Industrial production of α-amylase by genetically engineered Bacillus

Industrial production of -amylase by genetically engineered Bacillus J. Vehmaanper/i*, P. M. A. Nyberght, R. Tanner, E. Pohjonen, R. Bergelin and M. Korhola Research Laboratories of the Finnish State Alcohol Company, Alko Ltd, POB 350, SF-00101 Helsinki 10, Finland (Received 2 April 1987; revised 30 April 1987)

A genetically engineered Bacillus subtilis strain (A LKO 84) has been introduced for industrial production o f zt-amylase. This strain carries the ~-amylase gene from a traditionally developed production strain B. amyloliquefaciens (ALKO 89) on the muhicopy plasmid pUBIlO. 8 At laboratory scale the recombinant strain ALKO 84 produced in industrial medium about twice as much ~-amylase as the traditional strain ALKO 89. The process for production o f the enzyme was scaled-up to 60 m 3. At this scale B. subtilis ALKO 84 retained its relative superiority to B. amyloliquefaciens ALKO 89, producing about 85% o f the activity obtained at laboratory scale. Stability o[the recombinant plasmid was fimnd acceptable during the large-scale cuhivations with over 90% o f cells retaining plasmid-encoded characteristics throughout.

Key~ords: Bacillus genetic engineering: ~-amylase; enzyme production

Introduction Bacillus strains are widely used for production of industrial enzymes, particularly exoenzymes such as ztamylases and proteases. ~ Strain development has been traditionally carried out by mutagenesis of naturally good producers in order to enhance enzyme yields.-' Modern gene technology now provides the possibility of specifically increasing the dosage ofa gene of interest by cloning it into a muiticopy plasmid. In recombinant DNA research, B. suhtilis has served as a representative of the genus, because of its well-established genetics, 3 and a number of genes encoding industrially important enzymes have already been cloned into this organismff '~ However, to our knowledge there has been no report on industrial-scale applications of Bacillus recombinant strains. Some results may not have been disclosed for proprietary reasons, and in some countries large-scale cultivations have been delayed owing to strict legislation concerning the use of organisms containing recombinant rnolecules. The instability of recombinant plasmids in Bacillustn ~5 has probably further discouraged investigators from proceeding toward an industrial-scale proccss based on a genetically engineered strain. "['he recombinant plasmid p K T H I 0 containing the gene encoding x-amylase from our traditionally developed production strain B. amyloliquefaciens ALKO 89 has been found to bc highly stable in a wild-type B. subtilis strain (ALKO 84). ~' Since this recombinant strain also

* t o whom correspondence should be addressed + Present address: Technology Development Centre. TEK ES, POB 69, SF-(X)IOI Helsinki I0, Finland

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proved to bc able to produce about twice the amount of:tamylase as ALKO 89 at laboratory scale in media suitable for industrial applications, we were encouraged to make an effort to introduce the strain for industrial :t-amylase production. The recombinant strain was found to retain its superiority in :~-amylase production up to the industrial scale (60m3), and the stability of p K T H I 0 remained acceptable under the process conditions used.

Materials and methods Bacterial strains and plasmid B. subtilis (ALKO 84) and B. amylolique/'aciens (ALKO 89) were used throughout in the study. A L K O 84 is an a-amylase-positive prototrophic derivative of B. subtilis 168 strain BGSC (Bacillus Genetic Stock Center, Ohio University. Ohio, USA) 1A289 and contains the recombinant plasmid pKTHI0:8 the a-amylase production by the strain without the recombinant plasmid is insignificant compared to that of the pKTH 10-carrying strain. 8 AI,KO 89 was originally developed for ~-amylase production by five successive steps of mutagenesis and has formerly been known as B. subtilis V'l"['197 t'~ and B. amyhHique/~u'iens E188 or VTT-E-73018. Media and culture conditions The solid media consisted of LB-broth (10 g tryptone, 5 g yeast extract, 5 g NaCI per liter) supplemented with 15 g agar/1 ; starch (10 g/l) and kanamycin (10/~g/ml) were added, when appropriate. The shake flask cultivations for screening of suitable production media were grown at 3 T C in triplicate for 3 days. The inoculum (200 ml) for laboratory fermentations was cultivated in LB-broth 0141 0229/87/090546- 04 $03.00 ,. 1987 Butterworth Publishers

