Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle (Saale), Germany.
Institute of Chemistry, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Straße 2, 06120 Halle (Saale), Germany.
ACS Synth Biol. 2021 Jun 18;10(6):1360-1372. doi: 10.1021/acssynbio.0c00641. Epub 2021 Jun 2.
Fungal peroxygenases (UPOs) have emerged as oxyfunctionalization catalysts of tremendous interest in recent years. However, their widespread use in the field of biocatalysis is still hampered by their challenging heterologous production, substantially limiting the panel of accessible enzymes for investigation and enzyme engineering. Building upon previous work on UPO production in yeast, we have developed a combined promoter and signal peptide shuffling system for episomal high throughput UPO production in the industrially relevant, methylotrophic yeast . Eleven endogenous and orthologous promoters were shuffled with a diverse set of 17 signal peptides. Three previously described UPOs were selected as first test set, leading to the identification of beneficial promoter/signal peptide combinations for protein production. We applied the system then successfully to produce two novel UPOs: UPO from and UPO from . To demonstrate the feasibility of the developed system to other enzyme classes, it was applied for the industrially relevant lipase CalB and the laccase Mrl2. In total, approximately 3200 transformants of eight diverse enzymes were screened and the best promoter/signal peptide combinations studied at various cofeeding, derepression, and induction conditions. High volumetric production titers were achieved by subsequent creation of stable integration lines and harnessing orthologous promoters from . In most cases promising yields were also achieved without the addition of methanol under derepressed conditions. To foster the use of the episomal high throughput promoter/signal peptide system, we made all plasmids available through Addgene.
真菌过氧化物酶（UPOs）近年来作为氧化功能化催化剂引起了极大的关注。然而，它们在生物催化领域的广泛应用仍然受到其异源生产的挑战的阻碍，这大大限制了可用于研究和酶工程的酶的选择。在先前关于酵母中 UPO 生产的工作基础上，我们开发了一种组合启动子和信号肽改组系统，用于在工业相关的甲基营养酵母中进行质体高通量 UPO 生产。11 个内源和直系同源启动子与一组多样化的 17 个信号肽进行改组。选择了三种先前描述的 UPO 作为第一个测试集，从而确定了有利于蛋白质生产的启动子/信号肽组合。然后，我们成功地将该系统应用于生产两种新型 UPO：来自 的 UPO 和来自 的 UPO。为了证明所开发的系统对其他酶类的可行性，我们将其应用于工业相关的脂肪酶 CalB 和漆酶 Mrl2。总共筛选了大约 3200 个来自 8 种不同酶的转化体，并在各种共喂养、去阻遏和诱导条件下研究了最佳的启动子/信号肽组合。通过随后创建稳定的整合线并利用 的同源启动子，实现了高体积生产滴度。在大多数情况下，在去阻遏条件下无需添加甲醇也可以实现有希望的产率。为了促进质体高通量启动子/信号肽 系统的使用，我们通过 Addgene 提供了所有质粒。
