RESUMO
Increasing demands for bioactive compounds have motivated researchers to employ micro-organisms to produce complex natural products. Currently, Bacillus subtilis has been attracting lots of attention to be developed into terpenoids cell factories due to its generally recognized safe status and high isoprene precursor biosynthesis capacity by endogenous methylerythritol phosphate (MEP) pathway. In this review, we describe the up-to-date knowledge of each enzyme in MEP pathway and the subsequent steps of isomerization and condensation of C5 isoprene precursors. In addition, several representative terpene synthases expressed in B. subtilis and the engineering steps to improve corresponding terpenoids production are systematically discussed. Furthermore, the current available genetic tools are mentioned as along with promising strategies to improve terpenoids in B. subtilis, hoping to inspire future directions in metabolic engineering of B. subtilis for further terpenoid cell factory development.
Assuntos
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Vias Biossintéticas , Microbiologia Industrial , Engenharia Metabólica , Terpenos/metabolismo , Alquil e Aril Transferases , Butadienos , Eritritol/análogos & derivados , Eritritol/metabolismo , Hemiterpenos , Fosfatos Açúcares/metabolismoRESUMO
Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a death ligand cytokine known for its cytotoxic activity against malignantly transformed cells. TRAIL induces cell death through binding to death receptors DR4 and DR5. The inhibitory decoy receptors (DcR1 and DcR2) co-expressed with death receptor 4 (DR4)/DR5 on the same cell can block the transmission of the apoptotic signal. Here, we show that DcRs also regulate TRAIL sensitivity at a supracellular level and thus represent a mechanism by which the microenvironment can diminish tumour TRAIL sensitivity. Mathematical modelling and layered or spheroid stroma-extracellular matrix-tumour cultures were used to model the tumour microenvironment. By engineering TRAIL to escape binding by DcRs, we found that DcRs do not only act in a cell-autonomous or cis-regulatory manner, but also exert trans-cellular regulation originating from stromal cells and affect tumour cells, highlighting the potent inhibitory effect of DcRs in the tumour tissue and the necessity of selective targeting of the two death-inducing TRAIL receptors to maximise efficacy.
Assuntos
Membro 10c de Receptores do Fator de Necrose Tumoral/metabolismo , Células Estromais/patologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Receptores Chamariz do Fator de Necrose Tumoral/metabolismo , Linhagem Celular Tumoral , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Membro 10c de Receptores do Fator de Necrose Tumoral/genética , Células Estromais/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/química , Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores Chamariz do Fator de Necrose Tumoral/genéticaRESUMO
BACKGROUND: Tumour cell-selective activation of apoptosis by recombinant human TNF-related apoptosis-inducing ligand (rhTRAIL) is enhanced through co-activation of p53 by chemotherapeutic drugs. The novel anticancer agent nutlin-3 provides a promising alternative for p53 activation by disrupting the interaction between p53 and its negative feedback regulator MDM2. METHODS: We examined whether nutlin-3 enhances apoptosis induction by rhTRAIL and the DR5-selective TRAIL variant D269H/E195R in wild-type p53-expressing ovarian, colon and lung cancer cell lines and in an ex vivo model of human ovarian cancer. RESULTS: Nutlin-3 enhanced p53, p21, MDM2 and DR5 surface expression. Although nutlin-3 did not induce apoptosis, it preferentially enhanced D269H/E195R-induced apoptosis over rhTRAIL. Combination treatment potentiated the cleavage of caspases 8, 9, 3 and PARP. P53 and MDM2 siRNA experiments showed that this enhanced apoptotic effect was mediated by wild-type p53. Indeed, nutlin-3 did not enhance rhTRAIL-induced apoptosis in OVCAR-3 cells harbouring mutant p53. Addition of the chemotherapeutic drug cisplatin to the combination further increased p53 and DR5 levels and rhTRAIL- and D269H/E195R-induced apoptosis. As a proof of concept, we show that the combination of D269H/E195R, nutlin-3 and cisplatin induced massive apoptosis in ex vivo tissue slices of primary human ovarian cancers. CONCLUSION: Nutlin-3 is a potent enhancer of D269H/E195R-induced apoptosis in wild-type p53-expressing cancer cells. Addition of DNA-damaging agents such as cisplatin further enhances DR5-mediated apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Imidazóis/farmacologia , Neoplasias/patologia , Piperazinas/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Proteínas Recombinantes/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Substituição de Aminoácidos , Apoptose/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Genes p53 , Humanos , Neoplasias/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteínas Recombinantes/genética , Especificidade por Substrato , Ligante Indutor de Apoptose Relacionado a TNF/genética , Células Tumorais CultivadasRESUMO
