Enzyme In Biosynthesis Pathway Research Articles

The Research Progress of Taxol in Taxus.

Taxus is a valuable woody species with important medicinal value. The bark of Taxus can produce taxol, a natural antineoplastic drug that is widely used in the treatment of breast, ovarian and lung cancers. However, the low content of taxol in the bark of Taxus can not meet the growing clinical demands, so the current research aims at finding ways to increase taxol production. In this review, the research progress of taxol including the factors affecting the taxol content, biosynthesis pathway of taxol, production of taxol in vitro and the application of multi-omics approaches in Taxus as well as future research prospects will be discussed. The taxol content is not only dependent on the species, age and tissues but is also affected by light, moisture levels, temperature, soil fertility and microbes. Most of the enzymes in the taxol biosynthesis pathway have been identified and characterized. Total chemical synthesis, semi-synthesis, plant cell culture and biosynthesis in endophytic fungi have been explored to product taxol. Multi-omics have been used to study Taxus and taxol. Further efforts in the identification of unknown enzymes in the taxol biosynthesis pathway, establishment of the genetic transformation system in Taxus and the regulatory mechanism of taxol biosynthesis and Taxus cell growth will play a significant role in improving the yield of taxol in Taxus cells and plants.

Maternal cariprazine exposure inhibits embryonic and postnatal brain cholesterol biosynthesis

Cariprazine (CAR) is a strong inhibitor of the Dhcr7 enzyme, the last enzyme in the cholesterol biosynthesis pathway. We assessed the effects of CAR on maternally exposed Dhcr7+/− and wild-type mouse offspring, and tested the biochemical effects of CAR in human serum samples. Dhcr7+/− and wild-type time-pregnant mice were exposed to vehicle or 0.2 mg/kg CAR from E12 to E19. Levels of CAR, CAR metabolites, sterols, and oxysterols were measured in the brain of maternally exposed offspring at various time points using LC-MS/MS. Embryonic exposure to CAR significantly increased levels of 7-DHC in all organs of exposed embryos, with a particularly strong effect in the brain. Detectable levels of CAR and elevated 7-DHC were observed in the brain of newborn pups 14 days after drug exposure. In addition, CAR altered sterol metabolism in all animals analyzed, with the strongest effect on the brain of Dhcr7+/− pups born to Dhcr7+/− dams. Furthermore, CAR elevated toxic oxysterols in the brain of maternally exposed Dhcr7+/− offspring to levels approaching those seen in a mouse model of Smith–Lemli–Opitz syndrome. Finally, we observed that patients taking CAR have elevated 7-DHC in their serum. In summary, maternal DHCR7 heterozygosity, combined with offspring DHCR7 heterozygosity might represent a vulnerability factor to medications that interfere with sterol biosynthesis. Due to the conserved sterol biosynthesis between mice and humans, we suggest that the 1–3% of patient population with single-allele DHCR7 mutations might not be ideal candidates for CAR use, especially if they are nursing, pregnant or plan to become pregnant.

Construction of a linkage map flanking the I locus controlling dominant white bulb color and analysis of differentially expressed genes between dominant white and red bulbs in onion (Allium cepa L.)

White bulb colors of onion (Allium cepa L.) are determined by the C and I loci which control the so-called recessive and dominant white bulb colors, respectively. To identify the causal gene responsible for the I locus, a combined approach of bulked segregant analysis and RNA-Seq was used in the present study. A total of 68 contigs containing homozygous single nucleotide polymorphisms (SNPs) between red and dominant white bulked RNAs were identified. The position of I locus was found to be located at chromosome 3 by performing comparative analysis of these contigs and using a previously constructed linkage map. After verification of homozygous SNPs by sequencing of PCR products, 12 high resolution melting, one cleaved amplified polymorphic sequence, and one InDel markers were developed. A linkage map flanking the I locus was constructed using these markers. Two tightly linked markers (DW51596 and DW35019) flanking the I locus were identified by analyzing 1457 F4 individuals. A total of 104 and 39 contigs showing more than tenfold increase of expression in red and dominant white bulks, respectively, were identified. Transcriptions of all structural genes encoding enzymes in flavonoid biosynthesis pathway were significantly reduced in the dominant white bulk. Transcription levels of most contigs showing more than tenfold reduced expression in dominant white were also significantly reduced in the recessive white bulbs controlled by the C locus. Genomic DNA sequences of 12 genes encoding transcription factors assumed to regulate flavonoid biosynthesis were analyzed. However, the causal gene for the I locus could not be identified.

