Improvement of cis, cis-Muconic Acid Production in Saccharomyces cerevisiae through Biosensor-Aided Genome Engineering

Abstract

Muconic acid is a potential platform chemical for the production of nylon, polyurethanes, and terephthalic acid. It is also an attractive functional copolymer in plastics due to its two double bonds. At this time, no economically viable process for the production of muconic acid exists. To harness novel genetic targets for improved production of cis,cis-muconic acid (CCM) in the yeast Saccharomyces cerevisiae, we employed a CCM-biosensor coupled to GFP expression with a broad dynamic response to screen UV-mutagenesis libraries of CCM-producing yeast. Via fluorescence activated cell sorting we identified a clone Mut131 with a 49.7% higher CCM titer and 164% higher titer of biosynthetic intermediate-protocatechuic acid (PCA). Genome resequencing of the Mut131 and reverse engineering identified seven causal missense mutations of the native genes (PWP2, EST2, ATG1, DIT1, CDC15, CTS2, and MNE1) and a duplication of two CCM biosynthetic genes, encoding dehydroshikimate dehydratase and catechol 1,2-dioxygenase, which were not recognized as flux controlling before. The Mut131 strain was further rationally engineered by overexpression of the genes encoding for PCA decarboxylase and AROM protein without shikimate dehydrogenase domain (Aro1p^ΔE), and by restoring URA3 prototrophy. The resulting engineered strain produced 20.8 g/L CCM in controlled fed-batch fermentation, with a yield of 66.2 mg/g glucose and a productivity of 139 mg/L/h, representing the highest reported performance metrics in a yeast for de novo CCM production to date and the highest production of an aromatic compound in yeast. The study illustrates the benefit of biosensor-based selection and brings closer the prospect of biobased muconic acid.

Keywords: Saccharomyces cerevisiae; biosensor; muconic acid; mutagenesis; reverse engineering.

Figure 1

Biosensor-controlled GFP responds to in vivo cis,cis-muconic acid (CCM) production and enables fluorescence-based selection of improved CCM producing strains. (a) Metabolic pathway for muconic acid biosynthesis in S. cerevisiae. Native enzymes are in blue, heterologous in red. PaAroZ, DHS dehydratase from Podospora anserina; KpAroY.B, KpAroY.Ciso, subunits of functional PCA decarboxylase from Klebsiella pneumoniae; CaCatA, catechol 1,2-dioxygenase from Candida albicans; (b) CCM production and fluorescence output of BenM variant MP02_D04 biosensor response in strains that produce CCM at different levels on YPD medium; (c) flow cytometric analysis of yeast CCM-producing strains implemented with a biosensor on YPD medium. Data shown are mean values ± SDs of triplicates (b) and one representative of each strains (c). Glu, glucose; G6P, glucose 6-phosphate; GL6P, gluconolactone-6-phosphate; 6PG, gluconate-6-phosphate; Ru5P, ribulose 5-phosphate; X5P, xylulose 5-phosphate; R5P, ribose 5-phosphate; G3P, glyceraldehyde 3-phosphate; S7P, sedoheptulose 7-phosphate; E4P, erythrose 4-phosphate; F6P, fructose 6-phosphate; PEP, phosphoenolpyruvate; PYR, pyruvate; DAHP, 3-deoxyarabinoheptulosonate 7-phosphate; 3-DHQ, 3-dehydroquinic acid; 3-DHS, 3-dehydroshikimate; PCA, protocatechuic acid; CCM, cis,cis-muconic acid; AA, amino acid.

Figure 2

A S. cerevisiae mutant with improved CCM production was identified via biosensor-aided fluorescence-based selection. Ultraviolet (UV) light-treated ST8424 cells were subjected to two rounds of fluorescence activated cell sorting to select the single cells (defined with P1 gate) with the lowest (Min, defined with P2 gate) or highest (Max, defined with P3 gate) fluorescence output (a). CCM production of the sorted cell population (b) and individual cells (c) on YPD medium are evaluated, and potential improved CCM producer strains (marked as triangle) are validated on mineral medium (MM) (d), a mineral medium, and mimicked fed-batch/feed-in-time (FIT) medium (d) with uracil supplementation. All cultivations were performed in batch, and the mimicked glucose-limited fed-batch was realized via an enzymatic digestion of a polysaccharide source, therefore ensuring a continuous glucose feed. Data shown are from single replicates of mutants in panel c, and mean values ± SDs of triplicates for panel b and d and control strains in panel c.

Figure 3

Reverse engineering of identified mutations for improved CCM production. All tests were performed on 72 h culture on mineral medium without uracil (a) or supplemented with 20 mg/L uracil (b). The specific yield (mg/L/OD₆₀₀) and titer are shown. The CCM and PCA values for mutants, are all shown as fold changes in relation to the CCM values of the control strains. Data shown are mean values ± SDs of triplicate. Statistical difference between control and indicated strains (a and b), as well as that between the indicated strains (b) was determined by two-tailed Student’s t test (*p < 0.05). Gene-dis, strain carrying indicated gene(s) with a designed point mutation; pm, point mutation.

Figure 4

CCM production of rationally engineered strains (a) and CCM toxicity to S. cerevisiae strain CEN.PK113–7D (b). (a) CCM production of engineered strains on FIT medium with (ST8424, ST8918, ST8919, ST8920) or without uracil supplementation (ST8942, ST8943) for 72h. (b) Maximum specific growth rate of CEN.PK113-7D on mineral medium with CCM supplementation and initial pH unadjusted or adjusted. In tests with ≥5 g/L CCM supplementation and initial pH unadjusted, CCM is largely in the insoluble form, whereas, it is all soluble in the remainder of the tests. Data shown are mean values ± SDs of triplicates.

Figure 5

Controlled fed-batch fermentation for CCM production. Fermentations BD3 (a), BD4 (b), and BD5 (c) of ST8943 strain were performed with different feed solutions. The feed solution for BD4 contained additional salt, yeast extract, and trace metal compared to that of BD3. Feed solution for BD5 contained all components for BD4 except yeast extract. CCM titers (g/L), yields (mg/g glucose (Glu)), and productivities (mg/L/h) indicated were for 72 h (a), 72 h (b), 73 h (c), and 143 h (c), respectively. Data shown are from a single replicate.

Figure 6

Controlled fed-batch fermentation for CCM production. Fermentation BD6 of ST8943 strain was performed by feeding with 465.32 mL of 1× feed solution (200 g/L glucose, 15 g/L KH₂PO₄, 8 g/L MgSO₄, 10 g/L (NH₄)₂SO₄, 0.4 g/L CaCl₂, and trace metal) and 1399 mL of 3× feed solution into 1.3 L starting fermentation broth during 0–48 h and 48–151 h, respectively. CCM titers (g/L), yields (mg/g glucose (Glu)) and productivities (mg/L/h) indicated were for 123.5 and 149.5 h, respectively. These two time-points correspond to the highest yield/productivity and titer achieved in this run. Data shown are from a single replicate.