Impact of irradiance and inorganic carbon availability on heterologous sucrose production in Synechococcus elongatus PCC 7942.

Cyanobacteria have been proposed as a potential alternative carbohydrate feedstock and multiple species have been successfully engineered to secrete fermentable sugars. To date, the most productive cyanobacterial strains are those designed to secrete sucrose, yet there exist considerable differences in reported productivities across different model species and laboratories. In this study, we investigate how cultivation conditions (specifically, irradiance, CO2, and cultivator type) affect the productivity of sucrose-secreting Synechococcus elongatus PCC 7942. We find that S. elongatus produces the highest sucrose yield in irradiances far greater than what is often experimentally utilized, and that high light intensities are tolerated by S. elongatus, especially under higher density cultivation where turbidity may attenuate the effective light experienced in the culture. By increasing light and inorganic carbon availability, S. elongatus cscB/sps produced a total of 3.8 g L-1 of sucrose and the highest productivity within that period being 47.8 mg L-1 h-1. This study provides quantitative description of the impact of culture conditions on cyanobacteria-derived sucrose that may assist to standardize cross-laboratory comparisons and demonstrates a significant capacity to improve productivity via optimizing cultivation conditions.

Population-level heterogeneity complicates utilization of Synechococcus elongatus PCC 7942 surface display platforms.

Surface display technologies have been primarily developed for heterotrophic microbes, leaving photosynthetic counterparts like cyanobacteria with limited molecular tools. Here, we expanded upon surface display systems in Synechococcus elongatus PCC 7942 by modifying two outer-membrane proteins, SomA and Intimin, to display tags ( e.g. , SpyTag) to mediate physical interactions of living cyanobacteria with other biotic and abiotic targets. While re-engineered SomA constructs successfully translocated to the cell surface and could bind to compatible ligands, the efficacy of the best-performing designs was limited by a poorly-understood heterogeneity in the accessibility of the tags in living cells, resulting in low attachment penetrance.

Cyanobacteria as cell factories for the photosynthetic production of sucrose.

Biofuels and other biologically manufactured sustainable goods are growing in popularity and demand. Carbohydrate feedstocks required for industrial fermentation processes have traditionally been supplied by plant biomass, but the large quantities required to produce replacement commodity products may prevent the long-term feasibility of this approach without alternative strategies to produce sugar feedstocks. Cyanobacteria are under consideration as potential candidates for sustainable production of carbohydrate feedstocks, with potentially lower land and water requirements relative to plants. Several cyanobacterial strains have been genetically engineered to export significant quantities of sugars, especially sucrose. Sucrose is not only naturally synthesized and accumulated by cyanobacteria as a compatible solute to tolerate high salt environments, but also an easily fermentable disaccharide used by many heterotrophic bacteria as a carbon source. In this review, we provide a comprehensive summary of the current knowledge of the endogenous cyanobacterial sucrose synthesis and degradation pathways. We also summarize genetic modifications that have been found to increase sucrose production and secretion. Finally, we consider the current state of synthetic microbial consortia that rely on sugar-secreting cyanobacterial strains, which are co-cultivated alongside heterotrophic microbes able to directly convert the sugars into higher-value compounds (e.g., polyhydroxybutyrates, 3-hydroxypropionic acid, or dyes) in a single-pot reaction. We summarize recent advances reported in such cyanobacteria/heterotroph co-cultivation strategies and provide a perspective on future developments that are likely required to realize their bioindustrial potential.

An Integrated Assessment of Emissions, Air Quality, and Public Health Impacts of China's Transition to Electric Vehicles.

