ACS Synthetic Biology最新文献

{"title":"Engineering Auronidin Production in Marchantia polymorpha Enables a New Class of Plant-Derived Textile Pigments","authors":"Aurore Lormet,&nbsp;Jim Haseloff,&nbsp;Facundo Romani","doi":"10.1021/acssynbio.6c00118","DOIUrl":"10.1021/acssynbio.6c00118","url":null,"abstract":"<p>Plant-derived pigmentsoffer sustainable alternatives to syntheticcolorants, yet their practical deployment in textiles is limited byrestricted chemical diversity and low abundance. Liverworts representa source of diverse chemical compounds, and the model liverworts <em>Marchantia polymorpha</em> are emerging as chassis for bioengineeringand synthetic biology. Here, we report the biotechnological applicationof auronidins, a rare class of flavonoid pigments, as textile dyes.Using <em>M. polymorpha</em>, we engineered enhanced auronidinproduction through controlled expression of the R2R3-MYB transcriptionfactor Mp<em>MYB14</em>. We systematically benchmarked constitutiveand inducible gene expression systems, including heat-shock, glucocorticoidreceptor, and β-estradiol (XVE) circuits, identifying induciblestrategies that decouple biomass accumulation from secondary metaboliteproduction while achieving high pigment yields. Extracted auronidinswere used to dye cotton yarn directly, demonstrating the feasibilityof auronidins for textile dyeing. Our results establish <em>M. polymorpha</em> as a versatile plant chassis for programmablesecondary metabolite production and introduce auronidins as a promisingnatural pigment platform for sustainable textile biotechnology.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"15 8","pages":"3139–3145"},"PeriodicalIF":4.5,"publicationDate":"2026-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148496858","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Complete Utilization of Poly(butylene Adipate-co-terephthalate) Monomers and Production of Polyhydroxybutyrate by a Levulinic Acid-Mediated Microbial Consortium of Pseudomonas putida and Cupriavidus necator","authors":"Gaeun Lim,&nbsp;Yunhee Jeong,&nbsp;Seung Hun Lee,&nbsp;Tae Yun Kim,&nbsp;Gun Woo Doh,&nbsp;Hee Taek Kim,&nbsp;Jae-Seok Kim,&nbsp;Shashi Kant Bhatia,&nbsp;Hyung Chul Kim,&nbsp;Yung-Hun Yang","doi":"10.1021/acssynbio.6c00263","DOIUrl":"10.1021/acssynbio.6c00263","url":null,"abstract":"<p>Poly(butylene adipate-<em>co</em>-terephthalate)(PBAT)is a biodegradable plastic with enhanced mechanical properties, whichcan serve as a substitute for conventional plastics. However, themonomers released during its biodegradation, such as adipic acid (AA),1,4-butanediol (1,4-BDO), and terephthalic acid (TPA), accumulateinto the environment or are not simply consumed through central metabolism,limiting their resource value. Therefore, designing a single strainthat can efficiently consume all monomers is challenging. Here, aconsortium comprising <em>Pseudomonas putida</em> KT2440 harboring <em>tpaK</em>, <em>tphA</em>, and <em>tphB</em> which metabolizes 1,4-BDO and TPA and <em>Cupriavidus necator</em> H16, which converts AA into polyhydroxybutyate(PHB) via β-oxidation was constructed to convert all PBAT-derivedmonomers into PHB. To enhance polyhydroxyalkanoate production, a levulinicacid (LA)-producing strain GP101 was added to the consortium, resultingin 0.75 g/L PHB and 0.20 g/L 4-hydroxybutyrate (4HB) production andrepresenting a 6.25-fold increase in PHB production compared to thatby the consortium containing <em>P. putida</em> KT2440 that could not produce LA. Optimizing the strain ratio of <em>P. putida</em> KT2440 GP101 to <em>C. necator</em> H16 to 2:1 resulted in maximum production of 0.98 g/L PHB and 0.52g/L 4HB, and all PBAT-derived monomers were completely disappearedwithin 30 h. This is the first study to demonstrate complete utilizationof PBAT monomers and valorization into PHB by an LA-mediated microbialconsortium. This approach reveals controllable and reproducible consortiabeyond the complex design of a single strain and simple treatmentof each monomer, demonstrating the feasibility of converting PBAT-derivedmonomers into value-added products.