SBIR PHASE I: Cloning and Automated Screening of Genes Encoding Cold-Adapted Lipases From Bacteria Inhabiting Lipid-Rich Whale Skeletons

9561829 Stein这个小企业创新研究第一阶段项目将确定使用一种新的克隆和自动筛选方法来组装编码冷适应脂肪酶的基因库的可行性。该方法是基于“环境文库”的构建，这些文库代表了保存在克隆载体中的自然出现的原核生物的集体基因组，这些克隆载体可以在大肠杆菌或其他合适的宿主中繁殖。因为克隆的DNA最初是直接从环境样本中提取的，所以文库不仅限于可以在纯培养中生长的一小部分原核生物，也不偏向于少数快速生长的物种。然后使用氟连接底物和高通量机器人对这些文库进行筛选，以确定表达感兴趣的活性的克隆。此外，还将使用专门的克隆载体，以便快速鉴定含有针对出口基因产品的信号序列的克隆DNA片段。这类信号经常先于编码脂肪酶等水解酶的基因。这些方法将被用来筛选从一个独特的环境中提取的DNA构建的环境文库：深海盆地中包裹着富含脂肪的鲸鱼骨骼的细菌垫。初步工作表明，这些地点有一个多样化的细菌群落，这些细菌群落依靠鲸鱼骨骼中渗出的大量复杂脂肪生存多年。通过将环境文库方法与高通量自动筛选相结合，将对数十万个鲸骨细菌重组克隆进行脂肪酶活性筛选。编码脂肪酶的候选基因在低温下表现出高活性，但在中等温度下保持稳定，将被过度表达，以作为洗涤剂添加剂进行评估。此外，还将评估发现的其他独特脂肪酶在其他工业过程中的使用情况，包括食品和饮料、制药、废物系统、化工和医疗行业中的脂肪酶。

9561829 Stein This Small Business Innovation Research Phase I project will determine the feasibility of using a novel cloning and automated screening approach to assemble a library of genes encoding cold-adapted lipase. The approach is based on the construction of "environmental libraries" which represent the collective genomes of naturally occurring prokaryotes archived in cloning vectors that can be propagated in E. coli or other suitable host. Because the cloned DNA is initially extracted directly from environmental samples, the libraries are not limited to the small fraction of prokaryotes that can be grown in pure culture nor are they biased towards a few rapidly growing species. These libraries are then screened using fluor-linked substrates and high throughput robotics to identify clones expressing the activity of interest. In addition, specialized cloning vectors will be employed that allow the rapid identification of cloned DNA fragments containing signal sequences which target the gene product for export. Such signals frequently precede genes encoding hydrolytic enzymes such as lipases. These approaches will be used to screen environmental libraries constructed from DNA extracted from a unique environment: the bacterial mats encrusting lipid-rich whale skeletons in deep sea basins. Preliminary work suggests that these sites harbor a diverse bacterial community that subsist on the copious amounts of complex lipids that exude from the whale bones over a period of many years. By coupling the environmental library approach to high throughput automated screening, hundreds of thousands of recombinant clones from the whale bone bacteria will be screened for lipase activity. Candidate genes encoding lipases that display high activity at low temperatures yet remain stable at moderate temperatures will be overexpressed for evaluation as detergent additives. In addition, other unique lipases found will be evaluated for use in other industrial processes which include thos e in food and beverage, pharmaceutical, waste systems, chemical and medical industries.