Metabolic engineering for p‐coumaryl alcohol production in Escherichia coli by introducing an artificial phenylpropanoid pathway

Abstract

The plant polymer lignin is the greatest source of aromatic chemical structures on earth. Hence, the chemically diverse lignin monomers are valuable raw materials for fine chemicals, materials synthesis, and food and flavor industries. However, extensive use of this natural resource is hampered by the large number of different lignin monomers and the complex and irregular structure of lignin, which renders current processes for its chemical or enzymatic degradation inefficient. The microbial production of lignin monomers from renewable resources represents a promising alternative to lignin degradation, which could meet the demand for aromatic chemical structures. In this study, we describe the functional introduction of an artificial phenylpropanoid pathway into Escherichia coli, achieved by transferring several genes from plants and microbes. The established chimeric pathway efficiently converts l-tyrosine into the lignin precursor molecule p-coumaryl alcohol.

Paving the Way for Lignin Valorisation: Recent Advances in Bioengineering, Biorefining and Catalysis

“…[75] Phenylalanine is first deaminated to cinnamate (by the enzyme phenylalanine ammonia-lyase,P AL), and cinnamate is then hydroxylated to p-coumarate (by cinnamate 4-hydroxylase, C4H). If tyrosine is used instead as the starting point, this twostage enzymatic process is circumvented and Ty ri sd irectly converted into p-coumarate via deamination (by tyrosine ammonia-lyase,T AL, but also by PA Lwhich is not absolutely specific for Phe).…”

“…Incorporation of Ty r, as an alternative starting point into the phenylpropanoid pathway,may explain this result. [75] 3-Hydroxylation of p-coumarate by C3H was originally thought to occur at either the acid or the CoA level, until researchers showed the presence of an ew enzyme,H CT,i n various plants that produce p-coumaroyl shikimic or quinic acid conjugates that are the preferred substrates of C3H. Following the hydroxylation, HCT was conjectured to return the product to the CoA level as caffeoyl-CoA.…”

Combinatorial optimization of synthetic operons for the microbial production of p-coumaryl alcohol with Escherichia coli

“…Thus, balancing of the synthetic pathway already more than doubled the overall product titer compared to the previously engineered E. coli strain expressing the same genes, as this strain accumulated only up to 22 mg/L p-coumaryl alcohol without precursor feeding [20]. The modularity and simplicity of the Operon-PLICing method also provides the opportunity to generate the whole operon library of 81 variants in a single reaction tube by simply combining the 12 amplified and cleaved gene fragments and the equally treated plasmid fragment prior to hybridization and transformation of E. coli.…”

Microbial production of plant specialized metabolites

“…Very recently, 4-coumaryl alcohol production was reported in E. coli (Jansen et al 2014). They incorporated Rhodobacter sphaeroides TAL, Pteroselinum crispum 4CL, Zea mays CCR and CAD into E. coli cells.…”

“…However, the level of production of resveratrol is usually lower in yeast than in E. coli (Jeandet et al 2012). For example, Beekwilder et al (2006) integrated N. tabacum 4CL and V. vinifera STS into LEU2 locus of S. cerevisiae and obtained resveratrol (6 mg L −1 ) from 4-coumaric acid.…”