Figure 2 Biosynthesis of benzoate and derivatives by strains ofP. taiwanensis GRC3 ∆8∆pykA -tapattTn7 ::P14f-phdBCDE-4cl-pal with ΔbenABCD deletion for the production of benzoate (A,B) or ΔcatBCA deletion for the production of catechol (C,D) in shake flask in 50 ml MSM with 20 mM glucose (A,C) or 40 mM glycerol (B,D). Fed batch fermentations of the ΔcatB strain controlled at pH 7 for the production of cis,cis -muconate from glucose (E). Error bars represent the standard error of the mean. Shake flask cultivations were performed in triplicates, fed batch fermentations in duplicates.
P. taiwanensis is natively able to assimilate benzoate viathe intermediates catechol and cis,cis -muconate. Targeted disruption of this pathway thus allows the synthesis of these derivatives (Figure 1). Due to their potential as bio-based building blocks, the microbial production of these molecules is intensively studied [13-17]. 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 (P14f-phdBCDE-4cl-pal) was integrated.
On both carbon sources, this strain grew to an OD600 of ~3.1 (Figure 2C,D). By the end of cultivation, 0.43 ± 0.01 mM catechol were produced from glucose, and 0.67 ± 0.01 mM from glycerol. 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]. However, Pseudomonads were reported to tolerate higher amounts of catechol than produced during these experiments [19]. The concentrations produced by P. taiwanensis GRC3 Δ8ΔpykA -tap ΔcatBCA attTn7 ::P14f-phdBCDE-4cl-pal should thus not yet lead to a high impairment of cellular fitness, especially considering that this strain is more solvent-tolerant[7]. Alternatively, the low titers of catechol may be due to its instability in the presence of oxygen and water [18, 20].
The quantification of cis,cis -muconate produced by strainP. taiwanensis GRC3 Δ8ΔpykA -tap ΔcatBattTn7 ::P14f -phdBCDE-4cl-pal in shake flasks was complicated by its isomerization. During HPLC analysis of supernatants, peaks appeared broadened and split, thereby impairing reliable quantification. This effect is due to the isomerization ofcis,cis -muconate into cis,trans -muconate and subsequent lactonization, which occurs readily under acidic conditions [21]. Already a slight pH shift occurring during cultivation from initially pH 7.0 to 6.8 leads to isomerization. A five-fold increase of the medium’s buffer capacity did not reduce this effect. To avoid this,P. taiwanensis GRC3 Δ8ΔpykA -tap ΔcatBattTn7 ::P14f -phdBCDE-4cl-pal was cultivated in dO2-stat fed batch fermentations with strict pH control (>7; Figure 2E). Indeed, no isomerization was observed in the course of the fermentation and a titer of 7.2 ± 0.4 mM of cis,cis -muconate was achieved after 170 h. However, around 0.2 mM of catechol started to accumulate after 52h, followed by strong accumulation of trans -cinnamate to up to 2.9 ± 0.0 mM by the end of cultivation, indicating inhibition of the downstream pathway. ThecatBCA cluster is subject to Crc regulation [22] possibly creating a bottleneck of the CatA-catalyzed reaction once a certaincis,cis -muconate concentration is reached. Furthermore, P. taiwanensis harbors one copy of the catA gene, whileP. putida KT2440 holds a second chromosomal copy (catA2 ), offering a “safety valve” in the presence of high catechol concentrations [19, 23]. An overexpression of a modified CatA could thus not only counteract Crc regulation, but also enhance catechol conversion, thereby limiting the accumulation of this toxic intermediate.