Rebekah McKenna

Styrene Biosynthesis from Glucose by Engineered E. coli

Styrene is a large volume, commodity petrochemical with diverse commercial applications, including as a monomer building-block for the synthesis of many useful polymers. Here we demonstrate how, through the de novo design and development of a novel metabolic pathway, styrene can alternatively be synthesized from renewable substrates such as glucose. The conversion of endogenously synthesized l-phenylalanine to styrene was achieved by the co-expression of phenylalanine ammonia lyase and trans-cinnamate decarboxylase. Candidate isoenzymes for each step were screened from bacterial, yeast, and plant genetic sources. Finally, over-expression of PAL2 from Arabidopsis thaliana and FDC1 from Saccharomyces cerevisiae (originally classified as ferulate decarboxylase) in an l-phenylalanine over-producing Escherichia coli host led to the accumulation of up to 260 mg/L in shake flask cultures. Achievable titers already approach the styrene toxicity threshold (determined as ~300 mg/L). To the best of our knowledge, this is the first report of microbial styrene production from sustainable feedstocks.

Engineering microbial chemical factories to produce renewable “biomonomers”

By applying metabolic engineering tools and strategies to engineer synthetic enzyme pathways, the number and diversity of commodity and specialty chemicals that can be derived directly from renewable feedstocks is rapidly and continually expanding. This of course includes a number of monomer building-block chemicals that can be used to produce replacements to many conventional plastic materials. This review aims to highlight numerous recent and important advancements in the microbial production of these so-called “biomonomers.” Relative to naturally-occurring renewable bioplastics, biomonomers offer several important advantages, including improved control over the final polymer structure and purity, the ability to synthesize non-natural copolymers, and allowing products to be excreted from cells which ultimately streamlines downstream recovery and purification. To highlight these features, a handful of biomonomers have been selected as illustrative examples of recent works, including polyamide monomers, styrenic vinyls, hydroxyacids, and diols. Where appropriate, examples of their industrial penetration to date and end-product uses are also highlighted. Novel biomonomers such as these are ultimately paving the way toward new classes of renewable bioplastics that possess a broader diversity of properties than ever before possible.

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

(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.

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

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−1, respectively, at yields of 60.6 and 38.8 mg g−1. Following optimization of induction timing and initial glucose loading, 2PE titers by the styrene‐derived pathway approached 2 g L−1 – nearly a two‐fold twofold increase over prior reports for 2PE production by E. coli employing the Ehrlich pathway.