生物工程强化微生物电合成转化CO_2的研究进展

微生物电合成(Microbial electrosynthesis,MES)可直接利用电能驱动微生物还原固定CO_2合成多碳化合物,为可再生新能源转化、精细化学品制备和生态环境保护提供新机遇。但是,微生物吸收胞外电极电子速率慢、产物合成效率低和产品品位不高,限制了MES实现工业化应用。在概述阴极电活性微生物吸收胞外电子的分子机制的基础上,重点综述近5年应用生物工程的理论和技术强化MES用于CO_2转化的策略与研究进展,包括改造和调控胞外电子传递通路和胞内代谢途径以及定向构建有限微生物混合培养菌群三方面,阐明了生物工程可有效突破MES中电子传递慢和可用代谢途径相对单一等瓶颈。针对目前生物工程在改进MES所面临的主要问题,从胞外电子传递机理研究、基因工具箱开发、组学技术与现代分析技术联用等角度展望了今后的研究方向。     

Enhanced acetate utilization for value-added chemicals production in Yarrowia lipolytica by integration of metabolic engineering and microbial electrosynthesis

The limited supply of reducing power restricts the efficient utilization of acetate in Yarrowia lipolytica. Here, microbial electrosynthesis (MES) system, enabling direct conversion of inward electrons to NAD(P)H, was used to improve the production of fatty alcohols from acetate based on pathway engineering. First, the conversion efficiency of acetate to acetyl-CoA was reinforced by heterogenous expression of ackA-pta genes. Second, a small amount of glucose was used as cosubstrate to activate the pentose phosphate pathway and promote intracellular reducing cofactors synthesis. Third, through the employment of MES system, the final fatty alcohols production of the engineered strain YLFL-11 reached 83.8 mg/g dry cell weight (DCW), which was 6.17-fold higher than the initial production of YLFL-2 in shake flask. Furthermore, these strategies were also applied for the elevation of lupeol and betulinic acid synthesis from acetate in Y. lipolytica, demonstrating that our work provides a practical solution for cofactor supply and the assimilation of inferior carbon sources.     

Redox and ATP control of photosynthetic cyclic electron flow in Chlamydomonas reinhardtii (I) aerobic conditions

Assimilation of atmospheric CO₂ by photosynthetic organisms such as plants, cyanobacteria and green algae, requires the production of ATP and NADPH in a ratio of 3:2. The oxygenic photosynthetic chain can function following two different modes: the linear electron flow which produces reducing power and ATP, and the cyclic electron flow which only produces ATP. Some regulation between the linear and cyclic flows is required for adjusting the stoichiometric production of high-energy bonds and reducing power. Here we explore, in the green alga Chlamydomonas reinhardtii, the onset of the cyclic electron flow during a continuous illumination under aerobic conditions. In mutants devoid of Rubisco or ATPase, where the reducing power cannot be used for carbon fixation, we observed a stimulation of the cyclic electron flow. The present data show that the cyclic electron flow can operate under aerobic conditions and support a simple competition model where the excess reducing power is recycled to match the demand for ATP.     

Microbial utilization of electrically reduced neutral red as the sole electron donor for growth and metabolite production.

Electrically reduced neutral red (NR) served as the sole source of reducing power for growth and metabolism of pure and mixed cultures of H2-consuming bacteria in a novel electrochemical bioreactor system. NR was continuously reduced by the cathodic potential (-1.5 V) generated from an electric current (0.3 to 1.0 mA), and it was subsequently oxidized by Actinobacillus succinogenes or by mixed methanogenic cultures. The A. succinogenes mutant strain FZ-6 did not grow on fumarate alone unless electrically reduced NR or hydrogen was present as the electron donor for succinate production. The mutant strain, unlike the wild type, lacked pyruvate formate lyase and formate dehydrogenase. Electrically reduced NR also replaced hydrogen as the sole electron donor source for growth and production of methane from CO2. These results show that both pure and mixed cultures can function as electrochemical devices when electrically generated reducing power can be used to drive metabolism. The potential utility of utilizing electrical reducing power in enhancing industrial fermentations or biotransformation processes is discussed.     

