Mutant Library Research Articles

Mining the Penicillium expansum Genome for Virulence Genes: A Functional-Based Approach to Discover Novel Loci Mediating Blue Mold Decay of Apple Fruit.

Blue mold, a postharvest disease of pome fruits, is caused by the filamentous fungus Penicillium expansum. In addition to the economic losses caused by P. expansum, food safety can be compromised, as this pathogen is mycotoxigenic. In this study, forward and reverse genetic approaches were used to identify genes involved in blue mold infection in apple fruits. For this, we generated a random T-DNA insertional mutant library. A total of 448 transformants were generated and screened for the reduced decay phenotype on apples. Of these mutants, six (T-193, T-275, T-434, T-588, T-625, and T-711) were selected for continued studies and five unique genes were identified of interest. In addition, two deletion mutants (Δt-625 and Δt-588) and a knockdown strain (t-434KD) were generated for three loci. Data show that the ∆t-588 mutant phenocopied the T-DNA insertion mutant and had virulence penalties during apple fruit decay. We hypothesize that this locus encodes a glyoxalase due to bioinformatic predictions, thus contributing to reduced colony diameter when grown in methylglyoxal (MG). This work presents novel members of signaling networks and additional genetic factors that regulate fungal virulence in the blue mold fungus during apple fruit decay.

Analysis of random mutations in Salmonella Gallinarum dihydropteroate synthase conferring sulfonamide resistance.

In bacteria and primitive eukaryotes, sulfonamide antibiotics block the folate pathway by inhibiting dihydropteroate synthase (FolP) that combines para-aminobenzoic acid (pABA) and dihydropterin pyrophosphate (DHPP) to form dihydropteroic acid (DHP), a precursor for tetrahydrofolate synthesis. However, the emergence of resistant strains has severely compromised the use of pABA mimetics as sulfonamide drugs. Salmonella enterica serovar Gallinarum (S. Gallinarum) is a significant source of antibiotic-resistant infections in poultry. Here, a sulfonamide-resistant FolP mutant library of S. Gallinarum was generated through random mutagenesis. Among resistant strains, substitution of amino acid Arginine 171 with Proline (R171P) in the FolP protein conferred the highest resistance against sulfonamide. Substitution of Phe28 with Leu or Ile (F28L/I) led to modest sulfonamide resistance. Structural modeling indicates that R171P and Phenylalanine 28 with leucine or isoleucine (F28L/I) substitution mutations are located far from the substrate-binding site and cause insignificant conformational changes in the FolP protein. Rather, in silico studies suggest that the mutations altered the stability of the protein, potentially resulting in sulfonamide resistance. Identification of specific mutations in FolP that confer resistance to sulfonamide would contribute to our understanding of the molecular mechanisms of antibiotic resistance.

UBC6, a ubiquitin-conjugating enzyme, participates in secondary cell wall thickening in the inflorescence stem of Arabidopsis

Secondary cell wall (SCW) thickening in plant inflorescence stems is a complicated cellular process that is essential for stem strength and biomass. Although Arabidopsis NAC transcription factor (TF) 1 (NST1) regulates the SCW thickening in anther walls, the single T-DNA-insertion mutant (nst1) does not show disrupted SCW thickening in anther endothecium, interfascicular fibers or xylem. To better understand the regulatory mechanism of this process, we generated an ethyl methanesulfonate (EMS)-mutagenized Arabidopsis population with the nst1 background. scd5 (SCW-defective mutant 5) was isolated in a forward genetic screen from the EMS mutant library, which displayed not only less lignin deposition in the interfascicular fiber and xylem than the wild type but also a pendent inflorescence stem. The EMS-induced mutation associated with the scd5 phenotype was found in the 5th exon of At2G46030 that encodes a ubiquitin-conjugating enzyme (UBC6), we thereby renamed the allele nst1 ubc6. Overexpressing UBC6 in nst1 ubc6 rescued the defective SCW, whereas disrupting UBC6 in nst1 by the CRISPR/Cas9 system caused a phenotype similar to that observed in nst1 ubc6. UBC6 was localized to the nucleus and plasma membrane, and possessed E2 ubiquitin-conjugating activity in vitro. MYB7 and MYB32 are considered as transcription repressors in the phenylpropanoid pathway and are involved in NAC TF-related transcriptional regulation in SCW thickening. UBC6 can interact with MYB7 and MYB32 and positively mediate the degradation of MYB7 and MYB32 by the 26S proteasome. Overall, these results indicated the contribution of UBC6 to SCW thickening in Arabidopsis inflorescence stems.

