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N-Butanol Production from S. Cerevisiae

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The energy consumption grows year by year with the economical development of the countries such as China and Japan despite the boisterous events. With the ‘Arab Spring’ in 2011, energy market was shook in terms of both production and consumption of the oil with the loss of one of the suppliers, also the massive earthquake and tsunami that hit Japan coast caused immediate action for nuclear power and other fuels around the world. Due to those events, oil prices reached an all-time record high [BP Statistical Review of World Energy, 2012]. When the situation, which revolves around mostly economical, environmental and geopolitical issues, in fuel industry is like this, the consumers head towards to renewable energy such as biotechnology. An entire branch of biotechnology embraces the bioproduction of fuels and chemicals from renewable sources. These technologies use living cells and enzymes to synthesize products that are easily (bio)degradable, require less energy and create less waste during their production or use than those produced from fossil resources [Dellomonaco et al, 2010].

However fossil fuels still dominate energy consumption, with a market share of 87%. Renewable energy continues to gain but today accounts for only 2% of energy consumption globally. Meanwhile, the fossil fuel mix is changing as well. Oil, still the leading fuel, has lost market share for 12 consecutive years. Coal was once again the fastest growing fossil fuel, with predictable consequences for carbon emissions [BP Statistical Review of World Energy, 2012]. Ethanol fuel is the most common biofuel worldwide, particularly in Brazil and USA which is produced from several biomass feedstocks and different technologies [].The ethanol production methods are respectively, enzyme digestion (to release sugars from stored starches), fermentation of the sugars, distillation and drying. The distillation process requires significant energy input for heat, often unsustainable natural gas fossil fuel, but cellulosic biomass such as bagasse, the waste left after sugar cane is pressed to extract its juice, can also be used more sustainably [Knothe and Gerhard,2010]. Bioethanol has relatively large octane rating with 116 while ordinary petrol has 91, which allows an increase of an engine’s compression ratio for increased thermal efficiency. Yet the energy yield is nearly four times smaller than usual petrol which makes it inefficient.

The other disadvantage of the ethanol is cannot be piped which makes it hard to be processed and transported. Along with the many advantages of biofuels there are still some disadvantages exist, thus second generation biofuels are developed from the simple biomass consuming biofuels. Biomass is a wide-ranging term meaning any source of organic carbon that is renewed rapidly as part of the carbon cycle. First generation biofuels are made from the sugars and vegetable oils found in arable crops, which can be easily extracted using conventional technology but limited in most cases such as threatening the food supplies and biodiversity. In comparison, second generation biofuels are made from lingo-cellulosic biomass or woody crops, agricultural residues or waste, which makes it harder to extract the required fuel. Second generation biofuel technologies have been developed because first generation biofuels manufacture has important limitations [Evans, 2008].

Butanol has a 4-carbon structure and the carbon atoms can form either a straight-chain or a branched structure, resulting in different properties. The molecular structure and the main applications of butanol isomers are listed in Table 1. 1-butanol, also better known as n-butanol, has a straight-chain structure with the –OH at the terminal carbon. 2-butanol, also known as sec-butanol, is also a straight-chain alcohol but with the -OH group at an internal carbon. Iso-butanol is a branched isomer with the -OH group at the terminal carbon and tert-butanol refers to the branched isomer with the -OH group at an internal carbon. The different structures of butanol isomers have a direct impact on the physical properties, which are summarized in Table 1. Although the properties of butanol isomers are different in octane number, boiling point, viscosity, etc., the main applications are similar in some aspects, such as being used as solvents, industrial cleaners, or gasoline additives. All these butanol isomers can be produced from fossil fuels by different methods [Jin, et al., 2011]. But butanol produced from bioprocesses usually assumes a straight-chain molecule structure, n-butanol, which is also examined in this coursework. | 1-Butanol| 2-Butanol| Iso-Butanol| Tert-Butanol|

