Production of active bovine cathepsin C (dipeptidyl aminopeptidase I) in the methylotrophic yeast Candida boidinii

The heterologous production of active bovine cathepsin C (CTC; dipeptidyl aminopeptidase I) was investigated. Attempts to express CTC in Escherichia coli were hampered by formation of inclusion bodies that were partially degraded. To overcome this impediment, secretion of recombinant CTC was attempted in the methylotrophic yeast Candida boidinii. A DNA fragment encoding bovine procathepsin C was synthesized based on preferred codon usage in C. boidinii and placed downstream of the C. boidinii proteinase A signal sequence resulting in secretion of active CTC into the culture medium. The gene was expressed under the control of the methanol-inducible formate dehydrogenase gene promoter. Production levels were significantly improved by using a protease-deficient strain, changing medium composition, and by lowering the temperature of induction. When the recombinant C. boidinii was grown for 90 h in a jar-fermenter, active CTC was secreted with a yield of up to approximately 12 mg/l.     

Proteolytic Function of GPI-Anchored Plasma Membrane Protease Yps1p in the Yeast Vacuole and Golgi

Yps1p is a member of the GPI-anchored aspartic proteases which reside at the plasma membrane of Saccharomyces cerevisiae. Here we show that in Δerg6 cells, where a late biosynthetic step of the membrane lipid ergosterol is blocked, part of Yps1p was targeted to the vacuole. There it overtook proteolytic functions of the Pep4p protease, resulting in processing of pro-CPY to CPY in cells lacking the PEP4 gene. Yps1p was enriched in membrane microdomains, as it could be isolated in detergent-insoluble complexes from both normal and Δerg6 cells. Vacuolar Yps1 caused degradation of a mammalian sialyltransferase ectodomain fusion protein (ST6Ne), which was directed from the Golgi to the vacuole in both normal and Δerg6 cells. Unexpectedly, ST6Ne was degraded also when arrested in the Golgi in a temperature-sensitive sec7–1 mutant. Newly synthesized Yps1p, in transit to the plasma membrane, was also involved in the Golgi-associated degradation. These data show that GPI-anchored proteases, whose biological roles are unknown, may reside and function in different subcellular locations.     

Construction of second generation protease-deficient hosts of Bacillus subtilis for secretion of foreign proteins

Although one of the major factors limiting the application of Bacillus subtilis as an expression host has been its production of at least eight extracellular proteases, researchers have also noticed that some proteases benefited the secretion of foreign proteins at times. Therefore, to maximize the yield of a foreign protein, the proteases should be selectively inactivated. This raises a new question that how to identify the favorable and unfavorable proteases for a target protein. Here, an evaluation system containing nine mutant strains of B. subtilis 168 was developed to address this question. The mutant strain PD8 has all the eight proteases inactivated whereas each of the other eight mutant strains expresses only one kind of these eight proteases. The target protein is secreted in these nine mutant strains; if the production of target protein in a mutant strain is higher than that in strain PD8, the corresponding protease is regarded as favorable. Accordingly, the optimal protease-deficient host is constructed through inactivating the unfavorable proteases. The effectiveness of this system was confirmed by expressing three foreign proteins. This study provides a strategy for improving the secretion of a foreign protein in B. subtilis through tailoring a personalized protease-deficient host.     

Reduced proteolysis of secreted gelatin and Yps1-mediated alpha-factor leader processing in a Pichia pastoris kex2 disruptant

Heterologous proteins secreted by yeast and fungal expression hosts are occasionally degraded at basic amino acids. We cloned Pichia pastoris homologs of the Saccharomyces cerevisiae basic residue-specific endoproteases Kex2 and Yps1 to evaluate their involvement in the degradation of a secreted mammalian gelatin. Disruption of the P. pastoris KEX2 gene prevented proteolysis of the foreign protein at specific monoarginylic sites. The S. cerevisiae alpha-factor preproleader used to direct high-level gelatin secretion was correctly processed at its dibasic site in the absence of the prototypical proprotein convertase Kex2. Disruption of the YPS1 gene had no effect on gelatin degradation or processing of the alpha-factor propeptide. When both the KEX2 and YPS1 genes were disrupted, correct precursor maturation no longer occurred. The different substrate specificities of both proteases and their mutual redundancy for propeptide processing indicate that P. pastoris kex2 and yps1 single-gene disruptants can be used for the alpha-factor leader-directed secretion of heterologous proteins otherwise degraded at basic residues.     

