A phycocyanin-deletion mutant of Synechocystis (cyanobacteria) was generated upon replacement of the CPC-operon with a kanamycin resistance cassette. The Δcpc transformant strains (Δcpc) exhibited a green phenotype, compared to the blue-green of the wild type (WT), lacked the distinct phycocyanin absorbance at 625nm, and had a lower Chl per cell content and a lower PSI/PSII reaction center ratio compared to the WT. Molecular and genetic analyses showed replacement of all WT copies of the Synechocystis DNA with the transgenic version, thereby achieving genomic DNA homoplasmy. Biochemical analyses showed the absence of the phycocyanin α- and β-subunits, and the overexpression of the kanamycin resistance NPTI protein in the Δcpc. Physiological analyses revealed a higher, by a factor of about 2, intensity for the saturation of photosynthesis in the Δcpc compared to the WT. Under limiting intensities of illumination, growth of the Δcpc was slower than that of the WT. This difference in the rate of cell duplication diminished gradually as growth irradiance increased. Identical rates of cell duplication of about 13h for both WT and Δcpc were observed at about 800μmolphotonsm(-2)s(-1) or greater. Culture productivity analyses under simulated bright sunlight and high cell-density conditions showed that biomass accumulation by the Δcpc was 1.57-times greater than that achieved by the WT. Thus, the work provides first-time direct evidence of the applicability of the Truncated Light-harvesting Antenna (TLA)-concept in cyanobacteria, entailing substantial improvements in the photosynthetic efficiency and productivity of mass cultures upon minimizing the phycobilisome light-harvesting antenna size.

The phycobilisome is an elaborate antenna that is responsible for light-harvesting in cyanobacteria and red-algae. This large macromolecular complex captures incident sunlight and transfers the energy via a network of pigment molecules called bilins to the photosynthetic reaction centers. The phycobilisome of the model organism Synechocystis PCC 6803 consists of a core to which six rods are attached but its detailed molecular architecture and regulation in response to environmental conditions is not well understood. Here we present cryo-electron microscopy structures of the 6.2 MDa phycobilisome from Synechocystis PCC 6803 resolved at 2.1 Å (rods) to 2.7 Å (core), revealing three distinct conformations, two previously unknown. We found that two of the rods are mobile and can switch conformation within the complex, revealing a layer of regulation not described previously. In addition, we found a novel linker protein in the structure, that may represent a long-sought subunit that tethers the phycobilisome to the thylakoid membrane. Finally, we show how excitation energy is transferred within the phycobilisome and correlate our structures with known spectroscopic properties. Together, our results provide detailed insights into the biophysical underpinnings of cyanobacterial light harvesting and lay the foundation for bioengineering of future phycobilisome variants and artificial light harvesting systems.

The truncated light-harvesting antenna size3 (tla3) DNA insertional transformant of Chlamydomonas reinhardtii is a chlorophylldeficient mutant with a lighter green phenotype, a lower chlorophyll (Chl) per cell content, and higher Chl a/b ratio than corresponding wild-type strains. Functional analyses revealed a higher intensity for the saturation of photosynthesis and greater light-saturated photosynthetic activity in the tla3 mutant than in the wild type and a Chl antenna size of the photosystems that was only about 40% of that in the wild type. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western-blot analyses showed that the tla3 strain was deficient in the Chl a/b light-harvesting complex. Molecular and genetic analyses revealed a single plasmid insertion in chromosome 4 of the tla3 nuclear genome, causing deletion of predicted gene g5047 and plasmid insertion within the fourth intron of downstream-predicted gene g5046. Complementation studies defined that gene g5047 alone was necessary and sufficient to rescue the tla3 mutation. Gene g5047 encodes a C. reinhardtii homolog of the chloroplast-localized SRP43 signal recognition particle, whose occurrence and function in green microalgae has not hitherto been investigated. Biochemical analysis showed that the nucleus-encoded and chloroplast-localized CrCpSRP43 protein specifically operates in the assembly of the peripheral components of the Chl a/b light-harvesting antenna. This work demonstrates that cpsrp43 deletion in green microalgae can be employed to generate tla mutants with a substantially diminished Chl antenna size. The latter exhibit improved solar energy conversion efficiency and photosynthetic productivity under mass culture and bright sunlight conditions.

