Chloroplast factories for sustainable and high yield production of biotech products

Abstract

World demand for energy has been projected to double by 2050 and be more than triple by the end of the century. Since industrial revolution in the 1850s, the human consumption of fossil fuels has been one of the growing causes of international concern and unease among some industrial nations. The reasons for which can be attributed to the rapidly depleting reserves of fossil fuels. Over the past few decades, with the successes achieved in genetic engineering technology, advances made in the field of biofuels offer the only immediate solution to fossil fuels.

Presently, most of the ethanol in use is produced either from starch or sugar, but these sources have not proven to be sufficient to meet the growing global fuel requirements. However, conversion of abundant and renewable cellulosic biomass into alternative sources of energy seems to be an effective and promising solution. But for this technology to become viable there is a need to develop cheap and sustainable sources of cellulases along with eliminating the need for pretreatment processes.

The review thus aims to provide a brief overview about the need and importance of biofuels particularly bioethanol with respect to the growing environmental concerns along with an urgent need to address the existing problems about cost-optimisation and large scale production of biofuels.

1.0 Introduction

Biofuels are liquid fuels derived from plants. Currently, first generation biofuels are extensively being produced and used. These are generated using starch, sugar, vegetable oils and animal fats using fairly expensive conventional technology. In recent years, the fact that production of ethanol from cellulosic and lignocellulosic material is being hindered due to inadequate technology to enable efficient and economically viable methods to break down the multipolymeric raw material has gained wide popularity (Verma et al, 2010). Therefore, there is a need to develop efficient systems for the production of cellulases and other cellulose degrading enzymes. Lignocellulosic biofuels are thus likely to be seen as a part of the portfolio of solutions being offered to reduce high energy prices, including more efficient energy use along with the use of other alternative fuels (Coyle, 2007).

1.1 Importance of biofuels:

Factors like the finite petroleum reserves and constantly rising demands for energy by the industrialised as well as the highly populated countries (on their Way to industrialisation) like India and china have made it absolutely necessary to look into alternate and efficient methods to replace these fuels in future (Stephanopoulos, 2008). Also, concerns like steep rise in fossil fuel prices in the recent years, increasing concerns about climate change like global warming, insecurity and unrest among governments due to their depleting natural reserves are just a few factors that define an urgent need for a sustainable path towards renewable fuel technology development (Stephanopoulos, 2008). Among the various types of alternative fuels considered (liquid fuels from coal and/or biomass with and without carbon capture and storage (CCS)), biofuels derived from lignocellulosic biomass offer the most clean and sustainable alternative to fossil fuels essentially because of their cost competitiveness as opposed to the current expensive methods of ethanol production from sugarcane and corn (Stephanopoulos, 2008) (Shen and Gnanakaran, 2009).

The global production and use of biofuels has increased tremendously in recent years, from 18.2 billion litres in 2000 to about 60.6 billion litres in 2007. It has been estimated that about 85% of this amount is bioethanol (Coyle, 2007). This increase is primarily a result of the reasons stated above along with rising concerns about global warming and greenhouse gas emissions due to excessive fossil fuels usage since biofuels are carbon-neutral and reduce green house emissions (Sainz, 2009). Also, one of the factors contributing to the viability of biofuels as an alternative transportation fuel is their ease of compatibility with our existing liquid fuel infrastructure (Sainz, 2009).

An important step in the production of biofuels is the breakdown of cellulose fibres by the enzymes capable of degrading it. But the production of these enzymes is still an expensive task due to their production in large microorganism bioreactors. One method for the inexpensive production of these enzymes is the use of transgenic plants as heterologous protein production systems (Danna, 2001; Kusnadi et al., 1997; Twyman et al., 2003). Plant based enzyme production offers advantages over the traditional bacterial and fungal cultures by being commercially viable and particularly attractive since in plants, the desired protein can be made to accumulate at high levels i.e. at even greater levels than 10% of total soluble protein (Gray et al, 2008).

Another major economic advantage of plant-based protein production over one that is microorganism-based is in the scale-up of protein expression. Whereas scale-up of microbial systems implies large purchase and maintenance costs for large fermentors and related equipment, scale-up of plant-based protein product would only require planting of more seeds and harvesting of a larger area (Gray et al, 2008). Cellulase expressing transgenic plants may thus offer significant capital cost savings over more traditional cellulase production via cellulolytic fungi or bacteria (Gray et al, 2008).