A CRISPR/Cas9-Based Mutagenesis Protocol for Brachypodium distachyon and Its Allopolyploid Relative, Brachypodium hybridum.

The CRISPR/Cas9 system enables precise genome editing and is a useful tool for functional genomic studies. Here we report a detailed protocol for targeted genome editing in the model grass Brachypodium distachyon and its allotetraploid relative B. hybridum, describing gRNA design, a transient protoplast assay to test gRNA efficiency, Agrobacterium-mediated transformation and the selection and analysis of regenerated plants. In B. distachyon, we targeted the gene encoding phytoene desaturase (PDS), which is a crucial enzyme in the chlorophyll biosynthesis pathway. The albino phenotype of mutants obtained confirmed the effectiveness of the protocol for functional gene analysis. Additionally, we targeted two genes related to cell wall maintenance, encoding a fasciclin-like arabinogalactan protein (FLA) and a pectin methylesterase (PME), also in B. distachyon. Two genes encoding cyclin-dependent kinases (CDKG1 and CDKG2), which may be involved in DNA recombination were targeted in both B. distachyon and B. hybridum. Cas9 activity induces mainly insertions or deletions, resulting in frameshift mutations that, may lead to premature stop codons. Because of the close phylogenetic relationship between Brachypodium species and key temperate cereals and forage grasses, this protocol should be easily adapted to target genes underpinning agronomically important traits.

Brahma Related Gene 1 (Brg1) Regulates Cellular Cholesterol Synthesis by Acting as a Co-factor for SREBP2.

Hepatocyte is a hub for cholesterol metabolism. Augmented synthesis of cholesterol in the liver is associated with hypercholesterolemia and contributes to the pathogenesis of a host of cardiovascular and metabolic diseases. Sterol response element binding protein 2 (SREBP2) regulates hepatic cholesterol metabolism by activating the transcription of rate-limiting enzymes in the cholesterol biosynthesis pathway. The underlying epigenetic mechanism is not well understood. We report here that mice with hepatocyte-specific knockout (CKO) of Brg1, a chromatin remodeling protein, exhibit reduced levels of hepatic cholesterol compared to the wild type (WT) littermates when placed on a high-fact diet (HFD) or a methionine-and-choline-deficient diet (MCD). Down-regulation of cholesterol levels as a result of BRG1 deficiency was accompanied by attenuation of cholesterogenic gene transcription. Likewise, BRG1 knockdown in hepatocytes markedly suppressed the induction of cholesterogenic genes by lipid depletion formulas. Brg1 interacted with SREBP2 and was recruited by SREBP2 to the cholesterogenic gene promoters. Reciprocally, Brg1 deficiency dampened the occupancies of SREBP2 on target promoters likely through modulating H3K9 methylation on the cholesterogenic gene promoters. Mechanistically, Brg1 recruited the H3K9 methyltransferase KDM3A to co-regulate pro-cholesterogenic transcription. KDM3A silencing dampened the cholesterogenic response in hepatocytes equivalent to Brg1 deficiency. In conclusion, our data demonstrate a novel epigenetic pathway that contributes to SREBP2-dependent cholesterol synthesis in hepatocytes.

