Gene, 100 (1991) 85-93 0 1991 Elsevier Science Publishers B.V. 0378-l 119/91/$03.50
85
GENE 04064
Co-expression of a Saccharomyces diastaticus glucoamylase-encoding faciens a-amylase-encoding gene in Saccharomyces cerevisiae (Recombinant DNA; plasmid; yeast transformation;
gene and a Bacillus amylolique-
starch degradation; amylolytic enzymes)
Andries J.C. Steyn and Isak S. Pretorius Department of Microbiology and Institutefor Biotechnology, University of Stellenbosch, Stellenbosch 7600 (South Africa) Received by J. Marmur: 13 December 1990 Revised: 31 December 1990 Accepted: 28 January 1991
SUMMARY
A glucoamylase-encoding
gene (STAZ) from Saccharomyces diastaticus and an a-amylase-encoding gene (AMY) from were cloned separately into a yeast-integrating shuttle vector (YIPS), generating recombinant plasmids pSP1 and pSP2, respectively. The STA2 and AMY genes were jointly cloned into YIPS, generating plasmid pSP3. Subsequently, the dominant selectable marker APHI, encoding resistance to Geneticin G418 (GtR), was cloned into pSP3, resulting in pSP4. For enhanced expression of GtR, the APHl gene was fused to the GAL10 promoter and terminated by the URA3 terminator, resulting in pSP5. Plasmid pSP5 was converted to a circular minichromosome (pSP6) by the addition of the ARSI and CEM sequences. Laboratory strains of Saccharomyces cerevkiae transformed with plasmids pSP1 through pSP6, stably produced and secreted glucoamylase and/or a-amylase. Brewers’ and distillers’ yeast transformed with pSP6 were also capable of secreting amylolytic enzymes. Yeast transformants containing pSP1, pSP2 and pSP3 assimilated soluble starch with an efficiency of 69%, 84% and 93%, respectively. The major starch hydrolysis products produced by crude amylolytic enzymes found in the culture broths of the pSPl-, pSP2- and pSP3-containing transformants, were glucose, glucose and maltose (1: l), and glucose and maltose (3 : l), respectively. These results confirmed that co-expression of the STA2 and AMY genes synergistically enhanced starch degradation. Bacillus amyloliquefaciens
Correspondence to: Dr. IS. Pretorius, Department of Microbiology, University of Stellenbosch, Stellenbosch 7600 (South Africa) Tel. (27)2231-774730; Fax (27)2231-773611. Abbreviations: aa, amino acid(s); A546, absorbance at 546 nm; AMY, a-amylase (EC 3.2.1.1); AMY, gene encoding AMY; Ap, ampicillin; APHl, gene encoding aminoglycoside phosphotransferase-3’(I); ARSl, autonomously replicating sequence 1; B., Bacillus; bp, base pair(s); CEN4, centromere 4; chr., chromosome; CIAP, calf intestinal alkaline phosphatase; Cm, chloramphenicol; DNS, dinitrosalicylic acid; EtdBr, ethidium bromide; FID, flame ionization detector; GAI, GA11 and GAIII, extracellular glucoamylase (EC 3.2.1.3) isozymes I, II and III, respectively; GALIO, gene encoding uridine diphosphoglucose 4-epimerase EC 5.1.3.2; Gt, Geneticin (G418); kb, kilobase or 1000 bp; Km, kanamycin; LB, 1% Bacto tryptone/0.5% yeast extract/O.5% NaCl pH 7.0; LBP, LB/IO PAT tablets per 100 ml; LBS, LB/l % starch; LEUZ, gene encoding b-isopropylmalate dehydrogenase (EC 1.1.1.85); ODAD, O-dianisidine; ori, origin of replication; PAT, Phadebas Amylase Test;
PGO, peroxidase-glucose oxidase; PolIk, Klenow (large) fragment of E. coli DNA polymerase I; pYC, S. ceretiiae-E. coli shuttle cosmid vector; R, resistance/resistant; S., Saccharomyces; SC, 0.67% nitrogen base without aa/2% glucose plus all the required growth factors; SCeUra, SC lacking uracil; SCeL”“, SC lacking Leu; SGA, sporulation-specific intracellular glucoamylase EC 3.2.1.3; SGAl, gene encoding SGA; STAl, STA2 and STA3, genes encoding GAI, GA11 and GAIII, respectively; STAlO, gene encoding glucoamylase repressor; TAFE, transverse alternating-field electrophoresis; TAFE buffer, 20 mM Tris .HCl/7.8 mM EDTA/78 mM glacial acetic acid pH 8.0; Tc, tetracycline; u, unit(s); UR43, gene encoding orotidine-5’-phosphate carboxylyase (EC 4.11.23); YCp, yeast centromeric plasmid; YEp, yeast episomal plasmid; YIP, yeast integrating plasmid; YP, 1% yeast extract/2% Bacto peptone; YPD, YP/2% glucose; YPGal, YP/2% galactose; YPGE, YP/3% glycerol/2% ethanol; YPP, YP/l y0 starch/l0 PAT tablets per 100 ml; YPS, YP/l% starch; YPSB, YP/3% starch/0.003% bromocresol purple; [ 1, denotes plasmid-carrier state; ( ), plasmid integrated into yeast genome; : :, novel joint (fusion).
