I-Bacillus: Adapting Bacillus licheniformis for 21st century IB applications

Lead Research Organisation: Newcastle University
Department Name: Inst for Cell and Molecular Biosciences

Abstract

The soil bacterium Bacillus licheniformis is a preferred host for the production of a variety of industrial enzymes, including proteases, amylases and cellulases for washing detergents, food processing and the production of biofuels made from sustainable plant waste materials. To advance its genetics and utility, our key objective is to deploy novel Synthetic Biology tools to improve the production of its native enzymes and those from other sources so as to improve the economics of using B. licheniformis for exploitation by commercial end users such as detergent manufacturers, food processes etc. Technologies will be developed for modifying the genome of this bacterium so as to amplify genes encoding useful enzymes, targeting them to specific locations (genomic landing pads) that are validated for high-level and predictable expression, overcoming issues associated with non-targeted integration. Novel tools for genome editing will a) address aspects of the bacterium's metabolism that limit enzyme yield of under conditions found in industrial bioreactors, as identified using systems biology techniques and b) capitalize on in situ protein engineering to improve functionalities of industrial enzymes, including thermotolerance and optimal activity under operating conditions, that important for end users. Success in these areas will reduce the cost and improve the versatility and efficiency of industrial enzyme production by B. licheniformis.

Technical Summary

Technically, the project combines systems and synthetic biology methods demonstrated in other areas and recently developed tools, alongside more traditional approaches such as biocatalysis, fermentation and process engineering to provide novel and improved upstream and downstream processes for a well proven industrial host. These are further supplemented by theory and modelling to overcome challenges of technology deployment and scale-up, also consistent with competition scope. This project will be a collaboration between a leading European Bacillus research group at Newcastle University and a leading IB SME: Ingenza. Two market-facing customers are primed to commercialise project outcomes. Success will create new jobs at Ingenza, increase the UK's competitiveness in industrial enzyme-mediated bioprocessing, and further enable the UK to deliver on environmental, social and economic benefits of IB. This Industrial Research project combines knowledge and technologies from established disciplines to translate academic research into commercial products and enables rapid and adaptable deployment of an innovative and efficient manufacturing route to a growing and diversifying IB market (i.e. industrial enzymes and biologics) that now exceeds 8% CARG. By improving bioprocess efficiency, the project will reduce dependency on oil, further de-carbonising the UK's industrial base.

Planned Impact

EU IB currently has an turnover of >60 euros bn/yr. The industrial enzymes sector is currently valued at $4.8bn (2013) with an annual growth rate of 8% and a predicted value of $7.1bn by 2018. The world demand for enzymes used in the $7b "biological" laundry detergent market amount to ~10,000 mt/yr, corresponding to a market value of ~$300 million, while the market for biowaste conversion and biofuels is likely to be even larger. Key industrial enzyme market growth constraints directly addressed by this project involve the deployment of novel technologies aimed at: a) improving industrial enzyme yield (productivity and cost efficiency), b) enhancing enzyme function (protein engineering) and c) developing versatile production host plaforms for the next generation of industrial enzymes. Given its expertise in the academic and industrial partners of this project, there is great scope to develop the UK's enzyme production capacity and associated technology/knowledge base through the more effective exploitation of novel production hosts. We aim to revolutionise industrial enzyme production, make more efficient use of production inputs and increase the performance of manufactured outputs to help develop a more sustainable bioeconomy. We will initially achieve this in collaboration with established industrial enzyme producers in emerging markets, while expanding Ingenza's own capacity for industrial enzyme production. The tools and technology so developed will be of value to other industrial enzyme producers and will contribute a reduction in the use of fossil-fuel based processes.
 
