Engineering Chlorophyll, Bacteriochlorophyll, and Carotenoid Biosynthetic Pathways in Escherichia coli.

The biosynthesis of chlorophylls (Chls) and bacteriochlorophylls (BChls) represents a key aspect of photosynthesis research. Our previous work assembled the complete pathway for the synthesis of Chl a in Escherichia coli; here we engineer the more complex BChl a pathway in the same heterotrophic host. Coexpression of 18 genes enabled E. coli to produce BChl a, verifying that we have identified the minimum set of genes for the BChl a biosynthesis pathway. The protochlorophyllide reduction step was mediated by the bchNBL genes, and this same module was used to modify the Chl a pathway previously constructed in E. coli, eliminating the need for the light-dependent protochlorophyllide reductase. Furthermore, we demonstrate the feasibility of synthesizing more than one family of photosynthetic pigments in one host by engineering E. coli strains that accumulate the carotenoids neurosporene and ß-carotene in addition to BChl a.

How the O2-dependent Mg-protoporphyrin monomethyl ester cyclase forms the fifth ring of chlorophylls.

Mg-protoporphyrin IX monomethyl ester (MgPME) cyclase catalyses the formation of the isocyclic ring, producing protochlorophyllide a and contributing substantially to the absorption properties of chlorophylls and bacteriochlorophylls. The O2-dependent cyclase is found in both oxygenic phototrophs and some purple bacteria. We overproduced the simplest form of the cyclase, AcsF, from Rubrivivax gelatinosus, in Escherichia coli. In biochemical assays the di-iron cluster within AcsF is reduced by ferredoxin furnished by NADPH and ferredoxin:NADP+ reductase, or by direct coupling to Photosystem I photochemistry, linking cyclase to the photosynthetic electron transport chain. Kinetic analyses yielded a turnover number of 0.9 min-1, a Michaelis-Menten constant of 7.0 µM for MgPME and a dissociation constant for MgPME of 0.16 µM. Mass spectrometry identified 131-hydroxy-MgPME and 131-keto-MgPME as cyclase reaction intermediates, revealing the steps that form the isocyclic ring and completing the work originated by Sam Granick in 1950.

Evolution of Ycf54-independent chlorophyll biosynthesis in cyanobacteria.

Chlorophylls (Chls) are essential cofactors for photosynthesis. One of the least understood steps of Chl biosynthesis is formation of the fifth (E) ring, where the red substrate, magnesium protoporphyrin IX monomethyl ester, is converted to the green product, 3,8-divinyl protochlorophyllide a In oxygenic phototrophs, this reaction is catalyzed by an oxygen-dependent cyclase, consisting of a catalytic subunit (AcsF/CycI) and an auxiliary protein, Ycf54. Deletion of Ycf54 impairs cyclase activity and results in severe Chl deficiency, but its exact role is not clear. Here, we used a Δycf54 mutant of the model cyanobacterium Synechocystis sp. PCC 6803 to generate suppressor mutations that restore normal levels of Chl. Sequencing Δycf54 revertants identified a single D219G amino acid substitution in CycI and frameshifts in slr1916, which encodes a putative esterase. Introduction of these mutations to the original Δycf54 mutant validated the suppressor effect, especially in combination. However, comprehensive analysis of the Δycf54 suppressor strains revealed that the D219G-substituted CycI is only partially active and its accumulation is misregulated, suggesting that Ycf54 controls both the level and activity of CycI. We also show that Slr1916 has Chl dephytylase activity in vitro and its inactivation up-regulates the entire Chl biosynthetic pathway, resulting in improved cyclase activity. Finally, large-scale bioinformatic analysis indicates that our laboratory evolution of Ycf54-independent CycI mimics natural evolution of AcsF in low-light-adapted ecotypes of the oceanic cyanobacteria Prochlorococcus, which lack Ycf54, providing insight into the evolutionary history of the cyclase enzyme.

Protochlorophyllide synthesis by recombinant cyclases from eukaryotic oxygenic phototrophs and the dependence on Ycf54.

