Introduction and Expression of PEP Synthase in Synechocystis sp. PCC 6803 Paweł Piątek Degree project in applied biotechnology, Master of Science (2 years), 2013 Examensarbete i tillämpad bioteknik 45 hp till masterexamen, 2013 Biology Education Centre and the Department of Photochemistry and Molecular Science, Ångstrom, Uppsala University, Uppsala University Academic Supervisor: Prof. Peter Lindblad Laboratory Supervisors: Dr. Anja Nenninger and Elias Englund Contents Abstract 2 Introduction 3 Establishing synthetic pathways in cyanobacteria 3 Synthetic Pathway Design 5 The MOG pathway 6 Phosphoenolpyruvate Synthase 8 Pyruvate Phosphate Dikinase 8 Experimental Aims 10 Materials and Methods 10 Results 13 Construction of the pEERM[PEPS] plasmid 13 PEPS light regulation 13 Correct PEPS recombination 14 Examining overexpression of PEPS 15 Discussion 16 Plasmid Selection 16 Antibiotic Resistance Cassette 17 Light regulation of PEPS 17 RNA Extraction & cDNA Synthesis 18 Overexpression of PEPS and further experimentation 19 Conclusion 19 Appendix 20 Section 1: Colorimetric Determination of PEPS activity 20 Introduction and Theory 20 Materials and Methods 21 Results and Discussion 21 Section 2: Materials 23 Section 3: Primers 24 Acknowledgments 25 References 26 Websites/Databases 26 Literature 26 Abstract In recent years photosynthetic cyanobacteria have become the focus in high-value compound production through genetically engineered means. Through manipulation of both native and exogenous DNA, it has been shown that it is possible to construct synthetic pathways within cyanobacteria. However some of these constructed pathways are faced with natural bottle-necks found in the organism’s metabolic pathways. Therefore to produce useful compounds, the demands for the organism to fix carbon from the atmosphere greatly increase. This demand is restricted by the enzyme Rubisco, a native carbon-fixing enzyme found within cyanobacteria and plants alike which has been regarded as inefficient. The aim of this thesis is to highlight an alternate synthetic carbon-fixation pathway one could introduce into the cyanobacterium, Synechocystis sp. PCC 6803. This would be shown through the overexpression of phosphoenolpyruvate synthase (PEPS), the first enzyme of the malonyl-CoA-oxaloacetate-glyoxylate pathway. The initial step involved amplifying the native gene coding for PEPS and cloning it into a plasmid which was introduced into Synechocystis sp. PCC 6803. RNA was extracted and used in preparation for semi-quantitative reverse-transcriptase PCR, which detected whether PEPS is expressed alongside its native counterpart. Reverse-transcriptase PCR was similarly used to detect if wild type PEPS is light regulated. Results showed that there is PEPS overexpression in the desired locus alongside the native PEPS, as well as confirmation that wild type PEPS expression is influenced by light. Future experiments may include assays that detect the level of activity of the overexpressed enzyme in comparison to its wild-type equivalent. 2 Introduction Establishing synthetic pathways in cyanobacteria Metabolically engineered cyanobacteria have become an attractive organism which to produce high- value compounds, chiefly in regards to alternative biofuel production. Through genetically manipulating Synechocystis sp. PCC 6803, it has been shown that its oxygenic photosynthetic properties can provide a practical solution to this challenge, (Rosgaard et al., 2012). Over the course of the last decade, there have been significant strides in this direction with encouraging results illustrating cyanobacteria as excellent candidates for metabolic engineering, Synechocystis sp. PCC 6803 in particular, (Lindberg et al., 2010; Robertson et al., 2011; Skjanes et al., 2013). By capturing light energy and fixing CO2, Synechocystis sp. PCC 6803 (referred to as PCC 6803 thereon after), is able to produce organic compounds through its native pathways. This activity has been of primary interest to many researchers, with examples of such products including; plastic precursors, pharmaceuticals and biofuels, (Lindberg et al., 2010; Ducat et al., 2011; Skjanes et al., 2013; Wang et al., 2013). Some of these compounds can be naturally derived from PCC 6803, although inefficiently, and not on an industrially viable scale. Despite this, there is a wealth of knowledge pointing to the potential of introducing numerous enzymes derived from foreign organisms into PCC 6803 and redesigning entire pathways into producing desirable products, fig. 1, (Rosgaard et al., 2012). Fig. 1; Metabolic pathways in cyanobacteria; Metabolic pathways that have been engineered (orange) to produce products (blue). Several enzymes have been omitted from the Isobutanol and 1-butanol pathway. Abbreviations; 3PGA, 3-phosphoglyceric acid; GAP, glyceraldehyde-3-phosphate; 2PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate; DXP, 1-deoxyxylulose-5-phosphate; HMBPP, 1-hydroxy-2-methyl-2-butenyl-4- pyrophosphate; DMAPP, dimethylallyl-pyrophosphate. Enzyme abbreviations; IspS, isoprene synthase; LdhA, lactate dehydrogenase; Pdc, pyruvate decarboxylase; KivD, oxoacid decarboxylase; 1-butanol pathway enzymes can be found in Rosegaard et. al 2012’s study. Fig. 1, partially adapted from Rosgaard et al., 2012 and Ducat et al., 2011. 