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| {{Microbial production of chymosin}} | | {{Microbial production of chymosin}} |
| ==Introduction==
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| [[Image:Sin_titulo.jpg|thumb|300px|right|Process of cheese production. Curdling (clotting) is done in the early stages of production. Casein is broken in smaller fragments by the action of chymosin.]]
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| Cheese production requires the use of a 323 aminoacids protein called chymosin (also known as rennin). Chymosin (36 kDA) is a proteolitic enzyme which is usually obtained from calf stomachs. The role of this enzyme is to coagulate the milk, which is very important for digestion of milk in young animals. Initially, chymosin is secreted as inactive prochymosin and then activated at low pH. Once it is active, it breaks milk protein k-casein in a specific point and effects clotting which is the first step in cheese production. By clotting the milk, a solid product called curd is obtained. Curd is then processed to make cheese[1,2].
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| The increasing world production of cheese, coupled with a decline in the number of slaughtered calves, has stimulated a search for alternative sources of chymosin. One of these alternative sources is the use of recombinant chymosin produced by microorganisms.
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| Recombinant chymosin is mainly produced by funghi, but it can also be produced using bacteria.
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| ==Chymosin mechanism of action==
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| [[Image:break.jpg|thumb|300px|right|Mechanism of action of chymosin. Chymosin breaks κ-casein's peptide bond between Phe 105 and Met 106. Hydrophobic part is shown in blue while hydrophilic part is shown in green.
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| http://dwb.unl.edu/Teacher/NSF/C08/C08Links/www.fst.rdg.ac.uk/courses/fs560/topic1/t1g/t1g.htm]]
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| Milk consists of water, fat, protein, phosphate, lactose, citric acid and inorganics such as calcium phosphate. The protein component of milk can be divided into two groups, the casein fraction (α<sub>s1</sub>-casein, α<sub>s2</sub>-casein, β-casein and κ-casein) and the whey proteins (β-lactoglobulin, β-lactoalbumin, immunoglobulins and serum albumin). The casein proteins are the ones that will form the curd during cheese making, specifically κ-casein [3].
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| [[Image:chim.jpg|thumb|300px|left|3D structure of chymosin.http://dwb.unl.edu/Teacher/NSF/C08/C08Links/www.fst.rdg.ac.uk/courses/fs560/topic1/t1g/t1g.htm]]
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| Chymosin, commonly known as rennin, is the main milk-coagulating enzyme that consists of a single polypeptide chain of 323 amino acids with intramolecular disulfide linkages. Preparations of calf rennet contain two forms of chymosin, A and B. The only difference between chymosins A and B is one amino acid in the polypeptide chain; the chymosin A contains an aspartic acid residue at position 286, whereas the chymosin B contains a glycine residue at the same position [2].
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| Like other acidic proteases from the gastric juice, chymosin is secreted as an inactive precursor, prochymosin. The precursor is stable at weakly alkaline pH whereas the active enzyme is rapidly denatured at pH values above 7. Below pH 5 prochymosin is converted into chymosin by a limited proteolysis during which a peptide segment is cleaved from the N-terminus. The proteolytic activity of chymosin has optimum pH about pH 3-5.
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| The milk-clotting activity of chymosin is due to proteolysis of the κ-casein. κ-Casein consists of two parts: one hydrophobic and one hydrophilic and, in milk, this protein stabilizes the casein micelles against aggregation. During milk-clotting, a Phe-Met bond is hydrolysed, the hydrophilic part of the κ-casein is liberated and aggregation occurs[3]. Chymosin specifically recognises the sequence from His 98 to Lys 111 and cleaves the peptide bond between Phe 105 and Met 106 in the κ-casein chain[4].
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| ==Production of recombinant chymosin==
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| [[Image:recomb.jpg|thumb|300px|right|Process of production of recombinant chymosin. http://www.ncbe.reading.ac.uk/ncbe/gmfood/chymosin.html]]
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| Recombination is the process by which genetic material is broken and then joined to a new genetic material. In this case, the chymosin gene from the cow is taken out of the genome and then introduced into a plasmid. The plasmid is then introduced into a microorganism which will start producing the chymosin by transcribing and translating the gene from the plasmid.
