蛋白质组学和代谢组学在微生物代谢工程中的应用

构建微生物细胞工厂是化学品、生物能源以及药物分子可持续生产的可行性策略。然而，微生物的代谢复杂、调控严谨，制约着目标产物高效合成。蛋白质组学和代谢组学可以从系统生物学角度分析酶和代谢物组分，从而理解复杂的生物系统，为微生物代谢工程改造提供重要线索。该文介绍了蛋白质组学和代谢组学在微生物代谢工程中的应用，包括基因组尺度代谢模型构建、菌株生物合成优化、指导菌株耐受性改造、限速步骤预测、植物次级代谢途径挖掘，从而为微生物合成天然产物提供新的基因或途径。在此基础上，该文还展望了生物大数据未来的发展方向。     

Assimilation, dissimilation, and detoxification of formaldehyde, a central metabolic intermediate of methylotrophic metabolism

Methanol is a valuable raw material used in the manufacture of useful chemicals as well as a potential source of energy to replace coal and petroleum. Biotechnological interest in the microbial utilization of methanol has increased because it is an ideal carbon source and can be produced from renewable biomass. Formaldehyde, a cytotoxic compound, is a central metabolic intermediate in methanol metabolism. Therefore, microorganisms utilizing methanol have adopted several metabolic strategies to cope with the toxicity of formaldehyde. Formaldehyde is initially detoxified through trapping by some cofactors, such as glutathione, mycothiol, tetrahydrofolate, and tetrahydromethanopterin, before being oxidized to CO2. Alternatively, free formaldehyde can be trapped by sugar phosphates as the first reaction in the C1 assimilation pathways: the xylulose monophosphate pathway for yeasts and the ribulose monophosphate (RuMP) pathway for bacteria. In yeasts, although formaldehyde generation and consumption takes place in the peroxisome, the cytosolic formaldehyde oxidation pathway also plays a role in formaldehyde detoxification as well as energy formation. The key enzymes of the RuMP pathway are found in a variety of microorganisms including bacteria and archaea. Regulation of the genes encoding these enzymes and their catalytic mechanisms depend on the physiological traits of these organisms during evolution.     

甲醇选择氧化金属氧化物催化剂的结构与其催化性能的关系

甲醇是一个重要的平台分子, 实现其高效转化为能源和化学品的关键是揭示相关反应过程中催化剂的结构与催化性能之间的关系和反应机理. 围绕这个关键问题, 以甲醇选择氧化为探针反应, 本文总结了负载氧化钼、负载氧化钒和杂多酸等典型催化剂体系以及近年来发展的氧化铼、氧化钌等新催化剂体系在认识催化活性中心结构和反应机理, 进而调控它们的氧化中心和酸中心等方面所取得的进展. 这些认识将有助于设计制备性能优异的新催化剂和实现甲醇到目标氧化产物的定向转化.     

Useful Products from Complex Starting Materials: Common Chemicals from Biomass Feedstocks

A rapidly expanding area of inquiry is the use of plant biomass for the industrial production of organic compounds for which there is high demand. This interest is fuelled largely by the anticipated decline in the supply of petroleum, and the inevitable concomitant rise in cost. Over the past 30 years, significant progress has been made toward the large‐scale conversion of plant biomass to common chemicals such as methanol, ethanol, glycerol, substituted furans, and carboxylic acids. However, examination of the list of top production organic chemicals reveals numerous opportunities for future development, including simple halocarbons, alkenes and arenes. Progress toward efficient and economical production of these challenging targets from biomass has recently been reported, and future success is likely to continue through academic and industrial collaboration.     

Engineered Artificial Membraneless Organelles in Saccharomyces cerevisiae To Enhance Chemical Production

Microbial cell factories provide a green and sustainable opportunity to produce value-added products from renewable feedstock. However, the leakage of toxic or volatile intermediates decreases the efficiency of microbial cell factories. In this study, membraneless organelles (MLOs) were reconstructed in Saccharomyces cerevisiae by the disordered protein sequence A-IDPs. A regulation system was designed to spatiotemporally regulate the size and rigidity of MLOs. Manipulating the MLO size of strain ZP03-FM, the amounts of assimilated methanol and malate were increased by 162 % and 61 %, respectively. Furthermore, manipulating the MLO rigidity in strain ZP04-RB made acetyl-coA synthesis from oxidative glycolysis change to non-oxidative glycolysis; consequently, CO₂ release decreased by 35 % and the n-butanol yield increased by 20 %. This artificial MLO provides a strategy for the co-localization of enzymes to channel C₁ starting materials into value-added chemicals.     

