Archaeal Lipid Biosynthesis: exploring the biosynthetic pathway towards complex membrane phospholipids

Archaeal glycerophospholipids are the main constituents of the cytoplasmic membrane in the archaeal kingdom of life and fundamentally differ in chemical composition compared to bacterial or eukaryotic phospholipids. Archaeal membrane phospholipids consist of two isoprenyl chains ether-bound to glycerol-1-phosphate. In contrast, bacterial and eukaryotic glycerophospholipids typically are composed of two fatty acyl chains ester bonded to glycerol-3-phosphate. This largely domain-distinguishing feature between the domain of archaea and the domains of bacteria and eukarya has been termed the “lipid-divide”. The chemical composition of archaeal membranes contributes to the ability of archaea to survive and thrive in extreme environments. However, ether-bonded glycerophospholipids are not only limited to extremophiles and found also in mesophilic archaea. Resolving the structural basis of glycerophospholipid biosynthesis and the biochemical characterization of participating enzymes is a key objective to provide insights in the early evolution of membrane formation and to deepen our understanding of the molecular basis of extremophilicity

The catalytic and structural basis of archaeal glycerophospholipid biosynthesis

Archaeal glycerophospholipids are the main constituents of the cytoplasmic membrane in the archaeal domain of life and fundamentally differ in chemical composition compared to bacterial phospholipids. They consist of isoprenyl chains ether-bonded to glycerol-1-phosphate. In contrast, bacterial glycerophospholipids are composed of fatty acyl chains ester-bonded to glycerol-3-phosphate. This largely domain-distinguishing feature has been termed the "lipid-divide". The chemical composition of archaeal membranes contributes to the ability of archaea to survive and thrive in extreme environments. However, ether-bonded glycerophospholipids are not only limited to extremophiles and found also in mesophilic archaea. Resolving the structural basis of glycerophospholipid biosynthesis is a key objective to provide insights in the early evolution of membrane formation and to deepen our understanding of the molecular basis of extremophilicity. Many of the glycerophospholipid enzymes are either integral membrane proteins or membrane-associated, and hence are intrinsically difficult to study structurally. However, in recent years, the crystal structures of several key enzymes have been solved, while unresolved enzymatic steps in the archaeal glycerophospholipid biosynthetic pathway have been clarified providing further insights in the lipid-divide and the evolution of early life

Rieske Oxygenases and other Ferredoxin‐dependent Enzymes: Electron Transfer Principles and Catalytic Capabilities

Enzymes that depend on sophisticated electron transfer via ferredoxins (Fds) exhibit outstanding catalytic capabilities, but despite decades of research, many of them are still not well understood nor exploited for synthetic applications. This review aims to provide a general overview of the most important Fd-dependent enzymes and the electron transfer processes involved. While several examples are discussed, we focus in particular on the family of Rieske non-heme iron-dependent oxygenases (ROs). In addition to illustrating their electron transfer principles and catalytic potential, the current state of knowledge on structure-function relationships and the mode of interaction between the redox partner proteins is reviewed. Moreover, we highlight several key catalyzed transformations, but also take a deeper dive into their engineerability for biocatalytic applications. The overall findings from these case studies highlight the catalytic capabilities of these biocatalysts and may stimulate future interest in developing additional Fd-dependent enzyme classes for synthetic applications

Biochemical characterization of two GH70 family 4,6-α-glucanotransferases with distinct product specificity from Lactobacillus aviarius subsp. aviarius DSM 20655

Nine GtfB-like 4,6-α-glucanotransferases (4,6-α-GTs) (represented by GtfX of L. aviarius subsp. aviarius DSM 20655) were identified to show distinct characteristics in conserved motifs I-IV. In particular, the "fingerprint" Tyr in motif III of these nine GtfB-type 4,6-α-GTs was found to be replaced by a Trp. In L. aviarius subsp. aviarius DSM20655, a second GtfB-like protein (GtfY), containing the canonical GtfB Tyr residue in motif III, was located directly upstream of GtfX. Biochemical characterization revealed that both GtfX and GtfY showed GtfB-like 4,6-α-GT activity, cleaving (α1→4) linkages and catalyzing the synthesis of (α1→6) linkages. Nonetheless, they differ in product specificity; GtfY only synthesizes consecutive (α1→6) linkages, yielding linear α-glucan products, but GtfX catalyzes the synthesis of (α1→6) linkages predominantly at branch points (22%) rather than in linear segments (10%). The highly branched α-glucan produced by GtfX from amylose V is resistant to digestion by α-amylase, offering great potential as dietary fibers

A promiscuous archaeal cardiolipin synthase enables construction of diverse natural and unnatural phospholipids

