Yeast Expression Research Articles

Genome-Wide Identification of the Salvia miltiorrhiza SmCIPK Gene Family and Revealing the Salt Resistance Characteristic of SmCIPK13

Members of the CIPK (CBL-interacting protein kinases) gene family play important roles in calcium (Ca2+) signaling pathway-regulated plant resistance to abiotic stresses. Salvia miltiorrhiza, which is widely planted and grown in complex and diverse environments, is mainly focused on the transcriptional regulation of enzyme genes related to the biosynthesis of its bioactive components. However, the excavation of the genes related to the resistance of S.miltiorrhiza and the involved signaling pathways have not been deeply studied. In this study, 20 SmCIPK genes were identified and classified into two families and five subfamilies by biochemical means. Sequence characteristics and conserved motif analysis revealed the conservation and difference of SmCIPK protein in plants. Expression pattern analysis showed that SmCIPKs were mainly expressed in flowers and roots, and more than 90% of gene expression was induced by SA (salicylic acid), and MeJA (methyl jasmonate). Furthermore, the expression level of SmCIPK13 could be significantly increased after stress treatment with NaCl. SmCIPK13 expression in yeast reduces sensitivity to salt, while overexpression of it in Arabidopsis has the same effect and was localized in the cytoplasm, cell membrane and nucleus. In conclusion, the identification of the SmCIPK gene family and the functional characterization of the SmCIPK13 gene provides the basis for clarification of key genes in the Ca2+ signaling pathway and abiotic stress in S.miltiorrhiza.

The tomato cytochrome P450 CYP712G1 catalyses the double oxidation of orobanchol en route to the rhizosphere signalling strigolactone, solanacol.

Summary Strigolactones (SLs) are rhizosphere signalling molecules and phytohormones. The biosynthetic pathway of SLs in tomato has been partially elucidated, but the structural diversity in tomato SLs predicts that additional biosynthetic steps are required. Here, root RNA‐seq data and co‐expression analysis were used for SL biosynthetic gene discovery.This strategy resulted in a candidate gene list containing several cytochrome P450s. Heterologous expression in Nicotiana benthamiana and yeast showed that one of these, CYP712G1, can catalyse the double oxidation of orobanchol, resulting in the formation of three didehydro‐orobanchol (DDH) isomers.Virus‐induced gene silencing and heterologous expression in yeast showed that one of these DDH isomers is converted to solanacol, one of the most abundant SLs in tomato root exudate. Protein modelling and substrate docking analysis suggest that hydroxy‐orbanchol is the likely intermediate in the conversion from orobanchol to the DDH isomers.Phylogenetic analysis demonstrated the occurrence of CYP712G1 homologues in the Eudicots only, which fits with the reports on DDH isomers in that clade. Protein modelling and orobanchol docking of the putative tobacco CYP712G1 homologue suggest that it can convert orobanchol to similar DDH isomers as tomato.

Utilizing RNA origami scaffolds in Saccharomyces cerevisiae for dCas9-mediated transcriptional control.

Designer RNA scaffolds constitute a promising tool for synthetic biology, as they can be genetically expressed to perform specific functions in vivo such as scaffolding enzymatic cascades and regulating gene expression through CRISPR-dCas9 applications. RNA origami is a recently developed RNA design approach that allows construction of large RNA nanostructures that can position aptamer motifs to spatially organize other molecules, including proteins. However, it is still not fully understood how positioning multiple aptamers on a scaffold and the orientation of a scaffold affects functional properties. Here, we investigate fusions of single-guide RNAs and RNA origami scaffolds (termed sgRNAO) capable of recruiting activating domains for control of gene expression in yeast. Using MS2 and PP7 as orthogonal protein-binding aptamers, we observe a gradual increase in transcriptional activation for up to four aptamers. We demonstrate that different aptamer positions on a scaffold and scaffold orientation affect transcriptional activation. Finally, sgRNAOs are used to regulate expression of enzymes of the violacein biosynthesis pathway to control metabolic flux. The integration of RNA origami nanostructures at promoter sites achieved here, can in the future be expanded by the addition of functional motifs such as riboswitches, ribozymes and sensor elements to allow for complex gene regulation.

Absence of the Rpb9 subunit of RNA polymerase II reduces the chronological life span in fission yeast.