Electric vehicles (EVs) are a promising pathway to providing cleaner personal mobility. China provides substantial supports to increase EV market share. This study provides an extensive analysis of the currently unclear environmental and health benefits of these incentives at the provincial level. EVs in China have modest cradle-to-gate CO2 benefits (on average 29%) compared to conventional internal combustion engine vehicles (ICEVs), but have similar carbon emissions relative to hybrid electric vehicles. Well-to-wheel air pollutant emissions assessment shows that emissions associated with ICEVs are mainly from gasoline production, not the tailpipe, suggesting tighter emissions controls on refineries are needed to combat air pollution problems effectively. By integrating a vehicle fleet model into policy scenario analysis, we quantify the policy impacts associated with the passenger vehicles in the major Chinese provinces: broader EV penetration, especially combined with cleaner power generation, could deliver greater air quality and health benefits, but not necessarily significant climate change mitigation. The total value to society of the climate and mortality benefits in 2030 is found to be comparable to a prior estimate of the EV policy's economic costs.

China's vehicle electrification impacts on sales, fuel use, and battery material demand through 2050: Optimizing consumer and industry decisions.

The promotion of plug-in electric vehicles (PEVs) is pivotal to China's carbon neutrality strategy. Therefore, it is important to understand the vehicle market evolution and its impacts in terms of costs, sales, industry fuel economy, and PEV's battery material demand. By examining vehicle technologies, cost, policy incentives, infrastructure, and driver behavior, this study quantitatively projects the dynamics of China's passenger vehicle market from 2020 to 2050 under multiple technology evolution scenarios. By 2050, battery electric vehicles could gain significant market share-as much as 30.4%-64.6%; and the industry's sales-weighted average fuel consumption could reach 1.81-3.11 L/100 km. Cumulative battery demand from PEVs could soar to over 700 GWh by 2050, whereas battery recycling alone could satisfy about 60% of the demand by 2050. The key metal supplies-lithium, cobalt, and nickel-for China's PEV market are projected, and nickel should be concerned more over the coming decades.

Evaluation of non-exudative microcystoid macular abnormalities secondary to retinal vein occlusion.

PURPOSE: We aimed to investigate non-exudative microcystoid macular abnormalities for visual and anatomical outcome in patients with retinal vein occlusion (RVO) with and without glaucomatous optic neuropathy (GON). METHODS: Medical records of 124 eyes (105 patients) with RVO were reviewed and analyzed. Eyes demonstrating microcystoid macular abnormalities were divided into 2 groups, those with evidence of glaucoma (group A) and those without glaucoma (group B). Best-corrected visual acuity (BCVA), the prevalence and number of microcystoid macular abnormalities, and number of intravitreal anti-vascular endothelial growth factor (anti-VEGF) injections were compared at baseline and follow-up. RESULTS: Seventy-one out of 105 eyes (67.6%) with RVO displayed microcystoid macular abnormalities. Thirty-eight out of 71 eyes (53.5%) presented with concomitant glaucoma (group A), while the remaining 33 eyes (42.6%) had no history of glaucoma (group B). At the end of the follow-up period, mean BCVA was worse in group A versus group B (20/80 versus 20/40, respectively; p = .003). The mean number of anti-VEGF injections was 10.1 ± 9.2 in group A versus 5.9 ± 6.9 in group B (p = .03). CONCLUSION: Eyes with RVO and concomitant glaucoma exhibited a significantly higher number of microcystoid macular abnormalities and worse BCVA versus eyes with RVO without glaucoma.

LAMELLAR MACULAR HOLES IN THE PRESENCE OF AGE-RELATED MACULAR DEGENERATION.

PURPOSE: To investigate whether age-related macular degeneration (AMD) has an influence on the prevalence and anatomical characteristics of lamellar macular holes (LMHs). METHODS: Clinical records and spectral-domain optical coherence tomography images of 756 eyes of 423 consecutive patients diagnosed with AMD were reviewed and analyzed. Spectral-domain optical coherence tomography was used to identify degenerative or tractional LMH subtypes and assess their morphology. The clinical and optical coherence tomography findings of AMD eyes with LMH were compared with those of a control group of eyes with LMH without AMD from a previously published report. RESULTS: Lamellar macular holes were identified in 25 eyes of 23 patients (3.3%; 25 of 756). Seventeen of 25 eyes (68%) presented with degenerative LMH and underlying late neovascular AMD. Mean best-corrected visual acuity was worse in eyes with AMD and LMH eyes than in those with AMD and no LMH (20/230 vs. 20/98; P = 0.02). The mean outer diameter was greater in the group with degenerative LMH with concomitant AMD than in the control group of degenerative LMH without AMD (1,323.9 ± 999.1 µm vs. 905.9 ± 356.8 µm, respectively; P = 0.01). CONCLUSION: The incidence of degenerative LMH increased in advanced forms of AMD, whereas the presence of tractional LMH subtype may be unrelated to AMD evolution.

