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. 2024 Feb 7;9(3):848–863. doi: 10.1038/s41564-023-01596-4

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a, Diagram of two-member cross-feeding co-cultures M1M2. The two members were labelled with blue fluorescent protein mTagBFP2 and red fluorescent protein mScarlet-I, respectively. Each member is auxotrophic to one exchanged metabolite (em1) and overproduced another exchanged metabolite (em2). b, The phenotype of each member and the strain combination for co-cultures are listed in the table. Val-Lys^v1, cell culture using SM medium; Val-Lys^v2, cell culture using SM plus isoleucine; Trp+LysΔ^v1 (BFP-tagged) contains the native Trp pathway; Trp+LysΔ^v2 (BFP-tagged) does not contain the native Trp pathway. c, Maximal OD₇₀₀ values of two-member co-cultures and monoculture controls within 72 h. 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 and P values are noted. d, The diagram of three-member co-cultures via one-way (top) and two-way (bottom) communication; each member was labelled with one fluorescent protein mScarlet-I, mTagBFP2 or sfGFP, respectively. In one-way-communicated three-member co-cultures (MMM_1), each member is auxotrophic to one exchanged metabolite (em1 or em2) and overproduced another exchanged metabolite (em2 or em3). In two-way-communicated three-member co-cultures (MMM_2), each member is auxotrophic to two exchanged metabolites (em12, em13 or em23) and overproduced another exchanged metabolite (em3, em2 or em1). e, The strain combination table of 9 pairs of three-member co-cultures. We labelled three-member co-cultures and controls (monoculture and two-member co-cultures) using target gene abbreviations. For example, in three-member co-culture ade-Lys-Trp (labelled as AKW_I), monoculture controls of each member of adeΔLys+ (RFP-tagged), trpΔade+ (BFP-tagged), lysΔTrp+ (GFP-tagged) are labelled as K_I, A_I and W_I; controls of two-member co-cultures are labelled as AK_I, AW_I, KW_I. f, Maximal OD₇₀₀ values of the three-member co-cultures and the controls of monocultures and two-member co-cultures within 72 h. In these two- and three-member co-cultures, the initial OD₇₀₀ was 0.078 for each member, and the initial ratios were 1:1 and 1:1:1, respectively. 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 and P values are noted.

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