3.1. Construction of whole-cell systems for l-PA
production and selection of expression strain and vector
To construct the l-PA production system, pipA fromStreptomyces pristinaespiralis encoding LCD was introduced into
the MCS-1 site of each duet vector. pCDF duet-1, pRSF duet-1, pACYC
duet-1, and pET duet-1 were used as expression vectors. To identify the
most effective expression vector, the E. coli BL21 (DE3) protein
expression strain was transformed with these recombinant vectors.
Expression of pipA was induced, and the whole-cell reaction was
performed directly. In the whole-cell reaction, 200 mM l-lysine
was used as substrate, and the reaction proceeded at 37 °C for 1 day.
Only the strains containing the pCDF duet-1 and pET duet-1 vector
produced l-PA at 50 mM and 11 mM, respectively (Fig. 2A).
l-PA was not produced from the other strains. pCDF duet-1 was
more efficient than pET duet-1, with about 3.3-fold higher productivity.
Thus, the pCDF vector was deemed the most suitable for l-PA
production. Using this expression vector, other DE3 expression strains
were tested. As the control strain, BL21 was compared to BW25113 and
BL21 Star (DE3). As shown in Figure 2B, production in the Star strain
was 1.5-fold higher than the BL21 control strain. The BW25113 strain
also produced l-PA; however, conversion was lower than the
other strains. As a result, the Star strain containing the recombinant
pCDF duet-1 (yhPA001) was used for further experiments.