Fission yeast RNA polymerase II consists of 12 subunits, Rpb1-Rpb12. Among these subunits, Rpb9 is the only subunit whose absence does not cause lethality under optimum growth conditions in fission yeast. However, an rpb9 null fission yeast mutant exhibits a slow-growth phenotype under optimum growth conditions and a defect in survival under environmental and genotoxic stress conditions. To further gain an understanding of its physiological roles, in the present study we have elucidated the role of the Rpb9 subunit in chronological aging using fission yeast as the model organism. Our results provide evidence that the absence of Rpb9 reduces the chronological life span in fission yeast. Our data further shows that lack of Rpb9 in fission yeast causes oxidative stress sensitivity and accumulation of reactive oxygen species during the stationary phase. Our domain mapping experiments have demonstrated that the Rpb9 region encompassing its amino-terminal zinc finger domain and the central linker region is important for the role of Rpb9 in chronological aging. Finally, we also show that expression of the budding yeast or human Rpb9 ortholog can functionally complement the reduced chronological life span phenotype of the fission yeast rpb9 deletion mutant. Taken together, our study has identified a new role of the Rpb9 subunit in chronological aging.

Identification of Saccharum CaM gene family and functioncharacterization of ScCaM1 during cold and oxidant exposure in Pichia pastoris.

Calmodulin (CaM) plays an essential role in binding calcium ions and mediating the interpretation of Ca2+signals in plants under various stresses. However, the evolutionary relationship of CaM family proteins inSaccharumhas not been elucidated. To deduce and explore the evolution and function ofSaccharumCaM family. A total of 104 typical CaMs were obtained fromSaccharum spontaneumand other 18 plant species. The molecular characteristics and evolution of those CaM proteins were analyzed. A typicalCaMgene,ScCaM1, was subsequently cloned from sugarcane (Saccharumspp. hybrid). Its expression patterns in different tissues and under various abiotic stresses were assessed by quantitative real-time PCR. Then the green fluorescent protein was used to determine the subcellular localization of ScCaM1. Finally, the function ofScCaM1was evaluated via heterologous yeast expression systems. Three typical CaM members (SsCaM1, SsCaM2, and SsCaM3) were identified from theS. spontaneumgenome database.CaMs were originated from the two last common ancestors before the origin of angiosperms. The number of CaM family members did not correlate to the genome size but correlated with allopolyploidization events. TheScCaM1was more highly expressed in buds and roots than inother tissues. The expression patterns ofScCaM1suggested that it was involved in responses to various abiotic stresses in sugarcane via different hormonal signaling pathways. Noteworthily, its expression levels appeared relatively stable during the cold exposure in the cold-tolerant variety but significantly suppressed in the cold-susceptible variety. Moreover,therecombinant yeast (Pichia pastoris) overexpressingScCaM1grew better than the wild-type yeast strain under cold and oxidative stresses. It was revealed that theScCaM1played a positive role in reactive oxygen species scavenging and conferred enhanced cold and oxidative stress tolerance to cells. This study provided comprehensive information on the CaM gene family inSaccharumand would facilitate further investigation of their functional characterization.

Genome-Wide Identification and Expression Analysis of AMT Gene Family in Apple (Malus domestica Borkh.)

Ammonium is one of the prevalent nitrogen sources for growth and development of higher plants. Ammonium acquisition from soil is facilitated by ammonium transporters (AMTs), which are plasma membrane proteins that exclusively transport ammonium/ammonia. However, the functional characteristics and molecular mechanisms of AMTs in apple remain unclear. In this work, 15 putative AMT genes were identified and classified into four clusters (AMT1–AMT4) in apple. According to expression analysis, these AMTs had varying expressions in roots, leaves, stems, flowers and fruits. Some of them were strongly affected by diurnal cycles. AMT genes showed multiple transcript patterns to N regimes and were quite responsive to osmotic stress. In addition, phosphorylation analysis revealed that there were some conserved phosphorylation residues within the C-terminal of AMT proteins. Furthermore, detailed research was conducted on AMT1;2 functioning by heterologous expression in yeast. The present study is expected to provide basic bioinformatic information and expression profiles for the apple AMT family and to lay a basis for exploring the functional roles and regulation mechanisms of AMTs in apple.