Enzyme production by recombinant Bacillus: J Vehmaanper~ et al. supplemented with 5 #g kanamycin/ml in a shake flask on a rotary shaker. For large-scale cultivations of A L K O 84 the first seed cultures werc inoculated with 1 ml (about 4 × 108 viable cells) of frozen ( - 8 0 ° C ) stock culture originally grown in LB supplemented with 5/~g kanamycin/ml. The seed cultures (1 i, 301) and the production phase of the 900 1 fermentation of A L K O 84 were grown in a rich medium consisting of double-strength LB to which hydrolysed starch (dextrose equivalent value [DE] about 30) (8% [w/v]) was added; the 1 liter seed culture contained 10#g kanamycin/ml but the following steps were cultivated without antibiotic. The first seed culture (3 liters) for the 60 m 3 cultivation was grown in doublestrength LB with 2 % (w/v) glucose, whereas the following cultures (1501, 2.7m 3) and the production phase were grown in an industrial medium, consisting of 6 % (w/v) hydrolysed starch (DE 30-50) and 4 % (w/v) corn steep liquor. For cultivation of A L K O 84, the 31 and 1501 cultures were supplemented with 101tg kanamycin/ml. The bacteria were cultivated at 37 '(7 throughout.

Fermentations Laboratory-scale fermentations were performed in a Bioengineering Laboratory fermenter L1523 at a working volume of 81. The stirring rate was 525 rev min-1 (tipspeed 2.2 m/s), aeration 1.0 vvm, temperature 37°C, pHcontrol from 6.5 (NH3) to 7.5 (HaPO4). SAG 5693 (Union Carbide, USA) was used as antifoam. The larger-scale fermentations were run in conventional stirred-tank fermenters. For the 9001 cultivation the stirring rate was 250 rev min-~ (tip-speed 4.3 m/s), aeration 2.1 vvm and the antifoam was Silicon RD (Dow Coming, USA). At 60m 3 the stirring rate was 60rev m i n - t , (tip-speed 4.7 m/s), aeration 0.67 vvm and Berol 740 (Berol Kemi, Sweden) was used as antifoam. Beroi 740 was used on the industrial scale, because Silicon RD proved to be unsuitable for downstream processing of cultures grown in the industrial medium, and SAG 5693 was not available to us in large quantities at the time of the industrial fermentations; shake flask and laboratory fermenter tests indicated that there is no significant difference in ~-amylase productivity with the different antifoams. Other large-scale conditions were similar to those on the laboratory scale.

Plas'mid stahilit)' estimation The cells were plated on LB-agar and the resulting colonies were screened for the cloned :~-amylase production and kanamycin resistance on starch 1,B-plates containing kanamycin.

~-Amylase assay The :~-amylase activity was assayed as described earlier. 1~' The activity is presented in katals (kat); 1 kat produces 1 mole of reducing sugars (calculated as maltose) from starch per second under the determination conditions (pH 5.9, 30'~C).

Biosq/~,ty considerations The large-scale cultivations were performed according to the recommendations of the National Board of Health of Finland, essentially following the NIH Guidelincs for Research Involving Recombinant DNA Molecules. The pilot-scale (900 I) cultivation were carried out under PILS containment (Federal Register, 1980, vol. 45, No. 72.

Part II, VII-B) with some minor modifications. Later on strain constructions like ALKO 84 were exempted from the NIH Guidelines (I-'ederal Register, 1982, vol. 47, No. 167, part IV, III-D-4 and appendix A, sublist B). The possible release of the recombinant strain was followed by collecting samples on 1,B-plates from the outcoming air of the fermenters and from the air in the downstream processing hall using an Andersen analyzer. TM The colonies were screened on starch-containing LB-plates supplemented with kanamycin.

