No abstract

Multifactorial approaches can quickly and efficiently model complex, interacting natural or engineered biological systems in a way that traditional one-factor-at-a-time experimentation can fail to do. We applied a Design of Experiments (DOE) approach to model ethanol biosynthesis in yeast, which is well-understood and genetically tractable, yet complex. Six alcohol dehydrogenase (ADH) isozymes catalyze ethanol synthesis, differing in their transcriptional and post-translational regulation, subcellular localization, and enzyme kinetics. We generated a combinatorial library of all ADH gene deletions and measured the impact of gene deletion(s) and environmental context on ethanol production of a subset of this library. The data were used to build a statistical model that described known behaviors of ADH isozymes and identified novel interactions. Importantly, the model described features of ADH metabolic behavior without explicit a priori knowledge. The method is therefore highly suited to understanding and optimizing metabolic pathways in less well-understood systems.

Graphical abstract

Biofuels are a commercial reality with ethanol comprising approximately 10% of the US retail fuel market, and biodiesels contribute a little under 5% to the EU retail fuel market. These biofuels molecules are derived from the fermentation of sugars by yeast (ethanol), and from the chemical modification of animal fats and plant oils (biodiesel). However, these biofuel molecules are chemically distinct from the petroleum fuels that they are blended with. Petroleum-based fuels are predominantly composed of alkane and alkene hydrocarbons. These differences impact on fuel properties and infrastructure compatibility resulting in a 'blend wall' that -without significant infrastructure re-alignment and associated costs -limits the use of biofuels. For this reason, there is great interest in biosynthetic routes for alkane and alkene production. Here we will review the known biological routes to alka/ene biosynthesis with a focus on bacterial alkane and alkene biosynthetic pathways. Specifically, we will review pathways for which the underlying genetic components have been identified. We will also investigate the development of engineered metabolic pathways that permit the production of alkanes and alkenes that are not Page 2 of 19 naturally synthesised in bacteria (heterologous production), but are suitable for industrial commercial application.Finally, we will highlight some of the challenges facing this research area as it moves from proof-of-principle studies towards industrialisation.

Biofuels are a commercial reality with ethanol comprising approximately 10% of the US retail fuel market, and biodiesels contribute a little under 5% to the EU retail fuel market. These biofuels molecules are derived from the fermentation of sugars by yeast (ethanol), and from the chemical modification of animal fats and plant oils (biodiesel). However, these biofuel molecules are chemically distinct from the petroleum fuels that they are blended with. Petroleum-based fuels are predominantly composed of alkane and alkene hydrocarbons. These differences impact on fuel properties and infrastructure compatibility resulting in a 'blend wall' that -without significant infrastructure re-alignment and associated costs -limits the use of biofuels. For this reason, there is great interest in biosynthetic routes for alkane and alkene production. Here we will review the known biological routes to alka/ene biosynthesis with a focus on bacterial alkane and alkene biosynthetic pathways. Specifically, we will review pathways for which the underlying genetic components have been identified. We will also investigate the development of engineered metabolic pathways that permit the production of alkanes and alkenes that are not Page 2 of 19 naturally synthesised in bacteria (heterologous production), but are suitable for industrial commercial application.Finally, we will highlight some of the challenges facing this research area as it moves from proof-of-principle studies towards industrialisation.