An integrated omics analysis reveals the gene expression profiles of rapeseed, castor bean, and maize for seed oil biosynthesis

Background Seed storage lipids are valuable for human diet and for the sustainable development of mankind. In recent decades, many lipid metabolism genes and pathways have been identified, but the molecular mechanisms that underlie species differences in seed oil biosynthesis are not fully understood. Results To investigate the molecular mechanisms of seed oil accumulation in different species, we performed comparative genome and transcriptome analyses of rapeseed and castor bean, which have high seed oil contents, and maize, which has a low seed oil content. The results uncovered the molecular mechanism of the low and high seed oil content in maize and castor bean, respectively. Transcriptome analyses showed that more than 61% of the lipid- and carbohydrate-related genes were regulated in rapeseed and castor bean, but only 20.1% of the lipid-related genes and 22.5% of the carbohydrate-related genes were regulated in maize. Compared to rapeseed and castor bean, fewer lipid biosynthesis genes but more lipid metabolism genes were regulated in the maize embryo. More importantly, most maize genes encoding lipid-related transcription factors, triacylglycerol (TAG) biosynthetic enzymes, pentose phosphate pathway (PPP) and Calvin Cycle proteins were not regulated during seed oil synthesis, despite the presence of many homologs in the maize genome. These results revealed the molecular underpinnings of the low seed oil content in maize. In castor bean, we observed differential regulation of vital oil biosynthetic enzymes and extremely high expression levels of oil biosynthetic genes, which were consistent with the rapid accumulation of oil in castor bean developing seeds. Conclusions Compared to oil seed (rapeseed and castor bean), less oil biosynthetic genes were regulated during the seed development in non-oil seed (maize). These results shed light on molecular mechanisms of lipid biosynthesis in rapeseed, castor bean, and maize. They can provide information on key target genes that may be useful for future experimental manipulation of oil production in oilseed crops.

Biosynthetic enzymes of the SARS-CoV-2 as potential targets for the discovery of new antiviral drugs

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has raised a major global health concern. This urgent situation is pressing the world to respond with the development of novel vaccine or small molecule therapeutics for SARS-CoV-2. Drug repurposing screening is regarded as one of the most practical and rapid approaches for the discovery of such therapeutics. Direct-acting agents, targeting specific viral enzymes that play an essential role in viral replication, represent a milestone in antiviral therapy. Several biosynthetic enzymes of the SARS-CoV-2 were analyzed as potential targets to develop new therapeutic drugs. This work provides a basis and directions for future drug development and reuse on the protein level of COVID-19.

Plant height heterosis is quantitatively associated with expression levels of plastid ribosomal proteins

The use of hybrids is widespread in agriculture, yet the molecular basis for hybrid vigor (heterosis) remains obscure. To identify molecular components that may contribute to trait heterosis, we analyzed paired proteomic and transcriptomic data from seedling leaf and mature leaf blade tissues of maize hybrids and their inbred parents. Nuclear- and plastid-encoded subunits of complexes required for protein synthesis in the chloroplast and for the light reactions of photosynthesis were expressed above midparent and high-parent levels, respectively. Consistent with previous reports in Arabidopsis, ethylene biosynthetic enzymes were expressed below midparent levels in the hybrids, suggesting a conserved mechanism for heterosis between monocots and dicots. The ethylene biosynthesis mutant, acs2/acs6, largely phenocopied the hybrid proteome, indicating that a reduction in ethylene biosynthesis may mediate the differences between inbreds and their hybrids. To rank the relevance of expression differences to trait heterosis, we compared seedling leaf protein levels to the adult plant height of 15 hybrids. Hybrid/midparent expression ratios were most positively correlated with hybrid/midparent plant height ratios for the chloroplast ribosomal proteins. Our results show that increased expression of chloroplast ribosomal proteins in hybrid seedling leaves is mediated by reduced expression of ethylene biosynthetic enzymes and that the degree of their overexpression in seedlings can quantitatively predict adult trait heterosis.

Extraction of ADP-Heptose and Kdo2-Lipid A from E. coli Deficient in the Heptosyltransferase I Gene

The enzymes involved in lipopolysaccharide (LPS) biosynthesis, including Heptosyltransferase I (HepI), are critical for maintaining the integrity of the bacterial cell wall, and therefore these LPS biosynthetic enzymes are validated targets for drug discovery to treat Gram-negative bacterial infections. Enzymes involved in the biosynthesis of lipopolysaccharides (LPSs) utilize substrates that are synthetically complex, with numerous stereocenters and site-specific glycosylation patterns. Due to the relatively complex substrate structures, characterization of these enzymes has necessitated strategies to generate bacterial cells with gene disruptions to enable the extraction of these substrates from large scale bacterial growths. Like many LPS biosynthetic enzymes, Heptosyltransferase I binds two substrates: the sugar acceptor substrate, Kdo2-Lipid A, and the sugar donor substrate, ADP-l-glycero-d-manno-heptose (ADPH). HepI characterization experiments require copious amounts of Kdo2-Lipid A and ADPH, and unsuccessful extractions of these two substrates can lead to serious delays in collection of data. While there are papers and theses with protocols for extraction of these substrates, they are often missing small details essential to the success of the extraction. Herein detailed protocols are given for extraction of ADPH and Kdo2-Lipid A (KLA) from E. coli, which have had proven success in the Taylor lab. Key steps in the extraction of ADPH are clearing the extract through ultracentrifugation and keeping all water that touches anything in the extraction, including filters, at a pH of 8.0. Key steps in the extraction of KLA are properly lysing the dried down cells before starting the extraction, maximizing yield by allowing precipitate to form overnight, appropriately washing the pellet with phenol and dissolving the KLA in 1% TEA using visual cues, rather than a specific volume. These protocols led to increased yield and a higher success rate of extractions thereby enabling the characterization of HepI.

