Dissection of the Hydrogen Metabolism of the EnterobacteriumTrabulsiella guamensis: Identification of a Formate-Dependent and Essential Formate Hydrogenlyase Complex Exhibiting Phylogenetic Similarity to Complex I

Mapping Intimacies ◽

10.1101/563510 ◽

2019 ◽

Author(s):

Ute Lindenstrauß ◽

Constanze Pinske

Keyword(s):

Fossil Fuels ◽

Model Organism ◽

Attractive Alternative ◽

Hydrogen Metabolism ◽

Vacuum Cleaner ◽

Production Methods ◽

E Coli ◽

Formate Hydrogenlyase ◽

Biochemical Characterisation ◽

Alternative Carbon Source

AbstractTrabulsiella guamensisis a non-pathogenic enterobacterium that was isolated from a vacuum cleaner on the island of Guam. It has one H2-oxidizing Hyd-2-type hydrogenase (Hyd), and encodes a H2-evolving Hyd that is most similar to the uncharacterizedEscherichia coliformate hydrogenlyase (FHL-2Ec) complex. The FHL-2Tgcomplex is predicted to have 5 membrane-integral and between 4-5 cytoplasmic subunits. We could show that FHL-2Tgcomplex catalyses the disproportionation of formate to CO2and H2. FHL-2Tghas an activity similar to theE. coliFHL-1Eccomplex in H2-evolution from formate, but the complex appears more labile upon cell lysis. Cloning of the entire 13 kbp FHL-2Tgoperon in the heterologousE. colihost has now enabled us to prove FHL-2Tgactivity unambiguously and allowed us to characterize the FHL-2Tgcomplex biochemically. Although the formate dehydrogenase (FdhH) genefdhFis not encoded in the operon, the FdhH is part of the complex and FHL-2Tgactivity was dependent on the presence ofE. coliFdhH. Also, in contrast toE. coli, T. guamensiscan ferment the alternative carbon source cellobiose, and we further investigated the participation of both the H2-oxidizing Hyd-2Tgand the H2-forming FHL-2Tgunder these conditions.ImportanceBiological H2-production presents an attractive alternative for fossil fuels. But in order to compete with conventional H2-production methods, the process requires our understanding on the molecular level. FHL complexes are efficient H2-producers and the prototype FHL-1Eccomplex inE. coliis well studied. This paper presents the first biochemical characterisation of an FHL-2-type complex. The data presented here will enable us to solve the long-standing mystery of the FHL-2Eccomplex, allow a first biochemical characterisation ofT. guamensis’s fermentative metabolism and establish this enterobacterium as model organism for FHL-dependent energy conservation.

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Dissection of the Hydrogen Metabolism of the EnterobacteriumTrabulsiella guamensis: Identification of a Formate-Dependent and Essential Formate Hydrogenlyase Complex Exhibiting Phylogenetic Similarity to Complex I

Journal of Bacteriology ◽

10.1128/jb.00160-19 ◽

2019 ◽

Vol 201 (12) ◽

Cited By ~ 3

Author(s):

Ute Lindenstrauß ◽

Constanze Pinske

Keyword(s):