Molecular Structure| | | | |
Density(kg/m3)| 809.8| 806.3| 801.8| 788.7|
Research octane number| 96| 101| 113| 105|
Motor octane number| 78| 32| 94| 89|
Boiling temperature (°C)| 117.7| 99.5| 108| 82.4|

Flammability limits vol %| 1.4-11.2| 1.7-9.8| 1.2-10.9| 2.4-8| Table 1. Molecular structure and comparison of butanol isomers The main advantage of the butanol is its higher heating value which helps the reduction of fuel usage and greater mileage due to its four carbon structure. It is known that the saturation pressure of alcohols decreases with the increase of carbon atom number. This means that n-butanol will have less tendency towards cavitations and vapour lock problem, which can eliminate the need for very special blends during the summer and winter months as the gasoline [Jin, et al., 2011]. The heat of vaporization of butanol is less than half of that of ethanol, an engine running on butanol should be easier to start in cold weather than one running on ethanol or methanol. In addition, the autoignition temperature of the n-butanol is lower than ethanol or methanol, which also results in less ignition problems at cold start or low load conditions.

Some other advantages can be listed as good intersolubility with diesel, higher viscosity than ethanol but nearly the same as diesel fuel and it is relatively safer to handle with low vapour pressure and high flash point. Also butanol better tolerates water contamination and is less corrosive than ethanol which makes it easy to handle and distribute among the pipelines [Jin, et al., 2011]. Although the n-butanol has many benefits than the low-carbon alcohols such as ethanol and methanol as an engine fuel, there are still some potential issues with the direct use of butanol. The main disadvantage of butanol is its quite low production. Compared butanol yield by acetone butanol ethanol (ABE) fermentation to that of the yeast ethanol fermentation process, the yeast process yields of ethanol has a 10–30 times higher production rate. Also in spite of higher heating value than low-carbon alcohols, butanol still has slightly smaller heating value than gasoline which can cause problems with the engine performance. Since it is alcohol-based fuel, it is not compatible with some engine components and may cause incorrect readings in gauging and other parameters [Jin, et al., 2011]. Central Carbon Metabolism in Saccharomyces cerevisiae

Saccharomyces cerevisiae is chosen to be host cell to produce n-butanol in this coursework. It has several advantages such as it is genetically
tractable, well-characterized organism that is used for years in baking, brewing, wine making and research activities. The current industrial usage of S. cerevisiae includes pharmaceutical protein production, fermentative production of industrially relevant biochemicals, e.g., glycerol, propanediol, organic acids, sugar alcohols, l-glycerol-3-phosphate (l-G3P), steroids, and isoprenoids and bioethanol. Also it has been previously manipulated to produce other heterologous metabolites such as artemisinic acid. Since n-butanol and ethanol only differ by two carbons, S. cerevisiae may be able to tolerate high concentrations of n-butanol by the same mechanisms it tolerates ethanol. Recently, S. cerevisiae has been demonstrated to have tolerance to n-butanol [Fischer, Klein-Marcuschamer and Stephanopoulos, 2008].

Figure 1. Pathways for the metabolic conversion of biomass, which convert carbohydrate biomass into the central metabolic intermediates pyruvate and acetyl-CoA [Fischer, Klein-Marcuschamer and Stephanopoulos, 2008].

Figure 2. Product pathways, which convert central intermediates into fuels. Reducing equivalents, shown with yellow arrows, are generated in Figure 1 and consumed in Figure 2 [Fischer, Klein-Marcuschamer and Stephanopoulos, 2008].

