Synthetic Biology is a new discipline of research that combines biological science and engineering in order to design and build novel biological functions and systems. An engineer’s viewpoint is that Synthetic Biology includes the broad redefinition and expansion of biotechnology, with the ultimate goals of being able to design and build engineered biological systems that process information, manipulate chemicals, fabricate materials and structures, produce energy, provide food, and maintain and enhance human health and our environment. This very concept is being initiated at the DBT-ICT-Centre for Energy Biosciences. The general plan of work is as follows:
First, large-scale genome sequencing efforts of naturally occurring organisms will be accessed for continued wealth of information which will be used to design DNA fragments and plasmid constructs.
Second, the newly designed part will be sequenced to verify that the fabricated system is appropriate.
Third, the newly fabricated system will be used for fast, cheap and reliable method of generating designer enzyme molecules such as lipases, cellulases, glycosidases, proteases, etc.
The aim of the program in this area is to identify technology-oriented problems, explore the relevant literature; design molecules based on the available information and end use, and finally apply conventional as well as high-throughput molecular, genetic and metabolic engineering methods to bring about a solution. The following projects are underway in Synthetic Biology:
Conversion of sugars (glucose and xylose) to ethanol by improved strains of yeast and Pichia stipitis
Conversion of sugars (glucose and xylose) to butanol by improved strains of Clostridium acetobutylicum
Scaled up production of recombinant gluycosidases, lipases and proteases.
Scaled production of secondary metabolites like valine, vitamin B12 and vanillin. Engineering algal strains to produce biomass and natural products.
Fermentation along with separation is the final and the determining step for facilitating industrial production of biotechnologically important compounds and therefore is often considered as the ‘heart’ of any biotech production process. The primary area of activity in area of fermentation at the Centre includes biofuels (Ethanol & Butanol) production. Significant success has been achieved in the production of these liquid biofuels options using batch fermentations and theoretical yields at viable productivities have been achieved for both Ethanol & Butanol. Other active areas in fermentation are production of industrially important biotechnological products like amino acids like valine, vanillin, vitamin B12. The objective always is to develop cost efficient industrial processes and hence the efforts encompass development of processes and products from bench scale to pilot scale and through to industrial scale pilot plants and technology transfer to industry.
Adsorptive and Chromatographic purification of molecules
Adsorptive and chromatographic separations are almost indispensible in production of therapeutic biomolecules. These technologies are also expected to occupy centre-stage in the coming years for selective separations of many naturally derived as well synthetically prepared high value products. Development of an adsorptive technique requires two things: 1. Development of the most suitable and selective adsorbent (called Selectivity engineering); and 2. Design of a suitable operation protocol on an optimally designed equipment for optimum performance (called Process engineering). Work carried out at the Centre in this area is focused on the design of adsorbents for selective separation technologies of biomolecules like monoclonal antibodies and antibody fragments, blood proteins, plasmid DNA, cells, therapeutic proteins and peptides, and industrial and laboratory enzymes.) The basic approach towards designing desired selectivity is based on exploiting the principle of pseudo-bioaffinity.
AbSep, a rationally designed affinity adsorbent has been developed to purify polyclonal IgG from human plasma, goat serum and equine serum with a high recovery and purity similar to Protein A based matrices. AbSep has been successfully used in selective purification of monoclonal IgG and IgM from cell culture supernatants and can provides a robust and economic alternative for purification of polyclonal IgG from plasma and monoclonal antibodies from cell culture supernatants.
Chromatographic purification of highly pure Zirconium from trace metal impurities like Co, Sb, Sn has also been demonstrated.
Work carried out over last couple of years has also addressed adsorbent design for chromatographic separations. Major collaborations with Resindion SRL, Italy (subsidiary of Mitsubishi Chemical Corporation , Japan) and BioRad Laboratories, India/USA have focussed on design of adsorbents and columns, and generation of industrially scalable purification technologies. Indigenously developed based on cellulose that are super porous have been surface modified using appropriate chemistries to yield various functionalized matrices for general and specific applications. A number of important proteins/enzymes and other biomolecules (e.g. antibiotics and vitamins) have been purified using adsorption technology and these technologies have been or are being transferred to the industry.
Besides adsorptive separations, other techniques like affinity precipitation have been used as a useful concept to isolate and concentrate proteins more specifically in the initial stages of downstream processing. Charged ‘smart polymers’ can also be used to form poly-complexes with oppositely charged polymers in aqueous solutions wherein one of the components can be solublized depending on environmental factors. While a number of synthetic polymers were used to different degrees of success, an altogether new reversible polymer system based on the readily available carboxy-methyl cellulose has been developed. In addition, there has been work on a system for thermo-reversible precipitation of proteins on Eudragit Sl00, a methacrylate-methyl methacrylate co-polymer. Peroxidases and dehydrogenases have been purified from their crude extracts to near homogeneity in a single step of hetero-bifunctional affinity precipitation.
