(28d) Using Biopolymer to Improve Microbial Production of Hydrophobic Compounds

Cellular export of hydrophobic molecules by microbes is frequently reliant on active transport mechanisms, which do not recognize non-native molecules produced by the cells. Consequently, high yielding production of these hydrophobic molecules during engineered biosynthesis is often hindered by toxicity issues from compound accumulation. We hypothesize that sequestering of these molecules using intracellular vesicles may provide a solution to improve microbial production of hydrophobic compounds. “It has been shown recently that lipid droplets in oleaginous yeast can indeed store hydrophobic compounds and improve their production. It was, however, difficult to apply the same strategy in Escherichia coli, which is a workhorse species in this field but cannot be engineered to form lipid droplets efficiently. We were interested in replacing lipid droplets with a different artificial compartment in E. coli for storage of hydrophobic compounds, and had selected polyhydroxybutyrate (PHB) bodies that can be easily constructed in E. coli. The unique amorphous property of these intracellular PHBranules serve as a potential storage system for hydrophobic compounds.

To assess the feasibility of this approach, we tested the in vitro binding efficiency of crystalline PHB granules to selected compounds (lycopene, β-carotene, indigo, quercetin) that represent a range of logP values and colors. Despite the fact that crystalline PHB will likely exhibit weaker physical interactions than amorphous PHB, we obtained encouraging results that demonstrated that binding efficiency was positively correlated to hydrophobicity of the target compound. Subsequently, we proceeded to engineer E. coli strains for co-production of both PHB and terpenoid compounds. So far, preliminary data suggested that intracellularly-produced lycopene might bind to the PHB bodies in vivo. Under some experimental conditions, co-production of PHB bodies also substantially improved lycopene synthesis. Further characterization experiments are ongoing and the results will be presented during the conference.