Truncating variants in PAPSS2 gene: A cause of early prenatal onset brachyolmia?

Brachyolmia is a rare form of skeletal dysplasia characterized by a wide genetic and clinical heterogeneity. This condition is usually diagnosed postnatally, and very few cases of prenatal diagnosis have been described so far. Here, we report a case of a pregnant woman at 20 weeks' gestation referred to our center because of fetal short long bones. On targeted ultrasound, mild bowing of the femurs and fibulae and mild micrognathia were also observed. Exome sequencing analysis showed the presence in compound heterozygosity of two pathogenic variants-both truncating variants-in the 3-prime-phosphoadenosine 5-prime-phosphosulfate synthase 2 (PAPSS2) gene, known to cause brachyolmia type 4 (OMIM #612847). Of note, all of the few cases reported prenatally have indeed truncating variants. Hence, we speculate this kind of variant is likely responsible for a complete loss of function of the protein leading to an earlier and more severe phenotype.

Identification of ascorbate- and salicylate-responsive miRNAs and verification of the spectral control of miR395 in Arabidopsis.

We assumed that miRNAs might regulate the physiological and biochemical processes in plants through their effects on the redox system and phytohormones. To check this hypothesis, the transcriptome profile of wild-type Arabidopsis and lines with decreased ascorbate (Asc), glutathione (GSH), or salicylate (Sal) levels were compared. GSH deficiency did not influence the miRNA expression, whereas lower levels of Asc and Sal reduced the accumulation of 9 and 44 miRNAs, respectively, but only four miRNAs were upregulated. Bioinformatics analysis revealed that their over-represented target genes are associated with the synthesis of nitrogen-containing and aromatic compounds, nucleic acids, and sulphate assimilation. Among them, the sulphate reduction-related miR395 - ATP-sulfurylase couple was selected to check the assumed modulating role of the light spectrum. A greater induction of the Asc- and Sal-responsive miR395 was observed under sulphur starvation in far-red light compared to white and blue light in wild-type and GSH-deficient Arabidopsis lines. Sal deficiency inhibited the induction of miR395 by sulphur starvation in blue light, whereas Asc deficiency greatly reduced it independently of the spectrum. Interestingly, sulphur starvation decreased only the level of ATP sulfurylase 4 among the miR395 target genes in far-red light. The expression level of ATP sulfurylase 3 was higher in far-red light than in blue light in wild-type and Asc-deficient lines. The results indicate the coordinated control of miRNAs by the redox and hormonal system since 11 miRNAs were affected by both Asc and Sal deficiency. This process can be modulated by light spectrum, as shown for miR395.

ATP sulfurylase atypical leucine zipper interacts with Cys3 and calcineurin A in the regulation of sulfur amino acid biosynthesis in Cryptococcus neoformans.

Fungal pathogens are a major cause of death, especially among immunocompromised patients. Therapies against invasive fungal infections are restricted to a few antifungals; therefore, novel therapies are necessary. Nutritional signaling and regulation are important for pathogen establishment in the host. In Cryptococcus neoformans, the causal agent of fungal meningitis, amino acid uptake and biosynthesis are major aspects of nutritional adaptation. Disruptions in these pathways lead to virulence attenuation in an animal model of infection, especially for sulfur uptake and sulfur amino acid biosynthesis. Deletion of Cys3, the main transcription factor that controls these pathways, is the most deleterious gene knockout in vitro and in vivo, making it an important target for further application. Previously, we demonstrated that Cys3 is part of a protein complex, including calcineurin, which is necessary to maintain high Cys3 protein levels during sulfur uptake and sulfur amino acid biosynthesis. In the current study, other aspects of Cys3 regulation are explored. Two lines of evidence suggest that C. neoformans Cys3 does not interact with the F-box WD40 protein annotated as Met30, indicating another protein mediates Cys3 ubiquitin degradation. However, we found another level of Cys3 regulation, which involves protein interactions between Cys3 and ATP sulfurylase (MET3 gene). We show that an atypical leucine zipper at the N-terminus of ATP sulfurylase is essential for physical interaction with Cys3 and calcineurin. Our data suggests that Cys3 and ATP sulfurylase interact to regulate Cys3 transcriptional activity. This work evidences the complexity involved in the regulation of a transcription factor essential for the sulfur metabolism, which is a biological process important to nutritional adaptation, oxidative stress response, nucleic acid stability, and methylation. This information may be useful in designing novel therapies against fungal infections.

