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Photosynthetic complex I enables cyclic electron flow around photosystem I, a regulatory mechanism for photosynthetic energy conversion. We report a 3.3-angstrom-resolution cryo-electron microscopy structure of photosynthetic complex I from the cyanobacterium Thermosynechococcus elongatus. The model reveals structural adaptations that facilitate binding and electron transfer from the photosynthetic electron carrier ferredoxin. By mimicking cyclic electron flow with isolated components in vitro, we demonstrate that ferredoxin directly mediates electron transfer between photosystem I and complex I, instead of using intermediates such as NADPH (the reduced form of nicotinamide adenine dinucleotide phosphate). A large rate constant for association of ferredoxin to complex I indicates efficient recognition, with the protein subunit NdhS being the key component in this process., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

In comparison to crude oil, biorefinery raw materials are challenging in concerns of transport and storage. The plant raw materials are more voluminous, so that shredding and compacting usually are necessary before transport. These mechanical processes can have a negative influence on the subsequent biotechnological processing and shelf life of the raw materials. Various approaches and their effects on renewable raw materials are shown. In addition, aspects of decentralized pretreatment steps are discussed. Another important aspect of pretreatment is the varying composition of the raw materials depending on the growth conditions. This problem can be solved with advanced on-site spectrometric analysis of the material. Graphical Abstract.

Here, we have compiled a nuclear magnetic resonance (NMR)-derived set of nuclear Overhauser enhancement (NOE) distance and dihedral angle restraints that allow for the calculation of the structure of the NDH-1 complex subunit CupS from Thermosynechococcus elongatus in solution. These restraints to calculate the structure in solution of CupS have been deposited to the Protein Data Bank (www.rcsb.org) under PDB-ID accession number 2MXA. This is the first experimental data set published to compute the three-dimensional structure of CupS. This structure is presented in the research article "Solution structure of the NDH-1 complex subunit CupS from Thermosynechococcus elongatus" published by Korste et al. in Biochim. Biophys. Acta 1847(2015)1212-1219 [1]. The cyanobacterial multi-subunit membrane protein complex NDH-1 structurally and functionally relates to Complex I of eubacteria and mitochondria. The NDH-1 complex is mechanistically involved in respiration and cyclic electron transfer around photosystem I (PSI) as well as in a unique mechanism for inorganic carbon concentration.

The cyanobacterial multi-subunit membrane protein complex NDH-1 is both structurally and functionally related to Complex I of eubacteria and mitochondria. In addition to functions in respiration and cyclic electron transfer around photosystem I (PSI), the cyanobacterial NDH-1 complex is involved in a unique mechanism for inorganic carbon concentration. Although the crystal structures of the similar respiratory Complex I from Thermus thermophilus and Escherichia coli are known, atomic structural information is not available for the cyanobacterial NDH-1 complex yet. In particular, the structures of those subunits that are not homologous to Complex I will help to understand their distinct functions. The 15.7kDa protein CupS is a small soluble subunit of the complex variant NDH-1MS, which is thought to play a role in CO2 conversion. Here, we present the NMR structure of CupS from Thermosynechococcus elongatus, which is the very first structure of a specific cyanobacterial NDH-1 complex subunit. CupS shares a structural similarity with members of the Fasciclin protein superfamily. The structural comparison to Fasciclin type proteins based on known NMR structures and protein sequences of human TGFBIp, MPB70 from Mycobacterium bovis, and Fdp from Rhodobacter sphaeroides, together with a virtual docking model of CupS and NdhF3, provide first insight into the specific binding of CupS to the NDH-1MS complex at atomic resolution., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The cyanobacterial NDH-1 complex is involved in respiratory as well as in cyclic electron transfer around photosystem I. Here, we report both backbone and side chain chemical shift assignments of CupS, a small subunit of the multisubunit membrane protein complex NDH-1 from Thermosynechococcus elongatus. The construct contains 159 amino acids including a Strep-tag and two additional amino acids.

The cyanobacterial NADPH:plastoquinone oxidoreductase complex (NDH-1), that is related to Complex I of eubacteria and mitochondria, plays a pivotal role in respiration as well as in cyclic electron transfer (CET) around PSI and is involved in a unique carbon concentration mechanism (CCM). Despite many achievements in the past, the complex protein composition and the specific function of many subunits of the different NDH-1 species remain elusive. We have recently discovered in a NDH-1 preparation from Thermosynechococcus elongatus two novel single transmembrane peptides (NdhP, NdhQ) with molecular weights below 5 kDa. Here we show that NdhP is a unique component of the ∼ 450 kDa NDH-1L complex, that is involved in respiration and CET at high CO2 concentration, and not detectable in the NDH-1MS and NDH-1MS' complexes that play a role in carbon concentration. C-terminal fusion of NdhP with his-tagged superfolder GFP and the subsequent analysis of the purified complex by electron microscopy and single particle averaging revealed its localization in the NDH-1L specific distal unit of the NDH-1 complex, that is formed by the subunits NdhD1 and NdhF1. Moreover, NdhP is essential for NDH-1L formation, as this type of NDH-1 was not detectable in a ΔndhP::Km mutant.

