Screening Of Mutant Libraries Research Articles

Droplet-Based Microfluidic High-Throughput Screening of Enzyme Mutant Libraries Secreted by Yarrowia lipolytica.

Yarrowia lipolytica has emerged as an attractive solution for screening enzyme activities thanks to the numerous tools available for heterologous protein production and its strong secretory ability. Nowadays, activity screening for improved enzymes mostly relies on the evaluation of independent clones in microtiter plates. However, even with highly robotized screening facilities, the relatively low throughput and high cost of the technology do not enable the screening of large diversities, which significantly reduce the probability of isolating improved variants. Droplet-based microfluidics is an emerging technology that allows the high-throughput and individual picoliter droplets manipulation and sorting based on enzymatic substrate fluorescence. This technology is an attractive alternative to microtiter plate screenings with higher throughputs and drastic reduction of working volume and cost.Here, we present a droplet-based microfluidic platform for the screening of libraries expressed in the yeast Y. lipolytica, from the generation of a random mutagenesis library of a heterologous enzyme and its expression in Y. lipolytica to the droplet-based microfluidic procedures composed of cell encapsulation and growth and activity screening or sorting of improved clones.

Fluorimetric high-throughput screening method for polyester hydrolase activity using polyethylene terephthalate nanoparticles.

Biocatalysis has recently emerged as a powerful and eco-friendly technology in waste plastic recycling, especially for the widely used polyethylene terephthalate (PET). So far, however, a high-throughput screening assay specifically toward PET-hydrolyzing activity has rarely been applied. This hinders the identification of new polyester hydrolases and their variants with adequate activities fulfilling the requirements for industrial applications. This chapter describes the detailed procedure for assaying terephthalate as a major product of enzymatic PET hydrolysis in a 96-well microtiter plate format. Using PET nanoparticles derived readily from waste food packaging as a substrate, an active thermophilic PET hydrolase was clearly distinguished from an inactive variant by a Fenton chemistry-mediated fluorimetric detection. The assay uses enzymes in crude cell lysates, obtained by a simple freeze-thaw protocol. The experimental work validates the applicability of this method for screening mutant libraries of novel PET hydrolases and will thus facilitate the identification of promising variants useful for effective plastic waste recycling.

Engineering Stem Cell Factor Ligands with Different c-Kit Agonistic Potencies.

Receptor tyrosine kinases (RTKs) are major players in signal transduction, regulating cellular activities in both normal regeneration and malignancy. Thus, many RTKs, c-Kit among them, play key roles in the function of both normal and neoplastic cells, and as such constitute attractive targets for therapeutic intervention. We thus sought to manipulate the self-association of stem cell factor (SCF), the cognate ligand of c-Kit, and hence its suboptimal affinity and activation potency for c-Kit. To this end, we used directed evolution to engineer SCF variants having different c-Kit activation potencies. Our yeast-displayed SCF mutant (SCFM) library screens identified altered dimerization potential and increased affinity for c-Kit by specific SCF-variants. We demonstrated the delicate balance between SCF homo-dimerization, c-Kit binding, and agonistic potencies by structural studies, in vitro binding assays and a functional angiogenesis assay. Importantly, our findings showed that a monomeric SCF variant exhibited superior agonistic potency vs. the wild-type SCF protein and vs. other high-affinity dimeric SCF variants. Our data showed that action of the monomeric ligands in binding to the RTK monomers and inducing receptor dimerization and hence activation was superior to that of the wild-type dimeric ligand, which has a higher affinity to RTK dimers but a lower activation potential. The findings of this study on the binding and c-Kit activation of engineered SCF variants thus provides insights into the structure–function dynamics of ligands and RTKs.

