Tuning for light and more : engineering phototrophy and membrane proteins in Escherichia coli
Claassens, Nicolaas J.H.P. - \ 2017
Wageningen University. Promotor(en): John van der Oost; Willem de Vos, co-promotor(en): Vitor Martins dos Santos. - Wageningen : Wageningen University - ISBN 9789463430920 - 328
escherichia coli - phototropism - membranes - proteins - light - photosystem i - gene expression - escherichia coli - fototropie - membranen - eiwitten - licht - fotosysteem i - genexpressie
The application of microbial and plant photosynthesis for biobased production on the one hand has a huge potential but on the other hand photosynthesis has serious limitations regarding its efficiency. Hence, studying both fundamental features of photosynthetic processes and engineering of photosystems is of paramount interest, exploring the engineering of photosystems is the overarching aim of this thesis. As described in Chapter 1, natural photosystems may be modified or transplanted to allow for more efficient conversion of solar light energy into biochemical energy. Hereto ambitious proposals to engineer photosystems have been made, and to realize those endeavors the disciplines of synthetic and systems biology are required. To explore how to apply and improve those disciplines hereto, the work described in this thesis has focused on the transplantation of simple photosystems (proton-pumping rhodopsins; PPRs) into the cell membrane of the heterotrophic model bacterium Escherichia coli. Both in silico analyses, including metabolic and thermodynamic modeling (Chapter 3) and a series of experimental studies on transplanting PPR photosystems (Chapters 4,6 and 7) were performed, which identified several challenges, limitations and most importantly opportunities. This thesis also describes the application of novel tools to substantially improve the functional production of PPRs and a variety of other membrane proteins in E. coli.
Chapter 2 provides more details on previously reported examples of heterologous expression of PPRs in several hosts, and on the physiological impact of these transplanted photosystems. Based on this evaluation, some suggestions are made to improve and further exploit the transplantation of these photosystems.
In Chapter 3 a systematic, integrated in silico analysis is made of anaerobic, photo-electro-autotrophic synthetic metabolism in E. coli, consisting of (i) a PPR photosystem for ATP regeneration, (ii) an electron uptake pathway, and (iii) a natural or synthetic carbon fixation pathway. Constraint-based metabolic modelling of E. coli central metabolism is used, in combination with kinetic and thermodynamic pathway analyses. The photo-electro-autotrophic designs are predicted to have a limited potential for anaerobic, autotrophic growth of E. coli, given the relatively low ATP regenerating capacity of the PPR photosystems, and the relatively high ATP consumption due to maintenance. In general these analyses illustrate the potential of in silico analyses to identify potential bottlenecks and solutions in complex designs for autotrophic and photosynthetic metabolism, as a basis for subsequent experimental implementation.
To tackle a main bottleneck of PPR systems: their functional membrane-embedded production level, the heterologous production in E. coli of the proton-pumping rhodopsins from Gloeobacter violaceus (GR) and from Thermus thermophilus JL18 (TR) is quantified and experimentally optimized in Chapter 4. High constitutive production of both rhodopsin proteins is achieved by fine-tuning transcription and translation. Besides the canonical retinal pigment, the GR system has the ability to bind a light-harvesting antennae pigment, echinenone. After optimization of the heterologous pigment biosynthesis pathways for either retinal or echinenone production, appropriate quantities of retinal or echinenone for PPR reconstitution were detected in E. coli. Association of echinenone with GR broadens its absorption spectrum in E. coli, broadening the potential for light-harvesting also to blue light. Optimization of the branched pathway for simultaneous biosynthesis of both retinal and echinenone has been attempted by using a smart library of variable Ribosome Binding Sites (RBSs) with varying strengths (RedLibs). In general, the here described approaches towards improved functional production of rhodopsin photosystems in E. coli and their pigments may prove more widely applicable for heterologous production of more complex photosystems and other systems.
In Chapter 5 an up-to-date overview is provided on how codon usage can influence functional protein production. The fact that all known organisms have an incomplete set of tRNAs, indicates that biased codon usage could act as a general mechanism that allows for fine-tuning the translation speed. Although translation initiation is the key control step in protein production, it is broadly accepted that codon bias, especially in regions further downstream of the start codon, can contribute to the translation efficiency by tuning the translation elongation rate. Modulation of the translation speed depends on a combination of factors, including the secondary structure of the transcript (more or less RNA hairpins), the codon usage landscape (frequent and more rare codons) and for bacteria also RBS-like sequences at which ribosomes can pause. The complex combination of interdependent factors related to codon usage that can influence translation initiation and elongation. This complexity makes that the design of synthetic genes for heterologous expression is still in its infancy, and despite the availability of some codon usage algorithms, it is often as well a matter of trial and error.