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"15 8","pages":"3369–3381"},"PeriodicalIF":4.5,"publicationDate":"2026-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148519591","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Breaking the Stability-Activity-Selectivity Trilemma in Unspecific Peroxygenase through Computation-Based Cross-Regional Combinatorial Mutagenesis","authors":"Ruichen Gao,&nbsp;Fenglin Ye,&nbsp;Zesen Deng,&nbsp;Huayi Liu,&nbsp;Xuemei Zeng,&nbsp;Wenyong Lou,&nbsp;Xiaoling Wu","doi":"10.1021/acssynbio.6c00196","DOIUrl":"10.1021/acssynbio.6c00196","url":null,"abstract":"<p>The dynamic coupling relationship between the distalregion andthe region adjacent to the active site in enzymes is still poorlyunderstood. The resultant trade-off among stability, catalytic activity,and enantioselectivity remains a big challenge in developing robustenzymes in agrochemistry. Here, FoldX-based global saturation mutagenesispredictions with spatially targeted prioritization of functional regions,including the N-terminal domain, substrate access pocket, and dimerinterface of the enzyme, are developed and validated by experiment,which successfully overcomes the classical stability-activity-selectivitytrade-off in unspecific peroxygenase (UPO) from <em>Collariella virescens</em> (<em>Cvi</em>UPO). The mutation sites withhigh predicted stabilization probability and ultralow computed ΔΔ<em>G</em> values were selected. Among the eight candidate sitestested, the combinatorial variant M1_T9P_G161L showed a 36-fold extendedhalf-life and a 6-fold higher activity compared to M1, both maintainingexceptional enantioselectivity (&gt;99% <em>ee</em>). Thiscorrespondsto a total 75-fold activity improvement relative to the wild type.Semipreparative-scale synthesis of (<em>R</em>)-6-bromochroman-4-olwith 12 mM substrate is achieved with a conversion of 83% in 8 h.Furthermore, molecular dynamics (MD) simulations revealed how thecombinatorial mutations stabilized the global folding of the proteinmolecule and strategically reshaped the catalytic pocket microenvironment,which accounted for the enhanced catalytic performance. This cross-regionalcombinatorial mutagenesis computational strategy provides a new paradigmfor engineering metalloenzymes.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"15 8","pages":"3336–3346"},"PeriodicalIF":4.5,"publicationDate":"2026-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148366191","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineering a Cytochrome P450 O-Demethylase for the Bioconversion of Hardwood Lignin","authors":"Megan E. Wolf,&nbsp;Daniel J. Hinchen,&nbsp;Michael Zahn,&nbsp;John E. McGeehan,&nbsp;Lindsay D. Eltis","doi":"10.1021/acssynbio.6c00337","DOIUrl":"10.1021/acssynbio.6c00337","url":null,"abstract":"<p>Lignin is a sustainable alternativeto petroleum as a feedstockfor the chemical industry. Emergent strategies for lignin valorizationinvolve tandem processes in which biomass is chemo-catalytically fractionated,followed by bioconversion of the depolymerized lignin by microbialcell factories. A rate-limiting step in this bioconversion is <em>O</em>-demethylation of the lignin-derived monomers. The reductivecatalytic fractionation of hardwood biomass generates high yieldsof two classes of monomers: 4-alkylguaiacols and 4-alkylsyringols.The former are <em>O-</em>demethylated by AgcA, a cytochromeP450, and AgcB, the cognate reductase, but there are no known enzymesthat convert the latter. To develop a biocatalyst that can efficientlytransform these monomers, we studied and rationally engineered AgcAB.A 1.82 Å resolution crystal structure of AgcA_EP4 from <em>Rhodococcus rhodochrous</em> EP4 in complex with 4-ethylguaiacolidentified residues Leu78, Ala293, and Phe166 as potential specificitydeterminants. Substitution of Ala293 and Leu78 decreased the specificityof AgcA_EP4 for alkylguaiacols. Substitution of Phe166 yieldeda variant that bound 4-propylsyringol but did not transform it. Incontrast, the corresponding variant in the <em>Rhodococcus aromaticivorans</em> RHA1 homologue, AgcA_RHA1 Y166A, catalyzed the <em>O</em>-demethylation of both methoxygroups of 4-propylsyringol with a <em>k</em>_cat<em>/K</em>_m of 8500 M^–1 s^–1 for the first <em>O</em>-demethylation, nearly7-fold higher than WT AgcA_RHA1. Engineering RHA1 to expressthe variant yielded a strain that transformed 4-propylsyringol and4-propylguaiacol simultaneously. Moreover, the engineered strain convertedsome of the 4-propylsyringol to pentanoyl-CoA, consistent with catabolismvia the <em>meta</em>-cleavage pathway that catabolizes 4-alkylguaiacols.Exometabolomics validated the conversion of 4-propylsyringol via thispathway and identified <em>O</em>-demethylation and extradiolring cleavage as bottlenecks for its transformation. These studiesimprove our understanding of a critical lignin-degrading enzyme systemand significantly advance the development of a biocatalyst to convertthese monomers.