The tumour necrosis factor family member TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in a variety of cancer cells through the activation of death receptors 4 (DR4) and 5 (DR5) and is considered a promising anticancer therapeutic agent. As apoptosis seems to occur primarily via only one of the two death receptors in many cancer cells, the introduction of DR selectivity is thought to create more potent TRAIL agonists with superior therapeutic properties. By use of a computer-aided structure-based design followed by rational combination of mutations, we obtained variants that signal exclusively via DR4. Besides an enhanced selectivity, these TRAIL-DR4 agonists show superior affinity to DR4, and a high apoptosis-inducing activity against several TRAIL-sensitive and -resistant cancer cell lines in vitro. Intriguingly, combined treatment of the DR4-selective variant and a DR5-selective TRAIL variant in cancer cell lines signalling by both death receptors leads to a significant increase in activity when compared with wild-type rhTRAIL or each single rhTRAIL variant. Our results suggest that TRAIL induced apoptosis via high-affinity and rapid-selective homotrimerization of each DR represent an important step towards an efficient cancer treatment.
Assuntos
Antineoplásicos/química , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/química , Substituição de Aminoácidos , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Biologia Computacional , Feminino , Humanos , Cinética , Mutação , Neoplasias Ovarianas/tratamento farmacológico , Ligação Proteica , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
AIMS: Overproduced alpha-amylases in Bacillus subtilis provoke a specific stress response involving the CssRS two-component system, which controls expression of the HtrA and HtrB proteases. Previously, the B. subtilis TepA protein was implicated in high-level alpha-amylase secretion. Our present studies were aimed at investigating a possible role of TepA in secretion stress management, and characterizing the intensity of the secretion stress response in relation to alpha-amylase production. METHODS AND RESULTS: The expression of a transcriptional htrB-lacZ gene fusion, and the levels of alpha-amylase production were monitored simultaneously using tepA mutant B. subtilis strains. TepA was shown to be dispensable for secretion stress management. Importantly, however, the levels of htrB-lacZ expression can be correlated with the levels of alpha-amylase production. CONCLUSION: Our observations show that the secretion stress response can serve as an indicator for alpha-amylase production levels. SIGNIFICANCE AND IMPACT OF STUDY: Conceivably, this stress response can be employed to monitor the biotechnological production of various secretory proteins by the Bacillus cell factory.
Assuntos
Bacillus subtilis/enzimologia , Resposta ao Choque Térmico , alfa-Amilases/biossíntese , Aciltransferases/genética , Aciltransferases/metabolismo , Bacillus subtilis/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biotecnologia/métodos , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Óperon Lac , Mutação , Proteínas Periplásmicas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Serina Endopeptidases/metabolismoRESUMO
A new GC-MS method for monitoring lignans was developed to study the variation in plants and elucidate the biosynthetic steps. A simple and fast extraction procedure for lyophilised plant material was developed, giving a lignan-rich extract. A GC-MS method was set up using an apolar WCOT fused silica column using a high temperature programme (150 degrees C to 320 degrees C at 15 degrees C min(-1)). This new GC-MS method gave a clear lignan profile of plant material. It was possible to show the large variation in the concentrations of deoxypodophyllotoxin (DOP), yatein and anhydropodorhizol (AHP) in Anthriscus sylvestris (L.) Hoffm. plants growing on different locations using cinchonidin as an internal standard. In contrast with existing GC methods for lignan analysis no derivatisation is needed. It is also possible to use this method for the detection of different classes of lignans in biosynthetically related plant species.
