Production of Cinnamic and p-Hydroxycinnamic Acids in Engineered Microbes

Abstract

The aromatic compounds cinnamic and p-hydroxycinnamic acids (pHCAs) are phenylpropanoids having applications as precursors for the synthesis of thermoplastics, flavoring, cosmetic, and health products. These two aromatic acids can be obtained by chemical synthesis or extraction from plant tissues. However, both manufacturing processes have shortcomings, such as the generation of toxic subproducts or a low concentration in plant material. Alternative production methods are being developed to enable the biotechnological production of cinnamic and (pHCAs) by genetically engineering various microbial hosts, including Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, and Streptomyces lividans. The natural capacity to synthesize these aromatic acids is not existent in these microbial species. Therefore, genetic modification have been performed that include the heterologous expression of genes encoding phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities, which catalyze the conversion of l-phenylalanine (l-Phe) and l-tyrosine (l-Tyr) to cinnamic acid and (pHCA), respectively. Additional host modifications include the metabolic engineering to increase carbon flow from central metabolism to the l-Phe or l-Tyr biosynthetic pathways. These strategies include the expression of feedback insensitive mutant versions of enzymes from the aromatic pathways, as well as genetic modifications to central carbon metabolism to increase biosynthetic availability of precursors phosphoenolpyruvate and erythrose-4-phosphate. These efforts have been complemented with strain optimization for the utilization of raw material, including various simple carbon sources, as well as sugar polymers and sugar mixtures derived from plant biomass. A systems biology approach to production strains characterization has been limited so far and should yield important data for future strain improvement.

Pseudomonas putida as a platform for the synthesis of aromatic compounds

“…As previously demonstrated with the production of transcinnamic acid and p-hydroxycinnamic acid in P. putida S12 (Nijkamp et al, 2005(Nijkamp et al, , 2007Vargas-Tah & Gosset, 2015;Weber et al, 1993), chemical mutagenesis in the presence of high m-fluoro-phenylalanine concentrations, a toxic analogue to L-phenylalanine, led to the isolation of mutants able to accumulate higher amounts of L-phenylalanine and, in consequence, to produce higher concentrations of this amino acid. A combination of different strategies such as selection rounds in PFP and the use of EMS chemical mutagenesis allowed us to obtain P. putida CM12, a strain able to accumulate up to 250 mg l À1 of L-phenylalanine.…”

“…Although optimization of strains for L-phenylalanine production by selection in PFP and elimination of enzyme feedback repression have been performed previously (Dueñas-S anchez et al, 2014;Liu et al, 2014), to our knowledge, this is the first time that these optimizations were combined with the deletion of several genes involved in L-phenylalanine degradation. In future, further strain optimization will be focused on increasing central metabolism to enhance PEP and E4P levels (Vargas-Tah & Gosset, 2015). It is also interesting to note that an excess of glucose in the medium can cause growth inhibition in the chassis strain and therefore increasing the rate of glucose metabolism can relieve this inhibition and improve L-phenylalanine production.…”

Phenylalanine and Tyrosine as Exogenous Precursors of Wheat (Triticum aestivum L.) Secondary Metabolism through PAL-Associated Pathways

“…Since tyrosine was directly converted to PCA in TAL-mediated reactions and directly supplied the substrate for 4-coumarate: CoA ligase, the expression level of 4CL1 was higher in plants exposed for 4 h on tyrosine than in plants cultivated on phenylalanine. It could be assumed that TCA could act as a substrate for a number of substances, and not all of its pool goes to the synthesis of PCA [38]. P-coumarate 3-hydroxylase encoded by the C3H gene catalyzes the direct 3-hydroxylation of 4-coumarate to caffeate in lignin biosynthesis.…”

The Oleaginous Red YeastRhodotorula/Rhodosporidium: A Factory for Industrial Bioproducts

“…L-Phe, being an essential amino acid, is used in food formulations, feed for livestock, dietary supplements, and nutraceuticals [29]. L-PM is a precursor in the production of the sweetener aspartame [28], and p-HCA has utility in the cosmetic, health, and flavoring industries [30].…”