Fossil Fuels ◽

Biochemical Characterization ◽

Model Organism ◽

Attractive Alternative ◽

Hydrogen Metabolism ◽

Content Type ◽

E Coli ◽

Formate Hydrogenlyase ◽

Alternative Carbon Source

ABSTRACTTrabulsiella guamensisis a nonpathogenic enterobacterium that was isolated from a vacuum cleaner on the island of Guam. It has one H2-oxidizing Hyd-2-type hydrogenase (Hyd) and encodes an H2-evolving Hyd that is most similar to the uncharacterizedEscherichia coliformate hydrogenlyase (FHL-2Ec) complex. TheT. guamensisFHL-2 (FHL-2Tg) complex is predicted to have 5 membrane-integral and between 4 and 5 cytoplasmic subunits. We showed that the FHL-2Tgcomplex catalyzes the disproportionation of formate to CO2and H2. FHL-2Tghas activity similar to that of theE. coliFHL-1Eccomplex in H2evolution from formate, but the complex appears to be more labile upon cell lysis. Cloning of the entire 13-kbp FHL-2Tgoperon in the heterologousE. colihost has now enabled us to unambiguously prove FHL-2Tgactivity, and it allowed us to characterize the FHL-2Tgcomplex biochemically. Although the formate dehydrogenase (FdhH) genefdhFis not contained in the operon, the FdhH is part of the complex, and FHL-2Tgactivity was dependent on the presence ofE. coliFdhH. Also, in contrast toE. coli,T. guamensiscan ferment the alternative carbon source cellobiose, and we further investigated the participation of both the H2-oxidizing Hyd-2Tgand the H2-forming FHL-2Tgunder these conditions.IMPORTANCEBiological H2production presents an attractive alternative for fossil fuels. However, in order to compete with conventional H2production methods, the process requires our understanding on a molecular level. FHL complexes are efficient H2producers, and the prototype FHL-1Eccomplex inE. coliis well studied. This paper presents the first biochemical characterization of an FHL-2-type complex. The data presented here will enable us to solve the long-standing mystery of the FHL-2Eccomplex, allow a first biochemical characterization ofT. guamensis’s fermentative metabolism, and establish this enterobacterium as a model organism for FHL-dependent energy conservation.

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Identification and biochemical characterisation of tyrosine aminotransferase from Anthoceros agrestis unveils the conceivable entry point into rosmarinic acid biosynthesis in hornworts

Planta ◽

10.1007/s00425-021-03623-2 ◽

2021 ◽

Vol 253 (5) ◽

Author(s):

Tobias Busch ◽

Maike Petersen

Keyword(s):

Rosmarinic Acid ◽

Model Organism ◽

Initial Step ◽

Tyrosine Aminotransferase ◽

Terrestrial Plants ◽

Acid Biosynthesis ◽

E Coli ◽

Biochemical Characterisation ◽

Wide Range ◽

Amino Donor

Abstract Main conclusion Tyrosine aminotransferase (AaTAT) from the hornwort Anthoceros agrestis Paton (Anthocerotaceae) was amplified and expressed in E. coli. The active enzyme is able to accept a wide range of substrates with distinct preference for l-tyrosine, therefore, possibly catalysing the initial step in rosmarinic acid biosynthesis. Abstract The presence of rosmarinic acid (RA) in the hornwort A. agrestis is well known, and some attempts have been made to clarify the biosynthesis of this caffeic acid ester in lower plants. Parallel to the biosynthesis in vascular plants, the involvement of tyrosine aminotransferase (EC 2.6.1.5; TAT) as the initial step was assumed. The amplification of a nucleotide sequence putatively encoding AaTAT (Genbank MN922307) and expression in E. coli were successful. The enzyme proved to have a high acceptance of l-tyrosine (Km 0.53 mM) whilst slightly preferring 2-oxoglutarate over phenylpyruvate as co-substrate. Applying l-phenylalanine as a potential amino donor or using oxaloacetate or pyruvate as a replacement for 2-oxoglutarate as amino acceptor resulted in significantly lower catalytic efficiencies in each of these cases. To facilitate further substrate search, two methods were introduced, one using ninhydrin after thin-layer chromatography and the other using derivatisation with o-phthalaldehyde followed by HPLC or LC–MS analysis. Both methods proved to be well applicable and helped to confirm the acceptance of further aromatic and aliphatic amino acids. This work presents the first description of a heterologously expressed TAT from a hornwort (A. agrestis) and describes the possible entry into the biosynthesis of RA and other specialised compounds in a so far neglected representative of terrestrial plants and upcoming new model organism.

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Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris

Biotechnology for Biofuels ◽

10.1186/s13068-021-01936-8 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Laura Navone ◽

Thomas Vogl ◽

Pawarisa Luangthongkam ◽

Jo-Anne Blinco ◽

Carlos H. Luna-Flores ◽

...

