BackgroundOscillation is a special cell behavior in microorganisms during continuous fermentation, which poses threats to the output stability for industrial productions of biofuels and biochemicals. In previous study, a spontaneous oscillatory behavior was observed in Clostridium butyricum-intensive microbial consortium in continuous fermentation for 1,3-propanediol (1,3-PDO) production from glycerol, which led to the discovery of oscillation in species C. butyricum.ResultsSpontaneous oscillations by C. butyricum tended to occur under glycerol-limited conditions at low dilution rates. At a glycerol feed concentration of 88 g/L and a dilution rate of 0.048 h−1, the oscillatory behavior of C. butyricum was observed after continuous operation for 146 h and was sustained for over 450 h with an average oscillation period of 51 h. During oscillations, microbial glycerol metabolism exhibited dramatic periodic changes, in which productions of lactate, formate and hydrogen significantly lagged behind that of other products including biomass, 1,3-PDO and butyrate. Analysis of extracellular oxidation–reduction potential and intracellular ratio of NAD+/NADH indicated that microbial cells experienced distinct redox changes during oscillations, from oxidized to reduced state with decreasing of growth rate. Meanwhile, C. butyricum S3 exhibited periodic morphological changes during oscillations, with aggregates, elongated shape, spores or cell debris at the trough of biomass production. Transcriptome analysis indicated that expression levels of multiple genes were up-regulated when microbial cells were undergoing stress, including that for pyruvate metabolism, conversion of acetyl-CoA to acetaldehyde as well as stress response.ConclusionThis study for the first time systematically investigated the oscillatory behavior of C. butyricum in aspect of occurrence condition, metabolism, morphology and transcriptome. Based on the experimental results, two hypotheses were put forward to explain the oscillatory behavior: disorder of pyruvate metabolism, and excessive accumulation of acetaldehyde.

BackgroundApplication of raw starch-degrading enzymes (RSDEs) in starch processing for biofuel production can effectively reduce energy consumption and processing costs. RSDEs are generally produced by filamentous fungi, such as Penicillium oxalicum, but with very low yields, which seriously hampers industrialization of raw starch processing. Breeding assisted by random mutagenesis is an efficient way to improve fungal enzyme production.ResultsA total of 3532 P. oxalicum colonies were generated after multiple rounds of mutagenesis, by atmospheric and room-temperature plasma (ARTP) and/or ethyl methanesulfonate (EMS). Of these, one mutant A2-13 had the highest RSDE activity of 162.7 U/mL, using raw cassava flour as substrate, a yield increase of 61.1%, compared with that of the starting strain, OXPoxGA15A. RSDE activity of A2-13 further increased to 191.0 U/mL, through optimization of culture conditions. Increased expression of major amylase genes, including the raw starch-degrading glucoamylase gene, PoxGA15A, and its regulatory gene, PoxAmyR, as well as several single-nucleotide polymorphisms in the A2-13 genome, were detected by real-time reverse transcription quantitative PCR and genomic re-sequencing, respectively. In addition, crude RSDEs produced by A2-13, combined with commercial α-amylase, could efficiently digest raw corn flour and cassava flour at 40 °C.ConclusionsOverall, ARTP/EMS-combined mutagenesis effectively improved fungal RSDE yield. An RSDE-hyperproducing mutant, A2-13, was obtained, and its RSDEs could efficiently hydrolyze raw starch, in combination with commercial α-amylase at low temperature, which provides a useful RSDE resource for future starch processing.

