Journal: bioRxiv

Article Title: Synthetic biology-guided modular engineering of the secreted cutinase Fo Cut5a with a carbohydrate-binding module improves poly(ester-urethane) depolymerization in Yarrowia lipolytica

doi: 10.64898/2026.10.15.759203

Figure Lengend Snippet: Modular components used to construct the FoCut5a fusion library. The table lists the four signal peptides, four interdomain linkers, and six substrate-binding domains (SBDs) combined in this study, together with their source organisms and amino acid sequences. The catalytic module, FoCut5a, was common to all 96 possible module combinations generated in the library. Signal peptides were included to direct protein secretion in Y. lipolytica, whereas linkers and SBDs were selected to modulate domain spacing and substrate-binding properties, respectively. All SBDs were fused to a common C-terminal V5-His₆ tag (GKPIPNPLLGLDSTRTGHHHHHH). Amino acid sequences are reported using the standard one-letter code.

Article Snippet: The four esterases ( Fo Cut5a, Da PUase, Se1JFR, and Apar EST) and six SBDs were obtained as Y. lipolytica codon-optimized synthetic DNA fragments (Twist Bioscience, USA).

Techniques: Construct, Generated

Journal: bioRxiv

Article Title: Synthetic biology-guided modular engineering of the secreted cutinase Fo Cut5a with a carbohydrate-binding module improves poly(ester-urethane) depolymerization in Yarrowia lipolytica

doi: 10.64898/2026.10.15.759203

Figure Lengend Snippet: Selection of a secreted esterase in Y. lipolytica . (a) Schematic representation of the integration cassette containing promoter (pTEF1), a signal peptide (SigPep), the esterase coding sequence, terminator (tLIP2), and auxotrophic marker (URA3). The expression cassette is flanked by homology arms for site-specific integration into the F11 locus (IntF11 Up and IntF11 Rev) and by NotI restriction sites, which are used to linearize the cassette prior to transformation. Four esterases ( Fo Cut5a, Da PUase, Se1JFR, and Apar EST) were each combined with four candidate signal peptides (SigPep1-SigPep4; teal), generating four secretion variants per esterase. (b) Esterase activity in culture supernatants of individual transformants, quantified by p -nitrophenyl butyrate ( p NPB) hydrolysis and expressed as mU/mL, where 1 mU corresponds to the release of 1 nmol of p -nitrophenol ( p NP) per minute. Each dot represents an independent transformant and corresponds to the mean of two technical replicates (n = 12 transformants for Fo Cut5a, Da PUase and Se1JFR; n = 11 for Apar EST). Boxes indicate the interquartile range (IQR) with the median shown as a horizontal line; whiskers extend to 1.5 × IQR, and dots represent individual transformants. Statistical significance was assessed by two-sided Mann-Whitney U tests comparing Fo Cut5a with each of the other esterases, followed by Holm correction for multiple testing ( Fo Cut5a vs Da PUase: adjusted P = 0.068; vs Se1JFR: adjusted P = 0.091; vs Apar EST: adjusted P = 0.091).

Article Snippet: The four esterases ( Fo Cut5a, Da PUase, Se1JFR, and Apar EST) and six SBDs were obtained as Y. lipolytica codon-optimized synthetic DNA fragments (Twist Bioscience, USA).

Techniques: Selection, Sequencing, Marker, Expressing, Transformation Assay, Activity Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Synthetic biology-guided modular engineering of the secreted cutinase Fo Cut5a with a carbohydrate-binding module improves poly(ester-urethane) depolymerization in Yarrowia lipolytica

doi: 10.64898/2026.10.15.759203

Figure Lengend Snippet: (a,b) Release of poly(ester-urethane)-derived monomers from Impranil DLN-SD by Y. lipolytica strain YM4B5105 expressing Fo Cut5a. (a) Accumulation of adipic acid (blue) and neopentyl glycol (mauve), and (b) 1,6-hexanediol (violet) and 6-hydroxycaproate (teal), quantified by LC-MS after 0, 7, 24, 72, and 144 h of cultivation on 1% Impranil DLN-SD. Data points represent the mean of 2-5 independent measurements (n varied depending on the analyte and time point), and error bars indicate the standard error of the mean (SEM). (c) Proposed structure of Impranil DLN-SD, consisting of alternating polyester soft segments (y) and urethane hard segments (x) repeated along the poly(ester-urethane) chain (z). Monomeric units are colour-coded as in panels a and b; hexamethylene diisocyanate (amber) was not detected among the degradation products. Ester bonds (red) indicate the Fo Cut5a cleavage sites (arrows), releasing adipic acid, 1,6-hexanediol, and neopentyl glycol. The diol connecting soft and hard segments remains linked to the hard segment because it is flanked by an ester bond and a urethane bond. No carbamate bond hydrolysis was observed, consistent with the absence of hexamethylene diisocyanate in the culture supernatant. (d) Proposed metabolic conversion of 1,6-hexanediol to 6-hydroxycaproate via the intermediate 6-hydroxyhexanal.

Article Snippet: The four esterases ( Fo Cut5a, Da PUase, Se1JFR, and Apar EST) and six SBDs were obtained as Y. lipolytica codon-optimized synthetic DNA fragments (Twist Bioscience, USA).

Techniques: Derivative Assay, Expressing, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Synthetic biology-guided modular engineering of the secreted cutinase Fo Cut5a with a carbohydrate-binding module improves poly(ester-urethane) depolymerization in Yarrowia lipolytica

doi: 10.64898/2026.10.15.759203

Figure Lengend Snippet: Polyurethane degradation products released by engineered and control strains. Y. lipolytica strains were cultivated in YNB supplemented with 1% Impranil DLN-SD, and the concentrations of adipic acid (a) and 6-hydroxycaproate (b) in culture supernatants were monitored by HPLC over 144 h. The empty-backbone control strain YM4B5102 is shown in grey, while engineered strains YM4B5105, YM4B5106, YM4B5107, and YM4B5108 are shown in dark red, purple, green, and blue, respectively. Data points represent the mean of 2-5 independent measurements (n varied depending on the analyte and time point), and error bars indicate the standard error of the mean (SEM).

Article Snippet: The four esterases ( Fo Cut5a, Da PUase, Se1JFR, and Apar EST) and six SBDs were obtained as Y. lipolytica codon-optimized synthetic DNA fragments (Twist Bioscience, USA).

Techniques: Control