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| 2. |
- Guevara-Martínez, Mónica, 1989-, et al.
(författare)
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Regulating the production of (R)-3-hydroxybutyrate in Escherichia coli by N or P limitation
- 2015
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Ingår i: Frontiers in Microbiology. - : Frontiers Research Foundation. - 1664-302X. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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- Jarmander, Johan, 1983-, et al.
(författare)
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A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli
- 2012
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859 .- 1475-2859. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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- Jarmander, Johan, et al.
(författare)
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Cultivation strategies for production of (R)-3-hydroxybutyric acid from simultaneous consumption of glucose, xylose and arabinose by Escherichia coli
- 2015
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Ingår i: Microbial Cell Factories. - : BioMed Central. - 1475-2859 .- 1475-2859. ; 14:1, s. 51-
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundLignocellulosic 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.ResultsThe 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.ConclusionsIt 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.
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- Jarmander, Johan, 1983-
(författare)
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Improved detection and performance of surface expression from the AIDA-I autotransporter
- 2013
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Surface expression of recombinant proteins has attracted a lot of attention due to its potential in applications such as enzyme production, vaccine delivery and bioremediation. Autotransporters have been used for surface expression of a variety of proteins, but the expression systems reported in literature have typically been inflexible and incapable of detecting proteolysis, thereby limiting surface expression yield.In this thesis, a modular surface expression system, utilizing dual tag detection, was therefore created. It was based on the adhesin involved in diffuse adherence (AIDA-I) autotransporter, and was here used to express the model proteins SefA and H:gm on the cell surface of Escherichia coli. Due to the dual tag detection system, proteolysed H:gm could be successfully verified on the cell surface. By optimizing cultivation conditions, surface expression yield of SefA was increased by 300 %, and proteolysis reduced by 33 %. While proteolysis could not be eliminated completely, the work presented in this thesis is a major step towards a general system for surface expression of a wide range of proteins in varied applications.
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| 7. |
- Jarmander, Johan, et al.
(författare)
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Process optimization for increased yield of surface-expressed protein in Escherichia coli
- 2014
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Ingår i: Bioprocess and biosystems engineering (Print). - : Springer Science and Business Media LLC. - 1615-7591 .- 1615-7605. ; 37:8, s. 1685-1693
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 9. |
- Jarmander, Johan, et al.
(författare)
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Simultaneous Uptake of Lignocellulose- Based Monosaccharides by Escherichia Coli
- 2014
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 111:6, s. 1108-1115
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Tidskriftsartikel (refereegranskat)abstract
- 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%.
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| 10. |
- Jarmander, Johan
(författare)
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Strategies for improved Escherichia coli bioprocessing performance
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Escherichia coli has a proven track record for successful production of anything from small molecules like organic acids to large therapeutic proteins, and has thus important applications in both R&D and commercial production. The versatility of this organism in combination with the accumulated knowledge of its genome, metabolism and physiology, has allowed for development of specialty strains capable of performing very specific tasks, opening up opportunities within new areas. The work of this thesis has been devoted to alter membrane transport proteins and the regulation of these, in order for E. coli to find further application within two such important areas.The first area was vaccine development, where it was investigated if E. coli could be a natural vehicle for live vaccine production. The hypothesis was that the introduction and manipulation of a protein surface translocation system from pathogenic E. coli would result in stable expression levels of Salmonella subunit antigens on the surface of laboratory E. coli. While different antigen combinations were successfully expressed on the surface of E. coli, larger proteins were affected by proteolysis, which manipulation of cultivation conditions could reduce, but not eliminate completely. The surface expressed antigens were further capable of inducing proinflammatory responses in epithelial cells.The second area was biorefining. By altering the regulation of sugar assimilation, it was hypothesized that simultaneous uptake of the sugars present in lignocellulose hydrolyzates could be achieved, thereby improving the yield and productivity of important bio-based chemicals. The dual-layered catabolite repression was identified and successfully removed in the engineered E. coli, and the compound (R)-3-hydroxybutyric acid was produced from simultaneous assimilation of glucose, xylose and arabinose.
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