Department of Biocatalysis, Institute of Catalysis, CSIC, Madrid, Spain.
EvoEnzyme S.L., Madrid, Spain.
Appl Environ Microbiol. 2021 Sep 10;87(19):e0087821. doi: 10.1128/AEM.00878-21.
Fungal unspecific peroxygenases (UPOs) are emergent biocatalysts that perform highly selective C-H oxyfunctionalizations of organic compounds, yet their heterologous production at high levels is required for their practical use in synthetic chemistry. Here, we achieved functional expression of two new unusual acidic peroxygenases from () (UPO) in yeasts and their production at a large scale in a bioreactor. Our strategy was based on adopting secretion mutations from an Agrocybe aegerita UPO mutant, the PaDa-I variant, designed by directed evolution for functional expression in yeast, which belongs to the same phylogenetic family as UPOs, long-type UPOs, and shares 65% sequence identity. After replacing the native signal peptides with the evolved leader sequence from PaDa-I, we constructed and screened site-directed recombination mutant libraries, yielding two recombinant UPOs with expression levels of 5.4 and 14.1 mg/liter in Saccharomyces cerevisiae. These variants were subsequently transferred to Pichia pastoris for overproduction in a fed-batch bioreactor, boosting expression levels up to 290 mg/liter, with the highest volumetric activity achieved to date for a recombinant peroxygenase (60,000 U/liter, with veratryl alcohol as the substrate). With a broad pH activity profile, ranging from pH 2.0 to 9.0, these highly secreted, active, and stable peroxygenases are promising tools for future engineering endeavors as well as for their direct application in different industrial and environmental settings. In this work, we incorporated several secretion mutations from an evolved fungal peroxygenase to enhance the production of active and stable forms of two unusual acidic peroxygenases. The tandem-yeast expression system based on S. cerevisiae for directed evolution and P. pastoris for overproduction on an ∼300-mg/liter scale is a versatile tool to generate UPO variants. By employing this approach, we foresee that acidic UPO variants will be more readily engineered in the near future and adapted to practical enzyme cascade reactions that can be performed over a broad pH range to oxyfunctionalize a variety of organic compounds.
真菌非特异性过氧化物酶（UPO）是新兴的生物催化剂，能够对有机化合物进行高度选择性的 C-H 氧化官能化，但为了将其实际应用于合成化学，需要在异源系统中高水平生产。在这里，我们在酵母中实现了两种新型不寻常酸性过氧化物酶（UPO）的功能表达，并在生物反应器中大规模生产。我们的策略基于采用来自 Agrocybe aegerita UPO 突变体（UPO）的分泌突变，即通过定向进化设计用于在酵母中功能表达的 PaDa-I 变体，该变体属于与 UPOs、长型 UPOs 相同的系统发育家族，并共享 65%的序列同一性。在用 PaDa-I 的进化前导肽替换天然信号肽后，我们构建并筛选了定点重组突变文库，得到了两种在酿酒酵母中表达水平为 5.4 和 14.1 mg/L 的重组 UPO。然后，这些变体被转移到巴斯德毕赤酵母中，在分批补料生物反应器中进行过表达，表达水平提高到 290 mg/L，这是迄今为止重组过氧化物酶的最高体积活性（以藜芦醇为底物，达到 60,000 U/L）。这些高度分泌、活性和稳定的过氧化物酶具有广泛的 pH 活性谱，范围从 pH 2.0 到 9.0，是未来工程努力以及在不同工业和环境环境中直接应用的有前途的工具。在这项工作中，我们整合了来自进化真菌过氧化物酶的几种分泌突变，以提高两种不寻常酸性过氧化物酶的活性和稳定形式的产量。基于酿酒酵母的定向进化和巴斯德毕赤酵母的 300mg/L 规模的过表达的串联酵母表达系统是一种通用工具，可用于生成 UPO 变体。通过采用这种方法，我们预计在不久的将来，酸性 UPO 变体将更容易被工程化，并适应于在较宽 pH 范围内进行的实际酶级联反应，以氧化官能化各种有机化合物。