Neural network classification of myoelectric signal for prosthesis control

An alternate approach to deriving control for multidegree of freedom prosthetic arms is considered. By analyzing a single-channel myoelectric signal (MES), we can extract information that can be used to identify different contraction patterns in the upper arm. These contraction patterns are generated by subjects without previous training and are naturally associated with specific functions. Using a set of normalized MES spectral features, we can identify contraction patterns for four arm functions, specifically extension and flexion of the elbow and pronation and supination of the forearm. Performing identification independent of signal power is advantageous because this can then be used as a means for deriving proportional rate control for a prosthesis. An artificial neural network implementation is applied in the classification task. By using three single-layer perceptron networks, the MES is classified, with the spectral representations as input features. Trials performed on five subjects with normal limbs resulted in an average classification performance level of 85% for the four functions.     

Microbial Utilization of Electrically Reduced Neutral Red as the Sole Electron Donor for Growth and Metabolite Production

Electrically reduced neutral red (NR) served as the sole source of reducing power for growth and metabolism of pure and mixed cultures of H₂-consuming bacteria in a novel electrochemical bioreactor system. NR was continuously reduced by the cathodic potential (−1.5 V) generated from an electric current (0.3 to 1.0 mA), and it was subsequently oxidized by Actinobacillus succinogenes or by mixed methanogenic cultures. The A. succinogenes mutant strain FZ-6 did not grow on fumarate alone unless electrically reduced NR or hydrogen was present as the electron donor for succinate production. The mutant strain, unlike the wild type, lacked pyruvate formate lyase and formate dehydrogenase. Electrically reduced NR also replaced hydrogen as the sole electron donor source for growth and production of methane from CO₂. These results show that both pure and mixed cultures can function as electrochemical devices when electrically generated reducing power can be used to drive metabolism. The potential utility of utilizing electrical reducing power in enhancing industrial fermentations or biotransformation processes is discussed.     

Corn and kidney bean root endoplasmic reticulum vesicles reduce a fungal iron chelate,ferrirhodotorulic acid,in vitro

Summary Endoplasmic reticulum vesicles from both corn and kidney bean roots are capable of reducing ferrirhodotorulic acid, a fungal iron chelator, in vitro, using NADH as the reductant. In magnesium containing linear 15–45% sucrose density gradients, the activity was in a wide, high density band. The activity shifted in density to 1.07–1.08 when EDTA was included instead of magnesium. No plasma membrane reducing activity was found, even with iron deficient plants, using both NADH and NADPH as electron donors. It is speculated that the Eo value for ferrirhodotorulic acid, –0.36 V, may be too low for this chelate to be reduced by the iron deficiency induced iron chelate reductase found in the plasma membrane of nonpoaceous plants. Most of the activity was in the microsomal fraction rather than the soluble fraction.Abbreviations EDTA ethylene diamine tetraacetic acid - DTT dithiothreitol - PMSF phenylmethylsulfonyl fluoride - PVPP polyvinyl polypyrrolidone - BPDS bathophenanthroline disulfonate - MES 2-(n-morpholino)ethanesulfonic acid     

Identification of genes required for recycling reducing power during photosynthetic growth