2.0 Current sources of sugars and ethanol production

Ethanolis an alcohol fuel currently made from the sugars found in grains, such as corn, sorghum, and wheat, as well as potato skins, rice, sugar cane, sugar beets, molasses and yard clippings. There are two methods currently brought into use for the production of bioethanol. In the first one, sugar crops or starch are grown and through the process of fermentation, ethanol is produced. In the second method, plants are grown that naturally produce oil like jatropha and algae. These oils are heated to reduce their viscosity after which they are directly used as fuel for diesel engines.

However, currently, it is majorly being produced from starch (corn in US) and sugar (sugarcane in Brazil) sources. According to the latest statistics (in 2008), USA and Brazil (fig. 1) were the major producers of fuel ethanol by producing 51.9% and 37.3% of global bioethanol respectively (http://www.ethanolrfa.org/industry/statistics/#E). Brazil especially produces ethanol to a large extent from fermentation of sugarcane sugar to cater to one-fourth of its ground transportation needs (Sticklen, 2008).Similarly, to meet part of its own needs; United States produces ethanol from corn. Unfortunately, inspite of being breakthrough developments, the production of ethanol by this method is not cost-effective and barely manages to meet less than about 15 % of the country's demands (Sticklen, 2008). Their use as energy crops is thus posing to be inappropriate since these are primary food sources, and are unstable from the viewpoints of long-term supply and cost (Sainz, 2009).

The restrictions on available land and the rising price pressures would soon limit the production of grain and corn based ethanol to less than 8% in the US transport fuel mix (Tyner, 2008). Similarly, in spite of a predicted increase to 79.5 billion litres by 2022 in ethanol production from sugarcane in Brazil, this technology would eventually be limited by the same agro-economic factors affecting the grain and the corn based ethanol production (Sainz, 2009). For e.g. the use of corn for production of ethanol has led to an increase in the prices of livestock and poultry since it is the main starch component of the animal feed.

Therefore, there is an urgent need for new and sustainable technologies for a significant contribution of biofuels towards the progress of renewable sources of energy and the reduction of greenhouse gases (Sainz, 2009). Thus, the benefits of a high efficiency of carbohydrate recovery compared to other technologies and the possibilities of technology improvement due to breakthrough processes in biotechnology, offer cost-competitive solutions for bioethanol production, thus making the second generation or lignocellulosic sources the most attractive option the large scale production of biofuels (Wyman et al, 2005).

3.0 Potential of cellulosic bioethanol

Cellulosic ethanolis abiofuelproduced from wood, grasses, or the non-edible parts of plants. It is a type ofbiofuelproduced frombreaking down of lignocellulose, a tough structural material that comprises much of the mass of plants and provides them rigidity and structural stability (Coyle, 2007). Lignocellulose is composed mainly ofcellulose,hemicelluloseandlignin (Carroll and Sommerville, 2009).

Another factor that makes the production of cellulosic bioethanol a promising step in future is that unlike corn and sugarcane, its production is not dependent on any feedcrop since cellulose is the world's most widely available biological material that can be obtained from widely available low-value materials like wood waste, widely growing grasses and crop wastes and manures (Coyle, 2007). But production of ethanol from lignocellulose requires a greater amount of processing to make the sugar monomers available to the microorganisms that are typically used to produce ethanol by fermentation.

Bioethanol is one fuel that is expected to be in great global demand in the coming years since its only main requirement is the abundant supply of biomass either directly from plants or from plant derived materials including animal manures. It is also a clean fuel as it produces fewer air-borne pollutants than petroleum, has a low toxicity and is readily biodegradable. Furthermore, the use of cellulosic biomass allows bioethanol production in countries with climates that are unsuitable for crops such as sugarcane or corn. For example, the use of rice straw for the production of ethanol is an attractive goal given that it comprises 50% of the word's agronomic biomass (Sticklen, 2008).

Though cellulosic ethanol is a promising fuel from an environmental point of view, its industrial production and commercialisation has not been progressing successfully. This can mainly be attributed to the high cost of production of cellulose degrading enzymes -Cellulases (Lynd et.al, 1996). Yet another very important factor is the pretreatment of lignocellulosic content in the biomass to allow cellulases and hemicellulases to penetrate and break the cellulose in the cell wall. These two steps together incur very high costs and are a hindrance in efficient production of cellulosic bioethanol. Thus plant genetic engineering is the best alternative to bioreactors for an inexpensive production of these enzymes (cellulases and hemicellulases). It can also be used to modify the lignin content/amount to reduce the need for expensive pretreatment (Sticklen, 2008).

