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  • 1.
    Backlund, Emma
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Ignatushchenko, Marina
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Suppressing glucose uptake and acetic acid production increases membrane protein overexpression in Escherichia coli.2011In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 10, no 1, p. 35-Article in journal (Refereed)
    Abstract [en]

    The production of integral membrane spanning proteins (IMP's) constitutes a bottleneck in pharmaceutical development. It was long considered that the state-of-the-art was to produce the proteins as inclusion bodies using a powerful induction system. However, the quality of the protein was compromised and the production of a soluble protein that is incorporated into the membrane from which it is extracted is now considered to be a better method. Earlier research has indicated that a slower rate of protein synthesis might overcome the tendency to form inclusion bodies. We here suggest the use of a set of E. coli mutants characterized by a slower rate of growth and protein synthesis as a tool for increasing the amount of soluble protein in high- throughput protein production processes. RESULTS: A set of five IMP's was chosen which were expressed in three mutants and the corresponding WT cell (control). The mutations led to three different substrate uptake rates, two of which were considerably slower than that of the wild type. Using the mutants, we were able to express three out of the five membrane proteins. Most successful was the mutant growing at 50% of the wild type growth rate. A further effect of a low growth rate is a low acetic acid formation, and we believe that this is a possible reason for the better production. This hypothesis was further supported by expression from the BL21(DE3) strain, using the same plasmid. This strain grows at a high growth rate but nevertheless yields only small amounts of acetic acid. This strain was also able to express three out of the five IMP's, although at lower quantities. CONCLUSIONS: The use of mutants that reduce the specific substrate uptake rate seems to be a versatile tool for overcoming some of the difficulties in the production of integral membrane spanning proteins. A set of strains with mutations in the glucose uptake system and with a lower acetic acid formation were able to produce three out of five membrane proteins that it was not possible to produce with the corresponding wild type.

  • 2. Bostrom, M.
    et al.
    Larsson, Gen
    KTH, Superseded Departments, Biotechnology.
    Process design for recombinant protein production based on the promoter, P-malK2004In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 66, no 2, p. 200-208Article in journal (Refereed)
    Abstract [en]

    P-malK is induced through activation of MalT, by the formation of maltotriose and cyclic adenosine monophosphate ( cAMP). The possibility to influence endogenous inducer levels is used to vary the production rates in specifically designed production protocols. Induction based on a batch process protocol on maltose gives low production rates, as the result of a lack of cAMP, which is shown to be of major importance to fully induce this promoter. Two mechanisms are thus used to influence the levels of maltotriose and/or cAMP formation: ( 1) catabolite derepression achieved from low glucose concentration and ( 2) catabolite derepression/inducer exclusion from diauxic growth on glucose/maltose. Fed-batch processes based on limited amounts of glucose result in product accumulation of up to 10% of the total protein. Depending on the feed of limiting glucose, different production profiles are developed. The initial increase in the production rate is due to maltotriose formation from endogenous glycogen degradation while, later in the process, production can be further supported by elevated levels of cAMP, provided the feed rate is sufficiently low. The introduction of maltose after a preceding fed-batch process on glucose can be efficiently used to produce maltotriose in combination with cAMP formation in the event of catabolite derepression. This leads to higher production rates and a further increase in product accumulation of up to 30% of the total protein. The diauxic growth phase resulting from the shift in carbon source can be shortened and even avoided by the design of the preceding feed-rate of glucose. It is postulated that proper design of the inoculum and initial phases of production can reduce basal levels of product formation. With this promoter, the production rate can be as high as 65 units mg(-1) h(-1) and the time to reach a maximal production rate can be designed to take up to 8 h. Furthermore, the duration of the production rate can be as long as 7 h.

  • 3.
    Boström, Maria
    et al.
    KTH, School of Biotechnology (BIO).
    Markland, Katrin
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Sandén, Anna Maria
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Hedhammar, My
    KTH, School of Biotechnology (BIO), Proteomics. KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics. KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Effect of substrate feed rate on recombinant protein secretion, degradation and invlusion body formation in Escherichia coli2005In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 68, no 1, p. 82-90Article in journal (Refereed)
    Abstract [en]

    The effect of changes in substrate feed rate during fedbatch cultivation was investigated with respect to soluble protein formation and transport of product to the periplasm in Escherichia coli. Production was transcribed from the P-malK promoter; and the cytoplasmic part of the production was compared with production from the P-lacUV5 promoter. The fusion protein product, Zb-MalE, was at all times accumulated in the soluble protein fraction except during high-feed-rate production in the cytoplasm. This was due to a substantial degree of proteolysis in all production systems, as shown by the degradation pattern of the product. The product was also further subjected to inclusion body fori-nation. Production in the periplasm resulted in accumulation of the full-length protein; and this production system led to a cellular physiology where the stringent response could be avoided. Furthermore, the secretion could be used to abort the diauxic growth phase resulting from use of the P-malK promoter. At high feed rate, the accumulation of acetic acid, due to overflow metabolism, could furthermore be completely avoided.

  • 4.
    Bäcklund, Emma
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Markland, Katrin
    KTH, School of Biotechnology (BIO).
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Cell engineering of Escherichia coli allows high cell density accumulation without fed-batch process control2008In: Bioprocess and biosystems engineering (Print), ISSN 1615-7591, E-ISSN 1615-7605, Vol. 31, no 1, p. 11-20Article in journal (Refereed)
    Abstract [en]

    A set of mutations in the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was used to create Escherichia coli strains with a reduced uptake rate of glucose. This allows a growth restriction, which is controlled on cellular rather than reactor level, which is typical of the fed-batch cultivation concept. Batch growth of the engineered strains resulted in cell accumulation profiles corresponding to a growth rate of 0.78, 0.38 and 0.25 h(-1), respectively. The performance of the mutants in batch cultivation was compared to fed-batch cultivation of the wild type cell using restricted glucose feed to arrive at the corresponding growth profiles. Results show that the acetate production, oxygen consumption and product formation were similar, when a recombinant product was induced from the lacUV5 promoter. Ten times more cells could be produced in batch cultivation using the mutants without the growth detrimental production of acetic acid. This allows high cell density production without the establishment of elaborate fed-batch control equipment. The technique is suggested as a versatile tool in high throughput multiparallel protein production but also for increasing the number of experiments performed during process development while keeping conditions similar to the large-scale fed-batch performance.