Photosynthetic organisms have the unique ability to transform light energy into reducing power. We study the requirements for photosynthesis in the alpha-proteobacterium Rhodobacter sphaeroides. Global gene expression analysis found that approximately 50 uncharacterized genes were regulated by changes in light intensity and O\2 tension, similar to the expression of genes known to be required for photosynthetic growth of this bacterium. These uncharacterized genes included RSP4157 to -4159, which appeared to be cotranscribed and map to plasmid P004. A mutant containing a polar insertion in RSP4157, CT01, was able to grow via photosynthesis under autotrophic conditions using H2 as an electron donor and CO2 as a carbon source. However, CT01 was unable to grow photoheterotrophically in a succinate-based medium unless compounds that could be used to recycle reducing power (the external electron acceptor dimethyl sulfoxide (DMSO) or CO2 were provided. This suggests that the insertion in RSP4157 caused a defect in recycling reducing power during photosynthetic growth when a fixed carbon source was present. CT01 had decreased levels of RNA for genes encoding putative glycolate degradation functions. We found that exogenous glycolate also rescued photoheterotrophic growth of CT01, leading us to propose that CO2 produced from glycolate metabolism can be used by the Calvin cycle to recycle reducing power generated in the photosynthetic apparatus. The ability of glycolate, CO2, or DMSO to support photoheterotrophic growth of CT01 suggests that one or more products of RSP4157 to -4159 serve a previously unknown role in recycling reducing power under photosynthetic conditions.     

MES16, a member of the methylesterase protein family, specifically demethylates fluorescent chlorophyll catabolites during chlorophyll breakdown in Arabidopsis

During leaf senescence, chlorophyll (Chl) is broken down to nonfluorescent chlorophyll catabolites (NCCs). These arise from intermediary fluorescent chlorophyll catabolites (FCCs) by an acid-catalyzed isomerization inside the vacuole. The chemical structures of NCCs from Arabidopsis (Arabidopsis thaliana) indicate the presence of an enzyme activity that demethylates the C13(2)-carboxymethyl group present at the isocyclic ring of Chl. Here, we identified this activity as methylesterase family member 16 (MES16; At4g16690). During senescence, mes16 leaves exhibited a strong ultraviolet-excitable fluorescence, which resulted from large amounts of different FCCs accumulating in the mutants. As confirmed by mass spectrometry, these FCCs had an intact carboxymethyl group, which slowed down their isomerization to respective NCCs. Like a homologous protein cloned from radish (Raphanus sativus) and named pheophorbidase, MES16 catalyzed the demethylation of pheophorbide, an early intermediate of Chl breakdown, in vitro, but MES16 also demethylated an FCC. To determine the in vivo substrate of MES16, we analyzed pheophorbide a oxygenase1 (pao1), which is deficient in pheophorbide catabolism and accumulates pheophorbide in the chloroplast, and a mes16pao1 double mutant. In the pao1 background, we additionally mistargeted MES16 to the chloroplast. Normally, MES16 localizes to the cytosol, as shown by analysis of a MES16-green fluorescent protein fusion. Analysis of the accumulating pigments in these lines revealed that pheophorbide is only accessible for demethylation when MES16 is targeted to the chloroplast. Together, these data demonstrate that MES16 is an integral component of Chl breakdown in Arabidopsis and specifically demethylates Chl catabolites at the level of FCCs in the cytosol.     

Crystal structure of cytochrome P450 MoxA from Nonomuraea recticatena (CYP105)

Cytochrome P450 MoxA (P450moxA) from a rare actinomycete Nonomuraea recticatena belongs to the CYP105 family and exhibits remarkably broad substrate specificity. Here, we demonstrate that P450moxA acts on several luciferin derivatives, which were originally identified as substrates of the human microsomal P450s. We also describe the crystal structure of P450moxA in substrate-free form. Structural comparison with various bacterial and human microsomal P450s reveals that the P450moxA structure is most closely related to that of the fungal nitric oxide reductase P450nor (CYP55A1). Final refined model of P450moxA comprises almost all the residues, including the "BC-loop" and "FG-loop" regions pivotal for substrate recognition, and the current structure thus defines a well-ordered substrate-binding pocket. Clear electron density map reveals that the MES molecule is bound to the substrate-binding site, and the sixth coordination position of the heme iron is not occupied by a water molecule, probably due to the presence of MES molecule in the vicinity of the heme. The unexpected binding of the MES molecule might reflect the ability of P450moxA to accommodate a broad range of structurally diverse compounds.     