Screening of Beauveria bassiana with high biocontrol potential based on ARTP mutagenesis and high-throughput FACS

Beauveria bassiana is a promising biocontrol agent due to its entomopathogenic activities and residue-free characteristics. However, its susceptibility to abiotic stresses and naturally low virulence limit the effective application of this fungus. To effectively obtain fungal strains with high biocontrol potential, fluorescence-activated cell sorting (FACS) was used to screen mutant libraries generated by atmospheric and room temperature plasma (ARTP). Among about 8000 mutants obtained by ARTP mutagenesis, six candidate mutants were selected according to the forward scatter (FSC) signal readings of FACS. B6, with a 37.4% higher FSC reading than wild-type (WT), showed a 32.6% increase in virulence. It also presented a 13.5% decrease in median germinating time (GT50) and a 12.1% increase in blastospore production. Comparative analysis between insect transcriptional responses to B6 and WT infection showed that the immune response coupled with protein digestion and absorption progress was highly activated in B6-infected Galleria mellonella larvae, while fatty acid synthesis was suppressed after 3 days of infection. Our results confirmed the feasibility of sorting B. bassiana with high biocontrol potential via the combination of ARTP and FACS and facilitated the understanding of insect-pathogen interactions, highlighting a new strategy for modifying entomopathogenic fungi to improve the efficiency of biological control.

Mutant Construction and Integration Vector-Mediated Genetic Complementation in Listeria monocytogenes.

Genes that play a role in stress response mechanisms and other phenotypes of Listeria monocytogenes can be identified by construction and screening of mutant libraries. In this chapter, we describe the construction and screening of mutant libraries of L. monocytogenes using the plasmid pMC38, carrying a mariner-based transposon system (TC1/mariner) and constructed by Cao et al. (Appl Environ Microbiol 73:2758-2761, 2007). Following screening of mutant libraries, putative mutants are identified and the transposon is localized, leading to identification of the genes responsible for the phenotype of interest. To confirm the role of the transposon-harboring gene in the relevant phenotype, transposon mutants are genetically complemented with the wild-type gene using the site-specific temperature-sensitive integration vector pPL2, constructed by Lauer et al. (J Bacteriol 184:4177-4186, 2002).

Conditionally essential genes for survival during starvation in Enterococcus faecium E745

BackgroundThe nosocomial pathogen Enterococcus faecium can survive for prolonged periods of time on surfaces in the absence of nutrients. This trait is thought to contribute to the ability of E. faecium to spread among patients in hospitals. There is currently a lack of data on the mechanisms that are responsible for the ability of E. faecium to survive in the absence of nutrients.ResultsWe performed a high-throughput transposon mutant library screening (Tn-seq) to identify genes that have a role in long-term survival during incubation in phosphate-buffered saline (PBS) at 20 °C. A total of 24 genes were identified by Tn-seq to contribute to survival in PBS, with functions associated with the general stress response, DNA repair, metabolism, and membrane homeostasis. The gene which was quantitatively most important for survival in PBS was usp (locus tag: EfmE745_02439), which is predicted to encode a 17.4 kDa universal stress protein. After generating a targeted deletion mutant in usp, we were able to confirm that usp significantly contributes to survival in PBS and this defect was restored by in trans complementation. The usp gene is present in 99% of a set of 1644 E. faecium genomes that collectively span the diversity of the species.ConclusionsWe postulate that usp is a key determinant for the remarkable environmental robustness of E. faecium. Further mechanistic studies into usp and other genes identified in this study may shed further light on the mechanisms by which E. faecium can survive in the absence of nutrients for prolonged periods of time.

Multiplexed Enzyme Activity-Based Probe Display via Hybridization.

