Continuous expansion of a synthetic minimal cellular membrane

A critical aspect of a synthetic minimal cell is expansion of the surrounding boundary layer. This layer should consist of phospholipids (mimics) as these molecules assemble into a bilayer, creating a functional barrier with specific phospholipid species that are essential for membrane related processes. As a first step towards synthetic cells, an in vitro phospholipid biosynthesis pathway has been constructed that utilizes fatty acids as precursors to produce a wide variety of phospholipid species, thereby driving membrane growth. This now needs to be developed further into a sustainable expanding system, meanwhile keeping simplicity in mind. The non-enzymatic synthesis of phospholipid-like molecules forms a realistic alternative for natural enzymatic-based pathways, that nowadays can even support functional membrane proteins. Eventually, coupling to in vitro transcription/translation is required, for which efficient mechanisms of insertion and folding of the involved membrane proteins need to be developed. Such an integrated system will form a suitable foundation of a synthetic minimal cell that eventually can be coupled to other cellular processes such as division

Phospholipid dependency of membrane protein insertion by the Sec translocon

Membrane protein insertion into and translocation across the bacterial cytoplasmic membrane are essential processes facilitated by the Sec translocon. Membrane insertion occurs co-translationally whereby the ribosome nascent chain is targeted to the translocon via signal recognition particle and its receptor FtsY. The phospholipid dependence of membrane protein insertion has remained mostly unknown. Here we assessed in vitro the dependence of the SecA independent insertion of the mannitol permease MtlA into the membrane on the main phospholipid species present in Escherichia coli. We observed that insertion depends on the presence of phosphatidylglycerol and is due to the anionic nature of the polar headgroup, while insertion is stimulated by the zwitterionic phosphatidylethanolamine. We found an optimal insertion efficiency at about 30 mol% DOPG and 50 mol% DOPE which approaches the bulk membrane phospholipid composition of E. coli.</p

Phospholipid dependency of membrane protein insertion by the Sec translocon

Membrane protein insertion into and translocation across the bacterial cytoplasmic membrane are essential processes facilitated by the Sec translocon. Membrane insertion occurs co-translationally whereby the ribosome nascent chain is targeted to the translocon via signal recognition particle and its receptor FtsY. The phospholipid dependence of membrane protein insertion has remained mostly unknown. Here we assessed in vitro the dependence of the SecA independent insertion of the mannitol permease MtlA into the membrane on the main phospholipid species present in Escherichia coli. We observed that insertion depends on the presence of phosphatidylglycerol and is due to the anionic nature of the polar headgroup, while insertion is stimulated by the zwitterionic phosphatidylethanolamine. We found an optimal insertion efficiency at about 30 mol% DOPG and 50 mol% DOPE which approaches the bulk membrane phospholipid composition of E. coli.</p

Announcing Molecular Biomedicine

Molecular Biomedicine is a peer-reviewed and open- access journal launched by Springer Nature in 2020, publishing the pioneer works in molecular medicine. We appreciate for the sincere support of many talented sci- entists and academic institutions worldwide that inspired us to start this journal

Engineering of Pentose Transport in Saccharomyces cerevisiae for Biotechnological Applications

Lignocellulosic biomass yields after hydrolysis, besides the hexose D-glucose, D-xylose, and L-arabinose as main pentose sugars. In second generation bioethanol production utilizing the yeast Saccharomyces cerevisiae, it is critical that all three sugars are co-consumed to obtain an economically feasible and robust process. Since S. cerevisiae is unable to metabolize pentose sugars, metabolic pathway engineering has been employed to introduce the respective pathways for D-xylose and L-arabinose metabolism. However, S. cerevisiae lacks specific pentose transporters, and these sugars enter the cell with low affinity via glucose transporters of the Hxt family. Therefore, in the presence of D-glucose, utilization of D-xylose and L-arabinose is poor as the Hxt transporters prefer D-glucose. To solve this problem, heterologous expression of pentose transporters has been attempted but often with limited success due to poor expression and stability, and/or low turnover. A more successful approach is the engineering of the endogenous Hxt transporter family and evolutionary selection for D-glucose insensitive growth on pentose sugars. This has led to the identification of a critical and conserved asparagine residue in Hxt transporters that, when mutated, reduces the D-glucose affinity while leaving the D-xylose affinity mostly unaltered. Likewise, mutant Gal2 transporter have been selected supporting specific uptake of L-arabinose. In fermentation experiments, the transporter mutants support efficient uptake and consumption of pentose sugars, and even co-consumption of D-xylose and D-glucose when used at industrial concentrations. Further improvements are obtained by interfering with the post-translational inactivation of Hxt transporters at high or low D-glucose concentrations. Transporter engineering solved major limitations in pentose transport in yeast, now allowing for co-consumption of sugars that is limited only by the rates of primary metabolism. This paves the way for a more economical second-generation biofuels production process