Transposon-based identification of genes involved in the rimocidin biosynthesis in Streptomyces rimosus M527.

The transposon mutagenesis strategy has been employed to generate random insertion mutants and analyze the correlation between genes and secondary metabolites in the genus Streptomyces. In this study, our primary objective was to identify an unknown gene involved in rimocidin biosynthesis and elucidate its role in rimocidin production in Streptomyces rimosus M527. To achieve this, we established a random mutant library of S. rimosus M527 using a Tn5 transposon-mediated random mutagenesis strategy. Among the 137 isolated mutants, M527-G10 and M527-W5 exhibited the most significant variations in antagonistic activity against the plant pathogenic fungus Fusarium oxysporum f. sp. cucumerinum. Specifically, M527-G10 displayed a 72.93% reduction, while M527-W5 showed a 49.8% increase in rimocidin production compared to the wild-type (WT) strain S. rimosus M527. Subsequently, we employed a plasmid rescue strategy to identify the insertion loci of the transposon in the genomes of mutants M527-G10 and M527-W5, revealing a response regulator transcription factor (rrt) and a hypothetical protein (hyp), respectively. The roles of rrt and hyp in rimocidin biosynthesis were determined through gene deletion, overexpression in the WT strain, and complemented expression in the transposon mutants. Notably, the gene-deletion mutants M527-ΔRRT and M527-ΔHYP exhibited similar behavior in rimocidin production compared to the corresponding transposon mutants M527-G10 and M527-W5, suggesting that transposon insertions in genes rrt and hyp led to alterations in rimocidin production. Furthermore, both gene deletion and overexpression of rrt and hyp had no discernible effects on cell growth. These results reveal that genes rrt and hyp have positive and negative impacts on rimocidin production in S. rimosus M527, respectively.

A codon-based live-cell biomonitoring system for assessing intracellular phenylalanine bioavailability in cyanobacteria

Phenylalanine, as an essential aromatic amino acid, is not only needed for protein and vital molecules such as neurotransmitter and hormone synthesis but also a substrate for the biosynthesis of phenylpropanoids and various bioactive compounds. The metabolism of phenylalanine is dynamic and transitory, which would otherwise inhibit cell growth. Therefore, it is challenging and imperative to monitor intracellular phenylalanine bioavailability in real time, which has great significance for evaluating the effectiveness of introducing pathway-specific genetic modifications to enhance phenylalanine generation. In this study, we proposed a live-cell biomonitoring system to assess phenylalanine bioavailability in real time in cyanobacteria based on codon degeneracy and species-specific usage bias. The biomonitoring system was generated through genetic modification of phenylalanine codons in the chloramphenicol antibiotic resistance gene to wholly preferred and rare codons, in combination with an orthogonal constitutive promoter Trc to express these genes. Cyanobacterial cells equipped with a preferred codon-based gene showed a significant growth advantage over those with rare codons under antibiotic pressure, while the delayed growth caused by rare codon-based genes could be rescued by supplementing phenylalanine in the cultivation medium. Increasing intracellular phenylalanine bioavailability could promote rare codon-based gene containing cell growth to a similar level as wild-type strains harboring preferred codon-based gene, providing a live-cell visualized screening method to relatively define phenylalanine content from either random mutation libraries or pathway-specific engineering cyanobacterial chassis before conducting labor-intensive quantitative measurements.