  • 5.
    Bäcklund, Emma
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Markland, Katrin
    KTH, School of Biotechnology (BIO).
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Fedbatch design for periplasmic product retention in Escherichia coli2008In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 135, no 4, p. 358-365Article in journal (Refereed)
    Abstract [en]

    The feed profile of glucose during fedbatch cultivation could be used to influence the retention of the periplasmic product ZZ-cutinase. An increased feed rate led to a higher production rate but also to an increased specific leakage, which reduced the periplasmic retention. Three growth rates: 0.3, 0.2 and 0.1 h-1 where studied and resulted in 20, 9 and 6%, respectively, of the total ZZ-cutinase accumulating in the medium. It was also shown that leakage during fedbatch production of a Fab fragment was also influenced by the feed rate in a similar manner to ZZ-cutinase. If intracellular product accumulation is desired the advantage of a high productivity, resulting from a high substrate feed rate, is diminished because of a reduced product retention. Biochemical analysis revealed that the growth rate, resulting from a glucose limited feed, influenced the outer membrane protein compositions with respect to OmpF and LamB, whilst OmpA was largely unaffected. As the feed rate increased the amount of total outer membrane protein decreased. When ZZ-cutinase was produced there were further reductions in outer membrane protein accumulation, by 82, 100 and 22% for OmpF, LamB and OmpA, respectively, and the total reduction was almost 60% with a high product formation rate. We suggest that the reduced titre of the outer membrane proteins, OmpF and LamB, may have contributed to a reduced ability for the cell to retain recombinant protein secreted to the periplasm.

  • 6.
    Guevara, Monica
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Jarmander, Johan
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Perez-Zabaleta, Mariel
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Quillaguaman, Jorge
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Production of 3-hydroxybutyrate by E. coli: Application of Nitrogen and Phosphorous limitation to steer fluxes to product formation2014In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 31, p. S148-S148Article in journal (Other academic)
  • 7.
    Guevara-Martínez, Mónica
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. Faculty of Science and Technology, Center of Biotechnology, Universidad Mayor de San Simón, Cochabamba, Bolivia.
    Perez-Zabaleta, Mariel
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. Faculty of Science and Technology, Center of Biotechnology, Universidad Mayor de San Simón, Cochabamba, Bolivia.
    Gustavsson, Martin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Quillaguamán, Jorge
    Faculty of Science and Technology, Center of Biotechnology, Universidad Mayor de San Simón, Cochabamba, Bolivia.
    Larsson, Gen
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT. KTH, Superseded Departments (pre-2005), Biotechnology.
    van Maris, Antonius J. A.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    The role of the acyl-CoA thioesterase YciA in the production of (R)-3-hydroxybutyrate by recombinant Escherichia coli2019In: Applied Microbiology and Biotechnology, p. 1-12Article in journal (Refereed)
    Abstract [en]

    Biotechnologically produced (R)-3-hydroxybutyrate is an interesting pre-cursor for antibiotics, vitamins, and other molecules benefitting from enantioselective production. An often-employed pathway for (R)-3-hydroxybutyrate production in recombinant E. coli consists of three-steps: (1) condensation of two acetyl-CoA molecules to acetoacetyl-CoA, (2) reduction of acetoacetyl-CoA to (R)-3-hydroxybutyrate-CoA, and (3) hydrolysis of (R)-3-hydroxybutyrate-CoA to (R)-3-hydroxybutyrate by thioesterase. Whereas for the first two steps, many proven heterologous candidate genes exist, the role of either endogenous or heterologous thioesterases is less defined. This study investigates the contribution of four native thioesterases (TesA, TesB, YciA, and FadM) to (R)-3-hydroxybutyrate production by engineered E. coli AF1000 containing a thiolase and reductase from Halomonas boliviensis. Deletion of yciA decreased the (R)-3-hydroxybutyrate yield by 43%, whereas deletion of tesB and fadM resulted in only minor decreases. Overexpression of yciA resulted in doubling of (R)-3-hydroxybutyrate titer, productivity, and yield in batch cultures. Together with overexpression of glucose-6-phosphate dehydrogenase, this resulted in a 2.7-fold increase in the final (R)-3-hydroxybutyrate concentration in batch cultivations and in a final (R)-3-hydroxybutyrate titer of 14.3 g L-1 in fed-batch cultures. The positive impact of yciA overexpression in this study, which is opposite to previous results where thioesterase was preceded by enzymes originating from different hosts or where (S)-3-hydroxybutyryl-CoA was the substrate, shows the importance of evaluating thioesterases within a specific pathway and in strains and cultivation conditions able to achieve significant product titers. While directly relevant for (R)-3-hydroxybutyrate production, these findings also contribute to pathway improvement or decreased by-product formation for other acyl-CoA-derived products.

  • 8.
    Guevara-Martínez, Mónica
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Univ Mayor de San Simon, Fac Sci & Technol, Ctr Biotechnol.
    Sjöberg Gällnö, Karin
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Sjöberg, Gustav
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Jarmander, Johan
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Perez-Zabaleta, Mariel
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Univ Mayor de San Simon, Fac Sci & Technol, Ctr Biotechnol.
    Quillaguamán, Jorge
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Regulating the production of (R)-3-hydroxybutyrate in Escherichia coli by N or P limitation2015In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 6, article id 844Article in journal (Refereed)
    Abstract [en]

    The chiral compound (R)-3-hydroxybutyrate (3HB) is naturally produced by many wild type organisms as the monomer for polyhydroxybutyrate (PHB). Both compounds are commercially valuable and co-polymeric polyhydroxyalkanoates have been used e.g., in medical applications for skin grafting and as components in pharmaceuticals. In this paper we investigate cultivation strategies for production of 3HB in the previously described E. coil strain AF1000 pJBGT3RX. This strain produces extracellular 3HB by expression of two genes from the PHB pathway of Halomonas boliviensis. H. boliviensis is a newly isolated halophile that forms PHB as a storage compound during carbon excess and simultaneous limitation of another nutrient like nitrogen and phosphorous. We hypothesize that a similar approach can be used to control the flux from acetylCoA to 3HB also in E coli; decreasing the flux to biomass and favoring the pathway to the product. We employed ammonium- or phosphate-limited fed-batch processes for comparison of the productivity at different nutrient limitation or starvation conditions. The feed rate was shown to affect the rate of glucose consumption, respiration, 3HB, and acetic acid production, although the proportions between them were more difficult to affect. The highest 3HB volumetric productivity, 1.5 g L-1 h(-1), was seen for phosphate-limitation.