SAT-311 Role of SREBP in Regulation of Corticotroph Tumor ACTH Secretion and Cell Proliferation

Cushing Disease (CD) is a life-threatening condition with suboptimal medical treatment. To identify drugs that not only inhibit ACTH secretion to attain eucortisolemia but also inhibit tumor growth, we conducted a high throughput screen employing a novel “gain of signal” ACTH AlphaLISA assay. From a kinase inhibitor library containing 430 compounds, we identified the dual PI3K/HDAC inhibitor, CUDC-907, as a potent inhibitor of both in vitro and in vivo corticotroph tumor ACTH secretion and growth. By stepwise comparison of CUDC-907 with mono-functional PI3K and HDAC inhibitors, we demonstrated that CUDC-907 exerts its inhibitory effect on ACTH secretion primarily through its inhibition of HDAC activity at the POMC transcriptional level; while PI3K-mediated inhibition of corticotroph cell viability further contributes to reduced ACTH secretion. We also used RNA-seq to characterize the global transcriptiome changes associated with CUDC-907 treatment. Hierarchical clustering showed that 1432 differentially expressed genes (DEGs, p≤0.05 and fold-change≥1.5) were altered by CUDC-907 treatment in comparison to the vehicle-treated control cells. Gene ontology (GO) analysis of 456 downregulated and 976 upregulated DEGs revealed that the most enriched biological processes were cholesterol biosynthesis (GO:0006695, p=1.977e-17) and the type I interferon signaling pathway (GO: 0060337, p=4.928e-7) respectively. Further analysis demonstrated downregulation of the membrane-bound transcription factor sterol regulatory element binding proteins (SREBPs). Downregulation of SREBP-2 by CUDC-907 as well as the several other target enzymes in the cholesterol biosynthesis and uptake pathway including IDI2, NSDHL, MVD, and HMGCR, was confirmed by real-time PCR. To further characterize a role for SREBP-2 in regulation of corticotroph tumor ACTH secretion and proliferation, we employed siRNA targeting endogenous SREBP-2 (SREBP-2 mRNA, Control vs. siRNA 1±0.03 vs. 0.6±0.08, p<0.05), and demonstrated that knockdown of SREBP-2 not only inhibited POMC mRNA expression (POMC mRNA, 1±0.03 vs. 0.7±0.01, p<0.01), and ACTH secretion (ACTH (ng/mL) 29±0.4 vs. 23±0.3, p<0.005), but also suppressed cell proliferation (Relative Proliferation Rate, 1±0.01 vs. 0.7±0.01, p<0.005). This was further confirmed by overexpression of cleaved mature SREBP-2, which led to increased POMC expression and cell proliferation. We demonstrate for the first time the role of the SREBP-mediated cholesterol biosynthesis pathway in regulation of corticotroph tumor POMC regulation and growth. Our studies identify SREBP and cholesterol biosynthesis as a therapeutic target in CD.

Phase-Separated Multienzyme Biosynthesis.

Liquid-liquid phase separation forms condensates that feature a highly concentrated liquid phase, a defined yet dynamic boundary, and dynamic exchange at and across the boundary. Phase transition drives the formation of dynamic multienzyme complexes in cells, for example, the purinosome, which forms subcellular macrobodies responsible for de novo purine biosynthesis. Here, we construct synthetic versions of multienzyme biosynthetic systems by assembling enzymes in protein condensates. A synthetic protein phase separation system using component proteins from postsynaptic density in neuronal synapses, GKAP, Shank, and Homer provides the scaffold for assembly. Three sets of guest proteins: a pair of fluorescent proteins (CFP and YFP), three sequential enzymes in menaquinone biosynthesis pathway (MenF, MenD, and MenH), and two enzymes in terpene biosynthesis pathway (Idi and IspA) are assembled via peptide-peptide interactions in the condensate. First, we discover that coassembly of CFP and YFP exhibited a broad distribution of the FRET signal within the condensate. Second, a spontaneous enrichment of the rate-limiting enzyme MenD in the condensate is sufficient to increase the 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate production rate by 70%. Third, coassembly of both Idi and IspA in the protein condensate increases the farnesyl pyrophosphate production rate by more than 50%. Altogether, we show here that phase separation significantly accelerates the efficiency of multienzyme biocatalysis.