Enhanced Secretory Production of a Single-Chain Antibody Fragment from Bacillus subtilis by Coproduction of Molecular Chaperones

Formation of inclusion bodies is a major limiting factor for secretory production of an antidigoxin single-chain antibody (SCA) fragment from Bacillus subtilis. To address this problem, three new strains with enhanced production of molecular chaperones were constructed. WB600BHM constitutively produces the major intracellular molecular chaperones in an appropriate ratio without any heat shock treatment. This strain reduced the formation of insoluble SCA by 45% and increased the secretory production yield by 60%. The second strain, WB600B[pEPP], overproduces an extracytoplasmic molecular chaperone, PrsA. An increase in the total yield of SCA was observed. The third strain, WB600BHM[pEPP], coproduces both intracellular and extracytoplasmic molecular chaperones. This led to a further reduction in inclusion body formation and a 2.5-fold increase in the secretory production yield. SCA fragments secreted by this strain were biologically active and showed affinity to digoxin comparable to the affinity of those secreted by strains without overproduction of molecular chaperones. Interestingly, accumulation of a pool of periplasmic SCA was observed in the PrsA-overproducing strains. This pool is suggested to represent the secreted folding intermediates in the process of achieving their final configuration.     

The yeast GRD20 gene is required for protein sorting in the trans-Golgi network/endosomal system and for polarization of the actin cytoskeleton

The proper localization of resident membrane proteins to the trans-Golgi network (TGN) involves mechanisms for both TGN retention and retrieval from post-TGN compartments. In this study we report identification of a new gene, GRD20, involved in protein sorting in the TGN/endosomal system of Saccharomyces cerevisiae. A strain carrying a transposon insertion allele of GRD20 exhibited rapid vacuolar degradation of the resident TGN endoprotease Kex2p and aberrantly secreted approximately 50% of the soluble vacuolar hydrolase carboxypeptidase Y. The Kex2p mislocalization and carboxypeptidase Y missorting phenotypes were exhibited rapidly after loss of Grd20p function in grd20 temperature-sensitive mutant strains, indicating that Grd20p plays a direct role in these processes. Surprisingly, little if any vacuolar degradation was observed for the TGN membrane proteins A-ALP and Vps10p, underscoring a difference in trafficking patterns for these proteins compared with that of Kex2p. A grd20 null mutant strain exhibited extremely slow growth and a defect in polarization of the actin cytoskeleton, and these two phenotypes were invariably linked in a collection of randomly mutagenized grd20 alleles. GRD20 encodes a hydrophilic protein that partially associates with the TGN. The discovery of GRD20 suggests a link between the cytoskeleton and function of the yeast TGN.     

Fructose-1,6-bisphosphatase degradation in the methylotrophic yeast Komagataella phaffii occurs in autophagy pathway