  • 9.
    Gustavsson, Martin
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Bäcklund, Emma
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Optimisation of surface expression using the AIDA autotransporter2011In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 10Article in journal (Refereed)
    Abstract [en]

    Background: Bacterial surface display is of interest in many applications, including live vaccine development, screening of protein libraries and the development of whole cell biocatalysts. The goal of this work was to understand which parameters result in production of large quantities of cells that at the same time express desired levels of the chosen protein on the cell surface. For this purpose, staphylococcal protein Z was expressed using the AIDA autotransporter in Escherichia coli.

    Results: The use of an OmpT-negative E. coli mutant resulted in successful expression of the protein on the surface, while a clear degradation pattern was found in the wild type. The expression in the mutant resulted also in a more narrow distribution of the surface anchored protein within the population. Medium optimisation showed that minimal medium with glucose gave more than four times as high expression as LB-medium. Glucose limited fed-batch was used to increase the cell productivity and the highest protein levels were found at the highest feed rates. A maintained high surface expression up to cell dry weights of 18 g l(-1) could also be achieved by repeated glucose additions in batch cultivation where production was eventually reduced by low oxygen levels. In spite of this, the distribution in the bacterial population of the surface protein was narrower using the batch technique.

    Conclusions: A number of parameters in recombinant protein production were seen to influence the surface expression of the model protein with respect both to the productivity and to the display on the individual cell. The choice of medium and the cell design to remove proteolytic cleavage were however the most important. Both fed-batch and batch processing can be successfully used, but prolonged batch processing is probably only possible if the chosen strain has a low acetic acid production.

  • 10.
    Gustavsson, Martin
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Do, T. -H
    Lüthje, P.
    Tran, N. T.
    Brauner, A.
    Samuelson, Patrik
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Truong, N. H.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Improved cell surface display of Salmonella enterica serovar Enteritidis antigens in Escherichia coli2015In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 14, no 1, article id 47Article in journal (Refereed)
    Abstract [en]

    Background: Salmonella enterica serovar Enteritidis (SE) is one of the most potent pathogenic Salmonella serotypes causing food-borne diseases in humans. We have previously reported the use of the β-autotransporter AIDA-I to express the Salmonella flagellar protein H:gm and the SE serotype-specific fimbrial protein SefA at the surface of E. coli as live bacterial vaccine vehicles. While SefA was successfully displayed at the cell surface, virtually no full-length H:gm was exposed to the medium due to extensive proteolytic cleavage of the N-terminal region. In the present study, we addressed this issue by expressing a truncated H:gm variant (H:gmd) covering only the serotype-specific central region. This protein was also expressed in fusion to SefA (H:gmdSefA) to understand if the excellent translocation properties of SefA could be used to enhance the secretion and immunogenicity. Results: H:gmd and H:gmdSefA were both successfully translocated to the E. coli outer membrane as full-length proteins using the AIDA-I system. Whole-cell flow cytometric analysis confirmed that both antigens were displayed and accessible from the extracellular environment. In contrast to H:gm, the H:gmd protein was not only expressed as full-length protein, but it also seemed to promote the display of the protein fusion H:gmdSefA. Moreover, the epitopes appeared to be recognized by HT-29 intestinal cells, as measured by induction of the pro-inflammatory interleukin 8. Conclusions: We believe this study to be an important step towards a live bacterial vaccine against Salmonella due to the central role of the flagellar antigen H:gm and SefA in Salmonella infections and the corresponding immune responses against Salmonella.

  • 11.
    Gustavsson, Martin
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Hörnström, David
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Lundh, Susanna
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Belotserkovsky, Jaroslav
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Biocatalysis on the surface of Escherichia coli: melanin pigmentation of the cell exterior2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 36117Article in journal (Refereed)
    Abstract [en]

    Today, it is considered state-of-the-art to engineer living organisms for various biotechnology applications. Even though this has led to numerous scientific breakthroughs, the enclosed interior of bacterial cells still restricts interactions with enzymes, pathways and products due to the mass-transfer barrier formed by the cell envelope. To promote accessibility, we propose engineering of biocatalytic reactions and subsequent product deposition directly on the bacterial surface. As a proof-of-concept, we used the AIDA autotransporter vehicle for Escherichia coli surface expression of tyrosinase and fully oxidized externally added tyrosine to the biopolymer melanin. This resulted in a color change and creation of a black cell exterior. The capture of ninety percent of a pharmaceutical wastewater pollutant followed by regeneration of the cell bound melanin matrix through a simple pH change, shows the superior function and facilitated processing provided by the surface methodology. The broad adsorption spectrum of melanin could also allow removal of other micropollutants.

  • 12.
    Gustavsson, Martin
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Jarmander, Johan
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Arvestad, Lars
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Extended signal peptides in autotransporters are associated with large passenger proteinsManuscript (preprint) (Other academic)
  • 13.
    Gustavsson, Martin
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Muraleedharan, Madhu Nair
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Surface Expression of omega-Transaminase in Escherichia coli2014In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 80, no 7, p. 2293-2298Article in journal (Refereed)
    Abstract [en]

    Chiral amines are important for the chemical and pharmaceutical industries, and there is rapidly growing interest to use transaminases for their synthesis. Since the cost of the enzyme is an important factor for process economy, the use of whole-cell biocatalysts is attractive, since expensive purification and immobilization steps can be avoided. Display of the protein on the cell surface provides a possible way to reduce the mass transfer limitations of such biocatalysts. However, transaminases need to dimerize in order to become active, and furthermore, they require the cofactor pyridoxal phosphate; consequently, successful transaminase surface expression has not been reported thus far. In this work, we produced an Arthrobacter citreus omega-transaminase in Escherichia coli using a surface display vector based on the autotransporter adhesin involved in diffuse adherence (AIDA-I), which has previously been used for display of dimeric proteins. The correct localization of the transaminase in the E. coli outer membrane and its orientation toward the cell exterior were verified. Furthermore, transaminase activity was detected exclusively in the outer membrane protein fraction, showing that successful dimerization had occurred. The transaminase was found to be present in both full-length and proteolytically degraded forms. The removal of this proteolysis is considered to be the main obstacle to achieving sufficient whole-cell transaminase activity.