Evolution-Inspired Engineering of Anthracycline Methyltransferases

Streptomyces soil bacteria produce hundreds of anthracycline anticancer agents with a relatively conserved set of genes. This diversity depends on the rapid evolution of biosynthetic enzymes to acquire novel functionalities. Previous work has identified S-adenosyl-L-methionine -dependent methyltransferase-like proteins that catalyze either 4-O-methylation, 10-decarboxylation or 10-hydroxylation, with additional differences in substrate specificities. Here we focused on four protein regions to generate chimeric enzymes using sequences from four distinct subfamilies to elucidate their influence in catalysis. Combined with structural studies we managed to depict factors that influence gain-of-hydroxylation, loss-of-methylation and substrate selection. The engineering expanded the catalytic repertoire to include novel 9,10-elimination activity, and 4-O-methylation and 10-decarboxylation of unnatural substrates. The work provides an instructive account on how the rise of diversity of microbial natural products may occur through subtle changes in biosynthetic enzymes.

Coupled sterol synthesis and transport machineries at ER–endocytic contact sites

Sterols are unevenly distributed within cellular membranes. How their biosynthetic and transport machineries are organized to generate heterogeneity is largely unknown. We previously showed that the yeast sterol transporter Osh2 is recruited to endoplasmic reticulum (ER)–endocytic contacts to facilitate actin polymerization. We now find that a subset of sterol biosynthetic enzymes also localizes at these contacts and interacts with Osh2 and the endocytic machinery. Following the sterol dynamics, we show that Osh2 extracts sterols from these subdomains, which we name ERSESs (ER sterol exit sites). Further, we demonstrate that coupling of the sterol synthesis and transport machineries is required for endocytosis in mother cells, but not in daughters, where plasma membrane loading with accessible sterols and endocytosis are linked to secretion.

Establishment of Strigolactone-Producing Bacterium-Yeast Consortium

Strigolactones (SLs) are a class of phytohormones playing diverse roles in plant growth and development, yet the limited access to SLs is largely impeding SL-based foundational investigations and applications. Here, we developed Escherichia coli-Saccharomyces cerevisiae consortia to establish a microbial biosynthetic platform for the synthesis of various SLs, including carlactone, carlactonic acid, 5-deoxystrigol (5DS), 4-deoxyorobanchol (4DO), and orobanchol (OB). The SL-producing platform enabled us to conduct functional identification of CYP722Cs from various plants as either OB or 5DS synthase. It also allowed us to quantitatively compare known variants of plant SL biosynthetic enzymes in the microbial system. The titer of 5DS was further enhanced through pathway engineering to 0.0473 mg/L. This work provides a unique platform for investigating SL biosynthesis and evolution and lays the foundation for developing SL microbial production process.

Age-related changes in brain phospholipids and bioactive lipids in the APP knock-in mouse model of Alzheimer’s disease

Sustained brain chronic inflammation in Alzheimer’s disease (AD) includes glial cell activation, an increase in cytokines and chemokines, and lipid mediators (LMs), concomitant with decreased pro-homeostatic mediators. The inflammatory response at the onset of pathology engages activation of pro-resolving, pro-homeostatic LMs followed by a gradual decrease. We used an APP knock-in (App KI) AD mouse that accumulates β-amyloid (Aβ) and presents cognitive deficits (at 2 and 6 months of age, respectively) to investigate LMs, their precursors, biosynthetic enzymes and receptors, glial activation, and inflammatory proteins in the cerebral cortex and hippocampus at 2-, 4-, 8- and 18-month-old in comparison with wild-type (WT) mice. We used LC-mass-spectrometry and MALDI molecular imaging to analyze LMs and phospholipids, and immunochemistry for proteins. Our results revealed an age-specific lipid and cytokine profile, and glial activation in the App KI mice. Despite an early onset of Aβ pathology, pro-inflammatory and pro-resolving LMs were prominently increased only in the oldest age group. Furthermore, the LM biosynthetic enzymes increased, and their receptor expression decreased in the aged App KI mice. Arachidonic acid (AA)-containing phospholipid molecular species were elevated, correlating with decreased cPLA2 activity. MALDI molecular imaging depicted differential distribution of phospholipids according to genotype in hippocampal layers. Brain histology disclosed increased microglia proliferation starting from young age in the App KI mice, while astrocyte numbers were enhanced in older ages. Our results demonstrate that the brain lipidome is modified preferentially during aging as compared to amyloid pathology in the model studied here. However, alterations in phospholipids signal early pathological changes in membrane composition.