Insights into the role of N‐linked glycosylation in directing plasma membrane localization of GPCRs expressed in Saccharomyces cerevisiae

Glycosylation is the biochemical process of the attachment of sugar moieties or carbohydrates to proteins during co‐translational or post‐translational modification (PTM). It is one of many types of protein modifications that naturally occurs during the process of protein expression in organisms like Saccharomyces cerevisiae (Baker’s yeast). For natively expressed proteins, glycosylation can be critical for proper protein processing and can also be important for the production of proteins in a stable and biologically active state. For proteins that are non‐natively, or heterologously, expressed in these organisms, glycosylation can lead to improper processing and ultimately produce proteins with little to no biological relevance. This proves to be an undesirable effect for the elucidation of protein structure. However, large quantities of protein are required for structural analysis, presenting a significant obstacle for structural biologists and those who use heterologous expression as a means to acquire large quantities of protein. This problem becomes significantly more challenging for membrane proteins.Membrane proteins are an elusive class of biomolecules that reside or closely associate with the lipid bilayers of cells and encompass transporters, receptors and channel pores, to name a few. They play an intrinsic role in maintaining proper cell function including signaling and selective uptake of extracellular ions and small molecules. G‐protein coupled receptors (GPCRs) constitute one of the largest groups of membrane proteins across the plant and animal kingdoms. Despite being of scientific and medical interest, GPCRs and many integral membrane proteins are notoriously difficult to isolate and work with because of their hydrophobicity, structural flexibility and instability in the absence of the cell membrane. Optimized experimental conditions often vary from protein to protein and necessitate a systematic approach to achieve. This typically requires labor and resource intensive efforts. The goal of this work is to uncover new insights into the role of post‐translational processing and protein localization in the cell. Using a combination of fluorescence microscopy and bioinformatics we formulated an approach to investigate the effects of N‐linked glycosylation on plasma membrane localization of GPCRs expressed in S. cerevisiae. We postulate a critical link between plasma membrane localization and expression of “ligand active” or structurally relevant GPCRs. Previous studies of the human adenosine A2A receptor (hA2AR) show it readily localizes to the plasma membrane during expression in yeast, while the human dopamine D2 receptor (hD2R) does not. A sequence comparison of the two reveals three putative N‐linked glycosylation sites at the N‐terminal extracellular region of hD2R which is lacking in hA2AR. Using a chimera of these two receptors prepared with a C‐terminal GFP tag, we show the chimera does not traffic to the plasma membrane similarly to full‐length hA2AR, suggesting N‐linked glycosylation strongly influences functional high‐level expression of GPCRs and possibly other membrane proteins in yeast.

The transcription factor Atf1 lowers the transition barrier for nucleosome-mediated establishment of heterochromatin.

Transcription factors can exert opposite effects depending on the chromosomal context. The fission yeast transcription factor Atf1 both activates numerous genes in response to stresses and mediates heterochromatic gene silencing in the mating-type region. Investigating this context dependency, we report here that the establishment of silent heterochromatin in the mating-type region occurs at a reduced rate in the absence of Atf1 binding. Quantitative modeling accounts for the observed establishment profiles by a combinatorial recruitment of histone-modifying enzymes: locally by Atf1 at two binding sites and over the whole region by dynamically appearing heterochromatic nucleosomes, a source of which is the RNAi-dependent cenH element. In the absence of Atf1 binding, the synergy is lost, resulting in a slow rate of heterochromatin formation. The system shows how DNA-binding proteins can influence local nucleosome states and thereby potentiate long-range positive feedback on histone-modification reactions to enable heterochromatin formation over large regions in a context-dependent manner.

The Codependent Expression of Yeast RNA Polymerase I Rpa34/Rpa49 Heterodimer Subcomplex

Dysregulation of RNA polymerase I (Pol I) is a hallmark of many cancers and several developmental disorders. Pol I is a multi‐subunit complex that synthesizes ribosomal RNA (rRNA), the integral structural and catalytic component of the ribosome. A key yeast Pol I subcomplex that functions in nearly every step of the transcription cycle is the Rpa34/Rpa49 heterodimer. Recent studies with the heterodimer's human counterpart have shown species specific differences in terms of the heterodimer’s importance for cell growth and functional domain requirements. To further explore these differences, we used the yeast model system to show that similarly to humans, the yeast heterodimer subunits exhibit codependent expression. We further analyzed this codependence in terms of specific heterodimer domain requirements and the level of gene expression the codependence occurs. We found that Rpa34/Rpa49 codependence occurs at the post translational level in a manner dependent on Rpa49’s dimerization domain. Overall, our findings suggest that the codependent expression of yeast and human Rpa34/Rpa49 subcomplexes is an evolutionarily conserved feature that may apply to other Pol I, II, and III subcomplexes.

