Barley Viridis-k links an evolutionarily conserved C-type ferredoxin to chlorophyll biosynthesis.

Ferredoxins are single-electron carrier proteins involved in various cellular reactions. In chloroplasts, the most abundant ferredoxin accepts electrons from photosystem I and shuttles electrons via ferredoxin NADP+ oxidoreductase to generate NADPH or directly to ferredoxin dependent enzymes. In addition, plants contain other isoforms of ferredoxins. Two of these, named FdC1 and FdC2 in Arabidopsis thaliana, have C-terminal extensions and functions that are poorly understood. Here we identified disruption of the orthologous FdC2 gene in barley (Hordeum vulgare L.) mutants at the Viridis-k locus; these mutants are deficient in the aerobic cyclase reaction of chlorophyll biosynthesis. The magnesium-protoporphyrin IX monomethyl ester cyclase is one of the least characterized enzymes of the chlorophyll biosynthetic pathway and its electron donor has long been sought. Agroinfiltrations showed that the viridis-k phenotype could be complemented in vivo by Viridis-k but not by canonical ferredoxin. VirK could drive the cyclase reaction in vitro and analysis of cyclase mutants showed that in vivo accumulation of VirK is dependent on cyclase enzyme levels. The chlorophyll deficient phenotype of viridis-k mutants suggests that VirK plays an essential role in chlorophyll biosynthesis that cannot be replaced by other ferredoxins, thus assigning a specific function to this isoform of C-type ferredoxins.

Consumer acceptance of microalgae as a novel food - Where are we now? And how to get further.

Microalgae provide a potential new food resource for sustainable human nutrition. Many microalgae species can produce a high content of total protein with a balanced composition of essential amino acids, healthy oils rich in polyunsaturated fatty acids, carotenoids, fibers, and vitamins. These components can be made available via unprocessed microalgae or refined as individual ingredients. In either case, if added to foods, microalgae may affect taste, smell, texture, and appearance. This review focuses on how consumer acceptance of new foods - such as microalgae - can be accessed in the world of sensory science by bringing together examples from recent consumer surveys. The main aim is to obtain an overview of the attitude towards microalgae as a food ingredient in Europe. The overarching finding suggests that European consumers generally find microalgae acceptable as ingredients in food products. However, there is a prevailing preference for keeping inclusion levels low, primarily attributed to the vivid green color that algae impart to food items upon addition. Additionally, consumers tend to favor the taste of freshwater algae over marine species, often finding the latter's pronounced fishy flavor less appealing.

Tailoring regulatory components for metabolic engineering in cyanobacteria.

The looming climate crisis has prompted an ever-growing interest in cyanobacteria due to their potential as sustainable production platforms for the synthesis of energy carriers and value-added chemicals from CO2 and sunlight. Nonetheless, cyanobacteria are yet to compete with heterotrophic systems in terms of space-time yields and consequently production costs. One major drawback leading to the low production performance observed in cyanobacteria is the limited ability to utilize the full capacity of the photosynthetic apparatus and its associated systems, i.e. CO2 fixation and the directly connected metabolism. In this review, novel insights into various levels of metabolic regulation of cyanobacteria are discussed, including the potential of targeting these regulatory mechanisms to create a chassis with a phenotype favorable for photoautotrophic production. Compared to conventional metabolic engineering approaches, minor perturbations of regulatory mechanisms can have wide-ranging effects.

Ferredoxin C2 is required for chlorophyll biosynthesis and accumulation of photosynthetic antennae in Arabidopsis.

