JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 72, No. 5, 368-372. 1991

Secretory Overproduction of Streptomyces Cholesterol Oxidase by Streptomyces lividans with a Multi-Copy Shuttle Vector

ISTVAN MOLNAR, KWANG-PIL CHOI, NOBUYUKI HAYASHI, AND YOSHIKATSU MUROOKA

Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Kagamiyama 1, Higashi-Hiroshima 724, Japan

Received 5 April 1991/Accepted 9 August 1991

Deletion of a 1.2-kb fragment adjoined upstream of the choP-choA operon and subcloning of the operon into a multi-copy shuttle vector composed of pIJ702 and pUC19 in Streptomyces lividans resulted in the over- production of Streptomyces cholesterol oxidase extracellularly about 70-fold more than that of the original pro- ducer, Streptomyces sp. SA-COO. When the cells of S. Hvidans carrying the plasmid were grown in an ap- propriate medium, about 90 to 99% of the enzyme produced was secreted into the medium. Increased concen- tration of glucose in the culture medium resulted in a significant decrease in the production of the enzyme, while elevated peptone concentrations led to a further increase in the production. The level of the overproduc- tion of cholesterol oxidase was dependent on the copy numbers of the plasmids as well as the presence of the sequences derived from pUC19.

There has been a growing interest in recent years in the development of Streptomyces host-vector systems as use- ful alternatives to the more common expression systems of E. coli and Saccharomyces. Streptomyces species are espe- cially attractive as hosts for the secretory overproduction of heterologous gene products, since these species natu- rally secrete a large amount of protein directly into the culture fluid and the techniques for the cultivation of this organism are well established on an industrial scale. Ex- pression of Streptomyces genes propagated in multi-copy vectors allowed a 2 to 10-fold (in one case, 500- fold [1]) overproduction of the gene products in S. lividans (2-6). Low level productions of bovine growth hormone (7) and human interleukin 2 (8) in S. lividans cells were also achieved. The secretory synthesis of eukaryotic proteins, however, is often prevented by reduced translocation through the membrane, and the proteins are accumulated in the cells probably because of insufficient interaction of the Streptomyces signal peptide and the eukaryotic protein (9-11). In a recent example (12), however, efficient secre- tion of hirudin by S. lividans was achieved.

Recently, we have cloned in Streptomyces lividans an operon containing the genes for extracellular cholesterol oxidase (choA) (13, 14) and a cytochrome P450-1ike protein (choP) (15) from Streptomyces sp. SA-COO. Cholesterol oxidase (EC 1.1.3.6) catalyzes the oxidation of cholesterol (5-cholesten-3-fl-ol) to 4-cholesten-3-one, with the reduction of oxygen to hydrogen peroxide. The enzyme is used for the enzymatic transformation of cholesterol (16, 17), and by coupling with peroxidase, for the quantitation of cholesterol in clinical specimens (18).

During the course of our studies on cholesterol metab- olism in Streptomyces, we investigated the secretory overproduction of cholesterol oxidase in S. lividans by a multi-copy shuttle vector, the results of which are pre- sented in this paper.

* Corresponding author.

MATERIALS A N D METHODS

Bacterial strains and plasmids The Streptomyces strains used were Streptomyces sp. SA-COO, a strain which produces extracellular cholesterol oxidase (provided by Toyobo Co. Ltd., Tsuruga), S. lividans 1326 (19), and S. griseus HUT6037 (20). Escherichia coli XL1 Blue was purchased from Stratagene (La Jolla, CA, USA). Plasmids pUC19 (21), plJ702 (22), and pCO3 (13) were described previously.

Materials Restriction endonucleases, T4 DNA li- gase, the large fragment of DNA Polymerase I, and RNase A were purchased from Takara Shuzo Co. Ltd. (Kyoto), or Toyobo Co. Ltd. A Gene-Clean DNA purification kit was obtained from Biol01 Inc. (LaJolla, CA, USA). Hybond-N nylon membranes are products of Amersham Co. (Arlington Heights, I1, USA). A Genius TM non-radioac- tive DNA labeling and detection kit was purchased from Boehringer Mannheim GmbH (Mannheim, FRG). Thio- streptone was provided by Asahikasei Co. Ltd. (Tokyo).

