Progress and challenges in engineering cyanobacteria as chassis for light-driven biotechnology.

Cyanobacteria are prokaryotic phototrophs that, in addition to being excellent model organisms for studying photosynthesis, have tremendous potential for light-driven synthetic biology and biotechnology. These versatile and resilient microorganisms harness the energy of sunlight to oxidise water, generating chemical energy (ATP) and reductant (NADPH) that can be used to drive sustainable synthesis of high-value natural products in genetically modified strains. In this commentary article for the Synthetic Microbiology Caucus we discuss the great progress that has been made in engineering cyanobacterial hosts as microbial cell factories for solar-powered biosynthesis. We focus on some of the main areas where the synthetic biology and metabolic engineering tools in cyanobacteria are not as advanced as those in more widely used heterotrophic chassis, and go on to highlight key improvements that we feel are required to unlock the full power of cyanobacteria for future green biotechnology

Urban morphology and the post-industrial city: commercial space in Manchester

The contemporary post-industrial city has developed within a system where every square metre of its area might be assessed for its economic productivity and market value. Retail space, leisure space, even public open space, as well as housing and work environments are quantifiable and comparable in financial terms as the ultimate test of their value. This conception of urban space as units of capital has its origins in the industrial development of centres such as Manchester where, largely unencumbered by earlier urban patterns, the idea of the modern city could thrive. As a ‘shock city’ Manchester, during the peak of its industrial growth in the early nineteenth century was an object of fascination and repulsion to the visitors it attracted. Opinion and rhetoric dominated social, economic and political debate, but dispassionate spatial analysis was rare. In the view of contemporary authors the town had few significant public spaces, instead being largely comprised of the vast industrial structures that crowded around the roads and canals. The mills were assessed for legal and insurance purposes, however, at a time of rabid competition and the prevalence of industrial accidents. The surveys that have survived provide the first opportunities to assess these examples of new urban space. The image results of a settlement composed of a single type, the mill or warehouse. Ancillary structures, most especially the workers’ housing did not merit recording. In these products of spatial calculation the Manchester mill can be seen to set the pattern both for the productive spaces of industry and the spatial framework of the contemporary city, where the public space is one of consumption rather than community. The supervised and privatised public space of the contemporary city finds its genius loci in the industrial typology of its commercial origins

The morphology of the post-industrial city: the Manchester mill as ‘symbolic form’

The contemporary post-industrial city has developed within a system where every square metre of its area might be assessed for its economic productivity and market value. Retail space, leisure space, even public open space, as well as housing and work environments are quantifiable and comparable in financial terms as the ultimate test of their value. This conception of urban space as units of capital has its origins in the industrial development of centres such as Manchester where, largely unencumbered by earlier urban patterns, the idea of the modern city could thrive. As a ‘shock city’ Manchester, during the peak of its industrial growth in the early nineteenth century was an object of fascination and repulsion to the visitors it attracted. Opinion and rhetoric dominated social economic and political debate but dispassionate spatial analysis was rare. In the view of contemporary authors the town had few significant public spaces, instead being largely comprised of the vast industrial structures that crowded around the roads and canals. The mills were assessed for legal and insurance purposes, however, at a time of rabid competition and the prevalence of industrial accidents. The surveys that have survived provide the first opportunities to assess these examples of new urban space. The image results of a settlement composed of a single type, the mill or warehouse. Ancillary structure, most especially the workers’ housing did not merit recording. In these products of spatial calculation the Manchester mill can be seen to set the pattern both for the productive spaces of industry and the spatial framework of the contemporary city, where the public space is one of consumption rather than community. The supervised and privatised public space of the contemporary city finds its genius loci in the industrial typology of its commercial origins

Absence of the cbb3 terminal oxidase reveals an active oxygen-dependent cyclase involved in bacteriochlorophyll biosynthesis in Rhodobacter sphaeroides.

The characteristic green color associated with chlorophyll pigments results from the formation of an isocyclic fifth ring on the tetrapyrrole macrocyle 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 microoxic 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 (bacterio)chlorophyll 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

Learning from Letchworth and Welwyn Garden City: garden cities’ policies for the development of existing settlements in the contemporary world

So far, the garden city model has been analysed and adopted by scholars, urban planners, and institutions to design garden cities, new towns, and suburban communities as new settlements from scratch around the world. In contrast, this paper explores the potentialities of such a model to provide a multi-faceted approach based on territorial, socio-economic, and urban planning strategies, thus offering a series of policies to combat a wide range of issues in existing communities. For that purpose, this paper provides a new approach about the garden city model through the case studies of Letchworth and Welwyn Garden City and latest updated models to form a new scheme with the capability to encourage the decentralisation of population and means of production across the territory, develop cooperatives to achieve a self-financing system and affordable housing in existing communities, and implement the garden cities’ design principles for the regeneration and expansion of existing settlements towards a sustainable, attractive, self-reliant and prosperous future.Funding for open access charge: Universidad de Málaga / CBU

What is ‘research-led teaching’ and how can we deliver it in University STEM degrees?