In Chapter 6 the effect of different codon usage algorithms (optimization and harmonization) has been experimentally tested for heterologous production of membrane proteins. Apart from the codon usage algorithms also the combined effect of transcriptional fine-tuning in E. coli LEMO21(DE3) was assessed. The overproduction of 6 different membrane-embedded proteins, including 4 PPR variants (from bacteria, archaea and eukaryotes), was tested. For production of tested PPR variants, the different codon usage algorithms hardly influenced production, while transcriptional tuning had a large impact on production levels. Interestingly, for the other two tested non-PPR membrane proteins, some codon usage variants significantly improved production on top of transcriptional tuning. For both these proteins the codon-optimization algorithm reduced functional production below that of the wild-type codon variant, while the harmonization algorithm gave significantly higher production, equal or even higher than for the wild-type variant.
In Chapter 7 it is demonstrated that a translational-tuning system can be used to successfully optimize the expression of several membrane proteins, based on initial findings presented in Chapter 4. The employed, recently developed Bicistronic Design (BCD) system is based on translational coupling of a gene encoding a short leader peptide and the gene of interest that is under control of a variable ribosome binding site. A standardized library of 22 RBSs allows for precise, gene context-independent, fine-tuning of expression of this second gene, here encoding a membrane protein. For all four membrane proteins tested in this study the BCD approach resulted in 3 to 7-fold higher protein levels than those obtained by two other recently developed methods for optimizing membrane protein production. The presented approach allows for inducer-free, constitutive, high-level production of membrane proteins in E. coli, which can be widely applicable for both membrane protein purification studies as well as for synthetic biology projects involving membrane proteins.
In Chapter 8 a broad review and perspectives are provided on the potential of microbial autotrophs for the production of value-added compounds from CO2. Both photoautotrophic and chemolithoautotrophic production platforms are discussed, and recent progress in improving their efficiency and production potential is highlighted. Transplantation efforts for photosystems, but also for CO2 fixation pathways and electron uptake systems are discussed. An overview is provided on novel in silico and experimental approaches to engineer components related to autotrophy in heterotrophic and autotrophic model hosts, including approaches applied in this thesis. Future avenues are discussed for realizing more efficient autotrophic production platforms.
Finally, in Chapter 9 and 10 the work in this thesis is summarized and a general discussion is provided on future avenues for engineering of PPR photosystems, photosystems in general and on the optimization of membrane protein production.
Plant ‘voelt’ zwaartekracht vooral met speciale zetmeelkorrels : altijd interactie tussen geotropie en fototropie
Heuvelink, E. ; Kierkels, T. - \ 2016
Onder Glas 13 (2016)4. - p. 50 - 51.
tuinbouw - plantkunde - zwaartekracht - zetmeelkorrels - groeianalyse - gravitropie - fototropie - celmembranen - licht - auxinen - horticulture - botany - gravity - starch granules - growth analysis - gravitropism - phototropism - cell membranes - light - auxins
Charles Darwin is niet alleen bekend door de evolutietheorie. De negentiende eeuwse wetenschapper was namelijk de eerste die rond 1880 aantoonde dat planten reageren op de zwaartekracht. Daarna was het nog 125 jaar wachten voordat duidelijk werd dat planten de zwaartekracht ook echt nodig hebben voor een normale groei.
Phototrophic pigment production with microalgae
Mulders, K.J.M. - \ 2014
Wageningen University. Promotor(en): Rene Wijffels, co-promotor(en): Dirk Martens; Packo Lamers. - Wageningen : Wageningen University - ISBN 9789462571457 - 179
algen - fototropie - pigmenten - natuurlijke producten - metabolisme - carotenoïden - licht - gerichte mutagenese - algae - phototropism - pigments - natural products - metabolism - carotenoids - light - targeted mutagenesis
Microalgal pigments are regarded as natural alternatives for food colorants. To facilitate optimization of microalgae-based pigment production, this thesis aimed to obtain key insights in the pigment metabolism of phototrophic microalgae, with the main focus on secondary carotenoids.