Media Unless otherwise mentioned, YEME (23) and GMP (24) media, complemented with 10/zg/ml thiostrep- tone for the plasmid-containing strains, were used for the cultivation of Streptomyces cells. For E. coli, LB medium (25) was used.

Recombinant D N A technology Recombinant DNA techniques for E. coli and Streptomyces were done accord- ing to Maniatis et al. (25) and Hopwood et al. (23), respec- tively. "Total" DNA for determination of plasmid copy numbers was prepared from Streptomyces by the method of Kieser et al. (22), followed by RNase A treatment, ex- traction with phenol and chloroform, and precipitation by polyethylene glycol. The copy numbers of plasmids were measured by densitometric scanning of the negatives of gel photographs as described by Kieser et al. (22) and by a hybridization method. Digoxigenin-labeled probes for hybridizations were prepared and the hybrids formed were detected as recommended by the manufacturer of the Genius T M non-radioactive DNA labeling and detection kit. For the hybridization method, 10/zl of a series of dilutions

368

VOL. 72, 1991 SECRETORY OVERPRODUCTION OF CHOLESTEROL OXIDASE 369

of "total" DNA prepared from plasmid-containing Strep- tomyces strains was spotted onto a Hybond-N nylon mem- brane, and were then hybridized with a non-radioactive labeled probe fragment specific for the cholesterol oxidase gene. The plasmid concentrations were deduced from a comparison with a standard DNA fragment containing the choA gene on the same membrane. The two methods for copy number measurement yielded comparable results, which were subsequently averaged.

Cholesterol oxidase assay Cholesterol oxidase ac- tivity was measured according to the quality control assay for cholesterol oxidase in the manual of Boehringer Mannheim GmbH (26). One unit (U) of cholesterol oxidase was defined as the amount of enzyme oxidizing 1/~mol of cholesterol to cholestenone in 1 min at 25°C, pH 7.5, in the presence of 6 .9mg/ml Thesit R (Kao Chemicals, Tokyo). The culture broth was assayed for extracellular activity of cholesterol oxidase. Cell extracts obtained by sonication of mycelial pellets washed twice with potas- sium phosphate buffer containing Thesit R were assayed for intracellular activity.

RESULTS

Construct ion of shuttle vectors for the expression o f cholesterol oxidase For the secretory production of cholesterol oxidase from Streptomyces sp. SA-COO in S. lividans, we subcloned a 3.9-kb KpnI-KpnI fragment, con- taining the choP- choA operon from pC©3 (13), into a shuttle vector composed of plJ702 and pUC19 and used the resultant plasmids for E. coli and Streptomyces species (Fig. 1). The shuttle vectors pCO100A and pCO100B are identical except for the orientation of the 3.9-kb KpnI- KpnI fragment with regard to the vector sequences: in pCO100A, the lac promoter originating from pUC19 is located upstream of the choP-choA operon and the direc-

KpnI

~,gpnI , Sac] IKpnI , SacI 4.7 kb fraggment ~ I

SacI

lop _ ~

I KpnI ~ / Kpn]

3 . g k b fragment

s~c~ ~ , J J J

~," il.3kb} Kp.l

C#O ~ , ~ p n I

FIG. 1. Scheme for construction of shuttle vectors, pCOI00A and pCOI00B. Lines: --, pIJ702 fragment; --, pUC19 fragment; ~, Streptomyces sp. SA-COO fragment carrying the choA gene.

