Metabolic engineering of <i>Synechocystis </i>sp. PCC 6803 for production of the plant diterpenoid manoyl oxide

[Image: see text] Forskolin is a high value diterpenoid with a broad range of pharmaceutical applications, naturally found in root bark of the plant Coleus forskohlii. Because of its complex molecular structure, chemical synthesis of forskolin is not commercially attractive. Hence, the labor and resource intensive extraction and purification from C. forskohlii plants remains the current source of the compound. We have engineered the unicellular cyanobacterium Synechocystis sp. PCC 6803 to produce the forskolin precursor 13R-manoyl oxide (13R-MO), paving the way for light driven biotechnological production of this high value compound. In the course of this work, a new series of integrative vectors for use in Synechocystis was developed and used to create stable lines expressing chromosomally integrated CfTPS2 and CfTPS3, the enzymes responsible for the formation of 13R-MO in C. forskohlii. The engineered strains yielded production titers of up to 0.24 mg g(–1) DCW 13R-MO. To increase the yield, 13R-MO producing strains were further engineered by introduction of selected enzymes from C. forskohlii, improving the titer to 0.45 mg g(–1) DCW. This work forms a basis for further development of production of complex plant diterpenoids in cyanobacteria

Metabolic Engineering of Synechocystis sp. PCC 6803 for Terpenoid Production

In the Paris Agreement from 2015, nations agreed to limit the effects of global warming to well below 2°C. To be able to reach those goals, cheap, abundant and carbon neutral energy alternatives needs to be developed. The microorganisms that several billion years ago oxygenated the atmosphere; cyanobacteria, might hold the key for creating those energy technologies. Due to their capacity for photosynthesis, metabolic engineering of cyanobacteria can reroute the carbon dioxide they fix from the atmosphere into valuable products, thereby converting them into solar powered cell factories. Of the many products bacteria can be engineered to make, the production of terpenoids has gained increasing attention for their attractive properties as fuels, pharmaceuticals, fragrances and food additives. In this thesis, I detail the work I have done on engineering the unicellular cyanobacterium Synechocystis sp. PCC 6803 for terpenoid production. By deleting an enzyme that converts squalene into hopanoids, we could create a strain that accumulates squalene, a molecule with uses as a fuel or chemical feedstock. In another study, we integrated two terpene synthases from the traditional medical plant Coleus forskohlii, into the genome of Synechocystis. Expression of those genes led to the formation of manoyl oxide, a precursor to the pharmaceutically active compound forskolin. Production of manoyl oxide in Synechocystis was further enhanced by engineering in two additional genes from C. forskohlii that boosted the flux to the product. To learn how to increase the production of squalene, manoyl oxide or any other terpenoid, we conducted a detailed investigation of each step in the MEP biosynthesis pathway, which creates the two common building blocks for all terpenoids. Each enzymatic step in the pathway was overexpressed, and increased flux was assayed by using isoprene as a reporter and several potential targets for overexpression were identified. The final part of this thesis details the characterization of native, inducible promoters and ribosomal binding sites in Synechocystis.

Metabolic Engineering of Synechocystis sp. PCC 6803 for Terpenoid Production

In the Paris Agreement from 2015, nations agreed to limit the effects of global warming to well below 2°C. To be able to reach those goals, cheap, abundant and carbon neutral energy alternatives needs to be developed. The microorganisms that several billion years ago oxygenated the atmosphere; cyanobacteria, might hold the key for creating those energy technologies. Due to their capacity for photosynthesis, metabolic engineering of cyanobacteria can reroute the carbon dioxide they fix from the atmosphere into valuable products, thereby converting them into solar powered cell factories. Of the many products bacteria can be engineered to make, the production of terpenoids has gained increasing attention for their attractive properties as fuels, pharmaceuticals, fragrances and food additives. In this thesis, I detail the work I have done on engineering the unicellular cyanobacterium Synechocystis sp. PCC 6803 for terpenoid production. By deleting an enzyme that converts squalene into hopanoids, we could create a strain that accumulates squalene, a molecule with uses as a fuel or chemical feedstock. In another study, we integrated two terpene synthases from the traditional medical plant Coleus forskohlii, into the genome of Synechocystis. Expression of those genes led to the formation of manoyl oxide, a precursor to the pharmaceutically active compound forskolin. Production of manoyl oxide in Synechocystis was further enhanced by engineering in two additional genes from C. forskohlii that boosted the flux to the product. To learn how to increase the production of squalene, manoyl oxide or any other terpenoid, we conducted a detailed investigation of each step in the MEP biosynthesis pathway, which creates the two common building blocks for all terpenoids. Each enzymatic step in the pathway was overexpressed, and increased flux was assayed by using isoprene as a reporter and several potential targets for overexpression were identified. The final part of this thesis details the characterization of native, inducible promoters and ribosomal binding sites in Synechocystis.