Description We have extensively developed the genetic systems for B. licheniformis, allowing this bacterium to be developed as a host for the production of heterologous protein such as industrial enzymes and human therapeutic proteins and peptides. We have developed improved gene transfer systems using both conjugation and electrotransformation techniques to deliver plasmids into the host B. licheniformis strains, as well as a series of useful vectors. We have developed two widely used reporter genes for use in B. licheniformis, the Green Fluorescent Protein (GFP) and E. coli beta-galactosidase enzyme (LacZ); the latter involved excising the existing the gene encoding the bacterium's native beta-galactosidase gene. Both reporters work well. When bacteria are used for the production of useful products, current regulation require them not encode antibiotic resistance genes. We have also adapted a system to facilitate the precise excision of antibiotic resistance gene cassettes use as selective genes during the genetic manipulation of the host cells. The so-called XerCise system (developed and used in a previous BBSRC-funded project, works extremely efficiently, leaving just a 29-base pair scar. We have been able to adapt the plasmid-based Landing Pad identification cassette developed for Bacillus subtilis for Bacillus licheniformis. The transposase associated with the Mariner transposon works very efficiently in B. licheniformis and, in a single experiment, we have been able to isolate a large number of clones in which the reporter cassette has been translocated from the unstable carrier plasmid to the chromosome. At this stage it would be normal to back-cross the transposon into the host strain to ensure that only a single copy is present. However, the electrotransformation system is not sufficiently efficient to achieve this and consequently we are using Southern blotting. As anticipated the location of the reporter cassette at different locations in the chromosome leads to different levels of expression - maximally about 5-fold. We have used a combination of arbitrary PCR and DNA sequencing to identify these locations and developed vectors to deliver genes encoding recombinant proteins to these sites. We have also isolated strains deficient in the main alkaline protease and, to be able to use the the LacZ reporter, a strain lacking the beta-galactosidase.
Exploitation Route The identification of suitable sites in the chromosome for the expression of recombinant genes can lead to up to 5-fold increases in expression via a gene amplification mechanism and this has been confined using the GFP reporter. We anticipate that rather than using random integration sites for the expression of recombinant genes, the use of landing pads selected for a combination of expression and growth characteristics will provide production benefits. This, taken together with genetic tools, expression vectors, antibiotic resistance-free gene deletion system and developed strains will allow Ingenza to exploit this technology. We have also evaluated the toxicity of the antimicrobial compounds used by Bacillus licheniformis (mainly PKs and NRPs), which has important regulatory implications.
Sectors Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description They will be used by are partner company to negotiate a contract to improve the production of an industrial enzyme.
First Year Of Impact 2018
Sector Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Title Transconjugation cloning system for Bacillus licheniformis 
Description The application of a conjugation system for the manipulation of the industrial microbe, Bacillus licheniformis. We obtained a derivative of Bacillus licheniformis DSM13 that has two restriction endonuclease genes deleted and an E. coli/Bacillus conjugative shuttle vector. For commercial applications of Bacillus species, it is general preferred to express target heterologous proteins from genes integrated into the chromosome, rather than from an autonomous plasmid. This is because, during fermentation, the enzyme production phase can last for several days during fed-batch growth. Under these conditions, plasmid-based systems tend to be much less stable. In previous work on Bacillus subtilis we showed that the site of integration can be an important determinant of the level of expression of the target gene and therefore the production of its product.We therefore developed a plasmid that could be used in Bacillus licheniformis to identify efficient chromosomal integration sites (landing pads). This plasmid has a temperature sensitive origin of replication, a kanamycin resistance gene and a modified Mariner transposon with divergently expressed lacZ and sfGgp reporter genes. The transposase gene responsible for transposition, himar1, is located on the plasmid but outside the transposon itself, which is flanked by inverse terminal repeats. As a consequence, once the transposon has been transposed from the plasmid to the chromosome, and the plasmid itself can be cured following a shift in temperature from 30°C to 50°C, no further transposition can take place and the reporter cassette is found to be immobilised at random sites on the chromosome. Clones which have just copies of the transposon in the chromosome (KanR, ErmS) and be distinguished from those that still have the plasmid (KanR, ErmR) on the basis of their antibiotic resistance profile. Clones in which the transposon has been transposed to an efficient landing pad site can be identified by the production of bright blue colonies on plates containing X-gal and IPTG, and this can be confirmed by subsequent analysis of the Gfp signal. Spontaneous removal of genes used in selection processes. Since the transposon cassette uses the E. coli lacZ gene as the reporter for the initial selection of putatively efficient landing pads, we had to generate a Bacillus licheniformis strain in which one or more of the putative ß-galactosidase genes were deleted. To do this we developed a markerless gene knockout system for B. licheniformis, based on the Xer-cise system (Bloor and Cranenburgh, 2007) that would allow the generation of multiple deletions at different chromosomal locations without accumulating resistance markers. Once the beta-galactosidase gene was deleted we were able to use the transposon cassette to identify efficient landing pads. Colonies with different intensities of blue coloration, and therefore different levels of ß-galactosidase activities were identified in X-gal plates. Fifteen colonies showing a wide range of blue intensities were chosen for quantification of their LacZ and Gfp signals. This allowed use to identify, by arbitrary primer sequencing, a number of efficient landing pads. We then developed a delivery plasmid with the following properties: o A spectinomycin gene flanked by dif sites o A strong transcription terminator o A lacI gene encoding the lactose repressor o A multiple cloning site (MCS) o An appropriately orientated Phy_spank promoter As proof of principle, the ganA1 gene was cloned into the MCS to confirm that this gene could be delivered to the correct chromosomal location and induced as required. 
Type Of Material Biological samples 
Year Produced 2019 
Provided To Others? No  
Impact The research tool will facilitate the genetic manipulation of Bacillus licheniformis and other related and difficult to transform bacteria. We are currently adapting this transconjugation system for the identification of Landing Pads, preferential sites for the cloning and expression of recombinant proteins by Bacillus licheniformis. This working is written up in draft form, ready for the preparation of a manuscript once any IP issues are resolved. 
 
