Extended Data Fig. 7: Engineering C. glutamicum as living material for cellulose degradation and lycopene production.

(a) Construction of Δspa2Δdec chassis in which both spa2 gene (defective for ^CgCLP formation) and gene fragment between CEY17_RS03380 and CEY17_RS03560 (with a function of producing the precursor for lycopene production, resulting in the color change of the cells from yellow to white) were deleted. The colony PCR identification indicates that the spa2 gene and genes between CEY17_RS03380 and CEY17_RS03560 were markerless deleted. (b) Construction of P1 plasmid for lycopene production, and P2 and P3 plasmids for cellulose degradation. P1 plasmid co-expressed the dxs gene and crtEBI gene cluster under the control of an IPTG-inducible promoter; P2 plasmid simultaneously expressed Spa2 pilin fusion proteins of TrEgl-Spa2 and SdBgl-Spa2 under two independent expression cassettes with the same transcription and translation elements; P3 plasmid simultaneously expressed proteins of TrEgl and SdBgl under the same genetic parts in the P2 plasmid. (c) The red color of different engineered cells indicates the lycopene accumulation. The C001 strain showed white due to the deletion region between CEY17_RS03380 and CEY17_RS03560, which lacked the synthesis of decaprenoxanthin. The red cells of C002, C003, and C004 indicated that lycopene was successfully accumulated. The C002 cells harbor P1 plasmid; the C003 cells harbor both P1 and P2 plasmids; the C004 cells have both P1 and P3 plasmids. (d) TEM images show that cells of C003, which contain the P2 plasmid, enabled co-assembly of TrEgl and SdBgl into ^CgCLP structure, while the cells of C001, C002, and C004 did not. ^CgCLP was labeled with 10 nm gold particles by immunogold labelling. Scale bars, 200 nm.

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