NTRC new ways of using NADPH in the chloroplast

Despite being the primary source of energy in the biosphere, photosynthesis is a process that inevitably produces reactive oxygen species. Chloroplasts are a major source of hydrogen peroxide production in plant cells; therefore, different systems for peroxide reduction, such as ascorbate peroxidase and peroxiredoxins (Prxs), are found in this organelle. Most of the reducing power required for hydrogen peroxide reduction by these systems is provided by Fd reduced by the photosynthetic electron transport chain; hence, the function of these systems is highly dependent on light. Recently, it was described a novel plastidial enzyme, stated NTRC, formed by a thioredoxin reductase (NTR) domain at the N-terminus and a thioredoxin (Trx) domain at the C-terminus. NTRC is able to conjugate both NTR and Trx activities to efficiently reduce 2-Cys Prx using NADPH as a source of reducing power. Based on these results, it was proposed that NTRC is a new pathway to transfer reducing power to the chloroplast detoxification system, allowing the use of NADPH, besides reduced Fd, for such function. In this article, the most important features of NTRC are summarized and the implications of this novel activity in the context of chloroplast protection against oxidative damage are discussed.     

From microbial fuel cell (MFC) to microbial electrochemical snorkel (MES): maximizing chemical oxygen demand (COD) removal from wastewater

The paper introduces the concept of the microbial electrochemical snorkel (MES), a simplified design of a “short-circuited” microbial fuel cell (MFC). The MES cannot provide current but it is optimized for wastewater treatment. An electrochemically active biofilm (EAB) was grown on graphite felt under constant polarization in an urban wastewater. Controlling the electrode potential and inoculating the bioreactor with a suspension of an established EAB improved the performance and the reproducibility of the anodes. Anodes, colonized by an EAB were tested for the chemical oxygen demand (COD) removal from urban wastewater using a variety of bio-electrochemical processes (microbial electrolysis, MFC, MES). The MES technology, as well as a short-circuited MFC, led to a COD removal 57% higher than a 1000 Ω-connected MFC, confirming the potential for wastewater treatment.     

From microbial fuel cell (MFC) to microbial electrochemical snorkel (MES): maximizing chemical oxygen demand (COD) removal from wastewater

The paper introduces the concept of the microbial electrochemical snorkel (MES), a simplified design of a "short-circuited" microbial fuel cell (MFC). The MES cannot provide current but it is optimized for wastewater treatment. An electrochemically active biofilm (EAB) was grown on graphite felt under constant polarization in an urban wastewater. Controlling the electrode potential and inoculating the bioreactor with a suspension of an established EAB improved the performance and the reproducibility of the anodes. Anodes, colonized by an EAB were tested for the chemical oxygen demand (COD) removal from urban wastewater using a variety of bio-electrochemical processes (microbial electrolysis, MFC, MES). The MES technology, as well as a short-circuited MFC, led to a COD removal 57% higher than a 1000 Ω-connected MFC, confirming the potential for wastewater treatment.     

IgG and IgM responses in high and low antibody-selected lines of chickens

Kinetics of IgG and IgM as measured by 2-mercaptoethanol-resistant (MER) and susceptible (MES) antibodies to sheep erythrocytes, respectively, were determined as correlated responses in lines of chickens selected for high (HA) and low (LA) antibody response to sheep erythrocytes. Primary response patterns for total, MER, and MES antibody differed according to the genetic line. Total antibody increased rapidly, peaked, and persisted at moderate levels in line HA, whereas both peak and persistency were lower in line LA. Levels of MES peaked and then declined in line HA chickens but persisted at low levels throughout in line LA. Titers of MER antibody were considerably greater in line HA than in line LA both on an absolute basis and as a proportion of total antibody titer. Secondary total titers were greater at five days after injection than at three days and greater for line HA than for line LA chicks. The pattern observed for MER and MES in line LA was similar to that for total antibody, as was MER in line HA. For MES the pattern was reversed in line HA.     

