Exploring and Engineering Cyanobacterial Metabolism for Biofuel and Sugar Productions
- Author(s): Chuang, Shih-Wei
- Advisor(s): Liao, James
- et al.
Direct conversion of CO2 into chemical production through cyanobacteria is a desirable solution to utilizing the excess amount of greenhouse gases released by burning fossil fuel. Several important chemical products including iso-butanol, n-butanol and 3-hydroxypropionate have been demonstrated in our and other laboratories. However, several issues remain unsolved before practical deployment of such technologies. These problems require both scientific and engineering investigations. In this thesis, we start by reporting an effort on improving n-butanol production from Synechococcus elongatus PCC7942 by determining the bottleneck in a production strain. Then, we focus on determining the physiological role of phosphoketolase (Xpk), which is an enzyme that can be used in the synthetic pathway, non-oxidative glycolysis. Although this enzyme is prevalent in cyanobacteria, its physiological role or biochemical properties are still unknown. Therefore, the second part of my thesis describes the characterization of the biochemical and physiological roles of Xpk in cyanobacteria.To improve n-butanol production, pathway optimization for heterologous expression is one of the important strategies to enhance the carbon flux toward the targeted metabolite. Here, butyryl-CoA is identified as the bottlenecks in the original n-butanol producer through heterologous expression of Clostridial CoA dependent butanol production pathway in Synechococcus elongatus PCC7942. The combined strategy of enhancing the alcohol dehydrogenase (PduP) activity and overexpression of the single unit acetyl-CoA carboxylase from Yarrowia lipolytica, increases n-butanol production from 285mg/L in the original producer to 355mg/L in the strain DC11 after induction 10 days. To further improve metabolite production derived from acetyl-CoA, Xpk expression has been suggested to form the non-oxidative glycolysis pathway that conserves carbon. Xpk is an enzyme that links several primary metabolic pathways in cyanobacteria, including Calvin-Benson-Bassham (CBB) pathway, oxidative pentose phosphate (OPP) pathway, Embden–Meyerhof–Parnas (EMP) pathway and the tricarboxylic acid (TCA) cycle. Putative sequences annotated as Xpk are widespread in most well-studied cyanobacteria strains. However, little is known about its physiological functions. Here, we show that Xpk from S. elongatus PCC7942 is inhibited by ATP. Further surveying Xpk’s from different organisms indicates that ATP inhibition is conserved among cyanobacteria strains, suggesting its potential roles under low ATP conditions. Moreover, Xpk in S. elongatus PCC7942 and Cyanothece ATCC51142 show that they are dusk-peaked genes in day-night (12hr/12hr) cycle grown cultures, suggesting their roles in dark conditions. Finally, we find that knocking out xpk in S. elongatus PCC7942 abolished acetate production under dark and anaerobic condition. Interestingly, under this condition, Xpk pathway is particularly important in response to osmotic shock such as NaHCO3, KHCO3 and NaCl and its deletion causes viability decrease and glucose secretion as part of the incomplete glycogen degradation.