Lipid-anchor display on peptoid nanosheets via co-assembly for multivalent pathogen recognition.

Biological systems have evolved sophisticated molecular assemblies capable of exquisite molecular recognition across length scales ranging from angstroms to microns. For instance, the self-organization of glycolipids and glycoproteins on cell membranes allows for molecular recognition of a diversity of ligands ranging from small molecules and proteins to viruses and whole cells. A distinguishing feature of these 2D surfaces is they achieve exceptional binding selectivity and avidity by exploiting multivalent binding interactions. Here we develop a 2D ligand display platform based on peptoid nanosheets that mimics the structure and function of the cell membrane. A variety of small-molecule lipid-conjugates were co-assembled with the peptoid chains to create a diversity of functionalized nanosheet bilayers with varying display densities. The functional heads of the lipids were shown to be surface-exposed, and the carbon tails immobilized into the hydrophobic interior. We demonstrate that saccharide-functionalized nanosheets (e.g., made from globotriaosylsphingosine or 1,2-dipalmitoyl-sn-glycero-3-phospho((ethyl-1',2',3'-triazole)triethyleneglycolmannose)) can have very diverse binding properties, exhibiting specific binding to multivalent proteins as well as to intact bacterial cells. Analysis of sugar display densities revealed that Shiga toxin 1 subunit B (a pentameric protein) and FimH-expressing Escherichia coli (E. coli) bind through the cooperative binding behavior of multiple carbohydrates. The ability to readily incorporate and display a wide variety of lipidated cargo on the surface of peptoid nanosheets makes this a convenient route to soluble, cell-surface mimetic materials. These materials hold great promise for drug screening, biosensing, bioremediation, and as a means to combat pathogens by direct physical binding through a well-defined, multivalent 2D material.

Discovery of Stable and Selective Antibody Mimetics from Combinatorial Libraries of Polyvalent, Loop-Functionalized Peptoid Nanosheets.

The ability of antibodies to bind a wide variety of analytes with high specificity and high affinity makes them ideal candidates for therapeutic and diagnostic applications. However, the poor stability and high production cost of antibodies have prompted exploration of a variety of synthetic materials capable of specific molecular recognition. Unfortunately, it remains a fundamental challenge to create a chemically diverse population of protein-like, folded synthetic nanostructures with defined molecular conformations in water. Here we report the synthesis and screening of combinatorial libraries of sequence-defined peptoid polymers engineered to fold into ordered, supramolecular nanosheets displaying a high spatial density of diverse, conformationally constrained peptoid loops on their surface. These polyvalent, loop-functionalized nanosheets were screened using a homogeneous Förster resonance energy transfer (FRET) assay for binding to a variety of protein targets. Peptoid sequences were identified that bound to the heptameric protein, anthrax protective antigen, with high avidity and selectivity. These nanosheets were shown to be resistant to proteolytic degradation, and the binding was shown to be dependent on the loop display density. This work demonstrates that key aspects of antibody structure and function-the creation of multivalent, combinatorial chemical diversity within a well-defined folded structure-can be realized with completely synthetic materials. This approach enables the rapid discovery of biomimetic affinity reagents that combine the durability of synthetic materials with the specificity of biomolecular materials.

Engineering the S-Layer of Caulobacter crescentus as a Foundation for Stable, High-Density, 2D Living Materials.

