Rational Engineering of Phenylalanine Accumulation in Pseudomonas taiwanensis to Enable High-Yield Production of Trans-Cinnamate

Abstract

Microbial biocatalysis represents a promising alternative for the production of a variety of aromatic chemicals, where microorganisms are engineered to convert a renewable feedstock under mild production conditions into a valuable chemical building block. This study describes the rational engineering of the solvent-tolerant bacterium Pseudomonas taiwanensis VLB120 toward accumulation of L-phenylalanine and its conversion into the chemical building block t-cinnamate. We recently reported rational engineering of Pseudomonas toward L-tyrosine accumulation by the insertion of genetic modifications that allow both enhanced flux and prevent aromatics degradation. Building on this knowledge, three genes encoding for enzymes involved in the degradation of L-phenylalanine were deleted to allow accumulation of 2.6 mM of L-phenylalanine from 20 mM glucose. The amino acid was subsequently converted into the aromatic model compound t-cinnamate by the expression of a phenylalanine ammonia-lyase (PAL) from Arabidopsis thaliana. The engineered strains produced t-cinnamate with yields of 23 and 39% Cmol Cmol−1 from glucose and glycerol, respectively. Yields were improved up to 48% Cmol Cmol−1 from glycerol when two enzymes involved in the shikimate pathway were additionally overexpressed, however with negative impact on strain performance and reproducibility. Production titers were increased in fed-batch fermentations, in which 33.5 mM t-cinnamate were produced solely from glycerol, in a mineral medium without additional complex supplements. The aspect of product toxicity was targeted by the utilization of a streamlined, genome-reduced strain, which improves upon the already high tolerance of P. taiwanensis VLB120 toward t-cinnamate.

Benzoate synthesis from glucose or glycerol using engineered Pseudomonas taiwanensis

“…To prevent the degradation of benzoate by the engineered Pseudomonasstrain, the benABCD operon (PVLB 12215-12230) responsible for the conversion of benzoate to catechol (Figure 1) was deleted in a previously described phenylalanine-producing chassis [8] to yieldP. taiwanensis GRC3 Δ8ΔpykA -tap ΔbenABCD .…”

“…This corresponds to yields of 2.2 ± 0.04 and 3.3 ± 0.04% (Cmol Cmol -1 ), respectively. These catechol titers and yields are relatively low compared to those of trans -cinnamate [8] and benzoate (Figure 2A,B). One possible explanation might be the greater toxicity of catechol related to the formation of reactive oxygen species and protein damage [18].…”

“…Pseudomonas taiwanensis is a promising microbial cell factory, especially regarding the production of aromatics. This has been recently demonstrated by our group in multiple studies for de novosynthesis of phenol, 4-hydroxybenzoate, and trans -cinnamate [5][6][7][8]. In this study, a previously generated Pseudomonas taiwanensis trans -cinnamate overproducer was further engineered to enable benzoate production from renewable resources in a mineral medium without supplementation of complex substances or antibiotics.…”

“…Batch production experiments were performed in 500 mL Erlenmeyer flasks with a culture volume of 50 mL, cultivated in a rotary shaker with a frequency of 200 rpm and a throw of 50 mm. Fed batch fermentations were performed in DASbox(r) mini-bioreactors (Eppendorf) according to the setup and procedure described in Otto et al [8].…”

“…The pathway via l-phenylalanine described in this study is a novel strategy that adds a new aspect to this highly active field of research. To allow catechol orcis,cis -muconate accumulation, the genes catBCA(PVLB -12240-50) or catB (PVLB 12240) ( Figure 1) were deleted in the l-phenylalanine-overproducing Pseudomonas chassis [8], and the benzoate biosynthesis module (P 14f -phdBCDE-4cl-pal) was integrated.…”