Assuntos
Apiaceae , Lignanas/análise , Podofilotoxina/análogos & derivados , 4-Butirolactona/análogos & derivados , 4-Butirolactona/análise , Dioxóis/análise , Medicamentos de Ervas Chinesas , Cromatografia Gasosa-Espectrometria de Massas , Extratos Vegetais/química , Podofilotoxina/análiseRESUMO
Carboxylesterase NP of Bacillus subtilis Thai I-8, characterized in 1992 as a very enantioselective (S)-naproxen esterase, was found to show no enantiopreference towards (S)-1,2-O-isopropylideneglycerol (IPG) esters. The ybfK gene was identified by the B. subtilis genome project as an unknown gene with homology to carboxylesterase NP. The purpose of the present study was to characterize the ybfK gene product in order to determine whether this paralogue of carboxylesterase NP had an altered or enhanced stereospecificity. The ybfK gene was cloned and expressed in B. subtilis using a combination of two strong promoters in a multicopy vector. The enzyme was purified from the cytoplasm of B. subtilis by means of anion exchange and hydrophobic interaction chromatography. The purified YbfK is an enzyme of 296 amino acids and shows an apparent molecular mass of 32 kDa (SDS/PAGE). Comparison of the activities of YbfK and carboxylesterase NP towards caprylate esters of IPG revealed that YbfK produces (S)-IPG with 99.9% enantioselectivity. Therefore, we conclude that we have isolated a paralogue of carboxylesterase NP that can be used for the enantioselective production of (S)-IPG.
Assuntos
Bacillus subtilis/genética , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Genes Bacterianos , Bacillus subtilis/enzimologia , Carboxilesterase , Hidrolases de Éster Carboxílico/química , Clonagem Molecular , Homologia de Sequência de Aminoácidos , EstereoisomerismoRESUMO
Pseudomonas alcaligenes secretes a lipase with a high pH optimum, which has interesting properties for application in detergents. The expression of the lipase is strongly dependent on the presence of lipids in the growth medium such as soybean oil. The promoter of the gene was characterized and found to have resemblance to sigma54 controlled promoters, which are known to be tightly regulated. The transcription start was mapped precisely downstream of a sequence with close similarity to the -12/-24 consensus sequence of sigma54 controlled promoters. Interestingly, a hyperproducer mutant strain was isolated and found to have a C to T mutation in the -12/-24 promoter consensus region. In addition an Upstream Activating Sequence (UAS) with homology to sigma54 UAS consensus sequences was identified. It was demonstrated that an increase of the distance from the UAS to the transcription start or the deletion of the UAS results in significantly lower expression levels of lipase. A systematic mutational analysis of the UAS sequence has resulted in a variant with an increased lipase expression.
Assuntos
Lipase/genética , Regiões Promotoras Genéticas , Pseudomonas/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Sequência Consenso , DNA Recombinante , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Lipase/química , Lipase/metabolismo , Dados de Sequência Molecular , Mutagênese , Pseudomonas/genética , RNA Mensageiro/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sequências Reguladoras de Ácido Nucleico , Relação Estrutura-AtividadeRESUMO
The time course of the levels of artemisinin, its biosynthetic precursors and the biosynthetically related sesquiterpenes was monitored during a vegetation period of Artemisia annua plants of different geographical origin. Considerable differences in contents of artemisinin and its direct precursors artemisinic acid and dihydroartemisinic acid were found between these A. annua's. For the first time the A. annua plants of different geographical origin were found to belong to different chemotypes. A chemotype with a high artemisinin level was found to have also a high dihydroartemisinic acid level but a relatively low artemisinic acid level. Reversibly, a chemotype with low levels of artemisinin and dihydroartemisinic acid contained a high artemisinic acid level. Artemisinic acid is considered to be the direct precursor of dihydroartemisinic acid in the biosynthetic pathway of artemisinin. The observed accumulation of artemisinic acid in one of the A. annua chemotypes may indicate the presence of a rate-limiting step in the biosynthetic pathway of artemisinin. The enzymatic reduction of artemisinic acid into dihydroartemisinic acid is probably a "bottle neck" in the biosynthetic pathway of artemisinin in varieties with high artemisinic acid and consequentially low artemisinin levels. After a night-frost period, the level of artemisinin was increased, in the Vietnamese A. annua plants, while the dihydroartemisinic acid level was decreased. This phenomenon is in accordance with our hypothesis that stress triggers the conversion of dihydroartemisinic acid to artemisinin. It is suggested that the presence of high levels of dihydroartemisinic acid may be an adaptation to stress conditions (e.g., night-frost), during which relatively high levels of 1O2 are formed. Dihydroartemisinic acid gives the plant protection by reacting with these reactive oxygen species yielding artemisinin as stable end-product.
