The cloning and expression of chymosin (rennin) genes in microorganisms

Trends in Biotechnology, Vol. 1, No. 3, 1983

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The cloning and expression of chymosin (rennin) genes in microorganisms Teruhiko Beppu C h y m o s i n , also known as rennin, a milk-clotting e n z y m e obtained f r o m the s t o m a c h of calves, is used in the manufacture of cheese. The production of this e n z y m e by recombinant D N A technology is now b e c o m i n g possible. A new source of this e n z y m e to replace or supplement the animal product or similar, naturally occurring fungal e n z y m e s will be of great e c o n o m i c value. Chymosin (also called calf rennin, or rennet in the case of impure commercial preparations) is a protease obtained from the stomach of suckling calves. It has a high milk-clotting activity along with a very low proteolytic activity. These two properties make chymosin very suitable for the coagulation of milk, one of the processes in cheese manufacturing. The dairy industry requires large amounts of coagulant (estimated at about $120 million per Teruhiko Beppu is Professor of Fermentation and Microbiology at the Department of Agricultural Chemistry The University ofTokyo~ Yayoi 1-1-1, Bunkyo-ku, Tokyo, Japan.

year) to produce approximately 12 million tons of cheese in 1981. The supply of chymosin, however, is declining since most calves are now being raised to become mature animals. On the other hand, world production of cheese is constantly increasing, and thus a severe gap has opened between the demand for chymosin and its supply from traditional sources. The gap is currently closed by the development over the last 15 years of several substitute enzymes from fungi. Mucor rennins from two closely related species of fungi, Mucorpusillus and M. miehei, have sufficiently high milkclotting activity and are used to

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produce various types of cheese on a large commercial scale 1'2. Another enzyme from a fungus, Endothia parasitica, is also used for manufacturing cheese even though its clotting activity is relatively low. These microbial rennins are now replacing chymosin for more than half of the world production of cheese. A constant demand for real chymosin still exists, however, because of subtle but distinct differences between their properties and those of fungal rennins. The ratio of clotting activity to proteolytic activity is one and a half times that of the highest value for any microbial rennins. The heat stability of the Mucor rennins is slightly greater and thus their proteolytic activity remains after the milk curd has been harvested; this causes several secondary difficulties in the industrial processes. These differences, allied with the conservative attitude of traditional cheesemakers, provide an incentive to produce chymosin with the aid of recombinant DNA technology.

Characteristics of chymosin Chymosin is a special member of the acid protease group of enzymes which is characterized by high substrate

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Fig. 1. Fractionation of mRNA from the mucosa of calf stomach by sucrose gradient centrifugation (A), and the detection of the translational products coded by each fraction in SDS-gel electrophoresis (B). (A) Total mRNA encoding the incorporation of [3H]-leucine into the acid-insoluble fraction in reticulocyte lysate system ([--7)and mRNA encoding the incorporation of [3H]-leucineinto prochymosin which is adsorbed on the immobilized antibody ( I ) . (B) Autoradiogram of SDS-gel electrophoresis of translational products coded by each fraction. The arrow indicates the position of authentic prochymosin. Note abundant prochymosin in fractions 12-14 (B), in which 50% or more of the mRNA codes for prochymosin

(A).

© 1983, Elsewer Science Pubhshers B V , Amsterdam 0160 - 9430/83/$01 00

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Trends m Biotechnology, VoL 1, No. 3, 1988

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restriction enzymes are regenerated in the hybrid plasmid when the poly (C) tails of the vector and the poly (G) tails of the cDNA are ligated. specificity and extremely low proteolytic activity. A main protein component of milk, a-casein, is stably dispersed as colloidal micelles in which x-casein works as a stabilizer. Chymosin cleaves x-casein at only one peptide bond, between phenylalanine and methionine, and consequently destabilizes the micelles and causes milk to clot. The resultant curd is not further degraded since chymosin has a low proteolytic activity; this fact assures a high yield of curd and reduces the possibility that low molecular weight peptides with a bitter taste will be formed. Chemical modification

studies on chymosin have revealed the presence of two essential carboxyl groups on aspartyl residues at its catalytic site3. In addition, it has been suggested that a histidyl residue may be responsible for chymosin's characteristic catalytic properties 4'5. Chymosin is secreted from the mucosal tissue of the fourth stomach (abomasum) of suckling calves as an enzyme precursor (zymogen) called prochymosin (prorennin) which contains 365 amino acid residues and has a mol. wt of about 41 000. Its N-terminal peptide of 42 amino acids is cleaved autocatalyticalty under acidic conditions to form

the active enzyme (mol. wt approx 35 600). Chymosin and prochymosin share common antigenic determinants. Two chromatographically different forms of the enzyme, A and B, are known. Foltman et al. 3,6 have determined the entire amino acid sequence of prochymosin B and the partial sequence of A, and observed only a single amino acid substitution. Prochymosin seems to be a good target for cloning and expression by recombinant DNA technology, since possible lethal effect on bacterial host cells due to proteolytic activity may be avoided. Prochymosin produced in bacterial cells may be easily converted to chymosin by incubation of cell extracts at acidic pH.