Construction of a Protein Crystalline Inclusion-Based Enzyme Immobilization System for Biosynthesis of PAPS from ATP and Sulfate.

3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is the bioactive form of sulfate and is involved in all biological sulfation reactions. The enzymatic transformation method for PAPS is promising, but the low efficiency and high cost of enzyme purification and storage restrict its practical applications. Here, we reported PAPS biosynthesis with a protein crystalline inclusion (PCI)-based enzyme immobilization system. First, the in vivo crystalline inclusion protein CipA was identified as an efficient auto-assembly tag for immobilizing the bifunctional PAPS synthase (ASAK). After characterizing the pyrophosphokinase activity of a polyphosphate exonuclease PaPPX from Pseudomonas aeruginosa, and optimizing the linker fragment, auto-assembled enzymes ASAK-PT-CipA and PaPPX-PT-CipA were constructed. Then, the auto-assembled enzymes ASAK-PT-CipA and PaPPX-PT-CipA with high stability were co-expressed and immobilized for constructing a transformation system. The highest transformation rate of PAPS from ATP and sulfate reached 90%, and the immobilized enzyme can be reused 10 times. The present work provided a convenient, efficient, and easy to be enlarged auto-immobilization system for PAPS biosynthesis from ATP and sulfate. The immobilization system also represented a new approach to reduce the production cost of PAPS by facilitating the purification, storage, and reuse of related enzymes, and it would boost the studies on biotechnological production of glycosaminoglycans and sulfur-containing natural compounds.

Clinical Diagnostic Values of Transfer RNA-Derived Fragment tRF-41-YDLBRY73W0K5KKOVD and its Effects on the Growth of Gastric Cancer Cells.

Gastric cancer (GC) is a serious disease with high mortality and poor prognosis. It is known that tRNA halves play key roles in the progression of cancer. This study explored the function of the tRNA half tRF-41-YDLBRY73W0K5KKOVD in GC. Quantitative real-time reverse transcription-polymerase chain reaction was used to measure RNA levels. The level of tRF-41-YDLBRY73W0K5KKOVD in GC cells was regulated by its mimics or inhibitor. Cell proliferation was evaluated by using a Cell Counting Kit-8 and EdU cell proliferation assay. A Transwell assay was used to detect cell migration. Flow cytometry was used to measure cell cycle and apoptosis. The results showed that tRF-41-YDLBRY73W0K5KKOVD expression was decreased in GC cells and tissues. Functionally, overexpression of tRF-41-YDLBRY73W0K5KKOVD inhibited cell proliferation, reduced migration, repressed the cell cycle, and promoted cell apoptosis in GC cells. Based on RNA sequencing results and luciferase reporter assays, 3'-phosphoadenosine-5'-phosphosulfate synthase 2 (PAPSS2) was identified as a target gene of tRF-41-YDLBRY73W0K5KKOVD. These findings indicated that tRF-41-YDLBRY73W0K5KKOVD inhibited GC progression, suggesting that tRF-41-YDLBRY73W0K5KKOVD might be a potential therapeutic target in GC.

Crystal structure of adenosine 5'-phosphosulfate kinase isolated from Archaeoglobus fulgidus.

The 3'-phosphoadenosine-5'-phosphosulfate (PAPS) molecule is essential during enzyme-catalyzed sulfation reactions as a sulfate donor and is an intermediate in the reduction of sulfate to sulfite in the sulfur assimilation pathway. PAPS is produced through a two-step reaction involving ATP sulfurylase and adenosine 5'-phosphosulfate (APS) kinase enzymes/domains. However, archaeal APS kinases have not yet been characterized and their mechanism of action remains unclear. Here, we first structurally characterized APS kinase from the hyperthermophilic archaeon Archaeoglobus fulgidus, (AfAPSK). We demonstrated the PAPS production activity of AfAPSK at the optimal growth temperature (83 °C). Furthermore, we determined the two crystal structures of AfAPSK: ADP complex and ATP analog adenylyl-imidodiphosphate (AMP-PNP)/Mg2+/APS complex. Structural and complementary mutational analyses revealed the catalytic and substrate recognition mechanisms of AfAPSK. This study also hints at the molecular basis behind the thermal stability of AfAPSK.