2000

1500

o >,

1000 o

(_)

501]

O / O ~ •

/ /

/ / o _m---- m------m~m mU_

1 2 3 4 5 6 7 boys

FIG. 2. Production of extracellular cholesterol oxidase by the producing strain Streptomyces sp. SA-COO and S. lividans 1326 strain carrying various plasmids. Symbols: ©, Streptomyces sp. SA-COO; A, S. lividans 1326; ", S. lividans 1326 (pC©3); o, S. lividans 1326 (pCO100A).

tion of the lac promoter is identical with the direction of the cho operon; in pCO100B, the cho operon is inserted in the opposite direction to the E. coli promoter.

Product ion of cholesterol oxidase by S. lividans strains carrying recombinant plasmids Cells of Streptomyces sp. SA-COO and S. lividans 1326 with or without pC©3 or pCOI00A were cultivated aerobically at 28°C for 7 d in 100 ml of YEME medium, where 10 pg/ml of thiostrep- tone was supplemented for the plasmid-containing strains. The time courses of production of the extracellular (Fig. 2) and intracellular (Fig. 3) cholesterol oxidases are shown. No cholesterol oxidase activity was detected in S. lividans 1326. In Streptomyces sp. SA-COO, the activity of intracel- lular cholesterol oxidase increased slightly until the 3rd d and then slowly decreased, while the activity of the extracel-

20 / o •

• \ g ~

5

@---" -~ A ,k A A a. 1 2 3 4 5 G 7

Days

FIG. 3. Product ion of intracellular cholesterol o×idase by the producing strain Streptomyces sp. SA-COO and S. fividans 1326 strain carrying various plasmids. Symbols: ©, Streptomyces sp. SA-COO; z~, S. lividans 1326; " , S. lividans 1326 (pC©3); e , S. lividans 1326 (pC© 100A).

370 MOLN,4.R ET AL. J, FERMENT. BIOENG.,

lular enzyme reached a maximum on the 5th d, followed by a slight decrease. Most of the cholesterol oxidase produced (87 to 93%o) was found extracellularly at every time point. S. lividans 1326 cells carrying pCO3 produced both intracellular and extracellular cholesterol oxidases in parallel with Streptomyces sp. SA-COO. The maximum productions of the intracellular and extracellular enzymes by the cells carrying pCO3 were 1.4- and 9.2-fold higher than those by the strain SA-COO, respectively. The major- ity (94 to 99%o) of the cholesterol oxidase produced by pCO3 was exported into the culture medium. The highest yield of cholesterol oxidase was obtained by using S. lividans 1326 cells carrying pCO100A. The production of the intracellular enzyme reached its maximum on the 3rd d and then markedly decreased, while the production of the extracellular cholesterol oxidase attained a plateau in 6 d. The maximum product ion of the intracellular and ex- tracellular enzymes by the cells carrying pCO100A was 10- and 70-fold, respectively, relative to those by the parent strain, SA-COO. Throughout the experiment, 90-99°//oo of the total activity of cholesterol oxidase was detected in the culture medium.

Effect of host strains on the production of cholesterol oxidase Product ion of cholesterol oxidase by S. livi- clans 1326 and S. griseus HUT6037 cells carrying pCOI00A was compared (Fig. 4). Although the strain S. griseus HUT6037 carrying pCO100A grew faster and produced higher amounts of cell mass, the activity of extracellular cholesterol oxidase remained low, reaching only about 30°//oo of that of the S. lividans 1326 (pCO100A). S. griseus (pCO100A) exported 80-90% of the cholesterol oxidase produced into the culture medium.

Effect of growth media on the overproduction of cholesterol oxidase To optimize the secretory over- product ion of cholesterol oxidase by S. lividans ceils car- rying pCO100A, we examined the effects of a different medium as well as the variation of the concentrations of glucose and peptone in the trivial YEME medium (18). S. lividans strain carrying pCO100A produced only 1/9-fold

/

~soo 6 . . . .

u~ 1 0 0 { 5 " " . ~, , " A "S

C )

0 1 2 3 4 5 6

Days

FIG. 4. Production of extracellular cholesterol oxidase in strains S. lividans 1326 and S. griseus HUT6037 carrying pCO100A. Sym- bols: e , cholesterol oxidase activity in S. lividans (pCO100A); c3, cell growth of S. lividans (pCOI00A); A, cholesterol oxidase activity in S. griseus (pCOI00A); A, cell growth of S. griseus (pCOI00A).

