Lipid Membrane Engineering for Biotechnology

Abstract

The growing scientific knowledge about microorganisms has enabled the development of improved microbial strains currently used in biotechnology. The hyper Acetone-Butanol-Ethanol producer C. saccharoperbutylacetonicum is an attractive cell factory for butanol production. However, the organic acids and solvents accumulation during fermentation affects the membrane fluidity and the stress of the cells, lowering the yields and productivities. Butanol, for example, is thought to permeabilise the cell, compromising not only its viability, but the entire fermentation process. The membrane lipid composition and the stress response are two potential targets to optimise cell factories not only for butanol and weak acids production, but also more generally. With that in mind, C. saccharoperbutylacetonicum N1-4(HMT) was engineered by manipulating the genes involved in phospholipid synthesis, pssA, pgsA and cls. The mt1 gene, coding for an antioxidant enzyme, was also heterologous expressed in C. saccharoperbutylacetonicum N1-4(HMT) to combat any effects in the lipid membrane composition. Finally, the C. saccharoperbutylacetonicum N1-4(HMT) BCL strain, provided by Biocleave Ltd, is a mutant strain that displays a distinct membrane morphology, providing a butanol-producing strain with altered membrane characteristics. The strains in which pssA is knocked out or overexpressed have opposite lipid composition profiles. The strains in which pssA, pgsA and cls is overexpressed produce more phosphatidylethanolamine than the wild type. The strain overexpressing mt1 has significantly more lysophosphatidylethanolamine and less phosphatidylglycerol than the wild type, similarly to the strain with the pssA knockout. The BCL strain had a very similar lipidome as the wild type. The strains overexpressing pssA, pgsA, cls strains and the BCL strain produce similar concentrations of butanol to the wild type. The strains with the pssA knockout and overexpressing mt1 accumulate organic acids and do not produce butanol. Although, the butanol toxicity still needs optimisation, these results suggest a network between membrane composition, cell stress and ABE fermentation and reveal future routes for membrane engineering. However, it must be noted that effects of modulating lipid biosynthesis genes are not always predictable nor beneficial.