Many enzymes of methanol metabolism of methylotrophic yeasts are located in peroxisomes whereas some of them have the cytosolic localization. After shift of methanol-grown cells of methylotrophic yeasts to glucose medium, a decrease in the activity of cytosolic (formaldehyde dehydrogenase, formate dehydrogenase, and fructose-1,6-bisphosphatase [FBP]) along with peroxisomal enzymes of methanol metabolism is observed. Mechanisms of inactivation of cytosolic enzymes remain unknown. To study the mechanism of FBP inactivation, the changes in its specific activity of the wild type strain GS200, the strain with the deletion of the GSS1 hexose sensor gene and strain defected in autophagy pathway SMD1163 of Komagataella phaffii with or without the addition of the MG132 (proteasome degradation inhibitor) were investigated after shift of methanol-grown cells in glucose medium. Western blot analysis showed that inactivation of FBP in GS200 occurred due to protein degradation whereas inactivation in the strains SMD1163 and gss1Δ was negligible in such conditions. The effect of the proteasome inhibitor MG132 on FBP inactivation was insignificant. To confirm FBP degradation pathway, the recombinant strains with GFP-labeled Fbp1 of K. phaffii and red fluorescent protein-labeled peroxisomes were constructed on the background of GS200 and SMD1163. The fluorescent microscopy analysis of the constructed strains was performed using the vacuolar membrane dye FM4-64. Microscopic data confirmed that Fbp1 degrades by autophagy pathway in K. phaffii. K. phaffii transformants, which express heterologous β-galactosidase under FLD promoter, have been constructed.     

Use of the Bacillus subtilis subtilisin signal peptide for efficient secretion of TEM beta-lactamase during growth.

We report the development of an efficient Bacillus subtilis secretory system, with the secreted product stably maintained in the medium for 100 h. The system is based on characterization of the subtilisin signal peptidase cleavage site and promoters, catabolite repression of sporulation, presence of a vegetative secreting mechanism, and availability of a protease-deficient strain.     

Functional production and characterization of a fibrin-specific single-chain antibody fragment from Bacillus subtilis: effects of molecular chaperones and a wall-bound protease on antibody fragment production

To develop an ideal blood clot imaging and targeting agent, a single-chain antibody (SCA) fragment based on a fibrin-specific monoclonal antibody, MH-1, was constructed and produced via secretion from Bacillus subtilis. Through a systematic study involving a series of B. subtilis strains, insufficient intracellular and extracytoplasmic molecular chaperones and high sensitivity to wall-bound protease (WprA) were believed to be the major factors that lead to poor production of MH-1 SCA. Intracellular and extracytoplasmic molecular chaperones apparently act in a sequential manner. The combination of enhanced coproduction of both molecular chaperones and wprA inactivation leads to the development of an engineered B. subtilis strain, WB800HM[pEPP]. This strain allows secretory production of MH-1 SCA at a level of 10 to 15 mg/liter. In contrast, with WB700N (a seven-extracellular-protease-deficient strain) as the host, no MH-1 SCA could be detected in both secreted and cellular fractions. Secreted MH-1 SCA from WB800HM[pMH1, pEPP] could be affinity purified using a protein L matrix. It retains comparable affinity and specificity as the parental MH-1 monoclonal antibody. This expression system can potentially be applied to produce other single-chain antibody fragments, especially those with folding and protease sensitivity problems.     

Different substrate specificities of two triazine hydrolases (TrzNs) from Nocardioides species

Nocardioides sp. strain MTD22 degraded atrazine, ametryn and atraton, as did Arthrobacter aurescens strain TC1 and Nocardioides sp. strain C190. These strains contain trzN, a gene coding for TrzN, triazine hydrolase showing a broad substrate range. However, Nocardioides sp. strain AN3 degraded only atrazine despite containing trzN. These differences in s-triazine degradation are presumed to be due to differences in the amino acid sequences of TrzNs. Consequently, 1371 nucleotides of the trzN coding sequences of strains AN3 and MTD22 were determined. Comparisons of the amino acid sequences of TrzNs indicated that three residues of strain AN3 (Thr(214), His(215) and Gln(241)) were distinct from those of the other three strains (Pro(214), Tyr(215) and Glu(241)). To confirm the relationships between these amino acid sequences and the substrate specificities of TrzNs, wild and chimera trzN genes were constructed and expressed in Escherichia coli cells. Cells expressing wild MTD22 trzN (Pro(214)Tyr(215)Glu(241)) and chimera AN3-MTD22 trzN (Thr(214)His(215)Glu(241)) degraded all s-triazines, but the degradation rate was markedly decreased in AN3-MTD22 trzN. Wild AN3 trzN (Thr(214)His(215)Gln(241)) and chimera MTD22-AN3 trzN (Pro(214)Tyr(215)Gln(241)) degraded only atrazine. These results suggest that the substitution of Glu(241) for Gln(241) significantly decreases enzyme affinity for ametryn and atraton.     