Evaluation of the Neutralizing Antibody STE90-C11 against SARS-CoV-2 Delta Infection and Its Recognition of Other Variants of Concerns.

As of now, the COVID-19 pandemic has spread to over 770 million confirmed cases and caused approximately 7 million deaths. While several vaccines and monoclonal antibodies (mAb) have been developed and deployed, natural selection against immune recognition of viral antigens by antibodies has fueled the evolution of new emerging variants and limited the immune protection by vaccines and mAb. To optimize the efficiency of mAb, it is imperative to understand how they neutralize the variants of concern (VoCs) and to investigate the mutations responsible for immune escape. In this study, we show the in vitro neutralizing effects of a previously described monoclonal antibody (STE90-C11) against the SARS-CoV-2 Delta variant (B.1.617.2) and its in vivo effects in therapeutic and prophylactic settings. We also show that the Omicron variant avoids recognition by this mAb. To define which mutations are responsible for the escape in the Omicron variant, we used a library of pseudovirus mutants carrying each of the mutations present in the Omicron VoC individually. We show that either 501Y or 417K point mutations were sufficient for the escape of Omicron recognition by STE90-C11. To test how escape mutations act against a combination of antibodies, we tested the same library against bispecific antibodies, recognizing two discrete regions of the spike antigen. While Omicron escaped the control by the bispecific antibodies, the same antibodies controlled all mutants with individual mutations.

Analysing Megasynthetase Mutants at High Throughput Using Droplet Microfluidics.

Nonribosomal peptide synthetases (NRPSs) are giant enzymatic assembly lines that deliver many pharmaceutically valuable natural products, including antibiotics. As the search for new antibiotics motivates attempts to redesign nonribosomal metabolic pathways, more robust and rapid sorting and screening platforms are needed. Here, we establish a microfluidic platform that reliably detects production of the model nonribosomal peptide gramicidin S. The detection is based on calcein-filled sensor liposomes yielding increased fluorescence upon permeabilization. From a library of NRPS mutants, the sorting platform enriches the gramicidin S producer 14.5-fold, decreases internal stop codons 250-fold, and generates enrichment factors correlating with enzyme activity. Screening for NRPS activity with a reliable non-binary sensor will enable more sophisticated structure-activity studies and new engineering applications in the future.

Construction of transcription factor mutagenesis population in tomato using a pooled CRISPR/Cas9 plasmid library

Adequate mutant materials are the prerequisite for conducting gene function research or screening novel functional genes in plants. The strategy of constructing a large-scale mutant population using the pooled CRISPR/Cas9-sgRNA library has been implemented in several crops. However, the effective application of this CRISPR/Cas9 large-scale screening strategy to tomato remains to be attempted. Here, we identified 990 transcription factors in the tomato genome, designed and synthesized a CRISPR/Cas9 plasmid library containing 4379 sgRNAs. Using this pooled library, 487 T0 positive plants were obtained, among which 92 plants harbored a single sgRNA sequence, targeting 65 different transcription factors, with a mutation rate of 23%. In the T0 mutant population, the occurrence of homozygous and biallelic mutations was observed at higher frequencies. Additionally, the utilization of a small-scale CRISPR/Cas9 library targeting 30 transcription factors could enhance the efficacy of single sgRNA recognition in positive plants, increasing it from 19% to 42%. Phenotypic characterization of several mutants identified from the mutant population demonstrated the utility of our CRISPR/Cas9 mutant library. Taken together, our study offers insights into the implementation and optimization of CRISPR/Cas9-mediated large-scale knockout library in tomato.

