Journal: Synthetic and Systems Biotechnology

Article Title: AmXlnR, a transcription factor involved in xylan degradation and pentose catabolism, enhances pullulan production from xylose in Aureobasidium melanogenum

doi: 10.1016/j.synbio.2026.02.007

Figure Lengend Snippet: In vivo G FP expression analysis control of the native and mutated promoters of AmXLN2 , AmBXL2 and AmABF1 . ( a ) Schematic representation of the native promoters and their variants with deleted putative XlnR-binding sites: P XLN2 Δ:P XLN2 with 5′-GGCTGA-3′ deleted; P BXL2 Δ:P BXL2 with 5′-GGTTAA-3′ deleted; P ABF1 Δ:P ABF1 with 5′-GGCTAT-3′ deleted. ( b ) Relative fluorescence intensity, ( c ) relative GFP transcriptional level, and ( d ) bright-field and corresponding fluorescence images of the reporter strains. Three independent replicates were performed for the statistical analysis. ∗ P < 0.05,∗∗ P < 0.01, ns: no significance.

Article Snippet: GFP fluorescence intensity was visualized using an Olympus U-LH100HG fluorescent microscope and quantified using a BioTeK Synergy H1 Hybrid Reader (BioTek Instruments Inc., USA) (485 nm excitation and 520 nm emission).

Techniques: In Vivo, Expressing, Control, Binding Assay, Fluorescence

Journal: Nucleic Acids Research

Article Title: Dissecting host stress responses for predictable heterologous gene expression in E. coli

doi: 10.1093/nar/gkag256

Figure Lengend Snippet: Workflow for identifying cellular stress responses to heterologous gene expression. ( a ) Experimental workflow. Expression plasmids were transformed into E. coli and cultivated at 37°C. RNA-seq samples were collected following induction, here illustrated as a marked induction timepoint on a growth curve. iModulon signals were extracted from transcriptomes using ICA, enabling characterization of transcriptional, translational, and product-specific stress contributions, each illustrated as cell icons. ( b ) Design of protein library. The library consisted of 12 different heterologous proteins spanning 0–50 kDa with varying cysteine (0%–25%) and tyrosine (0%–25%) content. Proteins include whey/egg white, eGFP and eGFP fusions, cysteine-rich proteins, tyrosine-rich protein (MFP5), and proteins that fold well (MBP, MNEI). ( c ) ICA methodology. ICA decomposes a gene expression matrix ( X ) into gene weights ( M ) and iModulon activities ( A ), represented as blue, gray, and green rectangles. Gene weights define the membership of genes to each iModulon and relate gene expression to iModulon activities gene expression to iModulon activities across samples ( X = M*A). RNA-seq dataset from this study ( N = 74) was combined with the PRECISE-1K dataset ( N = 1035) to calculate 156 iModulons from 4257 genes.

Article Snippet: We correlated the activities of these responses with eGFP fluorescence intensity levels from our expression library (Fig. ) and found significant positive correlations for both ( r = 0.96 for both, RpoH: P = 1.88e-12, Proteostasis: P = 2.26e-12, Fig. ).

Techniques: Gene Expression, Expressing, Transformation Assay, RNA Sequencing

Journal: Nucleic Acids Research

Article Title: Dissecting host stress responses for predictable heterologous gene expression in E. coli

doi: 10.1093/nar/gkag256

Figure Lengend Snippet: Decoupling transcription and translation using RBS variants. ( a ) Expression plasmid designs. eGFP was expressed using either the native RNAP or T7 RNAP (T7 gene from a bacterial artificial chromosome). Five RBS variants were used to modulate translation efficiency, shown here with their corresponding sequences. Growth (OD 600 , left) and protein production (fluorescence intensity; median FITC-A, right) over time for ( b ) native RNAP and ( c ) T7 RNAP systems. Cultures were induced at OD ∼0.40 (induction line) and RNA-seq samples collected 2 h post-induction (sampling line). ( d ) eGFP mRNA levels (percent of total transcripts) and protein levels (median FITC-A) for each expression condition.

Article Snippet: We correlated the activities of these responses with eGFP fluorescence intensity levels from our expression library (Fig. ) and found significant positive correlations for both ( r = 0.96 for both, RpoH: P = 1.88e-12, Proteostasis: P = 2.26e-12, Fig. ).

