Pub Date : 2024-09-13DOI: 10.1101/2024.09.13.612811
Monami Kihara, Rio Okuda, Anri Okada, Teruyo Ojima-Kato, Hideo Nakano
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.
本研究以NZ-1抗体（靶向PA标记肽（GVAMPGAEDDVV））为模型，将PURE核糖体展示（利用纯化的无细胞蛋白质合成元件进行核糖体展示）与下一代测序（NGS）和Brevibacillus choshinensis分泌系统相结合，开发了一种高效富集和分析高亲和力抗体变体的方法。合成了编码 NZ-1 抗体单链结合片段（scFab）的人工 DNA，并通过无细胞蛋白合成系统（CFPS）进行了活性表达。根据 scFab 基因的结构信息，将特异性区域饱和突变引入 scFab 基因。通过两轮 PURE 核糖体展示筛选出与 PA 标记相对应的 scFab 文库，然后进行 Illumina 测序，以确定具有更高亲和力的潜在 scFab 变体。候选者利用 B. choshinensis 分泌系统表达为 Fab 片段。然后使用两步柱色谱法从培养上清液中纯化这些 Fab 片段。使用生物层干涉测定法评估了纯化 Fab 的结合亲和力，结果显示变体 Fab 的亲和力高于野生型 Fab。这些结果表明，将 PURE 核糖体展示与 NGS 分析和 B. choshinensis 分泌表达系统相结合，可以快速鉴定和分析高亲和力抗体变体。
{"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":"61 1","pages":""},"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}
Pub Date : 2024-09-13DOI: 10.1101/2024.09.13.612903
Adibvafa Fallahpour, Vincent Gureghian, Guillaume J. Filion, Ariel B. Lindner, Amir Pandi
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.
遗传密码是退化的，允许多种可能的 DNA 序列编码相同的蛋白质。由于每种生物的密码子使用偏好不同，这种退化性影响了异源蛋白质的生产效率。定制生物特异性同义密码子的过程被称为密码子优化，必须尊重超出全局密码子偏好的局部序列模式。因此，搜索空间面临着组合爆炸，不可能进行详尽的探索。然而，在地球上多种多样的生命中，自然选择已经对序列进行了优化，从而提供了丰富的数据源，使机器学习算法能够探索潜在的规则。在这里，我们介绍一个多物种深度学习模型--CodonTransformer，它是在来自164个生物体的100多万个DNA-蛋白质对上训练出来的，这些生物体横跨生命的所有领域。得益于我们使用的注意力机制和 Transformers 的双向性，以及结合了生物体、氨基酸和密码子编码的新型序列表示法，该模型展示了上下文感知能力。CodonTransformer 生成的宿主特异性 DNA 序列具有类似自然的密码子分布图和负顺式调控元素。这项工作引入了一种新颖的共享标记表示和编码与对齐多掩码（STREAM）策略，并提供了一个最先进的密码子优化框架，该框架具有可定制的开放存取模型和用户友好界面。
{"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":"52 1","pages":""},"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}
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.
真菌通常被用作同源和异源生产酶和代谢物的细胞工厂。获得高产菌株的一种策略是通过插入多份基因表达盒来提高负责生产产品的基因的表达水平。通常，这是通过用大量的 DNA 载体转化非同源末端连接熟练菌株来实现的，这种 DNA 载体在不同的基因座上随机整合多个拷贝，或者更常见的是整合到一个基因座上，拷贝排列成混合方向的重复序列。以这种方式生产的菌株大多不稳定，因此需要进行大量筛选，以确定产量高且稳定的菌株。此外，由于插入过程的随机性，很难确定拷贝在基因组中的位置和方式。为此，我们设想，用经典方法制造的基因簇的不稳定性主要是由于存在直接重复和倒置重复的混合物。在这种基因簇中，倒位重复序列形成的发夹可能会在复制过程中频繁发生重组病变，从而通过直接重复重组诱导基因表达盒拷贝丢失。因此，仅由直接重复序列构成基因表达盒簇的菌株可能更加稳定。我们以黑曲霉（Aspergillus nidulans）为模型，对这一想法进行了验证，并开发了一种简单高效的方法--RoCi，通过滚圆复制将多个直接重复的基因表达盒整合到一个确定的基因组位点，而无需预先设计菌株制备。此外，我们还证明了 RoCi 不需要基于大肠杆菌的克隆就能进行，从而使其与中高通量实验兼容。通过分析 RoCi 产生的菌株，我们构建了带有多达 68 个 mRFP GECs 的菌株，并证明 mRFP 多拷贝基因阵列可在固体培养基上支持至少约 150 代的高稳定 mRFP 生产。在液体培养中，我们观察到在 1 升的规模上有轻微的平均拷贝损失。通过使用残缺选择标记扩展基因表达盒，可以消除这种损失。为了证明这种方法的优势，我们用它来生产稳定、高产的细胞工厂，在固体培养基上生产特殊代谢物虫草素，在浸没培养中生产β-葡萄糖醛酸酶。最后，我们证明 RoCi 也可用于工业重型菌 A. niger 和 A. oryzae，这表明 RoCi 普遍适用于真菌。
{"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":"45 1","pages":""},"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}
Pub Date : 2024-09-12DOI: 10.1101/2024.09.12.612674
Jean-Loup Faulon, Paul Ahavi, An Hoang
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.
{"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":"18 1","pages":""},"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}
Pub Date : 2024-09-11DOI: 10.1101/2024.09.11.612432
Xiaozhi Fu, Oriol Gracia i Carmona, Gyorgy Abrusan, Xiang Jiao, Alexander Diaciuc, Mathias Gautel, Franca Fraternali, Aleksej Zelezniak
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.