Selective Inhibition of Plasmodium falciparum ATPase 6 by Artemisinins and Identification of New Classes of Inhibitors after Expression in Yeast.

ABSTRACTTreatment failures with artemisinin combination therapies (ACTs) threaten global efforts to eradicate malaria. They highlight the importance of identifying drug targets and new inhibitors and of studying how existing antimalarial classes work. Here, we report the successful development of a heterologous expression-based compound-screening tool. The validated drug target Plasmodium falciparum ATPase 6 (PfATP6) and a mammalian orthologue (sarco/endoplasmic reticulum calcium ATPase 1a [SERCA1a]) were functionally expressed in Saccharomyces cerevisiae, providing a robust, sensitive, and specific screening tool. Whole-cell and in vitro assays consistently demonstrated inhibition and labeling of PfATP6 by artemisinins. Mutations in PfATP6 resulted in fitness costs that were ameliorated in the presence of artemisinin derivatives when studied in the yeast model. As previously hypothesized, PfATP6 is a target of artemisinins. Mammalian SERCA1a can be mutated to become more susceptible to artemisinins. The inexpensive, low-technology yeast screening platform has identified unrelated classes of druggable PfATP6 inhibitors. Resistance to artemisinins may depend on mechanisms that can concomitantly address multitargeting by artemisinins and fitness costs of mutations that reduce artemisinin susceptibility.

Annotation survey and life cycle transcriptomics of transcription factors in rust fungi (Pucciniales) identify a possible role for cold shock proteins in dormancy exit

Fungi of the order Pucciniales are obligate plant biotrophs causing rust diseases. They exhibit a complex life cycle with the production of up to five spore types, infection of two unrelated hosts and an overwintering stage. Transcription factors (TFs) are key regulators of gene expression in eukaryote cells. In order to better understand genetic programs expressed during major transitions of the rust life cycle, we surveyed the complement of TFs in fungal genomes with an emphasis on Pucciniales. We found that despite their large gene numbers, rust genomes have a reduced repertoire of TFs compared to other fungi. The proportions of C2H2 and Zinc cluster - two of the most represented TF families in fungi - indicate differences in their evolutionary relationships in Pucciniales and other fungal taxa. The regulatory gene family encoding cold shock protein (CSP) showed a striking expansion in Pucciniomycotina with specific duplications in the order Pucciniales. The survey of expression profiles collected by transcriptomics along the life cycle of the poplar rust fungus revealed TF genes related to major biological transitions, e.g. response to environmental cues and host infection. Particularly, poplar rust CSPs were strongly expressed in basidia produced after the overwintering stage suggesting a possible role in dormancy exit. Expression during transition from dormant telia to basidia confirmed the specific expression of the three poplar rust CSP genes. Their heterologous expression in yeast improved cell growth after cold stress exposure, suggesting a probable regulatory function when the poplar rust fungus exits dormancy. This study addresses for the first time TF and regulatory genes involved in developmental transition in the rust life cycle opening perspectives to further explore molecular regulation in the biology of the Pucciniales.

ArabidopsisKCS5 and KCS6 Play Redundant Roles in Wax Synthesis.

3-ketoacyl-CoA synthases (KCSs), as components of a fatty acid elongase (FAE) complex, play key roles in determining the chain length of very-long-chain fatty acids (VLCFAs). KCS6, taking a predominate role during the elongation from C26 to C28, is well known to play an important role in wax synthesis. KCS5 is one paralog of KCS6 and its role in wax synthesis remains unknown. Wax phenotype analysis showed that in kcs5 mutants, the total amounts of wax components derived from carbon 32 (C32) and C34 were apparently decreased in leaves, and those of C26 to C32 derivatives were obviously decreased in flowers. Heterologous yeast expression analysis showed that KCS5 alone displayed specificity towards C24 to C28 acids, and its coordination with CER2 and CER26 catalyzed the elongation of acids exceeding C28, especially displaying higher activity towards C28 acids than KCS6. BiLC experiments identified that KCS5 physically interacts with CER2 and CER26. Wax phenotype analysis of different organs in kcs5 and kcs6 single or double mutants showed that KCS6 mutation causes greater effects on the wax synthesis than KCS5 mutation in the tested organs, and simultaneous repression of both protein activities caused additive effects, suggesting that during the wax biosynthesis process, KCS5 and KCS6 play redundant roles, among which KCS6 plays a major role. In addition, simultaneous mutations of two genes nearly block drought-induced wax production, indicating that the reactions catalyzed by KCS5 and KCS6 play a critical role in the wax biosynthesis in response to drought.

