Cofund on Biotechnologies

Critical Enzymes for Sustainable Biofuels from Cellulose

Results of CESBIC (Presentation)

Project coordinator:  Prof. Paul Howard Walton & Prof. Gideon John Davies - Department of Chemistry, University of York - UK

Project leaders:

Summary

Bioethanol produced from cellulose has the potential to transform the future of biofuels. It carries major and unique advantages in terms of carbon footprint, energy efficiency, use of low-value resources including waste, and economic viability. However, these advantages cannot be realised until an efficient means of overcoming the chemical recalcitrance of cellulose can be found.
In a recent (2011) major breakthrough it was shown that certain fungal enzymes are unprecedented copper-containing oxidases which have the capacity to breakdown cellulose into its constituent sugars. These enzymes have become the centre of worldwide attention as they likely hold the key to making cellulosic bioethanol a reality.
This proposal seeks to provide industry with an in-depth understanding of these enzymes, such that their commercial use can be maximised. It objectives are to:

• use modern genomics to catalogue the full range of cellulose-disrupting enzymes produced by fungi
• undertake a full structural, activity and mechanistic study of the enzymes
• perform pilot scale tests on the industrial production of the enzymes, and—critically—examine their efficacy in an industrial setting

Our expected results are a complete understanding of the enzymes‘ mechanism of action and a deep appreciation of their structural and functional aspects, thus creating a world-leading knowledge base. We also expect to have curated and made available a much expanded genomic database of these enzymes, including links to their activities and structures—i.e. a single web-based resource for both academic and industrial researchers. And, finally, we expect to have supported strongly European enzyme-producing industry through up-to-date knowledge on enzyme function and insight into the most promising enzymatic candidates for commercialisation.
Exploitation will be both direct and indirect. In the direct sense, the project has an industrial partner who will immediately take results from the project into an industrial context and undertake assessment trials. Indirectly the project aims to benefit the wider European and global bioethanol community through the CAZy database by the cataloguing and publication of integrated genomic, structural and activity data for this class of enzymes.

Overcoming metabolic stochasticity and population dynamics in microbial cell factories

Results of CONTIbugs (Presentation)

Project coordinator:  Dr. Katja Bühler and Prof. Dr. Andreas Schmid - Laboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering, TU Dortmund University - Germany

Project leaders:

Summary

The efficiency of industrial whole-cell production processes is most often afflicted by the formation of subpopulations in a microbial culture during biotransformations and fermentations. A bioreactor running in batch, fed-batch or continuous mode can be regarded as an artificial environment that is permanently changing [CurrOpMicrobiol 2000, 3:248] and thus creating a diversity of functional microhabitats which ultimately lead to the emergence of various pheno- and genotypes [BiotechBioeng 2010, 105:705]. Phenotypic heterogeneity and variability represents a key -and unwanted feature in the bacterial population that constitutes the biological catalyst. This project will investigate the phenomena of metabolic stochasticity and population dynamics in microbial cell factories using Pseudomonas sp. growing in suspended culture, as well as attached to surfaces as
catalytic biofilms. Biofilms are resilient to a wide variety of environmental stresses. This inherited robustness make biofilms desirable as potent biocatalysts, especially regarding reactions involving biological difficult substrates and/or products [TrendsBiotechnol 2009, 27:636].

This project addresses the phenomenon of catalytic heterogeneity of genetically identical bacterial cells using a controllable system composed of non-pathogenic Pseudomonas strains as host of reference. The biosynthesis of the short chain alcohol isobutanol will be employed as a model reaction system. On the technical side, cultivation systems and molecular tools will be developed for analyzing catalytic heterogeneity in bacterial cultures under process conditions (i). Following identification of process relevant subpopulations (ii), the responsible signals and molecular mechanisms controlling the formation of the respective mutations will be identified (iii) and strategies to guide phenotypic and genotypic heterogeneity throughout an entire population will be developed (iv). The main outcome of this project is a collection of Pseudomonas strains (CONTIbugs) optimized as cell factories for hosting and stably expressing heterologous genes and maintaining productive catalytic activities in controlled populations for industrial biotechnology.