Mutualism reduces the severity of gene disruptions in predictable ways across microbial communities

Predicting evolution in microbial communities is critical for problems from human health to global nutrient cycling. Understanding how species interactions impact the distribution of fitness effects for a focal population would enhance our ability to predict evolution. Specifically, does the type of ecological interaction, such as mutualism or competition, change the average effect of a mutation (i.e., the mean of the distribution of fitness effects)? Furthermore, how often does increasing community complexity alter the impact of species interactions on mutant fitness? To address these questions, we created a transposon mutant library in Salmonella enterica and measured the fitness of loss of function mutations in 3,550 genes when grown alone versus competitive co-culture or mutualistic co-culture with Escherichia coli and Methylorubrum extorquens. We found that mutualism reduces the average impact of mutations, while competition had no effect. Additionally, mutant fitness in the 3-species communities can be predicted by averaging the fitness in each 2-species community. Finally, we discovered that in the mutualism S. enterica obtained vitamins and more amino acids than previously known. Our results suggest that species interactions can predictably impact fitness effect distributions, in turn suggesting that evolution may ultimately be predictable in multi-species communities.

A Normalization Protocol Reduces Edge Effect in High-Throughput Analyses of Hydroxyurea Hypersensitivity in Fission Yeast.

Edge effect denotes better growth of microbial organisms situated at the edge of the solid agar media. Although the precise reason underlying edge effect is unresolved, it is generally attributed to greater nutrient availability with less competing neighbors at the edge. Nonetheless, edge effect constitutes an unavoidable confounding factor that results in misinterpretation of cell fitness, especially in high-throughput screening experiments widely employed for genome-wide investigation using microbial gene knockout or mutant libraries. Here, we visualize edge effect in high-throughput high-density pinning arrays and report a normalization approach based on colony growth rate to quantify drug (hydroxyurea)-hypersensitivity in fission yeast strains. This normalization procedure improved the accuracy of fitness measurement by compensating cell growth rate discrepancy at different locations on the plate and reducing false-positive and -negative frequencies. Our work thus provides a simple and coding-free solution for a struggling problem in robotics-based high-throughput screening experiments.

Organ agar serves as physiologically relevant alternative for in vivo bacterial colonization.

Animal models for host-microbial interactions have proven valuable, yielding physiologically relevant data that may be otherwise difficult to obtain. Unfortunately, such models are lacking or nonexistent for many microbes. Here, we introduce organ agar, a straightforward method to enable the screening of large mutant libraries while avoiding physiological bottlenecks. We demonstrate that growth defects on organ agar were translatable to bacterial colonization deficiencies in a murine model. Specifically, we present a urinary tract infection agar model to interrogate an ordered library of Proteus mirabilis transposon mutants, with accurate prediction of bacterial genes critical for host colonization. Thus, we demonstrate the ability of ex vivo organ agar to reproduce in vivo deficiencies. Organ agar was also useful for identifying previously unknown links between biosynthetic genes and swarming motility. This work provides a readily adoptable technique that is economical and uses substantially fewer animals. We anticipate this method will be useful for a wide variety of microorganisms, both pathogenic and commensal, in a diverse range of model host species.

Engineering and Expression Strategies for Optimization of L-Asparaginase Development and Production.

Genetic engineering for heterologous expression has advanced in recent years. Model systems such as Escherichia coli, Bacillus subtilis and Pichia pastoris are often used as host microorganisms for the enzymatic production of L-asparaginase, an enzyme widely used in the clinic for the treatment of leukemia and in bakeries for the reduction of acrylamide. Newly developed recombinant L-asparaginase (L-ASNase) may have a low affinity for asparagine, reduced catalytic activity, low stability, and increased glutaminase activity or immunogenicity. Some successful commercial preparations of L-ASNase are now available. Therefore, obtaining novel L-ASNases with improved properties suitable for food or clinical applications remains a challenge. The combination of rational design and/or directed evolution and heterologous expression has been used to create enzymes with desired characteristics. Computer design, combined with other methods, could make it possible to generate mutant libraries of novel L-ASNases without costly and time-consuming efforts. In this review, we summarize the strategies and approaches for obtaining and developing L-ASNase with improved properties.