3 The first step before introducing any enzymatic pathway is identifying natural bottlenecks in PCC 6803, and inquiring whether it is possible to alleviate or circumvent such obstacles. Carbon is the key element found in all organic compounds and therefore increasing its presence within the organism is vitally important in increasing the amount of desired product. PCC 6803 pumps carbon into the - cytosol in the form of HCO3 and CO2. Equilibrium inside the cytosol is reached with both compounds - present, to which HCO3 can rapidly convert into CO2. The enzyme, Carbonic Anhydrase (CA) found in - PCC 6803’s carboxysome micro-compartments, favours the conversion of HCO3 into CO2. Consequently, CO2 concentrates within the carboxysome and becomes fixed via Ribulose-1,5- bisphosphate carboxylase oxygenase (Rubisco) into Ribulose 1,5-bisphosphate (RuBP). This catalytic reaction ultimately results in 3-phosphoglyceric acid (3PGA) molecules being formed and released back into the cytosol fig. 2, (Badger et al., 2002; Espie and Kimber, 2011). - Fig. 2; Basic model of carbon concentration in cyanobacteria; Carbon in the form of CO2 and HCO3 is pumped - into the cytosol at the expense of ATP and NADH. This carbon accumulates in the form of HCO3 which passes - through the semi-permeable shell of the carboxysome. Carbonic anhydrase (CA) converts HCO3 into CO2 which cannot pass back through the shell. The CO2 is fixed by Rubisco and forms 3-phosphoglyceric acid (3PGA) that leaves the carboxysome to which some is converted into Ribulose 1,5-bisphosphate (RuBP) by the Calvin-Benson-Basham cycle. Fig. 2 partially adapted from Espie and Kimber, 2011 and Badger et al., 2002. This may describe an efficient carbon fixation system, exactly functioning as anticipated, but there are serious drawbacks with Rubisco with respect to metabolic efficiency. The carbon concentrating mechanism (CCM) was developed through evolutionary pressure to counteract the inefficient nature of Rubisco by increasing CO2 a 1000 times around the enzyme’s active site, (Badger and Price, 2003). A particular study pointed out two major flaws with Rubisco; specificity and catalytic turnover, (Marcus et al., 2011). 4 With regards to specificity, Rubisco is able to fix both O2 and CO2, as it is a reminder of an earlier era when O2 was rapidly increasing in the atmosphere and CO2 decreasing. This atmospheric shift resulted in CO2 becoming more scarce and thus a CCM system needed to be established, (Badger and Price, 2003). As there is a far higher abundance of O2 in the atmosphere than CO2, there is direct competition in terms of fixation with Rubisco. O2 fixation results in 2-phosphoglycolate formation that requires recycling back into the photorespiration system, resulting in lost energy and between 30-50% of potential carbon dioxide being fixed, (Raines, 2006; Bauwe et al., 2010). Catalytic rates within Rubisco have been described as slow which results from the kinetic relationship between CO2 and its intermediate transition product (3-keto-2-carboxyarabinitol 1,5- bisphosphate). In an attempt to highlight this relationship, a study has shown the nature of the transition product between CO2 and RuBP becoming tightly bound to the active site of the enzyme. This results in a slow cleavage into two units of RuBP and hinders efficient catalytic throughput, (Tcherkez et al., 2006). Moreover, it has been discussed in this study that Rubisco optimization may not be possible, as it has reached its full activity potential and any further modification would be marginal, if not negligible. Manipulating Rubisco into increased efficiency may be goals to aim for, but at this point, the complexity of the enzyme’s subunits prevent any progress to develop a novel Rubisco counterpart, (Raines, 2006). Therefore taking these considerations into account, one can start to think of ways of entirely avoiding Rubisco as a primary CO2 fixing enzyme. From literature and databases (see References; Websites/Databases) it has been established that there are such enzymes in existence and that lack the Rubisco’s drawbacks (Alber and Fuchs, 2002; Hugler et al., 2002). Moreover, one can manipulate said enzymes into a synthetic pathway which can directly produce the same labile