Ferredoxins (Fd) are small iron-sulphur proteins, with sub-types that have evolved for specific redox functions. Ferredoxin C2 (FdC2) proteins are essential Fd homologues conserved in all photosynthetic organisms and a number of different FdC2 functions have been proposed in angiosperms. Here we use RNAi silencing in Arabidopsis thaliana to generate a viable fdC2 mutant line with near-depleted FdC2 protein levels. Mutant leaves have ~50% less chlorophyll a and b, and chloroplasts have poorly developed thylakoid membrane structure. Transcriptomics indicates upregulation of genes involved in stress responses. Although fdC2 antisense plants show increased damage at photosystem II (PSII) when exposed to high light, PSII recovers at the same rate as wild type in the dark. This contradicts literature proposing that FdC2 regulates translation of the D1 subunit of PSII, by binding to psbA transcript. Measurement of chlorophyll biosynthesis intermediates revealed a build-up of Mg-protoporphyrin IX, the substrate of the aerobic cyclase. We localise FdC2 to the inner chloroplast envelope and show that the FdC2 RNAi line has a disproportionately lower protein abundance of antennae proteins, which are nuclear-encoded and must be refolded at the envelope after import.

Enhancing the textural and rheological properties of fermentation-induced pea protein emulsion gels with transglutaminase.

The aim of this study was to assess how transglutaminase (TG) impacts the microstructure, texture, and rheological properties of fermentation-induced pea protein emulsion gels. Additionally, the study examined the influence of storage time on the functional properties of these gels. Fermentation-induced pea protein gels were produced in the presence or absence of TG and stored for 1, 4, 8, 12, and 16 weeks. Texture analysis, rheological measurements, moisture content and microstructure evaluation with confocal laser scanning microscopy (CLSM) and 3D image analysis were conducted to explore the effects of TG on the structural and rheological properties of the fermented samples. The porosity of the protein networks in the pea gels decreased in the presence of TG, the storage modulus increased and the textural characteristics were significantly improved, resulting in harder and more springy gels. The gel porosity increased in gels with and without TG after storage but the effect of storage on textural and rheological properties was limited, indicating limited structural rearrangement once the fermentation-induced pea protein emulsion gels are formed. Greater coalescence was observed for oil droplets within the gel matrix after 16 weeks of storage in the absence of TG, consistent with these protein structures being weaker than the more structurally stable TG-treated gels. This study shows that TG treatment is a powerful tool to enhance the textural and rheological properties of fermentation-induced pea protein emulsion gels.

Development of a Highly Sensitive Luciferase-Based Reporter System To Study Two-Step Protein Secretion in Cyanobacteria.

Cyanobacteria, ubiquitous oxygenic photosynthetic bacteria, interact with the environment and their surrounding microbiome through the secretion of a variety of small molecules and proteins. The release of these compounds is mediated by sophisticated multiprotein complexes, also known as secretion systems. Genomic analyses indicate that protein and metabolite secretion systems are widely found in cyanobacteria; however, little is known regarding their function, regulation, and secreted effectors. One such system, the type IVa pilus system (T4aPS), is responsible for the assembly of dynamic cell surface appendages, type IVa pili (T4aP), that mediate ecologically relevant processes such as phototactic motility, natural competence, and adhesion. Several studies have suggested that the T4aPS can also act as a two-step protein secretion system in cyanobacteria akin to the homologous type II secretion system in heterotrophic bacteria. To determine whether the T4aP are involved in two-step secretion of nonpilin proteins, we developed a NanoLuc (NLuc)-based quantitative secretion reporter for the model cyanobacterium Synechocystis sp. strain PCC 6803. The NLuc reporter presented a wide dynamic range with at least 1 order of magnitude more sensitivity than traditional immunoblotting. Application of the reporter to a collection of Synechocystis T4aPS mutants demonstrated that the two-step secretion of NLuc is independent of T4aP. In addition, our data suggest that secretion differences typically observed in T4aPS mutants are likely due to a disruption of cell envelope homeostasis. This study opens the door to exploring protein secretion in cyanobacteria further. IMPORTANCE Protein secretion allows bacteria to interact and communicate with the external environment. Secretion is also biotechnologically relevant, where it is often beneficial to target proteins to the extracellular space. Due to a shortage of quantitative assays, many aspects of protein secretion are not understood. Here, we introduce an NLuc-based secretion reporter in cyanobacteria. NLuc is highly sensitive and can be assayed rapidly and in small volumes. The NLuc reporter allowed us to clarify the role of type IVa pili in protein secretion and identify mutations that increase secretion yield. This study expands our knowledge of cyanobacterial secretion and offers a valuable tool for future studies of protein secretion systems in cyanobacteria.