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| The first step in this process is to obtain a piece of tissue from the calf stomach and isolate the DNA from the cells. After isolating the DNA it is necessary to amplify the chymosyn gene using PCR to increase the amount of gene fragments compared to other sequences of the genome. Another option is to isolate the mRNA directly and turn it into cDNA using the enzyme retrotranscriptase; this way we would have the DNA fragment without the introns as genes are found in prokaryotic microbes.
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| The second step is to introduce the gene sequence into plasmids that can be then introduced in the target microbes. The gene is often introduced after a promoter sequence that we can control and increase its expression.
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| After introducing the gene in the plasmid and the plasmid in the microbe, it will start producing chymosin and the last step would be to purify it. The most common way of doing this is using a purification column in which chymosin would stick to the column due to its affinity to the matrix inside the column while the other proteins would not stick[2].
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| An important fact to be aware of while using recombination of genes from one species into a different one is the specific variation in codon usage. This problem usually causes low expression levels of the protein that we want to produce. To avoid this problem, the DNA sequence of the gene has to be modified and adapted to the codon usage of the specie in which the chymosin will be produced, this is called codon-optimization[5].
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| Another important step in the production is the secretion of the protein by the microbial cells, the sequence of the chymosin can be also changed to improve its secretion. The modifications are often additions of secretory control regions of the microbe that is going to be used to produce the gene.
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| One example of these strategies to improve production of chymosin is the addition in frame of the glucoamylase gente in <i>Aspergillus</i>, right after the prochymosin gene. Glucoamylase is a secreted protein that is secreted even with the chymosin protein attached to it. With this gene fusion a higher concentration of secreted prochymosin is obtained[6].
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| Some other useful tools to increase the production are the addition of transcriptional and translational control regions of the host specie to the gene which may be not only specific promotors (as mentioned above) but also specific transcription terminators[7].
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| A common way to control the production (and increase it) is to add a promotor that is inducible by some sort of molecule. When we add that molecule (inductor) to the culture media the microbe will start transcribing our gene and producing chymosin. One example of inductor that is used when working in <i>Escherichia coli</i> is the molecule IPTG, which is a lactose analog and binds to the represor protein in the lac operon, thus inducing it. Using the lac operon promotor in front of the chymosin gene and the IPTC as inductor increases the production of the protein[8].
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| ==Examples of microbes used in chymosin production==
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| [[Image:niger.jpg|thumb|300px|right|<i>Aspergillus niger</i>.
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| http://www.gefor.4t.com/hongos/aspergillusniger.html]]
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| The most common microbes used for the production of recombinant chymosin are funghi, but this protein can also be produced using bacteria. Three of the most used microbes are <i>Aspergillus niger, Kluyveromices lactis</i> and <i>Escherichia coli</i>. These three species has really well known genetics and metabolic pathways, specially <i>Aspergillus niger and Escherichia coli</i>, which make them very useful for recombination experiments like chymosin production.
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| ===<i>Aspergillus niger</i>===
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| <i>Aspergillus</i> is the name used for a genus of molds that reproduce only by asexual means. <i>Aspergillus</i> species are common and widespread. They are among the most successful groups of molds with important roles in natural ecosystems and the human economy[9].
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| <i>Aspergillus niger</i> is one of the most common species of this genus[10]. It is found in a large variety of environments, but especially on vegetation, decaying organic matter, and soil. In humans, an Aspergillus niger infection is usually only a problem with people who have a weakened immune system. It most commonly causes lung infections, but it can also spread to other organs and open wounds[11].
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| It first acquired practical importance in 1919, when its ability to produce citric acid was industrially exploited. Gluconic and fumaric acids have been produced with <i>A. niger</i>, although they are of less economic importance. However, since the 1960s, <i>A. niger</i> has become a source of a variety of enzymes that are well established as technical aids in fruit processing, baking, and in the starch and food industries. Gene technology has been successfully applied to improve production processes and to make use of <i>A. niger</i> as an expression system for foreign proteins[12]. That last part is the reason of using this microbe for the production of chymosyn, <i>A. niger</i> is used in a lot of different biotechnological processes, so the genetics are perfectly known.
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| Another reason for using <i>A. niger</i> is that many of the enzymes produced by A. niger, such as citric acid, amylases, lipases, cellulases, xylanases and proteases, are considered GRAS (generally recognized as safe) by the United States Food and Drug Administration[12], so it is not considered a risk for human health.
