3.2. Effect of tandem gene repeats on bioconversion of
l-PA
In a previous study, LCD in the whole-cell system produced 133 mM
l-PA from l-lysine (Ying
et al., 2015). Combination of expression vectors or reinforced whole
cells, which contain another plasmid with same gene or a duplicate copy
of the gene in the same plasmid, improves rapid production
(Y. G. Hong et al., 2019;
J. Kim et al., 2017). Thus, we combined
the effective expression vectors and performed additional gene
manipulation (Fig. 3). We constructed the Star strain harboring
recombinant pET duet-1::pipA (yhPA002) and the Star strain
harboring pCDF duet-1::pipA and pET duet-1::pipA(yhPA003). Comparing these strains, the yhPA001 (pCDF
duet-1::pipA ) strain had the highest conversion. Unexpectedly,
yhPA003 (pET duet-1::pipA + pCDF duet-1::pipA ), which
carries an extra vector, was less productive than yhPA001. To prepare
other combinations for the reinforced system, we inserted pipAinto the MCS-2 site of each recombinant vector. A reinforced gene
denotes that two or more copies were introduced into an expression
vector for overexpression (Y. G. Hong et
al., 2019), and this reinforced genetic material was used for the
transformation of expression strains which were named yhPA004 (pCDF
duet-1::pipA ::pipA ), yhPA005 (pET
duet-1::pipA ::pipA ), and yhPA006 (pET
duet-1::pipA ::pipA + pCDF
duet-1::pipA ::pipA ). The yhPA004 strain had the highest
production among all engineered strains. The yhPA006 strain containing
two reinforced two vectors had a lower conversion than the yhPA004 with
single vector. To determine whether chaperones could improve production,
we prepared whole cells with different chaperones and conducted the
whole-cell reaction (Supplementary Fig. 1A). The yhPA004 strain was used
as the control, and other strains were transformed with chaperone
vectors, including pGro7, pTF16, and pKJE7
(Nishihara, Kanemori, Kitagawa, Yanagi, &
Yura, 1998; Tian, Chen, Yu, & Shen,
2016). However, the activity of the control enzyme was higher than the
strains expressing the chaperones. We also tested whether lysine
permease improved lysine permeability. lysP was introduced and
expressed in the intact expression vector (pET duet-1) to avoid
overlapping vector use (Li et al., 2016;
Steffes, Ellis, Wu, & Rosen, 1992).
However, additional expression did not improve production (Supplementary
Fig. 1B).