The non-conventional yeast Kluyveromyces marxianus is a promising microbial host for industrial biomanufacturing. With the recent development of Cas9-based genome editing systems and other novel synthetic biology tools for K. marxianus, engineering of this yeast has become far more accessible. Enzyme colocalization is a proven approach to increase pathway flux and the synthesis of non-native products. Here, we engineer K. marxianus to enable peroxisomal surface display, an enzyme colocalization technique for displaying enzymes on the peroxisome membrane via an anchoring motif from the peroxin Pex15. The native KmPex15 anchoring motif was identified and fused to GFP, resulting in successful localization to the surface of the peroxisomes. To demonstrate the advantages for pathway localization, the Pseudomonas savastanoi IaaM and IaaH enzymes were co-displayed on the peroxisome surface; this increased production of indole-3-acetic acid 7.9-fold via substrate channeling effects. We then redirected pathway flux by displaying the violacein pathway enzymes VioE and VioD from Chromobacterium violaceum, increasing selectivity of proviolacein to prodeoxyviolacein by 2.5-fold. Finally, we improved direct access to peroxisomal acetyl-CoA and increased titers of the polyketide triacetic acid lactone (TAL) by 2-fold through concurrent display of the proteins Cat2, Acc1, and the type III PKS 2-pyrone synthase from Gerbera hybrida relative to the same three enzymes diffusing in the cytosol. We further improved TAL production by up to 2.1-fold through engineering peroxisome morphology and lifespan. Our findings demonstrate that peroxisomal surface display is an efficient enzyme colocalization strategy in K. marxianus and applicable for improving production of a wide range of non-native products.

中文翻译：

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工程过氧化物酶体表面显示以增强新兴酵母 Kluyveromyces marxianus 的生物合成


非常规酵母 Kluyveromyces marxianus 是一种很有前途的工业生物制造微生物宿主。随着最近基于 Cas9 的基因组编辑系统和其他用于 K. marxianus 的新型合成生物学工具的开发，这种酵母的工程改造变得更加容易。酶共定位是增加通路通量和非天然产物合成的成熟方法。在这里，我们设计了 K. marxianus 以实现过氧化物酶体表面显示，这是一种酶共定位技术，用于通过来自过氧化物酶 Pex15 的锚定基序在过氧化物酶体膜上显示酶。鉴定出天然 KmPex15 锚定基序并与 GFP 融合，从而成功定位到过氧化物酶体表面。为了证明通路定位的优势，Pseudomonas savastanoi IaaM 和 IaaH 酶共同显示在过氧化物酶体表面;这通过底物通道效应将吲哚-3-乙酸的产生增加了 7.9 倍。然后，我们通过显示来自紫罗兰色杆菌的紫罗兰素通路酶 VioE 和 VioD 来重定向通路通量，将紫罗兰素对原脱氧紫罗兰素的选择性提高 2.5 倍。最后，我们通过同时展示来自非洲菊的蛋白质 Cat2、Acc1 和 III 型 PKS 2-吡喃酮合酶，改善了对过氧化物酶体乙酰辅酶 A 的直接访问，并将聚酮三乙酸内酯 （TAL） 的滴度提高了 2 倍相对于在胞质溶胶中扩散的相同三种酶。我们通过工程过氧化物酶体形态和寿命，进一步将 TAL 产量提高了 2.1 倍。我们的研究结果表明，过氧化物酶体表面显示是 K 中一种有效的酶共定位策略。 Marxianus 并适用于改进各种非本地产品的生产。