bioRxiv - Synthetic Biology最新文献

{"title":"Improvement of Antibody Affinity and Using PURE Ribosome Display and Microbial Secretion System","authors":"Monami Kihara, Rio Okuda, Anri Okada, Teruyo Ojima-Kato, Hideo Nakano","doi":"10.1101/2024.09.13.612811","DOIUrl":"https://doi.org/10.1101/2024.09.13.612811","url":null,"abstract":"A method has been developed for efficiently enriching and analyzing high-affinity antibody variants by combining the PURE ribosome display (ribosome display using purified cell-free protein synthesis components) with next-generation sequencing (NGS) and the Brevibacillus choshinensis secretion system, using the NZ-1 antibody, which targets the PA tag peptide (GVAMPGAEDDVV), as a model antibody. An artificial DNA encoding the single-chain fragment of binding (scFab) of the NZ-1 antibody was synthesized and actively expressed by cell-free protein synthesis system (CFPS). Region-specific saturation mutations were introduced into the scFab gene based on its structural information. The resulting scFab library was selected against the PA tag through two rounds of PURE ribosome display, followed by Illumina sequencing to identify potential scFab variants with higher affinity. The candidates were expressed as Fab fragments using the B. choshinensis secretion system. These Fab fragments were then purified from the culture supernatant using two-step column chromatography. The binding affinity of the purified Fab was evaluated using a biolayer interferometry assay, revealing a variant Fab with higher affinity than the wild-type Fab. These results demonstrate that integrating PURE ribosome display with NGS analysis and the B. choshinensis secretion expression system enables the rapid identification and analysis of high-affinity antibody variants.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142226311","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CodonTransformer: a multispecies codon optimizer using context-aware neural networks","authors":"Adibvafa Fallahpour, Vincent Gureghian, Guillaume J. Filion, Ariel B. Lindner, Amir Pandi","doi":"10.1101/2024.09.13.612903","DOIUrl":"https://doi.org/10.1101/2024.09.13.612903","url":null,"abstract":"The genetic code is degenerate allowing a multitude of possible DNA sequences to encode the same protein. This degeneracy impacts the efficiency of heterologous protein production due to the codon usage preferences of each organism. The process of tailoring organism-specific synonymous codons, known as codon optimization, must respect local sequence patterns that go beyond global codon preferences. As a result, the search space faces a combinatorial explosion that makes exhaustive exploration impossible. Nevertheless, throughout the diverse life on Earth, natural selection has already optimized the sequences, thereby providing a rich source of data allowing machine learning algorithms to explore the underlying rules. Here, we introduce CodonTransformer, a multispecies deep learning model trained on over 1 million DNA-protein pairs from 164 organisms spanning all kingdoms of life. The model demonstrates context-awareness thanks to the attention mechanism and bidirectionality of the Transformers we used, and to a novel sequence representation that combines organism, amino acid, and codon encodings. CodonTransformer generates host-specific DNA sequences with natural-like codon distribution profiles and with negative cis-regulatory elements. This work introduces a novel strategy of Shared Token Representation and Encoding with Aligned Multi-masking (STREAM) and provides a state-of-the-art codon optimization framework with a customizable open-access model and a user-friendly interface.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142250836","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"RoCi - A Single Step Multi-Copy Integration System Based on Rolling-Circle Replication","authors":"Martzel Antsotegi-Uskola, Vasil D'Ambrosio, Zofia Dorota Jarczynska, Katherina Garcia Vanegas, Marti Morera-Gomez, Xinhui Wang, Thomas Ostenfeld Larsen, Jean-Marie Mouillon, Uffe Hasbro Mortensen","doi":"10.1101/2024.09.13.612835","DOIUrl":"https://doi.org/10.1101/2024.09.13.612835","url":null,"abstract":"Fungi are often used as cell factories for homologous and heterologous production of enzymes and metabolites. One strategy to obtain high yielding strains is to enhance the expression level of the gene(s) responsible for production of the product by inserting multiple copies of the gene-expression cassette. Typically, this is achieved by transforming non-homologous end-joining proficient strains with large amounts of a DNA vector, which randomly integrates in multiple copies at different loci, or more often, into a single