Determination o[antihacterial activity The antibacterial activity was determined from concentrated enzyme preparations as described earlier. ~'~

Results and discussion In rich medium (double-strengh LB with 10% [w/v] starch) the B. suhtilis strains transformed with pKTH 10 are known to produce up to five times as much 0c-amylase at laboratory scale as the traditionally developed production strain B. amyMiquefaciens A L K O 89." Since this medium is too expensive for industrial applications, the production of x-amylase by B. suhtilis ALKO 84, a stable pKTH 10-carrying strain, 10 was tested in shake cultures using media consisting of various cheap carbon and nitrogen sources (hydrolysed starch, whey, molasses, malt sprouts, soy flour, distiller's spent grain, corn steep liquor). Starch and corn steep liquor were chosen for laboratory fermentation tests on the basis of good productivity. Best results were obtained with the combination of 6 % (w."v) hydrolysed starch and 4 % (W/V) corn steep liquor, in which medium A L K O 84 retained its superiority in :~-amylasc production and produced about 40/.&at/ml compared with about 20 l&at/ml for ALKO 89 under similar conditions at similar cell densities (details not shown.) To evaluate the stability of the recombinant strain at larger scale, A L K O 84 was cultivated in 9001 of the rich medium with antibiotic selection applied up to the 1 I seed culture. The number of cell doublings in the batch was about 54 (counted from the single cell that produced the individual colony used for the inoculation of the stock cultures [see Materials and methods], the last 13 ofwhich were grown in the absence of kanamycin. Since previous results 16 suggested that instability of pKTH 10 is related to high expression of the cloned gene, and ~-amylase is maximally produced in Bacillus at stationary growth phase, ~ care was taken to transfer the seed cultures to the next stage at late exponential growth phase (cell density about 1 - 3 x 109 cells/ml). The amount of ~-amylase produced in the batch was approximately 70% of that obtained at laboratory scale in similar medium (48.5/,tkat/ml vs. 69.4 ~ukat/ml in 42-44 h). The decrease in ~-amylase production on the larger scale compared with the laboratory scale was expected (see below), and was of similar magnitude to that with our traditionally developed production strains during scaling-up from laboratory to industrial conditions (data not shown.). From about a thousand colonies plated from the stationary phase, no clones showed loss of plasmid-encoded characteristics demonstrating, in agreement with previous results, 16 the high stability of the strain. The recombinant strain A L K O 84 and the traditionally developed strain A L K O 89 were cultivated at industrial scale (60 m 3) in the industrial medium. Although

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experience with the 9001 cultivation (see above) verified the high stability of A L K O 84, the risk of losing the recombinant plasmid was further minimized by cultivating ALKO 84 up to the 1501 scale in the presence of kanamycin, thus reducing the number of cell doublings without antibiotic selection to about 12 (the total number of doublings in the batch was about 59). With this regimen, no antibacterial activity was detectable in the final product, the drug being diluted 400-fold by the culture dilutions (from 1501 to 60 m3), and removed during downstream processing (e.g., by ultrafiltration). The traditional strain ALKO 89 was treated in a similar way to ALKO 84, but without antibiotic at any stage. The recombinant strain ALKO 84 retained its superiority in ~-amylasc production at industrial scale and made about twice as much cnzyme as the traditional strain ALKO 89 (Figure 1), although with both strains the final :~-amylasc activity decreased to about 85 % of that obtained at laboratory scale the in same mcdium (from 37.7/~kat/ml to 31.8F&at/ml with ALKO 84, and from 22.2#kat/mi to 19.6/&at/ml with ALKO 89). Taking 24pkat/mg protein as the specific activity of the B. amyloliqmffaciens ~-amylase (1 l&at ~-amylase activity is equal to 62 U used by Ulmancn and coworkers; 2° the latter activity unit corresponds to mg maltose produced from starch in 3 min under similar assay conditions to ours), thc amount of enzyme produced by ALKO 84 at industrial scale equals about 1.3 g/l. Other cultivations with A L K O 84 at both semi-industrial and industrial scale under slightly modified conditions have resulted in similar enzymc yields; at 9 m 3 in medium consisting of 6.0% (w:v) hydrolyscd starch and 4.0% (w/v) corn steep liquor, ALKO 84 produccd 38.4/&at z~-amylasc/ml in 43 h, and at industrial scale (7.4% [w/v] hydrolysed starch and 3.5 % [w/v] corn steep liquor starting volume 50m 3) in fed-batch culturc (19m 3 of 11.75% [w/v] hydrolysed starch and 4.75% [w/v] corn steep liquor

