Bacillus subtilisspore surface display of photodecarboxylase for the transformation of lipids to hydrocarbons

Abstract

The display of enzymes on the surface of spores allows the rapid and very simple biotechnological production of immobilized enzymes. Here we describe the development of a Bacillus subtilis spore display platform and its application to produce hydrocarbons from lipids obtained from the oleaginous yeasts Yarrowia lipolytica, Cutaneotrichosporon oleaginosus as well as olive oil.Lipid hydrolysis was examined in a bienzymatic one-pot cascade using a commercially immobilized lipase (RO lipase) as well as spores with and without additional heterologous lipase expression. Decarboxylation of the released fatty acids was achieved displaying a photodecarboxylase (CvFAP) on the spore surface. Differences in composition of the formed hydrocarbons were observed depending on the lipids source. Using 3D printed lighting equipment titers of up to 64.0 ± 5.6 mg/L hydrocarbons were produced.

Implementation of a Lipase A spore display in Paenibacillus polymyxa.

“…Accordingly, we chose assay parameters as described for LipA from B. subtilis [32]. Sporulation is frequently induced via media change or the usage of sporulation media like SG-Media [30,31]. For our studies, we used minimal media based on the M9 media with additives described in the material and methods part giving us a faster sporulation without formation of cell-clumbs as frequently occurred with SG-media (data not shown).…”

“…Construction of strain and plasmids expressing the CotE-GFP and CotE-LipA fusion protein on the P. polymyxa spore surface The spore surface display was designed based on the previously generated knowledge of B. subtilis spore displays [24,26,30]. The display consists of an anchoring protein located in the coat of the spore, a linker for exibility and distance, and the protein to display.…”

“…However, the system's current cleavage e ciency is not optimal and requires further enhancements to achieve maximum e cacy. Future advancements could allow the simultaneous expression of multiple enzyme types, facilitating the engineering of enzymatic cascades on a single spore surface and creating versatile biocatalysts capable of multiple chemical transformations like it's already described for B. subtilis [30,62]. These developments are expected to contribute signi cantly to the eld of sustainable catalysis.…”

“…Enzymes such as lipase, xylanase, and cellulase have been successfully presented on the spore surface, highlighting the potential of spore display for biocatalytic applications [30][31][32]. While the effectiveness of spore surface display has been successfully demonstrated in Bacillus subtilis, its application in P. polymyxa is still in the early stages [33].…”

Chapter 9 Photocatalysis to promote cell-free biocatalytic reactions

“…CvFAP contains a flavin cofactor, which upon photoexcitation mediates the first single electron transfer from the enzyme-bound carboxylic acid initiating a sequence of CO 2 extrusion and back-transfer of the initially abstracted electron to the newly formed C-centered radical [137,139]. Soon, CvFAP and its mutants found broad interest as catalyst for the synthesis of (fuel) alkanes from carboxylic acids[ [140][141][142][143][144][145][146]] or for the synthesis of chiral compounds via kinetic resolution of racemic hydroxyl carboxylic acids [147]. In combination with other enzymes, CvFAP can also be applied to synthesize fine chemicals such as secondary alcohols [148] or polymer building blocks [149] from unsaturated fatty acids (Scheme 9.20).…”