Emulsions offer the means to miniaturize and parallelize high-throughput screening but require a robust method to localize activity-based fluorescent probes in each droplet. Multiplexing probes in droplets is impractical, though highly desirable for identifying library members that possess very specific activity. Here, we present multiplexed probe immobilization on library beads for emulsion screening. During library bead preparation, we quantitated ∼106 primers per bead by fluorescence in situ hybridization, however emulsion PCR yielded only ∼103 gene copies per bead. We leveraged the unextended bead-bound primers to hybridize complementary probe-oligonucleotide heteroconjugates to the library beads. The probe-hybridized bead libraries were then used to program emulsion in vitro transcription/translation reactions and analyzed by FACS to perform multiplexed activity-based screening of trypsin and chymotrypsin mutant libraries for novel proteolytic specificity. The approach's modularity should permit a high degree of probe multiplexing and appears extensible to other enzyme classes and library types.

Photodegradable Hydrogels for Rapid Screening, Isolation, and Genetic Characterization of Bacteria with Rare Phenotypes.

Screening mutant libraries (MLs) of bacteria for strains with specific phenotypes is often a slow and laborious process that requires assessment of tens of thousands of individual cell colonies after plating and culturing on solid media. In this report, we develop a three-dimensional, photodegradable hydrogel interface designed to dramatically improve the throughput of ML screening by combining high-density cell culture with precision extraction and the recovery of individual, microscale colonies for follow-up genetic and phenotypic characterization. ML populations are first added to a hydrogel precursor solution consisting of polyethylene glycol (PEG) o-nitrobenzyl diacrylate and PEG-tetrathiol macromers, where they become encapsulated into 13 μm thick hydrogel layers at a density of 90 cells/mm2, enabling parallel monitoring of 2.8 × 104 mutants per hydrogel. Encapsulated cells remain confined within the elastic matrix during culture, allowing one to track individual cells that grow into small, stable microcolonies (45 ± 4 μm in diameter) over the course of 72 h. Colonies with rare growth profiles can then be identified, extracted, and recovered from the hydrogel in a sequential manner and with minimal damage using a high-resolution, 365 nm patterned light source. The light pattern can be varied to release motile cells, cellular aggregates, or microcolonies encapsulated in protective PEG coatings. To access the benefits of this approach for ML screening, an Agrobacterium tumefaciens C58 transposon ML was screened for rare, resistant mutants able to grow in the presence of cell free culture media from Rhizobium rhizogenes K84, a well-known inhibitor of C58 cell growth. Subsequent genomic analysis of rare cells (9/28,000) that developed into microcolonies identified that seven of the resistant strains had mutations in the acc locus of the Ti plasmid. These observations are consistent with past research demonstrating that the disruption of this locus confers resistance to agrocin 84, an inhibitory molecule produced by K84. The high-throughput nature of the screen allows the A.tumefaciens genome (approximately 5.6 Mbps) to be screened to saturation in a single experimental trial, compared to hundreds of platings required by conventional plating approaches. As a miniaturized version of the gold-standard plating assay, this materials-based approach offers a simple, inexpensive, and highly translational screening technique that does not require microfluidic devices or complex liquid handling steps. The approach is readily adaptable to other applications that require isolation and study of rare or phenotypically pure cell populations.

An Invertebrate Burn Wound Model That Recapitulates the Hallmarks of Burn Trauma and Infection Seen in Mammalian Models.

The primary reason for skin graft failure and the mortality of burn wound patients, particularly those in burn intensive care centers, is bacterial infection. Several animal models exist to study burn wound pathogens. The most commonly used model is the mouse, which can be used to study virulence determinants and pathogenicity of a wide range of clinically relevant burn wound pathogens. However, animal models of burn wound pathogenicity are governed by strict ethical guidelines and hindered by high levels of animal suffering and the high level of training that is required to achieve consistent reproducible results. In this study, we describe for the first time an invertebrate model of burn trauma and concomitant wound infection. We demonstrate that this model recapitulates many of the hallmarks of burn trauma and wound infection seen in mammalian models and in human patients. We outline how this model can be used to discriminate between high and low pathogenicity strains of two of the most common burn wound colonizers Pseudomonas aeruginosa and Staphylococcus aureus, and multi-drug resistant Acinetobacter baumannii. This model is less ethically challenging than traditional vertebrate burn wound models and has the capacity to enable experiments such as high throughput screening of both anti-infective compounds and genetic mutant libraries.