4.0 The abundance and structure of cellulose

Photosynthetic organisms such as plants, algae and some bacteria produce more than 100 million tonnes of organic matter each year from the fixation of carbon dioxide. Half of this biomass is made up of the biopolymer cellulose which, as a result, is perhaps the most abundant It is the most common organic compound on Earth. Cellulose comprises about 33 percent of all plant matter, 90 percent of cotton is composed of cellulose and so is around 50 percent of wood (Britannica encyclopaedia, 2008). Higher plant tissues such as trees, cotton, flax, sugar beet residues, ramie, cereal straw, etc represent the main sources of cellulose. This carbohydrate macromolecule is the principal structural element of the cell wall of the majority of plants. Cellulose is also a major component of wood as well as cotton and other textile fibres such as linen, hemp and jute. Cellulose and its derivatives are one of the principal materials of use for industrial exploitation (paper, nitrocellulose, cellulose acetate, methyl cellulose, carboxymethyl cellulose etc.) and they represent a considerable economic investment (Pérez and Mackie, 2001).

Cellulose and lignin are the majorcombustiblecomponents of non-foodenergy crops. Some of the examples of non-feed industrial crops are tobacco, miscanthus, industrial hemp, Populus(poplar) species and Salix(willow).

Celluloseserves as one of the major resistance to external chemical, mechanical, or biological perturbations in plants. This resistance ofcelluloseto depolymerization is offered by its occurrence as highly crystalline polymer fibers (Shen and Gnanakaran, 2009).it occur in plants in two crystalline forms, I-αand I-β(Nishiyama et al, 2002) (Nishiyama et al, 2003). The crystal structures of both these forms suggest that hydrogen (H) bonding plays a key role in determining the properties ofcellulose (Shen and Gnanakaran, 2009).Thechemical formula of cellulose is(C6H10O5) n. It is apolysaccharideconsisting of a linear chain of several hundred to over ten thousand β (1→4) linkedD-glucoseunit (Crawford, 1981) (Updegraff, 1969). This tough crystalline structure of cellulose molecules is proving to be a critical roadblock in the production of cellulosic bioethanol as it is difficult to breakdown the microfibrils of crystalline cellulose to glucose (Shen and Gnanakaran, 2009).

4.1 Primary structure of cellulose

The main form of cellulose found in higher plants is I-β. The primary structure of cellulose as shown in figure 2, is a linear homopolymer of glucose residues having theDconfiguration and connected byβ-(1-4) glycosidic linkages (Sun et al, 2009). Essentially, the occurrence of intrachain and interchain hydrogen bonds (fig. 3) in cellulose structures has been known to provide thermostability to its crystal complex (Nishiyama, 2002). Intrachain hydrogen bonds are known to raise the strength and stiffness of each polymer while the interchain bonds along with weak Wander-Waal's forces hold the two sheets together to provide a 2-D structure. This arrangement makes the intrachain bonding stronger than that holding the two sheets together (Nishiyama, 2002).

The chain length and the degree of polymerisation of glucose units determine many properties of the cellulose molecule like its rigidity and insolubility compared to starch (Shigeru et al, 2006). Cellulose from different sources also varies in chain lengths, for e.g. cellulose from wood pulp has lengths between 300 and 1700 units while that from fibre plants and bacterial sources have chain lengths varying from 800 to 10,000 units (Klemm et al, 2005).

5.0 Methods to breakdown cellulose

Cellulose, a glucose polymer is the most abundant component in the cell wall. These cellulose molecules consist of long chains of sugar molecules. The process of breaking down these long chains to free the sugar is called hydrolysis. This is then followed by fermentation to produce bioethanol. Various enzymes are involved in the complex process of breaking down glycosidic linkages in cellulose (Verma et al, 2010). These are together known as glycoside hydrolases and include endo- acting cellulases and exo-acting cellulases or cellobiohydrolase along with β-glucosidase (Ziegelhoffer, 2001) (Ziegler, 2000).

In the cellulose hydrolysis process, endoglucanase first randomly cleaves different regions of crystalline cellulose producing chain ends. Exoglucanase then attaches to the chain end and threads it through its active site, cleaving off cellobiose units. The exoglucanse also acts on regions of amorphous cellulose with exposed chain ends without the need for prior endoglucanase activity. Finally β-glucosidase breaks the bonds between the two glucose sugars of cellobiose to produce monomers of glucose (Warren, 1996).