Keyword(s):

Pichia Pastoris ◽

Phytic Acid ◽

Disulfide Bond ◽

Wild Type ◽

Wild Type Enzyme ◽

E Coli ◽

Biochemical Characterisation ◽

Disulfide Bond Isomerase ◽

Microbial Systems ◽

Protein Disulfide

Abstract Background Phytases are widely used commercially as dietary supplements for swine and poultry to increase the digestibility of phytic acid. Enzyme development has focused on increasing thermostability to withstand the high temperatures during industrial steam pelleting. Increasing thermostability often reduces activity at gut temperatures and there remains a demand for improved phyases for a growing market. Results In this work, we present a thermostable variant of the E. coli AppA phytase, ApV1, that contains an extra non-consecutive disulfide bond. Detailed biochemical characterisation of ApV1 showed similar activity to the wild type, with no statistical differences in kcat and KM for phytic acid or in the pH and temperature activity optima. Yet, it retained approximately 50% activity after incubations for 20 min at 65, 75 and 85 °C compared to almost full inactivation of the wild-type enzyme. Production of ApV1 in Pichia pastoris (Komagataella phaffi) was much lower than the wild-type enzyme due to the presence of the extra non-consecutive disulfide bond. Production bottlenecks were explored using bidirectional promoters for co-expression of folding chaperones. Co-expression of protein disulfide bond isomerase (Pdi) increased production of ApV1 by ~ 12-fold compared to expression without this folding catalyst and restored yields to similar levels seen with the wild-type enzyme. Conclusions Overall, the results show that protein engineering for enhanced enzymatic properties like thermostability may result in folding complexity and decreased production in microbial systems. Hence parallel development of improved production strains is imperative to achieve the desirable levels of recombinant protein for industrial processes.

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Influence of Acetylcholine Esterase Inhibitors and Memantine, Clinically Approved for Alzheimer’s Dementia Treatment, on Intestinal Properties of the Mouse

International Journal of Molecular Sciences ◽

10.3390/ijms22031015 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1015

Author(s):

Vu Thu Thuy Nguyen ◽

Jason Sallbach ◽

Malena dos Santos Guilherme ◽

Kristina Endres

Keyword(s):

Calcium Influx ◽

Ex Vivo ◽

Model Organism ◽

Nmda Receptor Antagonist ◽

Acetylcholine Esterase ◽

Enteric Neurons ◽

Acetylcholine Esterase Inhibitors ◽

E Coli ◽

Esterase Inhibitors ◽

Intestinal Functions

Four drugs are currently approved for the treatment of Alzheimer’s disease (AD) by the FDA. Three of these drugs—donepezil, rivastigmine, and galantamine—belong to the class of acetylcholine esterase inhibitors. Memantine, a NMDA receptor antagonist, represents the fourth and a combination of donepezil and memantine the fifth treatment option. Recently, the gut and its habitants, its microbiome, came into focus of AD research and added another important factor to therapeutic considerations. While the first data provide evidence that AD patients might carry an altered microbiome, the influence of administered drugs on gut properties and commensals have been largely ignored so far. However, the occurrence of digestive side effects with these drugs and the knowledge that cholinergic transmission is crucial for several gut functions enforces the question if, and how, this medication influences the gastrointestinal system and its microbial stocking. Here, we investigated aspects such as microbial viability, colonic propulsion, and properties of enteric neurons, affected by assumed intestinal concentration of the four drugs using the mouse as a model organism. All ex vivo administered drugs revealed no direct effect on fecal bacteria viability and only a high dosage of memantine resulted in reduced biofilm formation of E. coli. Memantine was additionally the only compound that elevated calcium influx in enteric neurons, while all acetylcholine esterase inhibitors significantly reduced esterase activity in colonic tissue specimen and prolonged propulsion time. Both, acetylcholine esterase inhibitors and memantine, had no effect on general viability and neurite outgrowth of enteric neurons. In sum, our findings indicate that all AD symptomatic drugs have the potential to affect distinct intestinal functions and with this—directly or indirectly—microbial commensals.

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Complete biosynthesis of a sulfated chondroitin in Escherichia coli

Nature Communications ◽

10.1038/s41467-021-21692-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Abinaya Badri ◽

Asher Williams ◽

Adeola Awofiranye ◽

Payel Datta ◽

Ke Xia ◽

...

