Phospholipid biosynthesis regulation for improving pigment production by Monascus in response to ammonium chloride stress.

In the actual industrial production process, the efficient biosynthesis and secretion of Monascus pigments (MPs) tend to take place under abiotic stresses, which often result in an imbalance of cell homeostasis. The present study aimed to thoroughly describe the changes in lipid profiles in Monascus purpureus by absolute quantitative lipidomics and tandem mass tag-based quantitative proteomics. The results showed that ammonium chloride stress (15 g/L) increased MP production while inhibiting ergosterol biosynthesis, leading to an imbalance in membrane lipid homeostasis in Monascus. In response to the imbalance of lipid homeostasis, the regulation mechanism of phospholipids in Monascus was implemented, including the inhibition of lysophospholipids production, maintenance of the ratio of PC/PE, and improvement of the biosynthesis of phosphatidylglycerol, phosphatidylserine, and cardiolipin with high saturated and long carbon chain fatty acids through the CDP-DG pathway rather than the Kennedy pathway. The inhibition of lysophospholipid biosynthesis was attributed to the upregulated expression of protein and its gene related to lysophospholipase NTE1, while maintenance of the PC/PE ratio was achieved by the upregulated expression of protein and its gene related to CTP: phosphoethanolamine cytidylyltransferase and phosphatidylethanolamine N-methyltransferase in the Kennedy pathway. These findings provide insights into the regulation mechanism of MP biosynthesis from new perspectives.IMPORTANCEMonascus is important in food microbiology as it produces natural colorants known as Monascus pigments (MPs). The industrial production of MPs has been achieved by liquid fermentation, in which the nitrogen source (especially ammonium chloride) is a key nutritional parameter. Previous studies have investigated the regulatory mechanisms of substance and energy metabolism, as well as the cross-protective mechanisms in Monascus in response to ammonium chloride stress. Our research in this work demonstrated that ammonium chloride stress also caused an imbalance of membrane lipid homeostasis in Monascus due to the inhibition of ergosterol biosynthesis. We found that the regulation mechanism of phospholipids in Monascus was implemented, including inhibition of lysophospholipids production, maintenance of the ratio of PC/PE, and improvement of biosynthesis of phosphatidylglycerol, phosphatidylserine, and cardiolipin with high saturated and long carbon chain fatty acids through the CDP-DG pathway. These findings further refine the regulatory mechanisms of MP production and secretion.

Novel spiro[chromanone-2,4'-piperidine]-4-one derivatives as potential inhibitors of fatty acid synthesis in pathogens: Design, synthesis, and biological evaluation.

Bacterial survival depends on membrane lipid homeostasis that enables to regulate lipid composition to adapt and optimize their growth in diverse environments. Therefore, the development of inhibitors that interfere with the bacterial fatty acid synthesis process is considered to be a promising tactic. In this study, 58 novel spirochromanone derivatives were prepared and their structure-activity relationship (SAR) was investigated. The bioassay results showed that all most of the compounds showed excellent biological activities, exampled by compounds B14, C1, B15, and B13, which had outstanding inhibitory activities toward various pathogenic bacteria with EC50 values of 0.78 µg/mL ∼3.48 µg/mL. Preliminary antibacterial behavior was studied by a series of biochemical assays including, but not limited to, fluorescence imaging patterns, GC-MS analysis, TEM images, and fluorescence titration experiments. Notably, compound B14 decreased the lipid content of the cell membrane, and increased cell membrane permeability, thereby destroying the integrity of the bacterial cell membrane. Further qRT-PCR results indicated that compound B14 interfered with the mRNA expression levels of fatty acid synthesis process-related genes including ACC, ACP, and Fab family genes. Herein, we highlight the promising bactericidal skeleton based on the spiro[chromanone-2,4'-piperidine]-4-one as a potential inhibitor of fatty acid synthesis.

Regulation and functions of membrane lipids: Insights from Caenorhabditis elegans.

The Caenorhabditis elegans plasma membrane is composed of glycerophospholipids and sphingolipids with a small cholesterol. The C. elegans obtain the majority of the membrane lipids by modifying fatty acids present in the bacterial diet. The metabolic pathways of membrane lipid biosynthesis are well conserved across the animal kingdom. In C. elegans CDP-DAG and Kennedy pathway produce glycerophospholipids. Meanwhile, the sphingolipids are synthesized through a different pathway. They have evolved remarkably diverse mechanisms to maintain membrane lipid homeostasis. For instance, the lipid bilayer stress operates to accomplish homeostasis during any perturbance in the lipid composition. Meanwhile, the PAQR-2/IGLR-2 complex works with FLD-1 to balance unsaturated to saturated fatty acids to maintain membrane fluidity. The loss of membrane lipid homeostasis is observed in many human genetic and metabolic disorders. Since C. elegans conserved such genes and pathways, it can be used as a model organism.

Molecular species selectivity of lipid transport creates a mitochondrial sink for di-unsaturated phospholipids.