TABLE I. Effect of growth media on cholesterol oxidase overproduction"

Medium Relative activity of cholesterol oxidase

Addition (%) Extracellular lntracellular

YEME -- 89 11 GMP -- l I 101 YEME Glucose (1%) 48 ND b YEME Glucose (4O/oo) 8 ND YEME Glucose (9%) 4 ND YEME Peptone (0.5)~) 96 ND YEME Peptone (2.0%o) 117 ND YEME Peptone (4.5%0) 106 ND

a S. lividans 1326 cells carrying pCOI00A were cultivated in 100 ml of the above media complemented with 10/tg/ml of thiostreptone for 5 d at 28°C with shaking. Cholesterol oxidase activities were measured in the supernatant and in cell extracts as described in Materials and Methods. The total productivity in the standard YEME medium containing 1% glucose and 0.5o/oo peptone (18) was chosen as the basis of the comparison (100%).

b ND, Not determined.

amounts of extracellular cholesterol oxidase, but 10-fold amounts of the intracellular enzyme in GMP medium (Table 1). Increasing the concentrat ion of glucose as a carbon source in the standard YEME medium resulted in a prompt decrease in the production. Increasing the concen- tration of peptone in the YEME medium yielded a marked increase in the product ion of the extracellular cholesterol oxidase, with a maximum of 128°//00 when 2%o peptone was added to the standard YEME medium.

Effect of pUCl9-derived sequences on the production of cholesterol oxidase To clarify the role of the pUC19- derived sequences in the overproduction of cholesterol oxidase with the shuttle vector in S. lividans, we tested the product ion of cholesterol oxidase by S. lividans cells car- rying pCO100B. The S. lividans ceils carrying the shuttle vector with the choP-choA operon reversed with respect to the pUC19 sequences (see pCO100B in Fig. 1) produced a slightly lower amount of cholesterol oxidase, the extracellu- lar activity being more than 90%o of that of the cells car- rying pCO100A (Table 2). We also found that pCO100B was unstable during cultivation, frequently giving rise to deletion derivatives (results not shown). To investigate further the possible role of the pUC19-derived sequences, we deleted the whole pUC19 part and obtained pCO200. The S. liviclans cells carrying pCO200 produced extracel- lular cholesterol oxidase of about 33%o of that of the cells carrying pCO100A (Table 2). The plasmid pCO200 was

TABLE 2. Extracellular cholesterol oxidase production in S. lividans strains carrying various plasmids ~

Plasmid Copy number Relative activity of cholesterol oxidase (%)

pCOIOOA 180+30 100 pCO 100B ND b 90 pCO200 180 ± 30 33 pCO3 70± 15 12

a Cells were grown for 5 d in 10 ml of YEME medium at 28°C with shaking. Extracellular cholesterol oxidase activities and plasmid copy numbers were determined as described in Materials and Methods. The extracellular cholesterol oxidase activity of S. livictans 1326 (pCOI00A) strain was chosen as the basis of the comparison (100%).

b ND, Not determined, because the plasmid frequently formed dele- tion derivatives.

VOL. 72, 1991 SECRETORY OVERPRODUCTION OF CHOLESTEROL OXIDASE 371

stable during cultivation (results not shown). To assess the role of the gene dosage effects, we deter-

mined the copy numbers of the recombinant plasmids in S. lividans by the method of Kieser et al. (22) and the hybridi- zation method as described in Materials and Methods. pCO3 had a copy number of about 70, while the copy num- bers of pCO100A and pCO200 both proved to be about 180.