Construction of an Escherichia coli cell factory for de novo synthesis of tyrosol through semi-rational design based on phenylpyruvate decarboxylase ARO10 engineering

Tyrosol (2-(4-hydroxyphenyl) ethanol) is extensively used in the pharmaceutical industry as an important natural product from plants. In previous research, we constructed a recombinant Escherichia coli strain capable of de novo synthesis of tyrosol by integrating the phenylpyruvate decarboxylase ARO10 derived from Saccharomyces cerevisiae. Nevertheless, the insufficient catalytic efficiency of ARO10 required the insertion of multiple gene copies into the genome to attain enhanced tyrosol production. In this study, we constructed a mutation library of ARO10 based on a computer-aided semi-rational design strategy and developed a high-throughput screening method for selecting high-yield tyrosol mutants by introducing the heterologous hydroxylase complex HpaBC. Through multiple rounds of screening and site-saturation mutagenesis, we ultimately identified the two optimal ARO10 mutants, ARO10D331V and ARO10D331C, which respectively achieved a tyrosol titer of 2.02 g/L and 2.04 g/L in shake flasks, both representing more than 50 % improvement compared to the wild-type. Our study demonstrates the great potential of computer-based semi-rational enzyme design strategy in metabolic engineering. The high-throughput screening method for target compound derivative possesses a certain level of generality. Ultimately, we obtained promising mutants capable of achieving industrial-scale production of tyrosol, which also lays a solid foundation for the efficient synthesis of tyrosol derivatives.

Enhancing polyhydroxyalkanoate production in Cupriavidus sp. L7L through wcaJ gene deletion

Cupriavidus sp. L7L synthesizes a high content of ductile polyhydroxyalkanoate. However, during fermentation, the medium's viscosity gradually increases, eventually reaching a level similar to 93 % glycerol, leading to fermentation termination and difficulties in cell harvest. A non-mucoid variant was isolated from a mini-Tn5 mutant library with the transposon inserted at the promoter sequence upstream of the wcaJ gene. Deletion of wcaJ eliminated the mucoid-colony appearance. The complementation experiment confirmed the association between wcaJ gene expression and mucoid-colony formation. Additionally, the wild-type strain exhibited a faster specific growth rate than the deletion strain using levulinate (Lev) as a carbon source. In fed-batch fermentation, Cupriavidus sp. L7L∆wcaJ showed similar PHA content and monomer composition to the wild-type strain. However, the extended fermentation time resulted in a 42 % increase in PHA concentration. After fed-batch fermentation, the deletion strain's medium had only 8.75 % of the wild-type strain's extracellular polymeric substance content. Moreover, the deletion strain's medium had a much lower viscosity (1.04 mPa·s) than the wild-type strain (194.7 mPa·s), making bacterial cell collection easier through centrifugation. In summary, Cupriavidus sp. L7L∆wcaJ effectively addressed difficulties in cell harvest, increased PHA production, and Lev-to-PHA conversion efficiency, making these characteristics advantageous for industrial-scale PHA production.

Construction of Watermelon Mutant Library Based on 60Co γ-ray Irradiation and EMS Treatment for Germplasm Innovation