There is a need to develop renewable fuels and chemicals that will help meet global demands for energy and synthetic chemistry feedstock, without contributing to climate change or environmental degradation. Isoprene (C 5 H 8 ) is one such key chemical ingredient, required for the production of synthetic rubber or plastic products, and a potential biofuel. Enabling a sustainable microbial fermentation for the production of isoprene is an attractive alternative to a petroleum origin. This work demonstrates transgenic expression of the Pueraria montana (kudzu vine) isoprene synthase gene (kIspS) and heterologous isoprene production in Escherichia coli. Enhancements in the amount of E. coli isoprene production were achieved upon over-expression of the native 2-Cmethyl-D-erythritol-4-phosphate (MEP) biosynthetic pathway and, independently, upon heterologous over-expression of the entire mevalonic acid (MVA) pathway. A direct comparison of the efficiency of cellular organic carbon flux through the MEP and MVA pathways is provided, under conditions when these are expressed in the same host using the same plasmid, and same ribosome-binding sites (RBS). These alternative isoprenoid biosynthetic pathways were assembled in and expressed through a superoperon, suitable for transformation of E. coli. Introduction of specific RBS and nucleotide spacers between individual genes in the superoperon structure enabled maximal expression in E. coli batch cultures and translated to an improved production from 0.4 mg isoprene per liter of culture (control) to 5 mg isoprene per liter of culture (MEP superoperon transformants) and up to 320 mg isoprene per liter of culture (MVA superoperon transformants). This 800-fold increase in isoprene concentration from the MVA transformants and the attendant isoprene-to-biomass 0.78:1 carbon partitioning ratio suggested that the engineered MVA pathway introduces a bypass in the flux of endogenous substrate in E. coli to isopentenyl-diphosphate and dimethylallyl-diphosphate, thus overcoming flux limitations imposed upon the regulation of the native MEP pathway by the cell.

The truncated light-harvesting antenna2 (tla2) mutant of Chlamydomonas reinhardtii showed a lighter-green phenotype, had a lower chlorophyll (Chl) per-cell content, and higher Chl a/b ratio than corresponding wild-type strains. Physiological analyses revealed a higher intensity for the saturation of photosynthesis and greater P max values in the tla2 mutant than in the wild type. Biochemical analyses showed that the tla2 strain was deficient in the Chl a-b light-harvesting complex, and had a Chl antenna size of the photosystems that was only about 65% of that in the wild type. Molecular and genetic analyses showed a single plasmid insertion in the tla2 strain, causing a chromosomal DNA rearrangement and deletion/disruption of five nuclear genes. The TLA2 gene, causing the tla2 phenotype, was cloned by mapping the insertion site and upon complementation with each of the genes that were deleted. Successful complementation was achieved with the C. reinhardtii TLA2-CpFTSY gene, whose occurrence and function in green microalgae has not hitherto been investigated. Functional analysis showed that the nuclearencoded and chloroplast-localized CrCpFTSY protein specifically operates in the assembly of the peripheral components of the Chl a-b light-harvesting antenna. In higher plants, a cpftsy null mutation inhibits assembly of both the light-harvesting complex and photosystem complexes, thus resulting in a seedling-lethal phenotype. The work shows that cpftsy deletion in green algae, but not in higher plants, can be employed to generate tla mutants. The latter exhibit improved solar energy conversion efficiency and photosynthetic productivity under mass culture and bright sunlight conditions. Photosynthesis depends on the absorption of sunlight by chlorophyll (Chl) molecules in PSI and PSII. In higher plants and green algae, a completely functional but minimal PSI unit encompasses 95 Chl a molecules, while PSII functions with a minimal number of 37 Chl a molecules (Glick and Melis, 1988;Zouni et al., 2001). Increasing the number of light-harvesting pigments associated with each reaction center, upon the addition of peripheral Chl a and b molecules, is thought to afford a competitive advantage to the organism in an environment where sunlight is often limiting (Kirk, 1994). Photosynthetic organisms evolved a variety of such pigment-containing protein complexes associated peripherally with PSI and PSII. In higher plants and algae, these are referred to as Chl a-b light-harvesting complex (LHC)-I and LHC-II for photosystem-I (PSI) and photosystem-II (PSII) respectively. Photosystemperipheral LHCs serve as auxiliary antennae for the collection of sunlight energy and as a conducting medium for excitation energy migration toward a photochemical reaction center (Smith et al., 1990). The Chl a-b LHCs increase the number of pigment molecules that are associated with the reaction centers, normally up to 250 for PSI and 300 for PSII (Ley and Mauzerall, 1982;Melis and Anderson, 1983;Smith et al., 1990;Melis, 1991).The Chl antenna size...