Bacillus subtilis as cell factory for pharmaceutical proteins:a biotechnological approach to optimize the host organism

AbstractBacillus subtilis is a rod-shaped, Gram-positive soil bacterium that secretes numerous enzymes to degrade a variety of substrates, enabling the bacterium to survive in a continuously changing environment. These enzymes are produced commercially and this production represents about 60% of the industrial-enzyme market. Unfortunately, the secretion of heterologous proteins, originating from Gram-negative bacteria or from eukaryotes, is often severely hampered. Several bottlenecks in the B. subtilis secretion pathway, such as poor targeting to the translocase, degradation of the secretory protein, and incorrect folding, have been revealed. Nevertheless, research into the mechanisms and control of the secretion pathways will lead to improved Bacillus protein secretion systems and broaden the applications as industrial production host. This review focuses on studies that aimed at optimizing B. subtilis as cell factory for commercially interesting heterologous proteins

Characterization of an alkalophilic Bacillus brevis isolate with respect to its endo-(1,3-1,4)-β-glucanase gene, protein hyperproduction and the degS-degU operon

Bibliography: pages 127-144.Bacillus brevis Alk 36 was isolated from soil during a screening programme for the selection of extracellular enzyme producing strains. A gene coding for an endo(1,3- 1,4 )-.8-glucanase (or lichenase) was cloned from B. brevis Alk 36 and expressed in Escherichia coli. The nucleotide sequence of this gene was determined and found to encode a protein of 252 amino acid residues. The amino acid sequence of the B. brevis lichenase gene showed only a 50% similarity to previously published data for Bacillus endo-(1,3-1,4)-β-glucanases. The enzyme exhibited some unique properties. The optimum temperature and pH for enzyme activity were 65-70°C and 8-10, respectively. When held at 75°C for 1 h, 75% residual activity was measured. The molecular mass was estimated to be 29 kDa and the enzyme was found to be resistant to sodium dodecyl sulphate (SDS). B. brevis Alk 36 was evaluated as a potential host strain for the efficient production and secretion of foreign proteins and was found to grow optimally between pH 8.0 and pH 9.5 and between 42°C and 52°C. B. brevis was successfully transformed using vector DNA and was found to produce relatively low levels of protease. In addition, it was evaluated as a possible protein hyper-secreting strain. However, using PCR technology, the highly conserved cell wall protein genes could not be positively identified in B. brevis Alk 36

Effect of cerulenin on the production of alpha-lysin by Staphylococcus aureus Wood 46

This investigation was started with the intention of determining whether the secretion of haemolysins by Staphylococcus aureus took place in accordance with the signal hypothesis of Blobel and Dobberstein (1975 a,b). Before embarking on the main object of the investigation, various studies were made on the production and purification of the alpha-haemolysin of S.aureus Wood 46. The production of alpha-, beta- and delta-haemolysins and protease(s) by S.aureus Wood 46 was biphasic, in agreement with the published data (Duncan and Cho, 1971; McNiven and Arbuthnott, 1972). Purified alphalysin prepared by ammonium sulphate precipitation, chromatography on controlled-pore glass and electrofocusing had a pI of 8.5, a specific haemolytic activity of 1.63 x 10

Using synthetic biology to engineer bacterial cellulose to improve its industrial applications