In this work, a membrane-based downstream process for the recovery of glucose from cellulose hydrolysis is described and evaluated. The cellulose is pretreated with the ionic liquid 1,3-dimethyl-imidazolium dimethylphosphate to reduce its crystallinity. After enzymatic conversion of cellulose to glucose the hydrolysate is filtered with an ultrafiltration membrane to remove residual particulates and enzymes. Nanofiltration is applied to purify the glucose from molecular intermediates, such as cellobiose originating from the hydrolysis reaction. Finally, the ionic liquid is removed from the hydrolysate via electrodialysis. Technically, these process steps are feasible. An economic analysis of the process reveals that the selling price of glucose from this production process is about 2.75 €/kg which is too high as compared to the current market price., (Copyright © 2013. Published by Elsevier Ltd.)

A process concept combining pretreatment of wood in ionic liquids and subsequent enzymatic hydrolysis to sugars is herein investigated to identify operating conditions which allow for (i) the processing of larger wood chips of 10 mm length, (ii) low temperature, (iii) high sugar yield, and (iv) short processing time. A careful quantitative study of the interaction of pretreatment and hydrolysis reveals that hydrolysis is most effective if beech chips are first disintegrated in [EMIM][Ac] at 115 °C for 1.5 h. The cellulose conversion varies between 70.5 wt% and 90.2wt% for hydrolysis times between 5 h and 72 h. A complete recovery of cellulose and xylan resulting in a total saccharification of 65 wt% of the wood chips could be demonstrated. It is shown that short pretreatment times are required to enable high sugar yield as well as to limit product degradation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

New screening techniques for improved enzyme variants in turbid media are urgently required in many industries such as the detergent and food industry. Here, a new method is presented to measure enzyme activity in different types of substrate suspensions. This method allows a semiquantitative determination of protease activity using native protein substrates. Unlike conventional techniques for measurement of enzyme activity, the BioLector technology enables online monitoring of scattered light intensity and fluorescence signals during the continuous shaking of samples in microtiter plates. The BioLector technique is hereby used to monitor the hydrolysis of an insoluble protein substrate by measuring the decrease of scattered light. The kinetic parameters for the enzyme reaction (V(max,app) and K(m,app)) are determined from the scattered light curves. Moreover, the influence of pH on the protease activity is investigated. The optimal pH value for protease activity was determined to be between pH 8 to 11 and the activities of five subtilisin serine proteases with variations in the amino acid sequence were compared. The presented method enables proteases from genetically modified strains to be easily characterized and compared. Moreover, this method can be applied to other enzyme systems that catalyze various reactions such as cellulose decomposition., (Copyright © 2011 American Institute of Chemical Engineers (AIChE).)

The subunit composition of the NAD(P)H dehydrogenase complex of Thermosynechococcus elongatus was analyzed by different types of mass spectrometry. All 15 known subunits (NdhA-NdhO) were identified in the purified NDH-1L complex. Moreover, two additional intact mass tags of 4902.7 and 4710.5 Da could be assigned after reannotation of the T. elongatus genome. NdhP and NdhQ are predicted to contain a single transmembrane helix each, and homologues are apparent in other cyanobacteria. Additionally, ndhP is present in some cyanophages in a cluster of PSI genes and exhibits partial similarity to NDF6, a subunit of the plant NDH-1 complex.

A new prospective cellulase assay simultaneously combining high-throughput, online analysis and insoluble cellulosic substrates is described. The hydrolysis of three different insoluble cellulosic substrates, catalysed by a commercial cellulase preparation from Trichoderma reesei (Celluclast), was monitored using the BioLector - allowing online monitoring of scattered light intensities in a continuously shaken microtiter plate. Cellulase activities could be quantitatively assayed using the BioLector. At low cellulase/cellulose ratios, the Michaelis-Menten parameters of the cellulase mixture were mainly affected by the crystallinity index of the cellulose. Here, the apparent maximum cellulase activities inversely correlated with the crystallinity index of the cellulose. At high cellulase/cellulose ratios the particle size of the cellulose, defining the external surface area accessible to the cellulases, was the key determining factor for cellulase activity. The developed technique was also successfully applied to evaluate the pH optimum of cellulases. Moreover, the non-hydrolytic deagglomeration of cellulose particles was investigated, for the first time, using high-throughput scattered light detection. In conclusion, this cellulase assay ideally links high-throughput, online analysis and realistic insoluble cellulosic substrates in one simple system. It will considerably simplify and accelerate fundamental research on cellulase screening.