(472b) Engineering Peroxisomal Surface Display for Enhanced Product Biosynthesis in the Non-Conventional Yeast Kluyveromyces Marxianus

The non-conventional yeast Kluyveromyces marxianus is a promising microbial host for industrial biomanufacturing. Attributes include a rapid growth rate (twice that of Baker’s yeast), thermotolerance (to 52˚C), acid tolerance, and the ability to assimilate a wide range of low-cost feedstocks. Along with the development of Cas9-based genome editing systems, many novel synthetic biology tools in K. marxianus have been created, making the engineering of this yeast far more accessible. The colocalization of enzymes is a proven approach to increase pathway flux and the synthesis of non-native products. Previously, our group introduced peroxisomal surface display in Saccharomyces cerevisiae, a state-of-the-art technique that uses the peroxisomal membrane surface as an anchor point. Enzymes are fused to the native Pex15 anchoring motif to enable colocalization on the peroxisome surface facing the cytosol. This also allows access to the peroxisomal acetyl-CoA pool by intercepting exported acetyl-carnitine and converting it to acetyl-CoA (via display of the carnitine acetyl-transferase enzyme Cat2). Here, we expand this colocalization technique to K. marxianus, taking advantage of this yeast’s high flux through b-oxidation when grown on the pentose sugar xylose. Through comparative genomics and proteomics, we isolated the native KmPex15 anchoring motif, and demonstrated successful peroxisomal display of active enzymes via fluorescent microscopy. We then demonstrated this technique for production of a range of products. By displaying the Pseudomonas savastanoi IaaM and IaaH, we increased production of indole-acetic acid 8-fold, demonstrating general substrate channeling effects of this technique. We then redirected violacein pathway flux by displaying the pathway enzymes VioE and VioD from Chromobacterium violaceum, increasing selectivity of proviolacein to prodeoxyviolacein up to 2.5-fold. Finally, we improved access to peroxisomal acetyl-CoA and enhanced synthesis of the polyketide triacetic acid lactone (TAL) up to 4-fold by concurrently displaying the proteins Cat2, Acc1, and the type III PKS 2-pyrone synthase from Gerbera hybrida. We further improved TAL production up to 2.1-fold through engineering peroxisome morphology and longevity. Our findings reaffirm that peroxisomal surface display is an efficient enzyme colocalization strategy that is functional in multiple yeasts and applicable for improving production of a wide range of non-native products.