Microbial Cell Factories

L-Threonine is an essential amino acid widely used in the feed, food, and pharmaceutical industries, and microbial fermentation is now the dominant route for its production. Further increases in producer titers depend on random mutagenesis coupled with high-throughput screening (HTS), but progress has been limited by the lack of a biosensor with a broad operating range, high sensitivity, and target specificity. Here, we show that L-threonine induces the tryptophan-repressed trp promoter of Escherichia coli, a response that, to our knowledge, has not been described previously. Deletion analysis showed that both the trpO operator and the trpL attenuation element contribute to induction, and that the lowest basal expression occurs only when both elements are present. By optimizing the ribosome-binding site, the promoter -10 region, and TrpR expression, we constructed an L-threonine biosensor on a pACYC backbone. The sensor responded linearly to L-threonine from 0.1 to 30 g/L (R2 = 0.9895), with an induction ratio of up to 51.7-fold. Among the 20 proteinogenic amino acids tested, only L-threonine produced a detectable response. The biosensor distinguished strains with different L-threonine production capacities and guided the directed evolution of homoserine kinase (ThrB) at a conserved motif, yielding variants with brighter reporter fluorescence and increased L-threonine production. This work provides a high-performance biosensor based on native E. coli regulatory elements for screening L-threonine overproducers.

Glucoamylase is essential for industrial starch saccharification, but the limited thermostability and near-neutral pH tolerance of fungal glucoamylases necessitate cooling and acidification of liquefied starch. Here, we developed an artificial intelligence-guided strategy to simultaneously improve the thermostability, pH tolerance, and catalytic activity of glucoamylase from Penicillium oxalicum (PoGA). Two property-specific machine-learning models, CASPE-T and CASPE-A, identified substitutions associated with thermostability and pH tolerance, respectively. Experimental screening identified beneficial substitutions in 11 of 21 CASPE-T and 12 of 22 CASPE-A candidates. Folding-energy-guided recombination integrated the two traits while maintaining structural compatibility. The optimal variant, PoGA T513E/Q305N, exhibited 2.21-fold higher specific activity than the wild type, with half-life extended from 22.3 to 57.9 min at 60 degrees C and from 16.6 to 64.7 min at pH 8.0. Molecular dynamics simulations attributed these improvements to reinforcement of high-occupancy hydrogen-bonding networks, suppression of conformational fluctuations in the linker and carbohydrate-binding module, enhanced long-range dynamic coordination, and preservation of a compact catalytic architecture. At 60 degrees C and pH 6.5 without acidification, PoGA T513E/Q305N produced 219.9 g/L glucose and achieved 89.1% starch conversion, 31.4% higher than the wild type. This work provides an efficient framework for multi-objective enzyme engineering and sustainable starch biorefining.

Genome transplantation is a key technology for synthetic genomics, enabling entire genomes to be transferred into recipient cells. Despite its importance, genome transplantation remains confined to a small number of wall-less Mollicute species and is often characterized by low efficiencies, limiting the development and testing of synthetic genomes. Because genome transplantation relies on the same polyethylene glycol mediated DNA delivery process used for plasmid transformation, improving DNA uptake is an important step toward more efficient transplantation systems. Here, we systematically optimized polyethylene glycol-mediated transformation in Mesoplasma florum, a fast-growing, non-pathogenic Mollicute with a reduced genome that represents an attractive chassis for synthetic genomics. We evaluated 20 parameters spanning cell physiology, DNA preparation, membrane conditioning, and recovery conditions. DNA topology emerged as the strongest determinant of transformation efficiency, with highly compacted DNA preparations producing up to three orders of magnitude more transformants than conventional plasmid preparations. Growth phase and polyethylene glycol concentration also strongly influenced transformation outcomes. To identify potential recipients for future genome transplantation experiments, we further screened ten strains belonging to the Mesoplasma lineage. Transformation efficiencies varied widely among strains, and Mesoplasma entomophilum W17 emerged as a particularly promising candidate. Moreover, W17 supported replication of plasmids carrying the M. florum oriC, suggesting compatibility between the replication systems of the two species. Together, these results establish an improved transformation workflow for M. florum, identify DNA topology as a major determinant of DNA uptake, and reveal a new candidate recipient for future genome transplantation studies. These advances provide practical tools and biological insights for synthetic genome engineering in Mollicutes.