Materials synthesized by organisms, such as bones and wood, combine the ability to self-repair with remarkable mechanical properties. This multifunctionality arises from the presence of living cells within the material and hierarchical assembly of different components across nanometer to micron scales. While creating engineered analogues of these natural materials is of growing interest, our ability to hierarchically order materials using living cells largely relies on engineered 1D protein filaments. Here, we lay the foundation for bottom-up assembly of engineered living material composites in 2D along the cell body using a synthetic biology approach. We engineer the paracrystalline surface-layer (S-layer) of Caulobacter crescentus to display SpyTag peptides that form irreversible isopeptide bonds to SpyCatcher-modified proteins, nanocrystals, and biopolymers on the extracellular surface. Using flow cytometry and confocal microscopy, we show that attachment of these materials to the cell surface is uniform, specific, and covalent, and its density can be controlled on the basis of the insertion location within the S-layer protein, RsaA. Moreover, we leverage the irreversible nature of this attachment to demonstrate via SDS-PAGE that the engineered S-layer can display a high density of materials, reaching 1 attachment site per 288 nm2. Finally, we show that ligation of quantum dots to the cell surface does not impair cell viability, and this composite material remains intact over a period of 2 weeks. Taken together, this work provides a platform for self-organization of soft and hard nanomaterials on a cell surface with precise control over 2D density, composition, and stability of the resulting composite, and is a key step toward building hierarchically ordered engineered living materials with emergent properties.

Design, Synthesis, Assembly, and Engineering of Peptoid Nanosheets.

Two-dimensional (2D) atomically defined organic nanomaterials are an important material class with broad applications. However, few general synthetic methods exist to produce such materials in high yields and to precisely functionalize them. One strategy to form ordered 2D organic nanomaterials is through the supramolecular assembly of sequence-defined synthetic polymers. Peptoids, one such class of polymer, are designable bioinspired heteropolymers whose main-chain length and monomer sequence can be precisely controlled. We have recently discovered that individual peptoid polymers with a simple sequence of alternating hydrophobic and ionic monomers can self-assemble into highly ordered, free-floating nanosheets. A detailed understanding of their molecular structure and supramolecular assembly dynamics provides a robust platform for the discovery of new classes of nanosheets with tunable properties and novel applications. In this Account, we discuss the discovery, characterization, assembly, molecular modeling, and functionalization of peptoid nanosheets. The fundamental properties of peptoid nanosheets, their mechanism of formation, and their application as robust scaffolds for molecular recognition and as templates for the growth of inorganic minerals have been probed by an arsenal of experimental characterization techniques (e.g., scanning probe, electron, and optical microscopy, X-ray diffraction, surface-selective vibrational spectroscopy, and surface tensiometry) and computational techniques (coarse-grained and atomistic modeling). Peptoid nanosheets are supramolecular assemblies of 16-42-mer chains that form molecular bilayers. They span tens of microns in lateral dimensions and freely float in water. Their component chains are highly ordered, with chains nearly fully extended and packed parallel to one another as a result of hydrophobic and electrostatic interactions. Nanosheets form via a novel interface-catalyzed monolayer collapse mechanism. Peptoid chains first assemble into a monolayer at either an air-water or oil-water interface, on which peptoid chains extend, order, and pack into a brick-like pattern. Upon mechanical compression of the interface, the monolayer buckles into stable bilayer structures. Recent work has focused on the design of nanosheets with tunable properties and functionality. They are readily engineerable, as functional monomers can be readily incorporated onto the nanosheet surface or into the interior. For example, functional hydrophilic "loops" have been displayed on the surfaces of nanosheets. These loops can interact with specific protein targets, serving as a potentially general platform for molecular recognition. Nanosheets can also bind metal ions and serve as 2D templates for mineral growth. Through our understanding of the formation mechanism, along with predicted features ascertained from molecular modeling, we aim to further design and synthesize nanosheets as robust protein mimetics with the potential for unprecedented functionality and stability.