A Synthetic Biology Platform for the Optimization of Enzymic Biomass Processing

Results of Cellulect (Presentation)

Project coordinator:  Dr. Alistair Elflick - University of Edinburgh - UK

Project leaders:

Summary

IB Motivation
This proposal fits perfectly with the ERA-IB topics:

• Improved enzyme systems for new and more efficient bioprocesses;
• Biological processing (including separation and conversion) of biomass, including from side streams, and other renewable carbon sources into value added products.

Expected Results
The work proposed demonstrates novel technologies to screen biomass feedstock against a diverse library of enzymes, combined in thousands of discrete configurations using engineered microbes, to determine which gene combinations and concerted enzymatic activities give optimal degradation of diverse biomass sources. These combinations may then serve as a starting point to generate further valuable combinations in an iterative evolutionary process. The project output will create value for many end-users by improving the yield of biomass conversion to usable feedstock and will be applied to immediate industrial targets. In addition to enabling the optimisation of enzyme blends for any given application, analysis of the results will allow the team to develop heuristics that facilitate more rational design of biomass processing systems in future, leading to a deeper understanding of biomass degradation. The project results will also demonstrate the value of GSA, a novel combinatorial genetic approach that can greatly accelerate the normally time-consuming process ofachieving efficient, gene expression and concerted action of industrial enzymes.

Exploitation
The technology developed will be commercialised by Ingenza. There are a number of routes to value extraction including: i.) Biomanufacture and direct sale of the enzyme blend to biorefinery operators ii.) Licensing of microbes adapted for biomass usage to current manufacturing partners iii.) Provision of a contract service to customers wishing to have a bespoke digestion chassis implemented for the particular biomass of interest.

Improved Cellulosomes to Enhance Saccharification of Industrially-Suitable Lignocellulosic Biomass Residues

Results of FiberFuel (Presentation)

Project coordinator:  Staff scientist Mariano Carrión-Vázquez - Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC), Madrid - Spain

Project leaders:

1) The research in Poland is supported by the ERA-NET-IB/06/2013 Grant funded by the National Centre for Research and Development.

Summary

FiberFuel targets the rational design of optimized designer cellulosomes (DCs: cellulolytic enzyme systems based on a scaffolding protein) to overcome the major bottleneck in biomass industrial processing, namely saccharification (the conversion of cellulosic biomass to fermentable sugars). The goal is to improve the efficiency of the saccharafication process from low-value raw biomass materials (all of them renewable, sustainable and inexpensive) to produce industrial-value chemicals. Our cross-disciplinary approach includes bio-nanotechnology, structural biology, labon-a-chip and modeling.

Objectives

1.  Characterization of natural cellulosomes and candidate substrates. FiberFuel will produce the basic components of natural cellulosomes as well as other lignocellulosic enzymes and characterize two industrial substrates of interest (wheat straw and corn stover) to understand the architecture, nanomechanics and catalytic properties of cellulosomes, and the logic behind their construction.
2. Multi-scale modeling of the cellulosome for in silico knowledge integration. This will provide crucial support for the synthesis, assembly and characterization tasks, supplying detailed structural and energetic information to aid designing and interpreting experiments.
3. Rational design of DCs. Integration of acquired knowledge from 1 and 2 into DCs and subsequent activity screening will allow constructing DCs optimized for degrading selected industrial substrates, and validated at the laboratory scale.

Expected results

1.  A standard activity assay to monitor cellulosic substrate degradation.
2. Test of the mechanical hypothesis of the cellulosome.
3. Supramolecular DC structure characterized at the atomic and molecular levels.
4. Molecular and supra-molecular models of DCs.
5. Optimized DCs obtained based on rational design for the specific industrial substrates.

Exploitation
FiberFuel is very likely to generate new intellectual property covering the optimized
cellulosome derivatives and processes for biomass degradation into fermentable sugars. Both the
standard activity assay and DCs produced will be patented, which will be used and exploited by
ABNT and DesEn. The developed technology will be unique and as such is not expected to
conflict with other intellectual property.