86 INTRODUCTION
The budding yeast S. cerevisiae has been used widely both as a model system for unravelling the molecular, biochemical and genetic details of gene expression and the secretion process, and as a host for the production of heterologous proteins of biotechnological interest. The potential of starch as a renewable biological resource has stimulated research into amylolytic enzymes and the broadening of the substrate range of S. cerevisiae (for a review see Steyn and Pretorius, 1990). The enzymatic hydrolysis of starch, consisting of linear (amylose) and branched glucose polymers (amylopectin), is catalyzed by CI- and @nylases, glucoamylases and debranching
enzymes, e.g., pullulanases (Janse et al., 1990; Vihinen and MBnts&i, 1989). Although wild-type strains of S. cerevisiae lack extracellular amylolytic activity and are unable to utilize starch as a substrate, they do possess the ability to produce SGA (Clancy et al., 1982). Furthermore, S. cerevisiae is closely related to and can interbreed with the amylolytic yeast S. diastaticus. Strains of S. diastaticus carrying one of the unlinked, haploid-specific genes STAI, STA2 and STA3, produce the extracellular glucoamylase isozymes GAI, GA11 and GAIII, respectively (Lambrechts et al., 1991; Pretorius et al., 1986a; for a review see Pretorius et al., 1991). Some S. cerevisiae strains also carry the STAlO repressor gene that inhibits glucoamylase expression at the
TABLE I Saccharomyces
strains used in the present study Relevant genotype b
Source or reference
a a a a a
a sta” ura3::pSP4 his3 a sta” Ieu2:: pSP5 h&4 a sta” leu2 his4 [pSP6] Polyploid brewers’ yeast[pSP6] Polyploid distillers’ yeast[pSP6]
IS. Pretorius IS. Pretorius I.S. Pretorius This work This work This work This work This work This work This work This work
pAM13 pUS52 pET1 pSP1 pSP2 pSP3 pSP4 pSP5 pSP6
ApR LEU2 AMY ApR HIS3 STA2 ApR KmR TcR CmR Apu URA3 STA2 ApR VRA3 AMY ApR (IRA3 AMY STA2 ApR KmR UR43 AMY STA2 ApR KmR LEU2 AMY STA2 GALlOp ApR KmR LEU2 AMY STA2 GALlOp
Pretorius et al. (1988) Pretorius et al. (1986b) ATCC39566 This work This work This work This work This work This work
YEp62
ApR LEV2 GAL10 1acZ ApR TcR URA3 CEN4 ARGI ApR TcR URA3
Strain or plasmid” Yeast strains:
ISP52 ISP53 ISP55 ISP52(pSPl) ISP52(pSP2) ISP52(pSP3) ISP52(pSP4) ISP52(pSP5) ISPSZ[pSP6] RSAl[pSP6] RSA2[pSP6]
sta” wall his3 sta” leu2 hir4 STA2 stal0 arg4 sta” ura3 SGAI ::pSPI his3
sta” ura3 : : pSP2 hir3
a sta” ura3::pSP3
his3
Plasmids:
CEN4 ARSI
YCp50 YIp5
Broach et al. (1983) Johnston and Davis (1984) Struhl et al. (1979)
a Strains ISP52 and ISP53 are Sta- segregants of a cross between two strains carrying the STAZ and STA3 glucoamylase genes of S. diastaticus, respectively. Transformation of CaCl,-treated E. coli cells was performed according to Maniatis et al. (1982) and transformants were selected on media containing 50 pg/ml Ap (LB + Ap, LBS + Ap, LBP + Ap) or 20 &ml Km (LB + Km, LBS + Km, LBP + Km). Saccharomyces strains were transformed with linear or circular plasmid DNA by the lithium acetate method (Ito et al., 1983; Sakai and Yamamoto, 1986) and transformants were selected either on minimal media (SC- Ura, SC- ti”) or on complex media containing Gt (YPGal + Gt). Mitotic stability of markers transformed into S. cerevisiae was analyzed by culturing the yeast transformants in 2 liter of YPD until stationary phase. One ml of this yeast suspension was inoculated into fresh YPD (2 liter) and incubated until stationary phase. This procedure was repeated five times. Serial dilutions were made from the final culture and plated onto YPD agar plates and replica-plated onto selective media (SC- “ra, SC- Lcu,YPGal + Gt). The mitotic stability was determined by using the following equation: mitotic stability (%) = 100 x (number of colonies on selective medium / number of colonies on YPD). The mitotic stability was measured for two different transformants of each strain and the average recorded. b ::, indicates integration of the YIPS-derived plasmid into the genomes of the yeast strains. [ 1, indicates that the yeast strains harbour the YCpSO-derived centromeric plasmid. ( ), indicates that the plasmids were integrated into the yeast genomes.
87 transcriptional level (Pretorius et al., 1986d). Manipulation of S. cerevisiue to synthesize and secrete functional amylolytic enzymes would facilitate a direct, one-step bioconversion of starch-rich materials to ethanol (potable alcohol or a fuel extender) or single-cell protein (food and feed supplements). Amylolytic yeasts of genera other than Saccharomyces are generally not suitable for the production of bioethanol and single-cell protein. S. cerevisiue has a fast growth and fermentation rate, is ethanol-tolerant (De Mot et al., 1985), consists of 48% high-quality protein and has been associated with food and beverage production for centuries (De Mot and Verachtert, 1984). Heterologous amylase genes derived from various organisms have been expressed in S. cerevisiae. These include the a-amylase genes from wheat (Rothstein et al., 1984; 1987), mouse salivary glands (Thomsen, 1983) mouse pancreas (Filho et al., 1986), human salivary glands (Nakamura et al., 1986), human lung carcinoid tissue (Shiosaki et al., 1990), B. amylohquefaciens (Kovaleva et al., 1989; Pretorius et al., 1988; Ruohonen et al., 1987) B. stearothermophilus (Nonato and Shishido, 1988), Schwanniomyces occidentalis (Wang et al., 1989) and the glucoamylase genes from Aspergillus awamori (Cole et al., 1988; Inlow et al., 1988; Innis et al., 1985), Rhizopus oryzue (Ashikari et al., 1986; 1989), Succharomycopsis fibuligeru (Yamashita et al., 1985) and S. diustuticus (Erratt and Nasim, 1986; Meaden et al., 1985; Pardo et al., 1986; Pretorius et al., 1986b). Kim et al. (1988) described the construction of a hybrid yeast strain secreting both glucoamylase and AMY utilizing 93% of Lintner starch. This strain was obtained by transforming an S. diastaticus derivative with an episomal plasmid containing the mouse salivary AMY gene. However, the disadvantages of using this transformant for future industrial purposes are the high mitotic instability of the plasmid and the difficulty in further manipulation of the glucoamylase gene(s) located on the chromosome(s) of the recipient S. diustuticus hybrid strain. This report describes the construction of yeast integrating and centromeric plasmids containing the STA2 gene from S. diastaticus and/or an AMY gene from B. amyloliquefaciens. Laboratory and industrial strains of S. cerevisiae were transformed with these recombinant plasmids. End products of amylose hydrolysis in the culture broths of these transformants were analyzed and characterized by gas chromatography. Starch utilization by transformants was monitored and compared.
RESULTS
AND DISCUSSION
(a) Construction
of recombinant plasmids
Pretorius et al. (1986b) have previously cloned a STA2 gene from S. diastaticus into the E. coli-yeast shuttle cosmid
vector pYC1, resulting in pUS52. The STA2 gene was subcloned as an 8.3-kb BglII DNA fragment into the BamHI site of the yeast-integrating plasmid vector YIPS, generating recombinant plasmid pSP1 (Fig. 1A). Pretorius et al. (1988) described the cloning of an AMY gene from B. amyloliquefuciens (isolated from compost; Pretorius et al., 1986~) into the E. coli-yeast shuttle plasmid vector YEp13, resulting in pAM13. The AMY gene was expressed and AMY secreted in S. cerevisiae using the original AMY promoter and signal sequences. The AMY gene was subcloned as a 2.1-kb PvuII DNA fragment into the PvuII site of YIPS, generating pSP2 (Fig. 1B). Plasmid pSP3, containing both the STA2 and AMY genes, was constructed by the cloning of the 8.3-kb BgZII DNA fragment from pUS52 into the BamHI site of pSP2 (Fig. 1C). The dominant selectable marker APHl was added to pSP3, resulting in pSP4 (Fig. 1D). The APHl gene encodes aminoglycoside phosphotransferase-3’(I) that inactivates several different 2-deoxystreptamine aminoglycoside antibiotics, including Km and Gt. Plasmid pSP3 was linearized withXho1 and the cohesive termini were filled-in with PolIk and dephosphorylated with CIAP. A 1.7-kb PvuII DNA fragment, containing the APHI gene, was subcloned from pET1 (ATCC39566) into the linearized pSP3. For enhanced expression of GtR, the APHI gene was fused to the GAL10 promoter and terminated by the lJR43 transcriptional terminator, generating pSP5 (Fig. IE). A 1.2-kb XhoI-PvuII DNA fragment was subcloned from pET1 into plasmid YEp62, linearized with Sal1 + SmaI, fusing the APHl gene in frame to the GAL10 promoter. The resulting plasmid was designated pAJ418. Plasmid pSP3 was linearized with XhoI and NcoI and the sticky ends of the large (14-kb) fragment were filled-in with PolIk and dephosphorylated with CIAP. A 4.1-kb HpaI DNA fragment, containing the GAL10 promoter, APHI cassette and the LEU2 gene, was subcloned from pAJ418 into the linearized pSP3. The GAL10 promoter-APHI cassette was thus inserted upstream from the UBA3 terminator, thereby sandwiching the APHI gene between the GAL10 promoter and URA3 terminator in pSP5. A 4.3-kb NdeI DNA fragment, containing the ARSl and CEN4 sequences, was subcloned from the yeast centromeric plasmid YCp50 into the unique NdeI site of pSP5, thereby converting pSP5 into a circular minichromosome, pSP6 (Fig. 1F). (b) Yeast transformation
and marker stability
The yeast integrating plasmid, YIPS, is mitotically stable and was therefore used to construct pSP1, pSP2 and pSP3 to stably express GA11 and/or AMY in a laboratory yeast strain, ISP52. Chromoblotting was used to confirm the integration of these YIPS-derived plasmids into the genomes of the yeast transformants, containing pSP1, pSP2 and pSP3. The intact chromosomes of the respective Ura+
$x42, @Jggj ; LEu2, r-1 ~pro~~t&, * RR,
-
: C#!W#, m ; Genomic DNA fmm S.
