Stabilization of Photosystem II by the PsbT protein impacts photodamage, repair and biogenesis.

Photosystem II (PS II) catalyzes the light-driven process of water splitting in oxygenic photosynthesis. Four core membrane-spanning proteins, including D1 that binds the majority of the redox-active co-factors, are surrounded by 13 low-molecular-weight (LMW) proteins. We previously observed that deletion of the LMW PsbT protein in the cyanobacterium Synechocystis sp. PCC 6803 slowed electron transfer between the primary and secondary plastoquinone electron acceptors QA and QB and increased the susceptibility of PS II to photodamage. Here we show that photodamaged ∆PsbT cells exhibit unimpaired rates of oxygen evolution if electron transport is supported by HCO3- even though the cells exhibit negligible variable fluorescence. We find that the protein environment in the vicinity of QA and QB is altered upon removal of PsbT resulting in inhibition of QA- oxidation in the presence of 2,5-dimethyl-1,4-benzoquinone, an artificial PS II-specific electron acceptor. Thermoluminescence measurements revealed an increase in charge recombination between the S2 oxidation state of the water-oxidizing complex and QA- by the indirect radiative pathway in ∆PsbT cells and this is accompanied by increased 1O2 production. At the protein level, both D1 removal and replacement, as well as PS II biogenesis, were accelerated in the ∆PsbT strain. Our results demonstrate that PsbT plays a key role in optimizing the electron acceptor complex of the acceptor side of PS II and support the view that repair and biogenesis of PS II share an assembly pathway that incorporates both de novo synthesis and recycling of the assembly modules associated with the core membrane-spanning proteins.

Conserved Activity of Reassociated Homotetrameric Protein Subunits Released from Mesoporous Silica Nanoparticles.

Mesoporous silica nanoparticles (MSN) with enlarged pores were prepared and characterized, and reversibly dissociated subunits of concanavalin A were entrapped in the mesopores, as shown by multiple biochemical and material characterizations. When loaded in the MSN, we demonstrated protein stability from proteases and, upon release, the subunits reassociated into active proteins shown through mannose binding and o-phthalaldehyde fluorescence. We have demonstrated a versatile and facile method to load homomeric proteins into MSN with potential applications in enhancing the delivery of large therapeutic proteins.

Modulation of Medium-Chain Fatty Acid Synthesis in Synechococcus sp. PCC 7002 by Replacing FabH with a Chaetoceros Ketoacyl-ACP Synthase.

The isolation or engineering of algal cells synthesizing high levels of medium-chain fatty acids (MCFAs) is attractive to mitigate the high clouding point of longer chain fatty acids in algal based biodiesel. To develop a more informed understanding of MCFA synthesis in photosynthetic microorganisms, we isolated several algae from Great Salt Lake and screened this collection for MCFA accumulation to identify strains naturally accumulating high levels of MCFA. A diatom, Chaetoceros sp. GSL56, accumulated particularly high levels of C14 (up to 40%), with the majority of C14 fatty acids allocated in triacylglycerols. Using whole cell transcriptome sequencing and de novo assembly, putative genes encoding fatty acid synthesis enzymes were identified. Enzymes from this Chaetoceros sp. were expressed in the cyanobacterium Synechococcus sp. PCC 7002 to validate gene function and to determine whether eukaryotic enzymes putatively lacking bacterial evolutionary control mechanisms could be used to improve MCFA production in this promising production strain. Replacement of the Synechococcus 7002 native FabH with a Chaetoceros ketoacyl-ACP synthase III increased MCFA synthesis up to fivefold. The level of increase is dependent on promoter strength and culturing conditions.

Dynamics of Photosynthesis in a Glycogen-Deficient glgC Mutant of Synechococcus sp. Strain PCC 7002.

Cyanobacterial glycogen-deficient mutants display impaired degradation of light-harvesting phycobilisomes under nitrogen-limiting growth conditions and secrete a suite of organic acids as a putative reductant-spilling mechanism. This genetic background, therefore, represents an important platform to better understand the complex relationships between light harvesting, photosynthetic electron transport, carbon fixation, and carbon/nitrogen metabolisms. In this study, we conducted a comprehensive analysis of the dynamics of photosynthesis as a function of reductant sink manipulation in a glycogen-deficient glgC mutant of Synechococcus sp. strain PCC 7002. The glgC mutant showed increased susceptibility to photoinhibition during the initial phase of nitrogen deprivation. However, after extended periods of nitrogen deprivation, glgC mutant cells maintained higher levels of photosynthetic activity than the wild type, supporting continuous organic acid secretion in the absence of biomass accumulation. In contrast to the wild type, the glgC mutant maintained efficient energy transfer from phycobilisomes to photosystem II (PSII) reaction centers, had an elevated PSII/PSI ratio as a result of reduced PSII degradation, and retained a nitrogen-replete-type ultrastructure, including an extensive thylakoid membrane network, after prolonged nitrogen deprivation. Together, these results suggest that multiple global signals for nitrogen deprivation are not activated in the glgC mutant, allowing the maintenance of active photosynthetic complexes under conditions where photosynthesis would normally be abolished.