Ternary complex formation of AFN-1252 with Acinetobacter baumannii FabI and NADH: Crystallographic and biochemical studies.

Acinetobacter baumannii is an opportunistic Gram-negative bacterial pathogen, associated mostly with hospital-acquired infections. The emergence of drug resistance strains made it necessary to explore new pathways for the development of more effective antibiotics. Enoyl CoA reductase (FabI), a key enzyme in the fatty acid biosynthesis (FAS) pathway, has emerged as a potential target for antibacterial drug development. Earlier reports show that the lead SaFabI inhibitor AFN-1252 can inhibit FabI from other organisms including Escherichia coli and Burkholderia pseudomallei, but with differential potency. In the present work, we show that AFN-1252 is a moderate inhibitor of AbFabI with an IC50 of 216nM. AFN-1252 stabilized AbFabI with a 4.2°C increase in the melting temperature (Tm ) and, interestingly, the stabilization effect was significantly increased in presence of the cofactor NADH (∆Tm =17°C), suggesting the formation of a ternary complex AbFabI: AFN-1252: NADH. X-ray crystallography studies of AbFabI co-crystalized with AFN-1252 and NADH confirmed the ternary complex formation. The critical interactions of AFN-1252 with AbFabI and NADH identified from the co-crystal structure may facilitate the design and development of new drugs against A. baumannii infections by targeting the FAS pathway.

Disease pharmacokinetic-pharmacodynamic modelling in acute intermittent porphyria to support the development of mRNA-based therapies.

Acute intermittent porphyria (AIP) results from haplo-insufficiency of the porphobilinogen deaminase (PBGD) gene encoding the third enzyme in the haem biosynthesis pathway. As liver is the main organ of pathology for AIP, emerging therapies that restore enzyme hepatic levels are appealing. The objective of this work was to develop a mechanistic-based computational framework to describe the effects of novel PBGD mRNA therapy on the accumulation of neurotoxic haem precursors in small and large animal models. Liver PBGD activity data and/or 24-hr urinary haem precursors were obtained from genetic AIP mice and wild-type mice, rats, rabbits, and macaques. To mimic acute attacks, porphyrogenic drugs were administered over one or multiple challenges, and animals were used as controls or treated with different PBGD mRNA products. Available experimental data were sequentially used to build and validate a semi-mechanistic mathematical model using non-linear mixed-effects approach. The developed framework accounts for the different biological processes involved (i.e., mRNA sequence, release from lipid nanoparticle and degradation, mRNA translation, increased PBGD activity in liver, and haem precursor metabolism) in a simplified mechanistic fashion. The model, validated using external data, shows robustness in the extrapolation of PBGD activity data in rat, rabbit, and non-human primate species. This quantitative framework provides a valuable tool to compare PBGD mRNA drug products during early preclinical stages, optimize the amount of experimental data required, and project results to humans, thus supporting drug development and clinical dose and dosing regimen selection.

MicroRNA 27a Is a Key Modulator of Cholesterol Biosynthesis.