Inhibition of lytic polysaccharide monooxygenase by natural plant extracts.

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes of industrial and biological importance. In particular, LPMOs play important roles in fungal lifestyle. No inhibitors of LPMOs have yet been reported. In this study, a diverse library of 100 plant extracts was screened for LPMO activity-modulating effects. By employing protein crystallography and LC-MS, we successfully identified a natural LPMO inhibitor. Extract screening revealed a significant LPMO inhibition by methanolic extract of Cinnamomum cassia (cinnamon), which inhibited LsAA9A LPMO from Lentinus similis in a concentration-dependent manner. With a notable exception, other microbial LPMOs from families AA9 and AA10 were also inhibited by this cinnamon extract. The polyphenol cinnamtannin B1 was identified as the inhibitory component by crystallography. Cinnamtannin B1 was bound to the surface of LsAA9A at two distinct binding sites: one close to the active site and another at a pocket on the opposite side of the protein. Independent characterization of cinnamon extract by LC-MS and subsequent activity measurements confirmed that the compound inhibiting LsAA9A was cinnamtannin B1. The results of this study show that specific natural LPMO inhibitors of plant origin exist in nature, providing the opportunity for future exploitation of such compounds within various biotechnological contexts.

Outdoor cultivation of a novel isolate of the microalgae Scenedesmus sp. and the evaluation of its potential as a novel protein crop.

A Danish strain of the green microalgae Scenedesmus sp. was isolated, identified and characterized with respect to productivity under outdoor cultivation conditions at northern latitudes. The algae were cultivated outdoors in Denmark in closed tubular photobioreactors using only sunlight, simple inorganic nutrients and under ambient temperatures. The biomass composition was evaluated in terms of protein content and quality. The average volumetric and areal biomass productivity obtained for the Scenedesmus sp. isolate during outdoor cultivation was 0.083 g dry matter L-1 and 6.40 g dm m-2  day-1 , respectively. Thus, productivities are comparable to data reported in the literature under similar conditions. A strain-specific nitrogen to protein conversion factor of 5.5 was determined for the Scenedesmus sp. strain enabling more accurate protein estimations from simple nitrogen determination methods like Kjeldahl analysis in the future. The protein content was determined to be 52.4% of dried biomass for this Scenedesmus strain. The sum of essential amino acids was 42% which is high compared to other microalgae. The results are compared and discussed in comparison to other microalgae and soybean as a common plant protein source.

Clinicogenomic factors of biotherapy immunogenicity in autoimmune disease: A prospective multicohort study of the ABIRISK consortium.

