Biotransformations Near and Above 100 C: Mining Genomes of Hyperthermophiles for New Biocatalysts

生物催化剂的发现和表征仍然是一个关键的生物技术目标。高通量筛选和定向进化方法在过去的几年中已经发展起来，使得这样的生物催化剂能够被鉴定，然后就其生物化学、生物物理和生物加工性质进行优化。然而，目前的发现和优化方法严重依赖于基于酶的测定，由于化学限制或基因表达问题，这可能难以甚至不可行。此外，由于目前的方法集中在单个基因的产物上，因此这种方法不容易识别多酶转化。作为一种手段，规避与目前的方法的局限性，本研究的目标是发展功能基因组学为基础的发现方法，以确定新的生物催化剂。随着测量基因表达的高通量技术的出现，遗传水平上的细胞反应可以与环境条件相关联。这种酶发现的替代方法利用了对生物技术相关的环境挑战和生长条件的高度敏感的、基于转录的细胞反应。 在本项目期间，将广泛使用DNA微阵列来鉴定编码一系列重要生物转化新酶的基因。

Biocatalyst discovery and characterization remains a key biotechnological objective. High throughput screening and directed evolution approaches have been developed over the last few years that enable such biocatalysts to be identified and then optimized with respect to their biochemical, biophysical and bioprocessing properties. However, current methods for discovery and optimization rely heavily on enzyme-based assays, which may be difficult or even infeasible to implement because of chemical limitations or gene expression issues. Moreover, multienzyme transformations are not easily identified by this approach since current methods focus on the products of individual genes. As a means of circumventing the limitations with current approaches, the objective of this research is to develop functional genomics-based discovery methods to identify novel biocatalysts. With the advent of high-throughput technologies to measure gene expression, cellular responses at the genetic level can be correlated to environmental conditions. This alternative approach to enzyme discovery takes advantage of highly sensitive, transcriptional.based cellular responses to biotechnologically-related environmental challenges and growth conditions. In this project period, extensive use will be made of DNA microarrays to identify the genes that encode new enzymes for a range of important biotransformations.