Hypercholesterolemia is a strong predictor of cardiovascular diseases. The 3-hydroxy-3-methylglutaryl coenzyme A reductase gene (Hmgcr) coding for the rate-limiting enzyme in the cholesterol biosynthesis pathway is a crucial regulator of plasma cholesterol levels. However, the posttranscriptional regulation of Hmgcr remains poorly understood. The main objective of this study was to explore the role of microRNAs (miRNAs) in the regulation of Hmgcr expression. Systematic in silico predictions and experimental analyses reveal that miRNA 27a (miR-27a) specifically interacts with the Hmgcr 3' untranslated region in murine and human hepatocytes. Moreover, our data show that Hmgcr expression is inversely correlated with miR-27a levels in various cultured cell lines and in human and rodent tissues. Actinomycin D chase assays and relevant experiments demonstrate that miR-27a regulates Hmgcr by translational attenuation followed by mRNA degradation. Early growth response 1 (Egr1) regulates miR-27a expression under basal and cholesterol-modulated conditions. miR-27a augmentation via tail vein injection of miR-27a mimic in high-cholesterol-diet-fed Apoe-/- mice shows downregulation of hepatic Hmgcr and plasma cholesterol levels. Pathway and gene expression analyses show that miR-27a also targets several other genes (apart from Hmgcr) in the cholesterol biosynthesis pathway. Taken together, miR-27a emerges as a key regulator of cholesterol biosynthesis and has therapeutic potential for the clinical management of hypercholesterolemia.

CDPK6 phosphorylates and stabilizes MYB30 to promote hyperoside biosynthesis that prolongs the duration of full-blooming in okra.

The flowers of okra (Abelmoschus esculentus) open and wilt within only a few hours, and this is accompanied by accumulation of hyperoside, a secondary metabolite in the flavonoid pathway. However, little is known about the relationship between flavonoids and flowering. Here, we found that exogenous application of hyperoside extended the duration of the full-blooming period by more than 3-fold, and this was accompanied by a 14.7-fold increase in the expression of CALCIUM-DEPENDENT PROTEIN KINASE6 (AeCDPK6). Gene expression profiling indicated that the transcription factor AeMYB30 was co-expressed with AeCDPK6, and detailed protein interaction and phosphorylation experiments together with yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated an interaction between AeMYB30 and AeCDPK6. AeCDPK6 specifically phosphorylated AeMYB30S191, leading to increased protein stability and prevention of degradation. Furthermore, AeMYB30 directly bound to the promoter of AeUF3GaT1, a key enzyme in the hyperoside biosynthesis pathway. Analysis of transgenic plants showed that AeCDPK6 was required for the hyperoside-induced phosphorylation of AeMYB30 to enhance its stability and transcriptional activity. Ectopic expression of AeCDPK6 promoted hyperoside accumulation and prolonged the full-blooming period in an AeMYB30-dependent manner. Our results indicate the role of AeCDPK6-AeMYB30 in the molecular mechanism by which hyperoside regulates the period of full blooming in okra, a plant with a short duration of flowering.

Genome-wide analysis of Myo-inositol oxygenase gene family in tomato reveals their involvement in ascorbic acid accumulation

BackgroundAscorbic acid (Vitamin C, AsA) is an antioxidant metabolite involved in plant development and environmental stimuli. AsA biosynthesis has been well studied in plants, and MIOX is a critical enzyme in plants AsA biosynthesis pathway. However, Myo-inositol oxygenase (MIOX) gene family members and their involvement in AsA biosynthesis and response to abiotic stress remain unclear.ResultsIn this study, five tomato genes encoding MIOX proteins and possessing MIOX motifs were identified. Structural analysis and distribution mapping showed that 5 MIOX genes contain different intron/exon patterns and unevenly distributed among four chromosomes. Besides, expression analyses indicated the remarkable expression of SlMIOX genes in different plant tissues. Furthermore, transgenic lines were obtained by over-expression of the MIOX4 gene in tomato. The overexpression lines showed a significant increase in total ascorbate in leaves and red fruits compared to control. Expression analysis revealed that increased accumulation of AsA in MIOX4 overexpression lines is possible as a consequence of the multiple genes involved in AsA biosynthesis. Myo inositol (MI) feeding in leaf and fruit implied that the Myo-inositol pathway improved the AsA biosynthesis in leaves and fruits. MIOX4 overexpression lines exhibited a better light response, abiotic stress tolerance, and AsA biosynthesis capacity.ConclusionsThese results showed that MIOX4 transgenic lines contribute to AsA biosynthesis, evident as better light response and improved oxidative stress tolerance. This study provides the first comprehensive analysis of the MIOX gene family and their involvement in ascorbate biosynthesis in tomato.