A Missense Mutation (c.1037 G > C, p. R346P) in PAPSS2 Gene Results in Autosomal Recessive form of Brachyolmia Type 1 (Hobaek Form) in A Consanguineous Family.

BACKGROUND: Brachyolmia is a skeletal disorder with an autosomal mode of inheritance (both dominant and recessive) in which the patients have a short height, scoliosis and a reduced trunk size. METHODS: From the Muzaffargarh District in Pakistan, a consanguineous family with multiple Brachyolmia-affected subjects were enrolled in the present study. Basic epidemiological data and radiographs were collected for the subjects. Whole exome sequencing (WES) which was followed by Sanger sequencing was applied to report the geneticbasic of Brachyolmia. RESULTS: The WES identified a missense mutation (c.1037 G > C, p. R346P) in exon 9 of the PAPSS2 gene that was confirmed by the Sanger sequencing in the enrolled subjects. The mutation followed a Mendalian pattern with an autosomal recessive inheritance mode. Multiple sequence alignment by Clustal Omega indicated that the PAPSS2 mutation-containing domain is highly conserved. The HEK293T whole-cell extract that was transfected with the Myc-tagged PCMV6-PAPSS2 of both the wild and mutant constructs were resolved by SDS-PAGE as well as by a Western blot, which confirmed that there are different PAPSS2 protein expression patterns when they were compared between the control and Brachyolmia patients. This difference between the normal and mutated protein was not evident when the three-dimensional computational structures were generated using homology modeling. CONCLUSION: We report a missense mutation (c.1037 G > C, p. R346P) in the PAPSS2 gene that caused Brachyolmia in a consanguineous Pakistani family.

Gene Identification, expression analysis and molecular docking of ATP sulfurylase in the selenization pathway of Cardamine hupingshanensis.

BACKGROUND: ATP sulfurylase (ATPS) is a crucial enzyme for the selenate assimilation pathway in plants. RESULTS: In this study, genome-wide and comparative analyses of ATPS in Cardamine hupingshanensis, including sequence and structural analyses, were performed. The expression of ChATPS gene family members in C. hupingshanensis under selenium (Se) stress was also investigated, and our results suggest that ChATPS1-2 play key roles in the response to Se stress. Nine ATPS genes were found from C. hupingshanensis, which share highly conserved sequences with ATPS from Arabidopsis thaliana. In addition, we performed molecular docking of ATP sulfurylase in complex with compounds ATP, selenate, selenite, sulfate, and sulfite. ChAPS3-1 was found to have stronger binding energies with all compounds tested. Among these complexes, amino acid residues Arg, Gly, Ser, Glu, and Asn were commonly present. CONCLUSION: Our study reveals the molecular mechanism of C. hupingshanensis ATP sulfurylase interacting with selenate, which is essential for understanding selenium assimilation. This information will guide further studies on the function of the ChATPS gene family in the selenium stress response and lay the foundation for the selenium metabolic pathway in higher plants.

A biosynthetic pathway for the selective sulfonation of steroidal metabolites by human gut bacteria.

Members of the human gut microbiome enzymatically process many bioactive molecules in the gastrointestinal tract. Most gut bacterial modifications characterized so far are hydrolytic or reductive in nature. Here we report that abundant human gut bacteria from the phylum Bacteroidetes perform conjugative modifications by selectively sulfonating steroidal metabolites. While sulfonation is a ubiquitous biochemical modification, this activity has not yet been characterized in gut microbes. Using genetic and biochemical approaches, we identify a widespread biosynthetic gene cluster that encodes both a sulfotransferase (BtSULT, BT0416) and enzymes that synthesize the sulfonate donor adenosine 3'-phosphate-5'-phosphosulfate (PAPS), including an APS kinase (CysC, BT0413) and an ATP sulfurylase (CysD and CysN, BT0414-BT0415). BtSULT selectively sulfonates steroidal metabolites with a flat A/B ring fusion, including cholesterol. Germ-free mice monocolonized with Bacteroides thetaiotaomicron ΔBT0416 exhibited reduced gastrointestinal levels of cholesterol sulfate (Ch-S) compared with wild-type B. thetaiotaomicron-colonized mice. The presence of BtSULT and BtSULT homologues in bacteria inhibited leucocyte migration in vitro and in vivo, and abundances of cluster genes were significantly reduced in patients with inflammatory bowel disease. Together, these data provide a mechanism by which gut bacteria sulfonate steroidal metabolites and suggest that these compounds can modulate immune cell trafficking in the host.

