Enzymes in a box: Engineering bacterial microcompartments for biotechnological applications

Affordable & clean energy, sustainable cities & communities and climate action are three of the seventeen global goals for sustainable development by 2030. To achieve these goals a great focus is directed towards sustainable, renewable and clean alternatives to petroleum-based fuels and products. On these lines, the utilization of engineered microbes to produce bio-fuels and other bio-degradable chemicals from sustainable biomass is a promising alternative.

Background

Esters (e.g. ethyl acetate, butyl acetate, butyl butyrate) are very important molecules for the industry as they are used for many applications such as flavourings and fragrances and in myriads of products such as solvents, waxes, paints and coatings. Also, they have wide applications in cosmetic products and they are even used as biofuels.

Currently, production of ester compounds is heavily relied on petroleum-based feedstocks whereas only a small fraction is extracted from natural sources. The petroleum-based synthesis methods have many drawbacks, including harmful waste and by-products and high costs. Ester-producing industries are therefore looking for alternatives to switch from petrochemical to renewable feedstocks.

Aim of the project

We have recently identified a novel enzyme family called ethanol acetyltransferase 1 (Eat1) which is able to catalyse the condensation of an alcohol and acetyl-CoA. In this project we aim to use homologs of Eat1 for the in vivo/microbial production of different short-chain esters by combining different alcohols and acyl-CoA substrates. An ideal microbe for the production of such esters is Clostridium beijerinckii as it is a natural producer of alcohols (ethanol, butanol and isopropanol) and acyl-CoAs (acetyl-CoA and butyryl-CoA). CRISPR-Cas9 tools have been developed to engineer C. beijerinckii something that will allow us to proceed with further metabolic engineering in order to optimize the production of ester compounds by eliminating competing pathways and by increasing the carbon flux towards the Eat1 substrates.

Techniques

Multiple techniques are used during your project including in vitro enzymatic activities, protein engineering through random and/or directed mutagenesis, heterologous gene/protein expression in E. coli and C. beijerinckii, metabolic engineering using CRISPR-Cas9, multi-litre controlled fermentations and gas chromatography-mass spectrometry (GC-MS) measurements. In addition, due to the wide application of ester compounds in the industry, you may experience an entrepreneurial environment related to translating our research into business opportunities.