作为塑料污染的一个主要来源，PET 因其在循环经济中的潜力而引起了生物降解的极大兴趣。寻找有效的酶仍然是一项挑战，因为筛选方法受限于低通量或因 PET 的不溶性而依赖于替代性非 PET 底物。在此，我们报告了一种高活性、稳定和稳健的酶 Fast_2.9，该酶是在中温条件下利用液滴封装 PET 纳米颗粒直接筛选 PET 降解活性时发现的，通量超过 1 kHz。在对未经处理的消费后塑料进行测试时，我们发现了一个远端 S269T 突变，该突变提高了大多数已知 PET 酶的活性，是野生型的 400 倍，是已知工程 PET 酶的两倍多。微秒时间尺度的分子动力学分析表明，这种远距离突变可能通过一种跨 PET 酶的共同机制影响底物结合裂隙附近的残基。与最先进的 FastPETase 和 LCC_ICCG 酶相比，工程化的 Fast_2.9 酶达到相同酶活性所需的酶浓度分别低 8 倍和 42 倍，最终所需酶量大大减少。因此，我们的工程酶在 50 摄氏度的条件下，只需 0.72 mgenzyme/gPET 就能在短短两天内降解包括聚酯纺织品在内的多种消费后 PET 底物，对苯二甲酸转化率高达近 100%。
{"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":"71 1 1","pages":""},"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}
Pub Date : 2024-09-10DOI: 10.1101/2024.09.10.611283
Cody Kamoku, David R Nielsen
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.
{"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":"24 1","pages":""},"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}
Pub Date : 2024-09-09DOI: 10.1101/2024.09.09.612111
Rithu K Pattali, Izaiah J Ornelas, Carolyn D Nguyen, Da Xu, Nikita S Divekar, James K Nunez
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.
基于 CRISPR 的技术的出现，使细胞、组织和整个生物体内的可编程基因操作得以快速发展。表观遗传编辑是一种新兴的靶向基因扰动平台，它直接编辑 DNA 和组蛋白上的化学修饰，最终导致靶向基因的抑制或激活。与 CRISPR 核酸酶不同的是，表观遗传编辑调节基因表达，而不会诱导 DNA 断裂或改变宿主细胞的基因组序列。最近，我们开发出了 CRISPRoff 表观遗传编辑技术，它能同时在目标基因启动子上建立 DNA 甲基化和抑制性组蛋白修饰。在哺乳动物细胞中瞬时表达 CRISPRoff 和相应的引导 RNA 会导致目标基因的转录抑制，这种抑制会通过细胞分裂和分化被细胞遗传记忆。在这里，我们介绍了通过质粒 DNA 转染将 CRISPRoff 以及 CRISPRoff mRNA 导入转化人细胞系和原代免疫细胞的方案。我们还提供了评估目标基因沉默的指导，并强调了利用 CRISPRoff 进行基因扰乱时的主要注意事项。随着可编程基因组操作工具的快速发展，我们的方案也广泛适用于其他基于 CRISPR 的表观遗传编辑技术。
{"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":"63 1","pages":""},"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}
Pub Date : 2024-09-09DOI: 10.1101/2024.09.09.611992
Laura Roset Julia, Laura Grasemann, Francesco Stellacci, Sebastian Josef Maerkl
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.
无细胞系统是合成生物学的强大工具，具有多用途和广泛的应用。然而，这些系统（尤其是 PURE 无细胞系统）的一个重要瓶颈是反应寿命和产量有限。透析是延长反应寿命和提高产量的有效方法，但大多数定制透析系统需要使用 3D 打印机或微细加工工具等精密设备。在本研究中，我们利用市售元件，为无细胞反应设计了一种易于组装的中型透析系统。通过透析和定期更换进料溶液，我们在 PURE 系统中获得了 1.16 mg/mL GFP 的蛋白质产量，并将蛋白质合成时间延长了至少连续 12.5 天，证明了该系统出色的稳定性。
{"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":"9 1","pages":""},"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}
Pub Date : 2024-09-09DOI: 10.1101/2024.09.07.611833
Carly Retief, Sheemal Kumar, Kate Tepper, Maciej Maselko
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.
{"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":"255 1","pages":""},"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}
Pub Date : 2024-09-08DOI: 10.1101/2024.09.06.611674
Surendra Yadav, Alexander J. P. Perkins, Sahan B. W. Liyanagedera, Anthony Bougas, Nadanai Laohakunakorn
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.
利用重组元件合成蛋白质"（"PURE"）系统是一个最小的生化系统，能够利用确定的酶元件进行无细胞蛋白质合成。本研究通过整合基于丙酮酸氧化酶、乙酸激酶和过氧化氢酶的 ATP 再生系统对 PURE 进行了扩展。新途径从丙酮酸、磷酸盐和氧气中生成磷酸乙酰，用于原位再磷酸化 ATP。ATP 的成功再生需要约 10 mM 磷酸盐缓冲液的高初始浓度，但令人惊讶的是，这并不影响 PURE 的蛋白质合成活性。该途径可独立运行，也可与 PURE 中现有的肌酸系统结合使用；结合使用的系统可产生高达 233 微克/毫升的 mCherry，与单独使用肌酸系统相比提高了 78%。这些结果在多个批次的自制 PURE 中都具有可重复性，更重要的是，它们也适用于新英格兰生物实验室（New England Biolabs）的 PURExpress 等商业系统。这些结果展示了一种合理的自下而上的 PURE 工程方法，为无细胞合成生物学和合成细胞构建的应用铺平了道路。
{"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":"74 1","pages":""},"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}
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