S-Assimilation Influences in Carrageenan Biosynthesis Genes during Ethylene-Induced Carposporogenesis in Red Seaweed Grateloupia imbricata.

The synthesis of cell-wall sulfated galactans proceeds through UDP galactose, a major nucleotide sugar in red seaweed, whilst sulfate is transported through S-transporters into algae. Moreover, synthesis of ethylene, a volatile plant growth regulator that plays an important role in red seaweed reproduction, occurs through S-adenosyl methionine. This means that sulfur metabolism is involved in reproduction events as well as sulfated galactan synthesis of red seaweed. In this work we study the effects of methionine and MgSO4 on gene expression of polygalactan synthesis through phosphoglucomutase (PGM) and galactose 1 phosphate uridyltransferase (GALT) and of sulfate assimilation (S-transporter and sulfate adenylyltransferase, SAT) using treatment of ethylene for 15 min, which elicited cystocarp development in Grateloupia imbricata. Also, expressions of carbohydrate sulfotransferase and galactose-6-sulfurylase in charge of the addition and removal of sulfate groups to galactans backbone were examined. Outstanding results occurred in the presence of methionine, which provoked an increment in transcript number of genes encoding S-transporter and assimilation compared to controls regardless of the development stage of thalli. Otherwise, methionine diminished the transcript levels of PGM and GALT and expressions are associated with the fertilization stage of thalli of G. imbricata. As opposite, methionine and MgSO4 did not affect the transcript number of carbohydrate sulfotransferase and galactose-6-sulfurylase. Nonetheless, differential expression was obtained for sulfurylases according to the development stages of thalli of G. imbricata.

Identification of variants in ACAN and PAPSS2 leading to spondyloepi(meta)physeal dysplasias in four Chinese families.

BACKGROUND: Spondyloepi(meta)physeal dysplasias (SE[M]D) are a group of inherited skeletal disorders that mainly affect bone and cartilage, and next-generation sequencing has aided the detection of genetic defects of such diseases. In this study, we aimed to identify causative variants in four Chinese families associated with SE(M)D. METHODS: We recruited four unrelated Chinese families all displaying short stature and growth retardation. Clinical manifestations and X-ray imaging were recorded for all patients. Candidate variants were identified by whole-exome sequencing (WES) and verified by Sanger sequencing. Pathogenicity was assessed by conservation analysis, 3D protein modeling and in silico prediction, and was confirmed according to American College of Medical Genetics and Genomics. RESULTS: Three novel SE(M)D-related variants c.1090dupG, c.7168 T > G, and c.2947G > C in ACAN, and one reported variant c.712C > T in PAPSS2 were identified. Among them, c.1090dupG in ACAN and c.712C > T in PAPSS2 caused truncated protein and the other two variants led to amino acid alterations. Conservation analysis revealed sites of the two missense variants were highly conserved, and bioinformatic findings confirmed their pathogenicity. 3D modeling of mutant protein encoded by c.7168 T > G(p.Trp2390Gly) in ACAN proved the structural alteration in protein level. CONCLUSION: Our data suggested ACAN is a common pathogenic gene of SE(M)D. This study enriched the genetic background of skeletal dysplasias, and expanded the mutation spectra of ACAN and PAPSS2.

A Reference Range for Plasma Levels of Inorganic Pyrophosphate in Children Using the ATP Sulfurylase Method.