Integrative Approach to Promote Hydroxylations with Novel P450 Enzymes for Industrial Processes

Results of HyPerIn (Presentation)

Project coordinator:  Prof. Vlada B. Urlacher - Heinrich-Heine University Düsseldorf - Germany

Project leaders:

Summary

Functionalization of non-activated C-H bonds is one of the major challenges in chemistry yet this reaction-type is crucially important for the initial activation of simple starting molecules. Hydroxylation of C-H bonds can lead directly to the formation of high value chiral compounds in demand as specialty chemicals and pharmaceutical synthons. Cytochrome P450 enzymes remain unsurpassed in their targeted specificity and scope. Consequently, the application of P450 enzymes in synthetic organic chemistry is considered as “potentially the most useful of all biotransformations”. Despite this potential, the application of P450 reactions in industry has been hampered by several technical bottlenecks widely recognised as:

• access to P450 enzymes with regio- and stereoselective activity on diverse target compounds
• biocatalysts with high activity and sufficient operational stability
• efficient redox partner interaction
• biotransformation processes operating at high substrate concentrations
• biotransformation processes allowing sufficient oxygen supply.

In order to overcome these limitations a high level of multidisciplinary collaboration is required. This project addresses these bottlenecks by integrating expertise in novel enzyme recruitment, enzyme engineering, whole cell biocatalyst optimization, process development and scale-up. Each partner brings in leading techniques and develops them further to specifically address P450 processes. Examples include transcriptome analysis to identify active P450 genes, bioinformatics guided enzyme engineering to boost enzyme optimization, and high-throughput process development to optimize reactions and facilitate scale-up. The project will initially characterize a wild-type strain collection and then establish a platform for identification and optimization of new recombinant P450 expression systems. A particular focus will be set on the integration of high throughput microscale process development methods with biocatalyst development. This particular strength of the consortium is considered to be a necessary requirement to bring P450 systems into preparative scale. The project will illustrate the applicability of the developed systems at lab-scale for several pharmaceutical targets and at industrial scale with targets from fine chemistry and pharma. The suite of novel enzymes, host and process development platforms created will have broad impact across the fine chemistry, flavours, fragrances and pharma industry sectors. The overall aim of the project is thus to provide platforms of P450 biocatalysts for a range of target substrates with proven applications at industrial scale.

MICROscale downstream processing TOOLbox for Screening and process development

Results of MICROTOOLS (Presentation)

Project coordinator:  Dr Nicolas Szita - University College London (UCL) - United Kingdom

Project leaders:

Summary

Biocatalysis can replace traditional chemical catalysis based procesess resulting in a greener production process. However, the reaction step of such a biocatalysis process needs to be integrated with one or several purification steps to recover products and/or remove inhibitory substances. The economical feasibility of a bio-based process is typically dependent to a large extent on the efficiency and the cost of the subsequent separation steps. Consequently, a series of separation process candidates have to be investigated with the purpose of achieving the most efficient downstream processing configuration. The more rapidly such an investigation can be conducted, the faster critical decisions about economical feasibility of a bio-based process can be taken, and the faster one can bring a product on the market.  The main objective of this project is to establish a microscale downstream processing toolbox which can be used for rapid and high content screening or for process development. 
The following main results are expected to be obtained:   

• New and existing building blocks of a miniaturised downstream processing toolbox will be developed, standardized and evaluated. The toolbox will include separation processes based on extraction, pervaporation, adsorption, absorption and membrane technology.    
• The toolbox will be supplemented by advanced on-line measurements and rational experimentation protocols for rapid and accurate generation of data for downstream process characterization and development..  
• Scaling-up of the results obtained with the miniaturised downstream processing toolbox will be evaluated on the basis of lab-scale and pilot-scale experiments.  
• Separation process sequences will be developed for two challenging and industrially relevant case studies (transketolase, transaminase) in order to demonstrate the practical applicability of the miniaturised downstream processing toolbox.

At the end of the project, the toolbox will be useful for biocatalysis process development in general. Thus, the application potential is enormous, for example in view of the current focus on integrating biocatalysis in traditional chemical reaction sequences. Indeed, due to the relatively small scale of the equipment, the toolbox will be useful and affordable both for industry and academia.