; ARSl, m
diastatkus -
; GAL&I-P
; cm.43, uRA3-r([email protected]), ;ori,*;
R
Ap , -
; RNAsequences fmm Ylp5,
; KmR (= GtR), -
;
-
Fig. 1. Restrict&m endanuctease maps of recombiiztnt plasmids pSf1 (A), pSP2 (B), pSP3 (C), pSP4 (D}, pSP5 (E) and pSP6 (F). Pfasmids pSPl through pSP5 are yeast-i~te~ati~g plasmids, whereas pSP6 is a yeast-centromeric plasmid. Plasmids pSf3 throughpSf6 conttin both the STAZ andAMY genes, whereas pSP1 contains SE42 and pSF2 the AMY gene. Plasmids pSP4 through pSP6 carry the AfHI gene.
89 transformants were separated by pulsed-field gel electrophoresis (TAFE), blotted onto nylon filters, probed with the 1438-bp NdeI-PvuII [32P]DNA fragment (common to pSP1, pSP2 and pSP3) and autoradiographed (Fig. 2). By homologous recombination, plasmid pSP1 integrated into chr. IX, presumably at the SGAI locus, whereas pSP2 integrated into chr. V, presumably at the uru3 locus. Plasmid pSP3 integrated into one of the high-& c~omosomes that could not be identified on the chromoblot shown in Fig. 2. The mitotic stability of pSP1, pSP2 and pSP3 in these yeast transformants (grown in nonselective media) was calculated as 100 %. Polyploid brewing (RSAl) and distilling (RSA2) yeast strains lack selective genetic markers and could therefore only be transformed with plasmids containing the positive selectable marker, APHl. The laboratory strain, ISP53, was successfully transformed with pSP4 to GtR. The industrial yeast strains, RSAI and RSA2, could, however, not be ~~sformed to GtR and it was found that they were more sensitive to Gt (10 and 30 ,ug/ml, respectively) than the laboratory yeast strains (90 @g/ml). For enhanced expression of GtR, pSP5 was constructed in which APHl was linked to the galactose-inducible GAL10 promoter and the URA3 terminator. The GAL10 gene, encoding UDP galacA ISP52
HP52
ISP52
ISP52
IpSP2l LpSP11 LpSP31
Fig. 2. Integration of plasmids #PI, pSP2 and pSP3 into the chromosomes of S. cerevisiueISP52 transformants. (Panel A) Ethidium bromidestained agarose gel on which intact chromosomes of yeast transformants were resolved by TAFE (Geneline; Beckman Instruments, Palo Alto, CA). From left to right the lanes were loaded with indicated DNA samples. Standard electrophoretic conditions employed 1.25% agarose, 300 V, 14”C, 1 x TAFE buffer, switching interval of 55 s and running time of 18 h. Gels were stained afterwards with EtdBr to visualize the DNA. Yeast DNA samples were prepared as described by Smith and Cantor (1987). (Panel B) Southern blot probed with the 143%bp N&IPvuII [a-3ZP]DNA fragment. DNA transfer hyb~dization and chromosome identification were carried out as described by Pretorius and Marmur (1988).
tose-4-epimerase, is expressed at a level of approx. 1% of the total polyadenyiated RNA in a yeast cell (St. John and Davis, 1981). The laboratory strain, ISP53, was transformed with pSP5 to Leu+ (SC -Le” agar plates) and GtR (> 600 pg Gt/ml on YPGal -t G418 agar plates). However, efforts to transform industrial strains RSA 1 and RSA2 with pSP5 failed. To increase the transformation efficiency, pSP5 was converted to a centromeric plasmid. The circular m~ic~omosome, pSP6, was successfully transformed into the laboratory strain ISP53 as well as the brewing and distilling strains, RSAl and RSA2, rendering the transformants resistant to 6OOpg Gtjml on YPGal+ Gt agar plates. The presence of pSP6 in these transformants was confirmed by Southe~-blot hyb~dization (results not shown) and the formation of halos on YPP. (c) Synthesis and secretion of GA11 and/or AMY Competent E. coil HBlOl cells were transformed with the recombin~t plasmids and screened for starch hydrolysis. E. coli transformants producing AMY, containing pSP2 through pSP6, formed halos on LBS agar plates (using the iodine staining technique) and LBP agar plates (PAT; Pharmacia Diagnostics, Uppsala, Sweden). PAT starch is a water-insoluble cross-linked glucose polymer coupled to a blue dye and is resistant to the exo-activity of GA (Marciniak and Kula, 1982). Halo formation on LBP agar plates therefore specifically indicates AMY activity. Consistent with the findings of Pretorius et al. (1988), the AMY gene from B. ~~yloi~q~e~~i~ns (present on plasmids pSP2 through pSP6) conferred extracellul~ AMY activity to the E. co& transformants. Due to the inability of E. coli to recognize STA2 regulatory and/or secretory signals, E. coli transformants containing pSP1 did not form halos on LBS and LBP agar plates. S. cerevisiae strain ISP52 was tr~sfo~~ to Ura’ Sta+ with plasmid pSP1, to Ura+ Amy+ with pSP2, to Ura + Sta + Amy + with pSP3 and to Ura + Sta + Amy + and GtR with pSP4, respectively. Strain ISP53 was transformed to Leu + Sta” Amy + and GtR with plasmid pSP5. Laboratory strain ISP53 and industrial strains RSAl and RSA2 were transformed to Leu’ and GtR with pSP6 and exhibited extracellular amylolytic activity. Furthermore, the integrity of pSP6 in the yeast transformants was also confirmed by the transformation of E. coli HBlOl with extrachromosomal DNA from RSAl[pSP6] and RSA2[pSP6]. The E. coli tr~sfo~~ts cont~ning pSP6 formed halos on LBS and LBP agar plates. All the yeast transformants formed halos on YPSB (Pretorius et al., 1986a) and YPS agar plates. All yeast transformants except ISP52(pSPl) formed halos on YPP agar plates (Fig. 3A). Transformants ISP52(pSPl) formed a slightly turbid halo on YPS agar plates, whereas ISP52(pSP2) produced a clearer halo and ISP52(pSP3) a larger and clearer halo (Fig. 3B). These
90
Fig. 3. Secretion of amylolytic enzymes by yeast transformants. (Panel A) yeast transformants containing plasmids pSP1 through pSP3 were spotted on YPP plates. Halos only developed around colonies secreting AMY. (Panel B) The co-operative action of GA11 and AMY visualized on YPS plates after staining with an iodine solution (0.33% 1,/0.66% KI). Agar plates were incubated for live days at 30°C.
results indicate efficient starch degradation by the co-operative action of GA11 and AMY.