PURPOSE: Generalized arterial calcification of infancy, pseudoxanthoma elasticum, autosomal recessive hypophosphatemic rickets type 2, and hypophosphatasia are rare inherited disorders associated with altered plasma levels of inorganic pyrophosphate (PPi). In this study, we aimed to establish a reference range for plasma PPi in the pediatric population, which would be essential to support its use as a biomarker in children with mineralization disorders. METHODS: Plasma samples were collected from 200 children aged 1 day to 18 years who underwent blood testing for medical conditions not affecting plasma PPi levels. PPi was measured in proband plasma utilizing a validated adenosine triphosphate (ATP) sulfurylase method. RESULTS: The analytical sensitivity of the ATP sulfurylase assay consisted of 0.15 to 10 µM PPi. Inter- and intra-assay coefficients of variability on identical samples were below 10%. The standard range of PPi in the blood plasma of children and adolescents aged 0 to 18 years was calculated as 2.36 to 4.44 µM, with a median of 3.17 µM, with no difference between male and female probands. PPi plasma levels did not differ significantly in different pediatric age groups. MAIN CONCLUSIONS: Our results yielded no noteworthy discrepancy to the reported standard range of plasma PPi in adults (2-5 µM). We propose the described ATP sulfurylase method as a diagnostic tool to measure PPi levels in plasma as a biomarker in the pediatric population.

Structural basis for the substrate recognition mechanism of ATP-sulfurylase domain of human PAPS synthase 2.

Sulfation is an essential modification on biomolecules in living cells, and 3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is its unique and universal sulfate donor. Human PAPS synthases (PAPSS1 and 2) are the only enzymes that catalyze PAPS production from inorganic sulfate. Unexpectedly, PAPSS1 and PAPSS2 do not functional complement with each other, and abnormal function of PAPSS2 but not PAPSS1 leads to numerous human diseases including bone development diseases, hormone disorder and cancers. Here, we reported the crystal structures of ATP-sulfurylase domain of human PAPSS2 (ATPS2) and ATPS2 in complex with is product 5'-phosphosulfate (APS). We demonstrated that ATPS2 recognizes the substrates by using family conserved residues located on the HXXH and PP motifs, and achieves substrate binding and releasing by employing a non-conserved phenylalanine (Phe550) through a never observed flipping mechanism. Our discovery provides additional information to better understand the biological function of PAPSS2 especially in tumorigenesis, and may facilitate the drug discovery against this enzyme.

An epigenomic landscape of cervical intraepithelial neoplasia and cervical cancer using single-base resolution methylome and hydroxymethylome.

BACKGROUND: Cervical cancer (CC) is the second leading cause of cancer death among women worldwide. Epigenetic regulation of gene expression through DNA methylation and hydroxymethylation plays a pivotal role during tumorigenesis. In this study, to analyze the epigenomic landscape and identify potential biomarkers for CCs, we selected a series of samples from normal to cervical intra-epithelial neoplasia (CINs) to CCs and performed an integrative analysis of whole-genome bisulfite sequencing (WGBS-seq), oxidative WGBS, RNA-seq, and external histone modifications profiling data. RESULTS: In the development and progression of CC, there were genome-wide hypo-methylation and hypo-hydroxymethylation, accompanied by local hyper-methylation and hyper-hydroxymethylation. Hydroxymethylation prefers to distribute in the CpG islands and CpG shores, as displayed a trend of gradual decline from health to CIN2, while a trend of increase from CIN3 to CC. The differentially methylated and hydroxymethylated region-associated genes both enriched in Hippo and other cancer-related signaling pathways that drive cervical carcinogenesis. Furthermore, we identified eight novel differentially methylated/hydroxymethylated-associated genes (DES, MAL, MTIF2, PIP5K1A, RPS6KA6, ANGEL2, MPP, and PAPSS2) significantly correlated with the overall survival of CC. In addition, no any correlation was observed between methylation or hydroxymethylation levels and somatic copy number variations in CINs and CCs. CONCLUSION: Our current study systematically delineates the map of methylome and hydroxymethylome from CINs to CC, and some differentially methylated/hydroxymethylated-associated genes can be used as the potential epigenetic biomarkers in CC prognosis.

Changes in ATP Sulfurylase Activity in Response to Altered Cyanobacteria Growth Conditions.

We investigated variations in cell growth and ATP Sulfurylase (ATPS) activity when two cyanobacterial strains-Synechocystis sp. PCC6803 and Synechococcus sp. WH7803-were grown in conventional media, and media with low ammonium, low sulfate and a high CO2/low O2 atmosphere. In both organisms, a transition and adaptation to the reconstructed environmental media resulted in a decrease in ATPS activity. This variation appears to be decoupled from growth rate, suggesting the enzyme is not rate-limiting in S assimilation and raising questions about the role of ATPS redox regulation in cell physiology and throughout Earth history.

