Scoring changes in enzyme or pathway performance by their effect on growth behavior is a widely applied strategy for identifying improved biocatalysts. While in directed evolution this strategy is powerful in removing non-functional catalysts in selections, measuring subtle differences in growth behavior remains difficult at high throughput, as it is difficult to focus metabolic control on only one or a few enzymatic steps over the entire process of growth-based discrimination. Here, we demonstrate successful miniaturization of a growth-based directed enzyme evolution process. For cultivation of library clones we employed optically clear gel-like microcarriers of nanoliter volume (NLRs) as reaction vessels and used fluorescence-assisted particle sorting to estimate the growth behavior of each of the gel-embedded clones in a highly parallelized fashion. We demonstrate that the growth behavior correlates with the desired improvements in enzyme performance and that we can fine-tune selection stringency by including an antimetabolite in the assay. As a model enzyme reaction, we improve the racemization of ornithine, a possible starting block for the large-scale synthesis of sulphostin, by a broad-spectrum amino acid racemase and confirm the discriminatory power by showing that even moderately improved enzyme variants can be readily identified.

通过酶或途径性能对生长行为的影响来评分变化是鉴定改进的生物催化剂的广泛应用策略。尽管在定向进化中，该策略可有效去除选择中的非功能性催化剂，但在高通量下，测量生长行为的细微差异仍然很困难，因为很难将代谢控制重点放在整个发酵过程中的一个或几个酶促步骤上。基于增长的歧视。在这里，我们证明了基于生长的定向酶进化过程的成功小型化。对于文库克隆的培养，我们使用纳升体积（NLR）的光学透明凝胶状微载体作为反应容器，并使用荧光辅助颗粒分选以高度平行的方式估算每个凝胶包埋的克隆的生长行为。我们证明了生长行为与所需的酶性能改善相关，并且我们可以通过在测定中加入抗代谢物来微调选择严格性。作为模型酶反应，我们通过广谱氨基酸消旋酶改善鸟氨酸的消旋作用，鸟氨酸是大规模合成硫磺汀的可能起点，并通过证明即使适度改善的酶变体也可以容易地鉴别出鉴别力。确定。我们证明了生长行为与所需的酶性能改善相关，并且我们可以通过在测定中加入抗代谢物来微调选择严格性。作为模型酶反应，我们通过广谱氨基酸消旋酶改善鸟氨酸的消旋作用，鸟氨酸是大规模合成硫磺汀的可能起点，并通过证明即使适度改善的酶变体也可以容易地鉴别出鉴别力。确定。我们证明了生长行为与所需的酶性能改善相关，并且我们可以通过在测定中加入抗代谢物来微调选择严格性。作为一种典型的酶反应，我们通过广谱氨基酸消旋酶改善鸟氨酸的消旋作用，鸟氨酸是大规模合成硫磺汀的可能起点，并通过证明即使适度改善的酶变体也可以轻易地鉴别出鉴别力。确定。