Presently, two methods are widely used for cellulose degradation on an industrial scale:

* Chemical hydrolysis:

This is a traditional method in which, cellulose is broken down by the action of an acid, dilute and concentrated both acids can be used by varying the temperature and the pH accordingly. The product produced from this hydrolysis is then neutralised and fermented to produce ethanol. These methods are not very attractive due to the generation of toxic fermentation inhibitors.

· Enzymatic hydrolysis:

Due to the production of harmful by-products by chemical hydrolysis, the enzymatic method to breakdown cellulose into glucose monomers is largely preferred. This allows breaking down lignocellulosic material at relatively milder conditions (50οC and pH5), which leads to effective cellulose breakdown.

6.0 Steps involved in cellulosic ethanol (bioethanol) production process

The first step in the production of bioethanol, involves harvesting lignocellulose from the feedstock crops, compaction and finally its transportation to a factory for ethanol production where it is stored in a ready form for conversion. The second step is the removal of lignin present in the feedstock biomass by using heat or chemical pre-treatment methods. This step facilitates the breakdown of cell wall into intermediates and removes lignin so as to allow cellulose to be exposed to cellulases, which then break down cellulose into sugar residues.currently, cellulases are being produced as a combination of bacterial and fungal enzymes for such commercial purposes (Sticklen, 2008).

This is then followed by steps like detoxification, neutralisation and separation into solid and liquid components (Sticklen, 2008).

The hydrolysis of these components then takes place by the enzymes like cellulases and hemicellulases that are produced from micro-organisms in the bioreactors (Sticklen, 2008).and finally; ethanol is produced by sugar fermentation.

7.0 Major cell wall components and the key enzymes involved in their breakdown

6.1 Cellulose and cellulases:

About 180 billion tonnes of cellulose is produced per year by plants globally (Festucci et al, 2007). Cellulose makes up 15-30% of the dry mass of primary and up to 40% of the secondary cell walls (Sticklen, 2008). Till date, it is the only polysaccharide being used for commercial production of cellulosic ethanol because of the commercial availability of its deconstructing enzymes (Sticklen, 2008). As described above, three types of cellulases are involved in the breakdown of cellulose into sugars namely, endoglucanases, exoglucanasees and βglucosidase (Ziegler, 2000).

6.2 Hemicellulose and Xylanases:

xyloglucans and hemicelluloses surround the cellulose microfibrils. So in order to break cellulose units, specific enzymes are first required to first remove the hemicellulose polysaccharide. Hemicelluloses are diverse and amorphous and its main constituent is β-1, 4-xylan. Thus, xylanases re the most bundant type of hemicellulases required to cleave the endo-and exo-activity (Warren, 1996). These are mainly obtained from the fungi Trichoderma reesei , along witha a large number of bacteria, yeast and other fungi which have been reported to produce1.4 β-D xylanases.

6.3 Lignin and Laccasses:

The major constituent of plants' secondary cell wall is lignin. It accounts for nearly 10-25% of total plant dry matter (Sticklen, 2008). Unlike cellulose and hemicelluloses, the lignin polymer is not particularly linear and instead comprises of a complex of phenylpropanoid units which are linked in a 3-D network to cellulose and xylose with ester, phenyl and covalent bonds (Carpita, 2002). White rot fungi (esp. Phanerochaete chrysosporium and Trametes versicolour) are thought degrade lignin more efficiently and rapidly than any other studied microorganisms (D'Souza, 1999). P. Chrysosporium produces laccases like ligninases or lignin peroxidase, which initiate the process of degradation of lignin and manganese dependent peroxidises (Cullen, 1992).

8.0 Production of cellulases and hemicellulases in tobacco chloroplasts

Protein engineering methodologies provide the best answer to concerns regarding production of improved cellulases with reduced allosteric hindrance, improved tolerance to high temperatures and specific pH optima along with higher specific activity (Sainz, 2009).

The table below (table 1) lists different type of cellulases and hemicellulases that have been expressed in plant chloroplasts:

Enzyme

Enzyme source

Host

Comments

Reference

E1 Endoglucanase

Acidothermus cellulolyticus

Nicotiana tabacum cv petit Havana SR1

Normal growth photosynthesis;truncated;dry leaf activity

Dai et al. 2000

E1 & CD Endoglucanases

Acidothermus cellulolyticus

N.tabacum cv W38

Spacer length impacts chloroplast import

Jin et al.2003

Cel6A endoglucanse

Thermobifida fusca

Tobacco

Chloroplast transformation; homoplasmic events

Yu et al. 2007a, b

Cel6B cellobio-hydrolase

Thermobifida fusca

Nicotiana tabacum cv K327- non nicotine derivatie

Chloroplast transformation; homoplasmic events

Yu et al. 2007a, b

9.0 Use of chloroplasts to over-express recombinant proteins

Chloroplasts are green coloured plastids that have their own genome and are found in plant cells and other eukaryotic organisms like algae. The targeted expression of foreign genes in plant organelles can be used to introduce desired characteristics in a contained and economically sustainable manner (fig. 5). It also allows us to combine various other advantages like easy and efficient scalability along with being entirely free of animal pathogens.