Identification and purification of prochymosin mRNA In animal and other higher eukaryotic organisms, most chromosomal genes have intron sequences which interrupt the coding sequences for proteins. When these genes are transcribed in eukaryotic ceils, the introns are spliced out of the mRNA transcript and subsequent translation gives proteins with correct amino acid sequences. Since bacterial cells have no such splicing mechanism, genes obtained directly from eukaryotic chromosomes (genomic genes) cannot be correctly expressed in bacteria. One way to overcome this difficulty is to synthesize complementary DNA (cDNA) by reverse transcription of mRNA from which the introns have been spliced out. Highly specialized cells within tissues and organs frequently contain abundant amounts of the mRNA species encoding the polypeptides which they synthesize preferentially. This provides an opportunity to isolate the specific mRNA and to make the following cloning work easier. The mRNA for prochymosin is abundant in the mucosal tissue of calf abomasum. The detection and purification of this mRNA from the total RNA extracts of the tissue was first achieved by Uchiyama et al. 7,8By using standard procedures, i.e. poly(U) sepharose affinity chromatography for poly (A)-tailed mRNA and molecular size fractionation by sucrose density gradient sedimentation, they obtained fairly large amounts of mRNA. When tested in a cell-free protein synthesizing system (Fig. 1) this mRNA fraction was

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Trends in Bioteehnology, VoL 1, No. 3, 1983

found to code almost entirely for prochymosin. The relative abundance of the prochymosin mRNA in this tissue was also indicated by the observations by Harris et al. 9 and Moir et al. 1o that even non-fractionated mRNA preparations (poly (A)-enriched RNA) gave prochymosin as a major translational product in the cell-free protein synthesizing system. Cloning proehymosin eDNA Using the enriched mRNA as a template, double-stranded cDNA can be prepared by standard reverse transcription techniques and be introduced in Escherichia coli using plasmid or phage vectors (Fig. 2)*. Since the enriched mRNA preparation still contains various RNA species, the cDNA clones thus obtained will be heterogeneous (a 'cDNA library'). Two-step screening processes (primary by colony hybridization with radioactive mRNA or its cDNA transcript as a probe and secondary, more detailed, characterization) are usuaUy carried out to select the clone containing a specific cDNA sequence. Two different approaches have been used. Nishimori et al. i,.12 prepared a cDNA library by using the plasmid PBR322 as a vector. After first screening by colony hybridization, they carried out a hybrid-arrested translation assay (a negative mRNA hybrid selection, Fig. 3) to identify the prochymosin cDNA clones. The enriched mRNA for prochymosin was annealed with each cDNA insert obtained from the candidate clones and then introduced into the cell-free protein synthesizing system. SDSpolyacrylamide gel electrophoresis of the translational products revealed selective inhibition of prochymosin synthesis by the previous annealing with several cDNA inserts, which indicated the presence of the coding sequence for prochymosin in those inserts (Fig. 4). In a different approach Moir et al. lo carried out cloning with an fl bacteriophage vector and identified prochymosin cDNA clones by using positive mRNA hybrid selection (Fig. 3). Harris et aL 9 used a chemically synthesized oligodeoxynucleotide primer to obtain a specific probe for prochy*See also Trends in Biotechnology (1983) 1,

May/June, centre pages, for the principles of cDNAcloning.

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Procedures for positiveand negativehybrid selection of a specificmRNA.

mosin cDNA clones. Chymosin has an amino acid sequence (183-186) containing two adjacent methionines (Asn-Met-Met-Asn) and the degeneracy of the genetic code of this tetrapeptide is minimal (see Glossary). One of the synthetic dodecanucleotides with a sequence which codes for amino acids 183-186 was found to hybridize and specifically prime the synthesis of cDNA from mRNA of calf stomach which contained a part of the prochy-

mosin coding sequence. This probe was therefore used to identify prochymosin cDNA clones. The nucleotide sequence of 1095 base pairs from these clones was in substantial agreement with the reported amino acid sequence of prochymosin. The sequences by Nishimori et al. and Moir et al. have a codon for aspartate at the 286th position of the prochymosin polypeptide while that by Harris et aL has glycine at the same position. This is

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Trends in Biotechnology, VoL 1, No. 3, 1983

the single difference between the reported amino acid sequences ofprochymosin A (Asp) and B (Gly). The sequence also indicated the presence of the signal peptide with 16 amino acids of a hydrophobic nature at the N-terminus; this is assumed to be essential for the secretion of extracellular proteins. Prochymosin may, therefore, by synthesized in vivo as a precursor, preprochymosin, which contains the hydrophobic signal sequence.