Triple gene expressions in yeast, Escherichia coli, and mammalian cells by transferring DNA fragments amplified from a mother yeast expression plasmid

Escherichia coli, Saccharomyces cerevisiae, and mammalian culture cells are standard host organisms for genetic engineering and research, thus various plasmid vectors have been developed. However, the vectors are designed only for a single host owing to their host-specific genetic elements such as promoters and selection markers. In this study, we developed a yeast expression plasmid that enables the expression of the same gene in E.coli and mammalian cells via the transfer of PCR products amplified from the plasmid as a template. The yeast plasmid YHp26352 was constructed to contain the following regions sequentially: yeast TDH3 promoter (TDH3p), red fluorescent protein (eEmRFP), SV40 terminator (SVpA), E.coli origin (ori), ampicillin resistant gene (AmpR), mammalian cytomegalovirus promoter (CMVp), E.coli srlA promoter (srlAp), and yeast selection marker URA3, which expressed eEmRFP in yeast. To express eEmRFP in mammalian cells, an end-promoter DNA fragment encompassing the eEmRFP-SVpA-ori-AmpR-CMVp region was amplified by PCR and directly used for transfection to mammalian culture cells, resulting in gene expression in mammalian cells through non-homologous end joining. Homologous recombination-mediated circularization was carried out for E.coli cloning and expression by attaching a short overlapping sequence to the 5'-end of a PCR primer, which was used to amplify the eEmRFP-SVpA-ori-AmpR-CMVp-srlAp fragment, after which E.coli transformation was performed. Proof-of-concept experiments were performed by expressing GFP-fused human synaptobrevin VAMP1, and wild-type and codon-changed CLuc luciferase genes in yeast, E.coli, and HEK293cells. This is the first all-in-one plasmid applicable for expression in three host organisms.

EVALUATION OF RECOMBINANT GLUCOAMYLASE EXPRESSION BY A NATIVE AND α-MATING FACTOR SECRETION SIGNAL IN PICHIA PASTORIS

Raw starch degrading enzyme specially glucoamylase with starch binding domain (SBD) has great values in the starch processing industry because it digests the starch particles below the gelatinization temperature by releasing glucose from the non-reducing ends sequentially. The purpose of the study was to measure the secretion levels of recombinant glucoamylase from Pichia pastoris, by using the α-mating factor secretion signal peptide (α-MF) and the native signal peptide of glucoamylase from Aspergillus flavus NSH9. The Aspergillus flavus NSH9 gene (with and without native signal sequences), encoding a pH and thermostable glucoamylase with an SBD, was successfully cloned and expressed in Pichia pastoris to produce recombinant glucoamylases. The constructed recombinant plasmids pPICZB_GA2 (having a native signal peptides) and pPICZαC_GA2 (having the α-MF) were 5144 and 5356 bp in length respectively. Recombinant pichia having α-MF signal sequence (plasmid, pPICZαC_GA2) gave the highest level of secretions of recombinant glucoamylase after 6 days of incubation period with 0.5% methanol. In conclusion, yeast expression vector signal peptide is more efficient for heterologous expression/secretions of recombinant glucoamylase compared to its native signal sequences.

Artificial Intelligence: Bioengineers' Ultimate Best Friend

Artificial Intelligence: Bioengineers' Ultimate Best Friend

Identification of oxidosqualene cyclases associated with saponin biosynthesis from Astragalus membranaceus reveals a conserved motif important for catalytic function

