Materials and methods br Results br
Materials and methods
Discussion Microbial production of trans-4Hyp, an added-value amino Lesinurad for pharmaceuticals, offers significant advantages over conventional chemical extraction in economic and environmental aspects. However the low titer and productivity with the existing trans-P4Hs could not meet the requirements for efficient industrial production of trans-4Hyp 11, 12. For instance, the reported highest trans-4Hyp titer from glucose using an l-proline-producing recombinant E. coli strain overexpressing DaP4H, was only 25 g/L with a productivity of 0.26 g/L/h (11). In this study, three active trans-P4Hs (AlP4H, MiP4H and ScP4H) were identified after activity assay of ten candidate trans-P4Hs. The best strain SEcH (pTc-B74A-alp4h) produced approximately 45.83 g/L trans-4Hyp, 1.83-fold high of the titer 25 g/L reported in the literature (11) from glucose (Fig. 5). Importantly, the fermentation time of SEcH (pTc-B74A-alp4h) was dramatically shortened from 96 h to 36 h, resulting in a 4.88-fold high productivity, 1.27 g/L/h vs. 0.26 g/L/h in the literature (11). Considering the low soluble fraction of AlP4H (Figs. 2, S2 and S3), the performance of SEcH (pTc-B74A-alp4h) could be further improved by expression optimization of AlP4H, e.g., co-expression with chaperones like GroES/GroEL (17). To dismiss doubts of different expression and performance condition of AlP4H with DaP4H, the control strain SEcH (pTc-B74A-dap4h) was constructed under the same condition in this study. A 1.38-fold high titer of trans-4Hyp (45.83 g/L vs. 33.21 g/L) and 1.38-fold high trans-4Hyp productivity (1.27 g/L/h vs. 0.92 g/L/h) were obtained by the SEcH (pTc-B74A-alp4h) strain (Table 3). The higher productivity using SEcH (pTc-B74A-dap4h) in this study (0.92 g/L/h vs. 0.26 g/L/h in the literature (11)) might be caused by using a low-copy pTc plasmid vector in this study and different fermentation process. It has been reported that low-copy plasmids may perform better than high-copy plasmids for metabolic engineering of bacteria 29, 30. In this study, we surprisingly found that AlP4H displayed the lowest crude enzyme activities at 30°C (94.83 U/mg) and 33°C (60.88 U/mg) compared with other tested trans-P4Hs, even though the highest titer of trans-4Hyp (3.57 g/L at 30°C and 3.41 g/L at 33°C) were obtained (Fig. 3D and A). The highest titer of trans-4Hyp produced by AlP4H overexpression suggests its excellent enzymatic catalysis in vivo in the studied condition. The enzyme activity of AlP4H detected in vitro probably cannot reflect its real activity in vivo. The inconsistence has also been observed by Yi et al. (19). In their study, the trans-P4H from P. stutzer exhibited lower enzyme activity than the trans-P4H from B. bronchiseptica RB50 (22.2 U/mg wet cell weight vs. 50.0 U/mg wet cell weight), however, the former synthesized more trans-4Hyp than the latter (0.126 g/L vs. 0.115 g/L). The mechanism remains unclear, investigation on enzymatic property of AlP4H, such as enzymatic kinetics and stability, will be done in the future. Considering the performance of other two trans-P4Hs and the reported enzyme DaP4H, it seems that lower P4H activity did not favor the production of trans-4Hyp. In addition, the productivities of trans-4Hyp using different trans-P4Hs, including the commonly used DaP4H, were often significantly decreased at an elevated temperature of 37°C (Fig. 3), while the productivities using ScP4H kept unchanged. The result suggested that besides AlP4H, the ScP4H may also be a promising candidate for trans-4Hyp production and understanding of catalytic mechanisms. The four trans-P4Hs tested in this study were insoluble or low-level expression (Figs. 2B, S2 and S3), which could be caused by the differences in GC content, codon preference and incorrect protein folding. The GC contents of native genes (alp4h, mip4h and scp4h) are 38%, 63% and 66%, respectively, but the GC content of E. coli genome is 50%. Moreover, all three native genes contain high percent of rare codons of E. coli, suggesting codon optimization should be done in further expression improvement. Regarding to the poor soluble expression of DaP4H after codon optimization, protein folding should also be improved.