Assuntos
Artemisia/química , Artemisininas , Plantas Medicinais , Estações do Ano , Sesquiterpenos/análise , Cromatografia Líquida de Alta Pressão , Cromatografia Gasosa-Espectrometria de Massas , Espectrometria de MassasRESUMO
The increased insight into the mechanism of bacterial protein translocation has resulted in new concepts for the production of heterologous proteins. The periplasm of gram-negative bacteria is revealed to have a role as a 'protein construction compartment', which can be used to fold complex proteins. Passage across the outer membrane, however, remains a challenge due to the high selectivity of the outer membrane translocase. In gram-positive bacteria, slow folding at the membrane-cell-wall interface can make heterologous proteins vulnerable to degradation by wall-associated proteases. The recent identification of thiol-disulfide oxidoreductases in Bacillus subtilis might open the possibility of secreting proteins containing multiple disulfide bonds from this host.
Assuntos
Proteínas de Bactérias/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Transporte Biológico Ativo , Biotecnologia , Membrana Celular/metabolismo , Citosol/metabolismo , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Engenharia de ProteínasRESUMO
The in vivo formation of disulfide bonds, which is critical for the stability and/or activity of many proteins, is catalyzed by thiol-disulfide oxidoreductases. In the present studies, we show that the Gram-positive eubacterium Bacillus subtilis contains three genes, denoted bdbA, bdbB, and bdbC, for thiol-disulfide oxidoreductases. Escherichia coli alkaline phosphatase, containing two disulfide bonds, was unstable when secreted by B. subtilis cells lacking BdbB or BdbC, and notably, the expression levels of bdbB and bdbC appeared to set a limit for the secretion of active alkaline phosphatase. Cells lacking BdbC also showed decreased stability of cell-associated forms of E. coli TEM-beta-lactamase, containing one disulfide bond. In contrast, BdbA was not required for the stability of alkaline phosphatase or beta-lactamase. Because BdbB and BdbC are typical membrane proteins, our findings suggest that they promote protein folding at the membrane-cell wall interface. Interestingly, pre-beta-lactamase processing to its mature form was stimulated in cells lacking BdbC, suggesting that the unfolded form of this precursor is a preferred substrate for signal peptidase. Surprisingly, cells lacking BdbC did not develop competence for DNA uptake, indicating the involvement of disulfide bond-containing proteins in this process. Unlike E. coli and yeast, none of the thiol-disulfide oxidoreductases of B. subtilis was required for growth in the presence of reducing agents. In conclusion, our observations indicate that BdbB and BdbC have a general role in disulfide bond formation, whereas BdbA may be dedicated to a specific process.
