초록
<P><B>Background</B></P><P>Photosynthetic production of chemicals and fuels by recycling CO<SUB>2</SUB> 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 (<I>PDCzm</I>) and alcohol dehydrogenase II (<I>slr1192</I>). 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 <I>in vitro</I> reconstitution, genetic engineering and feeding-cultivation.</P><P><B>Results</B></P><P>We reconstituted the PDCzm-slr1192 pathway <I>in vitro</I> and performed specific titration assays for enzymes, substrates, cofactors, and metal ions. In the <I>in vitro</I> system, <I>K</I><SUB>50</SUB> of PDCzm was 0.326 μM, with a <I>V</I><SUB>max</SUB> 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. <I>K</I><SUB><I>m</I></SUB> values for NADPH, pyruvate, TPP, Mg<SUP>2+</SUP> and acetaldehyde were 0.136, 6.496, 0.011, 0.104, and 0.393 mM, respectively. We further constructed <I>Synechocystis</I> 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.</P><P><B>Conclusions</B></P><P>We systematically characterized and optimized the PDCzm-slr1192 pathway in engineered cyanobacteria for ethanol production. Information gained from <I>in vitro</I> 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.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (doi:10.1186/s13068-015-0367-z) contains supplementary material, which is available to authorized users.</P>