Rational and combinatorial approaches to engineering styrene production by Saccharomyces cerevisiae

Abstract

BackgroundStyrene is an important building-block petrochemical and monomer used to produce numerous plastics. Whereas styrene bioproduction by Escherichia coli was previously reported, the long-term potential of this approach will ultimately rely on the use of hosts with improved industrial phenotypes, such as the yeast Saccharomyces cerevisiae.ResultsClassical metabolic evolution was first applied to isolate a mutant capable of phenylalanine over-production to 357 mg/L. Transcription analysis revealed up-regulation of several phenylalanine biosynthesis pathway genes including ARO3, encoding the bottleneck enzyme DAHP synthase. To catalyze the first pathway step, phenylalanine ammonia lyase encoded by PAL2 from A. thaliana was constitutively expressed from a high copy plasmid. The final pathway step, phenylacrylate decarboxylase, was catalyzed by the native FDC1. Expression of FDC1 was naturally induced by trans-cinnamate, the pathway intermediate and its substrate, at levels sufficient for ensuring flux through the pathway. Deletion of ARO10 to eliminate the competing Ehrlich pathway and expression of a feedback-resistant DAHP synthase encoded by ARO4K229L preserved and promoted the endogenous availability precursor phenylalanine, leading to improved pathway flux and styrene production. These systematic improvements allowed styrene titers to ultimately reach 29 mg/L at a glucose yield of 1.44 mg/g, a 60% improvement over the initial strain.ConclusionsThe potential of S. cerevisiae as a host for renewable styrene production has been demonstrated. Significant strain improvements, however, will ultimately be needed to achieve economical production levels.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0123-2) contains supplementary material, which is available to authorized users.

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

“…In natural environments, Pad1 provides prFMN to allow for Fdc1 function in detoxification of phenylacrylic acids present in plant cell walls, such as ferulic, coumaric, or cinnamic acid, to more volatile compounds (15). In biotechnology, this reaction is relevant for decarboxylation of cinnamic acid to styrene, a building block required in plastics production (21,22). To test whether AroY-B can replace Pad1 in its function in providing the cofactor required for decarboxylase activity of Fdc1, we analyzed the impact of the coexpression of both enzymes on the detoxification of trans-cinnamic acid.…”

Structure and Mechanism of Ferulic Acid Decarboxylase (FDC1) from Saccharomyces cerevisiae

“…Related aromatic compounds are also natural components in wine and other fermented beverages and food (6)(7)(8). Similarly, other production routes for catechol and styrene, using microbes that metabolize benzenoid molecules by nonoxidative decarboxylation, have also been explored recently (8)(9)(10). For example, overexpression of phenylalanine ammonia lyase from Arabidopsis thaliana and of ferulic acid decarboxylase (FDC) from Saccharomyces cerevisiae in engineered Escherichia coli led to a strain that produced styrene (9,10).…”

Creating pathways towards aromatic building blocks and fine chemicals

“…Meanwhile, with conserved 4-hydroxyl functionality, phenolic products have typically been derived from the Tyr branch, including phenol [16], p-hydroxybenzoate [17 ], coumaric acid [18], caffeic acid [19,20], ferulic acid [20], p-hydroxystyrene [21], tyrosol [22], and hydroxytyrosol [23]. Stemming from the Phe branch, pathways have been engineered to 2-phenylethanol [24,25 ], cinnamic acid [18], styrene [14,26], (S)-styrene oxide [27], (R)-1,2-phenylethanediol [27], (S)-mandelic and (R)-mandelic acid [28], and benzaldehyde [29 ]. Although comparatively fewer routes have to date been constructed from the Trp branch, notable examples include indigo dye [30] and catechol (though subsequently converted to muconic acid) [31].…”

“…production of any aromatic target first requires metabolite flux to be enhanced through this otherwise tightly regulated pathway: a prerequisite condition that has been met in numerous microbial platforms, typically as a result of both combinatorial (e.g., metabolic evolution) and rational approaches [13 , 14,15 ]. A key and relatively unique factor enabling access to a broad range of established and putative final aromatic products is in fact the diverse pool of uniquely functionalized metabolites that can serve as pathway precursors (Figure 1) -namely those between 3-dehydroshikimate (3-DHS) and the terminal aromatic amino acid (AAA) products (i.e., tyrosine (Tyr), phenylalanine (Phe), and tryptophan (Trp)).…”