Different microalgal groups each possess their own set of primary pigments. Besides, a selected group of green algae (Chlorophytes) accumulate secondary pigments (secondary carotenoids) when exposed to oversaturating light conditions.
In this thesis it was found for the first time that nutrient-depleted Isochrysis. aff. galbana T-ISO (Haptophytes) accumulates 3-hydroxyechinenone, a precursor of astaxanthin. Besides, it was found that nitrogen-depleted Chromochloris (Chlorella) zofingiensis (Chlorophyes) accumulates astaxanthin, presumably synthesised via echinenone, and ketolutein.
Inhibition of production of β-carotene derivatives (e.g. echinenone and astaxanthin) did not lead to increased production of primary carotenoids (e.g. lutein) or ketolutein in this species, suggesting that the regulatory mechanisms controlling the flux towards ketolutein and primary carotenoids are not affected by the decreased levels of β-carotene derivatives.
Besides, optimal yields of secondary carotenoids and triacylglycerol (TAG) on light were reached with C. zofingiensis for a range of biomass concentrations at the moment of nitrogen depletion.
This indicated that the biomass-specific photon absorption rate did not affect the amounts of energy used for secondary carotenoid and TAG production, for the range of biomass concentrations tested.
It was also found that nitrogen-depleted C. zofingiensis resupplied with nitrogen hardly degraded astaxanthin, whereas the other major secondary metabolites were degraded rapidly.
This indicated that the overall carotenoid yield on light as well as its content may possibly be improved by applying a repeated batch instead of a series of single batch cultivations, which are traditionally applied.
Finally, it was discussed that the highest increases in carotenoid yield on light can be reached by optimizing strain performance (using targeted genetic engineering and/or random mutagenesis), rather than by optimizing the cultivation conditions/operation mode or reactor design.
Nutrient Removal and Biomass Production in an Outdoor Pilot-Scale Phototrophic Biofilm Reactor for Effluent Polishing
Boelee, N.C. ; Janssen, M. ; Temmink, H. ; Shrestha, R. ; Buisman, C.J.N. ; Wijffels, R.H. - \ 2014
Applied Biochemistry and Biotechnology 172 (2014)1. - ISSN 0273-2289 - p. 405 - 422.
afvalwaterbehandeling - biofilms - algen - biologische waterzuiveringsinstallaties - fototropie - nitraten - fosfaten - waste water treatment - biofilms - algae - biological water treatment plants - phototropism - nitrates - phosphates - waste-water treatment - rate algal pond - marine-phytoplankton - seasonal succession - phosphate-uptake - nitrogen uptake - nitrate uptake - phosphorus - growth - light
An innovative pilot-scale phototrophic biofilm reactor was evaluated over a 5-month period to determine its capacity to remove nitrogen and phosphorus from Dutch municipal wastewater effluents. The areal biomass production rate ranged between 2.7 and 4.5 g dry weight/m2/day. The areal nitrogen and phosphorus removal rates averaged 0.13 g N/m2/day and 0.023 g P/m2/day, which are low compared to removal rates achieved in laboratory biofilm reactors. Nutrient removal increased during the day, decreased with decreasing light intensity and no removal occurred during the night. Additional carbon dioxide supply was not requisite as the wastewater was comprised of enough inorganic carbon to sustain microalgal growth. The study was not conclusive for the limiting factor that caused the low nutrient removal rate, possibly the process was limited by light and temperature, in combination with pH increases above pH 9 during the daytime. This pilot-scale study demonstrated that the proposed phototrophic biofilm reactor is not a viable post-treatment of municipal wastewater effluents under Dutch climate conditions. However, the reactor performance may be improved when controlling the pH and the temperatures in the morning. With these adaptations, a phototrophic biofilm reactor could be feasible at lower latitudes with higher irradiance levels.
The effect of harvesting on biomass production and nutrient removal in phototrophic biofilm reactors for effluent polishing
Boelee, N.C. ; Janssen, M. ; Temmink, H. ; Taparaviciute, L. ; Khiewwijit, R. ; Janoska, A. ; Buisman, C.J.N. ; Wijffels, R.H. - \ 2014
Journal of Applied Phycology 26 (2014)3. - ISSN 0921-8971 - p. 1439 - 1452.
