Category Archives: NEWS

Biosynthesis of the antibiotic nonribosomal peptide penicillin in baker’s yeast

Biosynthesis of the antibiotic nonribosomal peptide penicillin in baker’s yeast

Fungi are a valuable source of enzymatic diversity and therapeutic natural products including antibiotics. Here we engineer the baker’s yeast Saccharomyces cerevisiae to produce and secrete the antibiotic penicillin, a beta-lactam nonribosomal peptide, by taking genes from a filamentous fungus and directing their efficient expression and subcellular localization. Using synthetic biology tools combined with long-read DNA sequencing, we optimize productivity by 50-fold to produce bioactive yields that allow spent S. cerevisiae growth media to have antibacterial action against Streptococcus bacteria. This work demonstrates that S. cerevisiae can be engineered to perform the complex biosynthesis of multicellular fungi, opening up the possibility of using yeast to accelerate rational engineering of nonribosomal peptide antibiotics.

Read the full article here.

Whole-cell biocatalysts by design

Whole-cell biocatalysts by design

Whole-cell biocatalysts provide unique advantages and have been widely used for the efficient biosynthesis of value-added fine and bulk chemicals, as well as pharmaceutically active ingredients. What is more, advances in synthetic biology and metabolic engineering, together with the rapid development of molecular genetic tools, have brought about a renaissance of whole-cell biocatalysis. These rapid advancements mean that whole-cell biocatalysts can increasingly be rationally designed. Genes of heterologous enzymes or synthetic pathways are increasingly being introduced into microbial hosts, and depending on the complexity of the synthetic pathway or the target products, they can enable the production of value-added chemicals from cheap feedstock. Metabolic engineering and synthetic biology efforts aimed at optimizing the existing microbial cell factories concentrate on improving heterologous pathway flux, precursor supply, and cofactor balance, as well as other aspects of cellular metabolism, to enhance the efficiency of biocatalysts. In the present review, we take a critical look at recent developments in whole-cell biocatalysis, with an emphasis on strategies applied to designing and optimizing the organisms that are increasingly modified for efficient production of chemicals.

Read the full article here.

WE NEED TO TALK ABOUT GENE TECH: CBMNet Management Board Member Prof Susan Molyneux Hodgson

 

CBMNet Management Board Member Professor Susan Molyneux Hodgson spoke at the Hay Litery festival last night in the session ‘We Need to talk about Gene Tech’.

Why does public debate and policy treat the application of genetic technology differently when we are discussing medicine and food? Why is our concept of what is ‘natural’ so controversial and the idea of GM food so alarming? Scientists and sociologists come together with Daniel Davis to discuss what’s being ventured and how it is perceived.

 

CBMNet Proof-of-Concept and Vacation Scholarship projects awarded

CBMNet Proof-of-Concept and Vacation Scholarship projects awarded

Following on from our latest Proof-of-Concept and Vacation Scholarship funding calls we are pleased to announce that we have funded 6 projects.


Proof-of-Concept: Newcastle University and Ingenza – L-form technologies: a novel platform for therapeutic protein production.

Many human proteins are used for the treatment of a wide variety of diseases and many more have the potential to be developed as drugs if they can be produced in sufficient quantities. To avoid contamination with agents (prions and viruses) that can cause serious diseases, these proteins are produced in bacterial or animal cells, rather than extraction from human tissue. There is therefore a need to produce sufficient amounts of novel human proteins for preliminary analyses and, if see to have therapeutic potential, for clinical trials. Production systems based on the bacterium, Escherichia coli, is the first choice system for producing such proteins. However, about one third of all human proteins are not capable of being synthesised in E. coli production systems and alternative systems have to be used. This particularly applied to proteins that are secreted from human cells and that have disulphide bonds in their final structure. Disulphide bonds are formed after synthesis and secretion from the cell and involve the formation of bonds between two amino acid residues (cysteine) in the protein. The collaboration between Newcastle University and Ingenza is aimed at relieving known bottlenecks in the production of therapeutic proteins by using a bacterium, Bacillus subtilis, that can be switched to a wall-less state that removes a major bottleneck to protein secretion. The project involves generating such strains and evaluating their performance under commercial biomanufacturing conditions. If successful, the strains could expand the range of therapeutic proteins available for the treatment of specific diseases.


 

Proof-of-Concept: University of Sheffield and Excivion – Viral antigen production for diagnosis and vaccine-mediated disease prevention by rational glycoengineering-mediated protein secretion in a mammalian cell factory.

