Doing synthetic biology with photosynthetic microorganisms.

The use of photosynthetic microbes as synthetic biology hosts for the sustainable production of commodity chemicals and even fuels has received increasing attention over the last decade. The number of studies published, tools implemented, and resources made available for microalgae have increased beyond expectations during the last few years. However, the tools available for genetic engineering in these organisms still lag those available for the more commonly used heterotrophic host organisms. In this mini-review, we provide an overview of the photosynthetic microbes most commonly used in synthetic biology studies, namely cyanobacteria, chlorophytes, eustigmatophytes and diatoms. We provide basic information on the techniques and tools available for each model group of organisms, we outline the state-of-the-art, and we list the synthetic biology tools that have been successfully used. We specifically focus on the latest CRISPR developments, as we believe that precision editing and advanced genetic engineering tools will be pivotal to the advancement of the field. Finally, we discuss the relative strengths and weaknesses of each group of organisms and examine the challenges that need to be overcome to achieve their synthetic biology potential.

Are synthetic biology standards applicable in everyday research practice?

The issue of standardization in synthetic biology is a recurring one. As a discipline that incorporates engineering principles into biological designs, synthetic biology needs effective ways to communicate results and allow different researchers (both academic and industrial) to build upon previous results and improve on existing designs. An aspect that is left out of the discussions, especially when they happen at the level of academic and industrial consortia or policymaking, is whether or not standards are applicable or even useful in everyday research practice. In this caucus article, we examine this particular issue with the hope of including it in the standardization discussions agenda and provide insights into a topic that synthetic biology researchers experience daily.

CyanoGate: A Modular Cloning Suite for Engineering Cyanobacteria Based on the Plant MoClo Syntax.

Recent advances in synthetic biology research have been underpinned by an exponential increase in available genomic information and a proliferation of advanced DNA assembly tools. The adoption of plasmid vector assembly standards and parts libraries has greatly enhanced the reproducibility of research and the exchange of parts between different labs and biological systems. However, a standardized modular cloning (MoClo) system is not yet available for cyanobacteria, which lag behind other prokaryotes in synthetic biology despite their huge potential regarding biotechnological applications. By building on the assembly library and syntax of the Plant Golden Gate MoClo kit, we have developed a versatile system called CyanoGate that unites cyanobacteria with plant and algal systems. Here, we describe the generation of a suite of parts and acceptor vectors for making (1) marked/unmarked knock-outs or integrations using an integrative acceptor vector, and (2) transient multigene expression and repression systems using known and previously undescribed replicative vectors. We tested and compared the CyanoGate system in the established model cyanobacterium Synechocystis sp. PCC 6803 and the more recently described fast-growing strain Synechococcus elongatus UTEX 2973. The UTEX 2973 fast-growth phenotype was only evident under specific growth conditions; however, UTEX 2973 accumulated high levels of proteins with strong native or synthetic promoters. The system is publicly available and can be readily expanded to accommodate other standardized MoClo parts to accelerate the development of reliable synthetic biology tools for the cyanobacterial community.

Terpenoid Metabolic Engineering in Photosynthetic Microorganisms.

Terpenoids are a group of natural products that have a variety of roles, both essential and non-essential, in metabolism and in biotic and abiotic interactions, as well as commercial applications such as pharmaceuticals, food additives, and chemical feedstocks. Economic viability for commercial applications is commonly not achievable by using natural source organisms or chemical synthesis. Engineered bio-production in suitable heterologous hosts is often required to achieve commercial viability. However, our poor understanding of regulatory mechanisms and other biochemical processes makes obtaining efficient conversion yields from feedstocks challenging. Moreover, production from carbon dioxide via photosynthesis would significantly increase the environmental and potentially the economic credentials of these processes by disintermediating biomass feedstocks. In this paper, we briefly review terpenoid metabolism, outline some recent advances in terpenoid metabolic engineering, and discuss why photosynthetic unicellular organisms-such as algae and cyanobacteria-might be preferred production platforms for the expression of some of the more challenging terpenoid pathways.

Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii.

Microalgae are regarded as promising organisms to develop innovative concepts based on their photosynthetic capacity that offers more sustainable production than heterotrophic hosts. However, to realize their potential as green cell factories, a major challenge is to make microalgae easier to engineer. A promising approach for rapid and predictable genetic manipulation is to use standardized synthetic biology tools and workflows. To this end we have developed a Modular Cloning toolkit for the green microalga Chlamydomonas reinhardtii. It is based on Golden Gate cloning with standard syntax, and comprises 119 openly distributed genetic parts, most of which have been functionally validated in several strains. It contains promoters, UTRs, terminators, tags, reporters, antibiotic resistance genes, and introns cloned in various positions to allow maximum modularity. The toolkit enables rapid building of engineered cells for both fundamental research and algal biotechnology. This work will make Chlamydomonas the next chassis for sustainable synthetic biology.