  • 14.
    Hörnström, David
    et al.
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Larsson, Gen
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    van Maris, Antonius J. A.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Gustavsson, Martin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Molecular optimization of autotransporter-based tyrosinase surface display2019In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1862, no 2, p. 486-494Article in journal (Refereed)
    Abstract [en]

    Display of recombinant enzymes on the cell surface of Gram-negative bacteria is a desirable feature with applications in whole-cell biocatalysis, affinity screening and degradation of environmental pollutants. One common technique for recombinant protein display on the Escherichia colt surface is autotransport. Successful autotransport of an enzyme largely depends on the following: (1) the size, sequence and structure of the displayed protein, (2) the cultivation conditions, and (3) the choice of the autotransporter expression system. Common problems with autotransporter-mediated surface display include low expression levels and truncated fusion proteins, which both limit the cell-specific activity. The present study investigated an autotransporter expression system for improved display of tyrosinase on the surface of E. coli by evaluating different variants of the autotransporter vector including: promoter region, signal peptide, the recombinant passenger, linker regions, and the autotransporter translocation unit itself. The impact of these changes on translocation to the cell surface was monitored by the cell-specific activity as well as antibody-based flow cytometric analysis of full-length and degraded passenger. Applying these strategies, the amount of displayed full-length tyrosinase on the cell surface was increased, resulting in an overall 5-fold increase of activity as compared to the initial autotransport expression system. Surprisingly, heterologous expression using 7 different translocation units all resulted in functional expression and only differed 1.6-fold in activity. This study provides a basis for broadening of the range of proteins that can be surface displayed and the development of new autotransporter-based processes in industrial-scale whole-cell biocatalysis.

  • 15.
    Jarmander, Johan
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Belotserkovsky, Jaroslav
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Sjöberg, Gustav
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Guevara-Martínez, Mónica
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Universidad Mayor de San Simón, Bolivia .
    Zabaleta, Mariel Perez
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Universidad Mayor de San Simón, Bolivia .
    Quillaguaman, Jorge
    Universidad Mayor de San Simón, Bolivia .
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Cultivation strategies for production of (R)-3-hydroxybutyric acid from simultaneous consumption of glucose, xylose and arabinose by Escherichia coli2015In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 14, no 1, p. 51-Article in journal (Refereed)
    Abstract [en]

    Background

    Lignocellulosic waste is a desirable biomass for use in second generation biorefineries. Up to 40 % of its sugar content consist of pentoses, which organisms either take up sequentially after glucose depletion, or not at all. A previously described Escherichia coli strain, PPA652ara, capable of simultaneous consumption of glucose, xylose and arabinose was in the present work utilized for production of (R)-3-hydroxybutyric acid (3HB) from a mixture of glucose, xylose and arabinose.

    Results

    The Halomonas boliviensis genes for 3HB production were for the first time cloned into E. coli PPA652ara leading to product secretion directly into the medium. Process design was based on comparisons of batch, fed-batch and continuous cultivation, where both excess and limitation of the carbon mixture was studied. Carbon limitation resulted in low specific productivity of 3HB (< 2 mg g-1 h-1) compared to carbon excess (25 mg g-1 h-1), but the yield of 3HB/cell dry weight (Y3HB/CDW) was very low (0.06 g g-1)during excess. Nitrogen-exhausted conditions could be used to sustain a high specific productivity (31 mg g-1 h-1) and to increase the yield of 3HB/cell dry weight to 1.38 g g-1. Nitrogen-limited fed-batch process design lead to further increased specific productivity (38 mg g-1 h-1) but also to additional cell growth (Y3HB/CDW = 0.16 g g-1). Strain PPA652ara did under all processing conditions simultaneously consume glucose, xylose and arabinose, which was not the case for a reference wild type E. coli, which also gave a higher carbon flux to acetic acid.

    Conclusions

    It was demonstrated that by using the strain E. coli PPA652ara it was possible to design a production process for 3HB from a mixture of glucose, xylose and arabinose where all sugars were consumed. An industrial 3HB production process is proposed to be divided into a growth and a production phase, and nitrogen depletion/limitation is a potential strategy to maximize the yield of 3HB/CDW in the latter. The specific productivity of 3HB by E. coli reported here from glucose, xylose and arabinose is further comparable to the current state of the art for production of 3HB from glucose sources.

  • 16.
    Jarmander, Johan
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Guevara, Mónica
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Zabaleta, Mariel Perez
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Sjöberg, Gustav
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Belotserkovsky, Jaroslav
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Quillaguaman, Jorge
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Production of 3-hydroxybutyrate from waste biomass by metabolically engineered Escherichia coli2014In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 31, p. S94-S95Article in journal (Other academic)
  • 17.
    Jarmander, Johan
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Do, Thi-Huyen
    Samuelson, Patrik
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli2012In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 11, article id 118Article in journal (Refereed)
    Abstract [en]

    Background: The discovery of the autotransporter family has provided a mechanism for surface expression of proteins in laboratory strains of Escherichia coli. We have previously reported the use of the AIDA-I autotransport system to express the Salmonella enterica serovar Enteritidis proteins SefA and H: gm. The SefA protein was successfully exposed to the medium, but the orientation of H:gm in the outer membrane could not be determined due to proteolytic cleavage of the N-terminal detection-tag. The goal of the present work was therefore to construct a vector containing elements that facilitates analysis of surface expression, especially for proteins that are sensitive to proteolysis or otherwise difficult to express. Results: The surface expression system pAIDA1 was created with two detection tags flanking the passenger protein. Successful expression of SefA and H:gm on the surface of E. coli was confirmed with fluorescently labeled antibodies specific for the N-terminal His(6)-tag and the C-terminal Myc-tag. While both tags were detected during SefA expression, only the Myc-tag could be detected for H: gm. The negative signal indicates a proteolytic cleavage of this protein that removes the His(6)-tag facing the medium. Conclusions: Expression levels from pAIDA1 were comparable to or higher than those achieved with the formerly used vector. The presence of the Myc- but not of the His(6)-tag on the cell surface during H:gm expression allowed us to confirm the hypothesis that this fusion protein was present on the surface and oriented towards the cell exterior. Western blot analysis revealed degradation products of the same molecular weight for SefA and H:gm. The size of these fragments suggests that both fusion proteins have been cleaved at a specific site close to the C-terminal end of the passenger. This proteolysis was concluded to take place either in the outer membrane or in the periplasm. Since H:gm was cleaved to a much greater extent then the three times smaller SefA, it is proposed that the longer translocation time for the larger H:gm makes it more susceptible to proteolysis.

