| Saccharomyces cerevisiae is a highly potential microbial cell factory, because it is a generally regarded as safe (GRAS) model eukaryote and has a favourable industrial application. It has an integrated secretory pathway for protein folding and modification, but the production of heterologous protein is still at a low level. However, efficient secretion of heterologous protein is essential for pursuing valuable applications. Heterologous cellulases expressing with high activity in S. cerevisiae is an important strategy to achieve consolidated bioprocess (CBP) for cellulosic ethanol production. CBP combines cellulases production, cellulose hydrolysis, and ethanol fermentation into a single process by one microorganism. Cellulosome from anaerobic bacteria is a multi-enzyme complex and the proximity of enzyme-enzyme allows the efficient hydrolysis of cellulose. Therefore, the surface assembly of cellulosome on S. cerevisiae is becoming a new hot spot for CBP construction. In this study, potential limiting steps were analyzed and engineered to optimize secretory pathway for high efficiency of heterologous protein secretion. In addition, a new minicellulosome assembled by disulfide bonds and complicated with two miniscafoldins was investigated in S. cerevisiae for cellulosic ethanol production. The research studies are as following:1. High ?-glucosidase secretion in S. cerevisiae improves the efficiency of cellulase hydrolysis and ethanol production in simultaneous saccharification and fermentationIn simultaneous saccharification and fermentation (SSF), intermediate cellobiose can inhibit the cellulases activity for lignocelluloses degradation and decrease bioethanol production. Converting cellobiose into glucose and fermenting into ethanol can abolish this inhabitation. However, cellulases from filamentous fungi are often short of ?-glucosidase which is responsible for cellobiose degradation. Thus, ?-glucosidase with high activity in S. cerevisiae is essential for enhancing efficiency of SSF. Among three ?-glucosidases, Saccharomycopsis fibuligera BGL1 showed the best extracellular activity and the signal peptide of inulinase from Kluyveromyces marxianus further enhanced its secretion, the maximum activity was 6.22 U/mL.BGL1 expressing strain metabolized cellobiose to produce ethanol with high efficiency, which was similar as glucose fermentation. However, Control strain with empty plasmid did not utilize cellobiose. BGL1 expressing strain showed higher hydrolysis efficiency in simultaneous saccharification and fermentation of cellulose, when using the Trichoderma reesei cellulase which is short of the ?-glucosidase activity. The final ethanol concentration was 110%(using Avicel) and 89%(using acid-pretreated corncob) higher than the control strain. These results demonstrated the efficient secretion of ?-glucosidase in S. cerevisiae covered the short of T. reesei cellulases and enhanced cellulosic ethanol conversion.2. Engineering protein folding and translocation improves heterologous protein secretion in S. cerevisiaeHeterologous proteins including ?-glucosidase (BGL1), endoglucanase (CelA) and a-amylase resulted in the accumulation of unfolded protein and decreased the protein secretion efficiency. Enhancement of protein folding capacity may be an effective strategy for high protein production. Overexpression of the endoplasmic reticulum (ER) chaperone protein Kar2p which assists in protein proper folding enhanced the extracellular activity of BGL1, but not CelA and ?-amylase. Overexpression of disulfide isomerase Pdilp which is responsible for proper disulfide bonds formation improved the secretion of all three heterologous proteins, although CelA did not have the modification of disulfide bonds.Translocation of nascent peptide chain into ER for folding is also important for efficient protein secretion. Components of signal recognition particle Srp14p and Srp54p, involving in co-translational translocation responsible for enlongation and location of nascent peptide chain, was overexpressed and the production of these three heterologous proteins was enhanced clearly. Overexpression of cytosolic chaperone Ssalp which assisted in nascent peptide chain folding and post-translational translocation enhanced the secretion of only BGL1. Signal peptides affect the protein translocation. In Srp54p, Srp14p, Kar2p and Pdilp expressing strain, the secretion of BGL1 with its native signal peptide SF, invertase signal peptide Suc2 and synthetic signal peptide Yap3 showed the similar trends. However, in Ssalp expressing strain, BGL1 secretion was improved with SF and Yap3, but not with Suc2. In addition, Ssalp enhanced the secretion of BGL1 with Yap3, but not the a-amylase with Yap3. These results demonstrated that optimization of co-translational translocation matched generous heterologous protein hyper-secretion and did not affect by signal peptide significantly, but post-translational translocation had a selective function and was affected by both signal peptide and protein.By engineering both translational translocation and protein folding, the secretion of heterologous proteins was further increased. Co-expression of Srp54p and Srp14p enhanced the production of BGL1, CelA and ?