Harnessing transcription for bioproduction in cyanobacteria.

Sustainable production of biofuels and other valuable compounds is one of our future challenges. One tempting possibility is to use photosynthetic cyanobacteria as production factories. Currently, tools for genetic engineering of cyanobacteria are not good enough to exploit the full potential of cyanobacteria. A wide variety of expression systems will be required to adjust both the expression of heterologous enzyme(s) and metabolic routes to the best possible balance, allowing the optimal production of a particular substance. In bacteria, transcription, especially the initiation of transcription, has a central role in adjusting gene expression and thus also metabolic fluxes of cells according to environmental cues. Here we summarize the recent progress in developing tools for efficient cyanofactories, focusing especially on transcriptional regulation.

Responses of Synechocystis sp. PCC 6803 to heterologous biosynthetic pathways.

BACKGROUND: There are an increasing number of studies regarding genetic manipulation of cyanobacteria to produce commercially interesting compounds. The majority of these works study the expression and optimization of a selected heterologous pathway, largely ignoring the wholeness and complexity of cellular metabolism. Regulation and response mechanisms are largely unknown, and even the metabolic pathways themselves are not fully elucidated. This poses a clear limitation in exploiting the rich biosynthetic potential of cyanobacteria. RESULTS: In this work, we focused on the production of two different compounds, the cyanogenic glucoside dhurrin and the diterpenoid 13R-manoyl oxide in Synechocystis PCC 6803. We used genome-scale metabolic modelling to study fluxes in individual reactions and pathways, and we determined the concentrations of key metabolites, such as amino acids, carotenoids, and chlorophylls. This allowed us to identify metabolic crosstalk between the native and the introduced metabolic pathways. Most results and simulations highlight the metabolic robustness of cyanobacteria, suggesting that the host organism tends to keep metabolic fluxes and metabolite concentrations steady, counteracting the effects of the heterologous pathway. However, the amino acid concentrations of the dhurrin-producing strain show an unexpected profile, where the perturbation levels were high in seemingly unrelated metabolites. CONCLUSIONS: There is a wealth of information that can be derived by combining targeted metabolite identification and computer modelling as a frame of understanding. Here we present an example of how strain engineering approaches can be coupled to 'traditional' metabolic engineering with systems biology, resulting in novel and more efficient manipulation strategies.

Photosynthetic fuel for heterologous enzymes: the role of electron carrier proteins.

Plants, cyanobacteria, and algae generate a surplus of redox power through photosynthesis, which makes them attractive for biotechnological exploitations. While central metabolism consumes most of the energy, pathways introduced through metabolic engineering can also tap into this source of reducing power. Recent work on the metabolic engineering of photosynthetic organisms has shown that the electron carriers such as ferredoxin and flavodoxin can be used to couple heterologous enzymes to photosynthetic reducing power. Because these proteins have a plethora of interaction partners and rely on electrostatically steered complex formation, they form productive electron transfer complexes with non-native enzymes. A handful of examples demonstrate channeling of photosynthetic electrons to drive the activity of heterologous enzymes, and these focus mainly on hydrogenases and cytochrome P450s. However, competition from native pathways and inefficient electron transfer rates present major obstacles, which limit the productivity of heterologous reactions coupled to photosynthesis. We discuss specific approaches to address these bottlenecks and ensure high productivity of such enzymes in a photosynthetic context.

The two Dps proteins, NpDps2 and NpDps5, are involved in light-induced oxidative stress tolerance in the N2-fixing cyanobacterium Nostoc punctiforme.

Cyanobacteria are photosynthetic prokaryotes that are considered biotechnologically prominent organisms for production of high-value compounds. Cyanobacteria are subject to high-light intensities, which is a challenge that needs to be addressed in design of efficient bio-engineered photosynthetic organisms. Dps proteins are members of the ferritin superfamily and are omnipresent in prokaryotes. They play a major role in oxidative stress protection and iron homeostasis. The filamentous, heterocyst-forming Nostoc punctiforme, has five Dps proteins. In this study we elucidated the role of these Dps proteins in acclimation to high light intensity, the gene loci organization and the transcriptional regulation of all five dps genes in N. punctiforme was revealed, and dps-deletion mutant strains were used in physiological characterization. Two mutants defective in Dps2 and Dps5 activity displayed a reduced fitness under increased illumination, as well as a differential Photosystem (PS) stoichiometry, with an elevated Photosystem II to Photosystem I ratio in the dps5 deletion strain. This work establishes a Dps-mediated link between light tolerance, H2O2 detoxification, and iron homeostasis, and provides further evidence on the non-redundant role of multiple Dps proteins in this multicellular cyanobacterium.

In vivo assembly of DNA-fragments in the moss, Physcomitrella patens.

