Identification of the autolysis sites in tbcf (K101A)
The SGT contains 16 potential autolysis residues (Arg, Lys). In previous study, the stability and production of SGT were improved with tbcf (K101A) mutant . For further investigating the autolysis of tbcf (K101A), its’ hydrolysate was analyzed by MALDI-TOF-MS. Based on the autolysis fragments of tbcf (K101A), five autolysis residues were identified as R21, R32, K122, R153, and R201 (Fig. 1A). This result indicated that the SGT slightly prefers to hydrolyze at R than K. Because the hydrogen bond interaction between R and substrate binding D189 was more stable than a water molecule bridged interaction between K and D189 . Štosová et al. significantly reduced the autolysis of SGT by modifying R or K residues with chemical reagents phenylglyoxal and formaldehyde, but the specific activity of SGT dropped to 12% of the parent enzyme . Because the R and K interacted with other residues to form hydrogen bond, salt bridge and π-interaction. These interactions played essential roles in SGT folding, three-dimensional structure, and catalytic activity. In secondary structure of SGT, except R201V (β-Sheet), the other four autolysis residues (R21, R32, K122, and R153) were located in the loop regions (Fig. 1B). Specifically, R21 interacted with Y131 by a hydrogen bond, which was also the case for R32-T55 and R32-Q128 interactions. Whereas K122 formed a salt bridge with D184 and E188, so as for R153 and D60. These results indicated that R21, R32, K122 and R153 residues helped lock the three-dimensional conformation.
Directed selection of SGT
The direct selection method was widely applied to engineer proteins for specific properties . With the help of online bioinformatic tools, bespoke candidate of mutations could be suggested according to the overall protein stability, residue physio-chemical environment, and interaction network of protein [27–29]. The SGT candidates were sorted for higher trypsin activity than parent tbcf (K101A) in flask culture. In the C-terminal residue R201, the tbcf (K101A, R201V) mutant showed the highest activity 60.85 ± 3.42 U·mL− 1, increased 1.5-fold than the parent (Fig. 2A). Among six mutations of R32, the R32A was outstanding with increased trypsin activity 45.15 ± 0.99 U·mL− 1 (Fig. 2B). While, the other mutations of R21, K122 and R153 decreased the trypsin activity (Fig. 2CDE).The R32A mutation was then added into tbcf (K101A, R201V) to afford tbcf (K101A, R201V, R32A) mutant. Even tbcf (K101A, R201V, R32A) showed higher expression level than tbcf (K101A, R201V) (Fig. S1), its activity decreased by 50% (30.67 ± 2.63 U·mL− 1). Further, the specific activity of trypsin mutants was compared, with substrate BAPNA using spectrophotometric assay. The mutant tbcf (K101A, R201V) showed the highest specific activity of 1527.96 ± 62.81 U·mg− 1, increased 13.76% than parent. Eventually, the mutant tbcf (K101A, R201V) was discovered with increased production of trypsin and its specific activity.
Enzyme kinetics and molecular modeling analysis of SGT mutant
The MD simulation was applied to analyze the three-dimensional structure of tbcf (K101A, R201V) compared with tbcf. The root-mean-square deviations (RMSD) of protein backbone atom indicated the stability of protein . After the 7 ns MD simulation, the backbone of tbcf (K101A, R201V) mutant showed the lower deviation of RMSD value (Fig. S2). This result indicated the K101A/R201V mutations could improve the stability of SGT backbone. Then, the internal interaction of catalytical triad (H57, D102 and S195) was analyzed. Interestingly, the tbcf (K101A, R201V) mutant showed a shorter distance between H57 and D102 (6.5 Å vs 7.0 Å) in the catalytic center (Fig. 3AB). Consequently, the tbcf (K101A, R201V) mutant, with a kcat/Km value of 1.53 × 107 min− 1·mM− 1, afforded higher catalytical activity than parent tbcf (K101A) ( Supplementary Table 3). Especially, the increased specific activity might attribute to shortened distance between D102 and H57 in catalytical triad, which could consolidate the hydrogen bond between carboxylic oxygen of D102 and δ-nitrogen of H57 . Because the hydrogen bond stabilized the structure of H57 in catalytical transient state, which facilitated H57 to accept the proton from S195 . Moreover, the Km value of tbcf (K101A, R201V) and tbcf were similar, which were 5.39 ± 0.36 × 10− 2 mM and 5.86 ± 0.16 × 10− 2 mM respectively. This result indicated that K101A/R201V mutations retained the conserved internal interaction at substrate binding domain.
