FIG 2 .

Construction of a lipoic acid-dependent, carnitine shuttle-constitutive S. cerevisiae strain and its laboratory evolution for lipoic acid-independent, carnitine-dependent growth. (A) In a previous study (33), the {PDHL} cluster, consisting of six cassettes required for cytosolic expression of a functional Enterococcus faecalis pyruvate dehydrogenase complex and flanked by 60-bp sequences, was assembled in vivo via homologous recombination (indicated with black crosses) and introduced in ACS2 after introduction of a Cas9-induced double-strand break. ACS1 was removed using a 120-bp DNA repair fragment (figure adapted from reference 33). (B) In this strain, the {CARN} cluster, consisting of six cassettes for constitutive expression of carnitine shuttle genes, was similarly in vivo assembled and introduced into the SGA1 locus, resulting in strain IMX745 (acs1Δ acs2Δ::{PDHL} sga1Δ::{CARN}). Activity of the E. faecalis PDH in the yeast cytosol is lipoic acid dependent (31). (C) As strain IMX745 did not show l-carnitine-dependent growth when lipoic acid was omitted from growth media, an evolution experiment was initiated using synthetic medium with 20 g ⋅ liter⁻¹ glucose (dextrose) (SMD) and 400 mg ⋅ liter⁻¹ l-carnitine. Abbreviations: chrI, chromosome I; chrIX, chromosome IX; chrXII, chromosome XII.