Carbohydrate inducible promoters on expression plasmids are widely used for biotechnology and basic science research. However, using carbohydrates for inducers has drawbacks including influencing bacterial metabolism, the inducer being used as a carbon source by the bacteria, high background expression, and only a subset of cells experiencing promoter activation. Furthermore, some bacteria including the pathogen Acinetobacter baumannii currently lack good carbohydrate-inducible promoters. To address this issue, a tetracycline-inducible promoter, PtetB, isolated from A. baumannii was cloned and evaluated in a number of important bacteria: Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Serratia marcescens. A highly sensitive luxCDABE reporter was used to evaluate promoter activity. S. marcescens strains with elevated and reduced cyclic-AMP levels were used to evaluate the impact of cAMP on PtetB function. The PtetB promoter was induced over 800-fold in A. baumannii and 10-750-fold inducible in the other tested species. Background expression was lower from PtetB in the pseudomonads than previously described carbohydrate inducible promoters. Data indicated that the PtetB promoter system may be a useful tool for future basic science research with the tested bacteria.

Influenza A virus (IAV) defective interfering particles (DIPs) are regarded as promising broad-spectrum antivirals due to their high efficacy demonstrated in animal trials. Previously, an OP7 chimera DIP was generated that showed markedly enhanced antiviral efficacy compared to conventional IAV DIPs. In addition, a high-yield cell culture-based production process for OP7 chimera DIPs was established in perfusion mode. However, due to the technical complexity of this technology, we aimed to combine the advantages of perfusion in terms of yield with that of a simple and less complex batch process. In the present work, we established a fed-batch production process for OP7 chimera DIPs that allows a more straightforward implementation into existing production facilities. Using a feeding regime that was able to overcome the cell density effect, we achieved productive virus infection in stirred tank reactors until a cell density of 10E+6 cells/mL. Furthermore, we obtained a high virus titer of 3.8 log10(HAU/100 uL), corresponding to a 3.4-fold increase compared to a reference batch production. Moreover, the produced material demonstrated a significantly increased interfering bioactivity shown by an in vitro potency assay. Interestingly, this efficacy was similarly high as compared to OP7 chimera DIP material previously produced in perfusion mode, despite twofold lower virus titers. Finally, we demonstrate robustness of OP7 chimera DIP seed virus trains, which is significant step towards later pharmaceutical production. Further, corresponding GMP MDCK-PB2(sus) cell and OP7 chimera virus banks are now available for later biomanufacturing further use. In conclusion, the established fed-batch process offers a valid n additional option for pharmaceutical grade OP7 chimera DIP GMP manufacturing production under GMP conditions required for subsequent clinical testing and for later large-scale manufacturing.

Bacterial genomes are dynamic and harbour accessory regions that can be gained, lost, or rearranged in response to selective pressures. Members of the genus Komagataeibacter are used for bacterial cellulose production and researched as platforms for engineered living materials. However, their genomes are rich in mobile genetic elements (MGEs), including insertion sequences (ISs), genomic islands and prophage remnants, which drive genome rearrangements under agitated cultivation conditions. Whether this genome architecture contributes to genomic changes during routine strain maintenance and distribution remains poorly understood. Comparative genome sequencing of independent Komagataeibacter rhaeticus iGEM stocks revealed recurrent loss of a 210 kbp chromosomal region flanked by homologous IS5 sequences. This deletion was identified in a stock received as an agar stab and reproduced from an intact-genome population subjected to simulated agar-stab handling. Phylogenomic analysis of the affected locus revealed gene-tree/species-tree discordance, heterogeneous GC content and multiple ISs, supporting its characterization as a dynamic accessory region. Populations carrying the larger genome reduction showed improved growth and cellulose production relative to the intact genome reference, whereas populations carrying a smaller nested deletion showed reduced performance. These findings demonstrate that routine strain handling is not necessarily genomically neutral and can expose structural genomic variabilities that generate population-level genomic heterogeneity with phenotypic consequences. For bacteria with MGE-rich genomes, handling history may represent an underappreciated source of genomic and phenotypic variation between laboratories. More broadly, identifying unstable accessory regions may highlight genomic content that is dispensable under laboratory conditions, providing candidates for genome streamlining and strain improvement.

