초록
<P><B>Abstract</B></P> <P>Cyanobacteria can be exploited as photosynthetic platforms for heterologous generation of terpene hydrocarbons with industrial applications. Transformation of <I>Synechocystis</I> and heterologous expression of the β-phellandrene synthase (<I>PHLS</I>) gene alone is necessary and sufficient to confer to <I>Synechocystis</I> the ability to divert intermediate terpenoid metabolites and to generate the monoterpene β-phellandrene during photosynthesis. However, terpene synthases, including the PHLS, have a slow <I>K</I> <SUB>cat</SUB> (low <I>V</I> <SUB>max</SUB>) necessitating high levels of enzyme concentration to enable meaningful rates and yield of product formation. Here, a novel approach was applied to increase the PHLS protein expression alleviating limitations in the rate and yield of β-phellandrene product generation. Different <I>PHLS</I> fusion constructs were generated with the <I>Synechocystis</I> endogenous <I>cpcB</I> sequence, encoding for the abundant in cyanobacteria phycocyanin β-subunit, expressed under the native <I>cpc</I> operon promoter. In one of these constructs, the CpcB·PHLS fusion protein accumulated to levels approaching 20% of the total cellular protein, i.e., substantially higher than expressing the PHLS protein alone under the same endogenous <I>cpc</I> promoter. The CpcB·PHLS fusion protein retained the activity of the PHLS enzyme and catalyzed β-phellandrene synthesis, yielding an average of 3.2mgproductg<SUP>−1</SUP> dry cell weight (dcw) versus the 0.03mgg<SUP>−1</SUP> dcw measured with low-expressing constructs, i.e., a 100-fold yield improvement. In conclusion, the terpene synthase fusion-protein approach is promising, as, in this case, it substantially increased the amount of the PHLS in cyanobacteria, and commensurately improved rates and yield of β-phellandrene hydrocarbons production in these photosynthetic microorganisms.</P> <P><B>Highlights</B></P> <P> <UL> <LI> •Cyanobacteria are suitable platforms for the generation of terpene hydrocarbons. </LI> <LI> •Rates and yield of terpene production are limited by the slow <I>K</I> <SUB>cat</SUB> of terpene synthases. </LI> <LI> •High amounts of heterologous terpene synthase are needed to enhance product yield. </LI> <LI> •A phycocyanin-terpene synthase fusion increased recombinant protein amount in cells. </LI> <LI> •A phycocyanin-phellandrene synthase fusion substantially enhanced product yields. </LI> </UL> </P>