Combinatory strategy for characterizing and understanding the ethanol synthesis pathway in cyanobacteria cell factories

Background Photosynthetic production of chemicals and fuels by recycling CO 2 in cyanobacteria is a promising solution facing energy shortage and resource declination. Ethanol is an attractive and demonstrative biofuel product, and ethanol synthesis in cyanobacteria has been achieved by assembling of a pathway consisting of pyruvate decarboxylase ( PDCzm ) and alcohol dehydrogenase II ( slr1192 ). For enabling more powerful ethanol photosynthetic production, an optimized and balanced catalyzing route was required. In this work, we provided a paradigm for systematically characterizing and optimizing the PDCzm-slr1192 pathway from engineered cyanobacteria strains, combining in vitro reconstitution, genetic engineering and feeding-cultivation. Results We reconstituted the PDCzm-slr1192 pathway in vitro and performed specific titration assays for enzymes, substrates, cofactors, and metal ions. In the in vitro system, K 50 of PDCzm was 0.326 μM, with a V max of 2.074 μM/s; while for slr1192, the values were 0.109 μM and 1.722 μM/s, respectively. Titration response discrepancy indicated that PDCzm rather than slr1192 was the rate-limiting factor for ethanol synthesis. In addition, a 4:6 concentration ratio of PDCzm-slr1192 would endow the reaction with a maximal specific catalytic activity. Titration assays for other components were also performed. K m values for NADPH, pyruvate, TPP, Mg 2+ and acetaldehyde were 0.136, 6.496, 0.011, 0.104, and 0.393 mM, respectively. We further constructed Synechocystis mutant strains with diverse PDCzm-slr1192 concentrations and ratios, and compared the growth and ethanol synthesis performances. The results revealed that activities of PDCzm indeed held control over the ethanol generation capacities. We performed pyruvate-feeding treatment with the newly developed Syn-YQ4 strain, and confirmed that improvement of pyruvate supply would direct more carbon flow to ethanol formation. Conclusions We systematically characterized and optimized the PDCzm-slr1192 pathway in engineered cyanobacteria for ethanol production. Information gained from in vitro monitoring and genetic engineering revealed that for further enhancing ethanol synthesis capacities, PDCzm activities needed enhancement, and the PDCzm-slr1192 ratio should be improved and held to about 1:1.5. Considering actual metabolites concentrations of cyanobacteria cells, enhancing pyruvate supply was also a promising strategy for further updating the current ethanol photosynthetic cell factories.