Firmenich SA, Corporate R&D, Route des Jeunes 1, CH-1211 Geneva 8, Switzerland.
J Biotechnol. 2013 Feb 10;163(3):339-45. doi: 10.1016/j.jbiotec.2012.11.005. Epub 2012 Nov 23.
The performance of a 13-hydroperoxide lyase from guava, an enzyme of the CYP74 family, which is of interest for the industrial production of saturated and unsaturated C6-aldehydes and their derivatives, was improved by directed evolution. Four rounds of gene shuffling and random mutagenesis improved the functional expression in E. coli by offering a 15-fold higher product yield factor. The increased product yield factor relates to an improved total turnover number of the variant enzyme, which also showed higher solubility and increased heme content. Thermal stability was also dramatically improved even though there was no direct selection pressure applied for evolving this trait. A structure based sequence alignment with the recently solved allene oxide synthase of Arabidopsis thaliana showed that most amino acid alterations occurred on the surface of the protein, distant of the active site and often outside of secondary structures. These results demonstrate the power of directed evolution for improving a complex trait such as the total turnover number of a cytochrome P450, a critical parameter for process performance that is difficult to predict even with good structural information at hand.
番木瓜中 13-羟过氧化物裂合酶（CYP74 家族的一种酶）的性能通过定向进化得到了改善，该酶对于工业生产饱和和不饱和 C6-醛及其衍生物具有重要意义。四轮基因改组和随机诱变提高了在大肠杆菌中的功能表达，提供了 15 倍更高的产物产率因子。增加的产物产率因子与变体酶的总周转率数的提高有关，该变体酶还表现出更高的溶解性和增加的血红素含量。尽管没有直接选择压力来进化这个特性，但热稳定性也得到了显著提高。与最近解决的拟南芥丙二烯氧化物合酶的基于结构的序列比对表明，大多数氨基酸改变发生在蛋白质的表面，远离活性位点，并且经常在二级结构之外。这些结果证明了定向进化在提高复杂特性方面的威力，例如细胞色素 P450 的总周转率，这是过程性能的关键参数，即使手头有良好的结构信息，也很难预测。
