The year 2017 marks the twentieth anniversary of terpenoid cyclase structural biology: a trio of terpenoid cyclase structures reported together in 1997 were the first to set the foundation for understanding the enzymes largely responsible for the exquisite chemodiversity of more than 80000 terpenoid natural products. Terpenoid cyclases catalyze the most complex chemical reactions in biology, in that more than half of the substrate carbon atoms undergo changes in bonding and hybridization during a single enzyme-catalyzed cyclization reaction. The past two decades have witnessed structural, functional, and computational studies illuminating the modes of substrate activation that initiate the cyclization cascade, the management and manipulation of high-energy carbocation intermediates that propagate the cyclization cascade, and the chemical strategies that terminate the cyclization cascade. The role of the terpenoid cyclase as a template for catalysis is paramount to its function, and protein engineering can...

Structural and Chemical Biology of Terpenoid Cyclases.

Global climate change linked to the accumulation of greenhouse gases has caused concerns regarding the use of fossil fuels as the major energy source. To mitigate climate change while keeping energy supply sustainable, one solution is to rely on the ability of microorganisms to use renewable resources for biofuel synthesis. In this Review, we discuss how microorganisms can be explored for the production of next-generation biofuels, based on the ability of bacteria and fungi to use lignocellulose; through direct CO2 conversion by microalgae; using lithoautotrophs driven by solar electricity; or through the capacity of microorganisms to use methane generated from landfill. Furthermore, we discuss how to direct these substrates to the biosynthetic pathways of various fuel compounds and how to optimize biofuel production by engineering fuel pathways and central metabolism.

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Fuelling the future: microbial engineering for the production of sustainable biofuels

https://www.cell.com/trends/biotechnology/pdf/S0167-7799(15)00071-2.pdf

Metabolic engineering of cyanobacteria for the synthesis of commodity products

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 spicata L-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 and 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.

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

The cyanobacterial tricarboxylic acid (TCA) cycle functions in both in biosynthesis and energy generation. However, it has until recently been generally considered to be incomplete1,2 with limited flux3,4, and few attempts have been made to draw carbon from the cycle for biotechnological purposes. We demonstrated that ethylene can be sustainably and efficiently produced from the TCA cycle of the recombinant cyanobacterium Synechocystis 6803 expressing the Pseudomonas ethylene-forming enzyme (Efe)5. A new strain with a modified ribosome binding site upstream of the efe gene diverts 10% of fixed carbon to ethylene and shows increased photosynthetic activities. The highest specific ethylene production rate reached 718 ± 19 μl l–1 h–1 per A730 nm. Experimental and computational analyses based on kinetic 13C-isotope tracer and liquid chromatography coupled with mass spectrometry (LC–MS) demonstrated that the TCA metabolism is activated by the ethylene forming reaction, resulting in a predominantly cyclic architecture. The outcome significantly enhanced flux through the remodelled TCA cycle (37% of total fixed carbon) compared with a complete, but bifurcated and low-flux (13% of total fixed carbon) TCA cycle in the wild type. Global carbon flux is redirected towards the engineered ethylene pathway. The remarkable metabolic network plasticity of this cyanobacterium is manifested by the enhancement of photosynthetic activity and redistribution of carbon flux, enabling efficient ethylene production from the TCA cycle. Expressing the Pseudomonas ethylene-forming enzyme (Efe) in Synechocystis 6803 causes it to produce ethylene. Tracer experiments and metabolic modelling show that this is achieved by plasticity of fluxes through the tricarboxylic acid cycle.

The plasticity of cyanobacterial metabolism supports direct CO2 conversion to ethylene

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.

Toward a photosynthetic microbial platform for terpenoid engineering

Photosynthetic microorganisms offer novel characteristics as synthetic biology chassis, and the toolbox of components and techniques for cyanobacteria and algae is rapidly increasing.

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The Synthetic Biology Toolkit for Photosynthetic Microorganisms.

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.

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

The cyanobacterium Synechococcus sp. PCC 7002 was genetically engineered to synthesize biofuel compatible medium-chain fatty acids 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 fatty acid in CO2-supplemented batch cultures. When grown at steady state over a range of light intensities in an LED turbidostat photobioreactor, the C12-secreting mutant exhibited a modest reduction in growth rate and increased O2 evolution relative to the wildtype. 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 fatty acid synthesis. Deletion of AGPase led to a ten-fold decrease in reducing carbohydrates and secretion of organic acids during nitrogen deprivation consistent with an energy spilling phenotype. When the carbohydrate-deficient background (∆glgC) 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 ∆glgC strain saturated at a lower light intensity than the wildtype, but O2 evolution was not compromised and became increasingly decoupled from growth rate with rising irradiance. Photophysiological properties of the ∆glgC strain suggest energy dissipation from photosystem II and reconfiguration of electron flow at the level of the plastoquinone pool.

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Fiona K. Davies

Papers

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

Toward a photosynthetic microbial platform for terpenoid engineering

The Synthetic Biology Toolkit for Photosynthetic Microorganisms.

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

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