Engineering the bilayer: Emerging genetic tool kits for mechanistic lipid biology

Abstract

The structural diversity of lipids underpins the biophysical properties of cellular membranes, which vary across all scales of biological organization. Because lipid composition results from complex metabolic and transport pathways, its experimental control has been a major goal of mechanistic membrane biology. Here, we argue that in the wake of synthetic biology, similar metabolic engineering strategies can be applied to control the composition, physicochemical properties, and function of cell membranes. In one emerging area, titratable expression platforms allow for specific and genome-wide alterations in lipid biosynthetic genes, providing analog control over lipidome stoichiometry in membranes. Simultaneously, heterologous expression of biosynthetic genes and pathways has allowed for gain-of-function experiments with diverse lipids in non-native systems. Finally, we highlight future directions for tool development, including recently discovered lipid transport pathways to intracellular lipid pools. Further tool development providing synthetic control of membrane properties can allow biologists to untangle membrane lipid structure-associated functions.

Keywords: Lipids; Membranes; Metabolic engineering; Synthetic biology.

Figure 1.. The metabolic engineering pipeline to investigate lipid function.

Once well characterized, lipid biosynthetic pathways can be predictably modulated through the titration of enzyme levels carrying out rate-limiting steps or knockdown (KOs) of genes encoding non-essential enzymes. The former is done through engineered control systems which allow for controllable expression levels, primarily through the replacement or targeting of the endogenous promoter. The chemical outputs of engineered systems are first characterized through mass spectrometry-based lipidomics. Lipid composition defines the resulting biophysical properties, which can also be measured by spectroscopic and imaging approaches. These systems can then be used to investigate the mechanisms by which lipid composition acts in cells. Examples of this approach include the elucidation of how acyl chain unsaturation controls cellular respiration and how headgroup composition controls TM protein topology .

Figure 2.. Examples of successful incorporation of heterologous lipid synthesis pathways in non-native hosts.

Row 1: Branched chain fatty acids from B. subtilis produced in E. coli next to native unbranched fatty acid . Row 2: Isoprenoid linked ether lipids from Archaea produced in E. coli next to native fatty acid ester . Row 3: Mono- and di-glucosyl glycerolipids from A. thaliana produced in E. coli next to native phosphatidylethanolamine [38]. Row 4: Campesterol and mannosyl glycosyl inositol phosphorylceramide (GIPC) [49] from A. thaliania produced in S. cerevisiae next to native ergosterol and mannosyl di-inositol phosphorylceramide (MIP₂C). Row 5: Polyunsaturated fatty acid from C. elegans produced in D. melanogaster next to a native unsaturated fatty acid . Row 6: Ganglio-series glycosphingolipid GM3 structure from M. musculus produced in D. melanogaster next to native arthro-series glycosphingolipid [53].