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Controlled intracellular trafficking alleviates an expression bottleneck in S. cerevisiae ester biosynthesis.

ABSTRACT: In metabolic engineering, most available pathway engineering strategies aim to control enzyme expression by making changes at the transcriptional level with an underlying assumption that translation and functional expression follow suit. In this work, we engineer expression of a key reaction step in medium chain ester biosynthesis that does not follow this common assumption. The native Saccharomyces cerevisiae alcohol acyltransferses Eeb1 and Eht1 condense acyl-CoAs with ethanol to produce the corresponding ester, a reaction that is rate limiting in engineering ester biosynthesis pathways. By changing the N- and C-termini of Eeb1 to those of Eht1, Eeb1 localization is changed from the mitochondria to lipid droplets. The change has no significant effect on transcription, but increases protein expression by 23-fold thus enabling a 3-fold increase in enzyme activity. This system demonstrates one example of the impact of protein trafficking on functional pathway expression, and will guide future metabolic engineering of ester biosynthesis and, potentially, other pathways with critical membrane-bound enzymes.

SUBMITTER: Zhu J

PROVIDER: S-EPMC6317282 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Controlled intracellular trafficking alleviates an expression bottleneck in <i>S. cerevisiae</i> ester biosynthesis.

Zhu Jie J Schwartz Cory C Wheeldon Ian I

Metabolic engineering communications 20181226

In metabolic engineering, most available pathway engineering strategies aim to control enzyme expression by making changes at the transcriptional level with an underlying assumption that translation and functional expression follow suit. In this work, we engineer expression of a key reaction step in medium chain ester biosynthesis that does not follow this common assumption. The native <i>Saccharomyces cerevisiae</i> alcohol acyltransferses Eeb1 and Eht1 condense acyl-CoAs with ethanol to produc ...[more]

PMID: 30622894

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Project description:BACKGROUND:Aroma is an important organoleptic quality for fruit and has a large influence on consumer preference. Kiwifruit esters undergo rapid and substantial changes contributing to the flavor during fruit ripening. Part of enzymes and their coding genes have been indicated potential candidates for flavor-related esters synthesis. However, there still exist obvious gaps in the biosynthetic pathways of esters and the mechanisms regulating ester biosynthesis in kiwifruit remain unknown. RESULTS:Using gas chromatography-mass spectrometry (GC-MS), volatile compounds of kiwifruit were quantified in response to ethylene (ETH, 100??l/l, 24?h, 20?°C) and 1-methylcyclopropene (1-MCP, 1??l/l, 24?h, 20?°C). The results indicated that esters showed the most substantial changes enhanced by ethylene and were inhibited by 1-MCP. Correlations between RNA-seq results and concentrations of esters, constructed using Weighted Gene Co-Expression Network Analysis (WGCNA) indicated that three structural genes (fatty acid desaturase, AdFAD1; aldehyde dehydrogenase, AdALDH2; alcohol acyltransferase, AdAT17) had similar expression patterns that paralled the changes in total ester content, and AdFAD1 transcripts exhibited the highest correlation. In order to search for potential regulators for ester biosynthesis, 14 previously reported ethylene-responsive transcription factors (TFs) were included in the correlation analysis with esters and their biosynthetic genes. Using dual-luciferase assay, the in vivo regulatory activities of TFs on ester biosynthetic gene promoters were investigated and the results indicated that AdNAC5 and AdDof4 (DNA binding with one finger) trans-activated and trans-suppressed the AdFAD1 promoter. CONCLUSIONS:The present study advanced the molecular basis of ripening-related ester biosynthesis in kiwifruit by identifying three biosynthetic related genes AdFAD1, AdALDH2 and AdAT17 by transcriptome analysis, and highlighted the function of two TFs by transactivation studies.

Dataset Information

Controlled intracellular trafficking alleviates an expression bottleneck in S. cerevisiae ester biosynthesis.

Publications

Controlled intracellular trafficking alleviates an expression bottleneck in <i>S. cerevisiae</i> ester biosynthesis.

Similar Datasets

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