Functional analysis of H + -pumping membrane-bound pyrophosphatase, ADP-glucose synthase, and pyruvate phosphate dikinase as pyrophosphate sources in Clostridium thermocellum

The atypical glycolysis of Clostridium thermocellum is characterized by the use of pyrophosphate (PP i ) as phosphoryl donor for phosphofructokinase (Pfk) and pyruvate phosphate dikinase (Ppdk) reactions. Previously, biosynthetic PP i was calculated to be stoichiometrically insufficient to drive glycolysis. This study investigates the role of a H + -pumping membrane-bound pyrophosphatase, glycogen cycling, a predicted Ppdk–malate shunt cycle and acetate cycling in generating PP i . Knockout studies and enzyme assays confirmed that clo1313_0823 encodes a membrane-bound pyrophosphatase. Additionally, clo1313_0717-0718 was confirmed to encode ADP-glucose synthase by knockouts, glycogen measurements in C. thermocellum and heterologous expression in E. coli . Unexpectedly, individually-targeted gene deletions of the four putative PP i sources did not have a significant phenotypic effect. Although combinatorial deletion of all four putative PP i sources reduced the growth rate by 22% (0.30±0.01 h −1 ) and the biomass yield by 38% (0.18±0.00 g biomass g substrate −1 ), this change was much smaller than what would be expected for stoichiometrically essential PP i -supplying mechanisms. Growth-arrested cells of the quadruple knockout readily fermented cellobiose indicating that the unknown PP i -supplying mechanisms are independent of biosynthesis. An alternative hypothesis that ATP-dependent Pfk activity circumvents a need for PP i altogether, was falsified by enzyme assays, heterologous expression of candidate genes and whole-genome sequencing. As a secondary outcome, enzymatic assays confirmed functional annotation of clo1313_1832 as ATP- and GTP-dependent fructokinase. These results indicate that the four investigated PP i sources individually and combined play no significant PP i -supplying role and the true source(s) of PP i , or alternative phosphorylating mechanisms, that drive glycolysis in C. thermocellum remain(s) elusive. IMPORTANCE Increased understanding of the central metabolism of C. thermocellum is important from a fundamental as well as from a sustainability and industrial perspective. In addition to showing that H + -pumping membrane-bound PPase, glycogen cycling, a Ppdk–malate shunt cycle, and acetate cycling are not significant sources of PP i supply, this study adds functional annotation of four genes and availability of an updated PP i stoichiometry from biosynthesis to the scientific domain. Together, this aids future metabolic engineering attempts aimed to improve C. thermocellum as a cell factory for sustainable and efficient production of ethanol from lignocellulosic material through consolidated bioprocessing with minimal pretreatment. Getting closer to elucidating the elusive source of PP i , or alternative phosphorylating mechanisms, for the atypical glycolysis is itself of fundamental importance. Additionally, the findings of this study directly contribute to investigations into trade-offs between thermodynamic driving force versus energy yield of PP i - and ATP-dependent glycolysis.

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Heterologous expression and site-directed mutagenesis of endoglucanase CelA from Clostridium thermocellum

Biotechnology Letters ◽

10.1007/s10529-007-9379-5 ◽

2007 ◽

Vol 29 (8) ◽

pp. 1243-1247 ◽

Cited By ~ 4

Author(s):

Qiang Yao ◽

Tingting Sun ◽

Guanjun Chen ◽

Weifeng Liu

Keyword(s):

Heterologous Expression ◽

Clostridium Thermocellum ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis

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Improved heterologous expression of the membrane-bound quinoprotein quinate dehydrogenase from Gluconobacter oxydans

Protein Expression and Purification ◽

10.1016/j.pep.2018.01.007 ◽

2018 ◽

Vol 145 ◽

pp. 100-107 ◽

Cited By ~ 5

Author(s):

Toshiharu Yakushi ◽

Kazutaka Komatsu ◽

Minenosuke Matsutani ◽

Naoya Kataoka ◽

Alisa S. Vangnai ◽

...