Expression of prochymosin eDNA in E. coli

Gene expression occurs in two stages, transcription and translation. To ensure the effective expression of a foreign gene in bacterial cells, the promotor sequence of a bacterial gene for transcription and sequences for the ribosome-binding site and AUG initiation codon of mRNA for translation should be present at positions upstream of the coding sequence. A simple way of achieving this is to fuse a foreign coding sequence (in the correct phase so that the codon reading frame is unaltered) to the proximal coding region of bacterial gene. By such a con-

struction, a foreign gene is expressed as a hybrid protein with a short length of bacterial protein at its N-terminus. This procedure was adopted by Nishimori et al. ~3to express prochymosin eDNA in E. coli in which the lac Z (/3-galactosidase) gene with a lac hyper promoter (lac UV 5) was used. The eDNA sequence for the peptide from the 5th arginine to the C-terminus of prochymosin, followed by a stop codon, was joined to the proximal coding sequence of the fl-galactosidase gene (Fig. 5). This construction yields a fused protein very like prochymosin, in which only the N-terminal 4 amino acids of prochymosin are missing; in their place is the short N-terminal peptide (10 amino acids) of flgalactosidase. This protein produced by E. coli carrying the hybrid plasmid reacts with anti-prochymosin antibody (Fig. 6). Amounts of the protein markedly increased when cells were cultivated in the presence of isoproply-flD-galactoside (IPTG), a/3-galactosidase inducer. The maximum yield was estimated to be more than 30 000 molecules per cell (approximately 0.5 per cent of total cell protein).

Glossary degeneracy of the genetic code - because most amino acids are speclfied by more than one codon (e.g. s~x codons for leuclne) Jt fs difficult to design a probe whlch wdl hybridlze well with the mRNA or gene sequence for a speclfic portion of a peptide. Worklng back from a known amlno acid sequence usually gives a large number of possible nucleottde sequences. However, methlonJne has only one codon and asparaglne has only two. Hence, there are only 4 possible ways in whlch the cell can code for the sequence Asn-Met-MetASh, which occurs In chymos~n. Thus only four dodecanucleotldes need be synthesized to be sure of producing one that will hybndize completely with a nucleotlde sequence encoding these amino acids (see foot of page) poly(A) - the 3' end of most eukaryotlc mRNAs carnes a sequence of 150-200 adenine nucleotides whfch are added to the molecule after transcription. Poly(A) tails seem to enhance the stablhty of mRNA molecules. The presence of this sequence can be utlhzed by hybridizing the NH 2 - 5' 3'

183 Asn AA~: TI-~

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185 Met AUG TAC

tails to a complementary sequence of nucleotldes (either poly(U) or poly(T)) attached of beads of, for example, Sepharose. In this way the total population of actwe mRNAs can be separated from the other cell constituents, Includtng other nucleic acids. primer- a short sequence (approx I 0 base paws) of DNA which ts complementary to a sequence of mRNA. The pnmer allows reverse transcriptase to start copying the adjacent sequences of mRNA. signal sequence - most proteins which are transported across membranes (efther between cell compartments or across the cell membrane to the exterior) have a 'signal sequence' at their N-terminus. This consists of about 15-30, mainly hydrophobic amino acid residues. The presence of a s~gr,al sequence on a peptlde is often indicated by the prefix 'pre-'. The s~gnat sequence is removed by speofic proteolytic cleavage after passage across the membrane. Thus preprochymosln becomes prochymosln. 186 Asn - A/~-TI-~--

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a

b

c

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Fig. 4. Specific inhibition of in vitro

synthesis of prochymosin by hybridization with cloned cDNA. The arrow indicates the position of authentic prochymosin. (a) Blank ofreticulocyte lysate system without mRNA. (b) Hybridization with the vector, pBR322. (c-t) Hybridization with each cloned eDNA insert. Note specific inhibition of prochymosin was observed with clones c and e which are therefore deduced to carry the prochymosin eDNA gene. In general, the expression of cDNA as a fused product suffers from an inherent drawback; recovery of the desired protein becomes difficult. However, in the case of prochymosin, recovery of active chymosin from the fused product seems possible, since the short peptide replacement at the N-terminal region ofprochymosin does not affect its autocatatytic processing activity; the unwanted bacterial peptide would be discarded along with the 'pro' part ofprochymosin to yield chymosin itself. Another problem is that prochymosin synthesized in E. colicells has a marked tendency to be deposited in an insoluble form which is recovered from the membrane fraction of cell extracts]L This must be efficiently dissolved and chymosin recovered to ensure a high yield.