The production and usage of biomaterials is no new feat for humanity. We have constantly used the natural resources around us to progress as a species but now that we enter a more advanced age the reliance on primitive biomaterials and their extraction methods has started to become a threat to the way of life to our species. The unsustainable production of materials for use has led us to a resource and climate crisis and the need for more sustainable, renewable, and smarter biomaterials has become one of the priorities for our research and development sectors. Utilising Synthetic Biology and its tools to engineer novel biomaterials has recently started to progress. Engineering microorganisms to synthesis materials that can be used for applications such as sensing-and-responding to their environment to having unique filtration properties able to purify contaminated water sources has recently been achieved. Bacteria from the Acetobacter genus know to produce a biomaterial called bacterial cellulose (BC) have been of increasing interest. One specific Acetobacter species named Komagateaibacter rhaeticus iGEM is a model bacterial cellulose producing organism and is able to synthesis copious amounts of this biomaterial. As it is a genetically amenable strain it allows us to engineer in functionalities and increase the amounts of BC that the organism can produce making it a smarter, engineerable, living biomaterial. In this work we have set out to apply Synthetic Biology principles to K. rhaeticus iGEM strain. By metabolically engineering this organism to overexpress a number of genes in bacterial cellulose synthesis pathway and the fructose utilisation pathway we were able to increase the yield of BC produced by K. rhaeticus. Additionally, we investigated the secretome of the organism in order to utilise its native Sec and Tat secretion pathways in order to secrete reporter proteins to achieve proof-of-concept of the ability to functionalise BC using K. rhaeticus as a secretion platform. This would provide the basis for future work in which one could grow a functional biomaterial capable of bioremediation, biosensing and wound repair with a reduced burden on current energy demanding processes and at both a low and high scale depending on the needs of the situation.Open Acces

Kationisten antimikrobipeptidien aiheuttamat stressivasteet Gram-positiivisissa bakteereissa

As the resistance of bacteria to conventional antibiotics has become an increasing problem, new antimicrobial drugs are urgently needed. One possible source of new antibacterial agents is a group of cationic antimicrobial peptides (CAMPs) produced by practically all living organisms. These peptides are typically small, amphipathic and positively charged and contain well defined a-helical or b-sheet secondary structures. The main antibacterial action mechanism of CAMPs is considered to be disruption of the cell membrane, but other targets of CAMPs also exist. Some bacterial species have evolved defence mechanisms against the harmful effects of CAMPs. One of the most effective defence mechanisms is reduction of the net negative charge of bacterial cell surfaces. Global analysis of gene expression of two Gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus, was used to further study the stress responses induced by different types of CAMPs. B. subtilis cells were treated with sublethal concentrations of a-helical peptide LL-37, b-sheet peptide protegrin 1 or synthetic analogue poly-L-lysine, and the changes in gene expression were studied using DNA macroarrays. In the case of S. aureus, three different a-helical peptides were selected for the transcriptome analyses: temporin L, ovispirin-1 and dermaseptin K4-S4(1-16). Transcriptional changes caused by peptide stress were examined using oligo DNA microarrays. The transcriptome analysis revealed two main cell signalling mechanisms mediating CAMP stress responses in Gram-positive bacteria: extracytoplasmic function (ECF)sigma factors and two-component systems (TCSs). In B. subtilis, ECF sigma factors sigW and sigM as well as TCS LiaRS responded to the cell membrane disruption caused by CAMPs. In S. aureus, CAMPs caused a similar stress response to antibiotics interfering in cell wall synthesis, and TCS VraSR was strongly activated. All of these transcriptional regulators are known to respond to several compounds other than CAMPs interfering with cell envelope integrity, suggesting that they sense cell envelope stress in general. Among the most strongly induced genes were yxdLM (in B. subtilis) and vraDE (in S. aureus) encoding homologous ABC transporters. Transcription of yxdLM and vraDE operons is controlled by TCSs YxdJK and ApsRS, respectively. These TCSs seemed to be responsible for the direct recognition of CAMPs. The yxdLM operon was specifically induced by LL-37, but its role in CAMP resistance remained unclear. VraDE was proven to be a bacitracin transporter. We also showed that the net positive charge of the cell wall affects the signalrecognition of different TCSs responding to cell envelope stress. Inactivation of the Dlt system responsible for the D-alanylation of teichoic acids had a strong and differential effect on the activity of the studied TCSs, depending on their functional role in cells and the stimuli they sense.Tauteja aiheuttavien bakteerien lisääntyvä vastustuskyky perinteisiä antibiootteja vastaan on kasvava ongelma terveydenhuollossa. Yksi potentiaalinen uusi vaihtoehto bakteeri-infektioiden hoitoon saattavat olla pienet positiivisesti varautuneet peptidit (ns. kationiset antimikrobipeptidit). Näitä peptideitä tuottavat lähes kaikki elävät organismit aina yksisoluisista eliöistä kasveihin ja eläimiin saakka osana omaa puolustusjärjestelmäänsä. Vain harvat bakteerit ovat vastustuskykyisiä kationisille antimikrobipeptideille. Poikkeuksiakin on, kuten Staphylococcus aureus – bakteeri, joka on yleisin sairaalainfektioiden aiheuttaja Suomessa. S aureus on erittäin vastustuskykyinen useimmille ihmisen tuottamille kationisille antimikrobipeptideille, minkä on ajateltu olevan yksi syy siihen, että kyseinen bakteeri pystyy valloittamaan elintilaa ihmisen iholla ja limakalvoilla ja sopivien olosuhteiden vallitessa aiheuttamaan infektion elimistössä. Tässä tutkimuksessa S. aureus- bakteerit altistettiin erilaisille kationisille antimikrobipeptideille ja pyrittiin tunnistamaan niitä bakteerigeenejä, jotka osallistuvat puolustukseen peptidejä vastaan. Tästä saattaa olla apua tulevaisuudessa uusia lääkkeitä kehitettäessä. Vertailun vuoksi tutkittiin myös toisen mikrobin, Bacillus subtilis- bakteerin, vastetta peptidialtistukseen. Toisin kuin S. aureus, B. subtilis on ihmiselle täysin vaaraton maaperän normaali bakteeri. Näitä kahta bakteeria vertaamalla voitiin selvittää onko taudinaiheuttajille kehittynyt erityisiä mekanismeja kationisten peptidien tuhoamista varten. Tulokset osoittivat, että bakteerien soluseinällä on erittäin tärkeä rooli puolustautumisessa peptidejä vastaan. Myös erityisiä mekanismeja peptidien torjuntaan on olemassa, mutta nämä ovat yleensä erikoistuneet poistamaan lähisukuisten, samassa elinympäristössä olevien bakteerien tuottamia peptidejä. Tulokset antavat viitteitä siitä, että kationisilla antimikrobipeptideillä voisi olla käyttöä infektioiden hoidossa yhdessä perinteisten antibioottien kanssa