The global incidence of mosquito-borne flavivirus induced disease such as dengue has increased exponentially over the last four decades. Fuelled by conditioning factors such as rapid urbanisation, demographic change, large-scale migration, and travel, dengue is now endemic in most countries of the tropics, and about 925 million people now live in urban areas that are at high risk of dengue infection. More recently Zika, another mosquito-borne virus, has been implicated in the causation of microcephaly in infants, and encephalitis in adults. In conjunction with concerns about expanding mosquito habitats, and global movement of humans on significant scales, there is a compelling need to find new solutions for the prevention this family of diseases, as well as diagnoses of extent and type of exposure, as this information has significant implications for subsequent treatment and strategies for prevention. A particular challenge in this family of diseases concerns the fact that while initial exposure gives rise to mild disease, subsequent exposure to other related viral strains can result in severe illness and death, as a consequence of known mechanisms associated with the immune system, which act to make the disease symptoms considerably worse. It follows that both specific diagnosis and new vaccine designs will be required to control these diseases effectively. This project aims to exploit specific cell factories, together with our understanding of key protein structures in flaviviruses, to generate novel non–natural proteins which will have the capability both of enabling strain-specific diagnosis, as well as inducing protection in vaccination programs of carefully screened individuals, without predisposing to haemorrhagic fever (which is a recognised risk for existing vaccine designs).


Vacation Scholarship: University of Kent – Functional characterisation of a putative succinate efflux pump from Corynebacterium glutamicum.

Succinate is a key precursor in the production of biodegradable plastics and fabrics. The majority of industrially produced succinate is derived from petrochemical precursors. However, several microbial species have been engineered to maximise succinate production during fermentation. A succinate efflux pump, SucE, was recently identified in C. glutamicum, which substantially increases succinate production when overexpressed. However, the structure, mechanism, energetics and substrate specificity of this transporter remain unknown. A comprehensive understanding of SucE’s transport mechanism could allow us to manipulate this transporter and/or it’s energy source to make succinate (and possibly other dicarboxylic acid) efflux more efficient, potentially increasing the succinate yield of C. glutamicum. This project fits perfectly within the remit of CBMNet as it is centred on understanding how an industrially important chemical is transported across the bacterial membrane. The aims of this project are to; 1) clone sucE from C. glutamicum into an E. coli expression vector, 2) optimise the expression and purification conditions, 3) assay SucE function using in vivo succinate accumulation assays, and 4) reconstitute SucE into liposomes for in vitro transport assays.


Vacation Scholarship: University of Leeds – Durable vesicles for stabilisation of membrane proteins in biotechnology

Hybrid vesicles, which combine the biofunctionality of phospholipids with the stability of block copolymer membranes, can enhance the functional durability of membrane proteins. We have demonstrated that hybrid vesicles extend the functional half-life of cytochrome bo3 from 1-2 weeks in proteoliposomes to 4-6 months in hybrids (Chem. Commun. 52, 11020, 2016). The student will aim to:
1. Successfully reproduce functional reconstitution of cytochrome bo3 into hybrid vesicles (training).
2. Characterise proton pumping by cytochrome bo3 in hybrid vesicles using a pH-sensitive fluorophore.
3. Test whether (i) using a different triblock copolymer, or (ii) protein reconstitution using SMALPs has advantages over our existing materials and protocols.
4. (If time permits) co-reconstitute cytochrome bo3 with F-ATPase to create a proto-organelle to generate ATP.


Vacation Scholarship: University of Nottingham – Purification of membrane transporters to identify topology and binding sites by mass spectrometry.

Multidrug (MDR) pump show an unusually broad substrate specificity, which is poorly understood. This lack of knowledge undermines their potential use in IBBE applications where this polyspecitify may be harnessed in engineered microorganisms. Our group has an excellent track record in working with MDR pumps and now wish to harness recent advances in protein labelling and mass spectrometry (MS) to see if we can map the polyspecific binding sites of MDR pumps using MS.


Vacation Scholarship: University of Kent – Vitamin B12 into yeast.

We will determine if Saccharomyces cerevisiae can transport vitamin B12 (cobalamin) into the cell. A recent paper concerning the production of butanol isomers suggests that this is possible (see Curr Opin Biol 2015, 33: 1-7) but we would like to provide definitive evidence and quantify the levels of cobalamin that can be accumulated. We will investigate the ability of S. cerevisiae to absorb B12 using two complementary approaches. Firstly, we will grow the yeast in the presence of a range of different concentrations of vitamin B12. After growth, the cells will be thoroughly washed and then lysed. The supernatant from the lysed cells will then be applied to a very sensitive B12-dependent microbial plate assay. This will allow the student to compare B12 uptake against external concentrations of added B12. Secondly, we have made a number of B12 fluorophores, whereby we have attached a fluorophore to the B12 mainframe. These fluorescent probes are taken up by bacteria, algae and worms. With our new confocal microscope system we will be able to follow the movement and accumulation of the fluorophore within the S. cerevisiae also.

