Expanding Upon Styrene Biosynthesis to Engineer a Novel Route to 2‐Phenylethanol

Abstract

2-Phenylethanol (2PE) is a key molecule used in the fragrance and food industries, as well as a potential biofuel. In contrast to its extraction from plant biomass and/or more common chemical synthesis, microbial 2PE production has been demonstrated via both native and heterologous expression of the yeast Ehrlich pathway. Here, a novel alternative to this established pathway is systematically engineered in Escherichia coli and evaluated as a more robust and efficient route. This novel pathway is constructed via the modular extension of a previously engineered styrene biosynthesis pathway, proceeding from endogenous l-phenylalanine in five steps and involving four heterologous enzymes. This "styrene-derived" pathway boasts nearly a 10-fold greater thermodynamic driving force than the Ehrlich pathway, and enables reduced accumulation of acetate byproduct. When directly compared using a host strain engineered for l-phenylalanine over-production, preservation of phosphoenolpyruvate, and reduced formation of byproduct 2-phenylacetic acid, final 2PE titers via the styrene-derived and Ehrlich pathways reached 1817 and 1164 mg L , respectively, at yields of 60.6 and 38.8 mg g . Following optimization of induction timing and initial glucose loading, 2PE titers by the styrene-derived pathway approached 2 g L - nearly a two-fold twofold increase over prior reports for 2PE production by E. coli employing the Ehrlich pathway.

Metabolic Engineering of Escherichia coli for para-Amino-Phenylethanol and para-Amino-Phenylacetic Acid Biosynthesis

“…In our study, a combination of the pabAB fusion gene from C. glutamicum and of the codon-optimized genes of papB and papC from S. venezuelae was used to allow a conversion of chorismate to APP (Mohammadi Nargesi et al, 2018). We combined the APP biosynthesis pathway with the yeast Ehrlich pathway in E. coli by recruiting Aro10 to enable PAPE and 4-APA production (Figure 1) (Vuralhan et al, 2003, 2005; Vogt and Gerulis, 2005; Atsumi et al, 2008b; Kneen et al, 2011; Machas et al, 2017; Mohammadi Nargesi et al, 2018). Already the wild type strain E. coli LJ110 harboring pC53BCA or pC53BCAF was able to produce 11 ± 1.5 mg l −1 PAPE or 36 ± 5 mg l −1 4-APA; the correct mass of these products was confirmed by mass spectrometry (Figure S2).…”

Recent Advances in Metabolically Engineered Microorganisms for the Production of Aromatic Chemicals Derived From Aromatic Amino Acids

“…The co-overexpression of the four genes kdc, adh1, pheA fbr , and aroF improved 2PE production to 285 mg/L from glucose in shake flask cultures. A novel route (styrene-derived pathway) has been established for 2PE production in E. coli (Machas et al, 2017). The styrene-derived pathway comprised PAL2 from Arabidopsis thaliana, FDC1 from S. cerevisiae, and styABC from Pseudomonas putida S12.…”

Emerging tools, enabling technologies, and future opportunities for the bioproduction of aromatic chemicals

“…Microbial production of aromatic chemicals has largely been enabled via pathway engineering, generally consisting of either: (i) the functional reconstruction of naturally-occurring but non-native (often plant) pathways; or (ii) the bottom-up construction of novel pathways comprised of individual enzymes derived from a diversity of heterologous sources. Recent examples include the successful engineering of microbes capable of the de novo production of, in the first case, flavonoids (usually consisting of two phenyl groups and a heterocyclic ring), 10,11 stilbenes (ethylene moiety with two phenyl groups), 12,13 and coumarins (containing a 1,2-benzopyrone backbone), 14,15 and, in the second case, numerous aromatic aldehydes, alcohols, and acids, [16][17][18][19][20][21] styrenics, [22][23][24][25] and phenolics. [26][27][28][29][30][31][32][33] In most cases, these heterologous pathways stem from natively produced aromatic chemicals such as the aromatic amino acids (i.e.…”