Synthetic allopolyploidization is a powerful strategy for generating strains with novel industrially relevant traits. During the design of select, hybridize, evolved and learn cycle, the mitochondrial inheritance is considered neutral and most studies overcome their phenotypic impact. Here, we generated 168 synthetic allotetraploid strains representing all pairwise parental combinations of eight wild Saccharomyces species, and systematically controlled mitochondrial inheritance in these crosses. These allopolyploids were phenotyped across 31 environmental conditions relevant to biotechnology. In ~50% of all condition-strain combinations conditions, allotetraploids exhibited mid-parent heterosis. In approximately 25% of conditions, we found crosses with hybrid vigor, particularly in combinations involving Saccharomyces arboricola x Saccharomyces mikatae . However, allotetraploids were highly unstable and rapidly sporulated. Our analyses revealed that environmental conditions accounted for the largest effects, follow by its interaction with mitotype. However, mitotype alone showed small effects but significantly affected 42% of the tested conditions, underscoring the importance of mitochondrial inheritance as a key determinant of allopolyploid performance. These findings demonstrate that mitochondrial inheritance is a key, environment-dependent determinant of allopolyploid fitness and should be considered alongside nuclear genome composition in the rational design of industrial allopolyploid strains

Epidemiological studies associate replacing animal-sourced with plant-sourced protein with lower colorectal cancer (CRC) risk, but controlled feeding studies have rarely tested how this substitution alters the gut microbiota and CRC-related biomarkers. In a 12-week randomized controlled feeding study, 136 healthy adults consumed diets providing 30 energy percent (E%), 50 E%, or 70 E% of protein from plant sources. Change in gut microbiota composition was the primary outcome; stool characteristics, fecal N-nitroso compounds (NOCs), and fecal and plasma bile acids (BAs) were secondary outcomes. The 70% plant-protein diet significantly restructured the gut microbiota, increasing fiber-associated Lachnospiraceae and Roseburia while reducing Methanobrevibacter and several asaccharolytic taxa. Microbial functional potential shifted toward plant-carbohydrate degradation, with lower amino acid degradation and methanogenesis. Fecal pH decreased, stool characteristics suggested faster colonic transit, and potentially carcinogenic total and haem-derived NOCs declined progressively with plant-protein replacement. The 70% plant-protein diet also reduced fecal secondary and taurine-conjugated BAs, including the CRC-associated BAs DCA and LCA. Plasma glucuronidated BAs increased, expanding the circulating pool of readily excreted BA conjugates. Higher insoluble fiber intake was associated with greater microbiota restructuring and lower fecal NOCs. Methanogen carriers had higher baseline fecal NOCs and features of slower colonic transit, and showed weaker microbiota and less favorable BA responses to the 70% plant-protein diet. Baseline methanogen carriage thus could be exploited for participant stratification in future dietary interventions.

The unprecedented rise in greenhouse gases such as carbon dioxide (CO2), and their detrimental effects on the atmosphere have intensified the current climate emergency. This challenge has inspired the development of novel carbon capture and utilisation (CCU) technologies, specifically microbial electrosynthesis (MES), for the bioelectrochemical fixation of CO2 into commodity chemical compounds (CCCs) using microbial biocatalysts. In this study, we systematically enriched and maintained robust mixed microbial communities of electroactive bacteria (EAB) from wastewater treatment sludge to serve as biocatalysts for MES. The MES performance of these biocatalysts under different operational conditions in bioelectrochemical systems (BES) reactors were investigated. BES reactors with enriched biocatalysts incubated and maintained under controlled conditions produced higher CCCs yields than those developed at ambient temperatures, while strict anaerobic operations further improved the MES efficiency compared to aerobic conditions. At an applied potential of -1000 mV vs Ag/AgCl, CO2 conversion reached up to 88.11%, significantly outperforming the lower potential (-600 mV) operation. These findings underscore the importance of systematic development of robust and stable biocatalysts, as well as maintaining strict anaerobic conditions, for achieving efficient MES performance, thereby demonstrating the potential of our approach for scalable industrial CCU applications.