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| [[Image:kluy.jpg|thumb|300px|right|<i>Kluyveromyces lactis</i> cells. http://www-archbac.u-psud.fr/genomes/]]
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| ===<i>Kluyveromyces lactis</i>===
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| <i>Kluyveromyces lactis</i> is an ascomyceteous yeast (very similar to <i>Saccharomyces cerevisiae</i>) which has the ability to assimilate lactose and convert it into lactic acid, in fact, it has been isolated from milk and constitutes the predominant eukaryote during cheese production.
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| <i>K. lactis</i> is used for the production of beta-galactosidase and its fermentation properties are well understood. It shares most of the features that make <i>Saccharomyces cerevisiae</i> the best eukaryotic model organism (the existence of a well-established classical genetics, the availability of fast and efficient transformation procedures and its efficient homologous recombination machinery) and it is also important that this microbe is also used in other stages of the cheese production chain. It is also considered a GRAS microbe[13,14].
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| It was found that the chymosin could be produced in this host and good levels of secretion into the medium were achieved. The chymosin gene is inserted into the <i>K. lactis</i> chromosome and the yeast is grown by fed-batch fermentation. After fermentation, the yeast is killed by addition of benzoic acid and the chymosin is isolated by filtration[15,16].
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| [[Image:e.coli.jpg|thumb|300px|right|<i>Escherichia coli</i> cells.
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| http://cellbiology.med.unsw.edu.au/units/science/lecture0802.htm]]
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| ===<i>Escherichia coli</i>===
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| <i>Escherichia coli</i> is a bacterium that is a common inhabitant of the human colon. It also lives in the intestine of many other animals, wild as well as domestic. It is a gram negative, rod-shaped gammaproteobacteria. It is the model organism for the study of bacteria.
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| Normally, <i>Escherichia coli</i> does not cause disease although some strains frequently cause diarrhea, and it is the most common cause of urinary tract infections.
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| <i>Escherichia coli</i> is one of the most thoroughly studied of all living things. It is a favorite organism for genetic engineering since its genetics and metabolism are the best known. Another advantage of studying <i>Escherichia coli</i> is that it can be grown very easily and inexpensively in a laboratory setting.
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| The enzymatic properties of recombinant <i>E. coli</i> chymosin are indistinguishable from those of native calf chymosin[17] which is also important because the efficiency of the process of cheese production is the same.
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| The problem with <i>E. coli</i>, however, is that recombinant proteins are frequently synthesized as intracellular inclusion bodies and not secreted, increasing process costs considerably. After disintegration of cells, inclusion bodies are harvested by centrifugation. Another issue with <i>E. coli</i> is that it is not generally recognized as safe for human consumption[2].
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| ==Conclusion==
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| Due to the decrease in the number of slaughtered calves and the ethical problem that killing a huge amount of animals causes, it is important to have an alternative way of producing cheese that does not require the sacrifice of any animal.
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| Moreover, microorganism production of recombinant chymosin offers an easy way of increasing the production of chymosin compared to the amount that can be obtained from young calves.
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| It is important to consider that some modifications may be needed in the gene sequence depending on the microbe that is going to be used for production of any recombinant protein due to the variation between species of the codon usage. Taking this into account may increase dramatically the production of the desired protein.
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| It is also important to modify that sequence to achieve the higher production levels, and that is what current research is focusing on.
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| It is clear that microbes play a role in our lifes that is getting more important as biotechnology and microbiology develop.
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| ==References==
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| [1] [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC394112/?page=1 J S Emtage, S Angal, M T Doel, T J Harris, B Jenkins, G Lilley, and P A Lowe. "Synthesis of calf prochymosin (prorennin) in Escherichia coli". ''Proc Natl Acad Sci U S A''. 1983 June; 80(12): 3671–3675.]
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| [2] [http://www.academia.edu/214179/Cloning_and_In_Vitro-Transcription_of_Chymosin_Gene_in_E._coli S.A. El-Sohaimy Elsayed. E, Hafez,and M.A. El Saadani. "Cloning and In Vitro-Transcription of Chymosin Gene in E. coli". ''The Open Nutraceuticals Journal''. 2010, 3, 63-68.]
