Fig. 2: Modularity of yeast S. cerevisiae for co-culture toolkit development.
From: A molecular toolkit of cross-feeding strains for engineering synthetic yeast communities
a, Model yeast S. cerevisiae BY4741 has four auxotrophic markers of histidine, leucine, uracil and methionine, and the yeast prototrophy kit (pHLUM v.2 plasmids)35 can be used to complement the auxotrophic markers. b, Auxotrophic markers in BY4741 can be replaced with functional modules that facilitate co-culture design. We developed 15 knockout strains: ade8Δ, ura3Δ, his3Δ, trp1Δ, tyr1Δ, pha2Δ, aro7Δ, leu2Δ, ilv1Δ, arg4Δ, lys2Δ, ser1Δ, cys4Δ, met14Δ and thr4Δ, which are auxotrophic to adenine, uracil, histidine, tryptophan, tyrosine, phenylalanine, tyrosine and phenylalanine, leucine, valine and isoleucine, arginine, lysine, serine, cysteine, methionine and threonine, respectively (Supplementary Table 1). One marker such as leu2∆ can be used to express different fluorescent proteins such as mTagBFP2, mScarlet-I and sfGFP, which can be used as fluorescent markers. One marker such as his3∆ can be used to express the genes that help the production of exchanged metabolites (emy), which can be used as metabolite donor. One marker such as met15∆ or CRIPSR-cas9 tool can be used to express a heterologous high-value bioproduct synthesis pathway. c, An adenine auxotrophic strain was designed to pair with 13 other auxotrophic strains for co-culture potential in 4 different cross-feeding structures (ade-em I, ade-em II, ade-em III, ade-em IV). The auxotrophs for each metabolite, with and without adenine overproduction (ade4op overexpression) were co-cultured with the adenine auxotroph (ade8Δ) with and without the overexpression of genes involved in the overproduction of the metabolites to identify the syntrophic pairs. d, The maximal OD700 values of co-culture ade-em I and ade-em II within 72 h were ranked from strong to weak. N = 3 biologically independent samples and data are presented as mean ± s.d. One-way ANOVA, followed by Bonferroni’s multiple comparisons test with 95% confidence intervals were performed using GraphPad Prism 9.5.0 software and P values are noted.