A 1
z 100
_.-•-*~st'52[psP3]
(d) Starch utilization and hydrolysis
Because secreted amylolytic enzymes are present continuously during growth of yeast cells, the sugar uptake and carbohydrate content of the culture broth at a given time will reflect both the types and activities of the amylolytic enzymes present as well as the ability of the cells to assimilate various starch hydrolysis products. The utilization of starch by the different yeast strains and transformants were compared with each other (Fig. 4A). As expected, the control strain ISP52, secreting neither GA11 nor AMY, consumed almost no starch. Transformant ISP52(pSPl), secreting GAII, consumed 69% of the available starch, indicating the ineffectiveness of this strain to utilize starch efficiently. Transformant ISP52(pSP2), secreting AMY, utilized 84% of the available starch. The relatively low concentration of residual sugar left in the culture medium indicates rapid sugar hydrolysis and utilization. Transformant ISP52(pSP3), secreting both GA11 and AMY, assimilated more than 93% of the starch within 120 h. In fact, 80% of the starch was assimilated within 48 h. The yeast strains and transformants were further analyzed with respect to their ability to hydrolyze residual starch (based on the visual disappearance of the purple colour of the starch-iodine complex) during a five-day growth period. As shown in Fig. 4B, a significant fraction of the starch was not hydrolyzed by S. diastaticus strain ISP55 and S. cerevisiae transformant ISP52(pSPl). This illustrates the inability of the secreted GA11 to hydrolyze starch completely. The AMY-containing yeast transforquite differently. Transformants mants behaved
.e---~-~-.~sP52[psP2]
0
12 24 36 46 60 72 64 96 106120
TIME (hours)
B
0
ii 24 i6 4'6 sb i2 Sk 96 106120
TIME (hours)
Fig. 4. Starch utilization and hydrolysis. Time course of starch utilization (part A) and residual starch hydrolysis (part B) by yeast strains and transformants. To analyze residual starch in the culture medium, 1 ml of supernatants ofeach ofthe different strains grown in YPS was mixed with 1 ml of 2 M HCl and complete hydrolysis was accomplished by heating the mixture in a boiling water bath for 30 min. After neutralization of the hydrolysate with 1 ml 2 M NaOH, the reducing sugars released from starch was determined, using the DNS calorimetric method (Bergmeyer, 1974).
91 TABLE II
ISP52(pSP2) hydrolyzed 98% of the starch after five days. In the culture supernatant of transformant ISP52(pSP3), containing both STA2 and AMY, starch hydrolysis was complete after 72 h. Thus, ISP52(pSP3) hydrolyzes starch much faster than any of the other transformants, indicating a co-operative action of GA11 and AMY. To quantify the levels of extracellular amylolytic activity produced by the various yeast strains and transformants, cell-free culture fluids were assayed for AMY and GA11 activity. The PAT assay was used to specifically quantify AMY activity, the DNS method was used to measure the amount of reducing sugars released from starch and the glucose-peroxidase test was used to determine the amount of glucose released from starch (Table II). The recipient strain, ISP52, exhibited no amylolytic activity as measured by any of the mentioned techniques. AMY activity was only present in the culture supernatant of the AMY containing transformants, ISP52(pSP2) (860 u/liter) and ISP52(pSP3) (850 u/liter). The effect of the STA2 and AMY genes on the production of amylolytic activity by ISP52(pSP3) appears to be more or less additive when reducing sugar was assayed. The sum of the GA11 activity (48 u/ml/min) produced by ISP52(pSPl) and the AMY activity (20 u/ml/min) produced by ISP52(pSP2) was 68 u/ml/min, similar to the value of 61 u/mI/min obtained with ISP52(pSP3). However, when amylolytic activity was assayed by the glucose-peroxidase test, important differences were observed. Low amounts of glucose were liberated from starch by the action of AMY (10.5 u/ml/mm) present in the culture supernatant of ISP52(pSP2), whereas amylolytic enzymes in the culture supernatants of both ISP52(pSPl) and ISP52(pSP3) released high amounts of
A
Amylolytic activities of yeast strains and transformants Yeast strains*
Phadebas b (u/liter)
Reducing sugars c (u/ml/min)
Glucose d (u/ml/mm)
ISP52 ISP55 ISP52(pSPl) ISP52(pSP2) ISP52(pSP3)
0 0 0 860 850
0 36.80 48.00 20.00 61.00
0 36.40 50.30 10.50 53.30
a Yeast cultures (see Table I) were grown in YPGE medium for 72 h and the cells harvested by centrifugation. The culture supematants were concentrated 60-fold by ultrafiltration using the Millipore Minitan system, the Millipore filter type 1000 NMWL and the Millipore Durapore membrane PTGC 00001 (Millipore Corporation, Bedford, MA). Reactions for ISP55 and ISP52(pSPl) were carried out at pH 5.0 for ISP52(pSP2) at pH 6.0 and for ISP52(pSP3) at pH 5.5. b AMY activity in the culture supernatants was quantified with the PAT assay (Pharmacia Diagnostics, Uppsala, Sweden) using a 20 mM sodium acetate buffer (pH 5.0, pH 5.5 or pH 6.0) and incubation at 60°C for 10 min. Starch hydrolysis in the culture supernatant was assayed by the iodine reaction as described by Filho et al. (1986). ’ Amylolytic activity in the culture supernatants was quantified using the DNS calorimetric method that detects reducing sugars released from starch (Pretorius et al., 1988). Reaction tubes contained 500 nl of culture supematant and 500 ~1 of a 1y0 Lintner starch solution in a 20 mM Na . acetate buffer (pH 5.0, pH 5.5 or pH 6.0). Tubes were incubated at 60°C for 10 min. The reaction was stopped by adding 1 ml DNS solution, boiled for 10 min and chilled on ice. Absorbance was measured at 546 nm (A&. d Amylolytic activity in culture supernatants was also quantified with the PGO/ODAD assay (Sigma kit No. 510; Sigma Chemical Company, St. Louis, MO) that detects glucose released from starch. Standard curves were constructed using glucose and maltose (Sigma CAR-1 1). One unit of amylolytic activity was defined as the amount of enzyme that liberated 1 pmol of reducing sugar or glucose/min/ml at 60°C.
B
C
$ $4 8 18
1
ol
b t
c H L
&
0)
E
a
:!__JI_ b: ‘n ,
5
7
5
Time (min)
7
18
Time (min)
5
7
18
T!me (mm)
Fig. 5. Gas-chromatographic analysis of the starch hydrolysates from the supematants of ISP52(pSPl) (part A), ISP52(pSP2) (part B) and ISP52(pSP3) (part C). No glucose or maltose were detected in the supematant of ISP52. Samples were analyzed with a Varian Vista 6000 gas chromatograph equipped with FID. A 12 m x 0.32 mm ID SE30 fused silica capillary column was used. The following operating conditions were used: column temperature was increased from 130” to 230°C at S”C/min intervals; injector temperature 220°C; FID temperature 250°C; helium carrier linear flow velocity 100 cm/s. Maltotriose, maltotetraose and carbohydrate standard solutions (CAR-11; Sigma Chemical Company, St. Louis, MO) were used as standards. Results were analyzed with a Varian Vista 402 data system. The supematants of strains grown in YPS were incubated at 60°C for 3 h, using the appropriate buffer. The samples were then frozen and freeze-dried. The carbohydrates were subsequently silylated with Tri-sil Z (500 ~1) at 60-65°C for 15 min.