Watermelon is a crucial horticultural crop worldwide but its genetic base has become extremely narrow owing to long-term cultivation. Induced mutagenesis can create a range of variations with distinctive agricultural characteristics. To broaden the genetic diversity of watermelon, we established a mutagenesis library containing over 4000 M1 seeds from an inbred line ‘M08’, which was irradiated by 350 Gy of 60Co γ-rays for 3 h. The rates of germination, emergence, and survival of the M1 seeds were reduced by 5.88%, 18.66%, and 41.96%, respectively. After phenotypic screening, 20 and 10 types of morphological changes were observed in the M1 and M2 generations, with approximately 10.57% and 14.17% mutation frequencies, respectively. Six mutants with desirable horticultural alterations were selected for additional presentation, including the leaf color mutant C1-NO.1, the yellow peel mutant C1-NO.2, the pericarp thickening mutant C1-NO.3, the pericarp thinning mutant C1-NO.4, the seedless mutant C1-NO.5, and the C2-No.1 mutant with normal female flowers and malformed male flowers. Moreover, the three mutants M1-3, M2-1, and M1-5 were identified from our EMS-induced M2 library, exhibiting the fusiform fruit, the dark green peel, and the yellow leaves, respectively. Compared to the wild type (WT), the photosynthetic pigments and parameters were negatively impacted in the yellow-leaf mutant M1-5. For example, the total chlorophyll was 1.22 and 2.22 mg/g in the young and mature leaves of M1-5, respectively, which were significantly lower than those in the WT (2.58 and 2.90 mg/g, respectively). Notably, some mutagenesis phenotypes could be stably inherited, including traits such as yellow leaf color, fusiform fruit shape, and thickening and thinning pericarp. Taken together, these results indicate that these two mutant libraries serve as essential resources to discover new phenotypic germplasms, thereby facilitating the genetic breeding and functional gene exploration in watermelon.

Diamide-based screening method for the isolation of improved oxidative stress tolerance phenotypes in Bacillus mutant libraries.

The bacterium Bacillus subtilis is of high importance both as a model organism for Gram-positive bacteria and as an industrial workhorse in the production of biomolecules. In recent years, advancements have been made to engineer the bacterium even further toward industrial applications. In this study, we present a novel screening method for mutant libraries using diamide, an oxidizing agent that binds free thiols and creates disulfide bonds between them, thereby causing a so-called "disulfide stress" in bacteria. The method shows promise to selectively identify phenotypes in B. subtilis with improved tolerance toward oxidative and disulfide-associated stress. Phenotypes initially identified by transposon mutagenesis were recreated through targeted gene deletions. Among the resulting deletion mutants, the largest difference in diamide tolerance compared to the parental strain was observed for pfkA and ribT deletion strains. A proteomics analysis showed that diamide tolerance can be achieved through different routes involving increased expression of stress management proteins and reduced availability or activity of the RNA degradosome. We conclude that our screening method allows the facile identification of Bacillus strains with improved oxidative stress tolerance phenotypes. IMPORTANCE During their life cycle, bacteria are exposed to a range of different stresses that need to be managed appropriately in order to ensure their growth and viability. This applies not only to bacteria in their natural habitats but also to bacteria employed in biotechnological production processes. Oxidative stress is one of these stresses that may originate either from bacterial metabolism or external factors. In biotechnological settings, it is of critical importance that production strains are resistant to oxidative stresses. Accordingly, this also applies to the major industrial cell factory Bacillus subtilis. In the present study, we, therefore, developed a screen for B. subtilis strains with enhanced oxidative stress tolerance. The results show that our approach is feasible and time-, space-, and resource-efficient. We, therefore, anticipate that it will enhance the development of more robust industrial production strains with improved robustness under conditions of oxidative stress.

Pleiotropic effects of N-acylhomoserine lactone synthase ExpI on virulence, competition, and transmission in Pectobacterium carotovorum subsp. carotovorum Pcc21.