The MES-2/MES-3/MES-6 complex and regulation of histone H3 methylation in C. elegans

The C. elegans proteins MES-2 and MES-6, orthologs of the Polycomb group (PcG) chromatin repressors E(Z) and ESC, exist in a complex with their novel partner MES-3. The MES system participates in silencing the X chromosomes in the hermaphrodite germline. Loss of maternal MES function leads to germline degeneration and sterility. We report here that the MES complex is responsible for di- and trimethylation of histone H3 Lys27 (H3-K27) in the adult germline and in early embryos and that MES-dependent H3-K27 marks are concentrated on the X's. Another H3-K27 HMT functions in adult somatic cells, oocytes, and the PGCs of embryos. In PGCs, the MES complex may specifically convert dimethyl to trimethyl H3-K27. The HMT activity of the MES complex appears to be dependent on the SET domain of MES-2. MES-2 thus joins its orthologs Drosophila E(Z) and human EZH2 among SET domain proteins known to function as HMTs (reviewed in ). Methylation of histones is important for long-term epigenetic regulation of chromatin and plays a key role in diverse processes such as X inactivation and oncogenesis. Our results contribute to understanding the composition and roles of E(Z)/MES-2 complexes across species.     

USP21 promotes self-renewal and tumorigenicity of mesenchymal glioblastoma stem cells by deubiquitinating and stabilizing FOXD1

Recent studies suggest that Forkhead box D1 (FOXD1) plays an indispensable role in maintaining the mesenchymal (MES) properties of glioblastoma (GBM) stem cells (GSCs). Thus, understanding the mechanisms that control FOXD1 protein expression is critical for guiding GBM treatment, particularly in patients with therapy-resistant MES subtypes. In this study, we identify the ubiquitin-specific peptidase 21 (USP21) as a critical FOXD1 deubiquitinase in MES GSCs. We find that USP21 directly interacts with and stabilizes FOXD1 by reverting its proteolytic ubiquitination. Silencing of USP21 enhances polyubiquitination of FOXD1, promotes its proteasomal degradation, and ultimately attenuates MES identity in GSCs, while these effects could be largely restored by reintroduction of FOXD1. Remarkably, we show that disulfiram, a repurposed drug that could block the enzymatic activities of USP21, suppresses GSC tumorigenicity in MES GSC-derived GBM xenograft model. Additionally, we demonstrate that USP21 is overexpressed and positively correlated with FOXD1 protein levels in GBM tissues, and its expression is inversely correlated with patient survival. Collectively, our work reveals that USP21 maintains MES identity by antagonizing FOXD1 ubiquitination and degradation, suggesting that USP21 is a potential therapeutic target for the MES subtype of GBM.Subject terms: Cancer stem cells, CNS cancer     

Medial epithelial seam cell migration during palatal fusion

The mammalian secondary palate forms from two shelves of mesenchyme sheathed in a single-layered epithelium. These shelves meet during embryogenesis to form the midline epithelial seam (MES). Failure of MES degradation prevents mesenchymal confluence and results in a cleft palate. Previous studies indicated that MES cells undergo features of epithelial-to-mesenchymal transition (EMT) and may become migratory as part of the fusion mechanism. To detect MES cell movement over the course of fusion, we imaged the midline of fusing embryonic ephrin-B2/GFP mouse palates in real time using two-photon microscopy. These mice express an ephrin-B2-driven green fluorescent protein (GFP) that labels the palatal epithelium nuclei and persists in those cells through the time window necessary for fusion. We observed collective migration of MES cells toward the oral surface of the palatal shelf over 48 hr of imaging, and we confirmed histologically that the imaged palates had fused by the end of the imaged period. We previously reported that ephrin reverse signaling in the MES is required for palatal fusion. We therefore added recombinant EphA4/Fc protein to block this signaling in imaged palates. The blockage inhibited fusion, as expected, but did not change the observed migration of GFP-labeled cells. Thus, we uncoupled migration and fusion. Our data reveal that palatal MES cells undergo a collective, unidirectional movement during palatal fusion and that ephrin reverse signaling, though required for fusion, controls aspects of the fusion mechanism independent of migration.     