BACKGROUND: Biopharmaceutical products (BPs) are widely used to treat autoimmune diseases, but immunogenicity limits their efficacy for an important proportion of patients. Our knowledge of patient-related factors influencing the occurrence of antidrug antibodies (ADAs) is still limited. METHODS AND FINDINGS: The European consortium ABIRISK (Anti-Biopharmaceutical Immunization: prediction and analysis of clinical relevance to minimize the RISK) conducted a clinical and genomic multicohort prospective study of 560 patients with multiple sclerosis (MS, n = 147), rheumatoid arthritis (RA, n = 229), Crohn's disease (n = 148), or ulcerative colitis (n = 36) treated with 8 different biopharmaceuticals (etanercept, n = 84; infliximab, n = 101; adalimumab, n = 153; interferon [IFN]-beta-1a intramuscularly [IM], n = 38; IFN-beta-1a subcutaneously [SC], n = 68; IFN-beta-1b SC, n = 41; rituximab, n = 31; tocilizumab, n = 44) and followed during the first 12 months of therapy for time to ADA development. From the bioclinical data collected, we explored the relationships between patient-related factors and the occurrence of ADAs. Both baseline and time-dependent factors such as concomitant medications were analyzed using Cox proportional hazard regression models. Mean age and disease duration were 35.1 and 0.85 years, respectively, for MS; 54.2 and 3.17 years for RA; and 36.9 and 3.69 years for inflammatory bowel diseases (IBDs). In a multivariate Cox regression model including each of the clinical and genetic factors mentioned hereafter, among the clinical factors, immunosuppressants (adjusted hazard ratio [aHR] = 0.408 [95% confidence interval (CI) 0.253-0.657], p < 0.001) and antibiotics (aHR = 0.121 [0.0437-0.333], p < 0.0001) were independently negatively associated with time to ADA development, whereas infections during the study (aHR = 2.757 [1.616-4.704], p < 0.001) and tobacco smoking (aHR = 2.150 [1.319-3.503], p < 0.01) were positively associated. 351,824 Single-Nucleotide Polymorphisms (SNPs) and 38 imputed Human Leukocyte Antigen (HLA) alleles were analyzed through a genome-wide association study. We found that the HLA-DQA1*05 allele significantly increased the rate of immunogenicity (aHR = 3.9 [1.923-5.976], p < 0.0001 for the homozygotes). Among the 6 genetic variants selected at a 20% false discovery rate (FDR) threshold, the minor allele of rs10508884, which is situated in an intron of the CXCL12 gene, increased the rate of immunogenicity (aHR = 3.804 [2.139-6.764], p < 1 × 10-5 for patients homozygous for the minor allele) and was chosen for validation through a CXCL12 protein enzyme-linked immunosorbent assay (ELISA) on patient serum at baseline before therapy start. CXCL12 protein levels were higher for patients homozygous for the minor allele carrying higher ADA risk (mean: 2,693 pg/ml) than for the other genotypes (mean: 2,317 pg/ml; p = 0.014), and patients with CXCL12 levels above the median in serum were more prone to develop ADAs (aHR = 2.329 [1.106-4.90], p = 0.026). A limitation of the study is the lack of replication; therefore, other studies are required to confirm our findings. CONCLUSION: In our study, we found that immunosuppressants and antibiotics were associated with decreased risk of ADA development, whereas tobacco smoking and infections during the study were associated with increased risk. We found that the HLA-DQA1*05 allele was associated with an increased rate of immunogenicity. Moreover, our results suggest a relationship between CXCL12 production and ADA development independent of the disease, which is consistent with its known function in affinity maturation of antibodies and plasma cell survival. Our findings may help physicians in the management of patients receiving biotherapies.

Treatment- and population-specific genetic risk factors for anti-drug antibodies against interferon-beta: a GWAS.