Techniques: Expressing, Plasmid Preparation, Fluorescence, RNA Sequencing, Sampling

Journal: Nucleic Acids Research

Article Title: Dissecting host stress responses for predictable heterologous gene expression in E. coli

doi: 10.1093/nar/gkag256

Figure Lengend Snippet: Characterizing protein production stress responses and engineering strain and media improvements. ( a ) Scatterplots showing the correlation between eGFP protein levels (median FITC-A) and iModulon activities for RpoH (top) and Proteostasis (bottom). Pearson correlation coefficients ( r ) and P -values are shown; shaded regions indicate 95% confidence intervals. ( b ) Gene membership comparison between RpoH and Proteostasis iModulons. Gene weights are plotted for each iModulon ( x -axis: RpoH; y -axis: Proteostasis). Dashed lines indicate membership thresholds. Labeled genes exceed membership thresholds in their respective iModulons (green: RpoH genes; blue: Proteostasis genes). ( c ) Function of RpoH and Proteostasis iModulon genes. RpoH genes provide chaperone activity, disaggregation, proteolysis, and refolding. Proteostasis genes regulate osmotic stress and stationary phase adaptation. ( d ) Phase-plane plot comparing RpoH ( x -axis) and Proteostasis ( y -axis) iModulon activities across PRECISE-1K samples ( n > 1000; see “Materials and methods” section). Samples from heat shock experiments (yellow: 30°C; orange: 37°C; red: 44°C) and Protein Stress clusters #1 and #2 (green) are highlighted; remaining samples are gray. ( e ) Violin plots comparing mRNA levels (log-TPM) of rpoH, ibpA , and ibpB across control, protein production, and heat shock conditions. Statistical comparisons are shown with P -values (Student’s t -test). ( f ) Effect of rspAB modulation on eGFP protein production. Top schematics show design of base strain, rspAB overexpression (OE), and rspAB knockout (KO). Time courses show development of eGFP production (mean FITC-A, circles, left y -axis) and cell density (OD 600 , crosses, right y -axis). ( g ) Effect of media supplementation with osmolytes (5 mM choline or 5 mM betaine) in eGFP-producing strains. Mean percent changes (Δ) and P -values (Student’s t -test) are used in panels ( f ) and ( g ) to compare eGFP levels from final timepoint samples to base controls.

Article Snippet: We correlated the activities of these responses with eGFP fluorescence intensity levels from our expression library (Fig. ) and found significant positive correlations for both ( r = 0.96 for both, RpoH: P = 1.88e-12, Proteostasis: P = 2.26e-12, Fig. ).

Techniques: Comparison, Labeling, Activity Assay, Control, Over Expression, Knock-Out

Journal: Nucleic Acids Research

Article Title: Dissecting host stress responses for predictable heterologous gene expression in E. coli

doi: 10.1093/nar/gkag256

Figure Lengend Snippet: Cold shock as a signature stress response to transcriptional burden. ( a ) Correlation between eGFP levels (percent of total transcripts) across eGFP expression conditions. Pearson correlation coefficient ( r ) and P -value are shown; shaded region indicates 95% confidence interval. ( b ) Gene weights of Cold Shock iModulon genes plotted by position on genome. Positive weights indicate activation; negative weights indicate repression. Genes with weights above threshold are labeled and color-coded by COG functional annotation. ( c ) Schematic representations illustrating the cellular functions associated with cold shock response: DNA/RNA chaperones (CSPs) and membrane fluidity (LxpP). DEG scatterplots comparing samples in mRNA Stress cluster ( d ) and mRNA + Protein Stress cluster ( e ) to control cluster. Mean gene expression levels (log-TPM) in stress samples ( y -axis) are plotted against control samples ( x -axis). DEGs are defined by |Δ log-TPM| > 2 and q -value < 0.05. The top 25 most differentially expressed DEGs are outlined and labeled. Genes are colored by iModulon membership (Cold Shock: yellow, RpoH: green, Proteostasis: blue). ( f ) Schematic of the degradosome complex highlighting the cold shock RNA helicase DeaD.

Article Snippet: We correlated the activities of these responses with eGFP fluorescence intensity levels from our expression library (Fig. ) and found significant positive correlations for both ( r = 0.96 for both, RpoH: P = 1.88e-12, Proteostasis: P = 2.26e-12, Fig. ).

Techniques: Expressing, Activation Assay, Labeling, Functional Assay, Membrane, Control, Gene Expression