92 glucose from starch (50.3 and 53.3 u/ml/min, respectively). These results can be explained together with the gas chromatographic analyses of the starch hydrolysates (Fig. 5). The STA2-encoded GAII, secreted by ISP52(pSPl), is an a-1,4 glucanglucohydrolase, releasing glucose (Fig. 5A) from the nonreducing end of the starch molecule (Modena et al., 1986). AMY is an a-1,4 glucan 4-glucanohydrolase, hydrolyzing the linkages of starch in a random fashion, producing primarily glucose and maltose (1: 1) (Fig. 5B). The GA11 and AMY activities, secreted by ISP52(pSP3), cooperatively liberated glucose and maltose (3 : 1) from starch (Fig. 5C). Since glucoamylases have a low affinity for low-M, substrates (Modena et al., 1986; Tucker et al., 1984; Whitaker, 1972), only part of the maltose and oligomeric products of AMY action served as substrate for the STAZ-encoded enzyme. In conclusion, laboratory and industrial strains of 5’. cerevisiue transformed with recombinant plasmids carrying both the STA2 and AMY genes, were capable of secreting glucoamylase and a-amylase simultaneously, thereby mediating efficient one-step starch utilization.
ACKNOWLEDGEMENTS
The authors thank Desmond van Jaarsveld for helping us to ultraliltrate culture supernatants and Dr. Marita le Roux for help with the gas-chromatographic analysis of starch hydrolysates.
REFERENCES Ashikari, T., Nakamura, M., Tanaka, Y., Kiuchi, N., Shibano, Y., Tanaka, T., Amachi, T. and Yoshizumi, H.: Rhizopus raw-starchdegrading glucoamylase: its cloning and expression in yeast. Agric. Biol. Chem. 50 (1986) 957-964. Ashikari, T., Kiuchi-Goto, N., Tanaka, Y., Shibano, Y., Amachi, T. and Yoshizumi, H.: High expression and efficient secretion of Rhizopus oryzae glucoamylase in the yeast Saccharomyces cerevtiiae. Appl. Microbial. Biotechnol. 30 (1989) 5 15-520. Bergmeyer, H.U.: Methods of Enzymatic Analysis, Vol. 1. Verlag Chemie, Weinheim, 1974, p. 42. Broach, J.R.,Li, Y.Y., Wu, L.C.C. and Jayaram, M.: Vectors for high-level inducible expression of cloned genes in yeast. In: Inouye, M. (Ed.), Experimental Manipulation of Gene Expression. Academic Press, New York, 1983, pp. 83-117. Clancy, M.J., Smith, M. and Magee, P.T.: Developmental regulation of a sporulation-specific enzyme activity in Saccharomyces cerevisiae. Mol. Cell. Biol. 2 (1982) 171-178. Cole, G.E., McCabe, P.C., Inlow, D., Gelfand, D.H., Ben-Bassat, A. and Innis, M.A.: Stable expression ofAspergiIlus awamoriglucoamylase in distiller’s yeast. Biotechnology 6 (1988) 417-421. De Mot, R. and Verachtert, H.: Biocatalysis and biotechnology with yeast. ASM News 50 (1984) 526-531. De Mot, R., Van Dijck, K., Donkers, A. and Verachtert, H.: Potentialities and limitations of direct alcoholic fermentation of starchy material
with amylolytic yeast. Appl. Microbial. Biotechnol. 22 (1985) 222-226. Erratt, J.A. and Nasim, A.: Cloning and expression of a Saccharomyces diastaticus glucoamylase gene in Saccharomyces cerevtsiae and Schizosaccharomyces pombe. J. Bacterial. 166 (1986) 484-490. Filho, S.A., Galembeck, E.V., Faria, J.B. and Schenberg Frascino, AC.: Stable yeast transformants that secrete functional a-amylase encoded by cloned mouse pancreatic cDNA. Biotechnology 4 (1986) 3 11-315. Inlow, D., McRae, J. and Ben-Bassat, A.: Fermentation of corn starch to ethanol with genetically engineered yeast. Biotechnol. Bioeng. 32 (1988) 227-234. Innis, M.A., Holland, M.J., McCabe, P.C., Cole, G.E., Wittman, V.P., Tall, R., Watt, K.W.K., Gelfand, D.H., Holland, J.P. and Meade, J.H.: Expression, glycosylation and secretion of an Aspergillw glucoamylase by Saccharomyces cerevisiae. Science 228 (1985) 21-26. Ito, H., Fukuda, Y., Murata, K. and Kimura, A.: Transformation ofintact yeast cells treated with alkali cations. J. Bacterial. 153 (1983) 163-168. Janse, B.J.H., Green, D.A. and Pretorius, I.S.: Structure and regulation of genes involved in pullulan utilization in Klebsiellapneumoniae. Acta Varia 5 (1990) 29-68. Johnston, M. and Davis, R.W.: Sequences that regulate the divergent GALI-GAL10 promoter in Saccharomyces cerevisiae. Mol. Cell. Biol. 4 (1984) 1440-1448. Kim, K., Park, C.S. and Mattoon, J.R.: High-efficiency, one-step starch utilization by transformed Saccharomyces cells which secrete both yeast glucoamylase and mouse a-amylase. Appl. Environ. Microbial. 54 (1988) 966-971. Kovaleva, I.E., Novikova, L.A. and Luzikov, V.N.: Synthesis and secretion of bacterial a-amylase by the yeast Saccharomyces cerevtsiae. FEBS Lett. 251 (1989) 183-186. Lambrechts, M.G., Pretorius, IS., Sollitti, P. and Marmur, J.: Primary structure and regulation of a glucoamylase-encoding gene (STAZ) in Saccharomyces diastaticus. Gene 100 (1991) 000-000. Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. Marciniak, V.G.P. and Kula, M.R.: A comparative study of the methods for the determination of the activity of bacterial a-amylases. Starke (in German) 34 (1982) 422-430. Meaden, P., Ogden, K., Bussey, H. and Tib, R.S.: A DEX gene conferring production of extracellular amyloglucosidase on yeast. Gene 34 (1985) 325-344. Modena, D., Vanoni, M., Englard, S. and Marmur, J.: Biochemical and immunological characterization of the STA2-encoded extracellular glucoamylase from Saccharomyces diastaticus. Arch. Biochem. Biophys. 248 (1986) 138-150. Nakamura, Y., Sato, T., Emi, M., Miyanohara, A., Nishide, T. and Matsubara, K.: Expression of human salivary a-amylase gene in Saccharomyces cerevisiae and its secretion using the mammalian signal sequence. Gene 50 (1986) 239-245. Nonato, R.V. and Shishido, K.: a-Factor directed synthesis of Bacillus stearothermophilw a-amylase in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 152 (1988) 76-82. Pardo, J.M., Polaina, J. and Jimenez, A.: Cloning of the STA2 and SGA genes encoding glucoamylases in yeast and regulation of their expression by the STAlO gene of Saccharomyces cerevisiae. Nucleic Acids Res. 14 (1986) 4701-4718. Pretorius, IS. and Marmur, J.: Localization of yeast glucoamylase genes by PFGE and OFAGE. Curr. Genet. 14 (1988) 9-13. Pretorius, I.S., Chow, T. and Marmur, J.: Identification and physical characterization of yeast glucoamylases structural genes. Mol. Gen. Genet. 203 (1986a) 36-41.