Assuntos
Bacillus subtilis/enzimologia , Proteína Dissulfeto Redutase (Glutationa)/genética , Fosfatase Alcalina/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/genética , Quinases Ciclina-Dependentes/metabolismo , Dissulfetos/metabolismo , Precursores Enzimáticos/metabolismo , Estabilidade Enzimática , Escherichia coli/enzimologia , Proteínas de Escherichia coli , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Genes Bacterianos/genética , Óperon Lac , Dados de Sequência Molecular , Mutação , Dobramento de Proteína , Homologia de Sequência de Aminoácidos , beta-Lactamases/metabolismoRESUMO
The large heterodimeric penicillin G acylase from Alcaligenes faecalis was displayed on the surface of phage fd. We fused the coding sequence (alpha subunit-internal peptide-beta subunit) to the gene of a phage coat protein. A modified g3p signal sequence was used to direct the polypeptide to the periplasm. Here we show that a heterodimeric enzyme can be expressed as a fusion protein that matures to an active biocatalyst connected to the coat protein of phage fd, resulting in a phage to which the beta-subunit is covalently linked and the alpha-subunit is non-covalently attached. The enzyme can be displayed either fused to the minor coat protein g3p or fused to the major coat protein g8p. In both cases the penicillin G acylase on the phage has the same Michaelis constant as its freely soluble counterpart, indicating a proper folding and catalytic activity of the displayed enzyme. The display of the heterodimer on phage not only allows its further use in protein engineering but also offers the possibility of applying this technology for the excretion of the enzyme into the extracellular medium, facilitating purification of the protein. With the example of penicillin acylase the upper limit for a protein to become functionally displayed by phage fd has been further explored. Polyvalent display was not observed despite the use of genetic constructs designed for this aim. These results are discussed in relation to the pore size being formed by the g4p multimer.
Assuntos
Inovirus/enzimologia , Inovirus/genética , Penicilina Amidase/genética , Penicilina Amidase/metabolismo , Alcaligenes/enzimologia , Alcaligenes/genética , Sequência de Aminoácidos , Sequência de Bases , Capsídeo/química , Capsídeo/genética , Capsídeo/metabolismo , Códon de Terminação/genética , Primers do DNA/genética , DNA Recombinante/genética , Dimerização , Escherichia coli/genética , Cinética , Dados de Sequência Molecular , Penicilina Amidase/química , Processamento de Proteína Pós-Traducional , Estrutura Quaternária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Protein export in Escherichia coli is mediated by translocase, a multisubunit membrane protein complex with SecA as the peripheral subunit and the SecY, SecE, and SecG proteins as the integral membrane domain. In the gram-positive bacterium Bacillus subtilis, SecA, SecY, and SecE have been identified through genetic analysis. Sequence comparison of the Bacillus chromosome identified a potential homologue of SecG, termed YvaL. A chromosomal disruption of the yvaL gene results in mild cold sensitivity and causes a beta-lactamase secretion defect. The cold sensitivity is exacerbated by overexpression of the secretory protein alpha-amylase, whereas growth and beta-lactamase secretion are restored by coexpression of yvaL or the E. coli secG gene. These results indicate that the yvaL gene codes for a protein that is functionally homologous to SecG.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Genes Bacterianos , Proteínas de Membrana/genética , Sequência de Aminoácidos , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Transporte Biológico Ativo , Temperatura Baixa , Primers do DNA/genética , Escherichia coli/genética , Deleção de Genes , Teste de Complementação Genética , Dados de Sequência Molecular , Fenótipo , Canais de Translocação SEC , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Using colchicine we induced tetraploidy in Artemisia annua L. plants. During a vegetation period we monitored the time course of the levels of artemisinin, its direct precursors, the biosynthetically related sesquiterpenes and the essential oil content in the diploid (wild-type) and tetraploid A. annua plants. The averaged artemisinin level in tetraploids was 38% higher than that of the wild-type as measured over the whole vegetation period. In contrast, the averaged essential oil content of the tetraploids over this period was 32% lower. This might suggest a reciprocal correlation between artemisinin (sesquiterpenes) and the essential oil content (monoterpenes). The averaged biomass of the leaves of the tetraploid plants was lower compared to the wild-type plants. Therefore, the artemisinin yield per m2 tetraploids was decreased by 25%. Although the tetraploid plants were smaller than the wild-type plants, certain individual organs like the leaves were considerably larger, and seeds obtained by cross pollination between tetraploid A. annua plants had a spectacular size. In principle, tetraploid A. annua can be a useful starting material for a breeding program in order to obtain larger and faster growing plants, which produce higher levels of artemisinin.