Methanogenesis mediated by methylotrophic mixed culture

Enrichment of methanogenic cultures on methanol from the microbial population in the anaerobic digesters operated on agricultural wastes revealed a high rate of biomethanation efficiency. Routine maintenance of this enrichment in a minimal basal medium at room temperature resulted in maximal growth in 40–50 d, and indicated pigment production toward the end of the growth phase. The cultures grown in three different media, with different substrates under light and dark conditions, were analyzed for protein, pigment, and gaseous products, and morphological studies were carried out by light, phase-contrast, fluorescence, and electron microscopy. In different media with methanol as substrate, growth and pigment production were maximal for the light-grown cells, decreasing in the order: phototrophic (PS(m)) > mineral > basal medium. Methanation and phototrophic growth were inversely correlated under lightgrown conditions. In contrast, growth in the dark was predominently methanogenic in the decreasing order: mineral > basal > PS (m). Among other growth conditions tested, utilization of phototrophic substrates under light and dark conditions indicated the following:

1. Basal and mineral media were supportive of methanogenic growth under both light and dark conditions, although methane yields under light-grown conditions were low;
2. Among the different substrates tested, methanol-grown cells gave the highest methane yield in the dark and;
3. Phototrophic growth in PS medium with succinate, malate, and pyruvate was better than that with methanol.

Absorption spectra of light-grown cells indicated the presence of bacteriochlorophyll a (Bchl a), as a doublet in the 800–0 nm region, which was absent in the dark-grown cells. Spectra of extracted pigments confirmed the presence of Bchl a with a 770-nm peak and carotenoid absorption bands in the 400–500 nm region indicative of the presence of a pigment of the spirilloxanthin type. Collective evidence for the predominant growth of a phototrophic organism under lightgrown conditions and microscopic examination under all conditions indicated the possible presence in the mixed culture of purple nonsulfur bacteria of theRhodopseudomonas type. In addition, the enrichment culture was found to contain other morphological forms, such as short and long rods, both individually and in clusters and coccoid cells. The presence of such different forms of microbial population in a methylotrophic enrichment along with phototrophic bacteria is interesting and is of ecological significance. Considering the uphill task of methanol oxidation under anaerobic conditions, the studies on the present enrichment signify metabolic partnerships in the methylotrophic biochemical mechanisms operative toward energy recovery.     

Cre/loxP-Mediated Multicopy Integration of the Mevalonate Operon into the Genome of <Emphasis Type="Italic">Methylobacterium extorquens</Emphasis> AM1

Methylobacterium extorquens AM1 is the model strain for methylotrophic bacteria that metabolize methanol as the sole carbon and energy source. Genetically modified M. extorquens AM1 is used as a methylotrophic cell factory (MeCF) for high value-added chemical production. We tested the Cre-loxP recombination system for its ability to mediate multicopy gene integration of the mvt3 operon (mvt3) in M. extorquens AM1. mvt3 controls the expression of the first three enzymes of the mevalonate synthesis pathway. We assayed for Cre-mediated multigene integration by screening for multicopy mutants via their survival in culture with a high kanamycin concentration (600 μg/mL). We identified mutant strains in which the mevalonate titer was increased by up to 1.9-fold compared with M2 (M. extorquens AM1ΔcelABCΔattTn7::mvt3::loxP) and confirmed mvt3 integration at 2–3 copies per genome. This result demonstrates the feasibility of multicopy integration in M. extorquens AM1 mediated by Cre-loxP recombination and its potential for improving the output of M. extorquens AM1 metabolic pathways, e.g., optimization of terpenoid synthesis.     

Establishing a Klebsiella pneumoniae-Based Cell-Free Protein Synthesis System

Cell-free protein synthesis (CFPS) systems are emerging as powerful platforms for in vitro protein production, which leads to the development of new CFPS systems for different applications. To expand the current CFPS toolkit, here we develop a novel CFPS system derived from a chassis microorganism Klebsiella pneumoniae, an important industrial host for heterologous protein expression and the production of many useful chemicals. First, we engineered the K. pneumoniae strain by deleting a capsule formation-associated wzy gene. This capsule-deficient strain enabled easy collection of the cell biomass for preparing cell extracts. Then, we optimized the procedure of cell extract preparation and the reaction conditions for CFPS. Finally, the optimized CFPS system was able to synthesize a reporter protein (superfolder green fluorescent protein, sfGFP) with a maximum yield of 253 ± 15.79 μg/mL. Looking forward, our K. pneumoniae-based CFPS system will not only expand the toolkit for protein synthesis, but also provide a new platform for constructing in vitro metabolic pathways for the synthesis of high-value chemicals.     