The development of a flagellin surface display expression system in the gram-positive bacterium, Bacillus halodurans Alk36

Includes bibliographical references (leaves 103-126).This study relates to the development of an alkaliphilic, thermo-tolerant, Gram-positive isolate, Bacillus halodurans Alk36, for the over-production and surface display of chimeric gene products. This bacterium harbors the endogenous genetic background to over-produce flagellin protein continuously. In order to harness this ability, key genetic tools, such as gene targeted inactivation, were developed for this strain. The hag gene which codes for flagellin was inactivated on the chromosome giving rise to the B. halodurans BhFC0l mutant. This strain was non-motile as determined on motility plates and confirmed by PCR analysis. Motility was, however, restored through complementation of the expression vector carrying a functional hag gene. Polylinkers were inserted as in-frame, chimeric, flagellin sandwich fusions in order to identify the permissive insertion sites corresponding to the variable regions of the flagellin protein. Flagellin expression and motility were evaluated for these constructs. Two sites were identified for possible peptide insertion in the flagellin gene, one of which produced functional flagella and was able to restore the motility phenotype to a non-motile mutant. Peptides encoding a poly-histidine peptide and the HIV-l clade C gpl20 epitope were respectively incorporated into both of the permissive sites as in-frame fusions and found to be successfully displayed on the cell surface. The poly-His peptide was shown to be functional through metal binding and affinity purification studies. The display of the HIV-1 subtype C gp 120 V3 loop was also shown to be functional through immunological studies using peptide specific antibodies. Surface display of the poly-His and HIV-l epitope was shown to have improved metal binding and enhanced expression levels of the chimeric flagellin when the peptides were insel1ed at amino acid position 180 (pSECNC6). This specific site is the only insertion point that falls within the re-defined variable domain of the FliC protein from B. halodurans Alk36