Assuntos
Artemisininas , Plantas Medicinais/genética , Ploidias , Estações do Ano , Sesquiterpenos/análise , Cromatografia Líquida de Alta Pressão , Sesquiterpenos/químicaRESUMO
Pseudomonas alcaligenes M-1 has been selected from an intensive screening for micro-organisms that can naturally produce a lipase active in detergent formulations. The lipase expression has been increased to allow high level secretion from Pseudomonas alcaligenes, via the introduction of multi-copy plasmids. In order to improve the lipase yield further, the phenotype enhancement method has been developed. This idea comprises the reintroduction of a cosmid library with random chromosomal fragments in a P. alcaligenes strain with already high lipase productivity. One of the strains which showed an enhanced lipase production appeared to contain a cosmid encoding the outer membrane secretion genes. These xcp-genes are clustered in two divergently transcribed operons similar to the situation in Pseudomonas aeruginosa. Remarkably and dissimilar to P. aeruginosa, in between the two xcp gene clusters, two reading frames of unknown function--OrfV and OrfX--are present. For OrfX no equivalent can be found in the known protein data bases. On the other hand, OrfV shows homology to the regulatory proteins MalT and AcoK. Some evidence is provided that suggests that OrfV acts as a regulator of the xcp operons. A model is proposed for the regulation of the secretion system from P. alcaligenes.
Assuntos
Proteínas de Bactérias/metabolismo , Lipase/biossíntese , Proteínas de Membrana/metabolismo , Pseudomonas/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Cosmídeos , DNA Bacteriano , Escherichia coli/genética , Proteínas de Membrana/genética , Dados de Sequência Molecular , Óperon , Fenótipo , Pseudomonas/genética , Homologia de Sequência de AminoácidosRESUMO
Approximately 47% of the genes of the Gram-positive bacterium Bacillus subtilis belong to paralogous gene families. The present studies were aimed at the functional analysis of the sip gene family of B. subtilis, consisting of five chromosomal genes, denoted sipS, sipT, sipU, sipV, and sipW. All five sip genes specify type I signal peptidases (SPases), which are actively involved in the processing of secretory preproteins. Interestingly, strains lacking as many as four of these SPases could be obtained. As shown with a temperature-sensitive SipS variant, only cells lacking both SipS and SipT were not viable, which may be caused by jamming of the secretion machinery with secretory preproteins. Thus, SipS and SipT are of major importance for protein secretion. This conclusion is underscored by the observation that only the transcription of the sipS and sipT genes is temporally controlled via the DegS-DegU regulatory system, in concert with the transcription of most genes for secretory preproteins. Notably, the newly identified SPase SipW is highly similar to SPases from archaea and the ER membrane of eukaryotes, suggesting that these enzymes form a subfamily of the type I SPases, which is conserved in the three domains of life.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Membrana , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Archaea/enzimologia , Archaea/genética , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Sequência de Bases , Sequência Conservada , Primers do DNA/genética , Retículo Endoplasmático/enzimologia , Células Eucarióticas , Genes Bacterianos , Dados de Sequência Molecular , Família Multigênica , Mutação , Reação em Cadeia da Polimerase , Processamento de Proteína Pós-Traducional , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/classificação , Serina Endopeptidases/genéticaRESUMO
In the present studies, we show that the SecD and SecF equivalents of the Gram-positive bacterium Bacillus subtilis are jointly present in one polypeptide, denoted SecDF, that is required to maintain a high capacity for protein secretion. Unlike the SecD subunit of the pre-protein translocase of Escherichia coli, SecDF of B. subtilis was not required for the release of a mature secretory protein from the membrane, indicating that SecDF is involved in earlier translocation steps. Strains lacking intact SecDF showed a cold-sensitive phenotype, which was exacerbated by high level production of secretory proteins, indicating that protein translocation in B. subtilis is intrinsically cold-sensitive. Comparison with SecD and SecF proteins from other organisms revealed the presence of 10 conserved regions in SecDF, some of which appear to be important for SecDF function. Interestingly, the SecDF protein of B. subtilis has 12 putative transmembrane domains. Thus, SecDF does not only show sequence similarity but also structural similarity to secondary solute transporters. Our data suggest that SecDF of B. subtilis represents a novel type of the SecD and SecF proteins, which seems to be present in at least two other organisms.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Proteínas de Membrana Transportadoras , Sequência de Aminoácidos , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Escherichia coli/genética , Genes Bacterianos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutação , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
