Increased Microbial Butanol Tolerance by Exogenous Membrane Insertion Molecules

Abstract

Butanol is an ideal biofuel, although poor titers lead to high recovery costs by distillation. Fluidization of microbial membranes by butanol is one of the major factors limiting titers in butanol-producing bioprocesses. Starting with the hypothesis that certain membrane insertion molecules would stabilize the lipid bilayer in the presence of butanol, we applied a combination of in vivo and in vitro techniques within an in silico framework to describe a new approach to achieve solvent tolerance in bacteria. Single-molecule tracking of a model supported bilayer showed that COE1-5C, a five-ringed oligo-polyphenylenevinylene conjugated oligoelectrolyte (COE), reduced the diffusion rate of phospholipids in a microbially derived lipid bilayer to a greater extent than three-ringed and four-ringed COEs. Furthermore, COE1-5C treatment increased the specific growth rate of E. coli K12 relative to a control at inhibitory butanol concentrations. Consequently, to confer butanol tolerance to microbes by exogenous means is complementary to genetic modification of strains in industrial bioprocesses, extends the physiological range of microbes to match favorable bioprocess conditions, and is amenable with complex and undefined microbial consortia for biobutanol production. Molecular dynamics simulations indicated that the π-conjugated aromatic backbone of COE1-5C likely acts as a hydrophobic tether for glycerophospholipid acyl chains by enhancing bilayer integrity in the presence of high butanol concentrations, which thereby counters membrane fluidization. COE1-5C-mitigated E. coli K12 membrane depolarization by butanol is consistent with the hypothesis that improved growth rates in the presence of butanol are a consequence of improved bilayer stability.

Interactions of a paracyclophane-based conjugated oligoelectrolyte with biological membranes

“…COE1-5C, in contrast, has been shown to rigidify membranes. 5,7,8 Oligophenylenevinylene COEs studied thus far largely contain a planar, two-dimensional conjugated fragment. A molecular structural variation that has yet to be considered concerns introducing dimensionality to the conjugated segment in order to understand possible repercussion of this structural modication on the reorganization of the membrane lipids and the overall perturbation of the bilayer.…”

Influence of molecular structure on the antimicrobial function of phenylenevinylene conjugated oligoelectrolytes

“… 22 , 23 , 28 More recently certain COEs have been shown to stabilize microbial membranes that have been subjected to butanol exposure. 31 …”

A Chain‐Elongated Oligophenylenevinylene Electrolyte Increases Microbial Membrane Stability

“…An example is the fermentative production of butanol, which is economically limited by poor tolerance to butanol . Membrane integrity was previously seen to be improved by supplementation with 5C (see Figure a for chemical structure) using both in silico techniques and by the improved growth of butanol‐exposed cells . This was the first demonstration of a materials approach to engineer butanol tolerance, as opposed to conventional genetic engineering …”

“…To determine the extent to which S6 improves microbial butanol tolerance, E. coli K12 was incubated in Luria Bertani (LB) broth using standardized inoculum sizes of ≈4 × 10 6 CFU mL −1 with twofold increasing butanol concentrations from 0% to 1.8% (v/v), and growth was monitored based on increases in optical density at 600 nm (OD 600 , see Figures S6 and S7, Supporting Information). The results are summarized in Figure b and compared to what is observed with 5C . Negligible increase in the biomass of untreated or 5C ‐treated cells occurs after 15 hour incubation with butanol concentrations higher than 0.9%.…”

“…To the best of our knowledge, S6 contains the longest conjugated framework (≈3.4 nm, Figure S1, Supporting Information) for a COE with suitable topology for specific membrane intercalation . The additional phenylene‐vinylene repeat unit in S6 is expected to favor greater membrane stability over its five aromatic ring counterpart, 5C , and their relative comparison provides useful insight into the relevance of molecular design . To achieve sufficient solubility in aqueous media (>50 mg mL −1 ), six positively charged side chains were necessarily included as terminal groups.…”