locus with copies arranged as mixed orientation repeats. The majority of strains produced in this manner are unstable and substantial screening is necessary to identify strains with high and stable production. Moreover, the randomness of the insertion processes makes it difficult to determine how and where the copies are positioned in the genome. To this end, we envisioned that the instability of gene clusters made by the classical method is mostly due to the presence of a mixture of directly and inverted repeats. In such clusters, hairpins formed by inverted repeats may cause frequent recombinogenic lesions during replication to induce gene-expression cassette copy-loss by direct-repeat recombination. It is therefore possible that strains with gene-expression cassette clusters made solely by direct repeats would be more stable. Using Aspergillus nidulans as a model, we tested this idea and developed RoCi, a simple and efficient method to facilitate integration of multiple directly repeated gene-expression cassettes into a defined genomic locus through rolling-circle replication without pre-engineering requirements for strain preparation. In addition, we demonstrate that RoCi can be performed without E. coli based cloning, making it compatible with medium-high throughput experiments. Analyzing strains produced by RoCi, we have constructed strains bearing up to 68 mRFP GECs and we show that an mRFP multi-copy gene-array supports high and stable mRFP production for at least ~150 generations on solid medium. In liquid culture we observed a minor average copy loss at 1 L scale. This loss could be eliminated by extending the gene-expression cassette with a crippled selection marker. To demonstrate the strength of the method, we used it to produce stable and high yielding cell factories for production of the specialized metabolite cordycepin on solid medium and of the enzyme β-glucuronidase in submerged culture. Finally, we show that RoCi can also be applied in the industrial workhorses A. niger and A. oryzae indicating that RoCi is generally applicable in fungi.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206680","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Reservoir Computing with Bacteria","authors":"Jean-Loup Faulon, Paul Ahavi, An Hoang","doi":"10.1101/2024.09.12.612674","DOIUrl":"https://doi.org/10.1101/2024.09.12.612674","url":null,"abstract":"This study explores the use of bacterial strains in reservoir computing (RC) to solve regression and classification tasks. We employ an Escherichia coli K-12 MG1655 strain as the physical reservoir, training it on M9 minimal media supplemented with 28 metabolites, and measuring growth rates across various media compositions. Our physical RC system, using an Escherichia coli strain, demonstrates superior performance compared to multi-linear regression or support-vector machine and comparable performance to multi-layer perceptron in various regression and classification tasks. Additionally, the performances of RC based on genome-scale metabolic models for several bacterial species correlate with the diversity and complexity of phenotypes they produce. These findings highlight the potential of bacterial RC systems for complex computational tasks typically reserved for digital systems and suggest future research directions, including optimizing feature-to-nutrient mappings and integrating with emerging technologies for enhanced computing capabilities.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206681","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Single Distal Mutation Enhances Activity of known PETases via Stabilisation of PET-binding","authors":"Xiaozhi Fu, Oriol Gracia i Carmona, Gyorgy Abrusan, Xiang Jiao, Alexander Diaciuc, Mathias Gautel, Franca Fraternali, Aleksej Zelezniak","doi":"10.1101/2024.09.11.612432","DOIUrl":"https://doi.org/10.1101/2024.09.11.612432","url":null,"abstract":"As a major source of plastic pollution, PET has attracted significant interest for biodegradation due to its potential in the circular economy. Finding effective enzymes still remains a challenge as screening methods are limited by either the low throughput or dependence on alternative non-PET substrates due to PET's insolubility. Here, we report a highly active, stable and robust enzyme, Fast_2.9, identified while directly screening for PET-degrading activity in mesophilic conditions using droplet-based encapsulation of PET nanoparticles with the throughput above 1 kHz. We identified a distal S269T mutation that improves activity in the majority of all known PETases with up to 400 times over wildtype, and more than twice of known engineered PETases, as tested on untreated post-consumer plastics. Microsecond time scale