Dynamic regulation of fatty acid pools for improved production of fatty alcohols in Saccharomyces cerevisiae

Paulo Gonçalves Teixeira,Raphael Ferreira,Yongjin J Zhou,Verena Siewers,Jens Nielsen

doi:10.1186/s12934-017-0663-3

Abstract

BackgroundIn vivo production of fatty acid-derived chemicals in Saccharomyces cerevisiae requires strategies to increase the intracellular supply of either acyl-CoA or free fatty acids (FFAs), since their cytosolic concentrations are quite low in a natural state for this organism. Deletion of the fatty acyl-CoA synthetase genes FAA1 and FAA4 is an effective and straightforward way to disable re-activation of fatty acids and drastically increase FFA levels. However, this strategy causes FFA over-accumulation and consequential release to the extracellular medium, which results in a significant loss of precursors that compromises the process yield. In the present study, we aimed for dynamic expression of the fatty acyl-CoA synthetase gene FAA1 to regulate FFA and acyl-CoA pools in order to improve fatty alcohol production yields.ResultsWe analyzed the metabolite dynamics of a faa1Δ faa4Δ strain constitutively expressing a carboxylic acid reductase from Mycobacterium marinum (MmCAR) and an endogenous alcohol dehydrogenase (Adh5) for in vivo production of fatty alcohols from FFAs. We observed production of fatty acids and fatty alcohols with different rates leading to high levels of FFAs not being converted to the final product. To address the issue, we expressed the MmCAR + Adh5 pathway together with a fatty acyl-CoA reductase from Marinobacter aquaeolei to enable fatty alcohol production simultaneously from FFA and acyl-CoA, respectively. Then, we expressed FAA1 under the control of different promoters in order to balance FFA and acyl-CoA interconversion rates and to achieve optimal levels for conversion to fatty alcohols. Expressing FAA1 under control of the HXT1 promoter led to an increased accumulation of fatty alcohols per OD600 up to 41% while FFA levels were decreased by 63% compared with the control strain.ConclusionsFine-tuning and dynamic regulation of key metabolic steps can be used to improve cell factories when the rates of downstream reactions are limiting. This avoids loss of precursors to the extracellular medium or to competing reactions, hereby potentially improving the process yield. The study also provides knowledge of a key point of fatty acid regulation and homeostasis, which can be used for future design of cells factories for fatty acid-derived chemicals.

Highlights

In vivo production of fatty acid-derived chemicals in Saccharomyces cerevisiae requires strategies to increase the intracellular supply of either acyl-CoA or free fatty acids (FFAs), since their cytosolic concentrations are quite low in a natural state for this organism
YJZ08 was transformed with the 2 μm plasmid pAOH3 for constitutive strong expression of MmCAR [11] encoding a carboxylic acid reductase from Mycobacterium marinum, which converts long chain FFAs into the respective aldehydes, and ADH5, encoding a native alcohol dehydrogenase from S. cerevisiae, which efficiently reduces long chain aldehydes to the respective alcohols [5] (Fig. 1a)
The resulting strain YZFOH1 was cultivated for 72 h in minimal media with 2% glucose and analyzed by quantification of optical density (OD) and relevant internal and external metabolites, i.e. glucose, ethanol, total FFAs and fatty alcohols

Summary

Introduction

In vivo production of fatty acid-derived chemicals in Saccharomyces cerevisiae requires strategies to increase the intracellular supply of either acyl-CoA or free fatty acids (FFAs), since their cytosolic concentrations are quite low in a natural state for this organism. Deletion of the fatty acyl-CoA synthetase genes FAA1 and FAA4 is an effective and straightforward way to disable re-activation of fatty acids and drastically increase FFA levels This strategy causes FFA over-accumulation and consequential release to the extracellular medium, which results in a significant loss of precursors that compromises the process yield. Byproduct formation arises when fluxes are not properly balanced and pathway precursors or intermediates accumulate in the cell, becoming abundant substrates for side reactions and processes. For this reason, balancing fluxes through enzyme modulation and controlling metabolite pool levels usually play an important role with regard to the final product yield

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Conclusion