Keyword(s):

Heterologous Expression ◽

Gluconobacter Oxydans ◽

Membrane Bound

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Pyrophosphate as an alternative energy currency in plants

Biochemical Journal ◽

10.1042/bcj20200940 ◽

2021 ◽

Vol 478 (8) ◽

pp. 1515-1524

Author(s):

Abir U. Igamberdiev ◽

Leszek A. Kleczkowski

Keyword(s):

Metabolic Pathways ◽

Energy Efficient ◽

Alternative Energy ◽

Plant Cells ◽

Atp Synthesis ◽

Continuous Operation ◽

Low Oxygen ◽

Pyruvate Phosphate Dikinase ◽

Oxygen Stress ◽

Membrane Bound

In the conditions of [Mg2+] elevation that occur, in particular, under low oxygen stress and are the consequence of the decrease in [ATP] and increase in [ADP] and [AMP], pyrophosphate (PPi) can function as an alternative energy currency in plant cells. In addition to its production by various metabolic pathways, PPi can be synthesized in the combined reactions of pyruvate, phosphate dikinase (PPDK) and pyruvate kinase (PK) by so-called PK/PPDK substrate cycle, and in the reverse reaction of membrane-bound H+-pyrophosphatase, which uses the energy of electrochemical gradients generated on tonoplast and plasma membrane. The PPi can then be consumed in its active forms of MgPPi and Mg2PPi by PPi-utilizing enzymes, which require an elevated [Mg2+]. This ensures a continuous operation of glycolysis in the conditions of suppressed ATP synthesis, keeping metabolism energy efficient and less dependent on ATP.

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Collagenolytic Serine-Carboxyl Proteinase from Alicyclobacillus sendaiensis Strain NTAP-1: Purification, Characterization, Gene Cloning, and Heterologous Expression

Applied and Environmental Microbiology ◽

10.1128/aem.69.1.162-169.2003 ◽

2003 ◽

Vol 69 (1) ◽

pp. 162-169 ◽

Cited By ~ 39

Author(s):

Naoki Tsuruoka ◽

Toru Nakayama ◽

Masako Ashida ◽

Hisashi Hemmi ◽

Masahiro Nakao ◽

...

Keyword(s):

Heterologous Expression ◽

Molecular Mass ◽

Gene Cloning ◽

Collagen Type I ◽

Protein Sequencing ◽

Collagenolytic Activity ◽

Efficient Production ◽

Type I ◽

Calculated Molecular Mass ◽

Collagen Peptides

ABSTRACT Enzymatic degradation of collagen produces peptides, the collagen peptides, which show a variety of bioactivities of industrial interest. Alicyclobacillus sendaiensis strain NTAP-1, a slightly thermophilic, acidophilic bacterium, extracellularly produces a novel thermostable collagenolytic activity, which exhibits its optimum at the acidic region (pH 3.9) and is potentially applicable to the efficient production of such peptides. Here, we describe the purification to homogeneity, characterization, gene cloning, and heterologous expression of this enzyme, which we call ScpA. Purified ScpA is a monomeric, pepstatin-insensitive carboxyl proteinase with a molecular mass of 37 kDa which exhibited the highest reactivity toward collagen (type I, from a bovine Achilles tendon) among the macromolecular substrates examined. On the basis of the sequences of the peptides obtained by digestion of collagen with ScpA, the following synthetic peptides were designed as substrates for ScpA and kinetically analyzed: Phe-Gly-Pro-Ala*Gly-Pro-Ile-Gly (k cat, 5.41 s−1; Km , 32 μM) and Met-Gly-Pro-Arg*Gly-Phe-Pro-Gly-Ser (k cat, 351 s−1; Km , 214 μM), where the asterisks denote the scissile bonds. The cloned scpA gene encoded a protein of 553 amino acids with a calculated molecular mass of 57,167 Da. Heterologous expression of the scpA gene in the Escherichia coli cells yielded a mature 37-kDa species after a two-step proteolytic cleavage of the precursor protein. Sequencing of the scpA gene revealed that ScpA was a collagenolytic member of the serine-carboxyl proteinase family (the S53 family according to the MEROPS database), which is a recently identified proteinase family on the basis of crystallography results. Unexpectedly, ScpA was highly similar to a member of this family, kumamolysin, whose specificity toward macromolecular substrates has not been defined.

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Beyond the cell factory: Homeostatic regulation of and by the UPRER

Science Advances ◽

10.1126/sciadv.abb9614 ◽

2020 ◽

Vol 6 (29) ◽

pp. eabb9614

Author(s):

Melissa G. Metcalf ◽

Ryo Higuchi-Sanabria ◽

Gilberto Garcia ◽

C. Kimberly Tsui ◽

Andrew Dillin

Keyword(s):