The valorisation of biological polymers requires microbial systems that can both access recalcitrant substrates and convert the resulting carbon into useful products. Although microbial genome and metagenome resources provide an expanding reservoir of candidate depolymerizing and modifying enzymes, most discovery workflows remain disconnected from enzyme optimisation and host adaptation. Here we present a coupled sequence-based enzyme discovery, enzyme evolution and synthetic yeast chassis adaptation strategy for microbial biopolymer valorisation. Focusing on laccases for the depolymerisation of lignin as a proof of concept, we combine sequence data mining for enzyme discovery, modular yeast surface display for functional screening, directed evolution for enzyme optimisation and synthetic yeast genome diversification for chassis improvement. In our study, surface display enabled functional benchmarking and recovery of improved laccase variants and synthetic-genome-enabled diversification provided a route to explore host configurations that influence display and enzyme performance. By integrating enzyme-level and chassis-level optimisation, this framework addresses a central bottleneck in converting microbial biodiversity by computational sequence repository mining into deployable biomanufacturing systems. Our results establish laccases as tractable entry points for oxidative biopolymer conversion and provide a generalizable platform for engineering yeast systems for sustainable carbon valorisation.

Ugba, a traditional alkaline-fermented condiment produced from African oil bean seeds (Pentaclethra macrophylla Benth.), remains poorly characterised at genome resolution. This study characterised a single spontaneous ugba fermentation using genome-resolved shotgun metagenomics across Early (0-24 h), Mid (48-96 h), and Late (120-144 h) composite stages. Thirty metagenome-assembled genomes (MAGs) were recovered and grouped according to their temporal abundance patterns. The Early-stage retained MAG population was dominated by Bacteroidota-associated MAGs, which exhibited the highest carbohydrate-active enzyme density and broad glycoside hydrolase repertoires, consistent with substantial genome-encoded potential for utilisation of the plant-derived seed matrix. The Mid stage showed marked restructuring of MAG composition, while Core and Late-associated MAGs increased towards fermentation maturation. Functional differentiation was also evident among Corynebacterium MAGs: C. nuruki encoded a complete urease system, whereas C. phoceense_A encoded glutamate dehydrogenase and nitrate-reduction genes. These stage-associated changes coincided with progressive alkalinisation from pH 7.15 to 8.01. Together, the results show that temporal restructuring of reconstructed microbial populations was accompanied by differentiation in genome-encoded functional potential. This study provides a genome-resolved characterisation of microbial composition and functional potential during spontaneous ugba fermentation and expands current understanding of the microbial organisation of this traditional African fermented food.

Mesoplasma florum is a fast-growing, near-minimal bacterium and an emerging model for systems and synthetic biology. However, its dependence on complex serum-containing media limits experimental control and complicates the interpretation of cellular phenotypes. Here, we developed CMRL-AT, a serum-free, quasi-defined medium that supports rapid growth comparable to the commonly used ATCC 1161 medium. Despite supporting similar biomass, CMRL-AT profoundly reshaped the M. florum transcriptome, with approximately one-third of the annotated protein-encoding genes being differentially expressed relative to ATCC 1161. These changes revealed distinct physiological programs associated with rapid growth in complex medium and higher nutrient acquisition in CMRL-AT, illustrating how medium composition alters the functional priorities of a near-minimal cell. Transcriptome profiling across six energy sources further uncovered distinct sugar-responsive expression programs. Combining these responses with transcription-unit organization, protein-domain predictions, and metabolic context resolved fructose- and sucrose-responsive modules, and allowed the assignment of previously ambiguous phosphotransferase system components to specific sugar-utilization pathways. CMRL-AT provides an experimental framework to help resolving gene functions, refining metabolic models, and designing reduced genomes adapted to defined environments.