Steps to n-butanol from Acetyl-CoA

Figure 3. Conversion of Acetyl-CoA to Acetoacetyl-CoA by the enzyme thiolase and its isozymes The first step to reach n-butanol is converting Acetyl-CoA to Acetoacetyl-CoA with the help of thiolase enzymes which are ERG10 (S. cerevisiae), phaA (R. eutropha) and atoB (E. coli). Atsumi, et al. (2008) showed that atoB has the highest yield of n-butanol production in E. coli, which is a native enzyme of E. coli. AtoB was also engineered to give even more efficiency in that paper (Atsumi, et al., 2008). The enzyme phaA belongs to R. eutropha has been used in the production of hydroxycarboxylic acids and gave highly efficient results (Lee and Lee, 2003). According to these examples, using the enzyme that belongs to the original species increases the yield of the product. So in this case using ERG10 should give the highest amount of n-butanol in S. cerevisiae. Indeed in the paper of Steen, et al., (2008), it has been showed that the strains containing ERG10 and phaA gave the highest n-butanol concentration.

Figure 4. Conversion of Acetoacetyl-CoA to 3-hydroxybutyryl-CoA by the enzyme 3-hydroxybutryl-CoA hydrogenase and its isozymes The second step is converting Acetoacetyl-CoA to 3-hydroxybutryl-CoA with the enzyme 3-hydroxybutryl-CoA hydrogenase and its possible isozymes Hbd and phaB. One the isozymes uses NADPH as a cofactor whereas the other uses NADH as a cofactor. It is well known that acetyl-CoA is used in TCA cycle in aerobic conditions with NADH. Since consumption of acetyl-CoA in TCA cycle is not wanted fermentative grown conditions should be applied to reach higher n-butanol concentrations. Thus using the isozyme Hbd which processes NADH as a cofactor can give better results. On the other hand, phaB is obtained from R. eutropha and it is believed that it can work with phaA more effectively more than any other enzyme can due to their synchronization and optimization through evolution in R. eutropha. To understand this complexity, Steen, et al., (2008) prepared eleven different set of DNA strains including ERG10, phaA, phaB and Hbd combinations and it has been showed that combinations of ERG10 as thiolase and Hbd as 3-hydroxybutryl-CoA hydrogenase doubles the production of n-butanol from phaA and phaB combination.

Figure 5. Conversion of 3-hydroxybutyryl-CoA to crotoyl-CoA by the enzyme crotonase. In the third step 3-hydroxybutryl-CoA is converted into crotonoyl-CoA by the enzyme crotonase. The crotonase family comprises mechanistically diverse proteins that share a conserved trimeric quaternary structure, the core of which consists of 4 turns of a (beta/beta/alpha)n superhelix [Holden, et al., 2001]. Despite of all of literature search there are not many articles about possible isozymes of crotonase in butanol production thus crotonase from Clostridium beijerinckii is the only option for this reaction step.

Figure 6. Conversion of crotoyl-CoA to butyryl-CoA by the enzyme butyryl-CoA dehydrogenase and its isozymes. In this step crotonoyl-CoA is converted into butyryl-CoA by the enzyme butyryl-CoA dehydrogenase. This enzyme has two possible isozymes that are tried on n-butanol production from S. cerevisiae which are bcd&EtfAB complex from C. beijerinckii and Ccr from Streptomyces collinus. Also there are other isozymes from other microorganism that used as butyryl-CoA dehydrogenase in E. coli, C. acetobutylicum (Becker et al., 1993), S. coelicolor (O’Neill et al., 1998) and M. elsdenii (Wallace et al., 1995).However there is no published paper about production of n-butanol from S. cerevisiae with the enzymes of these alternative species. According to Steen, et al., (2008) Ccr from S. collinus slightly increases the yield of n-butanol than bcd/EtfAB complex does.

Figure 7. Conversion of butyryl-CoA to butrylaldehyde and n-butanol by the enzyme butanol dehydrogenase. These are the final steps to reach butanol from S. cerevisiae by the enzyme butanol dehydrogenase. AdhE2 is a modified isozyme of butanol dehydrogenase. Originally it belongs to C. beijerinckii and C. acetobutylicum that can produce butanol with ethanol and acetate in fermentative conditions. However the yield of butanol is lower than ethanol production in those species metabolism. Thus the enzyme Adh was engineered to increase the butanol productivity of the microorganism.