Maize ZmFNSI Homologs Interact with an NLR Protein to Modulate Hypersensitive Response.

Nucleotide binding, leucine-rich-repeat (NLR) proteins are the major class of resistance (R) proteins used by plants to defend against pathogen infection. The recognition between NLRs and their cognate pathogen effectors usually triggers a rapid localized cell death, termed the hypersensitive response (HR). Flavone synthase I (FNSI) is one of the key enzymes in the flavone biosynthesis pathway. It also displays salicylic acid (SA) 5-hydroxylase (S5H) activity. A close homolog of FNSI/S5H displays SA 3-hydroxylase (S3H) activity. Both FNSI/S5H and S3H play important roles in plant innate immunity. However, the underlying molecular mechanisms and the relationship between S5H and S3H with the NLR-mediated HR are not known in any plant species. In this study, we identified three genes encoding ZmFNSI-1, ZmFNSI-2 and ZmS3H that are significantly upregulated in a maize line carrying an autoactive NLR Rp1-D21 mutant. Functional analysis showed that ZmFNSI-1 and ZmFNSI-2, but not ZmS3H, suppressed HR conferred by Rp1-D21 and its signaling domain CCD21 when transiently expressed in N. benthamiana. ZmFNSI-1 and ZmFNSI-2 physically interacted with CCD21. Furthermore, ZmFNSI-1 and ZmFNSI-2 interacted with HCT, a key enzyme in lignin biosynthesis pathway, which can also suppress Rp1-D21-mediated HR. These results lay the foundation for the further functional analysis of the roles of FNSI in plant innate immunity.

Cloning, prokaryotic expression, and enzyme activity of a UDP-glucose flavonoid 3-o-glycosyltransferase from mulberry (Morus alba L.) leaves

Background: Mulberry leaves are traditional Chinese medicines, which have pharmacological activities such as anti-inflammation, anti-oxidation, antitumor, and hypoglycemic. Moreover, flavonol glycosides are one of the main functional ingredients. However, the biosynthetic pathway involved in mulberry flavonol glycosides has not been clear. UDP-glucose flavonoid 3-O -glycosyltransferase (UFGT) is a key enzyme in the biosynthesis pathway of flavonol glycosides, which can glycosylate unstable flavonols to form stable flavonol glycosides. Objectives: In this article, MaUFGT , a cDNA encoding the UFGT from mulberry leaves, was cloned, codon optimized, and expressed in Escherichia coli to study its effect in vitro . Materials and Methods: Mulberry UDP-Glucose flavonoid-3-O-glucanotransferase (MaUFGT) gene was reverse transcription-polymerase chain reaction amplified using the cDNA obtained from young leaves of mulberry, and the full-length MaUFGT gene was synthesized by codon-optimized whole-gene synthetic method. Then, the plasmid pCzn1/MaUFGT was constructed and heterologously expressed in E. coli . After denatured, renatured, and purified, the recombinant protein was used to evaluate its function in vitro by determining the final product by high-performance liquid chromatography. Results: The target protein was in the range of 45–66 KD and mainly present in the form of inclusion bodies. The obtained protein was found to transfer UDP-glucose glycosyl moieties to the 3-hydroxyl group of quercetin or kaempferol to form the corresponding products in vitro . Conclusion: The MaUFGT was preliminarily proved to be involved in flavonoid 3-O -glucoside biosynthesis.

