Systems-level engineering and characterisation of Clostridium autoethanogenum through heterologous production of poly-3-hydroxybutyrate (PHB)

Lemgruber, Renato de Souza Pinto; Valgepea, Kaspar; Tappel, Ryan C.; Behrendorff, James B. Y. H.; Palfreyman, Robin W.; Plan, Manuel R.; Hodson, Mark P.; Simpson, Séan D.; Nielsen, Lars K.; Köpke, Michael; Marcellin, Esteban

doi:10.1016/j.ymben.2019.01.003

Cited by 60 publications

(34 citation statements)

References 67 publications

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“…All measurements were performed in triplicate. To analyse the PHB content of the whole-cells and purified PHB nanobeads, the lyophilised samples were processed and subjected to crotonic analysis as previously described [42].…”

Section: Preparation and Characterisation Of Phb Nanobeadsmentioning

confidence: 99%

Electrochemical Detection of Global DNA Methylation Using Biologically Assembled Polymer Beads

Soda

Gonzaga

Pannu

et al. 2021

Cancers

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DNA methylation is a cell-type-specific epigenetic marker that is essential for transcriptional regulation, silencing of repetitive DNA and genomic imprinting. It is also responsible for the pathogenesis of many diseases, including cancers. Herein, we present a simple approach for quantifying global DNA methylation in ovarian cancer patient plasma samples based on a new class of biopolymer nanobeads. Our approach utilises the immune capture of target DNA and electrochemical quantification of global DNA methylation level within the targets in a three-step strategy that involves (i) initial preparation of target single-stranded DNA (ss-DNA) from the plasma of the patients’ samples, (ii) direct adsorption of polymer nanobeads on the surface of a bare screen-printed gold electrode (SPE-Au) followed by the immobilisation of 5-methylcytosine (5mC)-horseradish peroxidase (HRP) antibody, and (iii) immune capture of target ss-DNA onto the electrode-bound PHB/5mC-HRP antibody conjugates and their subsequent qualification using the hydrogen peroxide/horseradish peroxidase/hydroquinone (H2O2/HRP/HQ) redox cycling system. In the presence of methylated DNA, the enzymatically produced (in situ) metabolites, i.e., benzoquinone (BQ), binds irreversibly to cellular DNA resulting in the unstable formation of DNA adducts and induced oxidative DNA strand breakage. These events reduce the available BQ in the system to support the redox cycling process and sequel DNA saturation on the platform, subsequently causing high Coulombic repulsion between BQ and negatively charged nucleotide strands. Thus, the increase in methylation levels on the electrode surface is inversely proportional to the current response. The method could successfully detect as low as 5% methylation level. In addition, the assay showed good reproducibility (% RSD ≤ 5%) and specificity by analysing various levels of methylation in cell lines and plasma DNA samples from patients with ovarian cancer. We envision that our bioengineered polymer nanobeads with high surface modification versatility could be a useful alternative platform for the electrochemical detection of varying molecular biomarkers.

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Section: Preparation and Characterisation Of Phb Nanobeadsmentioning

confidence: 99%

Electrochemical Detection of Global DNA Methylation Using Biologically Assembled Polymer Beads

Soda

Gonzaga

Pannu

et al. 2021

Cancers

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“…With pathway enzymes identified, we next optimized our production strains for increased titers. Specifically, we used a genome-scale model 16,39,40 and evolutionary algorithm 41,42 to predict gene KOs that would increase flux to acetone and eliminate unwanted byproducts, particularly 2,3-butanediol (2,3-BDO) and 3-HB. These byproducts have high boiling points and would accumulate during continuous fermentation with a recycle loop, adding separation costs.…”

Section: Strain Optimizationmentioning

confidence: 99%

Carbon-negative production of acetone and isopropanol by gas fermentation at industrial pilot scale

et al. 2022

Self Cite

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Many industrial chemicals that are produced from fossil resources could be manufactured more sustainably through fermentation. Here we describe the development of a carbon-negative fermentation route to producing the industrially important chemicals acetone and isopropanol from abundant, low-cost waste gas feedstocks, such as industrial emissions and syngas. Using a combinatorial pathway library approach, we first mined a historical industrial strain collection for superior enzymes that we used to engineer the autotrophic acetogen Clostridium autoethanogenum. Next, we used omics analysis, kinetic modeling and cell-free prototyping to optimize flux. Finally, we scaled-up our optimized strains for continuous production at rates of up to ~3 g/L/h and ~90% selectivity. Life cycle analysis confirmed a negative carbon footprint for the products. Unlike traditional production processes, which result in release of greenhouse gases, our process fixes carbon. These results show that engineered acetogens enable sustainable, high-efficiency, high-selectivity chemicals production. We expect that our approach can be readily adapted to a wide range of commodity chemicals.

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“…This would entail glucose expenditure on N-lean biomass as a carbon sink. This carbon sink could consist of intracellular polymeric substances (IPS), such as ATP-independent poly-3-hydroxybutyrate [27], and/or EPS. This competing process was deemed a challenge for future experiments.…”

Section: Diazotrophic Growthmentioning

confidence: 99%

Diazotrophic Behaviour in a Non-Sterile Bioreactor: The Effect of O2-Availability

et al. 2021

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The behaviour of a locally isolated diazotrophic consortium was investigated with the prospect of agricultural applications. A repeatable culture was obtained in a non-sterile bioreactor. Metagenomic analysis indicated Chryseobacterium ssp. and Flavobacterium ssp. were the dominant species, making up approximately 50% of the microbial community. The oxygen supply was varied and mass-transfer limited growth was attained under all experimental conditions. Negligible amounts of aqueous metabolites were formed, indicating a high selectivity towards biomass production. High oxygen availability resulted in decreased growth efficiencies i.e., the specific energy requirements for biomass synthesis. This was attributed to reduced electron transport chain efficiencies and nitrogenase protection mechanisms. Mass and energy balances indicated that sessile biomass with a high C:N served as a carbon sink. The most efficient growth was measured at an aeration feed composition of 21% oxygen and 79% nitrogen. The study presents one of the only known investigations of operational conditions on diazotrophic growth in a non-sterile bioreactor. In addition, it provides a strong foundation for the development of a Biological Nitrogen Fixation process with scaling potential.

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Systems-level engineering and characterisation of Clostridium autoethanogenum through heterologous production of poly-3-hydroxybutyrate (PHB)

Abstract: Systems-level engineering and characterization of Clostridium autoethanogenum through heterologous production of poly-3-hydroxybutyrate (PHB), Metabolic Engineering,

Cited by 60 publications

References 67 publications

Electrochemical Detection of Global DNA Methylation Using Biologically Assembled Polymer Beads

Electrochemical Detection of Global DNA Methylation Using Biologically Assembled Polymer Beads

Carbon-negative production of acetone and isopropanol by gas fermentation at industrial pilot scale

Diazotrophic Behaviour in a Non-Sterile Bioreactor: The Effect of O2-Availability

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