Process Equipment design for Scaled up Adsorptive/Chromatographic Purifications
Many new products are emerging in pharma, food and nutraceutical markets that need to be manufactured to high purities at large scale. Such products include antibiotics, health proteins from soya, whey, potato etc., and food additives like antioxidants and functional sugars. These need almost essentially to be purified at large scale and require handling several cubic meters of feedstock per day. Consequently purification of these products requires large volumes of chromatographic adsorbents and large scale equipment.
Large scale chromatography today has only two options – parallelaly operating large batch columns or use of simulated moving bed (SMB). Batch system is expensive due to large adsorbent inventories, while SMB has several limitations use for binary separations alone, clean and defined feed requirement, dependence on mathematical simulation, and expensive control/operation equipment. Thus SMB cannot be used for varying quality natural feed solutions or solutions coming from fermentations, or feeds containing several solutes with the target solute being a minority fraction. Further, SMB cannot handle or recover multiple solutes, nor it can handle widely varying load, elution and regeneration buffers required in natural product or fermentation broth separations.
Work at the Centre has been carried out on Liquid-Solid Circulating fluidized bed (LSCFB). However, the drawbacks of LSCFB are : The precarious pressure drop balance between the riser and downcomer for required solid circulation; essentially inefficient co-current elution in riser; and no possibility of step or linear gradient elution, and lack of separate regeneration system.
A new multistage counter-current FMB (fluidized moving bed) has been designed and patented. The FMB removes almost all the limitations of SMB and LSCFB, and is as versatile and flexible as any batch chromatography system with added advantages of low adsorbent inventory and continuous operation at a much lower capital cost compared to SMB.
Applications of FMB will be in large scale separation problems like those in antibiotic industry and food industry. At present a pilot scale unit has been built for beta carotene separation from red palm oil; recovery of copper from effluent, and purification of plant proteins. Other potential areas are purification of PenG, CephC and other antibiotics.
The application of enzymes for numerous applications in the food, feed, agriculture, paper, leather, and textiles industries have been highly beneficial. This has created a stimulus for chemical and pharma industries to accept the advantages that biocatalysts offer and utilize enzymes for greener technologies with higher product quality. The search for better enzymes for targeted activities and integration of processes to harness its potential is the scope of our research. The area of research is divided into two main segments, namely; Enzyme Engineering and Process Engineering. These two approaches help integrate a rather unique approach in the field of Enzyme Technology, wherein, biotransformation is planned at the atomic scale, developed at the molecular scale and integrated at the laboratory and pilot scale. The strength of DBT-ICT-CEB in Enzyme technology lies in this integrated approach.
The Enzyme Engineering envisages on enzyme design improvement primarily by in silico techniques and applies them by in vitro approaches. To understand the physical conditions and chemical environments that play a crucial role in determining interactions between the enzyme and substrate, computational chemistry tools are being used and strategies are planned in silico for in vivo studies. The in vitro approaches include both pre-expression modifications- rational and directed evolution methodologies as well as post-expression modifications-physical and chemical methodologies. The work is primarily directed towards the development of enzymes with better physic-chemical properties and for targeted biotransformation problems. The Process Engineering is more of an engineering problem wherein reaction engineering, reactor design and process integration and intensification are envisaged and developed with reference to the biocatalyst and the product targeted. The enzymes catalyzing various chemical reactions are studied for their substrate selectivity and kinetic behavior in an attempt to understand the equilibrium parameters. Modeling studies are undertaken to study the varied kinetic behavior exhibited by biocatalysts when immobilized onto different adsorbents with emphasis on designing extractive biotransformation with continuous removal of products from the reaction system. This unique combination of basic science and engineering principles has helped develop enzyme technologies with better efficiencies and end-product qualities. At DBT-ICT-CEB, under Enzyme technology, we have developed a repertoire of enzyme preparations for targeted biotransformations, namely; Several immobilized Lipases – For hydrolysis and synthesis; Peroxyme 1 – For oxidations and nitrations; Peroxyme 2 – For epoxidations and chlorinations; Pepzyme – For hydrolysing IgG and proteins; Dextrizymes – For hydrolysis of starch; Trypzyme – For hydrolysing/synthesis of proteins/peptides; Peczyme – For hydrolysis of pectin; Neutrazyme – For hydrolysis of plant proteins; Tyrozyme – For hydroxylation and oxidation of phenols and Gloxyme – For oxidation of sugars. Many of these technologies are patented or under the process of being patented. They are also being successfully used by industrial houses for manufacture of commercial products.