Novel industrial bioprocesses for production of key valuable steroid precursors from phytosterol

Results of MySterI (Presentation)

Project coordinator:  Dr. Carlos Barreiro - Asociación de investigación-INBIOTEC-Instituto de Biotecnología de León - Spain

Project leaders:

Summary

Project MySterI (Mycobacterial Steroids for Industry) aims to produce high value steroid precursors using a novel bioconversion strategy resulting in lower production costs and less cost to the environment. The aim is to convert phytosterols in cheap waste plant material to desired steroid precursors with engineered strains of fast-growing, saprophytic mycobacteria in single fermentation steps. The bioconversion of phytosterols has not been widely adopted in the biotechnology industry because of problems with microbial strains, process efficiency and therefore poor yield. At the heart of project MySterI is the bioconversion of phytosterols to 3β-hydroxyandrost-5-ene-17-one (DHEA) or to androst-4-ene-3,17-dione (AD) (intermediary precursor) and then to 11α-hydroxyandrost-4-ene-3,17-dione (11-α-OH-AD) and testosterone. To achieve this goal, project MySterI has the following objectives

• Genome sequencing and annotation of Mycobacterium sp. NRRL B-3805 (AD-producer) to identify key bioconversion genes and to enable ‘omics tools.
• Understanding of phytosterol bioconversion by means of ‘omics technologies
• Development of the genetic engineering tools for Mycobacterium sp. NRRL B-3805
• Construction of mycobacterial strains capable of producing 11-α-OH-AD, DHEA and testosterone.
• Designing more efficient and eco-friendly methods of production and downstream processing for the three selected compounds.

Expected results
The outputs from MySterI will be: i) novel strains capable of producing three valuable C19-steroid precursors from phytosterol ii) knowledge of the biochemistry of steroid biotransformations iii) optimized fermentation and eco-friendly downstream processes for the single-step production of steroids precursors using Mycobacterium sp. NRRL B-3805.

Exploitation
Efficient, cheap and environmentally clean production of intermediate steroids will confer a highly competitive status to Gadea Pharmaceutical Group and Pharmins Ltd. (industrial partners) in the regulatory market of the steroids. Additional indirect outcomes will be: i) the improvement of the European biotechnological business network, and ii) the establishment of research and industrial collaborations with Russia (novel incorporation to the ERA-IB call).

Production of Organic Acids for Polyester Synthesis

Results of POAP (Presentation)

Project coordinator:  Dr. Marta Tortajada - BIOPOLIS, S.L. - Spain

Project leaders:

Summary

The project objective is the development of efficient processes for the obtention of D-lactic acid (D-LA) and itaconic acid (IA) from low-lignin agricultural wastes (LLW), such as citrus processing waste (CPW) and chaff. Both CPW and chaff are abundant, cheap, non-food competing and renewable polysaccharide-based feedstocks. Thanks to their low lignin content, pre-treatment and enzymatic hydrolysis will be advantageous when comparing to lignocellulosic substrates.
D-LA and IA are among the most interesting candidates to replace petroleum based monomers to synthesize novel polyesters. These acids can currently be obtained by fermentation from expensive glucose/sucrose/starch sources. Alternative raw materials such as lignocellulosic substrates have been assayed but high pre-treatment costs hinder the performance of such processes.
In this project, LLW such as CPW and chaff will be processed by enzymatic and/or chemical hydrolysis. CPW will be previously treated by state-of-the-art extraction technologies to remove orange oil. Novel enzymes will be evaluated for the disintegration of cellulose, hemicelluloses and pectin. As a complementary substrate, raw glycerol from biodiesel processing is also considered.
Most suitable microorganisms for D-LA and IA production using hydrolyzed LLW will be screened. Optimization of fermentation with both free and immobilized cells will be carried out. Simultaneous saccharification and fermentation process (SSF) preferentially by solid-state fermentation will be evaluated and optimized. Studies regarding suitable purification methods and catalytic upgrade to new bio-based monomers and polymers will be conducted.
Process sustainability will be evaluated through the realization of a complete Life Cycle Assessment (LCA). The most efficient and sustainable process will be identified and scaled-up. Material Flow Accounting (MFA) methods will be utilized by the help of an information system for the evaluation life cycle cost analyses and financial feasibility.
Project outcomes will be new biocatalysts and proficient processes selected through LCA for the transformation of LLW into valuable building blocks with a further upgrading to polymers and copolymers. These results shall be protected by patenting and exploited preferentially within the consortium.