93 Pretorius, I.S., Chow, T., Modena, D. and Marmur, J.: Molecular cloning and characterization of the ST’2 ghrcoamylase gene of Saccharom.vces diastaticus. Mol. Gen. Genet. 203 (1986b) 29-35. Pretorius, IS., De Kock, M.J., Britz, T.J., Potgieter, H.J. and Lategan, P.M.: Numerical taxonomy of a-amylase producing BaciNus species. J. Appl. Bacterial. 60 (1986~) 351-360. Pretorius, IS., Laing, E., Pretorius, G.H.J. and Marmur, J.: Expression of a Bacillus a-amylase gene in yeast. Curr. Genet. 14 (1988) 1-8. Pretorius, IS., Modena, D., Vanoni, M., Englard, S. and Marmur, J.: Transcriptional control of glucoamylase synthesis in vegetatively growing and sporulating Saccharomyces species. Mol. Cell. Biol. 6 (1986d) 3034-3041. Pretorius, IS., Lambrechts, M.G. and Marmur, J.: The glucoamylase multigene family in Saccharomyces cerevisiae var. diastaticus: an overview. Crit. Rev. Biochem. Mol. Biol. (1991) (in press). Rothstein, S.J., Lazarus, C.M., Smith, W.E., Baulcombe, D.C. and Gatenby, A.A.: Secretion of a wheat a-amylase expressed in yeast. Nature 308 (1984) 662-665. Rothstein, S.J., Lahners, K.N., Lazarus, CM., Baulcombe, D.C. and Gatenby, A.A.: Synthesis and secretion of wheat a-amylase in Saccharomyces cerevisiae. Gene 55 (1987) 353-356. Ruohonen, L., Hackman, P., Lehtovaara, P., Knowles, J.K.C. and Keranen, S.: Efftcient secretion of Bacillus amyloliquefaciens a-amylase cells by its own signal peptide from Saccharomyces cerevisiue host. Gene 59 (1987) 161-170. Sakai, K. and Yamamoto, M.: Transformation of the yeast, Succharomyces curlsbergensis, using an antibiotic resistance marker. Agric. Biol. Chem. 50 (1986) 1177-1182. Shiosaki, K., Takata, K., Omichi, K., Tomita, N., Horii, A., Ogawa, M. and Matsubara, K.: Identification of a novel a-amylase by expression
of a new cloned human amy cDNA in yeast. Gene 89 (1990) 253-258. Smith, CL. and Cantor, CR.: Purification, specific fragmentation and separation of large DNA molecules. Methods Enzymol. 155 (1987) 449-467. Steyn, A.J.C. and Pretorius, I.S.: Expression and secretion of amylolytic enzymes by Saccharomyces cerevisiue. Acta Varia 5 (1990) 76-126. StJohn, T.P. and Davis, R.W.: The organization and transcription of the galactose gene cluster of Saccharomyces cerevtiiae. J. Mol. Biol. 152 (1981) 285-315. Struhl, K., Stincomb, D.T., Scherer, S. and Davis, R.W.: High-frequency transformation of yeast: autonomously replication of hybrid DNA molecules. Proc. Natl. Acad. Sci. USA 76 (1979) 1035-1039. Tucker, M., Grohmann, K. and Himmel, M.: Isolation and characteristics of a glucoamylase from Saccharomyces diastaficus. Biotech. Bioeng. Symp. 14 (1984) 279-293. Thomsen, K.K.: Mouse a-amylase synthesized by Sacchnromyces cerevisiue is released into the culture medium. Carlsberg Res. Commun. 48 (1983) 545-555. Vihinen, M. and MantsZila, P.: Microbial amylolytic enzymes. Crit. Rev. Biochem. Mol. Biol. 24 (1989) 329-418. Wang, T.T., Lin, L.L. and Hsu, W.H.: Cloning and expression of a Schwanniomyces occidentalk a-amylase gene in Saccharomyces cerevi&e. Appl. Environ. Microbial. 55 (1989) 3167-3172. Whitaker, J.R.: Principles of Enzymology for the Food Sciences. Marcel Dekker, New York, 1972. Yamashita, I., Itoh, T. and Fukui, S.: Cloning and expression of the SaccharomycopsisjTbuligera glucoamylase gene in Saccharomyces cerevisiue. Appl. Microbial. Biotechnol. 23 (1985) 130-133.
85
GENE 04064
Co-expression of a Saccharomyces diastaticus glucoamylase-encoding faciens a-amylase-encoding gene in Saccharomyces cerevisiae (Recombinant DNA; plasmid; yeast transformation;
gene and a Bacillus amylolique-
starch degradation; amylolytic enzymes)
Andries J.C. Steyn and Isak S. Pretorius Department of Microbiology and Institutefor Biotechnology, University of Stellenbosch, Stellenbosch 7600 (South Africa) Received by J. Marmur: 13 December 1990 Revised: 31 December 1990 Accepted: 28 January 1991
SUMMARY
A glucoamylase-encoding
gene (STAZ) from Saccharomyces diastaticus and an a-amylase-encoding gene (AMY) from were cloned separately into a yeast-integrating shuttle vector (YIPS), generating recombinant plasmids pSP1 and pSP2, respectively. The STA2 and AMY genes were jointly cloned into YIPS, generating plasmid pSP3. Subsequently, the dominant selectable marker APHI, encoding resistance to Geneticin G418 (GtR), was cloned into pSP3, resulting in pSP4. For enhanced expression of GtR, the APHl gene was fused to the GAL10 promoter and terminated by the URA3 terminator, resulting in pSP5. Plasmid pSP5 was converted to a circular minichromosome (pSP6) by the addition of the ARSI and CEM sequences. Laboratory strains of Saccharomyces cerevkiae transformed with plasmids pSP1 through pSP6, stably produced and secreted glucoamylase and/or a-amylase. Brewers’ and distillers’ yeast transformed with pSP6 were also capable of secreting amylolytic enzymes. Yeast transformants containing pSP1, pSP2 and pSP3 assimilated soluble starch with an efficiency of 69%, 84% and 93%, respectively. The major starch hydrolysis products produced by crude amylolytic enzymes found in the culture broths of the pSPl-, pSP2- and pSP3-containing transformants, were glucose, glucose and maltose (1: l), and glucose and maltose (3 : l), respectively. These results confirmed that co-expression of the STA2 and AMY genes synergistically enhanced starch degradation. Bacillus amyloliquefaciens
Correspondence to: Dr. IS. Pretorius, Department of Microbiology, University of Stellenbosch, Stellenbosch 7600 (South Africa) Tel. (27)2231-774730; Fax (27)2231-773611. Abbreviations: aa, amino acid(s); A546, absorbance at 546 nm; AMY, a-amylase (EC 3.2.1.1); AMY, gene encoding AMY; Ap, ampicillin; APHl, gene encoding aminoglycoside phosphotransferase-3’(I); ARSl, autonomously replicating sequence 1; B., Bacillus; bp, base pair(s); CEN4, centromere 4; chr., chromosome; CIAP, calf intestinal alkaline phosphatase; Cm, chloramphenicol; DNS, dinitrosalicylic acid; EtdBr, ethidium bromide; FID, flame ionization detector; GAI, GA11 and GAIII, extracellular glucoamylase (EC 3.2.1.3) isozymes I, II and III, respectively; GALIO, gene encoding uridine diphosphoglucose 4-epimerase EC 5.1.3.2; Gt, Geneticin (G418); kb, kilobase or 1000 bp; Km, kanamycin; LB, 1% Bacto tryptone/0.5% yeast extract/O.5% NaCl pH 7.0; LBP, LB/IO PAT tablets per 100 ml; LBS, LB/l % starch; LEUZ, gene encoding b-isopropylmalate dehydrogenase (EC 1.1.1.85); ODAD, O-dianisidine; ori, origin of replication; PAT, Phadebas Amylase Test;
PGO, peroxidase-glucose oxidase; PolIk, Klenow (large) fragment of E. coli DNA polymerase I; pYC, S. ceretiiae-E. coli shuttle cosmid vector; R, resistance/resistant; S., Saccharomyces; SC, 0.67% nitrogen base without aa/2% glucose plus all the required growth factors; SCeUra, SC lacking uracil; SCeL”“, SC lacking Leu; SGA, sporulation-specific intracellular glucoamylase EC 3.2.1.3; SGAl, gene encoding SGA; STAl, STA2 and STA3, genes encoding GAI, GA11 and GAIII, respectively; STAlO, gene encoding glucoamylase repressor; TAFE, transverse alternating-field electrophoresis; TAFE buffer, 20 mM Tris .HCl/7.8 mM EDTA/78 mM glacial acetic acid pH 8.0; Tc, tetracycline; u, unit(s); UR43, gene encoding orotidine-5’-phosphate carboxylyase (EC 4.11.23); YCp, yeast centromeric plasmid; YEp, yeast episomal plasmid; YIP, yeast integrating plasmid; YP, 1% yeast extract/2% Bacto peptone; YPD, YP/2% glucose; YPGal, YP/2% galactose; YPGE, YP/3% glycerol/2% ethanol; YPP, YP/l y0 starch/l0 PAT tablets per 100 ml; YPS, YP/l% starch; YPSB, YP/3% starch/0.003% bromocresol purple; [ 1, denotes plasmid-carrier state; ( ), plasmid integrated into yeast genome; : :, novel joint (fusion).