BACKGROUND: Upon treatment with biopharmaceuticals, the immune system may produce anti-drug antibodies (ADA) that inhibit the therapy. Up to 40% of multiple sclerosis patients treated with interferon ß (IFNß) develop ADA, for which a genetic predisposition exists. Here, we present a genome-wide association study on ADA and predict the occurrence of antibodies in multiple sclerosis patients treated with different interferon ß preparations. METHODS: We analyzed a large sample of 2757 genotyped and imputed patients from two cohorts (Sweden and Germany), split between a discovery and a replication dataset. Binding ADA (bADA) levels were measured by capture-ELISA, neutralizing ADA (nADA) titers using a bioassay. Genome-wide association analyses were conducted stratified by cohort and treatment preparation, followed by fixed-effects meta-analysis. RESULTS: Binding ADA levels and nADA titers were correlated and showed a significant heritability (47% and 50%, respectively). The risk factors differed strongly by treatment preparation: The top-associated and replicated variants for nADA presence were the HLA-associated variants rs77278603 in IFNß-1a s.c.- (odds ratio (OR) = 3.55 (95% confidence interval = 2.81-4.48), p = 2.1 × 10-26) and rs28366299 in IFNß-1b s.c.-treated patients (OR = 3.56 (2.69-4.72), p = 6.6 × 10-19). The rs77278603-correlated HLA haplotype DR15-DQ6 conferred risk specifically for IFNß-1a s.c. (OR = 2.88 (2.29-3.61), p = 7.4 × 10-20) while DR3-DQ2 was protective (OR = 0.37 (0.27-0.52), p = 3.7 × 10-09). The haplotype DR4-DQ3 was the major risk haplotype for IFNß-1b s.c. (OR = 7.35 (4.33-12.47), p = 1.5 × 10-13). These haplotypes exhibit large population-specific frequency differences. The best prediction models were achieved for ADA in IFNß-1a s.c.-treated patients. Here, the prediction in the Swedish cohort showed AUC = 0.91 (0.85-0.95), sensitivity = 0.78, and specificity = 0.90; patients with the top 30% of genetic risk had, compared to patients in the bottom 30%, an OR = 73.9 (11.8-463.6, p = 4.4 × 10-6) of developing nADA. In the German cohort, the AUC of the same model was 0.83 (0.71-0.92), sensitivity = 0.80, specificity = 0.76, with an OR = 13.8 (3.0-63.3, p = 7.5 × 10-4). CONCLUSIONS: We identified several HLA-associated genetic risk factors for ADA against interferon ß, which were specific for treatment preparations and population backgrounds. Genetic prediction models could robustly identify patients at risk for developing ADA and might be used for personalized therapy recommendations and stratified ADA screening in clinical practice. These analyses serve as a roadmap for genetic characterizations of ADA against other biopharmaceutical compounds.

Metabolic engineering of Synechocystis sp. PCC 6803 for the production of aromatic amino acids and derived phenylpropanoids.

In light of the climate change challenge, the advantageous trait of using solar energy and carbon dioxide to produce organic molecules has granted cyanobacteria deserved interest as hosts for metabolic engineering. Importantly, these organisms do not directly compete with agricultural resources. Aromatic amino acids and derived phenylpropanoids are of high importance because they are used by the pharmaceutical, food, cosmetic, and agricultural industries as precursors of active ingredients. Amino acids are traditionally produced by extraction from protein hydrolysates, chemical synthesis or fermentation pathways using heterotrophic microorganisms. In this work we demonstrate for the first time the efficient overproduction of phenylalanine and tyrosine from CO2 in a Synechocystis sp. PCC 6803 strain heterologously expressing the feedback-inhibition-resistant AroG and TyrA enzymes from E. coli. Production titers reached 904 ±â€¯53 mg/gDW (580 ±â€¯34 mg/L) of phenylalanine and 64 ±â€¯3.7 mg/gDW (41 ±â€¯2.3 mg/L) of tyrosine after 10 days of photoautotrophic growth. We estimate that the production of the two amino acids corresponds to 56% of the total fixed carbon. Phenylalanine and tyrosine are the precursors for phenylpropanoids, thus, we tested the functionality of several phenylpropanoid biosynthetic enzymes in the generated cyanobacterium strains and successfully achieved the production of 470 ±â€¯70 mg/gDW (207 mg/L) of p-coumaric acid, 267 ±â€¯31 mg/gDW (114 mg/L) of cinnamic acid and 47.4 ±â€¯13.9 mg/gDW (12.6 mg/L) of caffeic acid after 6 days of photoautotrophic growth. All compounds were secreted to the growth medium. Our work enlarges the repertoire and yield of heterologous chemicals produced by Synechocystis and contributes to extend the molecular knowledge about this cyanobacterium.

Defining optimal electron transfer partners for light-driven cytochrome P450 reactions.