Evidence for a novel interaction between key enzymes in the lipid biosynthesis pathway in S. cerevisiae

Lipids are essential for energy storage and integrity of membranes in organisms ranging from humans to yeast. The biological properties of triglycerides and other lipids are determined by their composition and the order of assembly. Previous research has demonstrated that acyl‐CoA molecules are delivered to the enzymes involved in lipid biosynthesis in a specific order. It has been proposed that this ordered assembly is achieved by means of a membrane‐bound lipid synthesis complex in which the key enzymes interact through specific protein‐protein contacts. Preliminary data from yeast two‐hybrid and coimmunoprecipitation studies have revealed specific protein‐protein interactions between lipid synthesizing enzymes. Interactions were found between Ole1, the main desaturase in yeast, and Dga1, the terminal enzyme in the triglyceride synthesis pathway, as well as between Ole1 and Slc1, a protein that acylates lysophosphatidic acid. Future research aims to determine the other proteins in complex with Ole1 with a biotin ligase proximity assay and direct interactors via in vitro studies. This research is revealing the architecture of the enzyme complex and how it controls lipid synthesis and composition. Investigating the lipid biosynthesis complex will have implications for a wide variety of applied research, from treating lipid dysregulation in humans to biofuel production by microbes.Support or Funding InformationNSERC, BioIndustrial Innovates Canada, Alberta Innovates Biofutures, University of Alberta Faculty of Medicine and Dentistry

Genetic Analysis and Immunoelectron Microscopy of Wild and Mutant Strains of the Rubber Tree Endophytic Bacterium Serratia marcescens Strain ITBB B5-1 Reveal Key Roles of a Macrovesicle in Storage and Secretion of Prodigiosin.

Rubber tree is an economically important tropical crop. Its endophytic bacterial strain Serratia marcescens ITBB B5-1 contains an intracellular macrovesicle and red pigment. In this research, the red pigment was identified as prodigiosin by quadrupole time-of-flight mass spectrometry. Prodigiosin has a wide range of potential medical values such as anticancer and antiorgan transplant rejection. The strain ITBB B5-1 accumulated prodigiosin up to 2000 mg/L, which is higher production compared to most known Serratia strains. The formation of the macrovesicle and prodigiosin biosynthesis were highly associated and were both temporal- and temperature-dependent. A mutant strain B5-1mu that failed to produce prodigiosin was obtained by ultraviolet mutagenesis. Whole genome sequencing of wild-type and mutant strains indicated that the PigC gene encoding the last-step enzyme in the prodigiosin biosynthesis pathway was mutated in B5-1mu by a 17-bp deletion. Transmission electron microscopy analysis showed that the macrovesicle was absent in the mutant strain, indicating that formation of the macrovesicle relied on prodigiosin biosynthesis. Immunoelectron microscopy using prodigiosin-specific antiserum showed the presence of prodigiosin in the macrovesicle, the cell wall, and the extracellular vesicles, while immuno-reaction was not observed in the mutant cell. These results indicate that the macrovesicle serves as a storage organelle of prodigiosin, and secretes prodigiosin into cell envelop and culture medium as extracellular vesicles.

Considerable increase in Poly(3-hydroxybutyrate) production via phbC gene overexpression in Ralstonia eutropha PTCC 1615.

Introduction: Poly(3-hydroxybutyrate) (PHB) is a well-known biodegradable polymer produced by some microorganisms and can be a suitable alternative for petrochemical plastics. PHB synthase encoded by phb C gene is the main enzyme in PHB biosynthesis pathway in Ralstonia eutropha. The aim of current study was the transformation of R. eutropha PTCC 1615 with its own phb C gene and evaluation of the overexpression effect on PHB accumulation. Methods: DNA fragment including phbC gene and its promoter and terminator regions, was isolated from R. eutropha PTCC 1615, inserted into pET28a(+) vector, and transferred to the competent bacteria using calcium chloride and heat shock method. The effect of the cloned gene expression on PHB production was investigated with absorption of crotonic acid produced through PHB dehydration. Statistical analyses were carried out by SPSS software. Results: PHB content of cells of the engineered strain was 1.4 times more than that of the native bacteria. This significant difference can be an important finding for improvement of biopolymer production. Conclusion: Overexpression of phb C, the critical gene in PHB biosynthesis pathway, in R. eutropha PTCC 1615 had considerable effect on PHB accumulation.