Electromicrobial production (EMP) technologies aim to combine renewable electricity, CO2, and engineered microbes to make energy-dense molecules at efficiencies exceeding photosynthesis. CO2 can be electrochemically reduced to formate, which is far easier to handle at the bench than H2 or an electrode, but formate carries only two electrons per carbon against the six in a biofuel. The remaining electrons must come from oxidizing additional formate, from H2 oxidation, or from extracellular electron uptake (EEU), and no rigorous comparison of these options coupled to the choice of carbon assimilation pathway currently exists. We calculate upper-limit efficiencies for butanol production by six carbon assimilation pathways, each paired with all three electron delivery mechanisms, using electrochemical parameters drawn from a survey of the recent literature. Electrical to butanol energy conversion efficiencies range from 35.5 to 51.7%, corresponding to solar-to-fuel efficiencies of 11.7 to 17%, so even the least efficient route exceeds the 8% theoretical ceiling of algal photosynthesis. The serine variant of the reductive glycine pathway reaches an electrical energy conversion efficiency of when using H2 oxidation, within 1.9 points of the most efficient pathway, and is the only high-efficiency option that tolerates O2. This makes an EMP system that combines electron delivery by formate coupled with the serine variant of reductive glycine pathway highly attractive, as it presents few barriers to rapid, iterative engineering in the lab, and a high theoretical ceiling. Drawing both carbon and electrons from formate costs 6.2 points against H2 at a state-of-the-art whole-cell voltage (2.2 V). However, this small penalty is amplified three-fold by any rise in the CO2-to-formate cell voltage, and reaches 13.5 points at the highest whole-cell voltages reported for scaled-up CO2-to-formate electrolyzers, where formate-only operation falls to 11.2 electrical-to-fuel and 3.7% solar-to-fuel efficiency, below the ceiling of photosynthesis, against 24.7 and 8.1% for H2 (only just above algal photosynthesis). Our choice between a formate-only system and one coupled to H2 oxidation or EEU therefore depends on our belief about the trajectory of CO2 reduction technology. If whole-cell voltages continue to fall at the rate of the past decade, formate alone is the right target, and the simplicity of its workflow is bought at low cost. However, if that improvement plateaus, the electron delivery mechanism must be swappable, and a system should be designed from the outset so that it can be. At the US Department of Energy SunShot target of 2 cents per kilowatt hour, the electricity to make a US gallon of butanol costs $1.40 for a formate-only system at the state of the art, rising to $5.45 at the highest scaled-up electrolyzer voltage reported, against $1.23 and $2.47 for formate and H2 system.

Disruption of the intestinal microbiota increases susceptibility to Clostridioides difficile infection (CDI), creating an opportunity for prophylactic interventions before symptomatic disease develops. We investigated whether Escherichia coli Nissle 1917 (EcN) engineered to express the bacteriophage-derived endolysin CD27L could enhance pathogen inhibition and attenuate CDI. Using a plasmid-cured EcN chassis and pMUT2-derived vectors, we generated strains expressing CD27L under constitutive or anaerobically regulated promoter control. The constitutive strain, BioEcN1, reduced viable C. difficile recovery relative to parental EcN and was selected for animal studies. BioEcN1 remained recoverable from monoassociated mice without in vivo antibiotic selection, with similar recovery on selective and nonselective media supporting construct retention. Prophylactic administration reduced clinical disease, body-weight loss, and intestinal histopathological injury in germ-free mice. Observed survival was 100% in BioEcN1-treated challenged mice versus 50% in untreated challenged mice during the monitored period. Protection was accompanied by lower fecal and cecal pathogen burdens. In mice harboring a human-derived dysbiotic microbiota, prophylactic BioEcN1 also reduced clinical and stool-consistency scores, with lower early fecal pathogen recovery and less colon shortening. These findings establish preclinical proof of concept for prophylactic administration of an endolysin-expressing probiotic to attenuate CDI.