Reconstitution of supramolecular organization involved in energy metabolism at electrochemical interfaces for biosensing and bioenergy production

How the redox proteins and enzymes involved in bioenergetic pathways are organized is a relevant fundamental question, but our understanding of this is still incomplete. This review provides a critical examination of the electrochemical tools developed in recent years to obtain knowledge of the intramolecular and intermolecular electron transfer processes involved in metabolic pathways. Furthermore, better understanding of the electron transfer processes associated with energy metabolism will provide the basis for the rational design of biotechnological devices such as electrochemical biosensors, enzymatic and microbial fuel cells, and hydrogen production factories. Starting from the redox complexes involved in two relevant bacterial chains, i.e., from the hyperthermophile Aquifex aeolicus and the acidophile Acidithiobacillus ferrooxidans, examination of protein–protein interactions using electrochemistry is first reviewed, with a focus on the orientation of a protein on an electrochemical interface mimic of a physiological interaction between two partners. Special attention is paid to current research in the electrochemistry of essential membrane proteins, which is one mandatory step toward the understanding of energy metabolic pathways. The complex and challenging architectures built to reconstitute a membrane-like environment at an electrode are especially considered. The role played by electrochemistry in the attempt to consider full bacterial metabolism is finally emphasized through the study of whole cells immobilized at electrodes as suspensions or biofilms. Before the performances of biotechnological devices can be further improved to make them really attractive, questions remain to be addressed in this particular field of research. We discuss the bottlenecks that need to be overcome in the future.     

Transition-Metal-Catalyzed Utilization of Methanol as a C1 Source in Organic Synthesis

Methanol is used as a common solvent, cost-effective reagent, and sustainable feedstock for value-added chemicals, pharmaceuticals, and materials. Among the various applications, the utilization of methanol as a C₁ source for the formation of carbon–carbon, carbon–nitrogen, and carbon–oxygen bonds continues to be important in organic synthesis and drug discovery. In particular, the synthesis of C-, N-, and O-methylated products is of central interest because these motifs are found in a large number of natural products as well as fine and bulk chemicals. In this Minireview, we summarize the utilization of methanol as a C₁ source in methylation, methoxylation, formylation, methoxycarbonylation, and oxidative methyl ester formation reactions.     

Bioinduced Room‐Temperature Methanol Reforming

Imitating nature′s approach in nucleophile‐activated formaldehyde dehydrogenation, air‐stable ruthenium complexes proved to be exquisite catalysts for the dehydrogenation of formaldehyde hydrate as well as for the transfer hydrogenation to unsaturated organic substrates at loadings as low as 0.5 mol %. Concatenation of the chemical hydrogen‐fixation route with an oxidase‐mediated activation of methanol gives an artificial methylotrophic in vitro metabolism providing methanol‐derived reduction equivalents for synthetic hydrogenation purposes. Moreover, for the first time methanol reforming at room temperature was achieved on the basis of this bioinduced dehydrogenation path delivering hydrogen gas from aqueous methanol.     

Enhancing Production of l-Serine by Increasing the glyA Gene Expression in Methylobacterium sp. MB200

Microbial fermentation using methylotrophic bacteria is one of the most promising methods for l-serine production. Here we describe the metabolic engineering of a Methylobacterium strain to increase the production of l-serine. The glyA gene, encoding serine hydroxymethyltransferase (SHMT), was isolated from the genomic DNA of Methylobacterium sp. MB200, using a DNA fragment encoding Methylobacterium extorquens AM1 SHMT as a probe, and inserted into the vector pLAFR3. The resulting construct was transformed into Methylobacterium sp. MB200 using triparental mating. The genetic-engineered strain, designated as Methylobacterium sp. MB202, was shown to produce 11.4?±?0.6 mg/ml serine in resting cell reactions from 30 mg/ml wet cells, 20 mg/ml glycine, and 70 mg/ml methanol in 2 days, representing a 4.4-fold increase from that of the wild strain. The results demonstrated the potential for improving l-serine production by manipulating the glyA in bacteria and should facilitate the production of l-serine using Methylobacterium sp. strains.     

