Requirement of a Functional Flavin Mononucleotide Prenyltransferase for the Activity of a Bacterial Decarboxylase in a Heterologous Muconic Acid Pathway in Saccharomyces cerevisiae

Abstract

Biotechnological production of cis,cis-muconic acid from renewable feedstocks is an environmentally sustainable alternative to conventional, petroleum-based methods. Even though a heterologous production pathway for cis,cis-muconic acid has already been established in the host organism Saccharomyces cerevisiae, the generation of industrially relevant amounts of cis,cis-muconic acid is hampered by the low activity of the bacterial protocatechuic acid (PCA) decarboxylase AroY isomeric subunit C iso (AroY-C iso ), leading to secretion of large amounts of the intermediate PCA into the medium. In the present study, we show that the activity of AroY-C iso in S. cerevisiae strongly depends on the strain background. We could demonstrate that the strain dependency is caused by the presence or absence of an intact genomic copy of PAD1, which encodes a mitochondrial enzyme responsible for the biosynthesis of a prenylated form of the cofactor flavin mononucleotide (prFMN). The inactivity of AroY-C iso in strain CEN.PK2-1 could be overcome by plasmid-borne expression of Pad1 or its bacterial homologue AroY subunit B (AroY-B). Our data reveal that the two enzymes perform the same function in decarboxylation of PCA by AroY-C iso , although coexpression of Pad1 led to higher decarboxylase activity. Conversely, AroY-B can replace Pad1 in its function in decarboxylation of phenylacrylic acids by ferulic acid decarboxylase Fdc1. Targeting of the majority of AroY-B to mitochondria by fusion to a heterologous mitochondrial targeting signal did not improve decarboxylase activity of AroY-C iso , suggesting that mitochondrial localization has no major impact on cofactor biosynthesis. IMPORTANCEIn Saccharomyces cerevisiae, the decarboxylation of protocatechuic acid (PCA) to catechol is the bottleneck reaction in the heterologous biosynthetic pathway for production of cis,cis-muconic acid, a valuable precursor for the production of bulk chemicals. In our work, we demonstrate the importance of the strain background for the activity of a bacterial PCA decarboxylase in S. cerevisiae. Inactivity of the decarboxylase is due to a nonsense mutation in a gene encoding a mitochondrial enzyme involved in the biosynthesis of a cofactor required for decarboxylase function. Our study reveals functional interchangeability of Pad1 and a bacterial homologue, irrespective of their intracellular localization. Our results open up new possibilities to improve muconic acid production by engineering cofactor supply. Furthermore, the results have important implications for the choice of the production strain.KEYWORDS biotechnology, muconic acid, phenylacrylic acid decarboxylase, prenylated flavin mononucleotide, protocatechuic acid decarboxylase, Saccharomyces cerevisiae, styrene

An Engineered Aro1 Protein Degradation Approach for Increased cis,cis -Muconic Acid Biosynthesis in Saccharomyces cerevisiae

“…Because PCA decarboxylase enzymes require the prenylated form of flavin mononucleotide (prFMN) cofactor for functionality, we postulated that an insufficient supply of this cofactor constrains AroY activity in yeast. Moreover, recent studies (6,24) have shown that CEN.PK family strains of S. cerevisiae do not produce functional Pad1, which is responsible for activation of the endogenous yeast Fdc1 decarboxylase, as well as some bacterial decarboxylases, including AroY (24). We hypothesized that introducing Pad1 from S. cerevisiae strain S288C into our engineered CEN.PK strains would activate AroY, thus triggering conversion of PCA to catechol and MA.…”

“…Here we targeted the shikimate precursor DHS to serve as the basis for building a synthetic MA production strain. Engineered bioproduction of MA is not without precedent, as many recent reports have detailed the successful reconstitution of heterologous MA pathways in S. cerevisiae (4,8,(12)(13)(14)(15)(16)24). A major caveat of many of these approaches involves the generation of aromatic amino acid auxotrophy resulting from deleterious manipulation of the pentafunctional Aro1 protein.…”

Building microbial factories for the production of aromatic amino acid pathway derivatives: From commodity chemicals to plant-sourced natural products

“…In all these engineered yeast strains, the PCA decarboxylase AroY seems to be the bottleneck step, causing significant accumulation of PCA and thus, low MA titers. Some studies have suggested that this is due to the O 2 sensitivity of this enzyme (Suastegui et al, 2016b;Weber et al, 2012), while others believe that its functionality requires the prenylated form of flavin mononucleotide as the cofactor, which is insufficient or even missing in some strains, such as the CEN.PK family of S. cerevisiae (Weber et al, 2017). Supplying the strain with an overexpressed flavin mononucleotide prenyltransferase (Pad1) addresses this bottleneck.…”

Bacterial bifunctional chorismate mutase-prephenate dehydratase PheA increases flux into the yeast phenylalanine pathway and improves mandelic acid production

“…We constructed plasmids from which ARO7 fbr , PHA2 and hmaS from N. uniformis were expressed – either with or without a mitochondrial or a peroxisomal targeting sequence, MTS or ePTS1 , respectively (for codon-optimized sequences see Supplementary table S4 ). We chose the mitochondrial targeting sequence of N. crassa ATP synthase subunit 9, which was previously shown to efficiently localize proteins to the mitochondria of S. cerevisiae ( Benisch and Boles, 2014 , Weber et al, 2017 , Westermann and Neupert, 2000 ). For peroxisomal localization we chose the enhanced peroxisomal targeting sequence ePTS1 that was identified by DeLoache et al (2016) and shown to cause efficient protein relocation into peroxisomes.…”