynthesis to enhance isobutanol tolerance. Furthermore, the tolerance mechanism may also be linked to tryptophan downstream metabolism, including the kynurenine pathway and nicotinamide adenine dinucleotide biosynthesis. Both pathways are responsible for cellular redox balance and anti-oxidative ability. Our study highlights the central role of tryptophan in yeast’s isobutanol tolerance and offers new clues for engineering a yeast host with strong isobutanol tolerance.

Highlights

Isobutanol is considered a potential biofuel, thanks to its high-energy content and octane value, limited water solubility, and compatibility with gasoline
To test the importance of tryptophan biosynthesis, we studied whether exogenous tryptophan can recover isobutanol tolerance of tryptophan biosynthesis-mutant strains
An enrichment analysis (DAVID Bioinformatics Resources 6.8) led to cell functions enriched among the 161 genes, where the top 3 categories were genes engaged in tryptophan biosynthesis, ubiquitination, and the pentose phosphate pathway (Fig. 1a)

Summary

Introduction

Isobutanol is considered a potential biofuel, thanks to its high-energy content and octane value, limited water solubility, and compatibility with gasoline. As its biosynthesis pathway is known, a microorganism, such as Saccharomyces cerevisiae, that inherently produces isobutanol, can serve as a good engineering host. Isobutanol, a branched-chain alcohol, is superior to ethanol in properties such as a higher energy content, limited solubility in water and no stress cracking in pipelines. The well-established genetic tools in S. cerevisiae and modern synthetic biology techniques allow for the engineering of yeast hosts for bioproduction of chemicals. As the production of a novel compound creates a stress to the host, it becomes a hurdle in mass production [6] To solve this problem, genetic modifications have been made to obtain tolerant strains [7, 8]. Deletion of GLN3, a transcriptional activator nitrogen catabolite repression system, was found to increase isobutanol tolerance by blocking the nitrogen starvation signal induced by isobutanol, and the yield of isobutanol was increased [10]

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