High-level accumulation of a recombinant antibody fragment in the periplasm of Escherichia coli requires a triple-mutant (degP prc spr) host strain

During production of a humanized antibody fragment secreted into the periplasm of Escherichia coli, proteolytic degradation of the light chain was observed. In order to determine which protease(s) were responsible for this degradation, we compared expression of the F(ab')(2) antibody fragment in several E. coli strains carrying mutations in genes encoding periplasmic proteases. Analysis of strains cultured in high cell density fermentations showed that the combination of mutations in degP prc spr was necessary for the cells to produce high levels of the desired recombinant antibody fragment. In order to eliminate the possible effects of mutations in other genes, we constructed E. coli strains with protease mutations in isogenic backgrounds and repeated the studies in high cell density fermentations. Extensive light chain proteolysis persisted in degP strains. However, light chain proteolysis was substantially decreased in prc and prc spr strains, and was further decreased with the introduction of a degP mutation in prc and prc spr mutant strains. These results show that the periplasmic protease Prc (Tsp) is primarily responsible for proteolytic degradation of the light chain during expression of a recombinant antibody fragment in E. coli, and that DegP (HtrA) makes a minor contribution to this degradation as well. The results also show that spr, a suppressor of growth defects in prc strains, is required for a prc mutant to survive throughout high cell density fermentations.     

Genetic or chemical protease inhibition causes significant changes in the Bacillus subtilis exoproteome

Bacillus subtilis is a prolific producer of enzymes and biopharmaceuticals. However, the susceptibility of heterologous proteins to degradation by (extracellular) proteases is a major limitation for use of B. subtilis as a protein cell factory. An increase in protein production levels has previously been achieved by using either protease-deficient strains or addition of protease inhibitors to B. subtilis cultures. Notably, the effects of genetic and chemical inhibition of proteases have thus far not been compared in a systematic way. In the present studies, we therefore compared the exoproteomes of cells in which extracellular proteases were genetically or chemically inactivated. The results show substantial differences in the relative abundance of various extracellular proteins. Furthermore, a comparison of the effects of genetic and/or chemical protease inhibition on the stress response triggered by (over) production of secreted proteins showed that chemical protease inhibition provoked a genuine secretion stress response. From a physiological point of view, this suggests that the deletion of protease genes is a better way to prevent product degradation than the use of protease inhibitors. Importantly however, studies with human interleukin-3 show that chemical protease inhibition can result in improved production of protease-sensitive secreted proteins even in mutant strains lacking eight extracellular proteases.     

Inhibition of protease activity by antisense RNA improves recombinant protein production in Nicotiana tabacum cv. Bright Yellow 2 (BY‐2) suspension cells

Recombinant proteins produced in plant suspension cultures are often degraded by endogenous plant proteases when secreted into the medium, resulting in low yields. To generate protease‐deficient tobacco BY‐2 cell lines and to retrieve the sequence information, we cloned four different protease cDNAs from tobacco BY‐2 cells (NtAP, NtCP, NtMMP1, and NtSP), which represent the major catalytic classes. The simultaneous expression of antisense RNAs against these endogenous proteases led to the establishment of cell lines with reduced levels of endogenous protease expression and activity at late stages of the cultivation cycle. One of the cell lines showing reduced proteolytic activity in the culture medium was selected for the expression of the recombinant full‐length IgG1(κ) antibody 2F5, recognizing the gp41 surface protein of HIV‐1. This cell line showed significantly reduced degradation of the 2F5 heavy chain, resulting in four‐fold higher accumulation of the intact antibody heavy chain when compared to transformed wild type cells expressing the same antibody. N‐terminal sequencing data revealed that the antibody has two cleavage sites within the CDR‐H3 and one site at the end of the H4‐framework region. These cleavage sites are found to be vulnerable to serine proteases. The data provide a basis for further improvement of plant cells for the production of recombinant proteins in plant cell suspension cultures.     