afvalwaterbehandeling - biofilms - dikte - dichtheid - algen - biologische waterzuiveringsinstallaties - fototropie - stikstof - fosfor - verwijdering - biobased economy - waste water treatment - biofilms - thickness - density - algae - biological water treatment plants - phototropism - nitrogen - phosphorus - removal - biobased economy - waste-water treatment - photosynthetic efficiency - chlorella-sorokiniana - microalgal biofilms - phosphorus removal - mass-transport - fresh-water - light - growth
An increasing number of wastewater treatment plants require post-treatment to remove residual nitrogen and phosphorus. This study investigated various harvesting regimes that would achieve consistent low effluent concentrations of nitrogen and phosphorus in a phototrophic biofilm reactor. Experiments were performed in a vertical biofilm reactor under continuous artificial lighting and employing artificial wastewater. Under similar conditions, experiments were performed in near-horizontal flow lanes with biofilms of variable thickness. It was possible to maintain low nitrogen and phosphorus concentrations in the effluent of the vertical biofilm reactor by regularly harvesting half of the biofilm. The average areal biomass production rate achieved a 7 g dry weight m-2 day-1 for all different harvesting frequencies tested (every 2, 4, or 7 days), corresponding to the different biofilm thicknesses. Apparently, the biomass productivity is similar for a wide range of biofilm thicknesses. The biofilm could not be maintained for more than 2 weeks as, after this period, it spontaneously detached from the carrier material. Contrary to the expectations, the biomass production doubled when the biofilm thickness was increased from 130 µm to 2 mm. This increased production was explained by the lower density and looser structure of the 2 mm biofilm. It was concluded that, concerning biomass production and labor requirement, the optimum harvesting frequency is once per week.
Kendrick, R.E. ; Weller, J.L. - \ 2003
In: Encyclopedia of Applied Plant Sciences / Thomas, B., Murphy, D.J., Murray, B.G., Elsevier/Academic Press - ISBN 9780122270505 - p. 1069 - 1076.
fotoperiodiciteit - fototropie - fotoreceptoren - lichtregiem - fytochroom - zaadkieming - photoperiodism - phototropism - photoreceptors - light regime - phytochrome - seed germination
Phototropism in seedlings of sunflower, Helianthus annuus L.
Franssen, J.M. - \ 1980
Landbouwhogeschool Wageningen. Promotor(en): J. Bruinsma. - Wageningen : Franssen - 84
asteraceae - fototropie - planten - asteraceae - phototropism - plants
In this thesis the phototropic bending of hypocotyls of sunflower seedlings, Helianthus annuus L., is investigated.Chapter 1 gives the reasons for this project. Although phototropism has been studied extensively over the past 100 years, the understanding of the mechanism is far from clear. During this century two main hypotheses were developed, namely the theory of BLAAUW (1915), explaining phototropism as an effect of growth inhibition by light, and the CHOLODNY-WENT theory (1927, 1928), in which the lateral distribution and basipetal transport of the growth-promoting substance, auxin, are involved. Especially the latter received the attention of many investigators, probably because it deals with the first phytohormone discovered. The research on this subject has been mainly concentrated on the properties of auxin and the way in which it influences the pattern of curvature in etiolated coleoptiles. Much experimentation has been done with the exogenous application of diffusate from coleoptile tips or IAA, the auxin, to etiolated monocotyledonous seedlings, without measuring the endogenous levels of this growth substance in the plants themselves. In 1975 BRUINSMA et al., investigating the levels of IAA, in illuminated and shaded halves of phototropically stimulated, green sunflower seedlings, showed that this hormone is equally distributed between both halves. This discovery, and the fact that the evidence for both hypotheses mentioned above is still inconclusive, led to the present investigation.As experimental material sunflower seedlings, Helianthus annuus L., were chosen. Chapter 2 lists the material and methods used for the experiments described in the subsequent Chapters.Chapter 3 describes the influence of other processes on phototropism. It is shown that geotropism acts strongly on the deflected hypocotyl. Only the first hour after the beginning of the phototropic response can be considered to show pure phototropism. Because the delay in the onset of the response, the maximal degree of curvature is determined for each individual seedling during the first 2 hours of phototropic stimulation. The influences of the age of the seedling and length of the hypocotyl were studied as well. Seedlings of 4 to 5 days old and 40 to 60 mm long bend as uniformly as possible, and were used in the experiments described in this thesis. Careful manipulation of the seedlings did not influence the response.Chapter 4 deals with the influence of the other organs of the green seedling on the bending of the hypocotyl. It is concluded that neither the cotyledons and the shoot tip, nor the roots are essential for the