초록
<P>Efforts to heterologously produce quantities of isoprene hydrocarbons (C<SUB>5</SUB>H<SUB>8</SUB>) renewably from CO<SUB>2</SUB> and H<SUB>2</SUB>O through the photosynthesis of cyanobacteria face barriers, including low levels of recombinant enzyme accumulation compounded by their slow innate catalytic activity. The present work sought to alleviate the “expression level” barrier upon placing the isoprene synthase (IspS) enzyme in different fusion configurations with the cpcB protein, the highly expressed β-subunit of phycocyanin. Different <I>cpcB*IspS</I> fusion constructs were made, distinguished by the absence or presence of linker amino acids between the two proteins. Composition of linker amino acids was variable with lengths of 7, 10, 16, and 65 amino acids designed to test for optimal activity of the IspS through spatial positioning between the cpcB and IspS. Results showed that fusion constructs with the highly expressed cpcB gene, as the leader sequence, improved transgene expression in the range of 61 to 275-fold over what was measured with the unfused IspS control. However, the specific activity of the IspS enzyme was attenuated in all fusion transformants, possibly because of allosteric effects exerted by the leader cpcB fusion protein. This inhibition varied depending on the nature of the linker amino acids between the cpcB and IspS proteins. In terms of isoprene production, the results further showed a trade-off between specific activity and transgenic enzyme accumulation. For example, the <I>cpcB*L7*IspS</I> strain showed only about 10% the isoprene synthase specific-activity of the unfused <I>cpcB-IspS</I> control, but it accumulated 254-fold more IspS enzyme. The latter more than countered the slower specific activity and made the <I>cpcB*L7*IspS</I> transformant the best isoprene producing strain in this work. Isoprene to biomass yield ratios improved from 0.2 mg g<SUP>–1</SUP> in the unfused <I>cpcB-IspS</I> control to 5.4 mg g<SUP>–1</SUP> in the <I>cpcB*L7*IspS</I> strain, a 27-fold improvement.</P><P><B>Graphic Abstract</B><BR><IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/asbcd6/2017/asbcd6.2017.6.issue-12/acssynbio.7b00214/production/images/medium/sb-2017-00214p_0014.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/sb7b00214'>ACS Electronic Supporting Info</A></P>