Tailor-made expression hosts depleted in protease activity for recombinant protein production

Results of PRODuCE (Presentation)

Project coordinator:  Dr. rer. nat. Andreas Schiermeyer - Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Aachen - Germany

Project leaders:

Summary

The demand for biopharmaceuticals is high and predicted to increase further and as such efficient production processes are required that yield quality protein. Despite the improvements in the production of complex biopharmaceuticals in Chinese Hamster Ovary (CHO) cells a number of challenges remain. A key challenge is the degradation of protein products during fermentation or downstream processing steps.

Within the proposed project we will systematically evaluate the proteolytic activities that hamper the successful production and/or purification of selected target proteins (e. g. antibodies, erythropoietin). The project will include CHO cells as the current industry work horse for protein production but also plant suspension cells (tobacco, Medicago) as emerging alternative production platforms. Proteolysis will be blocked using selective protease inhibitors to classify proteases responsible for product degradation. Proteolytic activities will be determined in cells and spent culture medium by activitybased protein profiling (ABPP) using chemical probes for proteases. Libraries of these probes are available for cysteine-, serine- and metalloproteases and for the proteasome. Probes for aspartic proteases will be produced within the project. The knowledge gained about the nature of the proteases will be utilized to engineer cell lines with reduced endogenous protease activities. Different strategies will be used to suppress product degradation:

• Knock-out of protease genes by gene targeting
• Knock-down of protease genes by posttranscriptional gene silencing
• Co-expression of selective protease inhibitors together with the target protein
• Rational design of cell culture media to suppress proteolytic activities

With the project we expect to establish new production cell lines (CHO and plant suspension cells) with reduced endogenous protease activities and to develop novel CHO cell culture medium formulations.

The consortium will seek protection of intellectual property rights for the novel engineered cell lines because cell lines with reduced proteolytic activities are of great value for the industry to produce sensitive biopharmaceutical proteins (e.g. factor VIII). Patent applications for novel cell culture medium formulations are uncommon and therefore IP protection will only be sought for novel ingredient compounds.

Rational Engineering of Advanced Clostridia for Transformational Improvements in Fermentation

Results of REACTIF (Presentation)

Project coordinator:  Chief Scientific Officer Edward Green - Green Biologics Limited, Abingdon - UK

Project leaders:

Summary

Challenge & objectives
The production of n-butanol from fermentation of sugar using Clostridia has tremendous industrial potential (n-butanol is an important chemical intermediate for paints plastics, coatings and polymers). Butanol production via fermentation is sustainable, environmentally friendly and offers a lower cost route than synthetic production from oil. However, the conventional fermentation route using Clostridia suffers from low titres and volumetric productivities and a reliance on expensive (and edible) starch-based feedstocks. Commercial implementation requires novel strains with improved fermentation performance and strains capable of fermenting non-food, cellulosic feedstocks.
Our aim is to develop advanced Clostridia strains. Specific deliverables include:

• identification and characterisation of alleles (genes) responsible for the desired tolerance traits, using genomic approaches, both in historical strains that were used commercially over four decades and in current production strains following the implementation of novel directed evolution strategies;
• transfer of the identified alleles into current, robust production strains, together with rational metabolic engineering to improve product titres;
• assessment of strain(s) performance on cellulosic feedstocks at lab and pilot scale.

Expected results & exploitation
Significant project IP will be created during all stages of the project and in particular the identification and manipulation of novel, non-obvious gene targets. The project will deliver advanced Clostridia strains that offer a transformational change to fermentation performance and process economics (projected butanol cost reductions of 300 $/T and a 50% reduction in CAPEX). The strains will exhibit:

• enhanced growth rates and tolerance to deviations in commercial process conditions;
• increased tolerance to cellulosic feedstock inhibitors, and;
• improved tolerance to the end product (butanol).

The commercial outputs of the project will be exploited by Green Biologics Ltd. (GBL) and Weyland, who bridge the commercial supply chain from feedstock to butanol production. They will develop a design brief for an integrated fermentation plant with a cellulosic feedstock and determine production cost together with energy and carbon balances over the complete lifecycle.

