3.1 GDH overproduction triggers autolysis
GDH is an important industrial enzyme, but high level production of GDH in BL21 (DE3) still presents a great technical challenge. In this study, the GDH activity suddenly decreased from 217 U / mL at 31h to 37.5 U / mL at 43h (Fig. 1A). Therefore, the fermentation time must be shortened, making it impossible to continue fermentation to obtain higher yields. Moreover, the OD in the later fermentation stage also showed a decline, indicating that autolysis may have caused cell death and a release of GDH into the extracellular medium. As shown in Fig. 1B, the SDS-PAGE showed a reduction of the intracellular heterologous protein, while there was a lot of the target protein in the extracellular medium. In order to investigate if autolysis was caused by the enzyme activity of GDH, the loss-of–function mutant GDHTyr253Cyswas constructed (Makino et al., 1989), and the enzyme activity was decreased from 214.3 U / mL to 23.1 U / mL. However, the SDS-PAGE results showed that the mutant GDHTyr253Cys still caused autolysis (Fig. 1C), which suggested that the autolysis was not related to enzyme activity. In addition, a mutant plasmid without RBS site was constructed to investigate if autolysis was caused by transcription rather than translation. The results showed that the cell growth was inhibited even after removing RBS site (Fig. S1), but no autolysis observed, which suggested that the intense burst of mRNA synthesis by T7 RNAP also had some toxic effects on E. coli . Furthermore, the membrane structure of E. coli cells during lysis due to GDH overexpression was visually inspected by means of scanning electron microscopy (SEM). The images showed extensive membrane elongation and shrinking (Fig. 1D), which confirmed the phenomenon of autolysis. Importantly, when amide hydrolase, Cephalosporin C acylase, formate dehydrogenase, R-carbonyl reductase, L-lactate dehydrogenase, L-2-hydroxyisocaproate dehydrogenase, L-phenylalanine dehydrogenase, secondary alcohol dehydrogenase and other 10 enzymes were individually overexpressed each also induced autolysis of BL21 (DE3). Therefore, solving the problem of autolysis in this workhorse strain is of great importance for industrial production.
3.2GDH expression resulted in programmed cell death
The combination of deceased OD and concomitant phenotypic changes provided convincing evidence that autolysis induced by GDH overexpression may be caused by triggering of a programmed cell death (PCD) pathway. The three major PCD pathways in bacteria are apoptotic-like death, mazEF-mediated death, and the cidA/lrgA holin-antiholin system. The RecA meditated apoptosis-like death pathway is thought to be activated by the SOS response (Lee et al., 2019b). The cidA/lrgA loci are regulators of the holin-antiholin system in Staphylococcus aureus , and the homologous locus of yohJK locus of E.coli was considered to have the same effect in E. coli (Dewachter et al., 2015). As shown in Fig. 2A, mRNA expression analysis showed that the expression of recA, mazEF, and yohJK at 43h was increased by 2.5-3.6 folds over their respective expression at 24h, which implied that autolysis could be triggered by PCD. To inhibit PCD mediated by known pathways, corresponding single and multiple deletion mutants were constructed. As shown in Fig. 2B, the △recA strain can exhibited reduction of the extracellular fraction of GDH enzyme activity from 90 to 65%, but it did not markedly inhibit the autolysis. Similarly, deletion mazEF and yohJK did not alter the cell viability under GDH overexpression. Even the combined deletion of △recA- mazEF-and yohJK did not alleviate the PCD, suggesting that PCD induced by GDH overproduction was not triggered by the reported three pathways.
3.3 Development of the BL21 (DE3-lac1G) strain that is resistant toPCD
The autolysis of DE3 strains may be controlled by various fermentation strategies, but obtaining a strong expression host that is intrinsically resistant to PCD is more meaningful and convenient for industrialization. As a common solution, C41 (DE3) was chosen as expression host, and the results showed that the GDH activity was increased from 173.7 U / mL at 24h to 207.3 U / mL at 43h (Fig. S2). It is important to note that there was no significant autolysis in the C41 (DE3) strain, although there was no great increase in GDH activity. Compared to BL21 (DE3), an important difference of the C41 (DE3) strain is the exchange of lacUV5 into a weaker lac promoter. In fact, the lacUV5 and lac (also named lac-1A) promoters differ only in two places and three points, so other two different promoter variants lacUV5-1A and lac-1G was obtained by shuffling and recombining of lacUV5 and lac-1A (Fig. 3A). Interestingly, the GDH activity increased significantly in the new strain BL21 (DE3-lac1G), resulting in 452.0 U / mL enzyme activity at 43h, which was about 12-times higher than that of BL21 (DE3) (Fig. 3B). However, the enzyme activity of the BL21 (DE3-1A) strain was lower than that of the starting strain BL21 (DE3). It is worth noting that although the enzyme activity of BL21 (DE3-lac1A) was low, there was no decrease of enzyme activity (Fig. 3B). More importantly, a very low extracellular GDH activity was obtained in BL21 (DE3-lac1G) strain (Fig. 3C), which implied that an appropriate T7 RNAP expression level was realized using the lac-1G promoter. Further SEM images indicated that the membranes of BL21 (DE3-lac1G) cells were smooth and robust at 43h (Fig. 3D), indicating that no PCD occurred in this new host. In the scale-up fed-batch fermentation, the GDH activity of BL21 (DE3) peaked at 31h and remained nearly unchanged thereafter, while that of BL21 (DE3-lac1G) was enhanced during the entire fermentation period, reaching yields of up to 3142.4 U / mL (Fig. 4). Notably, with increasing culture time, the productivity of GDH in BL21 (DE3) and BL21 (DE3-lac1G) followed opposite trajectories. In the BL21 (DE3) strain, the highest productivity was obtained at 7-19h, and then declined to only 3 U/mL/h at 31-43h. By contrast, the productivity of BL21 (DE3-lac1G) was increased constantly, and reaching 77.68 U/mL/h at 43-50h.