The crystal structure of the tetrameric DABA-aminotransferase EctB, a rate-limiting enzyme in the ectoine biosynthesis pathway.

l-2,4-diaminobutyric acid (DABA) aminotransferases can catalyze the formation of amines at the distal ω-position of substrates, and is the intial andrate-limiting enzyme in the biosynthesis pathway of the cytoprotecting molecule (S)-2-methyl-1,4,5,6-tetrahydro-4-pyrimidine carboxylic acid (ectoine). Although there is an industrial interest in the biosynthesis of ectoine, the DABA aminotransferases remain poorly characterized. Herein, we present the crystal structure of EctB (2.45Å), a DABA aminotransferase from Chromohalobactersalexigens DSM 3043, a well-studied organism with respect to osmoadaptation by ectoine biosynthesis. We investigate the enzyme's oligomeric state to show that EctB from C.salexigens is a tetramer of two functional dimers, and suggest conserved recognition sites for dimerization that also includes the characteristic gating loop that helps shape the active site of the neighboring monomer. Although ω-transaminases are known to have two binding pockets to accommodate for their dual substrate specificity, we herein provide the first description of two binding pockets in the active site that may account for the catalytic character of DABA aminotransferases. Furthermore, our biochemical data reveal that the EctB enzyme from C.salexigens is a thermostable, halotolerant enzyme with a broad pH tolerance which may be linked to its tetrameric state. Put together, this study creates a solid foundation for a deeper structural understanding of DABA aminotransferases and opening up for future downstream studies of EctB's catalytic character and its redesign as a better catalyst for ectoine biosynthesis. In summary, we believe that the EctB enzyme from C.salexigens can serve as a benchmark enzyme for characterization of DABA aminotransferases. DATABASE: Structural data are available in PDB database under the accession number 6RL5.

DES1: A Key Driver of Lipotoxicity in Metabolic Disease.

Ceramides have emerged as important regulators of tissue metabolism that play essential roles in cardiometabolic disease. They are potent biomarkers of diabetes and heart disease and are now being measured clinically as predictors of major adverse cardiac events. Moreover, studies in rodents reveal that inhibitors of ceramide synthesis prevent or reverse the pathogenic features of type 2 diabetes, nonalcoholic fatty liver disease, atherosclerosis, and cardiomyopathy. Herein the authors discuss inhibition of dihydroceramide desaturase-1, the final enzyme in the ceramide biosynthesis pathway, as a potential therapeutic approach to lower ceramides and combat cardiometabolic disease.

A yeast-based complementation assay elucidates the functional impact of 200 missense variants in human PSAT1.

Defects in serine biosynthesis resulting from loss of function mutations in PHGDH, PSAT1, and PSPH cause a set of rare, autosomal recessive diseases known as Neu-Laxova syndrome (NLS) or serine-deficiency disorders. The diseases present with a broad range of phenotypes including lethality, severe neurological manifestations, seizures, and intellectual disability. However, because L-serine supplementation, especially if started prenatally, can ameliorate and in some cases even prevent symptoms, knowledge of pathogenic variants is medically actionable. Here, we describe a functional assay that leverages the evolutionary conservation of an enzyme in the serine biosynthesis pathway, phosphoserine aminotransferase, and the ability of the human protein-coding sequence (PSAT1) to functionally replace its yeast ortholog (SER1). Results from our quantitative, yeast-based assay agree well with clinical annotations and expectations based on the disease literature. Using this assay, we have measured the functional impact of the 199 PSAT1 variants currently listed in ClinVar, gnomAD, and the literature. We anticipate that the assay could be used to comprehensively assess the functional impact of all SNP-accessible amino acid substitution mutations in PSAT1, a resource that could aid variant interpretation and identify potential NLS carriers.