-amylase the by 72%,60% and 103%, respectively, compared to the controls. Our results show that protein translocation is a limiting factor for heterologous protein production and is important for the optimization of secretory pathway.3. N-hypermannose glycosylation disruption enhances recombinant protein production by regulating secretory pathway and cell wall integrity in S. cerevisiaeHeterologous proteins including P-glucosidase (BGL1), endoglucanase (CelA) and exoglucanase (Cel7A) were N-hyperglycosylated when they were expressed in S. cerevisiae. N-hyperglycosylation of heterologous proteins may affect their bioactivity and production, thus, studies of the impact of N-glycans on protein activity are important for optimization of secretory pathway. To block the formation of hypermannose glycan, these proteins were expressed in strains with deletions in key Golgi mannosyltransferases (Ochlp, Mnn9p and Mnnlp), respectively. Their molecular weights were decreased to the theoretical value approximately and extracellular activities were improved markedly in the OCH1 and MNN9 deletion strains. The deletion of OCH1 improved the activity of BGL1, CelA and Cel7A by 135%,102% and 144%, respectively, and the deletion of MNN9 by 156%,105% and 230%, respectively. However, the deletion of MNN1 did not decrease the protein molecular weights and only resulted in the improvement of BGL1 activity. Interestingly, truncation of the N-hypermannose glycan did not increase the specific activity of these proteins, but improved the secretion yield.Further analysis showed OCHl and MNN9 deletion up-regulated genes in the secretory pathway, such as KAR2 and SSA1 involving in protein folding, DERI and HRD3 involving in ERAD and BOSl, ERV25, SNC2 and SSOl involving in vesicular trafficking, but did not induce the unfolded protein response by IRE1/HAC1 pathway. In addition, the cell wall integrity was also affected by OCHl and MNN9 deletion, which contributed to the release of secretory protein extracellularly. Overexpression of RHO1 and PKC1 involving in cell wall integrity response partially suppressed the growth defect of OCH1 and MNN9 deletion strains. This study demonstrated that mannosyltransferases disruption improved protein secretion through up-regulating secretory pathway and affecting cell wall integrity and provided new insights into glycosylation engineering for protein secretion.4. Engineering vesicle trafficking improves the extracellular secretion and surface display efficiency of heterologous proteins in S. cerevisiaeIn S. cerevisiae, limitations of vesicle transport result in the retention and degradation of over-expressed heterologous protein intracellularly. Strengthening vesicle trafficking is an effective strategy for the improvement of heterologous protein production. Overexpression of Sec12p, Sec13p, Erv25p and Bos1p, involving in vesicle trafficking from ER to Golgi, enhanced the secretion of C. thermocellum CelA. The maximum activity was achieved in Erv25p-expressing strain and the activity was improved by 31%, compared with empty plasmid expressing strain. For S. fibuligera BGL1 secretion, only Secl3p increased its extracellular activity by 40%. Overexpression of key components Sso1p, Snc2p, Sec1p, Exo70p, Sec4p and Ypt32p, involving in vesicle trafficking from Golgi to plasma membrane increased the secretion of BGL1, especially in Sso1p and Snc2p expressing strain and the activity was improved by 53% and 61%, respectively. In Snc2p, Seclp, Sec4p andYpt32p overexpressing strains, the extracellular activity of CelA was also improved. These results demonstrated that modification of ER-Golgi trafficking was suitable for efficient secretion of CelA and Golgi-plasma membrane trafficking matched the high production of BGL1.CelA and BGL1 were displayed on the cell surface through yeast cell wall protein Agalp-Aga2p, respectively. Extracellular activity and flow cytometry (FASC) analysis showed that engineering trafficking components improved the display efficiency of CelA and overexpression of Bos1p, Exo70p and Sec4p improved the cell activity by 71%,90% and 51%, respectively. In addition, the cell activity of BGL1 was also increased in these engineering strains. This study demonstrated that engineering vesicle trafficking increased the secretion of heterologous protein and was firstly reported to improve the efficiency of surface-displayed heterologous protein.5. Cellulases optimization and self-surface assembly of minicellulosome in S. cerevisiaeCellulosome assembled on yeast cell surface has been played to sufficient attention in cellulosic ethanol production. In S. cerevisiae, construction of minicellulosome requires the heterologous expression of miniscaffoldin and three cellulases at least. Miniscaffoldin ScafCipA3 containing three cohesins and a cellulose binding module (CBM) was from C. thermocellum scaffoldin I and displayed on cell surface successfully through yeast cell wall protein a-agglutinin (Aga1p-Aga2p). The results of FASC analysis showed the percentage of positively staining cells was 40%. ?