Complete enzyme set for chlorophyll biosynthesis in Escherichia coli
Chlorophylls are essential cofactors for photosynthesis, which sustains global food chains and oxygen production. Billions of tons of chlorophylls are synthesized annually, yet full understanding of chlorophyll biosynthesis has been hindered by the lack of characterization of the Mg–protoporphyrin IX monomethyl ester oxidative cyclase step, which confers the distinctive green color of these pigments. We demonstrate cyclase activity using heterologously expressed enzyme. Next, we assemble a genetic module that encodes the complete chlorophyll biosynthetic pathway and show that it functions in Escherichia coli. Expression of 12 genes converts endogenous protoporphyrin IX into chlorophyll a, turning E. coli cells green. Our results delineate a minimum set of enzymes required to make chlorophyll and establish a platform for engineering photosynthesis in a heterotrophic model organism. Read the full article here.
(A) Overall reactions from PPIX to Chl a catalyzed by the enzymes introduced to E. coli. The insertion of Fe2+ into PPIX (not shown) creates a biosynthetic branchpoint (not shown) that yields heme. Colored shading denotes the chemical change(s) at each step. ATP, adenosine triphosphate; ADP, adenosine diphosphate; SAM, S-adenosine-l-methionine; SAH, S-adenosyl-l-homocysteine; NADP+, nicotinamide adenine dinucleotide phosphate; NADPH, reduced form of NADP+; Pi, inorganic phosphate; PPi, inorganic pyrophosphate. (B) Arrangement and relative size of each gene in the constructed plasmids. The chlI, chlD, chlH, gun4, chlM, acsF, dvr (bciB), and chlG genes were consecutively cloned into a modified pET3a vector with a single T7 promoter upstream of chlI and a ribosome-binding site upstream of each gene using the link-and-lock method (see fig. S2 for details) (40). Colors for genes correspond to those used in (A). Plasmid constructs and gene contents are IM (chlI–chlM), IA (chlI–acsF), ID (chlI–dvr), IG (chlI–chlG), BoP (BoWSCP and chlP), and DE (dxs and crtE). BoP is a pACYCDuet1-based plasmid containing a sequence encoding the BoWSCP protein with a C-terminal His10 tag (10) and the Synechocystis chlP gene. DE is a pCOLADuet1-based plasmid containing the E. coli dxs gene and the Rvi. gelatinosus crtE gene.