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Biological membranes: the basics and why they are important in IBBE “Thinking about membranes could improve your bioprocess” Membrane function: an evolutionary perspective • First arose as a free energy barrier between ‘outside’ and ‘inside’ (diffusion) • Keeps cell contents from leaking out and unwanted chemicals getting in • Evolved to permit and regulate the transfer of nutrients and waste products (channels) • Acquired the ability to achieve these functions against a concentration gradient (transporters) • Later developments: conversion of membrane potential to do work; signalling from outside to inside; cell recognition; movement of molecules in eukaryotic vesicles; compartmentalization (eukaryotes) Membrane engineering: opportunities for enhanced productivity Rosanese et al. (1999) J. Cell Biol. “Thinking about membranes could improve your bioprocess” Membrane composition: lipids and proteins • Phospholipid bilayer • Hopanoids act as stiffening agents • False impression: > half the volume of membrane is protein Fluid mosaic models • Flexible phospholipid bilayer interspersed with protein molecules • Fluid – some parts move freely, if not anchored by other cell components • Mosaic – patchwork networks of proteins Chemical toxicity: historical perspective • Damage to cell membrane function implicated in chemical toxicity • Toxicity correlated to hydrophobicity (partition into membrane and lower surface tension) • Membrane fluidity increases Stolte et al. (2007) Green Chem. 9, 1170. The barrier: phospholipids • PE is the major E. coli phospholipid Variation in fatty acid components: stiffening the membrane under stress • Adaptation to starvation, temperature increase and acid stress is associated with membrane stiffening • cis unsaturated FAs have ‘kinked’ chains and pack less well than saturated FAs – increased fluidity Modifying lipid profiles: enhanced tolerance to toxic chemicals • Evolved strains gain tolerance by changing lipid profiles • Maintain optimal membrane fluidity under stress • Unpredictable: E. coli - isobutanol tolerance increased U:S ratio; n-butanol tolerance decreased U:S ratio • Clostridium: solvent tolerance more consistent decreased U:S ratio (abolished U production) • Engineered strain • Pseudomonas cti gene into E. coli Modifying lipid profiles: enhanced tolerance to toxic chemicals Octanoic acid tolerance ‘sweet-spot’ Modifying lipid profiles: enhanced tolerance to toxic chemicals Not only increased tolerance but increased production of octanoic acid and styrene More radical adaptations: engineering membrane fluidity Control gene expression and integrate into cell regulatory networks Crossing Biological Membranes Crossing Biological Membranes Primary transporters Secondary transporters Transporter engineering: threonine producing E. coli Transporter engineering: threonine producing E. coli parent thrA* Dtdh DtdcC::rhtA23 sstT Lee et al. (2009) Microb Cell Fact 8:2 Energetics: membrane potential is crucial Thinking about membranes could improve your bioprocess Engineering the cell-environment interface to improve process efficiency