IntroductionTriterpenoids and saponins have a broad range of pharmacological activities. Unlike most legumes which contain mainly oleanane-type scaffold, Astragalus membranaceus contains not only oleanane-type but also cycloartane-type saponins, for which the biosynthetic pathways are unknown. ObjectivesThis work aims to study the function and catalytic mechanism of oxidosqualene cyclases (OSCs), one of the most important enzymes in triterpenoid biosynthesis, in A. membranaceus. MethodsTwo OSC genes, AmOSC2 and AmOSC3, were cloned from A. membranaceus. Their functions were studied by heterologous expression in tobacco and yeast, together with in vivo transient expression and virus-induced gene silencing. Site-directed mutagenesis and molecular docking were used to explain the catalytic mechanism for the conserved motif. ResultsAmOSC2 is a β-amyrin synthase which showed higher expression levels in underground parts. It is associated with the production of β-amyrin and soyasaponins (oleanane-type) in vivo. AmOSC3 is a cycloartenol synthase expressed in both aerial and underground parts. It is related to the synthesis of astragalosides (cycloartane-type) in the roots, and to the synthesis of cycloartenol as a plant sterol precursor. From AmOSC2/3, conserved triad motifs VFM/VFN were discovered for β-amyrin/cycloartenol synthases, respectively. The motif is a critical determinant of yield as proved by 10 variants from different OSCs, where the variant containing the conserved motif increased the yield by up to 12.8-fold. Molecular docking and mutagenesis revealed that Val, Phe and Met residues acted together to stabilize the substrate, and the cation-π interactions from Phe played the major role. ConclusionThe study provides insights into the biogenic origin of oleanane-type and cycloartane-type triterpenoids in Astragalus membranaceus. The conserved motif offers new opportunities for OSC engineering.

Integrated Metabolomic and Transcriptomic Analysis and Identification of Dammarenediol-II Synthase Involved in Saponin Biosynthesis in Gynostemma longipes.

Gynostemma longipes contains an abundance of dammarane-type ginsenosides and gypenosides that exhibit extensive pharmacological activities. Increasing attention has been paid to the elucidation of cytochrome P450 monooxygenases (CYPs) and UDP-dependent glycosyltransferases (UGTs) that participate downstream of ginsenoside biosynthesis in the Panax genus. However, information on oxidosqualene cyclases (OSCs), the upstream genes responsible for the biosynthesis of different skeletons of ginsenoside and gypenosides, is rarely reported. Here, an integrative study of the metabolome and the transcriptome in the leaf, stolon, and rattan was conducted and the function of GlOSC1 was demonstrated. In total, 46 triterpenes were detected and found to be highly abundant in the stolon, whereas gene expression analysis indicated that the upstream OSC genes responsible for saponin skeleton biosynthesis were highly expressed in the leaf. These findings indicated that the saponin skeletons were mainly biosynthesized in the leaf by OSCs, and subsequently transferred to the stolon via CYPs and UGTs biosynthesis to form various ginsenoside and gypenosides. Additionally, a new dammarane-II synthase (DDS), GlOSC1, was identified by bioinformatics analysis, yeast expression assay, and enzyme assays. The results of the liquid chromatography–mass spectrometry (LC–MS) analysis proved that GlOSC1 could catalyze 2,3-oxidosqualene to form dammarenediol-II via cyclization. This work uncovered the biosynthetic mechanism of dammarenediol-II, an important starting substrate for ginsenoside and gypenosides biosynthesis, and may achieve the increased yield of valuable ginsenosides and gypenosides produced under excess substrate in a yeast cell factory through synthetic biology strategy.

Sorghum Ionomics Reveals the Functional SbHMA3a Allele that Limits Excess Cadmium Accumulation in Grains.

Understanding uptake and redistribution of essential minerals or sequestering of toxic elements is important for optimized crop production. Although the mechanisms controlling mineral transport have been elucidated in rice and other species, little is understood in sorghum-an important C4 cereal crop. Here, we assessed the genetic factors that govern grain ionome profiles in sorghum using recombinant inbred lines (RILs) derived from a cross between BTx623 and NOG (Takakibi). Pairwise correlation and clustering analysis of 22 elements, measured in sorghum grains harvested under greenhouse conditions, indicated that the parental lines, as well as the RILs, show different ionomes. In particular, BTx623 accumulated significantly higher levels of cadmium (Cd) than NOG, because of differential root-to-shoot translocation factors between the two lines. Quantitative trait locus (QTL) analysis revealed a prominent QTL for grain Cd concentration on chromosome 2. Detailed analysis identified SbHMA3a, encoding a P1B-type ATPase heavy metal transporter, as responsible for low Cd accumulation in grains; the NOG allele encoded a functional HMA3 transporter (SbHMA3a-NOG) whose Cd-transporting activity was confirmed by heterologous expression in yeast. BTx623 possessed a truncated, loss-of-function SbHMA3a allele. The functionality of SbHMA3a in NOG was confirmed by Cd concentrations of F2 grains derived from the reciprocal cross, in which the NOG allele behaved in a dominant manner. We concluded that SbHMA3a-NOG is a Cd transporter that sequesters excess Cd in root tissues, as shown in other HMA3s. Our findings will facilitate the isolation of breeding cultivars with low Cd in grains or in exploiting high-Cd cultivars for phytoremediation.

