RNAi-Assisted Genome Evolution in <i>Saccharomyces cerevisiae</i> for Complex Phenotype Engineering

A fundamental challenge in basic and applied biology is to reprogram cells with improved or novel traits on a genomic scale. However, the current ability to reprogram a cell on the genome scale is limited to bacterial cells. Here, we report RNA interference (RNAi)-assisted genome evolution (RAGE) as a generally applicable method for genome-scale engineering in the yeast <i>Saccharomyces cerevisiae</i>. Through iterative cycles of creating a library of RNAi induced reduction-of-function mutants coupled with high throughput screening or selection, RAGE can continuously improve target trait(s) by accumulating multiplex beneficial genetic modifications in an evolving yeast genome. To validate the RNAi library constructed with yeast genomic DNA and convergent-promoter expression cassette, we demonstrated RNAi screening in <i>Saccharomyces cerevisiae</i> for the first time by identifying two known and three novel suppressors of a telomerase-deficient mutation <i>yku70</i>Δ. We then showed the application of RAGE for improved acetic acid tolerance, a key trait for microbial production of chemicals and fuels. Three rounds of iterative RNAi screening led to the identification of three gene knockdown targets that acted synergistically to confer an engineered yeast strain with substantially improved acetic acid tolerance. RAGE should greatly accelerate the design and evolution of organisms with desired traits and provide new insights on genome structure, function, and evolution

Fully Automated One-Step Synthesis of Single-Transcript TALEN Pairs Using a Biological Foundry

Transcription activator-like effector nuclease (TALEN) is a programmable genome editing tool with wide applications. Since TALENs perform cleavage of DNA as heterodimers, a pair of TALENs must be synthesized for each target genome locus. Conventionally, TALEN pairs are either expressed on separate vectors or synthesized separately and then subcloned to the same vector. Neither approach allows high-throughput construction of TALEN libraries for large-scale applications. Here we present a single-step assembly scheme to synthesize and express a pair of TALENs in a single-transcript format with the help of a P2A self-cleavage sequence. Furthermore, we developed a fully automated platform to custom manufacture TALENs in a versatile biological foundry. 400 pairs of TALENs can be synthesized with over 96.2% success rate at a material cost of $2.1/pair. This platform opens the door to TALEN-based genome-wide studies

Characterization of <i>Bacillus subtilis</i> Colony Biofilms via Mass Spectrometry and Fluorescence Imaging

Colony biofilms of <i>Bacillus subtilis</i> are a widely used model for studying cellular differentiation. Here, we applied matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to examine cellular and molecular heterogeneity in <i>B. subtilis</i> colony biofilms. From <i>B. subtilis</i> cells cultivated on a biofilm-promoting medium, we detected two cannibalistic factors not found in previous MALDI MSI studies of the same strain under different culturing conditions. Given the importance of cannibalism in matrix formation of <i>B. subtilis</i> biofilms, we employed a transcriptional reporter to monitor matrix-producing cell subpopulations using fluorescence imaging. These two complementary imaging approaches were used to characterize three <i>B. subtilis</i> strains, the wild type isolate NCIB3610, and two mutants, Δ<i>spo0A</i> and Δ<i>abrB</i>, with defective and enhanced biofilm phenotypes, respectively. Upon deletion of key transcriptional factors, correlated changes were observed in biofilm morphology, signaling, cannibalistic factor distribution, and matrix-related gene expression, providing new insights on cannibalism in biofilm development. This work underscores the advantages of using multimodal imaging to compare spatial patterns of selected molecules with the associated protein expression patterns, obtaining information on cellular heterogeneity and function not obtainable when using a single method to characterize biofilm formation

RhlA Exhibits Dual Thioesterase and Acyltransferase Activities during Rhamnolipid Biosynthesis

Rhamnolipids (RLs) are a desirable class of biosurfactants originating from Pseudomonas aeruginosa. Rhamnosyltransferase 1 chain A (RhlA) catalyzes the formation of β-3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) to constitute the RL lipid moiety, and the molecular structure of this moiety exerts major impacts on the physiochemical and biological properties of corresponding RLs. However, the catalytic mechanism and sequence-structure–function relationship of RhlA remain elusive. Here, we report the X-ray crystal structure of P. aeruginosa RhlA with an α/β-hydrolase fold and a canonical nucleophile/histidine/acidic catalytic triad. Unexpectedly, free 3-hydroxy fatty acids within a secondary ligand-binding pocket were observed in the crystal of RhlA, which is traditionally considered an acyltransferase that acts only on acyl carrier protein (ACP)-bound substrates. In vitro isotopic labeling, enzyme kinetics experiments, and QM/MM simulations confirmed that free β-hydroxy fatty acids are a reaction intermediate during HAA synthesis. Moreover, first-shell residue mutations that targeted different ligand-binding pockets resulted in distinct modulation patterns for the two acyl chain lengths of HAAs. In conclusion, the revealed biosynthetic mechanism may guide future engineering for the biosynthesis of designer RLs

