Rewiring carbon metabolism in yeast for high level production of aromatic chemicals

The production of bioactive plant compounds using microbial hosts is considered a safe, cost-competitive and scalable approach to their production. However, microbial production of some compounds like aromatic amino acid (AAA)-derived chemicals, remains an outstanding metabolic engineering challenge. Here we present the construction of a Saccharomyces cerevisiae platform strain able to produce high levels of p-coumaric acid, an AAA-derived precursor for many commercially valuable chemicals. This is achieved through engineering the AAA biosynthesis pathway, introducing a phosphoketalose-based pathway to divert glycolytic flux towards erythrose 4-phosphate formation, and optimizing carbon distribution between glycolysis and the AAA biosynthesis pathway by replacing the promoters of several important genes at key nodes between these two pathways. This results in a maximum p-coumaric acid titer of 12.5 g L−1 and a maximum yield on glucose of 154.9 mg g−1.

. Flowchart of yeast strain construction in this study. Supplementary Fig. 5. Effects on p-HCA production of overexpressing wild-type ARO4 via the PAL branch. Cells were grown in defined minimal medium with 6 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 using one-tailed 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. Source data are provided as a Source Data file. Supplementary Fig. 6. Introduction of the PHK pathway enables a substantial increase in p-HCA titers via the TAL branch. Cells were grown in defined minimal medium with 6 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 using one-tailed 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. Source data are provided as a Source Data file. Supplementary Fig. 7. Deletion of GPP1 gene reduces the accumulation of acetate with improved cell growth in PHK pathway-expressing strains. Optical density measurements (a) and time course of acetate accumulation (b) during the whole growth phase are shown for three engineered strains, including the PHK-negative strain QL04 (red symbols), the PHK-expressing strain QL24 (blue symbols) and strain QL32 (pink symbols) carrying both the integrated PHK pathway and the deletion of GPP1.
Strains were grown in shake flasks with defined minimal medium containing 2% glucose. All data represent the mean of n = 3 biologically independent samples and error bars show standard deviation. introducing an additional copy of phosphotransacetylase (CkPta), were compared. Cells were grown in defined minimal medium with 6 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 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. Source data are provided as a Source Data file. Supplementary Fig. 9. Increased p-HCA production via phosphoketolase pathway expression leads to decreased cell biomass. Open triangles indicate using constitutive strong promoters to control gene expression. Cells were grown in defined minimal medium with 6 tablets of FeedBeads as the sole carbon source, and cultures were sampled after 96 h of growth for optical density evaluation.
Statistical analysis was performed using one-tailed 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. Source data are provided as a Source Data file.
Supplementary Fig. 10. Dynamic control of biosynthetic genes retains cell growth capacity with increased p-HCA production. Open triangles indicate using constitutive strong promoters to control gene expression, while filled triangles indicate using galactose-induced promoters. Cells were grown in defined minimal medium with 6 tablets of FeedBeads as the sole carbon source and 1% galactose as inducer, and cultures were sampled after 96 h of growth for optical density evaluation. Statistical analysis was performed using one-tailed 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. Source data are provided as a Source Data file. glucose (purple symbols) as the sole carbon source, respectively. Statistical analysis was performed 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. Source data are provided as a Source Data file. Supplementary Fig. 18. Effect of PHA2 overexpression on p-HCA production. Cells were grown in defined minimal medium with 6 tablets of FeedBeads as the sole carbon source and 1% galactose as inducer, and cultures were sampled after 96 h of growth for p-HCA detection. Statistical analysis was performed 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. Source data are provided as a Source Data file. Supplementary Fig. 19. Enabling higher resveratrol production by engineered p-HCA biosynthesis.
(a) Schematic illustration of resveratrol biosynthetic pathway in the context of enhanced supply of precursor p-HCA. The resveratrol biosynthetic pathway consists of Arabidopsis thaliana 4coumarate-CoA ligase 1 (At4CL1) and Vitis vinifera stilbene synthase (VvSTS); the deregulated mutant ACC1 S659A, S1157A was overexpressed to increase the supply of another precursor malonyl-CoA.
Open triangles indicate use of constitutive strong promoters to control gene expression, while filled triangles indicate use of galactose-induced promoters. (b) Resveratrol and p-HCA titers obtained with engineered strains. Cells were grown in defined minimal medium with 6 tablets of FeedBeads as the sole carbon source and 1% galactose as inducer when required. For strains with deletion of GAL80, no galactose was supplemented. Cultures were sampled after 96 h of growth for resveratrol and p-HCA detection. Statistical analysis was performed using Student's t test (one-tailed; twosample 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 Supplementary Figure 19b are provided as a Source Data file.