The Transcriptomic Mechanism of a Novel Autolysis Induced by a Recombinant Antibacterial Peptide from Chicken Expressed in Pichia pastoris.

Autolysis is a common physiological process in eukaryotic cells that is often prevented or applied, especially in yeast expression systems. In this study, an antimicrobial peptide from chicken (AMP) was recombinantly expressed in the Pichia pastoris expression system, which induced a series of cellular autolysis phenotypes after methanol treatment, such as the aggregated, lysed, irregular, and enlarged cell morphology, while the cells expressing a recombinant aflatoxin-detoxifizyme (ADTZ) were not autolyzed. A comparative transcriptomic analysis showed that the transcriptomic profiles of cells derived from the autolysis and non-autolysis groups were well discriminated, suggesting that the mechanisms of autolysis were at the transcriptional level. A further differential expression gene (DEG) analysis showed that the DEGs from the two groups were involved mainly in autophagy, the MAPK signaling pathway, transcriptional factors, the central carbon metabolism, anti-stress functions, and so on. In the autolysis group, the cell activity was significantly reduced with the MAPK signaling pathway, the central carbon metabolism was down-regulated, and components of the cytoplasm-to-vacuole targeting (CVT) and mitophagy pathways were up-regulated, suggesting that the autophagy involved in the trafficking of intracellular molecules in the vacuole and mitochondrion contributed to autolysis, which was regulated by transcriptional factors and signal pathways at the transcriptional level. This study provides a theoretical basis for genetic modifications to prevent or utilize cell autolysis in the recombinant protein expression system.

Intracellular production of recombinant GnRH1 in yeast, Pichia pastoris, and its potential as oral treatment to advance gonadal development in juvenile orange-spotted grouper, Epinephelus coioides

Late maturation and large size at maturity are significant challenges when administering hormonal treatments in captive broodstock. Here, we report the potential of yeast, Pichia pastoris, as a vehicle to orally deliver a recombinant gonadotropin-releasing hormone (rGnRH1) aimed at stimulating gonadal development in juvenile orange-spotted grouper, Epinephelus coioides. Two recombinant GnRH1 constructs were designed utilizing pPIC3.5, the yeast expression vector lacking a signal sequence, hence retaining the recombinant within the yeast. The first construct (rGnRH1_1xGAP) comprised of GnRH1 decapeptide and the GnRH-associated peptide (GAP) separated by a cleavage site. The second construct (rGnRH1_10x) consisted of ten GnRH1 decapeptides, each separated by a cleavage site. Expression of the two recombinant peptides was confirmed by mass spectrometry. ELISA for the GnRH analogue (GnRHa) was validated to determine the level of rGnRH1 from the yeast extracts. Parallelism between the serially diluted GnRHa and serially diluted extracts from recombinant yeast confirmed validity of the assay. A luciferase reporter assay showed stimulation of tilapia GnRH type 3 receptor by the yeast extract, suggesting biological activity in vitro. In a short term experiment, lyophilised yeast loaded in gelatine capsules and fed once to juvenile orange-spotted grouper resulted in a significantly higher plasma GnRH compared with the control. In a 4-week experiment, lyophilized yeast containing either rGnRH1_1xGAP or rGnRH1_10x was incorporated in fish pellets and fed daily at a dose of 1 μg rGnRH1/kg fish body weight/day. The mean gonadosomatic index and oocyte diameter did not vary between the treated and control fish. The mean plasma levels of FSH, LH and GnRH in the rGnRH1-fed and control fish did not also vary significantly, however there were individuals in the rGnRH1-fed groups that had increased level of the hormones. Together with histological evidence showing oocyte development in a proportion of fish fed with the recombinant, results of the present study point to a biological activity in vivo of the rGnRH1 in yeast. With further refinement, this technology has the potential to provide a non-invasive method for broodstock management of large, late-maturing species of fish and of small, delicate endangered species that require breeding in captivity.