DISCUSSION

In this work, we have examined the secretory over- production of Streptomyees cholesterol oxidase in S. lividans by a multi-copy shuttle vector based on plJ702 and pUC19. By subcloning of the entire ehoP-choA ope- ron without significant upstream adjacent sequences into the shuttle vector, we constructed a S. lividans strain that overproduced the extracellular cholesterol oxidase by about 70-fold more than that of the original producer, Streptomyces sp. SA-COO, and about 8-fold more than that of S. lividans harboring pCO3 (13). The overproduc- tion proved to be a plasmid-borne phenomenon and not the result of a mutation of the host strains occurring dur- ing protoplast formation and regeneration, since after retransformation of pCO100A (isolated either from E. eoli or S. lividans) all the transformants became high- producers (results not shown).

Although S. griseus 6037 cells carrying pCO100A reached higher cell densities during cultivation, the strain produced cholesterol oxidase at only 30°//oo of that of S. lividans 1326 (pCO100A). The lower level of overproduc- tion in S. griseus may be a consequence of unknown differences in the transcriptional and/or translational machinery in the host strains, S. lividans, S. griseus, and Streptomyces sp. SA-COO. Changing the growth medium revealed three interesting facts (see Table I): (a) S. lividans 1326 (pCO100A) cells growing in GMP medium retained the cholesterol oxidase produced inside the cells; (b) ele- vated glucose concentrations resulted in a prompt decrease in the overproduction; (c) elevated peptone concentrations resulted in a maximum 28%o increase in the production. The main difference between media YEME and GMP is the substitution of malt extract for meat extract in the latter. It is possible that certain components of malt or meat extract could affect the localization of the enzyme in Strepto- myces. The elevated glucose concentrations may delay or inhibit the "metabolic switch" from glucose to other alter- native C-sources, which can affect the expression of genes of the secondary metabolism, of which the choP-choA ope- ron is an example. The elevated levels of peptone, on the other hand, may provide an abundant N-source base to reduce the costs of the production of the enzyme by the cells. The strong dependence of the secretory overproduc- tion of cholesterol oxidase on the composition of the medi- um calls attention to fermentation for the expression of heterologous genes in Streptomyees, an aspect generally overlooked in genetic studies.

Examination of the possible causes of the gross over- production of the cholesterol oxidase in S. lividans showed that the lac promoter located upstream of the ehoP-choA operon in pCO100A, or any other promoters present in the pUCI9 part of the shuttle vector, did not influence the en- zyme production. As shown in Table 2, cells harboring pCO100B, the vector that contains the choP-choA operon in reverse direction, produced cholesterol oxidase of com- parable amounts to cells carrying pCO 100A. We examined

whether the increase in the enzyme production in the case of pCO100A (70-fold more than that of Streptomyces sp. SA-COO, 7.5-fold more than that of S. lividans [pCO3]) could be attributed to a copy number increase. We found that the copy number of pCO100A is about 2.7 times higher than that of pCO3. This copy number increase in pCO100A was not connected to the presence of pUC19 se- quences, since pCO200 (the plasmid having the pUC19 se- quence of pCO100A deleted) had the same copy number. The only significant difference between pCO3 and pCO200 is the deletion of the 1.2-kb KpnI-SacI fragment located immediately upstream of the choP-choA operon in the Streptomyces sp. SA-COO genome, so the absence of this fragment has to play a role in the copy number increase. This increase is not a simple effect of the decrease in the overall plasmid size, since pCO100A, due to the presence of pUC19 sequences, is even longer than pCO3.

The 2.7-fold increase in copy number of pCO100A is not sufficient to explain the 7.5-fold increase in cholesterol oxidase production over that of cells carrying pCO3. Fur- ther examination of unknown effects on the overproduc- tion of cholesterol oxidase should provide more insight into the mechanism of the expression and secretion of het- erologous gene products in Streptomyces.

ACKNOWLEDGMENT

This work was supported by grant No. 01480036 to Y. M. from the Ministry of Education, Science, and Culture of Japan. I. M. and K-P. C. have been supported by Monbusho Scholarships.

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