Proteomics analysis of metabolically engineered yeast cells and medium-chained hydrocarbon biofuel precursors synthesis.

Xiang Li,Wei Ning Chen

doi:10.1186/s13568-014-0061-8

Abstract

Recently, various biofuels have been synthesized through metabolic engineering approaches to meet the exploding energy demands. Hydrocarbon biofuels, energy-equivalent to petroleum-based fuels, are identified as promising replacements for petroleum. Metabolically engineered Saccharomyces cerevisiae capable of synthesize precursors of medium-chained hydrocarbons is proposed in this study. The hydroperoxide pathway introduced in S. cerevisiae consisted of lipoxygenase (LOX) and hydroperoxide lyase (HPL) from almond, which catalyzes linoleic acid to 3(Z)-nonenal, the precursor for medium-chained hydrocarbon biofuels. Proteomics study showed that 31 proteins displayed different expression levels among four functional strains and most of them were related to carbohydrate metabolism and protein synthesis, suggested prospective capabilities of energy generation and exogenous protein synthesis. Biotransformation efficiency studies carried out by GC-FID were in accordance with the expectations. The highest yield of 3(Z)-nonenal was up to 1.21 ± 0.05 mg/L with the carbon recovery of up to 12.4%.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-014-0061-8) contains supplementary material, which is available to authorized users.

Highlights

Petroleum shortage and environmental concerns have emphasized the synthesis and utilization of renewable fuels (Atsumi et al 2008; Chang and Keasling 2006; Lennen et al 2010)
We used S. cerevisiae to construct whole-cell based catalyst which was capable of synthesize 3(Z)-nonenal through exogenous expressing of 9LOX and 9HPL from almond (Prunus dulcis)
Apart from wild type as control, we used Δpxa1, Δpxa2 and Δpxa1&2 mutants as the hosts to block the translocations of absorbed long-chained fatty acids (LCFAs) into peroxisomes and divert them to the exogenous hydroperoxide pathway

Summary

Introduction

Petroleum shortage and environmental concerns have emphasized the synthesis and utilization of renewable fuels (Atsumi et al 2008; Chang and Keasling 2006; Lennen et al 2010). Hydrocarbons are energy-equivalent to petroleum-based fuels and render no mileage penalty in the procedure of usage. With LOX catalyzing, one peroxy is inserted onto the backbone of linoleic acid and yield one unsaturated acid hydroperoxide (HPOD). We used S. cerevisiae to construct whole-cell based catalyst which was capable of synthesize 3(Z)-nonenal through exogenous expressing of 9LOX and 9HPL from almond (Prunus dulcis). The protein complex, Pxa1p-Pxa2p, which embeds in the peroxisomal membrane, functions as transporter and translocates activated fatty acids into peroxisomes for beta-oxidation, utilizing the energy of ATP hydrolysis. Exogenous genes 9LOX and 9HPL were expressed in S. cerevisiae. Apart from wild type as control, we used Δpxa, Δpxa and Δpxa1&2 mutants as the hosts to block the translocations of absorbed LCFAs into peroxisomes and divert them to the exogenous hydroperoxide pathway. The biotransformation efficiencies of the functional strains were characterized by GC-FID approach and the highest yield of 3(Z)-nonenal we achieved was up to 1.21 mg/L

Materials and methods

Results

Discussion