Control of metabolite efflux in microbial cell factories: current advances and future prospects

Abstract

The yield and ease of purification of biotechnological products are typically greatly enhanced if they can be secreted into the external medium. Although not universally appreciated, however, small molecule biotechnological products do not ‘float across’ the phospholipid bilayer portion of biological membranes, and thus they need transporters to assist their passage into the extramembrane and extracellular spaces. Some of these transporters may be reversible, equilibrative transporters that might more normally be used for uptake, while others may have an efflux directionality imposed on them via suitable energy coupling mechanisms. Despite the energetic costs of small molecule synthesis, natural evolution does in fact provide a number of mechanisms by which the secretion of such products can actually enhance fitness. Where available these provide useful starting points, and assaying for such activities is crucial. In particular, in a systems biology approach, we first need to identify such/suitable activities before we can seek to increase them. They may then be improved through promoter engineering, via manipulation of control elements, or by directed evolution of the transporter proteins themselves. Modern methods of synthetic biology provide enormous opportunities for all kinds of efflux transporter engineering; they are just beginning to be realised.

A palette of fluorophores that are differentially accumulated by wild-type and mutant strains ofEscherichia coli: surrogate ligands for bacterial membrane transporters

“…Many are driven by ATP hydrolysis [66] though others obtain free energy via electron-transport-linked membrane energisation. A variety of antiporters can also, under some circumstances, appear to act as 'effluxers' [30,67,68]. TolC is an outer membrane component that interacts with many inner-membrane-located transporters (exemplified by acrAB) [61, [69][70][71][72][73][74].…”

“…It is reasonable that the genetic knockout of an import transporter (including antiporters [30,68]) will tend to result in lower uptake of members of the palette than do knockouts of predominantly efflux transporters, that may be expected to have the opposite effect. We assessed this expectation using three transporters, with the expected general results.…”

Protein‐facilitated transport of hydrophobic molecules across the yeast plasma membrane

“…On the other hand, there might also be an active protein‐mediated process . However, as mentioned in the introduction, recent reviews were published that contradict this notion quite strongly, asserting that membrane passage is only carrier‐mediated . The concept of active protein‐mediated transport of free fatty acids is supported by several facts: (a) fatty acid transport is inducible; (b) it is protease sensitive, or can be blocked with antibodies; (c) the ‘flip’ between the two surfaces of the membrane is rate‐limiting; and (d) cell types with high levels of fatty acid metabolism transport at higher rates .…”

“…Although membrane transporters are involved in one‐third of the reconstructed metabolic networks in yeast, they are still widely neglected . Even in well‐studied model organisms, (predicted) transporters remain orphans in terms of experimental assessment . So far, only 26 crystal structures of the model yeast S. cerevisiae transporters have been obtained although they are predicted to make up 5% of the 6237‐members counting proteome (Transport Classification Database consulted for crystal structures at January 6, 2019) .…”

Structural similarities between some common fluorophores used in biology and marketed drugs, endogenous metabolites, and natural products

“…th ey a r e ca p a b le of efflu xin g a va r iety of flu or escen t p r ob es (e.g. [29][30][31][32][33][34][35][36]). How ever , given th a t m ost of th ese p r ob es a r e con tem p or a r y, syn th etic m olecu les, th e u p ta ke tr a n sp or ter s for w h ich th ey a r e su b str a tes m u st h a ve evolved in n a tu r e for oth er p u r p oses.…”