Homology-Integrated CRISPR–Cas (HI-CRISPR) System for One-Step Multigene Disruption in <i>Saccharomyces cerevisiae</i>

One-step multiple gene disruption in the model organism Saccharomyces cerevisiae is a highly useful tool for both basic and applied research, but it remains a challenge. Here, we report a rapid, efficient, and potentially scalable strategy based on the type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–CRISPR associated proteins (Cas) system to generate multiple gene disruptions simultaneously in S. cerevisiae. A 100 bp dsDNA mutagenizing homologous recombination donor is inserted between two direct repeats for each target gene in a CRISPR array consisting of multiple donor and guide sequence pairs. An ultrahigh copy number plasmid carrying iCas9, a variant of wild-type Cas9, trans-encoded RNA (tracrRNA), and a homology-integrated crRNA cassette is designed to greatly increase the gene disruption efficiency. As proof of concept, three genes, <i>CAN1</i>, <i>ADE2</i>, and <i>LYP1</i>, were simultaneously disrupted in 4 days with an efficiency ranging from 27 to 87%. Another three genes involved in an artificial hydrocortisone biosynthetic pathway, <i>ATF2</i>, <i>GCY1</i>, and <i>YPR1</i>, were simultaneously disrupted in 6 days with 100% efficiency. This homology-integrated CRISPR (HI-CRISPR) strategy represents a powerful tool for creating yeast strains with multiple gene knockouts

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<p>Systemic wound response (SWR), a well-characterized systemic signaling response, plays crucial roles in plant defense responses. Progress in understanding of the SWR in abiotic stress has also been aided by the researchers. However, the function of SWR in freezing stress remains elusive. In this study, we showed that local mild mechanical wounding enhanced freezing tolerance in newly occurred systemic leaves of wheat plants (Triticum aestivum L.). Wounding significantly increased the maximal photochemical efficiency of photosystem II, net photosynthetic rate, and the activities of the antioxidant enzymes under freezing stress. Wounding also alleviated freezing-induced chlorophyll decomposition, electrolyte leakage, water lose, and membrane peroxidation. In addition, wounding-induced freezing stress mitigation was closely associated with the ratio between reduced glutathione (GSH) and oxidized glutathione (GSSG), and the ratio between ascorbate (AsA) and dehydroascorbate (DHA), as well as the contents of total soluble sugars and free amino acids. Importantly, pharmacological study showed that wounding-induced freezing tolerance was substantially arrested by pretreatment of wheat leaves with the scavenger of hydrogen peroxide (H₂O₂) or the inhibitor of NADPH oxidase (RBOH). These results support the hypothesis that local mechanical wounding-induced SWR in newly occurred leaves is largely attributed to RBOH-dependent H₂O₂ production, which may subsequently induce freezing tolerance in wheat plants. This mechanism may have a potential application to reduce the yield losses of wheat under late spring freezing conditions.</p><p>Highlights:</p><p>In our previous research, we found that local mechanical wounding could induce freezing tolerance in the upper systemic leaves of wheat plants. Surprisingly, in this paper, we further demonstrated that local mechanical wounding could also increase freezing resistance in newly occurred leaves of wheat plants. RBOH mediated H₂O₂ and ascorbate–glutathione cycle participate in this systemic wound response.</p

Profiling of Microbial Colonies for High-Throughput Engineering of Multistep Enzymatic Reactions via Optically Guided Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry (MS) imaging has been used for rapid phenotyping of enzymatic activities, but is mainly limited to single-step conversions. Herein we report a label-free method for high-throughput engineering of multistep biochemical reactions based on optically guided MALDI-ToF MS analysis of bacterial colonies. The bacterial cells provide containment of multiple enzymes and access to substrates and cofactors via metabolism. Automated MALDI-ToF MS acquisition from randomly distributed colonies simplifies procedures to prepare strain libraries without liquid handling. MALDI-ToF MS profiling was utilized to screen both substrate and enzyme libraries for natural product biosynthesis. Computational algorithms were developed to process and visualize the resulting mass spectral data sets. For analogues of the peptidic antibiotic plantazolicin, multivariate analyses by t-distributed stochastic neighbor embedding were used to group similar spectra for rapid identification of nonisobaric variants. After MALDI-ToF MS screening, follow-up analyses using high-resolution MS and tandem MS were readily performed on the same sample target. Separately, relative ion intensities of rhamnolipid congeners with various lipid moieties were evaluated to engineer enzymatic specificity. The glycolipid profiles of each colony were overlaid with optical images to facilitate the recovery of desirable mutants. For both the antibiotic and rhamnolipid cases, large populations of colonies were rapidly surveyed at the molecular level, providing information-rich insights not easily obtained with traditional screening assays. Utilizing standard microbiological techniques with routine microscopy and MALDI-ToF MS instruments, this simple yet effective workflow is applicable for a wide range of screening campaigns targeting multistep enzymatic reactions