1. Introduction
l-pipecolic acid (l-PA), a non-proteinogenic compound,
is a precursor for ropivacaine, bupivacaine, and chloroprocaine, which
are widely used in local anesthetics. l-PA is also a precursor
for many macrolide-pipecolate natural products including rapamycin,
tacrolimus (FK-506), and ascomycin (FK-520), which are indispensable
immunosuppressors in the clinic. Furthermore, l-PA is a
precursor for the anticancer agent VX-710, piperidine alkaloids, and
peptide antibiotics (Cheng et al., 2018;
Gatto, Boyne, Kelleher, & Walsh, 2006;
Y. H. Kim et al., 2014;
Muramatsu et al., 2006;
Pérez-García, Max Risse, Friehs, &
Wendisch, 2017; Tani et al., 2015;
Ying et al., 2017). l-PA is a
key chiral intermediate in the synthesis of many important drugs as
mentioned above.
Chirality, which is naturally determined in organisms, plays a key role
in the pharmaceutical, agricultural, and chemical industries. Especially
in the pharmaceutical industry, production and preparation of single
enantiomers from chiral intermediates are important, since chiral drugs
account for more than half of the drugs produced
(Ying et al., 2017). Similar to the
preparation of many other enantiomeric organic compounds, a pure
enantiomer of l-PA has been prepared by stereoselective
transformation, biosynthesis, and chemical procedures, including
resolution of racemates and diastereoselective and enantioselective
syntheses (Fujii, Aritoku, Agematu, &
TSUNEKAWA, 2002; Tani et al., 2015;
Ying et al., 2015). However, the chemical
procedures for commercial-scale preparation of l-PA are
complex, tedious, expensive, low-yielding, and environmentally
unfriendly (Cheng et al., 2018;
Ying et al., 2017;
Ying et al., 2015). Compared to chemical
methods, biosynthesis of drug-related chiral intermediates is a more
environmentally friendly approach for the preparation of affordable
pharmaceuticals with higher efficiency
(Ying et al., 2017). Since the biological
activity of these compounds is dependent on the stereochemistry, there
is an increasing demand for an efficient and applicable method to
synthesize enantiomerically pure l-PA.
Previous studies have shown two basic routes from l-lysine to
l-PA: the d-1-piperidine-2-carboxylic acid (P2C)
pathway and the d-1-piperidine-6-carboxylic acid (P6C) pathway
(Fujii, Mukaihara, Agematu, & Tsunekawa,
2002; He, 2006;
Ying et al., 2017). In the P2C pathway,
the α-amino group of l-lysine is lost, and the ε-nitrogen is
incorporated into l-PA. In the P6C pathway, the ε-nitrogen is
lost, and the α-nitrogen is incorporated into l-PA
(Ying et al., 2017). Typically, five
major steps are involved in these two pathways (Fig. 1). The first
requires apoptosis-inducing protein (rAIP) and P2C reductase (DpkA)
(Cheng et al., 2018;
Muramatsu et al., 2006;
Tani et al., 2015). The second belonged
to P2C pathway involves the enzymes aminotransferase AGD2-like defense
response protein 1 (ALD1) and systemic acquired resistance- deficient 4
(SARD4) (Ding et al., 2016;
Hartmann et al., 2017;
Xu et al., 2018). The third one requires
lysine cyclodeaminase (LCD), and this enzyme is the focus of this study.
LCD alone can directly catalyze the conversion of l-lysine to
l-PA through the P6C pathway and has advantages, including
being more suitable for complicated upstream processes because of its
simplicity (Cheng et al., 2018;
Gatto et al., 2006;
Tsotsou & Barbirato, 2007;
Ying et al., 2017;
Ying et al., 2018;
Ying et al., 2015). The fourth one
involves l-lysine 6-aminotransferase (LAT) fromFlavobacterium lutescens (IFO 3084 strain) and
pyrroline-5-carboxylate (P5C) reductase encoded by proC gene ofE. coli through the P6C pathway
(Fujii, Aritoku, et al., 2002;
Fujii, Mukaihara, et al., 2002). Finally,
another route in the P6C pathway using P5C reductase requires lysine
dehydrogenase (LysDH) from Silicibacter pomeroyi . In this route,
LysDH converts l-lysine to α-aminoadipic semialdehyde. Then,
α-aminoadipic semialdehyde is spontaneously transformed to P6C by
cyclization with dehydration (Pérez-García
et al., 2017; Pérez-García,
Peters-Wendisch, & Wendisch, 2016).
Up to now, two of the five enzymes, rAIP/DpkA and LCD, have been used in
high production systems (Table 1). Tani et al. (2015) used purified
rAIP/DpkA enzymes to produce 45.1 g/L of pipecolic acid with racemic
catalysis. With the same enzymes, Cheng et al. (2018) applied the
fed-batch fermentation system using recombinant cadaverine decarboxylase
(cadA)-knockout strains containing lysine permease (LysP) and glucose
dehydrogenase (GDH) and reported a titer of 46.7 g/L pipecolic acid from
l-lysine HCl solution. Ying et al. (2015) produced 17.25 g/L of
pipecolic acid using E. coli with LCD as the whole-cell
biocatalyst and demonstrated that it has sustained enzymatic activity
and self-regenerates NAD+ by cyclization, offering a
better alternative to the two-step enzymatic process with rAIP and DpkA.
To overcome substrate inhibition, they manipulated the enzyme structure
related with the substrate and product delivery tunnels via saturation
mutagenesis, resulting in 73.4 g/L of pipecolic acid using whole-cell
reaction (Ying et al., 2019).
In this study, we selected LCD because of its advantages for
l-PA production and used a reinforced whole-cell system by
introducing multiple copies of pipA to improve overall conversion
without any complex manipulation of LCD itself. After optimization of
the reinforced whole-cell reaction, we report the highest titer (93.5
g/L) to date, increased 5.4-fold over using the same LCD reported
previously. A major factor improving l-PA production in the
whole-cell system was fine-tuning of gene copy numbers.