molecular dynamics analyses indicate that this distant mutation possibly influences residues near the substrate-binding cleft via a common mechanism across PETases. Compared to the state-of-the-art FastPETase and LCC_ICCG enzymes, the engineered Fast_2.9 enzyme requires up to 8 and 42 times lower enzyme concentrations to reach the same enzymatic activity, ultimately requiring significantly less enzyme. As such our engineered enzyme degrades multiple post-consumer PET substrates, including polyester textiles, within as least as just 2 days with up to nearly 100% terephthalic acid conversion using as little as 0.72 mgenzyme/gPET at 50 degrees C. Our study presents a universal methodology for direct screening of insoluble substrates at ultra-high-throughput and highlights the techno-economic potential of Fast_2.9 for PET depolymerisation.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206682","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Highly Iterated Palindrome 1 (HIP1) sequence improves Synechococcus sp. PCC 7002 transformation efficiencies in a homology- and methylation-dependent manner","authors":"Cody Kamoku, David R Nielsen","doi":"10.1101/2024.09.10.611283","DOIUrl":"https://doi.org/10.1101/2024.09.10.611283","url":null,"abstract":"The ability to precisely control cyanobacterial metabolism first requires the ability to efficiently deliver engineered DNA constructs. Here, we investigate how natural transformation efficiencies in Synechococcus sp. PCC 7002 can be greatly improved by leveraging the native and abundant cyanobacterial Highly Iterated Palindrome 1 (HIP1) sequence. While including at least one homologous HIP1 site within the homology arms of an integrating plasmid increased integration efficiency by up to 7-fold, methylation of those sites by HIP1 methyltransferase (encoded by slr0214 from Synechococcus sp. PCC 6803) boosted this to greater than a 100-fold improvement overall. Non-homologous HIP1 sites also improved transformation efficiencies of both integrating and replicating episomal plasmids (by up to 60- and 9-fold, respectively), but only when methylated. The collective data further reveal that HIP1 does not function as part of a native restriction enzyme system in PCC 7002, but rather may improve transformation efficiency via two complementary mechanisms: i) altering DNA binding/uptake/processing prior to homologous recombination, and ii) increasing the efficiency of homologous recombination in a manner reminiscent of a crossover hotspot instigator (Chi) site. Future studies are needed, however, to more clearly elucidate the specific role of HIP1 during natural transformation of cyanobacteria.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206683","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CRISPRoff epigenetic editing for programmable gene silencing in human cells without DNA breaks","authors":"Rithu K Pattali, Izaiah J Ornelas, Carolyn D Nguyen, Da Xu, Nikita S Divekar, James K Nunez","doi":"10.1101/2024.09.09.612111","DOIUrl":"https://doi.org/10.1101/2024.09.09.612111","url":null,"abstract":"The advent of CRISPR-based technologies has enabled the rapid advancement of programmable gene manipulation in cells, tissues, and whole organisms. An emerging platform for targeted gene perturbation is epigenetic editing, the direct editing of chemical modifications on DNA and histones that ultimately results in repression or activation of the targeted gene. In contrast to CRISPR nucleases, epigenetic editors modulate gene expression without inducing DNA breaks or altering the genomic sequence of host cells. Recently, we developed the CRISPRoff epigenetic editing technology that simultaneously establishes DNA methylation and repressive histone modifications at targeted gene promoters. Transient expression of CRISPRoff and the accompanying guide RNAs in mammalian cells results in transcriptional repression of targeted genes that is memorized heritably by cells through cell division and differentiation. Here, we describe our protocol for the delivery of CRISPRoff through plasmid DNA transfection, as well as the delivery of CRISPRoff mRNA, into transformed human cell lines and primary immune cells. We also provide guidance on evaluating target gene silencing and highlight key considerations when utilizing CRISPRoff for gene perturbations. Our protocols are broadly applicable to other CRISPR-based epigenetic editing technologies, as programmable genome manipulation tools continue to evolve rapidly.