In the paper of Atsumi, et al. (2008), the genes related with butanol production was found and competitive ones were eliminated with gene knock out. According to paper, knocking out only Adh erratically decrease the ethanol production while acetone production slightly increased in the E. coli. Thus an assay prepared to knock out unnecessary and competitive genes and enzymes to butanol and introduced to microorganism. Another AdhE2 is introduced to the microorganism from C. beijerinckii which was based on the relative affinities of each Adh enzyme towards acetyl-CoA and butyryl-CoA. While the activity of the E. coli Adh towards butyryl-CoA is not much less than the C. acetobutylicum Adh, its activity torwards acetyl-CoA is four times higher than the C. acetobutylicum ADH for the same substrate which favors adhE2 over adhE for 1-butanol production (Atsumi, et al., 2008). Methods

After completing correct media requirements, reagents and chemicals for standard molecular biology methods, plasmid and strain construction takes place in the studies. Generally, microorganisms that are used in experiments are purchased from ATCC according to their catalogue number. After growing the microorganisms in cultivation media (each species has specific grow conditions and reaching a high purity in microorganism concentrations is mandatory), strains that are needed to be changed are decided and suitable plasmids are prepared in convenient cultivation medium. In the work of Steen, et al., (2008), S. cerevisiae strain BY4742, a derivative of S288C, was used as the parent strain for all yeast strains. This strain was grown in rich YPD medium at 30°C. Engineered yeast strains were grown in SD medium with leucine, uracil, histidine, and/or methionine dropped out where appropriate. For induction of genes expressed from the GAL1 and GAL10 promoters, S. cerevisiae strains were grown in 2% galactose as the sole carbon source unless otherwise indicated Among strong constitutive promoters, the most commonly used ones for high-level gene expression in S. cerevisiae are pTEF1, pPGK1, GAL1 and GAL10 as well as promoters derived from various glycolytic pathway genes.

Since yeast prefers glucose as a carbon source, expression vectors which contain two constitutive promoters, pTEF1 and pPGK1 should be used which provide high levels of gene expression in glucose containing medium. The promoters GAL1 and GAL10 are tightly repressed by glucose and they are strongly induced by galactose. Since the expression from the very strong GALl promoter can lead to toxic effects, using pTEF1 and pPGK1 promoters can be better option (Mumberg, MulIer and Funk, 1994) The selected genes from microorganisms are cloned from genomic DNA, in this case hbd gene stands for 3-hydroxybutyryl-CoA dehydrogenase; crt, crotonase; bcd, butyryl-CoA dehydrogenase; and etfA &etfB, two-electron transferring flavoproteins A & B from C. beijerinckii. After identifying all of the genes and enzymes, all genes are PCR amplified with Phusion polymerase.

Primers should be designed to have flanking regions homologous to the plasmid insertion regions, either one of the promoters and the terminator. Then plasmid construction can be carried out using the Sequence and Ligation Independent Cloning (SLIC) method [Li and Elledge, 2007]. To summarize, the copy number of a given homologous or heterologous gene can be changed by introducing single- or multicopy plasmid vectors. Alternatively, genomic insertions or deletions via homologous recombination can be carried out. The second target for modifying the level of gene expression is the promoter upstream of a gene. For example, gene overexpression can be achieved by fusing a strong promoter upstream of the gene of interest. When combined with multicopy expression, this approach maximizes the expression level [Nevoigt, 2008]. Immobilization of the yeast

For immobilization of the engineered S. cerevisiae entrapment method within calcium alginate has been chosen due to its wide usage in this process. In this method, the porous matrix is synthesized in situ around the cells. Most often, natural and synthetic polymeric hydrogels such as Ca-alginate, j-carrageenan, agar, polyurethane, polystyrene and polyvinylalcohol are being used (Ramakrishna and Prakasham 1999). These polymeric beads are usually spherical with diameters ranging from 0.3 to 3 mm [Verbelen, et al., 2006]. This method is especially suited to living cells as it tends not to damage them. Since the engineered S. cerevisiae is relatively new approach for n-butanol production, the yeast cells cannot be found easily and processed.