  • 18.
    Jarmander, Johan
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Hallström, Björn M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Simultaneous Uptake of Lignocellulose- Based Monosaccharides by Escherichia Coli2014In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 111, no 6, p. 1108-1115Article in journal (Refereed)
    Abstract [en]

    Lignocellulosic waste is a naturally abundant biomass and is therefore an attractive material to use in second generation biorefineries. Microbial growth on the monosaccharides present in hydrolyzed lignocellulose is however associated with several obstacles whereof one is the lack of simultaneous uptake of the sugars. We have studied the aerobic growth of Escherichia coli on D-glucose, D-xylose, and L-arabinose and for simultaneous uptake to occur, both the carbon catabolite repression mechanism (CCR) and the AraC repression of xylose uptake and metabolism had to be removed. The strain AF1000 is a MC4100 derivative that is only able to assimilate arabinose after a considerable lag phase, which is unsuitable for commercial production. This strain was successfully adapted to growth on L-arabinose and this led to simultaneous uptake of arabinose and xylose in a diauxic growth mode following glucose consumption. In this strain, a deletion in the phosphoenolpyruvate:phosphotransferase system (PTS) for glucose uptake, the ptsG mutation, was introduced. The resulting strain, PPA652ara simultaneously consumed all three monosaccharides at a maximum specific growth rate of 0.59h(-1), 55% higher than for the ptsG mutant alone. Also, no residual sugar was present in the cultivation medium. The potential of PPA652ara is further acknowledged by the performance of AF1000 during fed-batch processing on a mixture of D-glucose, D-xylose, and L-arabinose. The conclusion is that without the removal of both layers of carbon uptake control, this process results in accumulation of pentoses and leads to a reduction of the specific growth rate by 30%.

  • 19.
    Jarmander, Johan
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Janoschek, Lars
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Lundh, Susanna
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Process optimization for increased yield of surface-expressed protein in Escherichia coli2014In: Bioprocess and biosystems engineering (Print), ISSN 1615-7591, E-ISSN 1615-7605, Vol. 37, no 8, p. 1685-1693Article in journal (Refereed)
    Abstract [en]

    The autotransporter family of Gram-negative protein exporters has been exploited for surface expression of recombinant passenger proteins. While the passenger in some cases was successfully translocated, a major problem has been low levels of full-length protein on the surface due to proteolysis following export over the cytoplasmic membrane. The aim of the present study was to increase the surface expression yield of the model protein SefA, a Salmonella enterica fimbrial subunit with potential for use in vaccine applications, by reducing this proteolysis through process design using Design of Experiments methodology. Cultivation temperature and pH, hypothesized to influence periplasmic protease activity, as well as inducer concentration were the parameters selected for optimization. Through modification of these parameters, the total surface expression yield of SefA was increased by 200 %. At the same time, the yield of full-length protein was increased by 300 %, indicating a 33 % reduction in proteolysis.

  • 20.
    Kårelid, Victor
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Björlenius, Berndt
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Effects of recirculation in a three-tank pilot-scale system for pharmaceutical removal with powdered activated carbon2017In: Journal of Environmental Management, ISSN 0301-4797, E-ISSN 1095-8630, Vol. 193, no May, p. 163-Environmental Impact Optimization of Reinforced Concrete Slab Frame BridgesArticle, review/survey (Refereed)
    Abstract [en]

    The removal of pharmaceutically active compounds by powdered activated carbon (PAC) in municipal wastewater is a promising solution to the problem of polluted recipient waters. Today, an efficient design strategy is however lacking with regard to high-level overall, and specific, substance removal in the large scale. The performance of PAC-based removal of pharmaceuticals was studied in pilot-scale with respect to the critical parameters; contact time and PAC dose using one PAC product selected by screening in bench-scale. The goal was a minimum of 95% removal of the pharmaceuticals present in the evaluated municipal wastewater. A set of 21 pharmaceuticals was selected from an initial 100 due to their high occurrence in the effluent water of two selected wastewater treatment plants (WWTPs) in Sweden, whereof candidates discussed for future EU regulation directives were included. By using recirculation of PAC over a treatment system using three sequential contact tanks, a combination of the benefits of powdered and granular carbon performance was achieved. The treatment system was designed so that recirculation could be introduced to any of the three tanks to investigate the effect of recirculation on the adsorption performance. This was compared to use of the setup, but without recirculation. A higher degree of pharmaceutical removal was achieved in all recirculation setups, both overall and with respect to specific substances, as compared to without recirculation. Recirculation was tested with nominal contact times of 30, 60 and 120 min and the goal of 95% removal could be achieved already at the shortest contact times at a PAC dose of 10–15 mg/L. In particular, the overall removal could be increased even to 97% and 99%, at 60 and 120 min, respectively, when the recirculation point was the first tank. Recirculation of PAC to either the first or the second contact tank proved to be comparable, while a slightly lower performance was observed with recirculation to the third tank. With regards to individual substances, clarithromycin and diclofenac were ubiquitously removed according to the set goal and in contrast, a few substances (fluconazole, irbesartan, memantine and venlafaxine) required specific settings to reach an acceptable removal.

  • 21.
    Kårelid, Victor
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Björlenius, Berndt
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Pilot-scale removal of pharmaceuticals in municipal wastewater: Comparison of granular and powdered activated carbon treatment at three wastewater treatment plants2017In: Journal of Environmental Management, ISSN 0301-4797, E-ISSN 1095-8630, Vol. 193, no -1, p. 491-502Article in journal (Refereed)
    Abstract [en]

    Adsorption with activated carbon is widely suggested as an option for the removal of organic micropollutants including pharmaceutically active compounds (PhACs) in wastewater. In this study adsorption with granular activated carbon (GAC) and powdered activated carbon (PAC) was analyzed and compared in parallel operation at three Swedish wastewater treatment plants with the goal to achieve a 95% PhAC removal. Initially, mapping of the prevalence of over 100 substances was performed at each plant and due to low concentrations a final 22 were selected for further evaluation. These include carbamazepine, clarithromycin and diclofenac, which currently are discussed for regulation internationally. A number of commercially available activated carbon products were initially screened using effluent wastewater. Of these, a reduced set was selected based on adsorption characteristics and cost. Experiments designed with the selected carbons in pilot-scale showed that most products could indeed remove PhACs to the target level, both on total and individual basis. In a setup using internal recirculation the PAC system achieved a 95% removal applying a fresh dose of 15–20 mg/L, while carbon usage rates for the GAC application were much broader and ranged from <28 to 230 mg/L depending on the carbon product. The performance of the PAC products generally gave better results for individual PhACs in regards to carbon availability. All carbon products showed a specific adsorption for a specific PhAC meaning that knowledge of the target pollutants must be acquired before successful design of a treatment system. In spite of different configurations and operating conditions of the different wastewater treatment plants no considerable differences regarding pharmaceutical removal were observed.