"It’s dangerous to live" : a study of after-school teachers and their approach to students in relation to boundary-setting outdoors

Gränssättning i fritidshemmet handlar om att skapa regler som ger barn trygghet och fördelaktiga förutsättningar för utveckling. Syftet med denna studie är att undersöka fritidshemslärares förhållningssätt till sina elever och hur detta påverkar deras syn på gränssättning i relation till aktiviteter i fritidshemmets utomhusmiljö. Studien använder sig av ett barndomssociologiskt teoretiskt perspektiv och dess relaterade begrepp. Studien använder svensk och internationell forskning och utgått från kvalitativa intervjuer av fritidshemslärare för att komma nära deras individuella synsätt på frågeställningarna. Intervjuerna spelades in för att senare transkriberas och analyseras. I denna studie framkommer det att fritidshemslärarna är en övergripande struktur som påverkar barns möjligheter till handling. I sin gränssättning sätter de barnets bästa främst och genom det skapa trygga miljöer som bidrar till elevernas utveckling och förhindrar trauma och onödig skada. Fritidshemslärarnas barnsyn visar att de ser eleverna som sårbara med behov av stöd samtidigt som de ger eleverna utrymme att vara kompetenta aktörer med möjlighet till eget handlingsutrymme, men att graden av självständighet beror elevernas ålder och mognad.Boundary-setting in leisure-time teaching is about creating rules giving pupils safe and favorable conditions for their development. The aim of this study is to shed light on leisure-time teachers' approach to their pupils and how this affects their outlook on boundary-setting in relation to leisure-time centres outdoor environment. The study’s outlook is based on the theoretical perspective of childhood sociology and its concepts. The study has used Swedish and international research and been based upon qualitative interviews of leisure-time teachers to get close to their individual approach to the questions. The interviews were recorded, transcribed, and then analyzed. This study shows that leisure-time teachers function as a comprehensive structure that affects children's possibilities for action. In their boundary-setting leisure-time teachers act in the interests of the children, which creates safe environments that will contribute to the pupil’s development and meanwhile prevent trauma as well as unnecessary injury. The leisure-time teachers' perspective on children presents them as vulnerable and in need of support while pupils are given room to function as competent agents on their own, though the degree of independence depends upon the pupils age and maturity

"It’s dangerous to live" : a study of after-school teachers and their approach to students in relation to boundary-setting outdoors

Gränssättning i fritidshemmet handlar om att skapa regler som ger barn trygghet och fördelaktiga förutsättningar för utveckling. Syftet med denna studie är att undersöka fritidshemslärares förhållningssätt till sina elever och hur detta påverkar deras syn på gränssättning i relation till aktiviteter i fritidshemmets utomhusmiljö. Studien använder sig av ett barndomssociologiskt teoretiskt perspektiv och dess relaterade begrepp. Studien använder svensk och internationell forskning och utgått från kvalitativa intervjuer av fritidshemslärare för att komma nära deras individuella synsätt på frågeställningarna. Intervjuerna spelades in för att senare transkriberas och analyseras. I denna studie framkommer det att fritidshemslärarna är en övergripande struktur som påverkar barns möjligheter till handling. I sin gränssättning sätter de barnets bästa främst och genom det skapa trygga miljöer som bidrar till elevernas utveckling och förhindrar trauma och onödig skada. Fritidshemslärarnas barnsyn visar att de ser eleverna som sårbara med behov av stöd samtidigt som de ger eleverna utrymme att vara kompetenta aktörer med möjlighet till eget handlingsutrymme, men att graden av självständighet beror elevernas ålder och mognad.Boundary-setting in leisure-time teaching is about creating rules giving pupils safe and favorable conditions for their development. The aim of this study is to shed light on leisure-time teachers' approach to their pupils and how this affects their outlook on boundary-setting in relation to leisure-time centres outdoor environment. The study’s outlook is based on the theoretical perspective of childhood sociology and its concepts. The study has used Swedish and international research and been based upon qualitative interviews of leisure-time teachers to get close to their individual approach to the questions. The interviews were recorded, transcribed, and then analyzed. This study shows that leisure-time teachers function as a comprehensive structure that affects children's possibilities for action. In their boundary-setting leisure-time teachers act in the interests of the children, which creates safe environments that will contribute to the pupil’s development and meanwhile prevent trauma as well as unnecessary injury. The leisure-time teachers' perspective on children presents them as vulnerable and in need of support while pupils are given room to function as competent agents on their own, though the degree of independence depends upon the pupils age and maturity

