Some bacteria belonging to the actinobacteria and proteobacteria groups can accumulate neutral lipids expressing enzymes of the wax ester synthase/acyl coenzyme A: diacylglycerol acyltransferase (WS/DGAT) family. tDGAT is a WS/DGAT-like enzyme from Thermomonospora curvata able to produce TAGs and WEs when heterologously expressed in Escherichia coli. In this study, a protocol for the directed evolution of bacterial lipid-producing enzymes based on fluorimetry is developed and tested. tDGAT has been successfully evolved towards the improvement of TAG production with an up to 2.5 times increase in TAG accumulation. Mutants with no ability to produce TAGs but able to accumulate waxes were also selected during the screening. The localization of the mutations that enhance TAG production in the outer surface of tDGAT points out possible new mechanisms that contribute to the activity of this family of enzymes. This Nile red-based high throughput screening provides an evolution platform for other WS/DGAT-like enzymes.

中文翻译：

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指导细菌WS / DGAT酰基转移酶的进化：从弯曲热单孢菌（thermomonospora curvata）改良tDGAT。

属于放线菌属和变形杆菌属的某些细菌可以积聚表达蜡酯合酶/酰基辅酶A：二酰基甘油酰基转移酶（WS / DGAT）家族的中性脂质。tDGAT是弯曲热单孢菌的一种类似于WS / DGAT的酶，当在大肠杆菌中异源表达时能够产生TAG和WEs。在这项研究中，开发并测试了基于荧光法的细菌脂质生产酶的定向进化协议。tDGAT已成功地朝着TAG产量提高的方向发展，其TAG积累增加了多达2.5倍。在筛选过程中还选择了不能产生TAGs但能够积累蜡的突变体。在tDGAT的外表面增强TAG产生的突变的定位指出了可能有助于该酶家族活性的新机制。这种基于尼罗河红的高通量筛选为其他类似WS / DGAT的酶提供了进化平台。