Structure of plant Photosystem I-plastocyanin complex reveals strong hydrophobic interactions

Photosystem I is defined as plastocyanin-ferredoxin oxidoreductase. Taking advantage of genetic engineering, kinetic analyses and cryo-EM, our data provide novel mechanistic insights into binding and electron transfer between PSI and Pc. Structural data at 2.74 Å resolution reveals strong hydrophobic interactions in the plant PSI-Pc ternary complex, leading to exclusion of water molecules from PsaA-PsaB / Pc interface once the PSI-Pc complex forms. Upon oxidation of Pc, a slight tilt of bound oxidized Pc allows water molecules to accommodate the space between Pc and PSI to drive Pc dissociation. Such a scenario is consistent with the six times larger dissociation constant of oxidized as compared to reduced Pc and mechanistically explains how this molecular machine optimized electron transfer for fast turnover.

Isobutanol Production by Autotrophic Acetogenic Bacteria

Two different isobutanol synthesis pathways were cloned into and expressed in the two model acetogenic bacteria Acetobacterium woodii and Clostridium ljungdahlii. A. woodii is specialized on using CO2 + H2 gas mixtures for growth and depends on sodium ions for ATP generation by a respective ATPase and Rnf system. On the other hand, C. ljungdahlii grows well on syngas (CO + H2 + CO2 mixture) and depends on protons for energy conservation. The first pathway consisted of ketoisovalerate ferredoxin oxidoreductase (Kor) from Clostridium thermocellum and bifunctional aldehyde/alcohol dehydrogenase (AdhE2) from C. acetobutylicum. Three different kor gene clusters are annotated in C. thermocellum and were all tested. Only in recombinant A. woodii strains, traces of isobutanol could be detected. Additional feeding of ketoisovalerate increased isobutanol production to 2.9 mM under heterotrophic conditions using kor3 and to 1.8 mM under autotrophic conditions using kor2. In C. ljungdahlii, isobutanol could only be detected upon additional ketoisovalerate feeding under autotrophic conditions. kor3 proved to be the best suited gene cluster. The second pathway consisted of ketoisovalerate decarboxylase from Lactococcus lactis and alcohol dehydrogenase from Corynebacterium glutamicum. For increasing the carbon flux to ketoisovalerate, genes encoding ketol-acid reductoisomerase, dihydroxy-acid dehydratase, and acetolactate synthase from C. ljungdahlii were subcloned downstream of adhA. Under heterotrophic conditions, A. woodii produced 0.2 mM isobutanol and 0.4 mM upon additional ketoisovalerate feeding. Under autotrophic conditions, no isobutanol formation could be detected. Only upon additional ketoisovalerate feeding, recombinant A. woodii produced 1.5 mM isobutanol. With C. ljungdahlii, no isobutanol was formed under heterotrophic conditions and only 0.1 mM under autotrophic conditions. Additional feeding of ketoisovalerate increased these values to 1.5 mM and 0.6 mM, respectively. A further increase to 2.4 mM and 1 mM, respectively, could be achieved upon inactivation of the ilvE gene in the recombinant C. ljungdahlii strain. Engineering the coenzyme specificity of IlvC of C. ljungdahlii from NADPH to NADH did not result in improved isobutanol production.

Direct Conversion of Food Waste Extract into Caproate: Metagenomics Assessment of Chain Elongation Process

In a circular economy strategy, waste resources can be used for the biological production of high added-value substances, such as medium chain fatty acids (MCFAs), thus minimising waste and favouring a sustainable process. This study investigates single-stage fermentation processes for the production of MCFAs in a semi-continuous reactor treating the extract of real food waste (FW), without the addition of external electron donors. Two sequential acidogenic fermentation tests were carried out at an organic loading rate (OLR) of 5 and 15 gCOD L−1d−1 with a hydraulic retention time of 4 days and pH controlled at 6 ± 0.2. The highest level of caproate (4.8 g L−1) was observed at OLR of 15 gCOD L−1d−1 with a microbiome mainly composed by lactate-producing Actinomyces, Atopobium, and Olsenella species and caproate-producing Pseudoramibacter. Metagenomic analysis revealed the presence of key enzymes for the production of lactate, such as lactate dehydrogenase and pyruvate ferredoxin oxidoreductase, as well as several enzymes involved in the reverse β-oxidation pathway, thus suggesting the occurrence of a lactate-based chain elongation process.

