In silico engineering of Pseudomonas metabolism reveals new biomarkers for increased biosurfactant production

Annalisa Occhipinti​, Filmon Eyassu​, Thahira J. Rahman, Pattanathu K. S. M. Rahman, Claudio Angione​ (2018)

Background: Rhamnolipids, biosurfactants with a wide range of biomedical applications, are amphiphilic molecules produced on the surfaces of or excreted extracellularly by bacteria including Pseudomonas aeruginosa. However, Pseudomonas putida is a non-pathogenic model organism with greater metabolic versatility and potential for industrial applications.

Methods: We investigate in silico the metabolic capabilities of P. putida for rhamnolipids biosynthesis using statistical, metabolic and synthetic engineering approaches after introducing key genes (RhlA and RhlB) from P. aeruginosa into a genome-scale model of P. putida. This pipeline combines machine learning methods with multi-omic modelling, and drives the engineered P. putida model toward an optimal production and export of rhamnolipids out of the membrane.

Results: We identify a substantial increase in synthesis of rhamnolipids by the engineered model compared to the control model. We apply statistical and machine learning techniques on the metabolic reaction rates to identify distinct features on the structure of the variables and individual components driving the variation of growth and rhamnolipids production. We finally provide a computational framework for integrating multi-omics data and identifying latent pathways and genes for the production of rhamnolipids in P. putida.

Conclusions: We anticipate that our results will provide a versatile methodology for integrating multi-omics data for topological and functional analysis of P. putida toward maximization of biosurfactant production.

Supported by CBMNet Business Interaction Vouchers and Proof-of-Concept Funding

Structural basis of malodour precursor transport in the human axilla

Gurdeep S Minhas, Daniel Bawdon, Reyme Herman, Michelle Rudden, Andrew P Stone, A Gordon James, Gavin H Thomas, Simon Newstead (2018)

Mammals produce volatile odours that convey different types of societal information. In Homo sapiens, this is now recognised as body odour, a key chemical component of which is the sulphurous thioalcohol, 3-methyl-3-sulfanylhexan-1-ol (3M3SH). Volatile 3M3SH is produced in the underarm as a result of specific microbial activity, which act on the odourless dipeptide-containing malodour precursor molecule, S-Cys-Gly-3M3SH, secreted in the axilla (underarm) during colonisation. The mechanism by which these bacteria recognise S-Cys-Gly-3M3SH and produce body odour is still poorly understood. Here we report the structural and biochemical basis of bacterial transport of S-Cys-Gly-3M3SH by Staphylococcus hominis, which is converted to the sulphurous thioalcohol component 3M3SH in the bacterial cytoplasm, before being released into the environment. Knowledge of the molecular basis of precursor transport, essential for body odour formation, provides a novel opportunity to design specific inhibitors of malodour production in humans.

Supported by a CBMNet Business Interaction Voucher

Tripartite ATP-Independent Periplasmic (TRAP) Transporters and Tripartite Tricarboxylate Transporters (TTT): From Uptake to Pathogenicity

Leonardo T. Rosa, Matheus E. Bianconi, Gavin H. Thomas, David J. Kelly (2018)

The ability to efficiently scavenge nutrients in the host is essential for the viability of any pathogen. All catabolic pathways must begin with the transport of substrate from the environment through the cytoplasmic membrane, a role executed by membrane transporters. Although several classes of cytoplasmic membrane transporters are described, high-affinity uptake of substrates occurs through Solute Binding-Protein (SBP) dependent systems. Three families of SBP dependant transporters are known; the primary ATP-binding cassette (ABC) transporters, and the secondary Tripartite ATP-independent periplasmic (TRAP) transporters and Tripartite Tricarboxylate Transporters (TTT). Far less well understood than the ABC family, the TRAP transporters are found to be abundant among bacteria from marine environments, and the TTT transporters are the most abundant family of proteins in many species of β-proteobacteria. In this review, recent knowledge about these families is covered, with emphasis on their physiological and structural mechanisms, relating to several examples of relevant uptake systems in pathogenicity and colonization, using the SiaPQM sialic acid uptake system from Haemophilus influenzae and the TctCBA citrate uptake system of Salmonella typhimurium as the prototypes for the TRAP and TTT transporters, respectively. High-throughput analysis of SBPs has recently expanded considerably the range of putative substrates known for TRAP transporters, while the repertoire for the TTT family has yet to be fully explored but both types of systems most commonly transport carboxylates. Specialized spectroscopic techniques and site-directed mutagenesis have enriched our knowledge of the way TRAP binding proteins capture their substrate, while structural comparisons show conserved regions for substrate coordination in both families. Genomic and protein sequence analyses show TTT SBP genes are strikingly overrepresented in some bacteria, especially in the β-proteobacteria and some α-proteobacteria. The reasons for this are not clear but might be related to a role for these proteins in signaling rather than transport.

