Membrane engineering via trans unsaturated fatty acids production improves Escherichia coli robustness and production of biorenewables

Abstract

Constructing microbial biocatalysts that produce biorenewables at economically viable yields and titers is often hampered by product toxicity. For production of short chain fatty acids, membrane damage is considered the primary mechanism of toxicity, particularly in regards to membrane integrity. Previous engineering efforts in Escherichia coli to increase membrane integrity, with the goal of increasing fatty acid tolerance and production, have had mixed results. Herein, a novel approach was used to reconstruct the E. coli membrane by enabling production of a novel membrane component. Specifically, trans unsaturated fatty acids (TUFA) were produced and incorporated into the membrane of E. coli MG1655 by expression of cis-trans isomerase (Cti) from Pseudomonas aeruginosa. While the engineered strain was found to have no increase in membrane integrity, a significant decrease in membrane fluidity was observed, meaning that membrane polarization and rigidity were increased by TUFA incorporation. As a result, tolerance to exogenously added octanoic acid and production of octanoic acid were both increased relative to the wild-type strain. This membrane engineering strategy to improve octanoic acid tolerance was found to require fine-tuning of TUFA abundance. Besides improving tolerance and production of carboxylic acids, TUFA production also enabled increased tolerance in E. coli to other bio-products, e.g. alcohols, organic acids, aromatic compounds, a variety of adverse industrial conditions, e.g. low pH, high temperature, and also elevated styrene production, another versatile bio-chemical product. TUFA permitted enhanced growth due to alleviation of bio-product toxicity, demonstrating the general effectiveness of this membrane engineering strategy towards improving strain robustness.

Improved n-butanol production via co-expression of membrane-targeted tilapia metallothionein and the clostridial metabolic pathway in Escherichia coli

“…Since the cellular membrane is a vital factor that allows cells to acclimate to external stresses and is also one of the components that is strongly affected by organic solvents [47], many studies have proposed that the plasma membrane is the most affected target of organic solvents and plays a significant role in adapting to stress. Additionally, the length of the carbon backbone of the organic solvent can alter the toxicity mechanism; increasing the hydrophobicity of the solvent increases the level of toxicity [48].…”

“…Long- and short-chain alcohols cause stress during biofuel production by altering membrane fluidity (also known as Overton’s Rule). Ethanol and n-butanol, respectively, decrease and increase membrane fluidity [47, 49, 50]. …”

Systematic engineering of pentose phosphate pathway improves Escherichia coli succinate production

“…With the goal of relieving the transcriptional repression and thus increasing expression levels of PPP enzymes, we next employed the RBSL approach to replace the native promoter of all PPP enzymes by the constitutive M1-93 artificial promoter [29, 30] with varied RBS sequence (Fig. 2; Additional file 2: Table S2).…”

Emerging tools, enabling technologies, and future opportunities for the bioproduction of aromatic chemicals

“…For instance, in addition to damaging cell membranes, p ‐coumaric acid is reported to also interact with DNA, negatively affecting replication and transcription . As has been done for other inhibitory bioproducts, progress is also being made towards engineering cell robustness towards aromatic bioproducts by modifying the cell envelope . In this review, we will discuss important cell envelope engineering strategies of general utility for increasing resistance to membrane disrupting compounds, particularly highlighting those studies specifically involving aromatic bioproducts.…”