Structure of plant PSI-plastocyanin complex reveals strong hydrophobic interactions

Photosystem I is defined as plastocyanin-ferredoxin oxidoreductase. Taking advantage of genetic engineering, kinetic analyses and cryo-EM, our data provide novel mechanistic insights into binding and electron transfer between PSI and Pc. Structural data at 2.74 Å resolution reveals strong hydrophobic interactions in the plant PSI-Pc ternary complex, leading to exclusion of water molecules from PsaA-PsaB / Pc interface once the PSI-Pc complex forms. Upon oxidation of Pc, a slight tilt of bound oxidized Pc allows water molecules to accommodate the space between Pc and PSI to drive Pc dissociation. Such a scenario is consistent with the six times larger dissociation constant of oxidized as compared to reduced Pc and mechanistically explains how this molecular machine optimized electron transfer for fast turnover.One Sentence SummaryGenetic engineering, kinetics and cryo-EM structural data reveal a mechanism in a major step of oxygenic photosynthesis

Dynamic control over feedback regulatory mechanisms improves NADPH fluxes and xylitol biosynthesis in engineered E. coli

We report improved NADPH flux and xylitol biosynthesis in engineered E. coli. Xylitol is produced from xylose via an NADPH dependent reductase. We utilize two-stage dynamic metabolic control to compare two approaches to optimize xylitol biosynthesis, a stoichiometric approach, wherein competitive fluxes are decreased, and a regulatory approach wherein the levels of key regulatory metabolites are reduced. The stoichiometric and regulatory approaches lead to a 16 fold and 100 fold improvement in xylitol production, respectively. Strains with reduced levels of enoyl-ACP reductase and glucose-6-phosphate dehydrogenase, led to altered metabolite pools resulting in the activation of the membrane bound transhydrogenase and a new NADPH generation pathway, namely pyruvate ferredoxin oxidoreductase coupled with NADPH dependent ferredoxin reductase, leading to increased NADPH fluxes, despite a reduction in NADPH pools. These strains produced titers of 200 g/L of xylitol from xylose at 86% of theoretical yield in instrumented bioreactors. We expect dynamic control over enoyl-ACP reductase and glucose-6-phosphate dehydrogenase to broadly enable improved NADPH dependent bioconversions.HighlightsDecreases in NADPH pools lead to increased NADPH fluxesPyruvate ferredoxin oxidoreductase coupled with NADPH-ferredoxin reductase improves NADPH production in vivo.Dynamic reduction in acyl-ACP/CoA pools alleviate inhibition of membrane bound transhydrogenase and improve NADPH fluxXylitol titers > 200g/L in fed batch fermentations with xylose as a sole feedstock.

Dynamic control over feedback regulation identifies pyruvate-ferredoxin oxidoreductase as a central metabolic enzyme in stationary phase E. coli

We demonstrate the use of two-stage dynamic metabolic control to manipulate feedback regulation in central metabolism and improve stationary phase biosynthesis in engineered E. coli. Specifically, we report the impact of dynamic control over two enzymes: citrate synthase, and glucose-6-phosphate dehydrogenase, on stationary phase fluxes. Firstly, reduced citrate synthase levels lead to a reduction in α-ketoglutarate, which is an inhibitor of sugar transport, resulting in increased stationary phase glucose uptake and glycolytic fluxes. Reduced glucose-6-phosphate dehydrogenase activity activates the SoxRS regulon and expression of pyruvate-ferredoxin oxidoreductase, which is in turn responsible for large increases in acetyl-CoA production. The combined reduction in citrate synthase and glucose-6-phosphate dehydrogenase, leads to greatly enhanced stationary phase metabolism and the improved production of citramalic acid enabling titers of 126±7g/L. These results identify pyruvate oxidation via the pyruvate-ferredoxin oxidoreductase as a “central” metabolic pathway in stationary phase E. coli, which coupled with ferredoxin reductase comprise a pathway whose physiologic role is maintaining NADPH levels.HighlightsDynamic reduction in α-keto-glutarate pools alleviate inhibition of PTS dependent transport improving stationary phase sugar uptake.Dynamic reduction in glucose-6-phosphate dehydrogenase activates pyruvate flavodoxin/ferredoxin oxidoreductase and improves stationary acetyl-CoA flux.Pyruvate flavodoxin/ferredoxin oxidoreductase is responsible for large stationary phase acetyl-CoA fluxes under aerobic conditions.Production of citramalate to titers 126 ± 7g/L at > 90 % of theoretical yield.