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"15 8","pages":"3449–3461"},"PeriodicalIF":4.5,"publicationDate":"2026-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148395511","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Vision and Development of a Design, Implementation, and Verification Automation (DIVA) Software Platform for DNA Construction","authors":"Hector Plahar,&nbsp;Stephen D. Lane,&nbsp;Nina Stawski,&nbsp;Lisa Simirenko,&nbsp;Manjiri Tapaswi,&nbsp;Rita Kuo,&nbsp;Garima Goyal,&nbsp;Nathan J. Hillson,&nbsp;Christopher J. Petzold","doi":"10.1021/acssynbio.6c00197","DOIUrl":"10.1021/acssynbio.6c00197","url":null,"abstract":"<p>DNA construction, while a prerequisite to many biologicalendeavors,is often a time-consuming distraction from an individual’sprimary research objectives. We envisioned that with the right softwareinfrastructure and cultural mindset, a single person could executein parallel the batched DNA construction tasks of an entire researchinstitute, at scales realizing efficiency gains through process andlaboratory automation. In pursuit of this vision, we developed theDesign, Implementation, and Verification Automation (DIVA) softwareplatform. DIVA’s web interface enables researchers to designDNA constructs (using visual biological computer-aided design toolsand biological parts repositories), submit designs for constructionto dedicated staff, and track DNA construction as it progresses. DIVAsupports the dedicated staff through the DNA construction processand records both successful and unsuccessful attempts toward improvingthe overall process. The platform is publicly available at public-diva.jbei.org andits open-source code through github.com/JBEI/DIVA.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"15 8","pages":"3498–3503"},"PeriodicalIF":4.5,"publicationDate":"2026-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148402969","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An Explainable Deep Learning Framework Integrating DNA Sequence and Transcription Initiation Signals for Gene Expression Prediction","authors":"Jianbo Qiao,&nbsp;Wenjia Gao,&nbsp;Ding Wang,&nbsp;Junru Jin,&nbsp;Siqi Chen,&nbsp;Zhongmin Yan,&nbsp;Leyi Wei","doi":"10.1021/acssynbio.6c00275","DOIUrl":"10.1021/acssynbio.6c00275","url":null,"abstract":"<p>Gene expression is a fundamental process in cellularfunction andorganismal phenotype formation, governed by intricate and finely tunedregulatory mechanisms. Deciphering the regulatory code of gene expressionand elucidating the transcriptional effects of the genome remain criticalchallenges in human genetics. While numerous deep learning approacheshave been developed to predict gene expression levels, most prioritizeDNA sequence information, often overlooking the impact of transcriptioninitiation signals and lacking interpretability regarding both modelbehavior and feature contributions. In this study, we present an interpretabledeep learning framework based on convolutional neural networks witha Gated Recurrent Unit. This method efficiently predicts gene expressionlevels by integrating DNA sequence data, mRNA half-life features,and transcription initiation signals. We further validate the modelon GM12878, HepG2, and K562 data sets, demonstrating its robustnessacross different human cell lines. Moreover, interpretability analysisdemonstrates that the model provides valuable insights into gene expressionmechanisms, underscoring its practical utility as a robust tool forunderstanding and predicting gene expression levels.