Schmid_2026_Metab.Eng__102487

Marker-free genome engineering remains an important objective in the metabolic engineering of Komagataella phaffii, particularly for the construction of stable and regulatory-compliant industrial production strains. Here, we present MAPS, a marker-free auxiliary plasmid-assisted strain engineering system for K. phaffii, enabling antibiotic-free strain engineering and rapid assembly of multi-gene production strains. MAPS achieved integration efficiencies of up to 1.8% and facilitated the generation of stable recombinant strains without permanent selection markers. As a proof-of-principle application, we established a co-production platform combining intracellular biosynthesis of natural pigments with secretion of an industrial enzyme in a single fermentation process. Using beetroot (Beta vulgaris) betalain pigments as model metabolites and Candida antarctica lipase B (CalB) as an enzyme, strains were engineered for simultaneous pigment biosynthesis and protein secretion. This work further establishes betanin biosynthesis in K. phaffii. Formation of fully glycosylated betanin was confirmed by absorption spectroscopy and HPLC-MS analysis and required co-overexpression of Dorotheanthus bellidiformis betanidin-5-O-glucosyltransferase (DbGT) together with ascorbic acid supplementation. Using a derepressible promoter, CalB secretion was assessed before and after methanol-induced pigment production. Bioreactor cultivations yielded up to 215 mg L(-1) betalain pigments in the culture supernatant. Importantly, betalain co-production did not adversely affect CalB secretion in shake-flask or bioreactor cultivations, and CalB could be efficiently purified from pigment-containing supernatants, demonstrating compatibility with downstream processing. Together, these results establish MAPS as a versatile platform for marker-free engineering of K. phaffii and its applicability for constructing multi-product production strains combining enzyme secretion and metabolite biosynthesis in a single microbial host.