Proteome Cold-Shock Response in the Extremely Acidophilic Archaeon, Cuniculiplasma divulgatum

The archaeon Cuniculiplasma divulgatum is ubiquitous in acidic environments with low-to-moderate temperatures. However, molecular mechanisms underlying its ability to thrive at lower temperatures remain unexplored. Using mass spectrometry (MS)-based proteomics, we analysed the effect of short-term (3 h) exposure to cold. The C. divulgatum genome encodes 2016 protein-coding genes, from which 819 proteins were identified in the cells grown under optimal conditions. In line with the peptidolytic lifestyle of C. divulgatum, its intracellular proteome revealed the abundance of proteases, ABC transporters and cytochrome C oxidase. From 747 quantifiable polypeptides, the levels of 582 proteins showed no change after the cold shock, whereas 104 proteins were upregulated suggesting that they might be contributing to cold adaptation. The highest increase in expression appeared in low-abundance (0.001–0.005 fmol%) proteins for polypeptides’ hydrolysis (metal-dependent hydrolase), oxidation of amino acids (FAD-dependent oxidoreductase), pyrimidine biosynthesis (aspartate carbamoyltransferase regulatory chain proteins), citrate cycle (2-oxoacid ferredoxin oxidoreductase) and ATP production (V type ATP synthase). Importantly, the cold shock induced a substantial increase (6% and 9%) in expression of the most-abundant proteins, thermosome beta subunit and glutamate dehydrogenase. This study has outlined potential mechanisms of environmental fitness of Cuniculiplasma spp. allowing them to colonise acidic settings at low/moderate temperatures.

Effects of High Forage/Concentrate Diet on Volatile Fatty Acid Production and the Microorganisms Involved in VFA Production in Cow Rumen

The objectives of this study were to investigate the difference in the mechanism of VFAs production combined with macrogenome technology under different forage-to-concentrate ratios and sampling times. Six ruminally cannulated Holstein cows were used in a randomized complete block design. The high forage (HF) and high concentrate (HC) diets contained 70 and 35% dietary forage, respectively. The results showed that pH was affected by sampling time, at 4 h after feeding had lower value. Excepted for acetate, the VFAs was increased with forage decreased. Propionate formation via the succinic pathway, in which succinate CoA synthetase (EC 6.2.1.5) and propionyl CoA carboxylase (EC 2.8.3.1) were key enzymes, and significantly higher in HC treatment than in HF treatment, Selenomonas, Ruminobacter, Prevotella, and Clostridium were the main microorganism that encodes these key enzymes. Butyrate formation via the succinic pathway, in which phosphate butyryltransferase (EC 2.3.1.19), butyrate kinase (EC 2.7.2.7) and pyruvate ferredoxin oxidoreductase (EC 1.2.7.1) are the important enzymes, Prevotella and Bacteroides played important role in encodes these key enzymes. This research gave a further explanation on the metabolic pathways of VFAs, and microorganisms involved in VFAs production under different F:C ration, which could further reveal integrative information of rumen function.

Effect of recombinant phycocyanin on photosynthetic system in Chlamydomonas reinhardtii

The phycobilisome is an important photosynthetic antenna in the photosynthetic cyanobacteria, and phycocyanin is one of the main components of phycobilisomes. It helps cells absorb green light that green-lineage photo-synthetic organisms cannot. In this work, phycocyanin, heme oxidase and ferredoxin oxidoreductase from Arthrospira platensis FACHB 314 were successfully expressed in the green algae Chlamydomonas reinhardtii. Then the effects of this expression on the photosynthesis and growth of C. reinhardtii were detected. Transcriptional level analysis showed that the phycocyanin gene was successfully expressed stably in the transgenic strains. The results of low-temperature fluorescence emission spectra and chlorophyll fluorescence showed that recombinant phycocyanin has considerable optical activity. The expression of phycocyanin, heme oxidase and ferredoxin oxidoreductase in low-light conditions is particularly evident in the promotion of photosynthesis in C. reinhardtii. The growth of transgenic strains was significantly promoted in the early growth phase under low-light conditions. However, the final growth and biomass accumulation of transgenic C. reinhardti were inhibited by this expression. In this paper, the possibility of photoenergy transfer between phycocyanin and heterologous host thylakoid membrane was researched, which provided a useful attempt for the construction of a new photosynthetic system using phycobiliprotein from cyanobacteria.One-sentence summaryPhycocyanin from Arthrospira platensis FACHB 314 expressed in Chlamydomonas reinhardtii can effect the photosynthetic system of C. reinhardtii.