Colocalization of enzymes is a proven approach to increase pathway flux and the synthesis of nonnative products. Here, we develop a method for enzyme colocalization using the yeast peroxisomal membrane as an anchor point. Pathway enzymes were fused to the native Pex15 anchoring motif to enable display on the surface of the peroxisome facing the cytosol. The peroxisome is the sole location of β-oxidation in Saccharomyces cerevisiae, and acetyl-CoA is a by-product that is exported in the form of acetyl-carnitine. To access this untapped acetyl-CoA pool, we surface-anchored the native peroxisomal/mitochondrial enzyme Cat2 to convert acetyl-carnitine to acetyl-CoA directly upon export across the peroxisomal membrane; this increased acetyl-CoA levels 3.7-fold. Subsequent surface attachment of three pathway enzymes – Cat2, a high stability Acc1 (for conversion of acetyl-CoA to malonyl-CoA), and the type III PKS 2-pyrone synthase – demonstrated the success of peroxisomal surface display for both enzyme colocalization and access to acetyl-CoA from exported acetyl-carnitine. Synthesis of the polyketide triacetic acid lactone increased by 21% over cytosolic expression at low gene copy number, and an additional 11-fold (to 766 mg/L) after further optimization. Finally, we explored increasing peroxisomal membrane area through overexpression of the peroxisomal biogenesis protein Pex11. Our findings establish peroxisomal surface display as an efficient strategy for enzyme colocalization and for accessing the peroxisomal acetyl-CoA pool to increase synthesis of acetyl-CoA-based products.

中文翻译：

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通过酿酒酵母过氧化物酶体表面展示提高乙酰辅酶 A 基产品的产量

酶的共定位是增加通路通量和非天然产物合成的一种行之有效的方法。在这里，我们开发了一种使用酵母过氧化物酶体膜作为锚点的酶共定位方法。将通路酶融合到天然 Pex15 锚定基序上，以便能够在面向胞质溶胶的过氧化物酶体表面上展示。过氧化物酶体是β-氧化的唯一场所酿酒酵母，乙酰辅酶 A 是一种以乙酰肉碱形式出口的副产品。为了访问这个未开发的乙酰辅酶 A 库，我们表面锚定了天然过氧化物酶体/线粒体酶 Cat2，以在通过过氧化物酶体膜输出时直接将乙酰肉碱转化为乙酰辅酶 A；这使乙酰辅酶 A 水平增加了 3.7 倍。随后三种途径酶的表面附着——Cat2、高稳定性 Acc1（用于将乙酰辅酶 A 转化为丙二酰辅酶 A）和 III 型 PKS 2-吡喃酮合酶——证明了酶共定位和访问的过氧化物酶体表面展示的成功从输出的乙酰肉碱到乙酰辅酶 A。在低基因拷贝数下，聚酮化合物三乙酸内酯的合成比胞质表达增加了 21%，进一步优化后增加了 11 倍（达到 766 mg/L）。最后，我们探索了通过过氧化物酶体生物发生蛋白 Pex11 的过表达来增加过氧化物酶体膜面积。我们的研究结果确立了过氧化物酶体表面展示作为酶共定位和访问过氧化物酶体乙酰辅酶 A 池以增加基于乙酰辅酶 A 的产物合成的有效策略。