 

Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli

Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli

The exploitation of the CRISPR/Cas9 machinery coupled to lambda (λ) recombinase-mediated homologous recombination (recombineering) is becoming the method of choice for genome editing in E. coli. First proposed by Jiang and co-workers, the strategy has been subsequently fine-tuned by several authors who demonstrated, by using few selected loci, that the efficiency of mutagenesis (number of mutant colonies over total number of colonies analyzed) can be extremely high (up to 100%). However, from published data it is difficult to appreciate the robustness of the technology, defined as the number of successfully mutated loci over the total number of targeted loci. This information is particularly relevant in high-throughput genome editing, where repetition of experiments to rescue missing mutants would be impractical. This work describes a “brute force” validation activity, which culminated in the definition of a robust, simple and rapid protocol for single or multiple gene deletions.

Read the full article here.

£25,000 Proof-of-Concept Available

£25,000 Proof-of-Concept Available

As a result of our last Proof-of-Concept call, and some under-spend, we have £25,000 of PoC funding remaining (@80%fEC). Therefore, we are inviting applications from our members for a final PoC project.

Details: To allow consortia to generate the preliminary information required to establish the feasibility of their proposed approaches, with the target of generating competitive bids to other relevant funding calls.
Important dates: CALL OPEN UNTIL 12 NOON MAY 31st 2017
Amount available: £25,000 (@80% fEC) to fund ONE project.
Further information

CBMNet goes International

We are pleased to announce that we have successfully been awarded funding from the BBSRC to expand CBMNet activities across the globe!


We are headed to New Zealand for a workshop aimed at ‘Exploiting Algae and marine biomass for Industrial Biotechnology and Bioenergy’

In August 2017, CBMNet and PHYCONET members are heading to the Cawthron Institute, Nelson, New Zealand. The workshop focuses on a deeper understanding of the bottlenecks in producing polysaccharides, other bioactives and functional food ingredients from marine biomass. We will identify the challenges in characterisation, production and commercialisation, with the aim of generating project ideas to overcome yield-restricting bottlenecks in this process. The workshop will provide a forum for leading academic and industrial practitioners to establish a strong evidence-based assessment of our current understanding of the challenges and these will be carried forward by establishing new partnerships and collaborations.


CBMNet Co-Director, Dr Gavin Thomas, is planning a UK-Taiwan exchange to understand the structure & function of bacterial transporters for Industrial Biotechnology & Bioenergy.

The transport of small molecules across bacterial membranes via active transport is an underexploited component of metabolic engineering and has great potential in improving processes in industrial biotechnology and bioenergy (IBBE). To be able to rationally improve transporter function, knowledge of the structure/function relationships within these proteins is crucial. In this network we seek support to build a UK-Taiwan network of researchers sharing expertise in the study of transporters relevant for IBBE. The main component of the collaboration is the exchange of knowledge, in terms of understanding the function and structure of membrane transport proteins, in particular in scoping new research into IBBE-relevant targets and developing new techniques and expertise using TRAP transporters. This will be supported through three main activities; A Kick-off meeting July in York, several research exchanges and a grant writing and future perspectives meeting.


We’re welcoming Canadian and European Colleagues to Establish International Partnerships in Industrial Biotechnology and Bioenergy in improved glycoform-based biopharmaceutical production in plants.

This September we are hosting a 3 day workshop which will provide a forum to facilitate collaboration between international partners in developing ‘non-traditional’ expression systems, focusing on plant and yeast cell factories, developing capabilities that can translate to existing and future platform technologies for production of biopharmaceuticals. A key goal of this meeting is to explore opportunities for collaboration and funding within the BBSRC priority funding area ‘New approaches to industrial biotechnology’. Specifically, this workshop will focus on a deeper understanding of the native glycosylation machinery and the manipulation thereof for the production of biopharmaceutical products with enhanced or even novel functions.  The programme will draw on the extensive experience in plant and yeast-based systems and approaches developed to achieve predictive modification of glycoform. Invited speakers have been chosen based on their expertise in different areas of plant and yeast biology, glycosylation, protein biochemistry and cell trafficking.  A key aim of the workshop is to develop a new network of research groups interested in industrial biotechnology and identify common research goals for responsive mode funding opportunities.


 

Microbial response to environmental stresses: from fundamental mechanisms to practical applications

Microbial response to environmental stresses: from fundamental mechanisms to practical applications

Environmental stresses are usually active during the process of microbial fermentation and have significant influence on microbial physiology. Microorganisms have developed a series of strategies to resist environmental stresses. For instance, they maintain the integrity and fluidity of cell membranes by modulating their structure and composition, and the permeability and activities of transporters are adjusted to control nutrient transport and ion exchange. Certain transcription factors are activated to enhance gene expression, and specific signal transduction pathways are induced to adapt to environmental changes. Besides, microbial cells also have well-established repair mechanisms that protect their macromolecules against damages inflicted by environmental stresses. Oxidative, hyperosmotic, thermal, acid, and organic solvent stresses are significant in microbial fermentation. In this review, we summarize the modus operandi by which these stresses act on cellular components, as well as the corresponding resistance mechanisms developed by microorganisms. Then, we discuss the applications of these stress resistance mechanisms on the production of industrially important chemicals. Finally, we prospect the application of systems biology and synthetic biology in the identification of resistant mechanisms and improvement of metabolic robustness of microorganisms in environmental stresses.