Pectobacterium species are necrotrophic phytopathogenic bacteria that cause soft rot disease in economically important crops. The successful infection of host plants relies on interactions among virulence factors, competition, and transmission within hosts. Pectobacteria primarily produce and secrete plant cell-wall degrading enzymes (PCWDEs) for virulence. The regulation of PCWDEs is controlled by quorum sensing (QS). Thus, the QS system is crucial for disease development in pectobacteria through PCWDEs. In this study, we identified a Tn-insertion mutant, M2, in the expI gene from a transposon mutant library of P. carotovorum subsp. carotovorum Pcc21 (hereafter Pcc21). The mutant exhibited reduced production and secretion of PCWDEs, impaired flagellar motility, and increased sensitivity to hydrogen peroxide, resulting in attenuated soft rot symptoms in cabbage and potato tubers. Transcriptomic analysis revealed the down-regulation of genes involved in the production and secretion in the mutant, consistent with the observed phenotype. Furthermore, the Pcc21 wild-type transiently colonized in the gut of Drosophila melanogaster within 12 h after feeding, while the mutant compromised colonization phenotype. Interestingly, Pcc21 produces a bacteriocin, carocin D, to compete with other bacteria. The mutant exhibited up-regulation of carocin D-encoding genes (caroDK) and inhibited the growth of a closely related bacterium, P. wasabiae. Our results demonstrated the significance of ExpI in the overall pathogenic lifestyle of Pcc21, including virulence, competition, and colonization in plant and insect hosts. These findings suggest that disease outcome is a result of complex interactions mediated by ExpI across multiple steps. © 2023 Society of Chemical Industry.

Improving the Enantioselectivity of CHMOBrevi1 for Asymmetric Synthesis of Podophyllotoxin Precursor.

(R)-β-piperonyl-γ-butyrolactones are key building blocks for the synthesis of podophyllotoxin, which have demonstrated remarkable potential in cancer treatment. Baeyer-Villiger monooxygenases (BVMOs)-mediated asymmetric oxidation is a green approach to produce chiral lactones. While several BVMOs were able to oxidize the corresponding cyclobutanone, most BVMOs gave the (S) enantiomer while Cyclohexanone monooxygenase (CHMO) from Brevibacterium sp. HCU1 gave (R) enantiomer, but with a low enantioselectivity (75 % ee). In this study, we use a strategy called "focused rational iterative site-specific mutagenesis" (FRISM) at residues ranging from 6 Å from substrate. The mutations by using a restricted set of rationally chosen amino acids allow the formation of a small mutant library. By generating and screening less than 60 variants, we achieved a high ee of 96.8 %. Coupled with the cofactor regeneration system, 9.3 mM substrate was converted completely in a 100-mL scale reaction. Therefore, our work reveals a promising synthetic method for (R)-β-piperonyl-γ-butyrolactone with the highest enantioselectivity, and provides a new opportunity for the chem-enzymatic synthesis of podophyllotoxin.

Coordinated interplay between palmitoylation, phosphorylation and SUMOylation regulates kainate receptor surface expression.

Kainate receptors (KARs) are key regulators of neuronal excitability and synaptic transmission. KAR surface expression is tightly controlled in part by post-translational modifications (PTMs) of the GluK2 subunit. We have shown previously that agonist activation of GluK2-containing KARs leads to phosphorylation of GluK2 at S868, which promotes subsequent SUMOylation at K886 and receptor endocytosis. Furthermore, GluK2 has been shown to be palmitoylated. However, how the interplay between palmitoylation, phosphorylation and SUMOylation orchestrate KAR trafficking remains unclear. Here, we used a library of site-specific GluK2 mutants to investigate the interrelationship between GluK2 PTMs, and their impact on KAR surface expression. We show that GluK2 is basally palmitoylated and that this is decreased by kainate (KA) stimulation. Moreover, a non-palmitoylatable GluK2 mutant (C858/C871A) shows enhanced S868 phosphorylation and K886 SUMOylation under basal conditions and is insensitive to KA-induced internalisation. These results indicate that GluK2 palmitoylation contributes to stabilising KAR surface expression and that dynamic depalmitoylation promotes downstream phosphorylation and SUMOylation to mediate activity-dependent KAR endocytosis.

An efficient method for constructing a random insertional mutant library for forward genetics in Nannochloropsis oceanica

An efficient method for constructing a random insertional mutant library for forward genetics in Nannochloropsis oceanica