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142226310","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Long-term protein synthesis with PURE in a mesoscale dialysis system","authors":"Laura Roset Julia, Laura Grasemann, Francesco Stellacci, Sebastian Josef Maerkl","doi":"10.1101/2024.09.09.611992","DOIUrl":"https://doi.org/10.1101/2024.09.09.611992","url":null,"abstract":"Cell-free systems are powerful tools in synthetic biology with versatile and wide-ranging applications. However, a significant bottleneck for these systems, particularly the PURE cell-free system, is their limited reaction lifespan and yield. Dialysis offers a promising approach to prolong reaction lifetimes and increase yields, yet most custom dialysis systems require access to sophisticated equipment like 3D printers or microfabrication tools. In this study, we utilized an easy-to-assemble, medium-scale dialysis system for cell-free reactions using commercially available components. By employing dialysis with periodic exchange of the feeding solution, we achieved a protein yield of 1.16 mg/mL GFP in the PURE system and extended protein synthesis for at least 12.5 consecutive days, demonstrating the system's excellent stability.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206685","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biomanufacturing of a functional microbial phytase in an insect host.","authors":"Carly Retief, Sheemal Kumar, Kate Tepper, Maciej Maselko","doi":"10.1101/2024.09.07.611833","DOIUrl":"https://doi.org/10.1101/2024.09.07.611833","url":null,"abstract":"Background: Insects, such as Black Soldier Flies (Hermetia illucens), are increasingly used as sustainable animal feed ingredients that can be reared on plentiful organic substrates such as agricultural residues and pre-consumer food waste. Genetically engineering insects to heterologously express feed additive enzymes has the potential to generate more value from organic waste, while improving livestock health and productivity. Phytases are widely used feed additive enzymes that hydrolyse the phosphate groups from the myo-inositol backbone of phytic acid, a phosphate rich antinutrient compound that monogastric animals cannot efficiently digest. Dietary phytase supplementation improves absorption of phosphorous, proteins, and cationic nutrients, while mitigating the negative environmental effects of phytic acid rich excreta. Results: We evaluated the potential of using insects to biomanufacture microbial feed additive enzymes by engineering the model insect, Drosophila melanogaster, to express phytases. One histidine acid phytase, three beta propellor phytases, three purple acid phosphatases, and one PTP-like phytase were selected for screening in D. melanogaster. Transgenic flies expressing the AppA histidine acid phytase from E. coli had 27.82 FTU/g of phytase activity, which exceeds the 0.5-1.0 FTU/g required in animal feed. Maximum activity from AppA phytase expressed by D. melanogaster was observed at pH 5 and 55 oC, however, more than 50% of phytase activity was present at 25 oC and pH 2. Here we demonstrate that insects may be suitable hosts for the heterologous expression of a microbial phytase enzyme with applications for improving animal feed nutrition and organic waste valorisation.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206684","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"ATP Regeneration from Pyruvate in the PURE System","authors":"Surendra Yadav, Alexander J. P. Perkins, Sahan B. W. Liyanagedera, Anthony Bougas, Nadanai Laohakunakorn","doi":"10.1101/2024.09.06.611674","DOIUrl":"https://doi.org/10.1101/2024.09.06.611674","url":null,"abstract":"The 'Protein synthesis Using Recombinant Elements' ('PURE') system is a minimal biochemical system capable of carrying out cell-free protein synthesis using defined enzymatic components. This study extends PURE by integrating an ATP regeneration system based on pyruvate oxidase, acetate kinase, and catalase. The new pathway generates acetyl phosphate from pyruvate, phosphate, and oxygen, which is used to rephosphorylate ATP in situ. Successful ATP regeneration requires a high initial concentration of ~10 mM phosphate buffer, which surprisingly does not affect the protein synthesis activity of PURE. The pathway can function independently or in combination with the existing creatine-based system in PURE; the combined system produces up to 233 ug/ml of mCherry, an enhancement of 78% compared to using the creatine system alone. The results are reproducible across multiple batches of homemade PURE, and importantly also generalise to commercial systems such as PURExpress from New England Biolabs. These results demonstrate a rational bottom-up approach to engineering PURE, paving the way for applications in cell-free synthetic biology and synthetic cell construction.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142226312","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}