response, although a stress effect due to the removal of these organs may cause a delay. The epidermis of the hypocotyl is indirectly involved; the peripheral layers are not necessary for the perception of the unilateral light, but probably control the growth, without which curvature cannot occur.In Chapter 5 the results of treatments with different wavelengths are shown. Unilateral blue light is the main active region, although the response caused by blue light is always slightly less than when caused by unilateral white light. In contrast to seedlings grown in white light, etiolated ones are not phototropically sensitive. Pretreatment of dark-grown seedlings with white or blue light renders them able to curve, and only illumination of the hypocotyl is necessary. The role of the cotyledons, that cannot be removed during this treatment without decreasing the bending, is probably to supply the substrates essential for the growth of the hypocotyl. Pretreatment with darkness of seedlings grown in white light makes them phototropically unresponsive. Again, only the hypocotyl needs this treatment and this indicates that the mechanism for phototropism is located within the hypocotyl. Green seedlings pretreated with red light only show poor curvature, but the growth rate of the seedlings is not related to the bending capacity.Chapter 6 presents the results of the effect of pretreatment with far-red irradiation on the phototropic response. It is shown that far red, more than a treatment in darkness, decreases the curvature in seedlings grown in white light and that the response is a low-energy reaction (L.E.R.). Red light did not affect the lack of bending of etiolated plants, but prolonged red illumination could reverse the effect of far-red irradiance in green seedlings. It is concluded that the perception of the far-red irradiance is located in both the hypocotyl and the cotyledons. Again, no correlation between the growth rate and the curvature of plants pretreated with far red can be detected.The involvement of hormones in the phototropic response is studied in Chapter 7. IAA is not unilaterally distributed in curving parts of the seedlings that were 30 to 45 min phototropically stimulated. No IAA could be detected in diffusates from the seedlings, and this absence is not due to destruction of IAA at the cut surface. However, a growth-inhibiting activity was found in the diffusates, both from hypocotyls and from cotyledons, and this activity was not asymmetrically distributed in bending plants.The role of the growth inhibitor, xanthoxin, was investigated. Experiments are described to show that xanthoxin is not contaminated with ABA after the extraction procedure, and that a bioassay can be properly used for the determination of xanthoxin. In straight hypocotyls xanthoxin is equally distributed, but in curving and curved ones more inhibitor can be extracted from the illuminated side than from the shaded one. From seedlings pretreated with white or blue light, which are phototropically sensitive, a higher amount of xanthoxin can be extracted than from etiolated, phototropically unresponsive, plants. In seedlings pretreated with red light, which show only a poor bending, a variable amount of xanthoxin was detected. The results of experiments in which xanthoxin was exogenously applied to seedlings were inconclusive.Gibberellic acid, GA 3 , applied to the seedlings pretreated with red light 1 hour before the onset of the phototropic experiment, increases the curvature, whereas GA 3 applied to plants grown in white light has no effect. The growth rate of the treated plants does not differ from that of the control group. Seedlings treated daily with various concentrations of the growth retardant CCC show different lengths after four days. Their phototropic curvature, however, is the same, again demonstrating that the extent of curvature is not related to the elongation growth.In the General Discussion (Chapter 8) the CHOLODNY-WENT theory is rejected as an explanation of phototropism in the sunflower seedling. Instead the theory of BLAAUW is modified by ascribing the phototropic reaction to a growth inhibition at the irradiated side, caused by light-induced relative accumulation of xanthoxin at that side. This may account for a curvature independent of the growth rate regulated by auxin and gibberellin.
CO2 - assimilation light response curves of leaves : some experimental data
Laar, H.H. van; Penning de Vries, F.W.T. - \ 1972
Wageningen : [s.n.] (Verslagen / Instituut voor biologisch en Scheikundig onderzoek no. 62) - 1972
fototropie - planten - fotosynthese - plantenfysiologie - plantenontwikkeling - bladeren - licht - fotoperiode - fotoperiodiciteit - schaduw - phototropism - plants - photosynthesis - plant physiology - plant development - leaves - light - photoperiod - photoperiodism - shade
|Oscillations in the redistribution of the growth substance naphthylacetic acid after phototropic induction
Haan, I. de - \ 1969
Wageningen : [s.n.] (Publication / Centre for plant physiological research no. 77) - 11
naa - metabolisme - herverdeling - fototropie - sapstroom - zea mays - maïs - naa - metabolism - redistribution - phototropism - sap flow - zea mays - maize