Unlike most other methods of plant genetic engineering, the major advantage with chloroplast transformation is their characteristic of “transgene containment” i.e. transgenes in these plastids are not spread through pollen (Verma and Daniell, 2007). This implies that chloroplast genetic transformation is fairly a safe one and does not pose the risk of producing herbicide resistant weeds (Ho and Cummins, 2005).

Chloroplast transformation involves homologous recombination. Thisnot only minimises the insertion of unnecessary DNA that accompaniestransformation of the nuclear genome, but also allows precisetargeting of inserted genes, thereby also avoiding theuncontrollable, unpredictable rearrangements and deletions oftransgene DNA as well as host genome DNA at the site of insertionthat characterises nuclear transformation (Nixon, 2001).

Another advantage of chloroplast transformation is that foreign genes can be over-expressed due to the high gene copy number, up to 100,000 compared with single-copy nuclear genes (Maliga, 2003). While nuclear transformants typically produce foreign protein up to 1%TSP in transformed leaf tissue, with some exceptional transformants producing protein at 5-10%TSP, chloroplast transformants often accumulate foreign protein at 5-10%TSP in transformed leaves, with exceptional transformants reaching as high as >40%TSP (Maliga, 2003).

Research is needed to determine the stability of the biological activity of extracted plant-produced hydrolysis enzymes in TSP when stored under freeze conditions for different periods of time before their use in hydrolysis (Sticklen, 2008). Two other important and related areas for further research are increasing the levels of production and the biological activity of the heterologous enzymes (Sticklen, 2008).Many cell wall deconstructing enzymes have been isolated and characterised and more need to be investigated for finding more enzymes that can resist higher conversion temperatures and a range of pHs during pretreatment.

Serious efforts to produce cellulosic ethanol on an industrial scale are already underway. Other than the Canadian Iorgen plant, no commercial cellulosic ethanol plant is yet in operation or under construction (Sticklen, 2008). However, research in this area is underway and funding is becom­ing available around the world for this purpose, from both governmental and commercial sources. For exam­ple, British Petroleum have donated half a billion dol­lars to US institutions to develop new sources of energy — primarily biofuel crops (Sticklen, 2008).

10.0 Conclusion

The fact that corn ethanol produces more green house gas emissions than gasoline and that cellulosic ethanol from non-food crops produces less green house gas emissions than electricity or hydrogen, is one of the factors that highly favour production of ethanol from cellulosic biomass (Verma, 2010). However, biofuel production from lignocellulosic materials is a challenging problem because of the multifaceted nature of raw materials and lack of technology to efficiently and economically release fermentable sugars from the complex multi-polymeric raw materials (Verma, 2010).

After decades of research aimed at reducing the costs of microbial cellulases, their production is still expen­sive (Sticklen and Oraby, 2005). One way of decreasing such costs is to produce these enzymes within crop biomass. Although some important advances have been made to lay the foundations for plant genetic engineering for biofuel production, this science is still in its infancy (Sticklen, 2008). A general challenge is to develop efficient systems for the genetic transformation of plant systems for the production of cellulose degrading enzymes. Research is particularly needed to focus on the target­ing of these enzymes to multiple subcellular locations in order to increase levels of enzyme production and produce enzymes with higher biological activities (Sticklen, 2008). A huge potential exists to produce larger amounts of these enzymes in chloroplasts, and exciting progress has been made in terms of the crops for which the chloroplast can now be genetically engineered. More efforts are however needed towards the development of systems to genetically engi­neer chloroplasts of biomass crops such as cereals and perennial grasses (Blaschke, 2006).

Some of the key aims of the project would be:

* To characterise cell wall degrading enzymes

* Overexpression of cellulose cDNA in pET30 vector systems

* Induction and characterisation of proteins in different conditions

· The use of tobacco plant as means of producing cellulases through chloroplast genetic engineering to simultaneously addresses the most important question of shifting the agricultural land from feed crops to biofuel crops (like corn and sugarcane at present) along with the cost-effective large scale production of cellulose degrading enzymes.

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