过氧化物酶体分隔化已成为重建复杂代谢途径的非常有前途的策略。近年来,通过利用前体池,过氧化物酶体取得了重大进展,规避代谢串扰,并将外源途径的细胞毒性降至最低。然而,重要的是要注意,在甲基营养酵母(例如巴斯德毕赤酵母)中,过氧化物酶体的丰度和蛋白质组成是高度可变的,特别是当过氧化物酶体增殖由特定碳源诱导时。复杂的天然蛋白质亚细胞定位,过氧化物酶体代谢途径的变异性,缺乏过氧化物酶体靶向信号的系统表征限制了过氧化物酶体区室化在巴斯德毕赤酵母中的应用。因此,本研究建立了基于β-胡萝卜素生物合成途径的高通量筛选方法,以评估PTS1s(过氧化物酶体靶向信号1型)在巴斯德毕赤酵母中的靶向效率。首先,对25个推定的内源性PTS1进行了表征,并鉴定了3个具有高靶向效率的PTS1。然后,通过构建两个PTS1突变体文库进行PTS1的定向进化,并确定了总共51个PTS1s(29个经典和22个非规范PTS1s),可能具有更高的过氧化物酶体靶向效率,其中一部分通过共聚焦显微镜进一步表征。最后,新鉴定的PTS1用于香叶醇生物合成途径的过氧化物酶体区室化,与该途径定位于胞质溶胶时相比,导致单萜滴度增加30%以上。本研究扩展了合成生物学工具包,为巴斯德毕赤酵母过氧化物酶体划分奠定了坚实的基础。
Peroxisomal compartmentalization has emerged as a highly promising strategy for reconstituting intricate metabolic pathways. In recent years, significant progress has been made in the peroxisomes through harnessing precursor pools, circumventing metabolic crosstalk, and minimizing the cytotoxicity of exogenous pathways. However, it is important to note that in methylotrophic yeasts (e.g. Pichia pastoris), the abundance and protein composition of peroxisomes are highly variable, particularly when peroxisome proliferation is induced by specific carbon sources. The intricate subcellular localization of native proteins, the variability of peroxisomal metabolic pathways, and the lack of systematic characterization of peroxisome targeting signals have limited the applications of peroxisomal compartmentalization in P. pastoris. Accordingly, this study established a high-throughput screening method based on β-carotene biosynthetic pathway to evaluate the targeting efficiency of PTS1s (Peroxisome Targeting Signal Type 1) in P. pastoris. First, 25 putative endogenous PTS1s were characterized and 3 PTS1s with high targeting efficiency were identified. Then, directed evolution of PTS1s was performed by constructing two PTS1 mutant libraries, and a total of 51 PTS1s (29 classical and 22 noncanonical PTS1s) with presumably higher peroxisomal targeting efficiency were identified, part of which were further characterized via confocal microscope. Finally, the newly identified PTS1s were employed for peroxisomal compartmentalization of the geraniol biosynthetic pathway, resulting in more than 30% increase in the titer of monoterpene compared with when the pathway was localized to the cytosol. The present study expands the synthetic biology toolkit and lays a solid foundation for peroxisomal compartmentalization in P. pastoris.