Pseudomonas alcaligenes M-1 secretes an alkaline lipase, which has excellent characteristics for the removal of fatty stains under modern washing conditions. A fed-batch fermentation process based on the secretion of the alkaline lipase from P. alcaligenes was developed. Due to the inability of P. alcaligenes to grow on glucose, citric acid and soybean oil were applied as substrates in the batch phase and feed phase, respectively. The gene encoding the high-alkaline lipase from P. alcaligenes was isolated and characterized. Amplification of lipase gene copies in P. alcaligenes with the aid of low- and high-copy-number plasmids resulted in an increase of lipase expression that was apparently colinear with the gene copy number. It was found that overexpression of the lipase helper gene, lipB, produced a stimulating effect in strains with high copy numbers (> 20) of the lipase structural gene, lipA. In strains with lipA on a low-copy-number vector, the lipB gene did not show any effect, suggesting that LipB is required in a low ratio to LipA only. During scaling up of the fermentation process to 100 m3, severe losses in lipase productivity were observed. Simulations have identified an increased level of dissolved carbon dioxide as the most probable cause for the scale-up losses. A large-scale fermentation protocol with a reduced dissolved carbon dioxide concentration resulted in a substantial elimination of the scale-up loss.
Assuntos
Proteínas de Bactérias/genética , Hidrolases de Éster Carboxílico/genética , Lipase/genética , Pseudomonas/enzimologia , Transativadores/genética , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Fermentação , Dosagem de Genes , Lipase/metabolismo , Dados de Sequência Molecular , Pseudomonas/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transativadores/metabolismoRESUMO
Alcaligenes faecalis penicillin G acylase is more stable than the Escherichia coli enzyme. The activity of the A. faecalis enzyme was not affected by incubation at 50 degrees C for 20 min, whereas more than 50% of the E. coli enzyme was irreversibly inactivated by the same treatment. To study the molecular basis of this higher stability, the A. faecalis enzyme was isolated and its gene was cloned and sequenced. The gene encodes a polypeptide that is characteristic of periplasmic penicillin G acylase (signal peptide-alpha subunit-spacer-beta subunit). Purification, N-terminal amino acid analysis, and molecular mass determination of the penicillin G acylase showed that the alpha and beta subunits have molecular masses of 23.0 and 62.7 kDa, respectively. The length of the spacer is 37 amino acids. Amino acid sequence alignment demonstrated significant homology with the penicillin G acylase from E. coli A unique feature of the A. faecalis enzyme is the presence of two cysteines that form a disulfide bridge. The stability of the A. faecalis penicillin G acylase, but not that of the E. coli enzyme, which has no cysteines, was decreased by a reductant. Thus, the improved thermostability is attributed to the presence of the disulfide bridge.
Assuntos
Alcaligenes/enzimologia , Alcaligenes/genética , Genes Bacterianos , Penicilina Amidase/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Cisteína/química , DNA Bacteriano/genética , Dissulfetos/química , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Dados de Sequência Molecular , Estrutura Molecular , Sondas de Oligonucleotídeos/genética , Penicilina Amidase/química , Penicilina Amidase/metabolismo , Conformação Proteica , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , TemperaturaRESUMO
The heterologous expression of proteins is without doubt one of the most fascinating applications of the recombinant DNA technique. Despite clear successes many attempts to produce a certain protein in a heterologous host cell have met with technical difficulties. Secretion from cells has been used as a solution to overcome the intracellular formation of inactive protein. Microorganisms with a history of use in the fermentation industry exhibit clear advantages over the frequently used Escherichia coli as host cells for secreted products. Interleukin-3, chymosin and phytase are examples of commercial products that are produced efficiently with the aid of industrial microorganisms.