86 INTRODUCTION
The budding yeast S. cerevisiae has been used widely both as a model system for unravelling the molecular, biochemical and genetic details of gene expression and the secretion process, and as a host for the production of heterologous proteins of biotechnological interest. The potential of starch as a renewable biological resource has stimulated research into amylolytic enzymes and the broadening of the substrate range of S. cerevisiae (for a review see Steyn and Pretorius, 1990). The enzymatic hydrolysis of starch, consisting of linear (amylose) and branched glucose polymers (amylopectin), is catalyzed by CI- and @nylases, glucoamylases and debranching
enzymes, e.g., pullulanases (Janse et al., 1990; Vihinen and MBnts&i, 1989). Although wild-type strains of S. cerevisiae lack extracellular amylolytic activity and are unable to utilize starch as a substrate, they do possess the ability to produce SGA (Clancy et al., 1982). Furthermore, S. cerevisiae is closely related to and can interbreed with the amylolytic yeast S. diastaticus. Strains of S. diastaticus carrying one of the unlinked, haploid-specific genes STAI, STA2 and STA3, produce the extracellular glucoamylase isozymes GAI, GA11 and GAIII, respectively (Lambrechts et al., 1991; Pretorius et al., 1986a; for a review see Pretorius et al., 1991). Some S. cerevisiae strains also carry the STAlO repressor gene that inhibits glucoamylase expression at the
TABLE I Saccharomyces
strains used in the present study Relevant genotype b
Source or reference
a a a a a
a sta” ura3::pSP4 his3 a sta” Ieu2:: pSP5 h&4 a sta” leu2 his4 [pSP6] Polyploid brewers’ yeast[pSP6] Polyploid distillers’ yeast[pSP6]
IS. Pretorius IS. Pretorius I.S. Pretorius This work This work This work This work This work This work This work This work
pAM13 pUS52 pET1 pSP1 pSP2 pSP3 pSP4 pSP5 pSP6
ApR LEU2 AMY ApR HIS3 STA2 ApR KmR TcR CmR Apu URA3 STA2 ApR VRA3 AMY ApR (IRA3 AMY STA2 ApR KmR UR43 AMY STA2 ApR KmR LEU2 AMY STA2 GALlOp ApR KmR LEU2 AMY STA2 GALlOp
Pretorius et al. (1988) Pretorius et al. (1986b) ATCC39566 This work This work This work This work This work This work
YEp62
ApR LEV2 GAL10 1acZ ApR TcR URA3 CEN4 ARGI ApR TcR URA3
Strain or plasmid” Yeast strains:
ISP52 ISP53 ISP55 ISP52(pSPl) ISP52(pSP2) ISP52(pSP3) ISP52(pSP4) ISP52(pSP5) ISPSZ[pSP6] RSAl[pSP6] RSA2[pSP6]
sta” wall his3 sta” leu2 hir4 STA2 stal0 arg4 sta” ura3 SGAI ::pSPI his3
sta” ura3 : : pSP2 hir3
a sta” ura3::pSP3
his3
Plasmids:
CEN4 ARSI
YCp50 YIp5
Broach et al. (1983) Johnston and Davis (1984) Struhl et al. (1979)
a Strains ISP52 and ISP53 are Sta- segregants of a cross between two strains carrying the STAZ and STA3 glucoamylase genes of S. diastaticus, respectively. Transformation of CaCl,-treated E. coli cells was performed according to Maniatis et al. (1982) and transformants were selected on media containing 50 pg/ml Ap (LB + Ap, LBS + Ap, LBP + Ap) or 20 &ml Km (LB + Km, LBS + Km, LBP + Km). Saccharomyces strains were transformed with linear or circular plasmid DNA by the lithium acetate method (Ito et al., 1983; Sakai and Yamamoto, 1986) and transformants were selected either on minimal media (SC- Ura, SC- ti”) or on complex media containing Gt (YPGal + Gt). Mitotic stability of markers transformed into S. cerevisiae was analyzed by culturing the yeast transformants in 2 liter of YPD until stationary phase. One ml of this yeast suspension was inoculated into fresh YPD (2 liter) and incubated until stationary phase. This procedure was repeated five times. Serial dilutions were made from the final culture and plated onto YPD agar plates and replica-plated onto selective media (SC- “ra, SC- Lcu,YPGal + Gt). The mitotic stability was determined by using the following equation: mitotic stability (%) = 100 x (number of colonies on selective medium / number of colonies on YPD). The mitotic stability was measured for two different transformants of each strain and the average recorded. b ::, indicates integration of the YIPS-derived plasmid into the genomes of the yeast strains. [ 1, indicates that the yeast strains harbour the YCpSO-derived centromeric plasmid. ( ), indicates that the plasmids were integrated into the yeast genomes.