Optimized Fed-Batch Fermentation of Scheffersomyces stipitis for Efficient Production of Ethanol from Hexoses and Pentoses

Scheffersomyces stipitis was cultivated in an optimized, controlled fed-batch fermentation for production of ethanol from glucose–xylose mixture. Effect of feed medium composition was investigated on sugar utilization and ethanol production. Studying influence of specific cell growth rate on ethanol fermentation performance showed the carbon flow towards ethanol synthesis decreased with increasing cell growth rate. The optimum specific growth rate to achieve efficient ethanol production performance from a glucose-xylose mixture existed at 0.1 h^?1. With these optimized feed medium and cell growth rate, a kinetic model has been utilized to avoid overflow metabolism as well as to ensure a balanced feeding of nutrient substrate in fed-batch system. Fed-batch culture with feeding profile designed based on the model resulted in high titer, yield, and productivity of ethanol compared with batch cultures. The maximal ethanol concentration was 40.7 g/L. The yield and productivity of ethanol production in the optimized fed-batch culture was 1.3 and 2 times higher than those in batch culture. Thus, higher efficiency ethanol production was achieved in this study through fed-batch process optimization. This strategy may contribute to an improvement of ethanol fermentation from lignocellulosic biomass by S. stipitis on the industrial scale.     

Minor Actinides Can Replace Essential Lanthanides in Bacterial Life**

Certain f-block elements—the lanthanides—have biological relevance in the context of methylotrophic bacteria. The respective strains incorporate these 4 f elements into the active site of one of their key metabolic enzymes, a lanthanide-dependent methanol dehydrogenase. In this study, we investigated whether actinides, the radioactive 5 f elements, can replace the essential 4 f elements in lanthanide-dependent bacterial metabolism. Growth studies with Methylacidiphilum fumariolicum SolV and the Methylobacterium extorquens AM1 ΔmxaF mutant demonstrate that americium and curium support growth in the absence of lanthanides. Moreover, strain SolV favors these actinides over late lanthanides when presented with a mixture of equal amounts of lanthanides together with americium and curium. Our combined in vivo and in vitro results establish that methylotrophic bacteria can utilize actinides instead of lanthanides to sustain their one-carbon metabolism if they possess the correct size and a +III oxidation state.     

How Lanthanide Ions Affect the Addition–Elimination Step of Methanol Dehydrogenases

The recently discovered methanol dehydrogenase, XoxF, is a widespread enzyme used by methylotrophic bacteria to oxidize methanol for carbon and energy, and requires lanthanide ions for its activity. This enzyme represents an essential component of methanol utilization by both methanol- and methane-utilizing bacteria. The present investigation looks on the electronic, energetic and geometrical behavior of the methanol dehydrogenase from Methylacidiphilum fumariolicum SolV, which is strictly dependent on early lanthanide metals with +3 oxidation states, by examining enzyme-substrate complexes of all the lanthanides. We focus on the catalytic reaction mechanism of two methanol dehydrogenases having as cofactor europium and ytterbium belonging to the mid- and later- series of lanthanides, in comparison with the methanol dehydrogenase containing the cerium, one early lanthanide. Our results provide evidence for the influence of the lanthanide contraction effect in all the elementary steps of the catalytic reaction mechanism. This indication may prove useful for developing new catalytic machineries of enzymes that adopt new-to-nature transformations.     

Phenotype MicroArray Profiling of Zymomonas mobilis ZM4

In this study, we developed a Phenotype MicroArray? (PM) protocol to profile cellular phenotypes in Zymomonas mobilis, which included a standard set of nearly 2,000 assays for carbon, nitrogen, phosphorus and sulfur source utilization, nutrient stimulation, pH and osmotic stresses, and chemical sensitivities with 240 inhibitory chemicals. We observed two positive assays for C-source utilization (fructose and glucose) using the PM screen, which uses redox chemistry and cell respiration as a universal reporter to profile growth phenotypes in a high-throughput 96-well plate-based format. For nitrogen metabolism, the bacterium showed a positive test results for ammonia, aspartate, asparagine, glutamate, glutamine, and peptides. Z. mobilis appeared to use a diverse array of P-sources with two exceptions being pyrophosphate and tripolyphosphate. The assays suggested that Z. mobilis uses both inorganic and organic compounds as S-sources. No stimulation by nutrients was detected; however, there was evidence of partial inhibition by purines and pyrimidines, NAD, and deferoxamine. Z. mobilis was relatively resistant to acid pH, tolerating a pH down to about 4.0. It also tolerated phosphate, sulfate, and nitrate, but was rather sensitive to chloride and nitrite. Z. mobilis showed resistance to a large number of diverse chemicals that inhibit most bacteria. The information from PM analysis provides an overview of Z. mobilis physiology and a foundation for future comparisons of other wild-type and mutant Z. mobilis strains.     