After consideration of the applications of this method which mainly are immobilisation of cells in bioreactors, entrapment of plant protoplasts and plant embryos (artificial seeds) for micro-propagation, immobilisation of hybridomas for the production of monoclonal antibodies, and the entrapment of enzymes and drugs, it has been found that it is the most versatile and suitable method to for n-butanol production [Madden, 2007]. The assumptions were made due to particle size, process conditions, yeast sizes and extraction method. Thus the assumptions are: * All the process conditions are controlled well to get the best possible yield, * The size of the particles is round 0.3 mm, the shape is spherical to get the best mass transfer rate, * The products can be extracted from matrix very quickly, * All the yeast cells are entrapped in the matrix.



Steen E. J, Chan R., Prasad N., Myers S., Petzold C. J., Alyssa Redding, Mario Ouellet and Keasling J.D., Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol. Microbial Cell Factories, 2008, 7:36. Atsumi S, Cann A. F., Connor M. R., Shen C. R., K. M. Smith, M. P. Brynildsen, K. J.Y. Chou, T. Hanai1, J. C. Liao, Metabolic engineering of Escherichia coli for 1-butanol production. Metabolic Engineering Volume 10,
Issue 6, 2008, Pages 305–311 Jina C, Yao M, Liu H, Chia-fon F. Lee, Jing Ji, Progress in the production and application of n-butanol as a biofuel. Renewable and Sustainable Energy Reviews Volume 15, Issue 8, 2011, Pages 4080–4106. G. Knothe, 2010, Progress in Energy and Combustion Science 36, pages 364–373. Evans, G. International Biofuels Strategy Project. Liquid Transport Biofuels-Technology Status Report, National Non-Food Crops Centre, 2008-04-14. Jin C., Yao M., Haifeng Liu, Chia-fon F. Leed, Jing Ji, Progress in the production and application of n-butanol as a biofuel, 2011. Renewable and Sustainable Energy Reviews Volume 15, Issue 8, Pages 4080–4106. Holden H. M., Benning M., Haller T., Gerlt J. A., The Crotonase Superfamily: Divergently Related Enzymes That Catalyze Different Reactions Involving Acyl-Coenzyme A Thioesters, 2001. Acc. Chem. Res., 34 (2), pp 145–157 Martin J.J, Pitera D.J, Withers S., Newman J. D., and Keasling D., Engineering a mevalonate pathway in Escherichia coli for production of terpenoids, 2003. Nature Biotechnology 21, p. 796 – 802. Fischer C. R., Klein-Marcuschamer D., Stephanopoulos G., Selection and optimization of microbial hosts for biofuels production, 2008. Metabolic Engineering, Volume 10, Issue 6, Pages 295–304 Mumberg, MulIer and Funk, 1994, Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression. Nucleic Acids Research, Vol. 22, No. 25. Pages. 5767-5768. Verbelen Pieter J., De Shutter David P., Delvaux Filip, Verstrepen Kevin J., Delvaux Freddy R. (2006) Immobilized yeast cell system for continuous fermentation applications Biotechnol Lett , 28:1515–1525 Li M. Z., Elledge S.J., 2007, Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods, 4(3), pages 251-6. Madden D., Immobilisation of yeast in calcium alginate beads, 2007. National Centre for Biotechnology Education, University of Reading. Nevoigt E., Progress in Metabolic Engineering of Saccharomyces cerevisiae, 2008. Microbiol Mol Biol Rev., 72(3) pages. 379–412. Ro, et al., 2006. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440, pages, 940-943.

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