The specific projects in this area on use of hydrolases and oxidases deal with developing solutions for the following problems:
Lipase-mediated hydrolysis/transesterification/Interesterification of vegetable oils
Lipase catalyzed chiral resolution of intermediates of synthetic optically active APIs
Catechol and derivatives by oxidation of phenols
Semi-synthetic processes for transpeptidation using proteases
Protease catalyzed synthesis of peptides
Integrated process of starch hydrolysis for production of maltodextrins
Integrated process for isolation and controlled hydrolysis of plant proteins
Ethanol derived from renewable biomass has emerged as a major contender expected to replace liquid petroleum fuel. Largely produced as a chemical feedstock from molasses over the past four decades in many sugar producing countries including India, ethanol is currently produced in Brazil at a mammoth scale directly from cane juice and in USA from starch making bioethanol production plants from grains and molasses operational. However, the lopsided energy balances and requirement of acres of agricultural land for the cultivation of these grain plants remains a strong inhibitor in this technology. This has amply led to the innovation of ‘fuel and food’ rather than ‘fuel for food’ based technology. With far less available land per capita compared to countries such as the USA and Brazil, India can ill afford to substitute its food related agriculture with energy-oriented farming. Lignocellulosic waste biomass (LBM) derived from agriculture and forests have a potential to become the truly renewable source of bioethanol. There is intense activity all around the world aimed at developing technologies for conversion of LBM to liquid or other forms of fuel. The overall technology comprising multiple steps is still far from being economically and ecologically sustainable and attractive. Much work needs to be done in areas of biomass feedstock improvement, technologies for pretreatment and fractionation to cellulose, lignin and hemicelluloses, and their conversion to alcohol and other valued added products in a complete, cost effective as well as eco-friendly manner. There is also a need to develop solar-efficient biomass varieties that grow on non-agricultural marginal lands with minimum external inputs. The DBT-ICT Center for Energy Biosciences aims at developing sustainable LBM to alcohol technologies through a four-pronged initiative:
Developing of technologies for fractionation of lignocelluloses in ways that facilitate subsequent bioconversion of cellulose and hemicelluloses to fermentable sugars that in turn will be converted to alcohol.
Development of crop varieties that are solar efficient and amenable to easier pretreatment and bioconversions.
Development of cost effective enzyme and microorganism systems capable of breaking down lignocelluloses components to fermentable sugars and alcohol.
Development of other technologies for conversion of biomass to fuels through biotechnology routes, as well as using all components of LBM to useful/viable products.
In addition to bioethanol, the centre is also poised to look at alternative sources of second, third and fourth generation biofuels. With the aim of developing efficient biodiesel production technologies, work is being carried out to develop strains that produce lipases with desirable characteristics of specificity and stability, immobilizing enzymes, building reactors and developing technologies for recovery of high value products from vegetable oil/s before subjecting to enzyme action. Incredible work in the area of biodiesel has being carried out using immobilized lipases and the laboratory has been successful in preparing several immobilized stable preparation of lipases for different applications. Rationale for effective lipase immobilization chemistry has been modeled and developed. Inexpensive immobilized lipase preparations have been used for both inter-esterification of triglycerides and for production of biodiesel.
Lignin to Syngas
Lignin fractionated and obtained from any biomass can be pyrolyzed into syngas that can lead to hydrocarbon fuel through Fischer-Tropsch synthesis route. Work has been initiated towards the development of this technology.
Hydrogen is slated to be the ultimate clean fuel of the future. Water splitting is the most potent technology for hydrogen production. Current state of technologies however makes hydrogen production net energy negative. Among the many technologies being studied are biological production of hydrogen. The technology of highest promise is algal biohydrogen production that combines light and dark reactions in sequential bioreactors to produce hydrogen. However, there are several technological obstacles that need to be overcome for making the technology a commercial reality. Work will be taken up, in collaboration with BARC, Mumbai to develop biohydrogen technologies at a pilot scale level and evaluate their techno-commercial aspects.
Bioethanol production is to be based on lignocellulosic biomass. Often, the biomass obtained may not be obtained in desirable dry form, and thus will contain many water soluble and biodegradable components. The process intended to fractionate lignocellulose will produce stream containing biodegradable matter (cellulose, hemicellulose and lignin are non-biodegradable). This stream will form a waste disposal problem and could be put to use through anaerobic methane production. This will provide the biorefinery with additional energy and make the plant energy surplus (discounting the ethanol produced). Though design of methane bioreactors involving methanogens that grow on biodegradable waste is well established, some work will be required to integrate the system into ethanol biorefinery, and will form the objective of this project.
Recent trend in the need for renewable fuel resources have led researchers to manoeuvre in areas that encompass waste management (use of biomass), ease of logistics (in situ generation of the fuel) as well as exploring systems that can produce non-polluting biofuels (biohydrogen). Several algal systems are now being explored as model organisms for a wide range of biofuels, such as bio-hydrogen, bio-methane and also as a source of biomass to produce alcohols. Present focus on the utilization of biomass for the production of ethanol and/or butanol as a source for biofuels is to convert lignocelluosic biomass to sugars which may be eventually utilized in the fermentation processes. The protocol that is currently being developed is to produce high amounts of fermentable sugars in the single-cells of alga. While there are very few laboratories (and no industries at all) working in this area, we have explored the possibility of using the wild type of a group of algal cells for this purpose. An involved, long-drawn exploration into the chemical composition of alga expressed on a percentage dry matter basis shows that the carbohydrate content are high in some algal species for example Spirogyra sp., Porphyridium cruentum, Scenedesmus dimorphus, Dunaliella salina, Prynesium parvum and Anabaena cylindrica.