Keyword(s):

Chondroitin Sulfate ◽

Scale Up ◽

Production Methods ◽

E Coli ◽

Microbial Cell Factories ◽

Cell Factories ◽

Microbial Product ◽

Dry Cell Weight ◽

One Step ◽

Dry Cell

AbstractSulfated glycosaminoglycans (GAGs) are a class of important biologics that are currently manufactured by extraction from animal tissues. Although such methods are unsustainable and prone to contamination, animal-free production methods have not emerged as competitive alternatives due to complexities in scale-up, requirement for multiple stages and cost of co-factors and purification. Here, we demonstrate the development of single microbial cell factories capable of complete, one-step biosynthesis of chondroitin sulfate (CS), a type of GAG. We engineer E. coli to produce all three required components for CS production–chondroitin, sulfate donor and sulfotransferase. In this way, we achieve intracellular CS production of ~27 μg/g dry-cell-weight with about 96% of the disaccharides sulfated. We further explore four different factors that can affect the sulfation levels of this microbial product. Overall, this is a demonstration of simple, one-step microbial production of a sulfated GAG and marks an important step in the animal-free production of these molecules.

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Droplet- Rather than Aerosol-Mediated Dispersion Is the Primary Mechanism of Bacterial Transmission from Contaminated Hand-Washing Sink Traps

Applied and Environmental Microbiology ◽

10.1128/aem.01997-18 ◽

2018 ◽

Vol 85 (2) ◽

Cited By ~ 9

Author(s):

Shireen M. Kotay ◽

Rodney M. Donlan ◽

Christine Ganim ◽

Katie Barry ◽

Bryan E. Christensen ◽

...

Keyword(s):

Patient Care ◽

Sampling Methods ◽

Fluorescent Protein ◽

Model Organism ◽

Air Sampling ◽

Multidrug Resistant ◽

Hand Washing ◽

Content Type ◽

E Coli ◽

Green Fluorescent

ABSTRACT An alarming rise in hospital outbreaks implicating hand-washing sinks has led to widespread acknowledgment that sinks are a major reservoir of antibiotic-resistant pathogens in patient care areas. An earlier study using green fluorescent protein (GFP)-expressing Escherichia coli (GFP-E. coli) as a model organism demonstrated dispersal from drain biofilms in contaminated sinks. The present study further characterizes the dispersal of microorganisms from contaminated sinks. Replicate hand-washing sinks were inoculated with GFP-E. coli, and dispersion was measured using qualitative (settle plates) and quantitative (air sampling) methods. Dispersal caused by faucet water was captured with settle plates and air sampling methods when bacteria were present on the drain. In contrast, no dispersal was captured without or in between faucet events, amending an earlier theory that bacteria aerosolize from the P-trap and disperse. Numbers of dispersed GFP-E. coli cells diminished substantially within 30 minutes after faucet usage, suggesting that the organisms were associated with larger droplet-sized particles that are not suspended in the air for long periods. IMPORTANCE Among the possible environmental reservoirs in a patient care environment, sink drains are increasingly recognized as a potential reservoir to hospitalized patients of multidrug-resistant health care-associated pathogens. With increasing antimicrobial resistance limiting therapeutic options for patients, a better understanding of how pathogens disseminate from sink drains is urgently needed. Once this knowledge gap has decreased, interventions can be engineered to decrease or eliminate transmission from hospital sink drains to patients. The current study further defines the mechanisms of transmission for bacteria that colonize sink drains.

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Droplet rather than Aerosol Mediated Dispersion is the Primary Mechanism of Bacterial transmission from Contaminated Hand Washing Sink Traps

10.1101/392431 ◽

2018 ◽

Author(s):

Shireen Kotay ◽

Rodney M. Donlan ◽

Christine Ganim ◽

Katie Barry ◽

Bryan E. Christensen ◽

...