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Figure 1 Production of =-amylase by the traditionally developed production strain Bacillus amyloliquefaciens ALKO 89 ( 0 , O) and the recombinant strain B. subtilisALKO 84 (A, A ) at industrial scale in industrial medium (6% (w/v) hydrolysed starch, 4% (w/v) corn steep liquor). The horizontal axis indicates the time starting from the inoculation of the production phase (60 m3). The inoculum for the production phase was prepared by successive cultivation of the strain at 31, 1501 and 2.7 m3. With ALKO 84, the two first seed cultures were supplemented with kanamycin.

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added gradually between 8 28h after inoculation), ALKO 84 produced 34.3 l&at ~-amylase/ml in 45 h. The decrease in s-amylase production at industrial scale may be due to less optimal gas transfer rates in the industrial fermenter than in the laboratory ones, or to industrial sterilization conditions. The 7-amylase production in Bacillus is greatly affected by the composition of the cultivation medium, zl and side-reactions (e.g., Maillard reactions) forming non-utilizable and inhibitory compounds are more extensive during industrial-scale sterilization, because longer sterilization times or higher temperatures are used than on the laboratory scale to minimize contamination risks. The enzyme-production phase for A L K O 84 was about 5h longer than for ALKO 89, and maximal ~amylase activity was reached about 40 h after inoculation of the production phase (Figure 1). The maximal viable count was similar for both strains (Figure 1) and comparable with that obtained at laboratory scale. In the example shown the viable count of ALKO 89 dropped later in the stationary phase (Figure 1), but this varies from batch to batch, and seems not to correlate with the final enzyme activity. No clones of A LKO 84 lacking plasmid-encoded characteristics were found among colonies plated from seed cultures; however, !.0% (2/200) of colonies plated from the production phase 10 h after of inoculation and 6.0% (12/200) of colonies plated after 33h behaved atypically on screening plates (showing only the residual starch-degrading activity characteristic to B. subtilis). Both clones from the 10-hour-old culture were kanamycin-resistant. However, seven (3.5 %) of the clones from the 33-hour-old culture were in addition kanamycinsensitive. It was assumed that the cells with the former phenotype contained pKTH 10 derivatives with deletions resulting in incapacity to produce active co-amylase, 16 whereas the latter phenotype was believed to result from segregational loss of the recombinant plasmid. Since the slight instability observed at the latest stage of the process did not result in significant reduction in s-amylase productivity, the stability of ALKO 84 was regarded as acceptable for industrial batch cultivations. The B. suhtilis recombinant strain ALKO 84 is regarded as non-hazardous (see Materials and methods, biosafety considerations). Nevertheless, possible risks involved were minimized by following the Good Manufacturing Practice 1'~ guidelines, and the possible release of the recombinant strain to the environment was checked. It was estimated that during the entire process the number of living ALKO 84 cells escaping to the surroundings (mainly in the outcoming air of the fermenters and during downstream processing) corresponded to less than 1 ml of the culture fluid at maximum cell density. After the cultivation the cell mass was separated, and the product was then sterilized by filtration. The cells removed were killed by steam sterilization. As far as we are aware, this is the first report of successful application of a recombinant Bacillus strain to industrial scale and demonstrates the possibility of using gene technology in strain improvement.