Cardiolipins (CL) are a class of lipids involved in the structural organization of membranes, enzyme functioning, and osmoregulation. Biosynthesis of CLs has been studied in eukaryotes and bacteria, but has been barely explored in archaea. Unlike the common fatty acyl chain-based ester phospholipids, archaeal membranes are made up of the structurally different isoprenoid-based ether phospholipids, possibly involving a different cardiolipin biosynthesis mechanism. Here, we identified a phospholipase D motif-containing cardiolipin synthase (MhCls) from the methanogen Methanospirillum hungatei. The enzyme was overexpressed in Escherichia coli, purified, and its activity was characterized by LC-MS analysis of substrates/products. MhCls utilizes two archaetidylglycerol (AG) molecules in a transesterification reaction to synthesize glycerol-di-archaetidyl-cardiolipin (Gro-DACL) and glycerol. The enzyme is non-selective to the stereochemistry of the glycerol-backbone and the nature of the lipid tail, as it also accepts phosphatidylglycerol (PG) to generate glycerol-di-phosphatidyl-cardiolipin (Gro-DPCL). Remarkably, in the presence of AG and PG, MhCls formed glycerol-archaetidyl-phosphatidyl-cardiolipin (Gro-APCL), an archaeal-bacterial hybrid cardiolipin species that so far has not been observed in nature. Due to the reversibility of the transesterification, in the presence of glycerol, Gro-DPCL can be converted back into two PG molecules. In the presence of other compounds that contain primary hydroxyl groups (e.g., alcohols, water, sugars), various natural and unique unnatural phospholipid species could be synthesized, including multiple di-phosphatidyl-cardiolipin species. Moreover, MhCls can utilize a glycolipid in the presence of phosphatidylglycerol to form a glycosyl-mono-phosphatidyl-cardiolipin species, emphasizing the promiscuity of this cardiolipin synthase, that could be of interest for bio-catalytic purposes

Author Correction:CRISPR-based transcriptional activation tool for silent genes in filamentous fungi (Scientific Reports, (2021), 11, 1, (1118), 10.1038/s41598-020-80864-3)

The Supplementary Information published with this Article contained errors. In Note S2, the text formatting including green italics, red bold, yellow underline, purple text and blue underline was omitted. The original Supplementary Information file is provided below. These errors have now been corrected in the Supplementary Information file that accompanies the original Article

A Unified Approach for the Total Synthesis of cyclo-Archaeol, iso-Caldarchaeol, Caldarchaeol, and Mycoketide

Ir-catalyzed asymmetric alkene hydrogenation is presented as the strategy par excellence to prepare saturated isoprenoids and mycoketides. This highly stereoselective synthesis approach is combined with an established 13 C-NMR method to determine the enantioselectivity of each methyl-branched stereocenter. It is shown that this analysis is fit for purpose and the combination allows the synthesis of the title compounds with a significant increase in efficiency

A versatile method to separate complex lipid mixtures using 1-butanol as eluent in a reverse-phase UHPLC-ESI-MS system

Simple, robust and versatile LC-MS based methods add to the rapid assessment of the lipidome of biological cells. Here we present a versatile RP-UHPLC-MS method using 1-butanol as the eluent, specifically designed to separate different highly hydrophobic lipids. This method is capable of separating different lipid classes of glycerophospholipid standards, in addition to phospholipids of the same class with a different acyl chain composition. The versatility of this method was demonstrated through analysis of lipid extracts of the bacterium Escherichia coli and the archaeon Sulfolobus acidocaldarius. In contrast to 2-propanol-based methods, the 1-butanol-based mobile phase is capable of eluting highly hydrophobic analytes such as cardiolipins, tetraether lipids and mycolic acids during the gradient instead of the isocratic purge phase, resulting in an enhanced separation of cardiolipins and extending the analytical range for RPLC

MiniBacillus PG10 as a Convenient and Effective Production Host for Lantibiotics

Efficient bacterial cell factories are important for the screening and characterization of potent antimicrobial peptides such as lantibiotics. Although lantibiotic production systems have been established in Lactococcus lactis and Escherichia coli, the industrial workhorse Bacillus subtilis has been left relatively unexplored as a lantibiotic production host. Therefore, we tested different B. subtilis strains for their ability to produce lantibiotic peptides by using the subtilin modification and transport enzymes derived from the natural subtilin producer B. subtilis ATCC 6633. Our study shows that although B. subtilis ATCC 6633 and 168 are able to produce various processed lantibiotic peptides, an evident advantage of using either the 8-fold protease-deficient strain WB800 or the genome-minimized B. subtilis 168 strain PG10 is the lack of extracellular serine protease activity. Consequently, leader processing of lantibiotic precursor peptides is circumvented and thus potential toxicity toward the production host is prevented. Furthermore, PG10 provides a clean secondary metabolic background and therefore appears to be the most promising B. subtilis lantibiotic production host. We demonstrate the production of various lantibiotic precursor peptides by PG10 and show different options for their in vitro activation. Our study thus provides a convenient B. subtilis-based lantibiotic production system, which facilitates the search for novel antimicrobial peptides