Evaluation of promoters and ribosome binding sites for biotechnological applications in the unicellular cyanobacterium Synechocystis sp. PCC 6803

For effective metabolic engineering, a toolbox of genetic components that enables predictable control of gene expression is needed. Here we present a systematic study of promoters and ribosome binding sites in the unicellular cyanobacterium Synechocystis sp. PCC 6803. A set of metal ion inducible promoters from Synechocystis were compared to commonly used constitutive promoters, by measuring fluorescence of a reporter protein in a standardized setting to allow for accurate comparisons of promoter activity. The most versatile and useful promoter was found to be PnrsB, which from a relatively silent expression could be induced almost 40-fold, nearly up to the activity of the strong psbA2 promoter. By varying the concentrations of the two metal ion inducers Ni(2+) and Co(2+), expression from the promoter was highly tunable, results that were reproduced with PnrsB driving ethanol production. The activities of several ribosomal binding sites were also measured, and tested in parallel in Synechocystis and Escherichia coli. The results of the study add useful information to the Synechocystis genetic toolbox for biotechnological applications

Engineered cyanobacteria with enhanced growth show increased ethanol production and higher biofuel to biomass ratio

The Calvin-Benson-Bassham (CBB) cycle is the main pathway to fix atmospheric CO2 and store energy in carbon bonds, forming the precursors of most primary and secondary metabolites necessary for life. Speeding up the CBB cycle theoretically has positive effects on the subsequent growth and/or the end metabolite(s) production. Four CBB cycle enzymes, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase), transketolase (TK) and aldolase (FBA) were selected to be co-overexpressed with the ethanol synthesis enzymes pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) in the cyanobacterium Synechocystis PCC 6803. An inducible promoter, PnrsB, was used to drive PDC and ADH expression. When PnrsB was induced and cells were cultivated at 65 µmol photons m−2 s−1, the RuBisCO-, FBP/SBPase-, TK-, and FBA-expressing strains produced 55%, 67%, 37% and 69% more ethanol and 7.7%, 15.1%, 8.8% and 10.1% more total biomass (the sum of dry cell weight and ethanol), respectively, compared to the strain only expressing the ethanol biosynthesis pathway. The ethanol to total biomass ratio was also increased in CBB cycle enzymes overexpressing strains. This study experimentally demonstrates that using the cells with enhanced carbon fixation, when the product synthesis pathway is not the main bottleneck, can significantly increase the generation of a product (exemplified with ethanol), which acts as a carbon sink

Introduction of a green algal squalene synthase enhances squalene accumulation in a strain of Synechocystis sp. PCC 6803

Squalene is a triterpene which is produced as a precursor for a wide range of terpenoid compounds in many organisms. It has commercial use in food and cosmetics but could also be used as a feedstock for production of chemicals and fuels, if generated sustainably on a large scale. We have engineered a cyanobacterium, Synechocystis sp. PCC 6803, for production of squalene from CO2. In this organism, squalene is produced via the methylerythritol-phosphate (MEP) pathway for terpenoid biosynthesis, and consumed by the enzyme squalene hopene cyclase (Shc) for generation of hopanoids. The gene encoding Shc in Synechocystis was inactivated (Delta shc) by insertion of a gene encoding a squalene synthase from the green alga Botryococcus braunii, under control of an inducible promoter. We could demonstrate elevated squalene generation in cells where the algal enzyme was induced. Heterologous overexpression of genes upstream in the MEP pathway further enhanced the production of squalene, to a level three times higher than the.shc background strain. During growth in flat panel bioreactors, a squalene titer of 5.1 mg/L of culture was reached