Future trends Chymosin production by recombinant DNA technology seems to be promising, but some problems remain. Firstly, there are competitors in the market, the microbial rennins. Promotors stronger than lac UV 5 or those that cause the products to be secreted from the host cells may enhance the productivity in E. coll. For example, expression under the control of the trp

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Trends in Biotechnology, VoL 1, No. 3, 1983 E

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Fig. 5. Construction of a hybrid plasmid expressing prochymosin cDNA. E, S, K and B indicate restriction sites for EcoRI, SalI, KpnI and BamHI, respectively. lacpo-z'is a truncated fl-galactosidasestructural gene (lacz) with its promoter, which causes expression in the direction indicated (~). The vector was prepared by cleavagewith EcoRI, followed by treatment with DNA polymerase to create a blunt end to which the BamHI linker was ligated; cleavage with BamHI and SalI then produced the linear vector DNA shown. The cloned prochymosin cDNA was cleaved with BamHI and KpnI to produce the small B-K fragment, and with KpnI and SalI to produce the large K-S fragment. Ligation ofthe vector with the B-K and K-S fragments generated the desiredhybrid plasmid, which codes for the first 10 amino acids offl-galactosidase, followed by all except the first four amino acids ofprochymosin (see the sequence at the bottom of the diagram).

promotor gives markedly high yields of prochymosin~h Another problem is severe regulations concerning the use of food additives. Several microorganisms which have been used in brewery or food processing may be more satisfactory 1981 hosts than E. coli. In Collaborative Research Inc. claimed a patent for the expression of prochymosin in yeast and clearly the development of new host/vector systems with increased yields will be of great importance for the economic future of this work.

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References 1 Arima, K., Yu, J., Iwasaki, S. and Tamura, G. (1968)Appl. Microbiol. 16, 1727-1733 2 Sternberg, M. (1976) in Advances in

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Applied Microbiology (Perlman, D., ed), Vol. 20, pp. 135-157 Foltmann, B., Pederson, V. B., Kauffman, D. and Wybrandt, G. (1979)J. Biol. Chem. 254, 8447-8456 Hill, R. D. and Laing, R. R. (1965) Biochim. Biophys. Acta 99, 352-359 Etoh, Y., Shoun, H., Arima, K. and Beppu, T. (1982)J. Biochem. (Tokyo) 91, 747-753 Fohman, B., Pederson, V. B., Jacobson, H., Kauffman, D. and Wybrandt, G. (1977)Proc. NatlAcad. Sci. USA 74, 2321-2324 Uchiyama, H., Uozumi, T., Beppu, T. and Arima, K. (1980)Agnc. Biol. Chem. 44, 1373-1381 Beppu, T., Nishimori, K., Kawaguchi, Y., Hidaka, M. and Uozumi, T. (1982) in Geneticsof lndustrial Microorganisms, 1982 (Ikeda, Y. and Beppu, T., eds), pp. 195-201 Harris, T. J. R., Lowe, P. A., Lyons,

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Fig. 6. Immunological detection of pmchymosin fused protein synthesized in E. coil Cell-free extracts of the bacteria carrying the expression plasmid were analysed by SDS-gel electrophoresis. Separated protein bands were blotted onto a nitrocellulose filter and then bands which reacted with anti-prochymosin antibody were detected. (1) Authentic prochymosin, 2/~g; (2) authentic prochymosin, 1 /~g; (3) extract of bacteria induced with IPTG; (4) extract of bacteria not induced with IPTG.

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A., Thomas, P. G., Eaton, M. A. W., Millican, T. A., Patel, T. P., Bose, C. C., Carey, N. H. and Doel, M. T. (1982) Nucleic Acid Res. 10, 2177-2187 Moir, D., Mao, J., Schumm, J. W., Vovis, G. F., Alford, B. L. and Taunton-Rigby, A. (1982) Gene 19, 127-138 Nishimori, K., Kawaguchi, Y., Hidaka, M., Uozumi, T. and Beppu, T. (1981) J. Biochem. (Tokyo) 90, 901-904 Nishimori, K., Kawaguchi, Y., Hidaka, M., Uozumi, T. and Beppu, T. (1982) J. Biochem. (Tokyo) 91, 1085-1088 Nishimori, K., Kawaguchi, Y., Hidaka, M., Uozumi, T. and Beppu, T. (1982) Gene 19, 337-344 Emptage, J. S., Angal, S., Doel, M. T., Harris, T. J. R., Jenkins, B., Lilley, G. and Lowe, P. A. (1983) Proc. NatIAcad. Sci. USA 80, 3671-3675