Polyhydroxyalkanoates (PHAs) are microbial polyesters that represent a sustainable alternative to fossil fuel-based plastics. These biodegradable polymers have properties similar to conventional plastics and can be produced from waste materials. The key enzyme in PHA synthesis is PHA synthase (PhaC) and despite its importance, only a few PhaCs from extremophilic microorganisms have been characterised in detail. Here we characterised PHA synthase enzyme from the thermophilic bacterium Caldimonas thermodepolymerans DSM15344, an attractive candidate for PHA production from lignocellulosic residues due to its unique properties such as utilisation of xylose-rich substrates and high PHA yields on this pentose sugar. PhaC was successfully expressed in the surrogate host E. coli BL21 (DE3) GOLD, purified to homogeneity and its enzymatic activity was confirmed in vitro. The optimal pH range and temperature were determined as 6.3-6.9 and 50 {degrees}C, respectively. The melting temperature of PhaC was detected at 57.83 {+/-} 0.04 {degrees}C and the enzyme storage stability was assessed. The tertiary structure, including the enzyme active site, was predicted using available bioinformatic tools and the predicted catalytic residues were supported by site-directed mutagenesis. This study represents the first biochemical characterisation of PHA synthase from C. thermodepolymerans, expanding the limited set of characterised thermophilic PhaC enzymes.

Plasmid copy number (PCN) is a central determinant of recombinant gene expression levels and metabolic burden in bacterial cell factories. However, dynamic real-time control of PCN during microbial cultivation remains largely unexplored. In the ColE1/pUC19 replication system, PCN is governed by an antisense RNAI/RNAII circuit, but this native mechanism provides no external handle for process-level intervention. Here, we engineer and characterize a dual-layer control architecture that combines two orthogonal perturbations to direct plasmid replication in Escherichia coli populations i.e., temperature shifts, which modulate the secondary structure of RNAII and, consequently, its binding to RNAI and its inhibitory potency (ex vivo control), and arabinose-inducible overexpression of a supplementary RNAI cassette, integrated either on a secondary low-copy plasmid or directly on the high-copy target plasmid (in vivo control). Using a superfolder GFP validated fluorescent reporter and automated flow cytometry in continuous cultures, we track PCN dynamics at the population level in real time under chemostat conditions. Preliminary screening in microplates high-throughput cultivation device across three strains, two carbon sources, and three temperatures (30, 37, and 42C) reveals that the two control layers act synergistically. Neither temperature alone nor inducible RNAI alone achieves the dynamic range obtained by their combination. This synergistic effect is sustained during continuous cultivation at a fixed dilution rate of 0.1/h, with carbon source and temperature transitions producing predictable and reproducible shifts in population-level PCN. These results establish a scalable, dual-input framework for on-demand PCN tuning in continuous bioprocesses, with direct relevance to biopharmaceutical plasmid production and synthetic biology applications.

The enzymatic oxidation of 5-hydroxymethylfurfural (HMF) to 2,5-furandicarboxylic acid (FDCA) is a key step in the sustainable production of polyethylene furanoate (PEF), yet the final oxidation of 5-formyl-2-furancarboxylic acid (FFCA) to FDCA remains the principal bottleneck for industrial application. Although hydroxymethylfurfural oxidases (HMFOs) can catalyze this transformation, naturally occurring enzymes with high FFCA-oxidizing activity remain largely unexplored. Here, we present an iterative computational bioprospecting campaign to identify natural HMFOs with improved activity toward this rate-limiting reaction. Across four successive rounds, the computational workflow evolved from conservative homology-based screening to large-scale sequence mining assisted by machine learning, with each iteration redesigned according to the principal limitations identified through molecular simulations and experimental characterization. This adaptive strategy led to the discovery of several natural HMFOs with substantially improved FFCA oxidation activity, including candidates whose performance under the tested conditions approached that of the engineered benchmark 8BxHMFO. Beyond the identification of promising biocatalysts, the study demonstrates the value of continuous integration of computational prediction and experimental feedback for refining enzyme discovery campaigns, and provides practical insights into balancing catalytic prioritization, sequence diversity, developability, and computational scalability in future simulation-guided bioprospecting efforts.