High-yield production of SGT with co-overexpression chaperones in P. pastoris
Protein expression was known to be regulated by UPR  or ERAD  in P. pastoris. And expression of trypsinogen triggered UPR and ERAD in P. pastoris, because of the unfolded trypsinogen in endoplasmic reticulum (ER) and peroxide toxicity by forming disulfide bond [23, 24, 35]. It was known that protein expression could be improved by upregulating the endogenous proteins . Therefore, twelve proteins were individually overexpressed, involved in transcription regulation, disulfide bond formation and protein secretion. The ER located chaperones processed diverse functions during polypeptides folding into the biologically active protein. And these chaperones included the oxidative reaction in protein folding (Ero1), disulfide bond forming (GLR1, PDI, and GSH2), and degradation of the unfolded protein (UBC1) [34, 35]. Interestingly, the production of trypsin was increased by 17.0% and 31.6% with overexpression of GSH2 and UBC1, respectively (Fig. 4). Moreover, the transport of polypeptides was known to be critical for secretory proteins. The Bip, SLY1 and SEC53 chaperones were responsible for transporting and recognizing of nascent polypeptides in ER and Golgi membrane . And, the SEC1 and SSO2 promoted the extracellular secretory of the folded protein [38, 39]. So, overexpression of SEC1 and SSO2 increased the trypsin activity by 24.1% and 41.5%, respectively. Then, the SEC1, SSO2 and UBC1 were co-overexpressed, because they contributed more than 20% increase of trypsin activity. Finally, the trypsin production of strain GS115-tbcf (K101A, R201V)_SU showed highest production 109.25 ± 4.76 U·mL− 1 (increased by 79.5% ), with co-overexpression of SSO2 and UBC1 in flask culture (Fig. 4). This indicated that the bottle-neck for high-yield production of SGT might be hindered by secretory transportation and degradation of unfolded trypsin in P. pastoris.
Application of high-yield trypsin to processing insulin precursor
For scale-up production of the trypsin, the strain GS115-tbcf (K101A, R201V)_SU was cultured in 3-L bio-reactor, according to the published method . After glycerol fed-batch cultivation, a higher density of cells (68.02 ± 1.5 g/L, DCW) was achieved with glycerol feeding and high agitation speed (850 r·min− 1) (Fig. 5). Then, the fermentation entered methanol-feeding cultivation phase, when the glycerol was depleted with the indication of increased DO (over 50%). After induction for 156 h, the trypsin production reached 689.47 ± 6.78 U·mL− 1, which was increased 302.8% than parent GS115-tbcf (K101A).
The mammalian trypsin was generally applied for the preparing insulin from its precursor, because of the canonically tryptic cleavage of lysine for removal of C-chain [4, 40]. While, the traditional method for preparing trypsin is extraction from the mammalian pancreas, which has the risk of the bioactive compound, infectious virus, and heath-harmful proteases . Although the heterologous expression of mammalian trypsin could avoid the aforementioned problems, it still suffered from immunogenicity issues, low expression level, activation of the zymogen, and autolysis [15, 42, 43]. Importantly, the SGT mutant tbcf (K101A, R201V) showed higher hydrolysis performance, with no immunogenicity, high production, autoactivation and stability against autolysis. The tbcf (K101A, R201V) was mixed with insulin precursor rPI to afford insulin precursor with Asp30 deleted B-chain (PI-BD30) (Fig. 6A). And the tbcf (K101A, R201V) was compared with commercial porcine trypsin at the identical condition. After hydrolysis for 19 h, the rPI was converted to PI-BD30 as demonstrated in the HPLC chromatograph. The elution time of rPI was 18.75 min (Fig. 6B). After cleavage by commercial porcine trypsin and tbcf (K101A, R201V), the rPI was converted to PI-BD30, which was eluted out at 21.40 min (Fig. 6CD). So, the engineered SGT mutant tbcf (K101A, R201V) performed the potential application in insulin manufacture, due to the same hydrolysis capacity with commercial porcine trypsin.