Read the full article here.

Improved n-butanol production via co-expression of membrane-targeted tilapia metallothionein and the clostridial metabolic pathway in Escherichia coli

Improved n-butanol production via co-expression of membrane-targeted tilapia metallothionein and the clostridial metabolic pathway in Escherichia coli

Background

N-Butanol has favorable characteristics for use as either an alternative fuel or platform chemical. Bio-based n-butanol production using microbes is an emerging technology that requires further development. Although bio-industrial microbes such as Escherichia coli have been engineered to produce n-butanol, reactive oxygen species (ROS)-mediated toxicity may limit productivity. Previously, we show that outer-membrane-targeted tilapia metallothionein (OmpC-TMT) is more effective as an ROS scavenger than human and mouse metallothioneins to reduce oxidative stress in the host cell.

Results

The host strain (BUT1-DE) containing the clostridial n-butanol pathway displayed a decreased growth rate and limited n-butanol productivity, likely due to ROS accumulation. The clostridial n-butanol pathway was co-engineered with inducible OmpC-TMT in E. coli (BUT3-DE) for simultaneous ROS removal, and its effect on n-butanol productivity was examined. The ROS scavenging ability of cells overexpressing OmpC-TMT was examined and showed an approximately twofold increase in capacity. The modified strain improved n-butanol productivity to 320 mg/L, whereas the control strain produced only 95.1 mg/L. Transcriptomic analysis revealed three major KEGG pathways that were significantly differentially expressed in the BUT3-DE strain compared with their expression in the BUT1-DE strain, including genes involved in oxidative phosphorylation, fructose and mannose metabolism and glycolysis/gluconeogenesis.

Conclusions

These results indicate that OmpC-TMT can increase n-butanol production by scavenging ROS. The transcriptomic analysis suggested that n-butanol causes quinone malfunction, resulting in oxidative-phosphorylation-related nuo operon downregulation, which would diminish the ability to convert NADH to NAD+ and generate proton motive force. However, fructose and mannose metabolism-related genes (fucA, srlE and srlA) were upregulated, and glycolysis/gluconeogenesis-related genes (pfkB, pgm) were downregulated, which further assisted in regulating NADH/NAD+ redox and preventing additional ATP depletion. These results indicated that more NADH and ATP were required in the n-butanol synthetic pathway. Our study demonstrates a potential approach to increase the robustness of microorganisms and the production of toxic chemicals through the ability to reduce oxidative stress.

Read the full article here.

CBMNet Sponsored Early Career Researcher wins Gordon Conference ‘Best Oral Presentation’ Prize

     

CBMNet Sponsored Early Career Researcher wins Gordon Conference ‘Best Oral Presentation’ Prize

We are pleased to announce that CBMNet member Arthur Neuberger, University of Cambridge, has just returned from the Gordon Conference on ‘Multi-Drug Efflux Systems’, where he won 1st Prize for ‘Best Oral Presentation’. CBMNet sponsored him through one of our Early Researcher Grants to attend the event.

The Gordon Research Seminar and Conference on “Multi-Drug Efflux Systems” is the most important specialists’ meeting in the field. One of the Gordon Conference policies is that presenters should be actively encouraged to show and discuss a considerable amount of unpublished data and work in progress (where possible). This makes Gordon Conferences not only the most interesting and exciting meetings in the field but also those that truly “operate” at the utmost frontier of science.

Arthur told us ”As a result of the vast interactions with my peers, I have discussed a future collaboration with Prof. Arthur Roberts (University of Georgia, USA) on functional analysis of PgP (a transporter involved in the failure of human cancer chemotherapy due to drug efflux). This was encouraged by another presentation, in which unpublished data motivated further investigation of a potential secondary activity for PgP.”

”The conference was also vital for career planning: As part of the GRS, an expert panel discussion was set up to answer career-related questions from young researchers. New this year was the “power hour”: a panel to discuss science career opportunities and challenges for female scientists. Besides science talking, conference attendees usually discussed career opportunities. In my case, a professor from Essex encouraged me to apply for a lectureship position at his university right after my PhD.”

”Both my presentation and my poster attracted much interest from both young and senior scientists. Moreover, I won the “best presentation” award for my talk.I would like to thank CBMNet for the great opportunity to attend and be part of this fantastic meeting!”

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