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"15 8","pages":"3403–3412"},"PeriodicalIF":4.5,"publicationDate":"2026-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148366188","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Heterologous Expression of an Abandoned Termite Mound Fungus Gene Cluster Reveals a Protective Aldehyde–Alcohol Cycle and a Candidate Termiticidal Metabolite","authors":"Yize Cai,&nbsp;Hongling Yan,&nbsp;Jiaxu Qin,&nbsp;Yaxiong Qiang,&nbsp;Guo-Qiang Lin,&nbsp;Haiying Wang,&nbsp;Qing-Li He,&nbsp;Qunfei Zhao","doi":"10.1021/acssynbio.6c00101","DOIUrl":"10.1021/acssynbio.6c00101","url":null,"abstract":"<p>The medicinal fungus Wulingshen, comprising multiple <em>Xylaria</em> species, inhabits deserted termite mounds as sclerotia.To explorethe molecular basis of its niche adaptation, we employed a syntheticbiology-driven approach. Metagenomic and transcriptomic mining ofwild specimens identified a conserved biosynthetic gene cluster. Itsheterologous reconstruction in the fungal host <em>Aspergillus oryzae</em> enabled the characterization of a family ofα-pyrone metabolites and, crucially, the elucidation of a spatiallyseparated aldehyde–alcohol cycle. In this self-protection system,an extracellular oxidase (WlsA) converts an alcohol precursor to areactive aldehyde, while an intracellular reductase (WlsE) catalyzesthe reverse reaction. The aldehyde product exhibits potent toxicityagainst termites <em>in vitro</em>, suggesting a potentialrole in ecological interactions. This work establishes a functionalgenomics platform that decodes cryptic ecological metabolism by integratingmultiomics with heterologous pathway expression, providing a generalizablestrategy for discovering and mechanistically understanding niche-specificnatural products.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"15 8","pages":"3206–3214"},"PeriodicalIF":4.5,"publicationDate":"2026-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148389389","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biosensor-Based Detection and Quantification of Arsenic in Drinking Water","authors":"Oskar S. Zeballos Huarachi,&nbsp;Andrea A. Tambo Santos,&nbsp;Jonatan J. Aliaga Rodriguez,&nbsp;Andy K. Porcel Velasquez,&nbsp;Karen Cabero,&nbsp;Cesar Perez-Fernandez,&nbsp;Yadira Boada,&nbsp;Noemi Tirado,&nbsp;Mauricio Ormachea,&nbsp;Maria Eugenia García,&nbsp;Cecilia Gonzalez,&nbsp;Maria Teresa Alvarez-Aliaga,&nbsp;Tania Pozzo","doi":"10.1021/acssynbio.6c00308","DOIUrl":"10.1021/acssynbio.6c00308","url":null,"abstract":"<p>Arsenic contamination of drinking water remains a persistentglobalhealth burden and an environmental justice challenge, particularlyfor low-resource communities that lack access to reliable monitoringtools. Synthetic-biology-driven biosensors offer a promising complementto conventional analytical methods by coupling arsenic-responsivegenetic circuits with portable, low-cost readouts suitable for fielddeployment. This review traces the evolution from the early ArsR-based <em>Escherichia coli</em> biosensor to modern whole-cell andcell-free platforms that approach World Health Organization–relevantdetection limits for arsenic in water under controlled conditions,emphasizing how signal amplification strategies intersect with shelflife, biosafety, and regulatory simplicity. The operational principlesof <em>ars</em> operon–derived modules are examinedacross detection, processing, and host-engineering layers that collectivelytune sensitivity, dynamic range, and robustness. Immobilization formats,microfluidic architectures, and transduction mechanisms─includingcolorimetric, fluorescent, bioluminescent, and electrochemical outputs─areanalyzed for their ability to integrate biological sensing with commodityoptics and electronics in portable devices. Building on this engineeringlandscape, the review highlights how biodesign automation, high-throughputDesign–Build–Test–Learn workflows, and emergingAI tools such as supervised learning and Bayesian optimization areaccelerating the construction and optimization of arsenic-responsivegenetic circuits. Biosafety and regulatory considerations, includingbiocontainment, standardized stress-testing, and community codesign,are discussed to position arsenic biosensors as candidates for integrationinto distributed water-quality monitoring networks that combine syntheticbiology, low-cost hardware, automation, and AI under robust governanceregimes.