Background. Vaccines have been an essential tool in bringing the world close to polio eradication, with over 99.9 percent of the global population free of poliovirus. No antiviral drugs or monoclonal antibody products, however, are currently licensed for treatment of polio. We previously isolated a human monoclonal antibody (9H2) with potent neutralizing activity against all three poliovirus serotypes. To advance 9H2 as a therapeutic candidate, we optimized its sequence to extend serum half-life and improve manufacturability. Methods. A Multi-Attribute Method under stress conditions identified post-translational modification sites, and Abacus (Trademark) ranked sequence liabilities. Six amino acid substitutions were introduced into the variable regions, generating 23 combinatorial variants. Codon-optimized genes were synthesized and engineered into a human immunoglobulin G1 backbone containing crystallizable fragment (Fc) mutations (M428L/N434S) to extend serum half-life. Constructs were transfected into CHO K1 cells to generate stable pools in 24 well plates. Protein A purified antibodies were characterized using biophysical assays and an in vitro poliovirus neutralization assay. Results. All variants retained high in vitro neutralizing activity against poliovirus. A lead candidate was selected based on integrated assessment of biophysical properties across eight assays and expression yield. Conclusions. Structure-guided engineering and experimental evaluation enabled optimization of the 9H2 antibody sequence and identification of a lead candidate suitable for clinical manufacturing as a potential anti-poliovirus immunotherapeutic agent.

Engineering proteins at the scale of modern high-throughput screening technologies remains challenging because most experimental libraries sample only limited regions of sequence space while maintaining relatively low levels of functional diversity. Although recombination-based strategies can introduce substantially larger sequence changes than conventional mutagenesis approaches, it is unclear whether highly recombined enzymes can retain catalytic competence when library sizes approach millions to billions of variants. To investigate this question, we constructed a recombination library by combining ten sequence blocks from ten bacterial PETases, generating a theoretical diversity of 10^8 chimeric enzymes. Screening of yeast-displayed libraries using a fluorophosphonate probe for catalytic-serine reactivity identified substantial populations of catalytically competent variants despite extensive sequence recombination and dozens of amino acid substitutions per chimera. Approximately 2% of sampled variants possessed active-site serine reactivity, corresponding to an estimated 10 catalytically competent enzymes within the full theoretical library. Deep sequencing showed that functional populations remained broadly distributed across sequence space following selection and contained multiple enriched recombination patterns spanning the PETase structure. Secondary lysate screening and enzyme-normalized activity measurements further identified multiple purified PETase chimeras with soluble esterase activity comparable to or greater than the wild type Ideonella sakaiensis PETase on a chromogenic surrogate substrate. Together, these results demonstrate that large-scale recombination can generate millions of diverse and catalytically competent enzymes while remaining compatible with modern high-throughput screening workflows. More broadly, this work establishes recombination-based diversity generation as a scalable strategy for protein engineering and large-scale sequence-function data generation.

The human gut microbiota has been explored for the last two decades and studies have uncovered its major role in the degradation of complex glycans from the diet as well as the host tissues. Strikingly, until very recently, no similar investigation has been carried out on the human oral cavity microbiota, which is increasingly recognized has an important parameter in gastrointestinal health. Prevotellaceae are an important and under-investigated family of bacteria in both the gut and the oral cavity microbiota. The oral cavity is particularly rich in glycosaminoglycans which could provide an abundant carbon source for the inhabiting microorganisms, and play a significant role in host-pathogen interactions. Hence, we analyzed Polysaccharide Utilization Loci in oral microbiota species and we identified a glycosaminoglycan-degrading Polysaccharide Utilization Locus in Segatella oris. We produced and characterized the four enzymes, confirmed their ability to degrade glycosaminoglycans, and we focused our efforts on the biochemical and structural characterization of the first reported Prevotellaceae endosulfatase.