Direct assembly of multiple linear DNA fragments via homologous recombination, a phenomenon known as in vivo assembly or transformation associated recombination, is used in biotechnology to assemble DNA constructs ranging in size from a few kilobases to full synthetic microbial genomes. It has also enabled the complete replacement of eukaryotic chromosomes with heterologous DNA. The moss Physcomitrella patens, a non-vascular and spore producing land plant (Bryophyte), has a well-established capacity for homologous recombination. Here, we demonstrate the in vivo assembly of multiple DNA fragments in P. patens with three examples of effective genome editing: we (i) efficiently deleted a genomic locus for diterpenoid metabolism yielding a biosynthetic knockout, (ii) introduced a salt inducible promoter, and (iii) re-routed endogenous metabolism into the formation of amorphadiene, a precursor of high-value therapeutics. These proof-of-principle experiments pave the way for more complex and increasingly flexible approaches for large-scale metabolic engineering in plant biotechnology.

Extending the biosynthetic repertoires of cyanobacteria and chloroplasts.

Chloroplasts in plants and algae and photosynthetic microorganisms such as cyanobacteria are emerging hosts for sustainable production of valuable biochemicals, using only inorganic nutrients, water, CO2 and light as inputs. In the past decade, many bioengineering efforts have focused on metabolic engineering and synthetic biology in the chloroplast or in cyanobacteria for the production of fuels, chemicals and complex, high-value bioactive molecules. Biosynthesis of all these compounds can be performed in photosynthetic organelles/organisms by heterologous expression of the appropriate pathways, but this requires optimization of carbon flux and reducing power, and a thorough understanding of regulatory pathways. Secretion or storage of the compounds produced can be exploited for the isolation or confinement of the desired compounds. In this review, we explore the use of chloroplasts and cyanobacteria as biosynthetic compartments and hosts, and we estimate the levels of production to be expected from photosynthetic hosts in light of the fraction of electrons and carbon that can potentially be diverted from photosynthesis. The supply of reducing power, in the form of electrons derived from the photosynthetic light reactions, appears to be non-limiting, but redirection of the fixed carbon via precursor molecules presents a challenge. We also discuss the available synthetic biology tools and the need to expand the molecular toolbox to facilitate cellular reprogramming for increased production yields in both cyanobacteria and chloroplasts.

Heterologous expression of the isopimaric acid pathway in Nicotiana benthamiana and the effect of N-terminal modifications of the involved cytochrome P450 enzyme.

BACKGROUND: Plant terpenoids are known for their diversity, stereochemical complexity, and their commercial interest as pharmaceuticals, food additives, and cosmetics. Developing biotechnology approaches for the production of these compounds in heterologous hosts can increase their market availability, reduce their cost, and provide sustainable production platforms. In this context, we aimed at producing the antimicrobial diterpenoid isopimaric acid from Sitka spruce. Isopimaric acid is synthesized using geranylgeranyl diphosphate as a precursor molecule that is cyclized by a diterpene synthase in the chloroplast and subsequently oxidized by a cytochrome P450, CYP720B4. RESULTS: We transiently expressed the isopimaric acid pathway in Nicotiana benthamiana leaves and enhanced its productivity by the expression of two rate-limiting steps in the pathway (providing the general precursor of diterpenes). This co-expression resulted in 3-fold increase in the accumulation of both isopimaradiene and isopimaric acid detected using GC-MS and LC-MS methodology. We also showed that modifying or deleting the transmembrane helix of CYP720B4 does not alter the enzyme activity and led to successful accumulation of isopimaric acid in the infiltrated leaves. Furthermore, we demonstrated that a modified membrane anchor is a prerequisite for a functional CYP720B4 enzyme when the chloroplast targeting peptide is added. We report the accumulation of 45-55 µg/g plant dry weight of isopimaric acid four days after the infiltration with the modified enzymes. CONCLUSIONS: It is possible to localize a diterpenoid pathway from spruce fully within the chloroplast of N. benthamiana and a few modifications of the N-terminal sequences of the CYP720B4 can facilitate the expression of plant P450s in the plastids. The coupling of terpene biosynthesis closer to photosynthesis paves the way for light-driven biosynthesis of valuable terpenoids.

Modeling the mechanisms of biological GTP hydrolysis.

Enzymes that hydrolyze GTP are currently in the spotlight, due to their molecular switch mechanism that controls many cellular processes. One of the best-known classes of these enzymes are small GTPases such as members of the Ras superfamily, which catalyze the hydrolysis of the γ-phosphate bond in GTP. In addition, the availability of an increasing number of crystal structures of translational GTPases such as EF-Tu and EF-G have made it possible to probe the molecular details of GTP hydrolysis on the ribosome. However, despite a wealth of biochemical, structural and computational data, the way in which GTP hydrolysis is activated and regulated is still a controversial topic and well-designed simulations can play an important role in resolving and rationalizing the experimental data. In this review, we discuss the contributions of computational biology to our understanding of GTP hydrolysis on the ribosome and in small GTPases.