A method for high-throughput screening hydrolase of lignin \u03b2-aryl ether linkage from directed evolution by glutathione (GSH) assay

BackgroundLignin’s valorization plays a critical role in refining the bioresource. Considering that the β-aryl ether linkage (β-O-4 bond) accounts for 50–70% of lignin chemical linkage between aromatic rings, the hydrolase of lignin β-aryl ether linkage, especially the β-etherase, provided a promising way for the lignin depolymerization and valorization. As a result, it is essential to develop the effective high-throughput methods for screening the mutant library of β-etherase from directed evolution.ResultsBased on the enzymatic mechanism of β-O-4 bond’s cleavage by β-etherase, the LigF was selected as the model to study high-throughput method by GSH assay for screening the mutant library of β-etherase from directed evolution. After the primary study with purified LigF and cell lysate, the GSH assay was used to screen mutant library of β-etherase. The study on screening the mutant library with about 600 colonies indicated that the selected transformants all have one or two mutated sites in the gene sequence of LigF, and the activities from GSH assay of most selected transformants were the same as their activities from HPLC assay.ConclusionsThe results from the high-throughput screening of mutant library demonstrated that GSH assay could be applied to screen β-etherase mutant from directed evolution.

Identification of Regulatory Genes and Metabolic Processes Important for Alginate Biosynthesis in Azotobacter vinelandii by Screening of a Transposon Insertion Mutant Library.

Azotobacter vinelandii produces the biopolymer alginate, which has a wide range of industrial and pharmaceutical applications. A random transposon insertion mutant library was constructed from A. vinelandii ATCC12518Tc in order to identify genes and pathways affecting alginate biosynthesis, and about 4,000 mutant strains were screened for altered alginate production. One mutant, containing a mucA disruption, displayed an elevated alginate production level, and several mutants with decreased or abolished alginate production were identified. The regulatory proteins AlgW and AmrZ seem to be required for alginate production in A. vinelandii, similarly to Pseudomonas aeruginosa. An algB mutation did however not affect alginate yield in A. vinelandii although its P. aeruginosa homolog is needed for full alginate production. Inactivation of the fructose phosphoenolpyruvate phosphotransferase system protein FruA resulted in a mutant that did not produce alginate when cultivated in media containing various carbon sources, indicating that this system could have a role in regulation of alginate biosynthesis. Furthermore, impaired or abolished alginate production was observed for strains with disruptions of genes involved in peptidoglycan biosynthesis/recycling and biosynthesis of purines, isoprenoids, TCA cycle intermediates, and various vitamins, suggesting that sufficient access to some of these compounds is important for alginate production. This hypothesis was verified by showing that addition of thiamine, succinate or a mixture of lysine, methionine and diaminopimelate increases alginate yield in the non-mutagenized strain. These results might be used in development of optimized alginate production media or in genetic engineering of A. vinelandii strains for alginate bioproduction.

Genetic Regulators and Physiological Significance of Glycogen Storage in Candida albicans.

The dimorphic human fungal pathogen C. albicans has broad metabolic flexibility that allows it to adapt to the nutrient conditions in different host habitats. C. albicans builds large carbohydrate stores (glycogen) at the end of exponential growth and begins consumption of stored carbohydrates when nutrients become limiting. The expression of genes required for the successful transition between host environments, including the factors controlling glycogen content, is controlled by protein kinase A signaling through the transcription factor Efg1. In addition to the inability to transition to hyphal growth, C. albicans efg1 mutants have low glycogen content and reduced long-term survival, suggesting that carbohydrate storage is required for viability during prolonged culture. To test this assumption, we constructed a glycogen-deficient C. albicans mutant and assessed its viability during extended culture. Pathways and additional genetic factors controlling C. albicans glycogen synthesis were identified through the screening of mutant libraries for strains with low glycogen content. Finally, a part of the Efg1-regulon was screened for mutants with a shortened long-term survival phenotype. We found that glycogen deficiency does not affect long-term survival, growth, metabolic flexibility or morphology of C. albicans. We conclude that glycogen is not an important contributor to C. albicans fitness.