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"15 8","pages":"3123–3138"},"PeriodicalIF":4.5,"publicationDate":"2026-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148467798","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Flavin-Decorated Proteins and Bacteria Enable Photoenzymatic Amine Valorisation","authors":"Vasco F. Batista,&nbsp;Christian Å. Ringsmose,&nbsp;Erik D. Hedegård,&nbsp;Henrik Karring,&nbsp;Changzhu Wu","doi":"10.1021/acssynbio.6c00218","DOIUrl":"10.1021/acssynbio.6c00218","url":null,"abstract":"<p>The search for more sustainable catalytic methods, suchas biocatalysisand photocatalysis, has greatly contributed to a sustainable futureof synthetic chemistry. Within photocatalysis, flavin-type heterocyclesare powerful tools for oxidative transformations but often sufferfrom poor water solubility, photostability, and limited reusability.Here, we provide a proof-of-concept strategy to bridge the gap betweenphotocatalysis and biocatalysis through the development of artificialflavin biomolecules. A 7-/8-carboxyflavin derivative was synthesizedand covalently attached to protein surfaces via a biocompatible amidecoupling reaction, creating an artificial flavin-decorated proteinwith oxidative properties. This biophotocatalyst operates efficientlyin water under mild conditions, enabling the photooxidation of primaryamines without external oxidants. It was also integrated into one-pot,sequential cascade reactions with whole-cell biocatalysts to achievethe enantioselective synthesis of (<em>R</em>)-benzoin andchiral secondary alcohols. Furthermore, the flavin photocatalyst wasimmobilized on the surface of <em>Escherichia coli</em> cells through a similar process, creating a recyclable, heterogeneouscellular photocatalyst. These modified bacteria exhibited high activityin amine oxidation, could be easily recycled, and maintained performanceover multiple reaction cycles without evidence of catalyst leaching.Overall, this work establishes a versatile framework for constructinghybrid biophotocatalytic systems and showcases how they can contributeto environmentally sustainable synthesis through reactions in waterand catalyst recycling.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"15 8","pages":"3347–3356"},"PeriodicalIF":4.5,"publicationDate":"2026-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148434259","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Combinatorial Optimization of Protein Systems in Synthetic Cells","authors":"Marijn van den Brink,&nbsp;Nico J. Claassens,&nbsp;Christophe Danelon","doi":"10.1021/acssynbio.6c00166","DOIUrl":"10.1021/acssynbio.6c00166","url":null,"abstract":"<p><em>In vitro</em> reconstitution of protein systems─e.g.,metabolic pathways, genetic circuits, or biosensors─often requiresoptimization to enhance their activity. Combinatorial DNA librariesthat simultaneously target multiple genes allow for a holistic optimizationstrategy by studying the interplay between the systems’ components,which may reveal DNA variants that would be hidden when testing eachelement in isolation. Here, we screen large populations of syntheticvesicles that express combinatorial DNA variants of a DNA self-replicatoror a phospholipid synthesis pathway. We simultaneously vary the strengthsof multiple RBSs or synonymously mutate the first codons of multiplegenes to explore the effects of the protein translation rates directlyon the functionality of the two core synthetic cell modules. We isolatedhigh performers through DNA self-selection or functional screeningby fluorescence-activated cell sorting. Long-read sequencing of thefittest variants revealed the optimal RBS strengths and base substitutionsin the first codons and indicated which genes were most impactfulin regulating the functionality of the protein systems. Single-mutationdata were used to predict the fitness of combinatorial variants, whichwas compared with the experimental fitness observed. The theoreticalfitness of combinatorial variants was extremely predictive for thetwo-gene library of the DNA replicator but less for the larger pathwaylibrary. Altogether, our approach exemplifies how combinatorial testingcan be expanded from single proteins to multiprotein systems, whichcan in the future be extended to the evolutionary engineering of evenlarger genetic and metabolic networks, and eventually an entire artificialcell.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"15 8","pages":"3285–3299"},"PeriodicalIF":4.5,"publicationDate":"2026-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148785399","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}