Microbial production of the aromatic building‐blocks (S)‐styrene oxide and (R)‐1,2‐phenylethanediol from renewable resources

Abstract

(S)-Styrene oxide and (R)-1,2-phenylethanediol are chiral aromatic molecular building blocks used commonly as precursors to pharmaceuticals and other specialty chemicals. Two pathways have been engineered in Escherichia coli for their individual biosynthesis directly from glucose. The novel pathways each constitute extensions of the previously engineered styrene pathway, developed by co-expressing either styrene monooxygenase (SMO) or styrene dioxygenase (SDO) to convert styrene to (S)-styrene oxide and (R)-1,2-phenylethanediol, respectively. StyAB from Pseudomonas putida S12 was determined to be the most effective SMO. SDO activity was achieved using NahAaAbAcAd of Pseudomonas sp. NCIB 9816-4, a naphthalene dioxygenase with known broad substrate specificity. Production of phenylalanine, the precursor to both pathways, was systematically enhanced through a number of mutations, most notably via deletion of tyrA and over-expression of tktA. As a result, (R)-1,2-phenylethanediol reached titers as high as 1.23 g/L, and at 1.32 g/L (S)-styrene oxide titers already approach their toxicity limit. As with other aromatics, product toxicity was strongly correlated with a model of membrane accumulation and disruption. This study additionally demonstrates that greater flux through the styrene pathway can be achieved if its toxicity is addressed, as achieved in this case by reacting styrene to less toxic products.

Comparing in situ removal strategies for improving styrene bioproduction

“…In contrast, during single-phase shake flask cultures maximal styrene titers reached just 260 mg/L or 2.50 mM [14]. Of particular interest, however, is the prior observation that, in the absence of the styrene pathway and under the same culture conditions, the host strain (i.e., E. coli NST74) is only capable of producing as much as 1.1 g/L or 6.60 mM Lphenylalanine, the immediate endogenous precursor to the styrene pathway [56]. This realization implies that, at least in the case of solvent extraction by BEHP, effective styrene removal from the culture not only enables its accumulation to above would be toxic levels, but doing so also enhances flux through the L-phenylalanine biosynthesis pathway.…”

Creating pathways towards aromatic building blocks and fine chemicals

“…For example, facile, step-wise extension of existing pathways can be performed to introduce additional product functionality. The authors have explored this prospect, for example, by demonstrating how the chiral aromatic fine chemicals (S)-styrene oxide and (R)-1,2-phenylethanediol can both be synthesized by single-step extensions of the previously-engineered styrene pathway [27]. More significantly, by leveraging common intermediates, the 'plug and play' assembly of different yet complementary pathway modules allows metabolite flux to be easily redirected towards range of alternative end products.…”

“…Toxicity is a common challenge for most aromatics, and one that can limit achievable product titers [27,52,53]. It may be possible to address this issue, however, by taking cues from nature.…”

“…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].…”

Herstellung von Grundchemikalien auf Basis nachwachsender Rohstoffe als Alternative zur Petrochemie?

“…Für Styrol gelingt es trotz seiner Toxizität, mit entsprechend modifizierten E. coli Produktkonzentrationen von 260 mg L -1 zu erreichen [77]. Für Styrol gelingt es trotz seiner Toxizität, mit entsprechend modifizierten E. coli Produktkonzentrationen von 260 mg L -1 zu erreichen [77].…”