Mitochondria depend on the import of phospholipid precursors for the biosynthesis of phosphatidylethanolamine (PE) and cardiolipin, yet the mechanism of their transport remains elusive. A dynamic lipidomics approach revealed that mitochondria preferentially import di-unsaturated phosphatidylserine (PS) for subsequent conversion to PE by the mitochondrial PS decarboxylase Psd1p. Several protein complexes tethering mitochondria to the endomembrane system have been implicated in lipid transport in yeast, including the endoplasmic reticulum (ER)-mitochondrial encounter structure (ERMES), ER-membrane complex (EMC), and the vacuole and mitochondria patch (vCLAMP). By limiting the availability of unsaturated phospholipids, we created conditions to investigate the mechanism of lipid transfer and the contributions of the tethering complexes in vivo. Under these conditions, inactivation of ERMES components or of the vCLAMP component Vps39p exacerbated accumulation of saturated lipid acyl chains, indicating that ERMES and Vps39p contribute to the mitochondrial sink for unsaturated acyl chains by mediating transfer of di-unsaturated phospholipids. These results support the concept that intermembrane lipid flow is rate-limited by molecular species-dependent lipid efflux from the donor membrane and driven by the lipid species' concentration gradient between donor and acceptor membrane.

Shortening of membrane lipid acyl chains compensates for phosphatidylcholine deficiency in choline-auxotroph yeast.

Phosphatidylcholine (PC) is an abundant membrane lipid component in most eukaryotes, including yeast, and has been assigned multiple functions in addition to acting as building block of the lipid bilayer. Here, by isolating S. cerevisiae suppressor mutants that exhibit robust growth in the absence of PC, we show that PC essentiality is subject to cellular evolvability in yeast. The requirement for PC is suppressed by monosomy of chromosome XV or by a point mutation in the ACC1 gene encoding acetyl-CoA carboxylase. Although these two genetic adaptations rewire lipid biosynthesis in different ways, both decrease Acc1 activity, thereby reducing average acyl chain length. Consistently, soraphen A, a specific inhibitor of Acc1, rescues a yeast mutant with deficient PC synthesis. In the aneuploid suppressor, feedback inhibition of Acc1 through acyl-CoA produced by fatty acid synthase (FAS) results from upregulation of lipid synthesis. The results show that budding yeast regulates acyl chain length by fine-tuning the activities of Acc1 and FAS and indicate that PC evolved by benefitting the maintenance of membrane fluidity.

Phospholipid turnover and acyl chain remodeling in the yeast ER.

The turnover of phospholipids plays an essential role in membrane lipid homeostasis by impacting both lipid head group and acyl chain composition. This review focusses on the degradation and acyl chain remodeling of the major phospholipid classes present in the ER membrane of the reference eukaryote Saccharomyces cerevisiae, i.e. phosphatidylcholine (PC), phosphatidylinositol (PI) and phosphatidylethanolamine (PE). Phospholipid turnover reactions are introduced, and the occurrence and important functions of phospholipid remodeling in higher eukaryotes are briefly summarized. After presenting an inventory of established mechanisms of phospholipid acyl chain exchange, current knowledge of phospholipid degradation and remodeling by phospholipases and acyltransferases localized to the yeast ER is summarized. PC is subject to the PC deacylation-reacylation remodeling pathway (PC-DRP) involving a phospholipase B, the recently identified glycerophosphocholine acyltransferase Gpc1p, and the broad specificity acyltransferase Ale1p. PI is post-synthetically enriched in C18:0 acyl chains by remodeling reactions involving Cst26p. PE may undergo turnover by the phospholipid: diacylglycerol acyltransferase Lro1p as first step in acyl chain remodeling. Clues as to the functions of phospholipid acyl chain remodeling are discussed.

Focal Targeting of the Bacterial Envelope by Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are utilized by both eukaryotic and prokaryotic organisms. AMPs such as the human beta defensins, human neutrophil peptides, human cathelicidin, and many bacterial bacteriocins are cationic and capable of binding to anionic regions of the bacterial surface. Cationic AMPs (CAMPs) target anionic lipids [e.g., phosphatidylglycerol (PG) and cardiolipins (CL)] in the cell membrane and anionic components [e.g., lipopolysaccharide (LPS) and lipoteichoic acid (LTA)] of the cell envelope. Bacteria have evolved mechanisms to modify these same targets in order to resist CAMP killing, e.g., lysinylation of PG to yield cationic lysyl-PG and alanylation of LTA. Since CAMPs offer a promising therapeutic alternative to conventional antibiotics, which are becoming less effective due to rapidly emerging antibiotic resistance, there is a strong need to improve our understanding about the AMP mechanism of action. Recent literature suggests that AMPs often interact with the bacterial cell envelope at discrete foci. Here we review recent AMP literature, with an emphasis on focal interactions with bacteria, including (1) CAMP disruption mechanisms, (2) delocalization of membrane proteins and lipids by CAMPs, and (3) CAMP sensing systems and resistance mechanisms. We conclude with new approaches for studying the bacterial membrane, e.g., lipidomics, high resolution imaging, and non-detergent-based membrane domain extraction.