Lauric Acid Production in a Glycogen-Less Strain of Synechococcus sp. PCC 7002.

The cyanobacterium Synechococcus sp. Pasteur culture collection 7002 was genetically engineered to synthesize biofuel-compatible medium-chain fatty acids (FAs) during photoautotrophic growth. Expression of a heterologous lauroyl-acyl carrier protein (C12:0-ACP) thioesterase with concurrent deletion of the endogenous putative acyl-ACP synthetase led to secretion of transesterifiable C12:0 FA in CO2-supplemented batch cultures. When grown at steady state over a range of light intensities in a light-emitting diode turbidostat photobioreactor, the C12-secreting mutant exhibited a modest reduction in growth rate and increased O2 evolution relative to the wild-type (WT). Inhibition of (i) glycogen synthesis by deletion of the glgC-encoded ADP-glucose pyrophosphorylase (AGPase) and (ii) protein synthesis by nitrogen deprivation were investigated as potential mechanisms for metabolite redistribution to increase FA synthesis. Deletion of AGPase led to a 10-fold decrease in reducing carbohydrates and secretion of organic acids during nitrogen deprivation consistent with an energy spilling phenotype. When the carbohydrate-deficient background (ΔglgC) was modified for C12 secretion, no increase in C12 was achieved during nutrient replete growth, and no C12 was recovered from any strain upon nitrogen deprivation under the conditions used. At steady state, the growth rate of the ΔglgC strain saturated at a lower light intensity than the WT, but O2 evolution was not compromised and became increasingly decoupled from growth rate with rising irradiance. Photophysiological properties of the ΔglgC strain suggest energy dissipation from photosystem II and reconfiguration of electron flow at the level of the plastoquinone pool.

Toward a photosynthetic microbial platform for terpenoid engineering.

Plant terpenoids are among the most diverse group of naturally-occurring organic compounds known, and several are used in contemporary consumer products. Terpene synthase enzymes catalyze complex rearrangements of carbon skeleton precursors to yield thousands of unique chemical structures that range in size from the simplest five carbon isoprene unit to the long polymers of rubber. Such chemical diversity has established plant terpenoids as valuable commodity chemicals with applications in the pharmaceutical, neutraceutical, cosmetic, and food industries. More recently, terpenoids have received attention as a renewable alternative to petroleum-derived fuels and as the building blocks of synthetic biopolymers. However, the current plant- and petrochemical-based supplies of commodity terpenoids have major limitations. Photosynthetic microorganisms provide an opportunity to generate terpenoids in a renewable manner, employing a single consolidated host organism that is able to use solar energy, H2O and CO2 as the primary inputs for terpenoid biosynthesis. Advances in synthetic biology have seen important breakthroughs in microbial terpenoid engineering, traditionally via fermentative pathways in yeast and Escherichia coli. This review draws on the knowledge obtained from heterotrophic microbial engineering to propose strategies for the development of microbial photosynthetic platforms for industrial terpenoid production. The importance of utilizing the wealth of genetic information provided by nature to unravel the regulatory mechanisms of terpenoid biosynthesis is highlighted.

Engineering Limonene and Bisabolene Production in Wild Type and a Glycogen-Deficient Mutant of Synechococcus sp. PCC 7002.

The plant terpenoids limonene (C10H16) and α-bisabolene (C15H24) are hydrocarbon precursors to a range of industrially relevant chemicals. High-titer microbial synthesis of limonene and α-bisabolene could pave the way for advances in in vivo engineering of tailor-made hydrocarbons, and production at commercial scale. We have engineered the fast-growing unicellular euryhaline cyanobacterium Synechococcus sp. PCC 7002 to produce yields of 4 mg L(-1) limonene and 0.6 mg L(-1) α-bisabolene through heterologous expression of the Mentha spicatal-limonene synthase or the Abies grandis (E)-α-bisabolene synthase genes, respectively. Titers were significantly higher when a dodecane overlay was applied during culturing, suggesting either that dodecane traps large quantities of volatile limonene or α-bisabolene that would otherwise be lost to evaporation, and/or that continuous product removal in dodecane alleviates product feedback inhibition to promote higher rates of synthesis. We also investigate limonene and bisabolene production in the ΔglgC genetic background, where carbon partitioning is redirected at the expense of glycogen biosynthesis. The Synechococcus sp. PCC 7002 ΔglgC mutant excreted a suite of overflow metabolites (α-ketoisocaproate, pyruvate, α-ketoglutarate, succinate, and acetate) during nitrogen-deprivation, and also at the onset of stationary growth in nutrient-replete media. None of the excreted metabolites, however, appeared to be effectively utilized for terpenoid metabolism. Interestingly, we observed a 1.6- to 2.5-fold increase in the extracellular concentration of most excreted organic acids when the ΔglgC mutant was conferred with the ability to produce limonene. Overall, Synechococcus sp. PCC 7002 provides a highly promising platform for terpenoid biosynthetic and metabolic engineering efforts.