Intestinal Sulfation Is Essential to Protect Against Colitis and Colonic Carcinogenesis.

BACKGROUND & AIMS: Sulfation is a conjugation reaction essential for numerous biochemical and cellular functions in mammals. The 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthase 2 (PAPSS2) is the key enzyme to generate PAPS, which is the universal sulfonate donor for all sulfation reactions. The goal of this study was to determine whether and how PAPSS2 plays a role in colitis and colonic carcinogenesis. METHODS: Tissue arrays of human colon cancer specimens, gene expression data, and clinical features of cancer patients were analyzed. Intestinal-specific Papss2 knockout mice (Papss2ΔIE) were created and subjected to dextran sodium sulfate-induced colitis and colonic carcinogenesis induced by a combined treatment of azoxymethane and dextran sodium sulfate or azoxymethane alone. RESULTS: The expression of PAPSS2 is decreased in the colon cancers of mice and humans. The lower expression of PAPSS2 in colon cancer patients is correlated with worse survival. Papss2ΔIE mice showed heightened sensitivity to colitis and colon cancer by damaging the intestinal mucosal barrier, increasing intestinal permeability and bacteria infiltration, and worsening the intestinal tumor microenvironment. Mechanistically, the Papss2ΔIE mice exhibited reduced intestinal sulfomucin content. Metabolomic analyses revealed the accumulation of bile acids, including the Farnesoid X receptor antagonist bile acid tauro-ß-muricholic acid, and deficiency in the formation of bile acid sulfates in the colon of Papss2ΔIE mice. CONCLUSIONS: We have uncovered an important role of PAPSS2-mediated sulfation in colitis and colonic carcinogenesis. Intestinal sulfation may represent a potential diagnostic marker and PAPSS2 may serve as a potential therapeutic target for inflammatory bowel disease and colon cancer.

Overexpression of ATP sulfurylase improves the sulfur amino acid content, enhances the accumulation of Bowman-Birk protease inhibitor and suppresses the accumulation of the ß-subunit of ß-conglycinin in soybean seeds.

ATP sulfurylase, an enzyme which catalyzes the conversion of sulfate to adenosine 5'-phosphosulfate (APS), plays a significant role in controlling sulfur metabolism in plants. In this study, we have expressed soybean plastid ATP sulfurylase isoform 1 in transgenic soybean without its transit peptide under the control of the 35S CaMV promoter. Subcellular fractionation and immunoblot analysis revealed that ATP sulfurylase isoform 1 was predominantly expressed in the cell cytoplasm. Compared with that of untransformed plants, the ATP sulfurylase activity was about 2.5-fold higher in developing seeds. High-resolution 2-D gel electrophoresis and immunoblot analyses revealed that transgenic soybean seeds overexpressing ATP sulfurylase accumulated very low levels of the ß-subunit of ß-conglycinin. In contrast, the accumulation of the cysteine-rich Bowman-Birk protease inhibitor was several fold higher in transgenic soybean plants when compared to the non-transgenic wild-type seeds. The overall protein content of the transgenic seeds was lowered by about 3% when compared to the wild-type seeds. Metabolite profiling by LC-MS and GC-MS quantified 124 seed metabolites out of which 84 were present in higher amounts and 40 were present in lower amounts in ATP sulfurylase overexpressing seeds compared to the wild-type seeds. Sulfate, cysteine, and some sulfur-containing secondary metabolites accumulated in higher amounts in ATP sulfurylase transgenic seeds. Additionally, ATP sulfurylase overexpressing seeds contained significantly higher amounts of phospholipids, lysophospholipids, diacylglycerols, sterols, and sulfolipids. Importantly, over expression of ATP sulfurylase resulted in 37-52% and 15-19% increases in the protein-bound cysteine and methionine content of transgenic seeds, respectively. Our results demonstrate that manipulating the expression levels of key sulfur assimilatory enzymes could be exploited to improve the nutritive value of soybean seeds.

Expression of enzymes for 3'-phosphoadenosine-5'-phosphosulfate (PAPS) biosynthesis and their preparation for PAPS synthesis and regeneration.