Protein Misfolding ◽

Lipid Synthesis ◽

Transcriptional Response ◽

Cell Factory ◽

Unfolded Protein ◽

Membrane Bound ◽

Emerging Themes ◽

The Unfolded Protein Response ◽

Protein Response ◽

Cellular Components

The endoplasmic reticulum (ER) is commonly referred to as the factory of the cell, as it is responsible for a large amount of protein and lipid synthesis. As a membrane-bound organelle, the ER has a distinct environment that is ideal for its functions in synthesizing these primary cellular components. Many different quality control machineries exist to maintain ER stability under the stresses associated with synthesizing, folding, and modifying complex proteins and lipids. The best understood of these mechanisms is the unfolded protein response of the ER (UPRER), in which transmembrane proteins serve as sensors, which trigger a coordinated transcriptional response of genes dedicated for mitigating the stress. As the name suggests, the UPRER is most well described as a functional response to protein misfolding stress. Here, we focus on recent findings and emerging themes in additional roles of the UPRER outside of protein homeostasis, including lipid homeostasis, autophagy, apoptosis, and immunity.

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Multi-Path Optimization for Efficient Production of 2′-Fucosyllactose in an Engineered Escherichia coli C41 (DE3) Derivative

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2020.611900 ◽

2020 ◽

Vol 8 ◽

Author(s):

Zhijian Ni ◽

Zhongkui Li ◽

Jinyong Wu ◽

Yuanfei Ge ◽

Yingxue Liao ◽

...

Keyword(s):

Escherichia Coli ◽

Batch Fermentation ◽

Infant Nutrition ◽

High Titer ◽

Production Performance ◽

Cell Factory ◽

Efficient Production ◽

Healthy Development ◽

Fucosyltransferase Gene ◽

Fed Batch Fermentation

2′-fucosyllactose (2′-FL), one of the simplest but most abundant oligosaccharides in human milk, has been demonstrated to have many positive benefits for the healthy development of newborns. However, the high-cost production and limited availability restrict its widespread use in infant nutrition and further research on its potential functions. In this study, on the basis of previous achievements, we developed a powerful cell factory by using a lacZ-mutant Escherichia coli C41 (DE3)ΔZ to ulteriorly increase 2′-FL production by feeding inexpensive glycerol. Initially, we co-expressed the genes for GDP-L-fucose biosynthesis and heterologous α-1,2-fucosyltransferase in C41(DE3)ΔZ through different plasmid-based expression combinations, functionally constructing a preferred route for 2′-FL biosynthesis. To further boost the carbon flux from GDP-L-fucose toward 2′-FL synthesis, deletion of chromosomal genes (wcaJ, nudD, and nudK) involved in the degradation of the precursors GDP-L-fucose and GDP-mannose were performed. Notably, the co-introduction of two heterologous positive regulators, RcsA and RcsB, was confirmed to be more conducive to GDP-L-fucose formation and thus 2′-FL production. Further a genomic integration of an individual copy of α-1,2-fucosyltransferase gene, as well as the preliminary optimization of fermentation conditions enabled the resulting engineered strain to achieve a high titer and yield. By collectively taking into account the intracellular lactose utilization, GDP-L-fucose availability, and fucosylation activity for 2′-FL production, ultimately a highest titer of 2′-FL in our optimized conditions reached 6.86 g/L with a yield of 0.92 mol/mol from lactose in the batch fermentation. Moreover, the feasibility of mass production was demonstrated in a 50-L fed-batch fermentation system in which a maximum titer of 66.80 g/L 2′-FL was achieved with a yield of 0.89 mol 2′-FL/mol lactose and a productivity of approximately 0.95 g/L/h 2′-FL. As a proof of concept, our preliminary 2′-FL production demonstrated a superior production performance, which will provide a promising candidate process for further industrial production.

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Electrodes modified with lipid membranes to study quinone oxidoreductases

Biochemical Society Transactions ◽

10.1042/bst0370707 ◽

2009 ◽

Vol 37 (4) ◽

pp. 707-712 ◽

Cited By ~ 7

Author(s):

Sophie A. Weiss ◽

Lars J.C. Jeuken

Keyword(s):

Lipid Membranes ◽

Biochemical Properties ◽

Model Membranes ◽

Water Soluble ◽

Enzyme Assays ◽

Membrane Enzymes ◽

Membrane Bound ◽

Hydrophobic Nature

Quinone oxidoreductases are a class of membrane enzymes that catalyse the oxidation or reduction of membrane-bound quinols/quinones. The conversion of quinone/quinol by these enzymes is difficult to study because of the hydrophobic nature of the enzymes and their substrates. We describe some biochemical properties of quinones and quinone oxidoreductases and then look in more detail at two model membranes that can be used to study quinone oxidoreductases in a native-like membrane environment with their native lipophilic quinone substrates. The results obtained with these model membranes are compared with classical enzyme assays that use water-soluble quinone analogues.

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