Enzymatic gene synthesis

下一代测序平台带来的革命导致了过去十年中可获得的DNA序列信息的大量增加。现在有一个前所未有的机会来设计代谢途径和生物体，改进工业过程，创造新的过程，设计具有新的和改进的特征的基因组，并使用DNA作为数字数据存储的媒介。所有上述情况都将对科学和工业产生根本性影响，并可能对数百万人的生活质量产生影响。这一迫在眉睫的研究和商业努力领域被称为“合成生物学”。然而，目前无法以低成本生产大量准确的长DNA分子，这限制了合成生物学产业的发展。目前的DNA合成方法包括生成寡核苷酸，主要使用亚磷酰胺化学，然后在池中退火和组装，以生成双链DNA。这在DNA长度在3千碱基或更少的情况下最为成功，其中累积的误差不需要耗时且昂贵的序列校正，而在更长的长度上，这变得至关重要。此外，目前基于喷墨和其他物理分离方式的平台在确保规模化DNA制造方面的能力极其有限。已经合成了超过10亿个碱基的双链DNA，根据合成后校正的要求，价格变化很大。此外，这类合成的交付时间已经大大延长，因此许多项目仍未完成。总部位于剑桥的初创公司Evonetix的目标是彻底改变新基因的合成。我们正在开发一种高度破坏性的DNA/基因合成平台，以满足日益增长的对低成本准确合成DNA的需求。我们相信，使用酶促寡核苷酸合成，与亚磷酰胺化学相比，在较温和的水溶液条件下操作，将为我们的平台的从头合成DNA/基因提供显著的商业优势；它将通过降低成本、更环保和进一步简化高保真DNA合成的工业化来实现这一点。众所周知，具有末端脱氧核苷酸转移酶/合成酶活性的酶无需模板链即可延伸单链DNA。这种产生DNA的独特能力使这些酶成为生产合成DNA的非常有价值的生物工具。我们项目的目的是开发新型的、修饰的核苷酸和具有末端脱氧核苷酸转移酶/合成酶活性的基因工程酶，作为成熟的亚磷酰胺化学的替代品，用我们的创新技术合成高质量的DNA/基因。

"The revolution generated by next-generation sequencing platforms led to a massive increase in DNA sequence information becoming available over the past ten years. There now exists an unprecedented opportunity to engineer metabolic pathways and organisms, improve industrial processes, create new processes, engineer genomes with new and improved traits and use DNA as a medium for digital data storage. All the foregoing will have a fundamental impact on science and industry, and potentially on the quality of life for millions of individuals. This impending area of research and commercial endeavour has been termed ""synthetic biology"".However, the current inability to produce vast amounts of accurate long, DNA molecules at low cost is limiting the growth of the synthetic biology industry. Current approaches to DNA synthesis involve the generation of oligonucleotides, mostly using phosphoramidite chemistry, followed by annealing and assembly in pools, to generate double-stranded DNA. This is most successful at DNA lengths up to 3 kilobases and less, where accumulated errors do not require the time-consuming and expensive correction of sequences that becomes essential at greater lengths. In addition, current platforms, based on ink-jet and other physical separation modalities, are extremely limited in their ability to secure scaled DNA manufacture. More than 1 billion bases of double-stranded DNA have been synthesised at hugely variable pricing depending on the requirement for post-synthesis correction. Moreover, delivery times for such syntheses have been highly extended and thus many projects remain unfinished.Evonetix, a Cambridge-based start-up, aims to revolutionise _de novo_ gene synthesis. We are developing a highly disruptive DNA/gene synthesis platform to address the increasing demand for accurate synthetic DNA at low cost.We believe the use of enzymatic oligonucleotide synthesis, which operates under milder aqueous conditions compared to phosphoramidite chemistry, will provide a significant commercial advantage for _de novo_ DNA/genes synthesis with our platform; it will achieve this by reducing cost, being more environmentally friendly and further streamlining the industrialisation of high-fidelity DNA synthesis.Enzymes with terminal deoxynucleotidyl transferase/synthetase activities are known to extend single-stranded DNA without the need for a template strand. This unique ability to produce DNA _de novo_ makes these enzymes highly valuable biological tools for the production of synthetic DNA.The aim of our project is to develop novel, modified nucleotides and genetically engineered enzymes with terminal deoxynucleotidyl transferase/synthetase activities as an alternative to the well-established phosphoramidite chemistry, to synthesise high-quality DNA/genes with our innovative technology."