3. Results and Discussion
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 .
Subsequently, the synthetic operon encoding the pathway from
l-phenylalanine to benzoate was integrated at theattTn7 -site under the control of the constitutive promoterP14f [11]. The phenylalanine ammonia-lyase
(PAL) deaminates l-phenylalanine to trans -cinnamate that
is subsequently CoA-activated by the 4‑coumarate CoA-ligase (4CL)
[10]. The resulting trans ‑cinnamoyl-CoA is converted into
benzoate by the enzymes encoded by the phd cluster [12]. The
Phd pathway from C. glutamicum was a key enabling factor for
benzoate production because it accepts non-hydroxylated cinnamoyl-CoA as
a substrate, unlike the ferulic acid pathway from P. putida,which only converts hydroxylated phenylpropanoids [5]. Shake flask
cultivations were performed to characterize benzoate production from
glucose and glycerol (Figure 2A,B).
The strain reached a final OD600 of ~3
while producing 1.9 ± 0.0 or 3.0 ± 0.0 mM benzoate from 20 mM glucose or
40 mM glycerol, respectively. Assuming complete carbon utilization, this
corresponds to yields of 10.8 ± 0.1 on glucose and 17.3 ± 0.1 % (Cmol
Cmol−1) on glycerol. In the course of the cultivation,
no accumulation of trans -cinnamate was observed, confirming the
efficient operation of the Phd pathway. To the best of our knowledge,
this is the first approach demonstrating de novo benzoate
biosynthesis applying a synthetic pathway. Moreover, high titers and
yields were achieved for a microbial benzoate production process with a
minimal medium, thereby producing benzoate solely from glucose or
glycerol.