PCE dechlorination by non-<Emphasis Type="Italic">Dehalococcoides</Emphasis> in a microbial electrochemical system

The bioremediation of tetrachloroethene (perchloroethene; PCE) contaminated sites generally requires a supply of some fermentable organic substrates as an electron donor. On the other hand, organic substrates can induce the massive growth of microorganisms around the injection wells, which can foul the contaminated subsurface environment. In this study, PCE dechlorination to ethene was performed in a microbial electrochemical system (MES) using the electrode (a cathode polarized at ?500 mV vs. standard hydrogen electrode) as the electron donor. Denaturing gel gradient electrophoresis and pyrosequencing revealed a variety of non-Dehalococcoides bacteria dominant in MES, such as Acinetobacter sp. (25.7 % for AS1 in suspension of M3), Rhodopseudomonas sp. (10.5 % for AE1 and 10.1 % for AE2 in anodic biofilm of M3), Pseudomonas aeruginosa (22.4 % for BS1 in suspension of M4), and Enterobacter sp. (21.7 % for BE1 in anodic biofilm of M4) which are capable of electron transfer, hydrogen production and dechlorination. The Dehalococcoides group, however, was not detected in this system. Therefore, these results suggest that a range of bacterial species outside the Dehalococcoides can play an important role in the microbial electrochemical dechlorination process, which may lead to innovative bioremediation technology.     

Anaerobic oxidation of p-cresol by a denitrifying bacterium.

Metabolism of p-cresol (pCr) under nitrate-reducing conditions is mediated by the denitrifying bacterial isolate PC-07. The methyl substituent of the substrate is oxidized anaerobically by whole-cell suspensions of PC-07 through a series of dehydrogenation and hydration reactions to yield p-hydroxybenzoate (pOHB) in stoichiometric proportions. The partially oxidized intermediates in the pathway p-hydroxybenzyl alcohol and p-hydroxybenzaldehyde can also serve as substrates for pOHB formation. Nitrate is required as the external electron acceptor and is reduced to molecular N2. Reduction of the nitrate is stoichiometric, with pCr serving as the electron donor. In addition, the molar relationship between the electron acceptor (NO3-) reduced to the electron donor oxidized decreased to approximately 2:3 and then to 1:3 when p-hydroxybenzyl alcohol or p-hydroxybenzaldehyde, respectively, served as substrates. The decreased ratios were to be expected when the partially oxidized intermediates served as substrates, because they provided correspondingly less reducing power for pOHB formation. The anaerobic oxidation of pCr by PC-07 demonstrates a mechanism whereby aromatic compounds can be transformed in anoxic environments.     

Anaerobic oxidation of p-cresol by a denitrifying bacterium

Metabolism of p-cresol (pCr) under nitrate-reducing conditions is mediated by the denitrifying bacterial isolate PC-07. The methyl substituent of the substrate is oxidized anaerobically by whole-cell suspensions of PC-07 through a series of dehydrogenation and hydration reactions to yield p-hydroxybenzoate (pOHB) in stoichiometric proportions. The partially oxidized intermediates in the pathway p-hydroxybenzyl alcohol and p-hydroxybenzaldehyde can also serve as substrates for pOHB formation. Nitrate is required as the external electron acceptor and is reduced to molecular N2. Reduction of the nitrate is stoichiometric, with pCr serving as the electron donor. In addition, the molar relationship between the electron acceptor (NO3-) reduced to the electron donor oxidized decreased to approximately 2:3 and then to 1:3 when p-hydroxybenzyl alcohol or p-hydroxybenzaldehyde, respectively, served as substrates. The decreased ratios were to be expected when the partially oxidized intermediates served as substrates, because they provided correspondingly less reducing power for pOHB formation. The anaerobic oxidation of pCr by PC-07 demonstrates a mechanism whereby aromatic compounds can be transformed in anoxic environments.