Fig. 4

Construction of crocetin pathway and crocetin production in flask and batch bioreactor fermentations. (A) HPLC and LG-MS analysis of an extract of the Z1pC strain expressing ALDH6803 (as pBBR-A6803) from Synechocystis sp. PCC6803 or ALDH7942 (pBBR-A7942) from S. elongates PCC7942. In the HPLC chromatogram, peak 1 corresponds to crocetin, and peak 2, to crocetin dialdehyde. (B) Quantifying crocetin production in the Z1pC strain expressing ALDH6803 or ALDH7942 in flask cultures. (C) Schematical description of individual modular (upper) and polycistronic (lower) expression systems of CsCCD2 and ALDH7942. (D) Quantifying crocetin production in Z1pC strain expressing ALDH7942 via polycistronic or modular mode in flask cultures. (E) mRNA expression levels of CsCCD2 and ALDH7942 via two plasmid systems (noted as Z1pC + pBBR-A7942) and one plasmid system with polycistronic (noted as Z1pCA7942(P)) or individual modular expression (noted as Z1pCA7942(M)). Relative expression was presented by normalizing with the expression level of the cysG gene (a reference gene). (F) Batch bioreactor fermentations of the crocetin-producing Z1pCA7942(P) strain at 20 ℃. All experiments, including bioreactor fermentation, were done in biological triplicate, and error bars represent mean ± SD.