Engineering peroxisomal surface display for enhanced biosynthesis in the emerging yeast Kluyveromyces marxianus

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.