Plants and cyanobacteria are promising heterologous hosts for metabolic engineering, and particularly suited for expression of cytochrome P450 (P450s), enzymes that catalyse key steps in biosynthetic pathways leading to valuable natural products such as alkaloids, terpenoids and phenylpropanoids. P450s are often difficult to express and require a membrane-bound NADPH-dependent reductase, complicating their use in metabolic engineering and bio-production. We previously demonstrated targeting of heterologous P450s to thylakoid membranes both in N. benthamiana chloroplasts and cyanobacteria, and functional substitution of their native reductases with the photosynthetic apparatus via the endogenous soluble electron carrier ferredoxin. However, because ferredoxin acts as a sorting hub for photosynthetic reducing power, there is fierce competition for reducing equivalents, which limits photosynthesis-driven P450 output. This study compares the ability of four electron carriers to increase photosynthesis-driven P450 activity. These carriers, three plant ferredoxins and a flavodoxin-like engineered protein derived from cytochrome P450 reductase, show only modest differences in their electron transfer to our model P450, CYP79A1 in vitro. However, only the flavodoxin-like carrier supplies appreciable reducing power in the presence of competition for reduced ferredoxin, because it possesses a redox potential that renders delivery of reducing equivalents to endogenous processes inefficient. We further investigate the efficacy of these electron carrier proteins in vivo by expressing them transiently in N. benthamiana fused to CYP79A1. All but one of the fusion enzymes show improved sequestration of photosynthetic reducing power. Fusion with the flavodoxin-like carrier offers the greatest improvement in this comparison - nearly 25-fold on a per protein basis. Thus, this study demonstrates that synthetic electron transfer pathways with optimal redox potentials can alleviate the problem of endogenous competition for reduced ferredoxin and sets out a new metabolic engineering strategy useful for producing valuable natural products.

Pausing of Chloroplast Ribosomes Is Induced by Multiple Features and Is Linked to the Assembly of Photosynthetic Complexes.

Many mRNAs contain pause sites that briefly interrupt the progress of translation. Specific features that induce ribosome pausing have been described; however, their individual contributions to pause-site formation, and the overall biological significance of ribosome pausing, remain largely unclear. We have taken advantage of the compact genome of chloroplasts to carry out a plastid genome-wide survey of pause sites, as a basis for studying the impact of pausing on posttranslational processes. Based on ribosomal profiling of Arabidopsis (Arabidopsis thaliana) chloroplast mRNAs, we demonstrate that a combination of factors-mRNA secondary structure, internal Shine-Dalgarno sequences, and positively charged amino acids in the nascent peptide chain-explains 95% of the major pause sites on plastid mRNAs, whereas codon usage has little impact. The distribution of the pause sites is nonrandom and conforms to distinct patterns in the vicinity of sequences coding for transmembrane domains, which depend on their orientation within the membrane as well as being next to sequences coding for cofactor binding sites. We found strong indications that the mechanisms causing ribosomal pausing and at least some of the ribosomes pause sites are conserved between distantly related plant species. In addition, the positions of features that cause pausing are well conserved in photoautotrophic plants, but less so in their nonphotosynthetic, parasitic relatives, implying that the synthesis and assembly of photosynthetic multiprotein complexes requires localized ribosome pausing.

A force awakens: exploiting solar energy beyond photosynthesis.

In recent years, efforts to exploit sunlight, a free and abundant energy source, have sped up dramatically. Oxygenic photosynthetic organisms, such as higher plants, algae, and cyanobacteria, can convert solar energy into chemical energy very efficiently using water as an electron donor. By providing organic building blocks for life in this way, photosynthesis is undoubtedly one of the most important processes on Earth. The aim of light-driven catalysis is to harness solar energy, in the form of reducing power, to drive enzymatic reactions requiring electrons for their catalytic cycle. Light-driven enzymes have been shown to have a large number of biotechnological applications, ranging from the production of high-value secondary metabolites to the development of green chemistry processes. Here, we highlight recent key developments in the field of light-driven catalysis using biological components. We will also discuss strategies to design and optimize light-driven systems in order to develop the next generation of sustainable solutions in biotechnology.

Prognostic value of cerebrospinal fluid neurofilament light chain and chitinase-3-like-1 in newly diagnosed patients with multiple sclerosis.