-glucosidase BGL1 from S. fibuligera showed a higher activity (abstract 1) and was fused with dockerin of C. thermocellum xylanase XynC for cellulosome construction. Extracellular activities of different exoglucanases were compared and exoglucanase CBH1 from Talaromyces emersonii showed the best activity. In addition, C. thermocellum CelA had a higher secretion activity in different endoglucanases. According to the interaction of dockerin and cohesin, three cellulases were successfully assembled to surface displayed miniscaffoldin. The percentage of CBH1, CelA and BGL1 assembled cells was 3.8%,19.3% and 2.5%, respectively, indicating that minicellulosome was formed. Moreover, concentrating cellulases and decreasing numbers of scaffoldin expressing strain improved the assembly percentage significantly, and CBH1, CelA and BGL1 was 7.4%,94.3% and 9.0%, indicating the short of cellulases secretion. Yeast self-surface assembly of minicellulosome fermented phosphoric-acid-swollen Avicel (PASC) and produced ethanol. This study demonstrated that short of heterologous cellulases secretion was one limitation of yeast cellulosome construction.6. Self-surface assembly and complication of novel minicellulosome through disulfide bonds in S. cerevisiaeGenerally, cellulosome assembly on S. cerevisiae cell surface was according to the interaction of dockerins from cellulases and cohesins from scaffoldin. Protein-protein interaction includes various styles, such as antibody-antigen interaction and the formation of disulfide bonds, which may be the potential new interaction for cellulosome assembly. Cell wall protein a-agglutinin is composed of two subunits Agalp and Aga2p which interact with each other by the formation of disulfide bonds. 1-149 amino acids of Agalp (named AGA) are responsible for interaction with Aga2p. Based on this, design of a novel minicellulosome assembled through disulfide bonds used AGA as cohesin and Aga2p as dockerin. Three AGAs fused with CBM from T. reesei exoglucanase CBH1 were used as miniscaffoldin ScafAGA3 and it successfully displayed on cell surface by the GPI domain of Aga1p. The display percentage of ScafAGA3 was 56% which was higher than ScafCipA3 (40%, seen in abstract5). Aga2p was fused to exoglucanase CBH1, endoglucanase CelA and ?-glucosidase BGL1 to form aCBHl, aCelA and aBGL1. These three cellulases also showed secretion activity which could be used for functional minicellulosome assembly. aCelA assembled to AGA which was anchored on cell surface by cell wall protein Sedlp with the assembly percentage of 11.7%, indicating that AGA can be used as the functional cohensin. The assembly percentage of aCBHl, aCelA and aBGL1 to scaffolfin ScafAGA3 was 12.1%,28.3% and 5.8%, respectively. Yeast surface assembly of this minicellulosome fermented PASC and produced 0.93 g/L ethanol, which was improved by 50% compared to control strain.Scaffoldin ScafAGA3 used as scaffoldin II and scaffoldin ScafCipA3 as scaffoldin I were co-expressed to form the interaction of two scaffoldins through disulfide bonds, then cellulases assembled on scaffodin I by dockerin-cohesin to construct a more complex minicellulosome. The assembly percentage of CBH1, CelA and BGL1 was relatively low, and concentrating cellulases and decreasing numbers of scaffoldin expressing strains increased the assembly percentage of cellulases, respectively. This complex minicellulosome fermented PASC and produced 0.5 g/L ethanol.In this study, engineering secretory pathway including protein folding, protein translocation, N-glycosylation and vesicle trafficking improved the secretion of heterologous protein such as cellulases. It is found that modification of N-glycosylation had a widely enhancement on the production of various heterologous proteins. Engineering protein folding, protein translocation and vesicle trafficking affected the secretion of heterologous proteins differently and key secretoty components were concluded to improve secretion of each protein based on its secreted limitations. In addition, Design and construction a novel minicellulosome through disulfide bonds assembly on yeast surface. The study of secretory pathway engineering also plays an essential role in improving the secretion of scaffodin and cellulase components of cellulosome. |
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