The unique isocyclic E ring of chlorophylls contributes to their role as light-absorbing pigments in photosynthesis. The formation of the E ring is catalyzed by the Mg-protoporphyrin IX monomethyl ester cyclase, and the O2-dependent cyclase in prokaryotes consists of a diiron protein AcsF, augmented in cyanobacteria by an auxiliary subunit Ycf54. Here, we establish the composition of plant and algal cyclases, by demonstrating the in vivo heterologous activity of O2-dependent cyclases from the green alga Chlamydomonas reinhardtii and the model plant Arabidopsis thaliana in the anoxygenic photosynthetic bacterium Rubrivivax gelatinosus and in the non-photosynthetic bacterium Escherichia coli. In each case, an AcsF homolog is the core catalytic subunit, but there is an absolute requirement for an algal/plant counterpart of Ycf54, so the necessity for an auxiliary subunit is ubiquitous among oxygenic phototrophs. A C-terminal ∼40 aa extension, which is present specifically in green algal and plant Ycf54 proteins, may play an important role in the normal function of the protein as a cyclase subunit.

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.

Three classes of oxygen-dependent cyclase involved in chlorophyll and bacteriochlorophyll biosynthesis.

The biosynthesis of (bacterio)chlorophyll pigments is among the most productive biological pathways on Earth. Photosynthesis relies on these modified tetrapyrroles for the capture of solar radiation and its conversion to chemical energy. (Bacterio)chlorophylls have an isocyclic fifth ring, the formation of which has remained enigmatic for more than 60 y. This reaction is catalyzed by two unrelated cyclase enzymes using different chemistries. The majority of anoxygenic phototrophic bacteria use BchE, an O2-sensitive [4Fe-4S] cluster protein, whereas plants, cyanobacteria, and some phototrophic bacteria possess an O2-dependent enzyme, the major catalytic component of which is a diiron protein, AcsF. Plant and cyanobacterial mutants in ycf54 display impaired function of the O2-dependent enzyme, accumulating the reaction substrate. Swapping cyclases between cyanobacteria and purple phototrophic bacteria reveals three classes of the O2-dependent enzyme. AcsF from the purple betaproteobacterium Rubrivivax (Rvi.) gelatinosus rescues the loss not only of its cyanobacterial ortholog, cycI, in Synechocystis sp. PCC 6803, but also of ycf54; conversely, coexpression of cyanobacterial cycI and ycf54 is required to complement the loss of acsF in Rvi. gelatinosus These results indicate that Ycf54 is a cyclase subunit in oxygenic phototrophs, and that different classes of the enzyme exist based on their requirement for an additional subunit. AcsF is the cyclase in Rvi. gelatinosus, whereas alphaproteobacterial cyclases require a newly discovered protein that we term BciE, encoded by a gene conserved in these organisms. These data delineate three classes of O2-dependent cyclase in chlorophototrophic organisms from higher plants to bacteria, and their evolution is discussed herein.

Absence of the cbb3 Terminal Oxidase Reveals an Active Oxygen-Dependent Cyclase Involved in Bacteriochlorophyll Biosynthesis in Rhodobacter sphaeroides.

UNLABELLED: The characteristic green color associated with chlorophyll pigments results from the formation of an isocyclic fifth ring on the tetrapyrrole macrocycle during the biosynthesis of these important molecules. This reaction is catalyzed by two unrelated cyclase enzymes employing different chemistries. Oxygenic phototrophs such as plants and cyanobacteria utilize an oxygen-dependent enzyme, the major component of which is a diiron protein named AcsF, while BchE, an oxygen-sensitive [4Fe-4S] cluster protein, dominates in phototrophs inhabiting anoxic environments, such as the purple phototrophic bacterium Rhodobacter sphaeroides We identify a potential acsF in this organism and assay for activity of the encoded protein in a strain lacking bchE under various aeration regimes. Initially, cells lacking bchE did not demonstrate AcsF activity under any condition tested. However, on removal of a gene encoding a subunit of the cbb3-type respiratory terminal oxidase, cells cultured under regimes ranging from oxic to micro-oxic exhibited cyclase activity, confirming the activity of the oxygen-dependent enzyme in this model organism. Potential reasons for the utilization of an oxygen-dependent enzyme in anoxygenic phototrophs are discussed. IMPORTANCE: The formation of the E ring of bacteriochlorophyll pigments is the least well characterized step in their biosynthesis, remaining enigmatic for over 60 years. Two unrelated enzymes catalyze this cyclization step; O2-dependent and O2-independent forms dominate in oxygenic and anoxygenic phototrophs, respectively. We uncover the activity of an O2-dependent enzyme in the anoxygenic purple phototrophic bacterium Rhodobacter sphaeroides, initially by inactivation of the high-affinity terminal respiratory oxidase, cytochrome cbb3 We propose that the O2-dependent form allows for the biosynthesis of a low level of bacteriochlorophyll under oxic conditions, so that a rapid initiation of photosynthetic processes is possible for this bacterium upon a reduction of oxygen tension.