Description I-Bacillus: Adapting Bacillus licheniformis for 21st century IB applications 
Organisation Ingenza Ltd
Country United Kingdom 
Sector Private 
PI Contribution The development of gene transfer, expression and secretion systems for industrial strains of Bacillus
Collaborator Contribution Landing pad technology and industrial formation expertise
Impact It is too early to repost outcomes other than the work is financed via a BBSRC/Innovate UK grant
Start Year 2016
 
Description 19th International Gram-positive Conference, Berlin 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This well-established series of meeting has been running for more than 40 years is the main meeting for researchers in the field of Bacillus and Gram-positive bacteria. Initially established for the Bacillus community, 20 years ago it was expanded to include other Gram-positive bacteria. Until recently it was organised by colleagues at the Scripps Institute at La Jolla, but due to the retirement of the organiser, a group of senior academics and industrialists in Europe, including myself, agreed to continue running this series of meetings.
Year(s) Of Engagement Activity 2017
URL https://www.bacillus-2017.de
 
Description 4th Global Synthetic Bbiology Meeting London 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Synthetic biology continues to be an exciting and rapidly developing area in the life sciences with the potential to revolutionize many aspects of society. As technologies and strategies continue to mature, this congress brings together experts in academia and industry to network and share ideas, and provides the opportunity for successful start-ups to showcase the commercial potential of synthetic biology. With a focus on healthcare and investment, this interactive meeting provides up to date information on cutting edge of research and tool development, access to case studies on drug discovery, therapeutics and technologies, and the opportunity to make connections between academics, entrepreneurs, investors and businesses.
Year(s) Of Engagement Activity 2017
URL http://www.global-engage.com/event/synthetic-biology-gene-editing/
 
Description Advances in Microbiology and Biotechnology. Lviv 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Special guest and invited speaker in this meeting aimed at the biotechnology of microorganisms. I presented a talk entitled "Bacillus subtilis as the model gram- positive industrial bacterium"
Year(s) Of Engagement Activity 2018
URL http://www.cellbiol.lviv.ua/2018/
 
Description Bacterial Protein Export Conference, Leuven 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact An invited speaker at this research meeting that brought together a high proportion of the world's leading experts on bacterial protein secretion to report on their current research. BPE2018 will provide an overview of recent exciting advances in the study of the various bacterial protein transport pathways, ranging from fundamentals to their impact on pathogenesis and biotechnology and the development of novel anti-bacterials.