Universities advertise STEM degrees as being delivered via ‘research-led teaching’. We worked with students at the University of Liverpool to ascertain what students understood ‘research-led teaching’ to mean, and how it could be improved. We found that most students considered it important to be taught by active researchers and expected that their lecturers be experts in their fields. We found that students craved a more practical, research-based curriculum. Students believed that a research-heavy degree would better prepare them for future employment and provide skills that are more difficult to attain through lecture-based courses, but those who had commitments outside of the University were more hesitant. Our findings highlight the importance of considering underserved communities such as carers and single parents in course design. Overall, we found that the way in which teaching is delivered has a critical impact on students’ confidence and future career choices. We also report that students’ confidence and perceived learning opportunities have been significantly affected by the COVID-19 pandemic. We propose that this is something that needs to be factored in when recruiting for postgraduate degrees and graduate jobs to protect future opportunities for students whose degrees were completed during the pandemic

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

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 pho- totrophs, carry an ethyl group at the C-8 position of the molecule, having undergone reduction of a vinyl group during biosyn- thesis. 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 delta bciB 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

Engineered biosynthesis of bacteriochlorophyll b in Rhodobacter sphaeroides.

Bacteriochlorophyll b has the most red-shifted absorbance maximum of all naturally occurring photopigments. It has a characteristic ethylidene group at the C8 position in place of the more common ethyl group, the product of a C8-vinyl reductase, which is carried by the majority of chlorophylls and bacteriochlorophylls used in photosynthesis. The subsequent and first step exclusive to bacteriochlorophyll biosynthesis, the reduction of the C7=C8 bond, is catalyzed by chlorophyllide oxidoreductase. It has been demonstrated that the enzyme from bacteriochlorophyll a-utilizing bacteria can catalyze the formation of compounds carrying an ethyl group at C8 from both ethyl- and vinyl-carrying substrates, indicating a surprising additional C8-vinyl reductase function, while the enzyme from organisms producing BChl b could only catalyze C7=C8 reduction with a vinyl substrate, but this product carried an ethylidene group at the C8 position. We have replaced the native chlorophyllide oxidoreductase-encoding genes of Rhodobacter sphaeroides with those from Blastochloris viridis, but the switch from bacteriochlorophyll a to b biosynthesis is only detected when the native conventional C8-vinyl reductase is absent. We propose a non-enzymatic mechanism for ethylidene group formation based on the absence of cellular C8-vinyl reductase activity

The role of the γ subunit in the photosystem of the lowest-energy phototrophs.

Purple phototrophic bacteria use a 'photosystem' consisting of light harvesting complex 1 (LH1) surrounding the reaction centre (RC) that absorbs far-red-near-infrared light and converts it to chemical energy. Blastochloris species, which harvest light >1000 nm, use bacteriochlorophyll b rather than the more common bacteriochlorophyll a as their major photopigment, and assemble LH1 with an additional polypeptide subunit, LH1γ, encoded by multiple genes. To assign a role to γ, we deleted the four encoding genes in the model Blastochloris viridis. Interestingly, growth under halogen bulbs routinely used for cultivation yielded cells displaying an absorption maximum of 825 nm, similar to that of the RC only, but growth under white light yielded cells with an absorption maximum at 972 nm. HPLC analysis of pigment composition and sucrose gradient fractionation demonstrate that the white light-grown mutant assembles RC-LH1, albeit with an absorption maximum blue-shifted by 46 nm. Wavelengths between 900-1000 nm transmit poorly through the atmosphere due to absorption by water, so our results provide an evolutionary rationale for incorporation of γ; this polypeptide red-shifts absorption of RC-LH1 to a spectral range in which photons are of lower energy but are more abundant. Finally, we transformed the mutant with plasmids encoding natural LH1γ variants and demonstrate that the polypeptide found in the wild type complex red-shifts absorption back to 1018 nm, but incorporation of a distantly related variant results in only a moderate shift. This result suggests that tuning the absorption of RC-LH1 is possible and may permit photosynthesis past its current low-energy limit

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 O-2-sensitive [4Fe-4S] cluster protein, whereas plants, cyanobacteria, and some phototrophic bacteria possess an O-2-dependent enzyme, the major catalytic component of which is a diiron protein, AcsF. Plant and cyanobacterial mutants in ycf54 display impaired function of the O-2-dependent enzyme, accumulating the reaction substrate. Swapping cyclases between cyanobacteria and purple phototrophic bacteria reveals three classes of the O-2-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 O-2-dependent cyclase in chlorophototrophic organisms from higher plants to bacteria, and their evolution is discussed herein