BACKGROUND: Neurofilament light chain (NFL) and chitinase-3-like-1 (CHI3L1) concentrations in cerebrospinal fluid (CSF) may have prognostic value in clinically isolated syndromes (CIS) and relapsing-remitting multiple sclerosis (RRMS). OBJECTIVES: To compare the prognostic value of CSF concentrations of NFL and CHI3L1 in newly diagnosed CIS and RRMS patients. METHODS: NFL and CHI3L1 were measured in CSF in 177 newly diagnosed patients with CIS or RRMS who were followed clinically for a mean of 5.7 years. RESULTS: At baseline CSF concentrations of NFL correlated with CSF concentrations of CHI3L1, relapses in the previous year, time from last relapse, and the Expanded Disability Status Scale (EDSS) score. CSF concentrations of NFL and CHI3L1 were both associated with increased relapse risk during the first 2 years in univariate analyses, but only the CSF concentration of NFL was independently associated with relapse risk in a multivariable analysis. There was no relationship between CSF concentrations of NFL or CHI3L1 and risk of conversion to secondary progressive MS or development of disability. CONCLUSION: CSF concentrations of NFL are associated with 2-year relapse risk but not with disease progression or clinical worsening in newly diagnosed CIS and RRMS patients. This may be due to confounding by the effect of disease-modifying therapies.

Prediction of natalizumab anti-drug antibodies persistency.

BACKGROUND: Anti-drug antibodies (ADA) against natalizumab develop early during treatment. ADA persistency is defined by two consecutive positive results as performed by the current qualitative ELISA assay (positive/negative). Very little is known about the magnitude of the natalizumab ADA response and persistency. DESIGN/METHODS: We developed a highly sensitive natalizumab ADA titration assay on the Meso Scale Discovery (MSD) platform and a pharmacokinetic (PK) assay. We included 43 patients with a positive ELISA-ADA result within 6 months of treatment initiation (baseline) of whom a follow-up serum sample was available 12-30 months after treatment start. MSD-ADA titres and drug levels were measured. RESULTS: Median MSD-ADA titre at baseline was 4881 and 303 at follow-up. A titre of >400 at baseline had a 94% sensitivity and 89% specificity to predict ADA persistency. Reversion to ADA negativity occurred in 10 patients with mean drug levels of 10.8 µg/mL. The median trough drug level in ADA-positive samples was 0 µg/mL. PK levels and ADA titres correlated strongly negatively ( r = -0.67). CONCLUSION: High baseline natalizumab ADA titres accurately predict persistency. Despite continuous treatment, the majority of patients with persistent ADA had no detectable drug levels indicating loss of efficacy in line with phase 3 study results.

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.

The Low Molecular Weight Protein PsaI Stabilizes the Light-Harvesting Complex II Docking Site of Photosystem I.

PsaI represents one of three low molecular weight peptides of PSI. Targeted inactivation of the plastid PsaI gene in Nicotiana tabacum has no measurable effect on photosynthetic electron transport around PSI or on accumulation of proteins involved in photosynthesis. Instead, the lack of PsaI destabilizes the association of PsaL and PsaH to PSI, both forming the light-harvesting complex (LHC)II docking site of PSI. These alterations at the LHCII binding site surprisingly did not prevent state transition but led to an increased incidence of PSI-LHCII complexes, coinciding with an elevated phosphorylation level of the LHCII under normal growth light conditions. Remarkably, LHCII was rapidly phosphorylated in ΔpsaI in darkness even after illumination with far-red light. We found that this dark phosphorylation also occurs in previously described mutants impaired in PSI function or state transition. A prompt shift of the plastoquinone (PQ) pool into a more reduced redox state in the dark caused an enhanced LHCII phosphorylation in ΔpsaI Since the redox status of the PQ pool is functionally connected to a series of physiological, biochemical, and gene expression reactions, we propose that the shift of mutant plants into state 2 in darkness represents a compensatory and/or protective metabolic mechanism. This involves an increased reduction and/or reduced oxidation of the PQ pool, presumably to sustain a balanced excitation of both photosystems upon the onset of light.

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.

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.