Metabolic engineering of cyanobacteria: developing molecular tools and characterizing strain performance in light:dark cycles

Abstract

The conversion of CO2 and light energy to biofuels holds promise for a renewable and environmentally responsible source of energy that could meet the growing demand for transportation fuels. However, early efforts to commercialize biofuels from plants were hampered by social, economic, and technological difficulties. Photosynthetic microbes present an opportunity for a more efficient conversion of fixed carbon to biofuels by bypassing the need of harvesting sugars from plants to be fermented by heterotrophic bacteria. More recently, cyanobacterial technologies have received considerable interest due to their ease of genetic manipulation that enables them to produce a myriad of biofuels and biochemicals directly from CO2. This relatively nascent technology needs to be developed in order to realize its commercial potential. Metabolic engineering is the systematic improvement of strains through the use of a variety of theoretical and experimental techniques. To date, heterologous pathways expression has been the most successful in model heterotrophic organisms (e.g. E. coli) and advances from these systems have to be carefully transferred over to cyanobacteria. Though several studies have demonstrated the capability of engineering cyanobacteria to produce biofuels, there is yet to be any commercially feasible production platform of fuels from CO2. Amongst the challenges is the need to improve yields and titers from recombinant strains. However, the physiology of cyanobacteria is distinct from that of heterotrophic organisms and therefore requires careful design and study in order to optimize for higher yields. This thesis contributes several technologies to foster the scale-up of cyanobacteria systems from the bench to industrial scale. We first developed a markerless chromosomal modification method in WT Synechocystis PCC6803 that could reduce the metabolic load and cultivation cost compared to plasmid-based expression methods. We established a counter-selection method that necessitates two rounds of modifications in order to screen for the desired mutant harboring the gene(s) of interest. In the first round, a synthetic circuit consisting of a nickel inducible toxin gene (mazF) and a kanamycin resistance marker is integrated into a specific locus in WT Synechocystis. In the second round, a construct harboring gene(s) of interest is transformed into the prerequisite strains and screen on Ni2+ to obtain the desired mutants. Next we established a free fatty acid (FFA) producing platform in Synechocystis PCC6803 by pursuing three goals: 1) deletion of acyl-acyl carrier protein (acyl-ACP) synthetase (aas), 2) optimize the expression of thioesterase I (TesA) with a promoter library and 3) examine the effects of light:dark cycles on FFA production in Synechocystis. For the first goal, we were successful in engineering an aas deletion strain that had increased FFA production. In the second goal, we developed four Synechocystis variants with increasing TesA expression strengths from the aas deletion strain. No increase in FFA production was observed between the TesA expressing strains (with aas deleted) compared to the baseline aas deletion strain. On the protein level, we found no evidence of TesA enzyme activity even though TESA peptides were detected in our Synechocystis strains. In the third goal, we learn that diel light:dark cycles causes a significant decrease in production of FFAs in FFA producing mutants of Synechocystis compared to continuous light. We did not observe any transcriptional changes in the fatty acid biosynthesis pathway between our WT and FFA producing strains to explain these changes. In summary, this thesis is impactful in two ways: 1) it entails the development of a markerless genetic modification method for use in cyanobacteria and 2) it characterizes the production of FFAs from engineered cyanobacteria under diel light:dark cycles. Overall, this thesis helps address the difficulties in the development of cyanobacteria systems for eventual use in an industrial setting.