Fig. 2
From: Rewiring carbon metabolism in yeast for high level production of aromatic chemicals
Relieving bottlenecks in the AAA biosynthesis pathway. a Overview of yeast metabolic pathway for p-HCA biosynthesis. Both the PAL branch (pink) consisting of Arabidopsis thaliana phenylalanine ammonia lyase (AtPAL2), cinnamic acid hydroxylase (AtC4H), P450 reductase (AtATR2), and yeast native cytochrome b5 (CYB5) and the TAL branch (blue) consisting of tyrosine ammonia lyase (FjTAL) from Flavobacterium johnsoniae were constructed for p-HCA production. Overexpressed yeast endogenous genes are shown in orange, including DAHP synthase (ARO3), l-tyrosine-feedback-insensitive DAHP synthase (ARO4K229L), pentafunctional aromatic protein (ARO1), chorismate synthase (ARO2), l-tyrosine-feedback-insensitive chorismate mutase (ARO7G141S), prephenate dehydratase (PHA2), and aromatic aminotransferase I (ARO8). In addition, the E. coli shikimate kinase (EcaroL) was expressed, and three heterologous l-tyrosine prephenate dehydrogenase encoding genes (ZmtyrC from Zymomonas mobilis, GmPDH1 from Glycine max, and MtPDH1 from Medicago truncatula) were evaluated for enhancing l-tyrosine supply. The dashed lines indicate feedback inhibition of Aro4 and Aro7 by l-tyrosine and feedback inhibition of Aro3 by l-phenylalanine. Removal of the AAA degradation pathway was enabled by deleting the corresponding genes PDC5 and ARO10 (marked with red cross) in the engineered strains. DHQ 3-dehydroquinate, DHS 3-dehydro-shikimate, S3P shikimate-3-phosphate, SHIK shikimate, EPSP 5-enolpyruvyl-shikimate-3-phosphate, PPY phenylpyruvate, HPP para-hydroxy-phenylpyruvate, CA cinnamic acid; see Fig. 1 legend regarding abbreviations of other metabolites. p-HCA titers obtained with engineered strains derived from the PAL branch b, the TAL branch c, and the combination of both branches and the removal of the AAA degradation pathway d, respectively. ePhe represents the combination of identified upstream beneficial manipulations (shown in Fig. 2b) for phenylalanine biosynthesis. Cells were grown in defined minimal medium with six tablets of FeedBeads as the sole carbon source, and cultures were sampled after 96 h of growth for p-HCA detection. Statistical analysis was performed by using Student’s t test (one-tailed; two-sample unequal variance; *p < 0.05, **p < 0.01, ***p < 0.001). All data represent the mean of n = 3 biologically independent samples and error bars show standard deviation. The source data underlying figures b–d are provided in a Source Data file