An output from the CBMNet event: Import and Export of Small Molecules for Biocatalysis, September 12-13th 2017, Edinburgh

A reconstitution method for integral membrane proteins in hybrid lipid-polymer vesicles for enhanced functional durability

Rashmi Seneviratne, Sanobar Khan, Ellen Moscrop, Michael Rappolt, Stephen P. Muench, Lars J.C. Jeuken, Paul A. Beales (2018)

Hybrid vesicles composed of lipids and block copolymers hold promise for increasing liposome stability and providing a stable environment for membrane proteins. Recently we reported the successful functional reconstitution of the integral membrane protein cytochrome bo3 (ubiquinol oxidase) into hybrid vesicles composed of a blend of phospholipids and a block copolymer (PBd-PEO). We demonstrated that these novel membrane environments stabilise the enzymes’ activity, prolonging their functional lifetime [Chem. Commun. 52 (2016) 11020–11023]. This approach holds great promise for applications of membrane proteins where enhanced durability, stability and shelf-life will be essential to creating a viable technology. Here we present a detailed account of our methods for membrane protein reconstitution into hybrid vesicles and discuss tips and challenges when using block copolymers compared to pure phospholipid systems that are more common materials for this purpose. We also extend the characterisation of these hybrid vesicles beyond what we have previously reported and show: (i) hybrid membranes are less permeable to protons than phospholipid bilayers; (ii) extended enzyme activity data is presented over a period of 500 days, which fully reveals the truly remarkable enhancement in functional lifetime that hybrid vesicles facilitate.

From CBMNet Vacation Scholarship: Durable vesicles for stabilisation of membrane proteins in biotechnology

Part by part: synthetic biology parts used in solventogenic Clostridia

Gyulev IS, Willson BJ, Hennessy RC, Krabben P, Jenkinson ER, Thomas GH (2018)

The solventogenic Clostridia are of interest to the chemical industry because of their natural ability to produce chemicals such as butanol, acetone and ethanol from diverse feedstocks. Their use as whole cell factories presents multiple metabolic engineering targets that could lead to improved sustainability and profitability of Clostridium industrial processes. However, engineering efforts have been held back by the scarcity of genetic and synthetic biology tools. Over the last decade, genetic tools to enable transformation and chromosomal modifications have been developed, and the lack of a broad palette of synthetic biology parts remains one of the last obstacles to the rapid engineered improvement of these species for bioproduction. We have systematically reviewed existing parts that have been used in the modification of solventogenic Clostridia and reveal particular categories where increased fundamental knowledge is needed to fully develop reliable parts, such as promoters, transcriptional terminators and ribosome binding sites. We also aim to define areas where improved toolboxes are needed in these industrially important bacteria.

From CBMNet Project DeTox

General calibration of microbial growth in microplate readers

Keiran Stevenson, Alexander F. McVey, Ivan B. N. Clark, Peter S. Swain, Teuta Pilizota (2016)

Optical density (OD) measurements of microbial growth are one of the most common techniques used in microbiology, with applications ranging from studies of antibiotic efficacy to investigations of growth under different nutritional or stress environments, to characterization of different mutant strains, including those harbouring synthetic circuits. OD measurements are performed under the assumption that the OD value obtained is proportional to the cell number, i.e. the concentration of the sample. However, the assumption holds true in a limited range of conditions, and calibration techniques that determine that range are currently missing. Here we present a set of calibration procedures and considerations that are necessary to successfully estimate the cell concentration from OD measurements.

Including reference to CBMNet funded Vacation Scholarship and Proof-of-Concept projects

Dynamics of Escherichia coli’s passive response to a sudden decrease in external osmolarity

Renata Buda, Yunxiao Liu, Jin Yang, Smitha Hegde, Keiran Stevenson, Fan Bai, Teuta Pilizota (2016)

Mechanosensation is central to life. Bacteria, like the majority of walled cells, live and grow under significant osmotic pressure. By relying on mechanosensitive regulation, bacteria can adapt to dramatic changes in osmotic pressure. Studying such mechanical sensing and control is critical for understanding bacterial survival in a complex host and natural environment. Here, we investigate the fundamental design principles of Escherichia coli’s passive mechanosensitive response to osmotic downshocks by implementing single-cell high-resolution imaging. We explain the observed cell volume changes by modeling flux of water and solutes across the cell membrane. A better characterization of bacterial mechanosensitive response can help us map their reaction to environmental threats.

Including reference to CBMNet funded Vacation Scholarship and Proof-of-Concept projects

Biosurfactants – E-book (Frontiers in Microbiology)

Pattanathu K.S.M. Rahman (2015)

Covers a compilation of original research articles, reviews and research commentary submitted by researchers enthusiastically working in the field of biosurfactants and highlights recent advances in our knowledge of the biosurfactants and understanding of the biochemical and molecular mechanisms involved in their production, scale-up and industrial applications. There are 11 manuscripts accepted for publication in this research topic contributed by 55 authors from UK, Denmark, Greece, Germany, South Africa, India, Brazil, Bahrain, Portugal, and China.

Including reference to CBMNet funded Proof-of-Concept project ‘Plants as Nanoparticle Producers’