Inactivation of the pgmA Gene in Streptococcus mutans Significantly Decreases Biofilm-Associated Antimicrobial Tolerance.

Screening of a Streptococcus mutans mutant library indicated that pgmA mutants displayed a reduced biofilm-associated tolerance toward gentamicin. The biofilms formed by the S. mutans pgmA mutant also displayed decreased tolerance towards linezolid and vancomycin compared to wild-type biofilms. On the contrary, the resistance of planktonic S. mutans pgmA cells to gentamycin, linezolid, and vancomycin was more similar to wild-type levels. Investigations of biofilms grown in microtiter trays and on submerged glass slides showed that pgmA mutants formed roughly the same amount of biofilm as the wild type, indicating that the reduced antimicrobial tolerance of these mutants is not due to diminished biofilm formation. The pgmA gene product is known to be involved in the synthesis of precursors for cell wall components such as teichoic acids and membrane glycolipids. Accordingly, the S. mutans pgmA mutant showed increased sensitivity to Congo Red, indicating that it has impaired cell wall integrity. A changed cell wall composition of the S. mutans pgmA mutant may play a role in the increased sensitivity of S. mutans pgmA biofilms toward antibiotics.

A deeper understanding of the spontaneous derepression of the URA3 gene in MaV203 Saccharomyces cerevisiae and its implications for protein engineering and the reverse two-hybrid system.

Within the field of protein-based biomaterials, the need exists for both covalent and oriented bioconjugation strategies for improved performance. Such bioconjugation reactions can be facilitated by engineering proteins with chemically activated amino acids at strategically chosen sites. The incorporation of these unnatural amino acids (uAAs) can be achieved by using the nonsense suppression technique. This requires an aminoacyl-tRNA-synthetase (aaRS) that exclusively recognizes the uAA and loads it to the corresponding tRNA. Appropriate (aaRS) mutants can be found through reverse engineering using the Saccharomyces cerevisiae strain MaV203. This strain contains a counterselectable, Gal4p-inducible SPAL10::URA3 fusion and deletions in the endogenous GAL80 and GAL4 genes. Therefore, it has been used extensively for the screening of aaRS mutant libraries. It is generally assumed that the SPAL10 promoter actively represses the URA3 gene in the absence of Gal4p, resulting in MaV203 cells with a Ura- phenotype. The current contribution reveals that in a small fraction of MaV203 cells, a basal expression of the URA3 gene occurs. The unexpected URA3 expression is reported for the first time, and the nature of the mutation causing this expression was identified as a spontaneous recessive mutation in a single gene of a protein involved in the repression of the SPAL10 promoter. The basal URA3 expression causes aaRS mutants to be missed, which affects the outcome of the library screening. It is demonstrated that the use of diploid cells can circumvent the MaV203 Ura+ phenotype, allowing for an optimization of S.cerevisiae library screening.

Repurposing the thrombopoietin receptor agonist eltrombopag as an anticryptococcal agent.