Acknowledgements The skillful technical assistance of Kari Makkonen, Sanna Hiljanen, Marja Kamila, Anna Micro and Annikki Vfilimfiki is greatly acknowledged. We are also grateful to

Enzyme production by recombinant Bacillus: J. Vehmaanper~ et al. the technical staffs of the Pilot-fermentor in the Biotechnicai Laboratory of the Technical Research Centre of Finland, and of the fermentation plant at the Koskenkorva factory of Alko Ltd. We also thank Dr. Roy Tubb and Dr. John Londesborough for valuable discussions during the preparation of the manuscript. References 1 Priest, F. G. Bacteriol. Rev. 1977, 41,711-753 2 Rowlands, R. T. Enzyme Microb. Technol. 1984, 6, 3-10 3 Gryczan, T. J. in 7he Molecular Biology of the Bacilli (Dubnau, D., ed.) Academic Press, NY, 1982, pp. 307 329 4 Aiba, S., Kitai, K. and Imanaka, T. Appl. Environ. Microbiol. 1983, 46, 1059-1065 5 Fujii, M., Takagi, M., Imanaka, T. and Aiba, S. J. BacterioL 1983, 154, 831-837 6 Jacobs, M., Eliasson, M., Uhl~n, M. and Flock, J-l. Nucleic Acid* Res. 1985, 13, 8913-8926 7 Ortlepp, S. A., Ollington, J. F. and McConnell, D. J. Gene 1983, 23, 267-276 8 Palva, I. Gene 1982, 19, 81-87 9 Wells, J. A., Ferrari, E., Henner, D. J., Estell, D. A. and Chen, E. Y. Nucleic Acid~ Res. 1983, 11,7911-7925 10 Bron, S. and Luxen, E. Plasmid 1985, 14, 235-244 I 1 Ehrlich, S. D., Niaudet, B. and Michel, B. in Curr. Top. MicrobioL

lmmunoL (Hofschneider, P. H. and Goebel, W., eds), Springer-

Verlag, NY, 9182, 96, pp. 19-29 Kreft, J. and Hughes, C. in Curr. Top. Microbiol. lmmunol. (Hofschneider, P. H. and Goebel, W., eds) Springer-Verlag, NY, 1982, 96, pp. 1-17 13 l,opez, P., Espinosa, M., Greenberg, B. and Lacks, S. A. Proc. Natl. Acad. Sci. USA 1984, 81, 5189-5193 14 Pinches, A., Louw, M. E. and Watson, T. G. Biotech. Lett. 1985, 7, 621~26 15 Saunders, C. W., Banner, C. D. B., Fahnestock, S. R., Lindberg, M., Mirot, M. S., Rhodes, C. S., Rudolph, C. F., Schmidt, B. J., Thompson, L. D., Uhlen, M. and Guyer, M. S. in Protein Transport 12

and ,Secretion. UCLA Symposium on Molecular and Cellular Biology (Oxender, D. L., ed.) Alan R. Liss, Inc., NY, 1984, pp.

329-339 16 Vehmaanper~i, J. O. and Korhola, M. P. Appl. Microbiol. Biotechnol. 1986, 23, 456-461 17 Bailey, M. J. and Markkanen, P. H. J. Appl. (;hem. Biotechnol. 1975, 25, 73-79 18 Andersen, A. A. J. BacterioL 1958, 76, 471-484 t9 Godfrey, T. and Reichelt, J. Industrial Enzymology - 7"he Application of Enzymes in Industry. The Nature Press, New York, 1983, pp. 151-153 20 Ulmancn, I., Lundstr6m, K., Lehtovaara, P., Sarvas, M., Ruohonen, M. and Palva, I. J. BacterioL 1985, 62, 176-182 21 Ingle, M. B. and Boyer, E. W. in Microbiology 1976(Schlessinger, I)., ed.) American Soc. Microbiology, Washington DC, 1976, pp. 420 426

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