Metabolic Engineering of Saccharomyces cerevisiae for High-Level Production of Chlorogenic Acid from Glucose

Abstract

Chlorogenic acid (CGA), a major dietary phenolic compound, has been increasingly used in the food and pharmaceutical industries because of its ready availability and extensive biological and pharmacological activities. Traditionally, extraction from plants has been the main approach for the commercial production of CGA. This study reports the first efficient microbial production of CGA by engineering the yeast, Saccharomyces cerevisiae, on a simple mineral medium. First, an optimized de novo biosynthetic pathway for CGA was reconstructed in S. cerevisiae from glucose with a CGA titer of 36.6 ± 2.4 mg/L. Then, a multimodule engineering strategy was employed to improve CGA production: (1) unlocking the shikimate pathway and optimizing carbon distribution; (2) optimizing the L-Phe branch and pathway balancing; and (3) increasing the copy number of CGA pathway genes. The combination of these interventions resulted in an about 6.4-fold improvement of CGA titer up to 234.8 ± 11.1 mg/L in shake flask cultures. CGA titers of 806.8 ± 1.7 mg/L were achieved in a 1 L fed-batch fermenter. This study opens a route to effectively produce CGA from glucose in S. cerevisiae and establishes a platform for the biosynthesis of CGA-derived valueadded metabolites.

Recent advance in the biological activity of chlorogenic acid and its application in food industry

“…This method is superior to the previous dual fermentation system. In addition to bacteria, by modifying the shikimic acid metabolic pathway of Saccharomyces cerevisiae and increasing the expression of key genes for CGA synthesis, the yield of CGA can be significantly increased by 6.4 times (Xiao et al ., 2022). This breaks through the food safety bottleneck of the inability of pre fermentation products to be used for food processing.…”

Advances in Production of Hydroxycinnamoyl-Quinic Acids: From Natural Sources to Biotechnology

“…Further improvements have been achieved by introducing into S. cerevisiae the de novo 5-CQA synthesis pathway, including PAL2, C4H, 4CL1, C3′H, CPR1, CPR2, HQT2, YdiB, CYB5 and implementing the following modifications ( Figure 6 ): (1) unlocking the shikimate pathway and optimizing carbon distribution by overexpressing the L-phenylalanine feedback-insensitive DAHP synthase (ARO3 K222L ), L-tyrosine feedback-insensitive DAHP synthase (ARO4 K229L ), pyruvate kinase 1 mutant with reduced catalytic activity (PYK1 D146N ) and transketolase (TKL1); (2) optimizing the L-phenylalanine branch and pathway balancing by overexpressing the l-tyrosine feedback-insensitive chorismate mutase (ARO7 G141S ), endogenous prephenate dehydratase (PHA2), NADH kinase (POS5), and cytochrome b5 (Cyb5); (3) increasing the copy number of 5-CQA pathway genes encoding hydroxycinnamoyl-CoA quinate transferase 2 (HQT2) and cytochrome P450 98A3 (C3′H) [ 151 ]. The engineered S. cerevisiae strain produced 5-CQA to 234.8 ± 11.1 mg/L in shake flask culture and 806.8 ± 1.7 mg/L in fed-batch fermentation [ 151 ].…”

De Novo Biosynthesis of Sakuranetin from Glucose by Engineered Saccharomyces cerevisiae

“…All strains used or constructed were listed in Table 1, gene knockout and insertion of DNA fragments in Saccharomyces cerevisiae were operated by CRISPR/Cas9 system (Stovicek et al, 2015). YT00 (CEN.PK2-1C, IX1 :: TEFp-SpCas9-ADH2t ) (Xiao et al, 2022) was adopted as the host strain for the integration of sakuranetin synthesis pathway genes. LiAc/ssDNA/PEG method was used to co-transform an equal amount of purified linearized fragments (50-100 ng/kb) with the corresponding gRNA plasmids (˜300-500 ng) into S. cerevisiae , and YPD agar plate containing 200 μg/mL G418 was used for the selection of the resulting strains.…”

“…The gene cluster of the abovementioned four enzymes under four constitutive promoters (Fig. 2A), was introduced into strain YT02 (Xiao et al, 2022) (Table 1). The resulting strain YHS01 (YT02, X3 ::PGK1p-At4CL1-HXT7t-TPI1p-PhCHS-TPI1t-ENO2p-MsCHI-PGK1t-TEF1p-OsNOMT-TEF1t ) produced 4.28 mg/L sakuranetin (Fig.…”