Effective production of γ-valerolactone from biomass-derived methyl levulinate over CuOx-CaCO3 catalyst

The production of γ-valerolactone (GVL) from lignocellulosic biomass has become a focus of research owing to its potential applications in fuels and chemicals. In this study, (n)CuO_x-CaCO₃ (where n is the molar ratio of Cu to Ca) compounds were prepared for the first time and shown to function as efficient bifunctional catalysts for the conversion of biomass-derived methyl levulinate (ML) into GVL, using methanol as the in-situ hydrogen source. Among the catalysts with varied Cu/Ca molar ratios, (3/2)CuO_x-CaCO₃ provided the highest GVL yield of 95.6% from ML. The incorporation of CaCO₃ with CuO resulted in the formation of Cu⁺ species in a CuO_x-CaCO₃ catalyst, which greatly facilitated the hydrogenation of ML. Notably, CuO_x-CaCO₃ also displayed excellent catalytic performance in the methanolysis products of cellulose, even in the presence of humins. Therefore, a facile two-step strategy for the production of GVL from cellulose could be developed over this robust and inexpensive catalyst, through the integration of cellulose methanolysis catalyzed by sulfuric acid, methanol reforming, and ML hydrogenation in methanol medium.     

Effect of Evolutionary Adaption on Xylosidase Activity in Thermotolerant Yeast Isolates <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> NIRE-K1 and NIRE-K3

Efficient use of xylose along with glucose is necessary for the economic production of lignocellulosic based biofuels. Xylose transporters play an important role in the microorganisms for efficient utilization of xylose. In the present study, a novel method has been developed for a rapid assay of xylose transport activity in the xylose-utilizing isolates and other known yeasts. An assay was conducted to compare the activity of β-xylosidase using p-nitrophenyl-β-d-xylopyranoside (pNPX) in the intact, intracellular, and extracellular yeasts cells showing xylose transporter. Saccharomyces cerevisiae (MTCC 170) showed no xylosidase activity, while little growth was observed in the xylose-containing medium. Although other yeasts, i.e., Kluyveromyces marxianus NIRE-K1 (MTCC 5933), K. marxianus NIRE-K3 (MTCC 5934), and Candida tropicalis (MTCC 230), showed xylosidase activity in intact, intracellular, and extracellular culture. The xylosidase activity in intact cell was higher than that of extracellular and intracellular activity in all the yeast cells. The enzyme activity was higher in case of K. marxianus NIRE-K1 and K. marxianus NIRE-K3 rather than the C. tropicalis. Further, better xylosidase activity was observed in adapted K. marxianus cells which were 2.79–28.46 % higher than that of native (non-adapted) strains, which indicates the significant improvement in xylose transportation.     

A Novel Methylotrophic Bacterial Consortium for Treatment of Industrial Effluents

Considering the importance of methylotrophs in industrial wastewater treatment, focus of the present study was on utilization of a methylotrophic bacterial consortium as a microbial seed for biotreatment of a variety of industrial effluents. For this purpose, a mixed bacterial methylotrophic AC (Ankleshwar CETP) consortium comprising of Bordetella petrii AC1, Bacillus licheniformis AC4, Salmonella subterranea AC5, and Pseudomonas stutzeri AC8 was used. The AC consortium showed efficient biotreatment of four industrial effluents procured from fertilizer, chemical and pesticide industries, and common effluent treatment plant by lowering their chemical oxygen demand (COD) of 950–2000 mg/l to below detection limit in 60–96 h in 6-l batch reactor and 9–15 days in 6-l continuous reactor. The operating variables of wastewater treatment, viz. COD, BOD, pH, MLSS, MLVSS, SVI, and F/M ratio of these effluents, were also maintained in the permissible range in both batch and continuous reactors. Therefore, formation of the AC consortium has led to the development of an efficient microbial seed capable of treating a variety of industrial effluents containing pollutants generated from their respective industries.