乙型肝炎病毒表面抗原前S区和谷胱甘肽巯基转移酶融合基因在大肠杆菌中的表达

我们构建了谷胱甘肽巯基转移酶（ＧＳＴ）和完整的或部分缺失的乙型肝炎病毒表面抗原前Ｓ区的融合基因,并在大肠杆菌中进行了表达。融合蛋白的产量随着前Ｓ区长度的增加而迅速降低,而且融合蛋白的前Ｓ区有严重的降解,主要降解位点在ｐｒｅＳ１区的ａ．ａ．７５和ｐｒｅＳ２区的ａ．ａ．１３０和ａ．ａ．１６５左右。利用蛋白降解酶系缺陷型菌株进行的研究表明,这种降解酶存在于多个大肠杆菌株中而且和大肠杆菌中的两个主要的蛋白降解酶系Ｌｏｎ和ｈｔｐＲ无关。具有重要生物学功能的前Ｓ区肽段（ｐｒｅＳ１ａ．ａ．１－６５）因含有阻止分泌的滞留顺序而无法在哺乳动物细胞和酵母中大量表达,但滞留顺序的存在并没有影响含有这一肽段的融合蛋白在大肠杆菌中的表达和产物的纯化。ＧＳＴ融合表达系统产量高,纯化快速简便。用这一方法大量表达并得到的这一肽段不仅是研究乙型肝炎病毒的分子生物学的重要材料,而且可以作为新一代乙型肝炎疫苗的主要组成成分。     

Generation of diploid Pichia pastoris strains by mating and their application for recombinant protein production

ABSTRACT: BACKGROUND: Yeast mating provides an efficient means for strain and library construction. However, biotechnological applications of mating in the methylotrophic yeast Pichia pastoris have been hampered because of concerns about strain stability of P. pastoris diploids. The aim of the study reported here is to investigate heterologous protein expression in diploid P. pastoris strains and to evaluate diploid strain stability using high cell density fermentation processes. RESULTS: By using a monoclonal antibody as a target protein, we demonstrate that recombinant protein production in both wild-type and glycoengineered P. pastoris diploids is stable and efficient during a nutrient rich shake flask cultivation. When diploid strains were cultivated under bioreactor conditions, sporulation was observed. Nevertheless, both wild-type and glycoengineered P. pastoris diploids showed robust productivity and secreted recombinant antibody of high quality. Specifically, the yeast culture maintained a diploid state for 240 h post-induction phase while protein titer and N-linked glycosylation profiles were comparable to that of a haploid strain expressing the same antibody. As an application of mating, we also constructed an antibody display library and used mating to generate novel full-length antibody sequences. CONCLUSIONS: To the best of our knowledge, this study reports for the first time a comprehensive characterization of recombinant protein expression and fermentation using diploid P. pastoris strains. Data presented here support the use of mating for various applications including strain consolidation, variable-region glycosylation antibody display library, and process optimization.     

Degradation of fluorobenzene by Rhizobiales strain F11 via ortho cleavage of 4-fluorocatechol and catechol

The aerobic metabolism of fluorobenzene by Rhizobiales sp. strain F11 was investigated. Liquid chromatography-mass spectrometry analysis showed that 4-fluorocatechol and catechol were formed as intermediates during fluorobenzene degradation by cell suspensions. Both these compounds, unlike 3-fluorocatechol, supported growth and oxygen uptake. Cells grown on fluorobenzene contained enzymes for the ortho pathway but not for meta ring cleavage of catechols. The results suggest that fluorobenzene is predominantly degraded via 4-fluorocatechol with subsequent ortho cleavage and also partially via catechol.     