BackgroundHemicellulose accounts for a significant part of plant biomass, and still poses a barrier to the efficient saccharification of lignocellulose. The recalcitrant part of hemicellulose is a serious impediment to the action of cellulases, despite the use of xylanases in the cellulolytic cocktail mixtures. However, the complexity and variety of hemicelluloses in different plant materials require the use of highly specific enzymes for a complete breakdown. Over the last few years, new fungal enzymes with novel activities on hemicelluloses have emerged. In the present study, we explored the synergistic relationships of the xylan-active AA14 lytic polysaccharide monooxygenase (LPMO), PcAA14B, with the recently discovered glucuronoxylan-specific xylanase TtXyn30A, of the (sub)family GH30_7, displaying xylobiohydrolase activity, and with commercial cellobiohydrolases, on pretreated natural lignocellulosic substrates.ResultsPcAA14B and TtXyn30A showed a strong synergistic interaction on the degradation of the recalcitrant part of xylan. PcAA14B was able to increase the release of xylobiose from TtXyn30A, showing a degree of synergism (DS) of 3.8 on birchwood cellulosic fibers, and up to 5.7 on pretreated beechwood substrates. The increase in activity was dose- and time- dependent. A screening study on beechwood materials pretreated with different methods showed that the effect of the PcAA14B–TtXyn30A synergism was more prominent on substrates with low hemicellulose content, indicating that PcAA14B is mainly active on the recalcitrant part of xylan, which is in close proximity to the underlying cellulose fibers. Simultaneous addition of both enzymes resulted in higher DS than sequential addition. Moreover, PcAA14B was found to enhance cellobiose release from cellobiohydrolases during hydrolysis of pretreated lignocellulosic substrates, as well as microcrystalline cellulose.ConclusionsThe results of the present study revealed a new synergistic relationship not only among two recently discovered xylan-active enzymes, the LPMO PcAA14B, and the GH30_7 glucuronoxylan-active xylobiohydrolase TtXyn30A, but also among PcAA14B and cellobiohydrolases. We hypothesize that PcAA14B creates free ends in the xylan polymer, which can be used as targets for the action of TtXyn30A. The results are of special importance for the design of next-generation enzymatic cocktails, able to efficiently remove hemicelluloses, allowing complete saccharification of cellulose in plant biomass.

BackgroundChromochloris zofingiensis, an oleaginous microalga, is a promising feedstock for the co-production of triacylglycerol (TAG)-based biodiesel and the high-value product astaxanthin. To reveal the molecular mechanism of TAG and astaxanthin biosynthesis during transitions of sulfur nutritional status, namely sulfur-starvation (SS) and sulfur-replenishment (SR), the physiological responses and the transcriptomic dynamics of C. zofingiensis were examined.ResultsThe results revealed a reversible TAG and astaxanthin accumulation under SS, which is correlated with the reduction of cell growth and protein content, indicating the reallocation of carbon. By correlating the data on the physiological and transcriptional responses to different sulfur nutritional status, a model for the underlying mechanism of TAG and astaxanthin accumulation in C. zofingiensis was postulated, which involved up-regulation of key genes including diacylglycerol acyltransferase (DGTT5) and beta-carotene ketolase (BKT1), increased energy and NADPH supply by elevating the tricarboxylic acid (TCA) cycle and the oxidative pentose phosphate (OPP) pathway, and the increased carbon precursors (pyruvate and acetyl-CoA) through central carbon metabolism. In addition, the net enhancement of the de novo biosynthesis of fatty acids and the re-direction of the terpenoid precursors toward the branch catalyzed by lycopene beta cyclase (LCYb) and BKT1 escalated the substrate availability for the biosynthesis of TAG and astaxanthin, respectively.ConclusionsIn this study, the time-resolved transcriptional analysis of C. zofingiensis under SS and SR conditions was reported for the first time to elucidate the regulatory roles of key enzymes, including DGTT5, BKT1 and LCYb, in the underlying mechanisms of TAG and astaxanthin accumulation.

BackgroundThe use of cyanobacteria and microalgae as cell factories to produce biofuels and added-value bioproducts has received great attention during the last two decades. Important investments have been made by public and private sectors to develop this field. However, it has been a challenge to develop a viable and cost-effective platform for cultivation of cyanobacteria and microalgae under outdoor conditions. Dealing with contamination caused by bacteria, weedy algae/cyanobacteria and other organisms is a major constraint to establish effective cultivation processes.ResultsHere, we describe the implementation in the cyanobacterium Synechococcus elongatus PCC 7942 of a phosphorus selective nutrition system to control biological contamination during cultivation. The system is based on metabolic engineering of S. elongatus to metabolize phosphite, a phosphorus source not normally metabolized by most organisms, by expressing a bacterial phosphite oxidoreductase (PtxD). Engineered S. elongatus strains expressing PtxD grow at a similar rate on media supplemented with phosphite as the non-transformed control supplemented with phosphate. We show that when grown in media containing phosphite as the sole phosphorus source in glass flasks, the engineered strain was able to grow and outcompete biological contaminants even when the system was intentionally inoculated with natural competitors isolated from an irrigation canal. The PtxD/phosphite system was successfully used for outdoor cultivation of engineered S. elongatus in 100-L cylindrical reactors and 1000-L raceway ponds, under non-axenic conditions and without the need of sterilizing containers and media. Finally, we also show that the PtxD/phosphite system can be used as selectable marker for S. elongatus PCC 7942 transgenic strains selection, eliminating the need of antibiotic resistance genes.ConclusionsOur results suggest that the PtxD/phosphite system is a stable and sufficiently robust strategy to control biological contaminants without the need of sterilization or other complex aseptic procedures. Our data show that the PtxD/phosphite system can be used as selectable marker and allows production of the cyanobacterium S. elongatus PCC 7942 in non-axenic outdoor reactors at lower cost, which in principle should be applicable to other cyanobacteria and microalgae engineered to metabolize phosphite.