In this study, a Food and Drug Administration (FDA)-approved drug with previously unreported antifungal activity was investigated for suitability for use as an anticryptococcal agent. First, we screened a compound library of 1018 FDA-approved drugs against Cryptococcus neoformans. Of 52 drugs possessing anti-Cryptococcus activity, eltrombopag was chosen due to its novel activity. The susceptibility of Cryptococcus against eltrombopag was then studied by determining the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC), while the synergy of eltrombopag with other drugs was tested by fractional inhibitory concentration index (FICI). Eltrombopag had a limited spectrum of antifungal activity against C. neoformans/C. gattii species complex (MICs of 0.125mg/l), Candida glabrata (MIC, 0.25mg/l), and Trichophyton rubrum (MIC, 0.5mg/l). Eltrombopag affected cryptococcal virulence factors, including capsule and biofilm formation, melanin production, and growth ability at 37°C. Further, RNA sequencing and deletion mutant library screening experiments revealed that genes involved in the calcineurin pathway, lipid biosynthesis, membrane component, and transporter genes were associated with eltrombopag. In addition, eltrombopag showed synergism with the calcineurin inhibitor FK506 (FICI < 0.5) against Cryptococcus species. In conclusion, eltrombopag exhibited excellent antifungal activity against Cryptococcus species potentially via a mode of action which interferes with virulence factors and the calcineurin pathway, indicating that eltrombopag might be usefully repurposed as an antifungal agent for treating cryptococcosis.

Displaced by Deceivers: Prevention of Biosensor Cross-Talk Is Pivotal for Successful Biosensor-Based High-Throughput Screening Campaigns

Transcriptional biosensors emerged as powerful tools for protein and strain engineering as they link inconspicuous production phenotypes to easily measurable output signals such as fluorescence. When combined with fluorescence-activated cell sorting, transcriptional biosensors enable high throughput screening of vast mutant libraries. Interestingly, even though many published manuscripts describe the construction and characterization of transcriptional biosensors, only very few studies report the successful application of transcriptional biosensors in such high-throughput screening campaigns. Here, we describe construction and characterization of the trans-cinnamic acid responsive transcriptional biosensor pSenCA for Escherichia coli and its application in a FACS based screen. In this context, we focus on essential methodological challenges during the development of such biosensor-guided high-throughput screens such as biosensor cross-talk between producing and nonproducing cells, which could be minimized by optimization of expression and cultivation conditions. The optimized conditions were applied in a five-step FACS campaign and proved suitable to isolate phenylalanine ammonia lyase variants with improved activity in E. coli and in vitro. Findings from this study will help researchers who want to profit from the unmatched throughput of fluorescence-activated cell sorting by using transcriptional biosensors for their enzyme and strain engineering campaigns.

Rapid and Quantitative Profiling of Substrate Specificity of ω‐Transaminases for Ketones

Abstractω‐Transaminases (ω‐TAs) have gained growing attention owing to their capability for asymmetric synthesis of chiral amines from ketones. Reliable high‐throughput activity assay of ω‐TAs is essential in carrying out extensive substrate profiling and establishing a robust screening platform. Here we report spectrophotometric and colorimetric methods enabling rapid quantitation of ω‐TA activities toward ketones in a 96‐well microplate format. The assay methods employ benzylamine, a reactive amino donor for ω‐TAs, as a cosubstrate and exploit aldehyde dehydrogenase (ALDH) as a reporter enzyme, leading to formation of benzaldehyde detectable by ALDH owing to concomitant NADH generation. Spectrophotometric substrate profiling of two wild‐type ω‐TAs of opposite stereoselectivity was carried out at 340 nm with 22 ketones, revealing subtle differences in substrate specificities that were consistent with docking simulation results obtained with cognate amines. Colorimetric readout for naked eye detection of the ω‐TA activity was also demonstrated by supplementing the assay mixture with color‐developing reagents whose color reaction could be quantified at 580 nm. The colorimetric assay was applied to substrate profiling of an engineered ω‐TA for 24 ketones, leading to rapid identification of reactive ketones. The ALDH‐based assay is expected to be promising for high‐throughput screening of enzyme collections and mutant libraries to fish out the best ω‐TA candidate as well as to tailor enzyme properties for efficient amination of a target ketone.

Systems Biology - A Guide for Understanding and Developing Improved Strains of Lactic Acid Bacteria.