  • 22.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Challenges in production of recombinant proteins2008In: Bioprocess and biosystems engineering (Print), ISSN 1615-7591, E-ISSN 1615-7605, Vol. 31, no 1, p. 1-2Article in journal (Refereed)
  • 23.
    Larsson, Gen
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO).
    Endotoxin analysis2005Patent (Other (popular science, discussion, etc.))
  • 24.
    Larsson, Gen
    et al.
    KTH, Superseded Departments, Biotechnology.
    Shokri, Atefeh
    KTH, Superseded Departments, Biotechnology.
    Characterisation of the Escherichia coli membrane during fedbatch cultivation2004In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 3, p. 9-Article in journal (Refereed)
    Abstract [en]

    Background: Important parameters during recombinant protein production in Escherichia coli, such as productivity and protein activity, are affected by the growth rate. This includes the translocation of protein over the membrane to gain better folding capacity or reduced proteolysis. To vary the growth rate two techniques are available: fedbatch and continuous cultivation, both controlled by the ingoing feed rate. Results: During fedbatch cultivation, E. coli contains phosphatidylethanolamine, phosphatidylglycerol, cardiolipin and saturated fatty acids in amounts which are stable with growth rate. However, the levels of cardiolipin are very high compared to continuous cultivation. The reason for fedbatch triggering of this metabolism is not known but hypothesised to result from an additional need for carbon and energy. The reason could be the dynamic and sometimes rapid changes in growth rate to which the fedbatch cell has at all times to adjust. The membrane flexibility, essential for translocation of various components, is however to some degree sustained by production of increased amounts of unsaturated fatty acids in phosphatidylglycerol. The result is a functionally stiff membrane which generally promotes low cell lysis and is constant with respect to protein leakage to the medium. At comparatively high growth rates, when the further stabilising effect of cyclic fatty acids is gone, the high level of unsaturated fatty acids results in a pronounced effect upon sonication. This is very much in contrast to the membrane function in continuous cultivation which shows very specific characteristics as a function of growth rate. Conclusions: The stiff and unchanging fedbatch membrane should promote a stable behaviour during downstream processing and is less dependent on the time of harvest. However, optimisation of protein leakage can only be achieved in the continuously cultivated cell where leakage is twice as high compared to the constant leakage level in fedbatch. If leakage is undesired, continuous cultivation is also preferred since it can be designed to lead to the lowest values detected. Induction at low growth rate (<0.2 h-1) should be avoided with respect to productivity, in any system, since the specific and total protein production shows their lowest values at this point.

  • 25.
    Lindroos, Magnus
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH). KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Hörnström, David
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
    van Maris, Antonius J. A.
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
    Continuous removal of the model pharmaceutical chloroquine from water using melanin-covered Escherichia coli in a membrane bioreactor2019In: Journal of Hazardous Materials, ISSN 0304-3894, E-ISSN 1873-3336, Vol. 365, p. 74-80Article in journal (Refereed)
    Abstract [en]

    Environmental release and accumulation of pharmaceuticals and personal care products is a global concern in view of increased awareness of ecotoxicological effects. Adsorbent properties make the biopolymer melanin an interesting alternative to remove micropollutants from water. Recently, tyrosinase-surface-displaying Escherichia coli was shown to be an interesting self-replicating production system for melanin-covered cells for batch-wise absorption of the model pharmaceutical chloroquine. This work explores the suitability of these melanin-covered E. coli for the continuous removal of pharmaceuticals from wastewater. A continuous-flow membrane bioreactor containing melanized E. coli cells was used for adsorption of chloroquine from the influent until saturation and subsequent regeneration. At a low loading of cells (10 g/L) and high influent concentration of chloroquine (0.1 mM), chloroquine adsorbed until saturation after 26 +/- 2 treated reactor volumes (39 +/- 3 L). The average effluent concentration during the first 20 h was 0.0018 mM, corresponding to 98.2% removal. Up to 140 +/- 6 mg chloroquine bound per gram of cells following mixed homo- and heterogeneous adsorption kinetics. In situ low pH regeneration released all chloroquine without apparent capacity loss over three consecutive cycles. This shows the potential of melanized cells for treatment of conventional wastewater or highly concentrated upstream sources such as hospitals or manufacturing sites.

  • 26. Mergulhao, F. J. M.
    et al.
    Monteiro, G. A.
    Larsson, Gen
    KTH, Superseded Departments, Biotechnology.
    Sanden, A. M.
    Farewell, A.
    Nystrom, T.
    Cabral, J. M. S.
    Taipa, M. A.
    Medium and copy number effects on the secretion of human proinsulin in Escherichia coli using the universal stress promoters uspA and uspB2003In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 61, no 06-maj, p. 495-501Article in journal (Refereed)
    Abstract [en]

    The use of the uspA and uspB promoters (universal stress promoters) for heterologous protein production in Escherichia coli is described. Best results were obtained with a moderate copy number vector (1560 copies) bearing the uspA promoter, reaching 4.6 mg/g dry cell weight (DCW) of ZZ-proinsulin secreted to the periplasm and 1.9 mg/g DCW secreted to the culture medium. These values are about 1.7-fold higher than those previously reported with the same ZZ fusion tag and the SpA leader peptide showing that these stress promoters are potentially valuable for recombinant protein secretion in E. coli. It is further demonstrated that the use of M9 minimal medium is advantageous for protein secretion as compared to LB rich medium.

  • 27. Nhan, Nguyen Thanh
    et al.
    Gonzalez de Valdivia, Ernesto
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Hai, Truong Nam
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Surface display of Salmonella epitopes in Escherichia coli and Staphylococcus carnosus2011In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 10, p. 22-Article in journal (Refereed)
    Abstract [en]

    Background: Salmonella enterica serotype Enteritidis (SE) is considered to be one of the most potent pathogenic Salmonella serotypes causing food-borne disease in humans. Since a live bacterial vaccine based on surface display of antigens has many advantages over traditional vaccines, we have studied the surface display of the SE antigenic proteins, H: gm and SefA in Escherichia coli by the beta-autotransporter system, AIDA. This procedure was compared to protein translocation in Staphylococcus carnosus, using a staphylococci hybrid vector earlier developed for surface display of other vaccine epitopes. Results: Both SefA and H: gm were translocated to the outer membrane in Escherichia coli. SefA was expressed to full length but H: gm was shorter than expected, probably due to a proteolytic cleavage of the N-terminal during passage either through the periplasm or over the membrane. FACS analysis confirmed that SefA was facing the extracellular environment, but this could not be conclusively established for H: gm since the N-terminal detection tag (His(6)) was cleaved off. Polyclonal salmonella antibodies confirmed the sustained antibody-antigen binding towards both proteins. The surface expression data from Staphylococcus carnosus suggested that the H: gm and SefA proteins were transported to the cell wall since the detection marker was displayed by FACS analysis. Conclusion: Apart from the accumulated knowledge and the existence of a wealth of equipment and techniques, the results indicate the selection of E. coli for further studies for surface expression of salmonella antigens. Surface expression of the full length protein facing the cell environment was positively proven by standard analysis, and the FACS signal comparison to expression in Staphylococcus carnosus shows that the distribution of the surface protein on each cell was comparatively very narrow in E. coli, the E. coli outer membrane molecules can serve as an adjuvant for the surface antigenic proteins and multimeric forms of the SefA protein were detected which would probably be positive for the realisation of a strong antigenic property. The detection of specific and similar proteolytic cleavage patterns for both the proteins provides a further starting point for the investigation and development of the Escherichia coli AIDA autotransporter efficiency.