We will discuss the underlying molecular mechanisms of all known and emerging protein translocation modes, the structure and assembly of secretion channels, the biogenesis of integral membrane proteins, the structural dynamics of exported proteins and their interaction with chaperones, the use of signals for targeting and specific sub-cellular localization, the regulation and energetics of protein transport. The conference will cover from fundamental bacterial genetics and biochemistry to structural biology, protein dynamics and advanced single molecule biophysics.
Year(s) Of Engagement Activity 2018
URL https://rega.kuleuven.be/bac/economou/bacterial-protein-export
 
Description CBMNet meeting Factories for advanced biomanufacturing, Sheffield 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact The imperative to revolutionise the chemicals industry by creating a sustainable bio-based future and the increasing importance of biologics in medicine pose major challenges to UK biotechnologists. The design and implementation of bespoke advanced microbial cell factories, that can reproducibly yield bio-based alternatives to the chemicals that underpin so much of modern infrastructure is a fundamental challenge. Chassis engineering represents the single most critical technology to revolutionise biomanufacturing by improving product yields, simplifying product recovery and improving sustainability through reduced materials use and waste, thereby enhancing process economics and commercial viability. With a strong research base already working on microbial chassis engineering, CBMNet is the natural progenitor for such an event centred on microbial chassis design.
Year(s) Of Engagement Activity 2017
URL https://cbmnetnibb.group.shef.ac.uk/members-forum/event-reports/december-2017-factories-for-advanced...
 
Description Cesar Meeting on Antibiotic Resistance Sveti Martin 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This meeting is part of the very active Croatian Microbiology Society. I gave a talk relating to the issue of non-expressed antibiotic resistance genes in Bacillus species and the regulatory implications in relation to release into the environment.
Year(s) Of Engagement Activity 2018
URL https://fems-microbiology.org/opportunities/central-european-symposium-antimicrobials-antimicrobial-...
 
Description European Bacillus meeting and Symposium on Central Carbon Metabolism, Paris 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This is a long-standing series of meeting that I helped to establish in the 1990s and are still organised on an annual basis. I organised the meeting in Newcastle in 2015. The mission of the meeting is to bring together researchers from the European Bacillus community as well as members from several European consortia that perform fundamental and applied research on Bacillus subtilis and related Gram-positive bacteria.
Year(s) Of Engagement Activity 2016
URL https://symposium.inra.fr/bacell2016/
 
Description European Bacillus meeting, Bath 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This was the latest meeting of the European Bacillus community (BACELL), that I helped to establish in 1990s. It particularly provided young researchers the opportunity to discuss their research work
Year(s) Of Engagement Activity 2018
URL https://www.facebook.com/events/2017588131854888/
 
Description Meeting of FEFANA (EU Association of Specialty Feed Ingredients and their Mixtures) concerning draft revised regulations from the European Food Standards Authority (EFSA) 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact The aim of this meeting was to provide a detailed response to the EFSA document entitled "Guidance on the characterisation of microorganisms 1 used as feed additives or as production organisms". My remit was to lead the response to EFSA in relation to antibiotic resistance genes.
Year(s) Of Engagement Activity 2017
URL https://www.wired-gov.net/wg/news.nsf/articles/Guidance+on+characterisation+of+microorganisms+used+a...
 
Description Research presentation at the 9th Recombinant Protein Production (RPP9) meeting, Croatia 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact RPP9 is part of a long-established series of meeting organised by the European Federation of Biotechnology. It beings together academic researchers and industrialists involved in the production of recombinant proteins.
Year(s) Of Engagement Activity 2017
URL https://fems-microbiology.org/opportunities/recombinant-protein-production-9-comparative-view-host-p...
 
Description SynGen - Genomics and Synthetic Biology Conference, London 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact The SynGen Series UK, with over 600 senior-level delegates representing internationally renowned research & academic institutions, clinical research institutions and pharmaceutical companies. Over 20 case studies and presentations demonstrating the latest synthetic biology tools and their therapeutic applications, including 2 interactive streams:
Synthetic Biology - Tool Development
Synthetic Biology - Applications
Co-located with the highly established Annual Next Generation Sequencing and Clinical Diagnostics Congress, Annual Single Cell Analysis Congress and the Annual Genome Editing Congress.
Invited talk on The Bacillus Cell Factory: Recent advances in novel tool development and session chair
Year(s) Of Engagement Activity 2018
URL https://www.oxfordglobal.co.uk/syntheticbiology-congress/