The synthesis of sulfated polysaccharides involves the sulfation of simpler polysaccharide substrates, through the action sulfotransferases using the cofactor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Three enzymes are essential for the in vitro synthesis of PAPS, namely, pyrophosphatase (PPA), adenosine 5'-phosphosulfate kinase (APSK), and ATP sulfurylase (ATPS). The optimized enzyme expression ratio and effect on PAPS synthesis were evaluated using ePathBrick, a novel synthetic biology tool that assemble multiple genes in a single vector. The introduction of multiple promoters and stop codons at different location enable the bacterial system to fine tune expression level of the genes inserted. Recombinant vectors expressing PPA (U39393.1), ATPS (CP021243.1), and PPA (CP047127.1) were used for fermentations and resulted in volumetric yields of 400-1380 mg/L with accumulation of 34-66% in the soluble fraction. The enzymes from soluble fraction, without any further purification, were used for PAPS synthesis. The PAPS was used for the chemoenzymatic synthesis of a heparan sulfate polysaccharide and coupled with a PAPS-ASTIV regeneration system. ASTIV catalyzes the regeneration of PAPS. A recombinant vector expressing the enzyme ASTIV (from Rattus norvegicus) was used for fermentations and resulted in volumetric yield of 1153 mg/L enzyme with accumulation of 48% in the soluble fraction. In conclusion, we have successfully utilized a metabolic engineering approach to optimize the overall PAPS synthesis productivity. In addition, we have demonstrated that the ePathBrick system could be applied towards study and improvement of enzymatic synthesis conditions. In parallel, we have successfully demonstrated an autoinduction microbial fermentation towards the production of mammalian enzyme (ASTIV). KEY POINTS : • ePathBrick used to optimize expression levels of enzymes. • Protocols have been used for the production of recombinant enzymes. • High cell density fed-batch fermentations with high yields of soluble enzymes. • Robust fermentation protocol successfully transferred to contract manufacturing and research facilities.

PoMet3 and PoMet14 associated with sulfate assimilation are essential for conidiogenesis and pathogenicity in Pyricularia oryzae.

Pyricularia oryzae is the causal agent of blast disease on staple gramineous crops. Sulphur is an essential element for the biosynthesis of cysteine and methionine in fungi. Here, we targeted the P. oryzae PoMET3 encoding the enzyme ATP sulfurylase, and PoMET14 encoding the APS (adenosine-5'-phosphosulphate) kinase that are involved in sulfate assimilation and sulphur-containing amino acids biosynthesis. In P. oryzae, deletion of PoMET3 or PoMET14 separately results in defects of conidiophore formation, significant impairments in conidiation, methionine and cysteine auxotrophy, limited invasive hypha extension, and remarkably reduced virulence on rice and barley. Furthermore, the defects of the null mutants could be restored by supplementing with exogenous cysteine or methionine. Our study explored the biological functions of sulfur assimilation and sulphur-containing amino acids biosynthesis in P. oryzae.

Sulfur application reduces cadmium uptake in edible parts of pakchoi (Brassica chinensis L.) by cadmium chelation and vacuolar sequestration.

Sulfur (S) application in pakchoi (Brassica chinensis L.) cultivation is vital for reducing cadmium (Cd) accumulation in the plants. However, the mechanism of S application on Cd uptake and translocation in pakchoi is unclear. In this study, a hydroponic experiment was performed to investigate the effects of S application on Cd accumulation in pakchoi at one Cd concentration (50 µM, in comparison to the control condition, 0 µM) and three S levels (0, 2, 4 mM). The results showed that excessive S application (4 mM) reduced Cd accumulation and alleviated pakchoi growth inhibition caused by Cd stress in shoots and roots. With increased S application, the proportion of Cd in the vacuolar fraction and the proportion of NaCl-extractable Cd increased in roots. Additionally, S application increased the content of glutathione (GSH) and phytochelatins (PCs). The reduced Cd uptake and accumulation in pakchoi shoots could have been due to increased Cd chelation and vacuolar sequestration in roots. In addition, sufficient S application (2 mM) increased the expression of γ-glutamylcysteine synthetase (GSH1) and nicotinamide synthase (NAS) in roots, and excessive S application upregulated the expression of ATP sulfurylase (ATPS) and phytochelatin synthase (PCs). This study provides evidence for the mechanism of mitigating Cd toxicity in pakchoi and will be helpful for developing strategies to reduce Cd accumulation in the edible parts of pakchoi through S fertilizer application.