  • 28.
    Perez-Zabaleta, Mariel
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
    Guevara-Martínez, Mónica
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
    Gustavsson, Martin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
    Quillaguamán, Jorge
    Larsson, Gen
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
    van Maris, Antonius J. A.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
    Comparison of engineered Escherichia coli AF1000 and BL21 strains for (R)-3-hydroxybutyrate production in fed-batch cultivation2019In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 103, no 14, p. 5627-5636Article in journal (Refereed)
    Abstract [en]

    Accumulation of acetate is a limiting factor in recombinant production of (R)-3-hydroxybutyrate (3HB) by E. coli in high-cell-density processes. To alleviate this limitation, this study investigated two approaches: (i) Deletion of phosphotransacetylase (pta), pyruvate oxidase (poxB) and/or the isocitrate-lyase regulator (iclR), known to decrease acetate formation, on bioreactor cultivations designed to achieve high 3HB concentrations. (ii) Screening of different E. coli strain backgrounds (B, BL21, W, BW25113, MG1655, W3110 and AF1000) for their potential as low acetate-forming, 3HB-producing platforms. Deletion of pta and pta-poxB in the AF1000 strain background was to some extent successful in decreasing acetate formation, but also dramatically increased excretion of pyruvate and did not result in increased 3HB production in high-cell-density fed-batch cultivations. Screening of the different E. coli strains confirmed BL21 as a low acetate forming background. Despite low 3HB titers in low-cell density screening, 3HB-producing BL21 produced 5 times less acetic acid per mol of 3HB, which translated into a 2.3-fold increase in the final 3HB titer and a 3-fold higher volumetric 3HB productivity over 3HB-producing AF1000 strains in nitrogen-limited fed-batch cultivations. Consequently, the BL21 strain achieved the hitherto highest described volumetric productivity of 3HB (1.52 g L-1 h-1) and the highest 3HB concentration (16.3 g L-1) achieved by recombinant E. coli. Screening solely for 3HB titers in low-cell-density batch cultivations would not have identified the potential of this strain, reaffirming the importance of screening with the final production conditions in mind.

  • 29.
    Perez-Zabaleta, Mariel
    et al.
    KTH, School of Biotechnology (BIO).
    Jarmander, Johan
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Guevara, Monica
    KTH, School of Biotechnology (BIO).
    Quillaguaman, Jorge
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Design and flux modelling for recombinant production of 3-Hydroxybutyrate in Escherichia coli2014In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 31, p. S167-S167Article in journal (Other academic)
  • 30.
    Perez-Zabaleta, Mariel
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Sjöberg, Gustav
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Guevara-Martínez, Mónica
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Universidad Mayor de San Simón, Bolivia.
    Jarmander, Johan
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Quillaguamán, Jorge
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Increasing the production of (R)-3-hydroxybutyrate in recombinant Escherichia coli by improved cofactor supply2016In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 15, no 1, article id 91Article in journal (Refereed)
    Abstract [en]

    Background: In a recently discovered microorganism, Halomonas boliviensis, polyhydroxybutyrate production was extensive and in contrast to other PHB producers, contained a set of alleles for the enzymes of this pathway. Also the monomer, (R)-3-hydroxybutyrate (3HB), possesses features that are interesting for commercial production, in particular the synthesis of fine chemicals with chiral specificity. Production with a halophilic organism is however not without serious drawbacks, wherefore it was desirable to introduce the 3HB pathway into Escherichia coli. Results: The production of 3HB is a two-step process where the acetoacetyl-CoA reductase was shown to accept both NADH and NADPH, but where the V-max for the latter was eight times higher. It was hypothesized that NADPH could be limiting production due to less abundance than NADH, and two strategies were employed to increase the availability; (1) glutamate was chosen as nitrogen source to minimize the NADPH consumption associated with ammonium salts and (2) glucose-6-phosphate dehydrogenase was overexpressed to improve NADPH production from the pentose phosphate pathway. Supplementation of glutamate during batch cultivation gave the highest specific productivity (q(3HB) = 0.12 g g(-1) h(-1)), while nitrogen depletion/zwf overexpression gave the highest yield (Y-3HB/CDW = 0.53 g g(-1)) and a 3HB concentration of 1 g L-1, which was 50 % higher than the reference. A nitrogen-limited fedbatch process gave a concentration of 12.7 g L-1 and a productivity of 0.42 g L-1 h(-1), which is comparable to maximum values found in recombinant E. coli. Conclusions: Increased NADPH supply is a valuable tool to increase recombinant 3HB production in E. coli, and the inherent hydrolysis of CoA leads to a natural export of the product to the medium. Acetic acid production is still the dominating by-product and this needs attention in the future to increase the volumetric productivity further.

  • 31.
    Prytz, Ingela
    et al.
    KTH, Superseded Departments, Biotechnology.
    Sandén, Anna Maria
    KTH, Superseded Departments, Biotechnology.
    Nyström, Thomas
    Farewell, Anne
    Wahlström, Åsa
    Förberg, Cecilia
    Pragai, Zoltan
    Barer, Mike
    Harwood, Colin
    Larsson, Gen
    KTH, Superseded Departments, Biotechnology.
    Fed-batch production of recombinant beta-galactosidase using the universal stress promoters uspA and uspB in high cell density cultivations2003In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 83, no 5, p. 595-603Article in journal (Refereed)
    Abstract [en]

    A high-level production system using the universal stress promoters uspA and uspB in a fed-batch cultivation based on minimal medium was designed. In development it was shown that a standard industrial fed-batch protocol could not be used for this purpose since it failed to induce the levels of product as compared to the basal level. Instead, a batch protocol followed by a low constant feed of glucose was shown to give full induction. The levels of the product protein, beta-galactosidase, corresponded to approximately 25% of the total protein. Higher levels were found using the uspA than uspB vectors where uspA showed considerably higher basal level. The data indicate that the sigma(70) regulated promoter, uspA, although affected by the alarmone guanosine tetraphosphate, ppGpp, worked partly in a similar manner to constitutive promoters. An industrial high cell density fedbatch cultivation on the basis of the suggested fed-batch protocol and the uspA promoter gave a final beta-galatosidase concentration of 7 g/L and a final cell concentration of 65 g/L. The heterogeneity in production of the individual cell was measured by fluorescence microscopy. The data show that there is a process time independent heterogeneity in production, which is suggested to be caused by heterogeneity in the substrate uptake rate of the individual cell.