Reduced Proteolysis of Secreted Gelatin and Yps1-Mediated α-Factor Leader Processing in a Pichia pastoris kex2 Disruptant

Heterologous proteins secreted by yeast and fungal expression hosts are occasionally degraded at basic amino acids. We cloned Pichia pastoris homologs of the Saccharomyces cerevisiae basic residue-specific endoproteases Kex2 and Yps1 to evaluate their involvement in the degradation of a secreted mammalian gelatin. Disruption of the P. pastoris KEX2 gene prevented proteolysis of the foreign protein at specific monoarginylic sites. The S. cerevisiae α-factor preproleader used to direct high-level gelatin secretion was correctly processed at its dibasic site in the absence of the prototypical proprotein convertase Kex2. Disruption of the YPS1 gene had no effect on gelatin degradation or processing of the α-factor propeptide. When both the KEX2 and YPS1 genes were disrupted, correct precursor maturation no longer occurred. The different substrate specificities of both proteases and their mutual redundancy for propeptide processing indicate that P. pastoris kex2 and yps1 single-gene disruptants can be used for the α-factor leader-directed secretion of heterologous proteins otherwise degraded at basic residues.     

Arginine and citrulline do not stimulate growth of two Oenococcus oeni strains in wine

Arginine metabolism by wine lactic acid bacteria (LAB) may lead to wine quality degradation. While arginine is essential for growth of the wine relevant LAB Oenococcus oeni , it remains unclear whether it also stimulates its growth. This study evaluated the effect of arginine and citrulline, the partially metabolized intermediate of the arginine deiminase pathway, on the growth of two commercial O. oeni strains in comparison with a Lactobacillus buchneri strain in wine and at wine pH values. Neither arginine nor citrulline increased growth of both O. oeni strains in comparison with the L. buchneri strain. However, arginine and citrulline were partially degraded in all incubations. The extent of citrulline degradation correlated with lower pH values in oenococcal cultivations but with higher pH values in those of the L. buchneri strain. The degradation kinetics of O. oeni and L. buchneri for malic acid and arginine differed and the latter grew in sterile filtered post-malolactic fermentation wine. This study shows that arginine and citrulline did not stimulate growth of the two O. oeni strains studied, and that their physiological role differed among the wine LAB considered. While arginine may play a role in wine microbiological stability, other nutrients should be investigated for their suitability to create a selective ecological advantage for O. oeni strains in wine.     

Characteristics of Saccharomyces cerevisiae gal1 Delta and gal1 Delta hxk2 Delta mutants expressing recombinant proteins from the GAL promoter

Galactose can be used not only as an inducer of the GAL promoters, but also as a carbon source by Saccharomyces cerevisiae, which makes recombinant fermentation processes that use GAL promoters complicated and expensive. To overcome this problem during the cultivation of the recombinant strain expressing human serum albumin (HSA) from the GAL10 promoter, a gal1 Delta mutant strain was constructed and its induction kinetics investigated. As expected, the gal1 Delta strain did not use galactose, and showed high levels of HSA expression, even at extremely low galactose concentrations (0.05-0.1 g/L). However, the gal1 Delta strain produced much more ethanol, in a complex medium containing glucose, than the GAL1 strain. To improve the physiological properties of the gal1 Delta mutant strain as a host for heterologous protein production, a null mutation of either MIG1 or HXK2 was introduced into the gal1 Delta mutant strain, generating gal1 Delta mig1 Delta and gal1 Delta hxk2 Delta double strains. The gal1 Delta hxk2 Delta strain showed a decreased rate of ethanol synthesis, with an accelerated rate of ethanol consumption, compared to the gal1 Delta strain, whereas the gal1 Delta mig1 Delta strain showed similar patterns to the gal1 Delta strain. Furthermore, the gal1 Delta hxk2 Delta strain secreted much more recombinant proteins (HSA and HSA fusion proteins) than the other strains. The results suggest that the gal1 Delta hxk2 Delta strain would be useful for the large-scale production of heterologous proteins from the GAL10 promoter in S. cerevisiae.