Lactic Acid Bacteria (LAB) are extensively employed in the production of various fermented foods, due to their safe status, ability to affect texture and flavor and finally due to the beneficial effect they have on shelf-life. More recently, LAB have also gained interest as production hosts for various useful compounds, particularly compounds with sensitive applications, such as food ingredients and therapeutics. As for all industrial microorganisms, it is important to have a good understanding of the physiology and metabolism of LAB in order to fully exploit their potential, and for this purpose, many systems biology approaches are available. Systems metabolic engineering, an approach that combines optimization of metabolic enzymes/pathways at the systems level, synthetic biology as well as in silico model simulation, has been used to build microbial cell factories for production of biofuels, food ingredients and biochemicals. When developing LAB for use in foods, genetic engineering is in general not an accepted approach. An alternative is to screen mutant libraries for candidates with desirable traits using high-throughput screening technologies or to use adaptive laboratory evolution to select for mutants with special properties. In both cases, by using omics data and data-driven technologies to scrutinize these, it is possible to find the underlying cause for the desired attributes of such mutants. This review aims to describe how systems biology tools can be used for obtaining both engineered as well as non-engineered LAB with novel and desired properties.

A Potential Antifungal Effect of Chitosan Against Candida albicans Is Mediated via the Inhibition of SAGA Complex Component Expression and the Subsequent Alteration of Cell Surface Integrity.

Due to the high incidence of nosocomial Candida albicans infection, the first-line drugs for C. albicans infection have been heavily used, and the emergence of drug-resistant strains has gradually increased. Thus, a new antifungal drug or therapeutic method is needed. Chitosan, a product of chitin deacetylation, is considered to be potentially therapeutic for fungal infections because of its excellent biocompatibility, biodegradability and low toxicity. The biocidal action of chitosan against C. albicans shows great commercial potential, but the exact mechanisms underlying its antimicrobial activity are unclear. To reveal these mechanisms, mutant library screening was performed. ADA2 gene, which encodes a histone acetylation coactivator in the SAGA complex, was identified. Transmission electronic microscopy images showed that the surface of chitosan-treated ada2Δ cells was substantially disrupted and displayed an irregular morphology. Interestingly, the cell wall of ada2Δ cells was significantly thinner than that of wild-type cells, with a thickness similar to that seen in the chitosan-treated wild-type strain. Although ADA2 is required for chitosan tolerance, expression of ADA2 and several Ada2-mediated cell wall-related genes (ALS2, PGA45, and ACE2) and efflux transporter genes (MDR1 and CDR1) were significantly inhibited by chitosan. Furthermore, GCN5 encoding a SAGA complex catalytic subunit was inhibited by chitosan, and gcn5Δ cells exhibited phenotypes comparable to those of ada2Δ cells in response to chitosan and other cell surface-disrupting agents. This study demonstrated that a potential antifungal mechanism of chitosan against C. albicans operates by inhibiting SAGA complex gene expression, which decreases the protection of the cell surface against chitosan.

A genome-wide algal mutant library and functional screen identifies genes required for eukaryotic photosynthesis.

Photosynthetic organisms provide food and energy for nearly all life on Earth, yet half of their protein-coding genes remain uncharacterized1,2. Characterization of these genes could be greatly accelerated by new genetic resources for unicellular organisms. Here, we generated a genome-wide, indexed library of mapped insertion mutants for the unicellular alga Chlamydomonas reinhardtii. The 62,389 mutants in the library, covering 83% of nuclear, protein-coding genes, are available to the community. Each mutant contains unique DNA barcodes, allowing the collection to be screened as a pool. We performed a genome-wide survey of genes required for photosynthesis, which identified 303 candidate genes. Characterization of one of these genes, the conserved predicted phosphatase-encoding gene CPL3, showed it is important for accumulation of multiple photosynthetic protein complexes. Notably, 21 of the 43 highest-confidence genes are novel, opening new opportunities for advances in our understanding of this biogeochemically fundamental process. This library will accelerate the characterization of thousands of genes in algae, plants and animals.