  • 32.
    Sandén, Anna Maria
    et al.
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Boström, Maria
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Markland, Katrin
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Solubility and proteolysis of the Zb-MaIE and Zb-MaIE31 proteins during overproduction in Escherichia coli2005In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 90, no 2, p. 239-247Article in journal (Refereed)
    Abstract [en]

    From the hypothesis that the rate of expression of a nascent polypeptide controls the accumulation of soluble full-length protein, accumulation of the model fusion proteins Zb-MalE and Zb-MalE31, were studied. MalE and MalE31 are two isoforms of the maltose binding protein, differing only in two consecutive amino acids. Parameters controlling the expression rate were the transcription rate, which was controlled by IPTG induction of the lacUV5 promoter and the substrate addition levels during fed-batch cultivation.

    Results show that the two product proteins appear in both soluble and insoluble fractions during cultivation and are both subjected to proteolysis. However, the accumulation of the soluble form of Zb-MalE31 protein is radically lower, at all conditions, due to the small difference in primary structure.

    It was shown that both proteolysis and inclusion body formation could be influenced by the selected parameters although a change in feed rate had a considerably higher effect. A high concentration of inducer and a "high" feed rate result in a low accumulation of soluble product, due to a high proteolysis. The concentration of inducer leading to different levels of transcription is, however, an efficient tool to influence inclusion body formation. At low IPTG concentrations (<= 5 mu M), this formation is almost abolished while at a comparatively high concentration (>= 300 mu M) 50% of the total product accumulated was in the form of inclusion bodies.

  • 33.
    Sandén, Anna Maria
    et al.
    KTH, Superseded Departments, Biotechnology.
    Prytz, Ingela
    KTH, Superseded Departments, Biotechnology.
    Larsson, Gen
    KTH, Superseded Departments, Biotechnology.
    The influence of time and method of induction on recombinant protein productivity and acetic acid formation in Escherichia coli2004In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290Article in journal (Other academic)
  • 34.
    Sandén, Anna Maria
    et al.
    KTH, Superseded Departments, Biotechnology.
    Prytz, Ingela
    KTH, Superseded Departments, Biotechnology.
    Tubulekas, I.
    Förberg, C.
    Le, H.
    Hektor, A.
    Neubauer, P.
    Pragai, Z.
    Harwood, C.
    Ward, A.
    Picon, A.
    de Mattos, J. T.
    Postma, P.
    Farewell, A.
    Nyström, T.
    Reeh, S.
    Pedersen, S.
    Larsson, Gen
    KTH, Superseded Departments, Biotechnology.
    Limiting factors in Escherichia coli fed-batch production of recombinant proteins2003In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 81, no 2, p. 158-166Article in journal (Refereed)
    Abstract [en]

    Fed-batch production of recombinant beta-galactosidase in E. coli was studied with respect to the specific growth rate at induction. The cultivations were designed to induce protein production by IPTG at a glucose feed rate corresponding to high (mu = 0.5 h(-1)) or low (mu = 0.1 h(-1)) specific growth rate. Protein production rate was approximately 100% higher at the higher specific growth rate, resulting in the accumulation of beta-galactosidase up to 30% of the total cell protein. Transcription analysis showed that beta-galactosidase-specific messenger RNA was immediately formed after induction (<5 min), but the amount was the same in both cases and was thus not the initial limiting factor. The content of ribosomes, as represented by rRNA, rapidly decreased with specific growth rate from a relative level of 100%, at the high specific growth rate, to 20% at the low specific growth rate. At high specific growth rate, ribosomes were additionally degraded upon induction due to the high production level. Translation therefore seemed to be the initial limiting factor of the protein synthesis capacity. The alarmone guanosine tetraphosphate increased at both high and low feed level inductions, indicating an induction-forced starvation of charged tRNA and/or glucose. The altered physiological status was also detected by the formation of acetic acid. However, the higher production rate resulted in high-level accumulation of acetic acid, which was absent at low feed rate production. Acetic acid production is thus coupled to the high product formation rate and is proposed to be due either to a precursor drain of Krebs cycle intermediates and a time lag before induction of the glyoxalate shunt, or to single amino acid overflow, since the model product is relatively poor in glycin and alanin. In conclusion, it is proposed that production at high specific growth rate becomes precursor-limited, while production at low specific growth rate is carbon- and/or energy-limited.

  • 35. Shokri, Atefeh
    et al.
    Veide, Andres
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    RelA1 gene control of Escherichia coli lipid structure and cell performace during glucose limited fed-batch conditions2006In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 73, no 2, p. 464-473Article in journal (Refereed)
    Abstract [en]

    At increasing glucose limitation, typical for fed-batch cultivation performance, cultivation of Escherichia coli (relA1) results in development of a lipid structure that radically differs from the wild type and is characterised by accumulation of neutral phospholipids and saturated fatty acids. The mutant can, furthermore, not change the level of cardiolipin, which is generally the hallmark of changes to severe glucose limitation. The result suggests an increased negative control in the mutant with respect to the flux to phosphatidyl glycerol and cardolipin as well as to unsaturated fatty acids. Opposite to the wild type, the cardiolipin-depleted membrane is more fragile with respect to sonication and osmotic chock, at severe limitation, and results in extensive foaming during the process. Protein leakage and cell lysis is, however, lower in the mutant most likely due to the increased amounts of saturated fatty acids, which might be a possible strategy to overcome the reduced amounts of membrane-strengthening cardiolipin. The membrane potential of the outer surface is negative, however less negative for the mutant. This was supported by aqueous two-phase extraction experiments which, furthermore indicated a difference in outer surface hydrofobicity. These findings suggest that the relA1 gene has a defined, but ppGpp-independent, role in cells with a slowly decreasing metabolism of glucose to control the membrane morphology.

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