Keyword(s):

Patient Care ◽

Sampling Methods ◽

Model Organism ◽

Air Sampling ◽

Multidrug Resistant ◽

Hand Washing ◽

Antibiotic Resistant ◽

E Coli ◽

Potential Reservoir ◽

Healthcare Associated

ABSTRACTAn alarming rise in hospital outbreaks implicating hand-washing sinks has led to widespread acknowledgement that sinks are a major reservoir of antibiotic resistant pathogens in patient-care areas. An earlier study using a GFP-expressing Escherichia coli (GFP-E. coli) as a model organism demonstrated dispersal from drain biofilm in contaminated sinks. The present study further characterizes the dispersal of microorganisms from contaminated sinks. Replicate hand-washing sinks were inoculated with GFP-E. coli, and dispersion was measured using qualitative (settle plates) and quantitative (air sampling) methods. Dispersal caused by faucet water was captured with settle plates and air sampling methods when bacteria were present on the drain. In contrast, no dispersal was captured without or in between faucet events amending earlier theory that bacteria aerosolize from P-trap and disperse. Numbers of dispersed GFP-E. coli diminished substantially within 30 minutes after faucet usage, suggesting that the organisms were associated with larger droplet-sized particles that are not suspended in the air for long periods.IMPORTANCEAmong the possible environmental reservoirs in a patient care environment, sink drains are increasingly recognized as potential reservoir of multidrug resistant healthcare-associated pathogens to hospitalized patients. With increasing antimicrobial resistance limiting therapeutic options for patients, better understanding of how pathogens disseminate from sink drains is urgently needed. Once this knowledge gap has decreased, interventions can be engineered to decrease or eliminate transmission from hospital sink drains to patients. The current study further defines the mechanisms of transmission for bacteria colonizing sink drains.

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Synthesis of high-titer alka(e)nes in Yarrowia lipolytica is enabled by a discovered mechanism

Nature Communications ◽

10.1038/s41467-020-19995-0 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Jingbo Li ◽

Yongshuo Ma ◽

Nian Liu ◽

Bekir E. Eser ◽

Zheng Guo ◽

...

Keyword(s):

Yarrowia Lipolytica ◽

Fossil Fuels ◽

Neutral Lipids ◽

High Titer ◽

Attractive Alternative ◽

Long Distance ◽

Long Distance Transport ◽

Net Zero ◽

Distance Transport ◽

Dense Liquid

AbstractAlka(e)nes are ideal fuel components for aviation, long-distance transport, and shipping. They are typically derived from fossil fuels and accounting for 24% of difficult-to-eliminate greenhouse gas emissions. The synthesis of alka(e)nes in Yarrowia lipolytica from CO2-neutral feedstocks represents an attractive alternative. Here we report that the high-titer synthesis of alka(e)nes in Yarrowia lipolytica harboring a fatty acid photodecarboxylase (CvFAP) is enabled by a discovered pathway. We find that acyl-CoAs, rather than free fatty acids (FFAs), are the preferred substrate for CvFAP. This finding allows us to debottleneck the pathway and optimize fermentation conditions so that we are able to redirect 89% of acyl-CoAs from the synthesis of neutral lipids to alka(e)nes and reach titers of 1.47 g/L from glucose. Two other CO2-derived substrates, wheat straw and acetate, are also demonstrated to be effective in producing alka(e)nes. Overall, our technology could advance net-zero emissions by providing CO2-neutral and energy-dense liquid biofuels.

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Different responses to nitrate and nitrite by the model organism Escherichia coli and the human pathogen Neisseria gonorrhoeae

Biochemical Society Transactions ◽

10.1042/bst0340111 ◽

2006 ◽

Vol 34 (1) ◽

pp. 111-114 ◽

Cited By ~ 7

Author(s):

R.N. Whitehead ◽

J.A. Cole

Keyword(s):

Escherichia Coli ◽

Neisseria Gonorrhoeae ◽

Model Organism ◽

Anaerobic Growth ◽

Human Pathogen ◽

Regulate Gene Expression ◽

E Coli ◽

Regulatory Systems ◽

Two Component ◽

Nitrate And Nitrite

The ability of Escherichia coli to use both nitrate and nitrite as terminal electron acceptors during anaerobic growth is mediated by the dual-acting two-component regulatory systems NarX-NarL and NarQ-NarP. In contrast, Neisseria gonorrhoeae responds only to nitrite: it expresses only NarQ-NarP. We have shown that although N. gonorrhoeae NarQ can phosphorylate E. coli NarL and NarP, the N. gonorrhoeae NarP is unable to regulate gene expression in E. coli. Mutagenesis experiments have revealed residues in E. coli NarQ that are essential for nitrate and nitrite sensing. Chimaeric proteins revealed domains of NarQ that are important for ligand sensing.

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