BackgroundThe simultaneous and effective conversion of both pentose and hexose in fermentation is a critical and challenging task toward the lignocellulosic economy. This study aims to investigate the feasibility of an innovative co-fermentation process featuring with a cell recycling unit (CF/CR) for mixed sugar utilization. A l-lactic acid-producing strain Enterococcus mundtii QU 25 was applied in the continuous fermentation process, and the mixed sugars were utilized at different productivities after the flowing conditions were changed. A mathematical model was constructed with the experiments to optimize the biological process and clarify the cell metabolism through kinetics analysis. The structured model, kinetic parameters, and achievement of the fermentation strategy shall provide new insights toward whole sugar fermentation via real-time monitoring for process control and optimization.ResultsSignificant carbon catabolite repression in co-fermentation using a glucose/xylose mixture was overcome by replacing glucose with cellobiose, and the ratio of consumed pentose to consumed hexose increased significantly from 0.096 to 0.461 by mass. An outstanding product concentration of 65.2 g L−1 and productivity of 13.03 g L−1 h−1 were achieved with 50 g L−1 cellobiose and 30 g L−1 xylose at an optimized dilution rate of 0.2 h−1, and the cell retention time gradually increased. Among the total lactic acid production, xylose contributed to more than 34% of the mixed sugars, which was close to the related contents in agricultural residuals. The model successfully simulated the transition of sugar consumption, cell growth, and lactic acid production among the batch, continuous process, and CF/CR systems.ConclusionCell retention time played a critical role in balancing pentose and hexose consumption, cell decay, and lactic acid production in the CF/CR process. With increasing cell concentration, consumption of mixed sugars increased with the productivity of the final product; hence, the impact of substrate inhibition was reduced. With the validated parameters, the model showed the highest accuracy simulating the CF/CR process, and significantly longer cell retention times compared to hydraulic retention time were tested.

BackgroundThe exposure of microalgae and plants to low UV-C radiation dosages can improve their biomass composition and stress tolerance. Despite UV-C sharing these effects with UV-A/B but at much lower dosages, UV-C sensing and signal mechanisms are still mostly unknown. Thus, we have described and integrated the proteometabolomic and physiological changes occurring in Chlamydomonas reinhardtii—a simple Plantae model—into the first 24 h after a short and low-intensity UV-C irradiation in order to reconstruct the microalgae response system to this stress.ResultsThe microalgae response was characterized by increased redox homeostasis, ROS scavenging and protein damage repair/avoidance elements. These processes were upregulated along with others related to the modulation of photosynthetic electron flux, carbon fixation and C/N metabolism. These changes, attributed to either direct UV-C-, ROS- or redox unbalances-associated damage, trigger a response process involving novel signaling intermediaries and effectors such as the translation modulator FAP204, a PP2A-like protein and a novel DYRK kinase. These elements were found linked to the modulation of Chlamydomonas biomass composition (starch accumulation) and proliferation, within an UV-C response probably modulated by different epigenetic factors.ConclusionChosen multiomics integration approach was able to describe many fast changes, including biomass composition and ROS stress tolerance, as a response to a low-intensity UV-C stress. Moreover, the employed omics and systems biology approach placed many previously unidentified protein and metabolites at the center of these changes. These elements would be promising targets for the characterization of this stress response in microalgae and plants and the engineering of more productive microalgae strains.