Systematic consideration of inhomogeneity at the large scale: towards a stringent development of industrial bioprocesses

Results of SCILS (Presentation)

Project coordinator:  Prof. Dr. Marco Oldiges - Institute of Bio- and Geosciences, IBG-1: Biotechnology / Forschungszentrum Jülich GmbH - Germany

Project leaders:

Summary

The project aims to systematically elucidate the influence of increasing bioreactor inhomogeneity which occurs in industrial-scale bioreactor, with respect to microbial physiology and production performance of Corynebacterium glutamicum, a microorganism with broad industrial applications. Early consideration of inhomogeneity issues during lab scale process development will facilitate the selection of the most potent production strain, accelerate the upscaling process and improve the performance at production scale. Such inhomogeneous conditions can be mimicked at lab scale by a so called scale-down simulator bioreactor, consisting of a well-mixed stirred tank reactor (STR) and a plug-flow reactor (PFR) connected in series to it. During operation the cultivation volume is continuously pumped from the STR through the PFR simulating zones of inhomogeneity known to be present at the large scale.

This central challenge of inhomogeneity is addressed, for the first time, by an effort bridging both, the cellular and the process level, enabled by scale-down simulator bioreactor technology, innovative process analytical technology, multi-omics analysis and genetic engineering.

General objectives of the project
scale-down simulator bioreactor studies for lab scale analysis of bioreactor inhomogeneity using process and multi-omics data
development of novel tools for advanced bioprocess characterization and analytics
engineering of microbial systems with improved robustness to bioreactor inhomogeneity
evaluation of bioreactor inhomogeneity by computational fluid dynamics (CFD) and linkage with metabolic network models

Expected results
knowledge about influence of inhomogeneous conditions on metabolic regulation, stress patterns and omics data and methods for improved scale-up
integration of novel in-situ optical sensor technology to study morphology and physiology aspects
framework for application of flow-following mobile sensors (sensor pill) in bioreactors and concepts for the harvesting and data collection procedures
novel robust strains which are less sensitive to inhomogeneous conditions at larger scale for more effective processes and accelerated upscaling
prediction of large scale process performance by linking CFD with metabolic network modeling and process data

Exploitation
Results and inventions will be translated into innovation and will attract attention from industry and the scientific community. Knowledge will be patented/licensed and proper dissemination of results will be achieved by high-quality peer-reviewed publications, presentation at conferences and industrial meetings. The project has the potential to lead to highly innovative industrial applications and to improve the competitiveness of European companies in the bioprocessing sector.

Novel thermostable enzymes for industrial biotechnology

Results of THERMOGENE (Presentation)

Project coordinator:  Professor of Biological Chemistry Jennifer Ann Littlechild - University of Exeter - UK

Project leaders:

Summary

The THERMOGENE project aims to identify transferase enzymes from microorganisms inhabiting natural hot environments. After ‘proof of concept’, the technologies based on new enzymes will be scaled-up in collaboration with biotech companies and commercialized. The project employs microbiology, large-scale genomics, bioinformatics, biochemistry and structural biology. It will be based on the following pipeline: sampling from natural thermal environments, isolation of phylogenetically diverse thermophilic species with desired enzymatic activities, sequencing of metagenomes or genomes of selected microorganisms, both new isolates and microorganisms from previously obtained culture collections of thermophiles, genome assembly, gene mining and functional classification of predicted proteins, high-throughput cloning, expression and activity screening, detailed biochemical and structural characterisation of selected novel enzymes.

Objectives
Identification and biochemical characterization of novel thermostable transferases with biotechnological potential. The project is within scope of this call "Improved enzyme systems for new and more efficient bioprocesses".

There is an increasing demand for new thermostable enzymes with enhanced performance and/or novel functionalities that provide savings in time, money and energy for industrial processes in the areas of high value chemical production and other "white" biotechnology applications.

Enzyme chemistry can make reactions feasible that are currently unavailable using conventional chemical methods. Use of enzymes for chemical processes is a route to lower energy consumption and reduced waste generation. The selectivity of enzymatic processes reduces raw material costs and the safety issues surrounding the production of harmful byproducts. Optimised enzyme production will lead to economically viable and cost effective, sustainable production.

THERMOGENE will focus on the discovery of selected transferase enzymes with known and potential commercial applications. These include enzymes which transfer 2-carbon units, transketolases; transfer amine groups, transaminases; transfer isoprenyl or prenyl groups, prenyltransferases and which transfer methyl and hydroxymethyl groups, methyl and hydroxymethyl transferases.