Synthesis of Chlorophyll-Binding Proteins in a Fully Segregated Δycf54 Strain of the Cyanobacterium Synechocystis PCC 6803.

In the chlorophyll (Chl) biosynthesis pathway the formation of protochlorophyllide is catalyzed by Mg-protoporphyrin IX methyl ester (MgPME) cyclase. The Ycf54 protein was recently shown to form a complex with another component of the oxidative cyclase, Sll1214 (CycI), and partial inactivation of the ycf54 gene leads to Chl deficiency in cyanobacteria and plants. The exact function of the Ycf54 is not known, however, and further progress depends on construction and characterization of a mutant cyanobacterial strain with a fully inactivated ycf54 gene. Here, we report the complete deletion of the ycf54 gene in the cyanobacterium Synechocystis 6803; the resulting Δycf54 strain accumulates huge concentrations of the cyclase substrate MgPME together with another pigment, which we identified using nuclear magnetic resonance as 3-formyl MgPME. The detection of a small amount (~13%) of Chl in the Δycf54 mutant provides clear evidence that the Ycf54 protein is important, but not essential, for activity of the oxidative cyclase. The greatly reduced formation of protochlorophyllide in the Δycf54 strain provided an opportunity to use (35)S protein labeling combined with 2D electrophoresis to examine the synthesis of all known Chl-binding protein complexes under drastically restricted de novo Chl biosynthesis. We show that although the Δycf54 strain synthesizes very limited amounts of photosystem I and the CP47 and CP43 subunits of photosystem II (PSII), the synthesis of PSII D1 and D2 subunits and their assembly into the reaction centre (RCII) assembly intermediate were not affected. Furthermore, the levels of other Chl complexes such as cytochrome b 6 f and the HliD- Chl synthase remained comparable to wild-type. These data demonstrate that the requirement for de novo Chl molecules differs completely for each Chl-binding protein. Chl traffic and recycling in the cyanobacterial cell as well as the function of Ycf54 are discussed.

Two Unrelated 8-Vinyl Reductases Ensure Production of Mature Chlorophylls in Acaryochloris marina.

UNLABELLED: The major photopigment of the cyanobacterium Acaryochloris marina is chlorophyll d, while its direct biosynthetic precursor, chlorophyll a, is also present in the cell. These pigments, along with the majority of chlorophylls utilized by oxygenic phototrophs, carry an ethyl group at the C-8 position of the molecule, having undergone reduction of a vinyl group during biosynthesis. Two unrelated classes of 8-vinyl reductase involved in the biosynthesis of chlorophylls are known to exist, BciA and BciB. The genome of Acaryochloris marina contains open reading frames (ORFs) encoding proteins displaying high sequence similarity to BciA or BciB, although they are annotated as genes involved in transcriptional control (nmrA) and methanogenesis (frhB), respectively. These genes were introduced into an 8-vinyl chlorophyll a-producing ΔbciB strain of Synechocystis sp. strain PCC 6803, and both were shown to restore synthesis of the pigment with an ethyl group at C-8, demonstrating their activities as 8-vinyl reductases. We propose that nmrA and frhB be reassigned as bciA and bciB, respectively; transcript and proteomic analysis of Acaryochloris marina reveal that both bciA and bciB are expressed and their encoded proteins are present in the cell, possibly in order to ensure that all synthesized chlorophyll pigment carries an ethyl group at C-8. Potential reasons for the presence of two 8-vinyl reductases in this strain, which is unique for cyanobacteria, are discussed. IMPORTANCE: The cyanobacterium Acaryochloris marina is the best-studied phototrophic organism that uses chlorophyll d for photosynthesis. Unique among cyanobacteria sequenced to date, its genome contains ORFs encoding two unrelated enzymes that catalyze the reduction of the C-8 vinyl group of a precursor molecule to an ethyl group. Carrying a reduced C-8 group may be of particular importance to organisms containing chlorophyll d Plant genomes also contain orthologs of both of these genes; thus, the bacterial progenitor of the chloroplast may also have contained both bciA and bciB.