BackgroundA precise map of the metabolic fluxome, the closest surrogate to the physiological phenotype, is becoming progressively more important in the metabolic engineering of photosynthetic organisms for biofuel and biomass production. For photosynthetic organisms, the state-of-the-art method for this purpose is instationary 13C fluxomics, which has arisen as a sibling of transcriptomics or proteomics. Instationary 13C data processing requires solving high-dimensional nonlinear differential equations and leads to large computational and time costs when its scope is expanded to a genome-scale metabolic network.ResultHere, we present a parallelized method to model instationary 13C labeling data. The elementary metabolite unit (EMU) framework is reorganized to allow treating individual mass isotopomers and breaking up of their networks into strongly connected components (SCCs). A variable domain parallel algorithm is introduced to process ordinary differential equations in a parallel way. 15-fold acceleration is achieved for constant-step-size modeling and ~ fivefold acceleration for adaptive-step-size modeling.ConclusionThis algorithm is universally applicable to isotope granules such as EMUs and cumomers and can substantially accelerate instationary 13C fluxomics modeling. It thus has great potential to be widely adopted in any instationary 13C fluxomics modeling.

BackgroundAcetylacetone is a commercially bulk chemical with diverse applications. However, the traditional manufacturing methods suffer from many drawbacks such as multiple steps, harsh conditions, low yield, and environmental problems, which hamper further applications of petrochemical-based acetylacetone. Compared to conventional chemical methods, biosynthetic methods possess advantages such as being eco-friendly, and having mild conditions, high selectivity and low potential costs. It is urgent to develop biosynthetic route for acetylacetone to avoid the present problems.ResultsThe biosynthetic pathway of acetylacetone was constructed by reversing its biodegradation route, and the acetylacetone was successfully produced by engineered Escherichia coli (E. coli) by overexpression of acetylacetone-cleaving enzyme (Dke1) from Acinetobacter johnsonii. Several promising amino acid residues were selected for enzyme improvement based on sequence alignment and structure analysis, and the acetylacetone production was improved by site-directed mutagenesis of Dke1. The double-mutant (K15Q/A60D) strain presented the highest acetylacetone-producing capacity which is 3.6-fold higher than that of the wild-type protein. Finally, the strain accumulated 556.3 ± 15.2 mg/L acetylacetone in fed-batch fermentation under anaerobic conditions.ConclusionsThis study presents the first intuitive biosynthetic pathway for acetylacetone inspired by its biodegradation, and shows the potential for large-scale production.

BackgroundCyanobacterial carbohydrates, such as sucrose, have been considered as potential renewable feedstock to support the production of fuels and chemicals. However, the separation and purification processes of these carbohydrates will increase the production cost of chemicals. Co-culture fermentation has been proposed as an efficient and economical way to utilize these cyanobacterial carbohydrates. However, studies on the application of co-culture systems to achieve green biosynthesis of platform chemicals are still rare.ResultsIn this study, we successfully achieved one-step conversion of sucrose derived from cyanobacteria to fine chemicals by constructing a microbial consortium consisting of the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 and Escherichia coli to sequentially produce sucrose and then the platform chemical 3-hydroxypropionic acid (3-HP) from CO2 under photoautotrophic growth conditions. First, efforts were made to overexpress the sucrose permease-coding gene cscB under the strong promoter Pcpc560 in S. elongatus UTEX 2973 for efficient sucrose secretion. Second, the sucrose catabolic pathway and malonyl-CoA-dependent 3-HP biosynthetic pathway were introduced into E. coli BL21 (DE3) for heterologous biosynthesis of 3-HP from sucrose. By optimizing the cultivation temperature from 37 to 30 °C, a stable artificial consortium system was constructed with the capability of producing 3-HP at up to 68.29 mg/L directly from CO2. In addition, cell growth of S. elongatus UTEX 2973 in the consortium was enhanced, probably due to the quick quenching of reactive oxygen species (ROS) in the system by E. coli, which in turn improved the photosynthesis of cyanobacteria.ConclusionThe study demonstrated the feasibility of the one-step conversion of sucrose to fine chemicals using an artificial consortium system. The study also confirmed that heterotrophic bacteria could promote the cell growth of cyanobacteria by relieving oxidative stress in this microbial consortium, which further suggests the potential value of this system for future industrial applications.