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).