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| [3] [http://www.aseanfood.info/articles/11021753.pdf E. Gastaldi, N. Trial, C. Guillaume, E. Bourret, N. Gontard, and J. L. Cuq. "Effect of Controlled κ-Casein Hydrolysis on Rheological Properties of Acid Milk Gels". ''J Dairy Sci''. 2003 Mar;86(3):704-11.]
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| [4] [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1185148/ B Foltmann. "Prochymosin and chymosin (prorennin and rennin)". ''Biochem J''. 1969 November; 115(3): 3P–4P.]
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| [5] [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3056764/ Hugo G Menzella. "Comparison of two codon optimization strategies to enhance recombinant protein production in ''Escherichia coli''". ''Microb Cell Fact''. 2011; 10: 15.]
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| [6] [http://www.ncbi.nlm.nih.gov/pubmed/1366537 Ward M, Wilson LJ, Kodama KH, Rey MW, Berka RM. "Improved production of chymosin in Aspergillus by expression as a glucoamylase-chymosin fusion". ''Biotechnology (N Y)''. 1990 May;8(5):435-40.]
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| [7] [http://www.ncbi.nlm.nih.gov/pubmed/12783481 Cardoza RE, Gutiérrez S, Ortega N, Colina A, Casqueiro J, Martín JF. "Expression of a synthetic copy of the bovine chymosin gene in Aspergillus awamori from constitutive and pH-regulated promoters and secretion using two different pre-pro sequences." ''Biotechnol Bioeng''. 2003 Aug 5;83(3):249-59.]
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| [8] [http://www.ncbi.nlm.nih.gov/pubmed/1823586 Zhang Y, Zhou Wei, Liu N, Yang K. "Expression of calf prochymosin gene in Escherichia coli". ''Chin J Biotechnol''. 1991;7(3):169-75.]
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| [9] [http://www.open-access-biology.com/aspergillus/aspergillusch1.pdf Joan W. Bennett. "An Overview of the Genus ''Aspergillus''".]
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| [10] [http://www.horizonpress.com/aspergillus Masayuki Machida and Katsuya Gom. "''Aspergillus'': Molecular Biology and Genomics". ''National Institute of Advanced Industrial Science and Technology''. January 2010.]
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| [11] [http://www.silvrstat.com/library-aspergillus-niger.php ''Aspergillus niger''.]
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| [12] Schuster, E., Dunn-Coleman, N., Frisvad, J., van Dijck, P. “On the safety of Aspergillus niger – a review”. Applied Microbiology and Biotechnology. 2002. Volume 59. p. 426-435.
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| [13] [http://www.ncbi.nlm.nih.gov/pubmed/23576126 Rodicio R, Heinisch JJ. "Yeast on the milky way: genetics, physiology and biotechnology of ''Kluyveromyces lactis''". ''Yeast''. 2013 Apr 11.]
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| [14] [http://www.ncbi.nlm.nih.gov/pubmed/21141120 Yuan W, Ke T, Du M, Chu X, Hu F, Hui F. "Gene synthesis of the bovine prochymosin gene and high-level expression in Kluyvermyces lactis". ''Sheng Wu Gong Cheng Xue Bao''. 2010 Sep;26(9):1281-6.]
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| [15] Wésolowski-Louvel, M., K.D. Breunig, and H. Fukuhara, Kluyveromyces lactis. In: K. Wolf, editor. Non-conventional yeast in biotechnology. Heidelberg: Springer- Verlag, 1996: p. 139-201.
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| [16] Lachance, M.-A., Current status of Kluyveromyces systematics. FEMS Yeast Research, 2007. 7(5): p. 642-645.]
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| [17] Meisel H, Frister H. "Chemical characterization of a caseinophosphopeptide isolated from in vivo digests of a casein diet". ''Biol ChemHoppe Seyler'' 1988; 369(12): 1275-9
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| Edited by Enrique Rodriguez, a student of [http://www.jsd.claremont.edu/faculty/profile.asp?FacultyID=254/ Nora Sullivan] in BIOL187S (Microbial Life) in [http://www.jsd.claremont.edu/ The Keck Science Department of the Claremont Colleges] Spring 2013.
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| <!--Do not edit or remove this line-->[[Category:Pages edited by students of Nora Sullivan at the Claremont Colleges]]
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