87 transcriptional level (Pretorius et al., 1986d). Manipulation of S. cerevisiue to synthesize and secrete functional amylolytic enzymes would facilitate a direct, one-step bioconversion of starch-rich materials to ethanol (potable alcohol or a fuel extender) or single-cell protein (food and feed supplements). Amylolytic yeasts of genera other than Saccharomyces are generally not suitable for the production of bioethanol and single-cell protein. S. cerevisiue has a fast growth and fermentation rate, is ethanol-tolerant (De Mot et al., 1985), consists of 48% high-quality protein and has been associated with food and beverage production for centuries (De Mot and Verachtert, 1984). Heterologous amylase genes derived from various organisms have been expressed in S. cerevisiae. These include the a-amylase genes from wheat (Rothstein et al., 1984; 1987), mouse salivary glands (Thomsen, 1983) mouse pancreas (Filho et al., 1986), human salivary glands (Nakamura et al., 1986), human lung carcinoid tissue (Shiosaki et al., 1990), B. amylohquefaciens (Kovaleva et al., 1989; Pretorius et al., 1988; Ruohonen et al., 1987) B. stearothermophilus (Nonato and Shishido, 1988), Schwanniomyces occidentalis (Wang et al., 1989) and the glucoamylase genes from Aspergillus awamori (Cole et al., 1988; Inlow et al., 1988; Innis et al., 1985), Rhizopus oryzue (Ashikari et al., 1986; 1989), Succharomycopsis fibuligeru (Yamashita et al., 1985) and S. diustuticus (Erratt and Nasim, 1986; Meaden et al., 1985; Pardo et al., 1986; Pretorius et al., 1986b). Kim et al. (1988) described the construction of a hybrid yeast strain secreting both glucoamylase and AMY utilizing 93% of Lintner starch. This strain was obtained by transforming an S. diastaticus derivative with an episomal plasmid containing the mouse salivary AMY gene. However, the disadvantages of using this transformant for future industrial purposes are the high mitotic instability of the plasmid and the difficulty in further manipulation of the glucoamylase gene(s) located on the chromosome(s) of the recipient S. diustuticus hybrid strain. This report describes the construction of yeast integrating and centromeric plasmids containing the STA2 gene from S. diastaticus and/or an AMY gene from B. amyloliquefaciens. Laboratory and industrial strains of S. cerevisiae were transformed with these recombinant plasmids. End products of amylose hydrolysis in the culture broths of these transformants were analyzed and characterized by gas chromatography. Starch utilization by transformants was monitored and compared.
RESULTS
AND DISCUSSION
(a) Construction
of recombinant plasmids
Pretorius et al. (1986b) have previously cloned a STA2 gene from S. diastaticus into the E. coli-yeast shuttle cosmid
vector pYC1, resulting in pUS52. The STA2 gene was subcloned as an 8.3-kb BglII DNA fragment into the BamHI site of the yeast-integrating plasmid vector YIPS, generating recombinant plasmid pSP1 (Fig. 1A). Pretorius et al. (1988) described the cloning of an AMY gene from B. amyloliquefuciens (isolated from compost; Pretorius et al., 1986~) into the E. coli-yeast shuttle plasmid vector YEp13, resulting in pAM13. The AMY gene was expressed and AMY secreted in S. cerevisiae using the original AMY promoter and signal sequences. The AMY gene was subcloned as a 2.1-kb PvuII DNA fragment into the PvuII site of YIPS, generating pSP2 (Fig. 1B). Plasmid pSP3, containing both the STA2 and AMY genes, was constructed by the cloning of the 8.3-kb BgZII DNA fragment from pUS52 into the BamHI site of pSP2 (Fig. 1C). The dominant selectable marker APHl was added to pSP3, resulting in pSP4 (Fig. 1D). The APHl gene encodes aminoglycoside phosphotransferase-3’(I) that inactivates several different 2-deoxystreptamine aminoglycoside antibiotics, including Km and Gt. Plasmid pSP3 was linearized withXho1 and the cohesive termini were filled-in with PolIk and dephosphorylated with CIAP. A 1.7-kb PvuII DNA fragment, containing the APHI gene, was subcloned from pET1 (ATCC39566) into the linearized pSP3. For enhanced expression of GtR, the APHI gene was fused to the GAL10 promoter and terminated by the lJR43 transcriptional terminator, generating pSP5 (Fig. IE). A 1.2-kb XhoI-PvuII DNA fragment was subcloned from pET1 into plasmid YEp62, linearized with Sal1 + SmaI, fusing the APHl gene in frame to the GAL10 promoter. The resulting plasmid was designated pAJ418. Plasmid pSP3 was linearized with XhoI and NcoI and the sticky ends of the large (14-kb) fragment were filled-in with PolIk and dephosphorylated with CIAP. A 4.1-kb HpaI DNA fragment, containing the GAL10 promoter, APHI cassette and the LEU2 gene, was subcloned from pAJ418 into the linearized pSP3. The GAL10 promoter-APHI cassette was thus inserted upstream from the UBA3 terminator, thereby sandwiching the APHI gene between the GAL10 promoter and URA3 terminator in pSP5. A 4.3-kb NdeI DNA fragment, containing the ARSl and CEN4 sequences, was subcloned from the yeast centromeric plasmid YCp50 into the unique NdeI site of pSP5, thereby converting pSP5 into a circular minichromosome, pSP6 (Fig. 1F). (b) Yeast transformation
and marker stability
The yeast integrating plasmid, YIPS, is mitotically stable and was therefore used to construct pSP1, pSP2 and pSP3 to stably express GA11 and/or AMY in a laboratory yeast strain, ISP52. Chromoblotting was used to confirm the integration of these YIPS-derived plasmids into the genomes of the yeast transformants, containing pSP1, pSP2 and pSP3. The intact chromosomes of the respective Ura+
$x42, @Jggj ; LEu2, r-1 ~pro~~t&, * RR,
-
: C#!W#, m ; Genomic DNA fmm S.
; ARSl, m
diastatkus -
; GAL&I-P
; cm.43, uRA3-r([email protected]), ;ori,*;
R
Ap , -
; RNAsequences fmm Ylp5,
; KmR (= GtR), -
;
-
Fig. 1. Restrict&m endanuctease maps of recombiiztnt plasmids pSf1 (A), pSP2 (B), pSP3 (C), pSP4 (D}, pSP5 (E) and pSP6 (F). Pfasmids pSPl through pSP5 are yeast-i~te~ati~g plasmids, whereas pSP6 is a yeast-centromeric plasmid. Plasmids pSf3 throughpSf6 conttin both the STAZ andAMY genes, whereas pSP1 contains SE42 and pSF2 the AMY gene. Plasmids pSP4 through pSP6 carry the AfHI gene.
89 transformants were separated by pulsed-field gel electrophoresis (TAFE), blotted onto nylon filters, probed with the 1438-bp NdeI-PvuII [32P]DNA fragment (common to pSP1, pSP2 and pSP3) and autoradiographed (Fig. 2). By homologous recombination, plasmid pSP1 integrated into chr. IX, presumably at the SGAI locus, whereas pSP2 integrated into chr. V, presumably at the uru3 locus. Plasmid pSP3 integrated into one of the high-& c~omosomes that could not be identified on the chromoblot shown in Fig. 2. The mitotic stability of pSP1, pSP2 and pSP3 in these yeast transformants (grown in nonselective media) was calculated as 100 %. Polyploid brewing (RSAl) and distilling (RSA2) yeast strains lack selective genetic markers and could therefore only be transformed with plasmids containing the positive selectable marker, APHl. The laboratory strain, ISP53, was successfully transformed with pSP4 to GtR. The industrial yeast strains, RSAI and RSA2, could, however, not be ~~sformed to GtR and it was found that they were more sensitive to Gt (10 and 30 ,ug/ml, respectively) than the laboratory yeast strains (90 @g/ml). For enhanced expression of GtR, pSP5 was constructed in which APHl was linked to the galactose-inducible GAL10 promoter and the URA3 terminator. The GAL10 gene, encoding UDP galacA ISP52
HP52
ISP52
ISP52
IpSP2l LpSP11 LpSP31
Fig. 2. Integration of plasmids #PI, pSP2 and pSP3 into the chromosomes of S. cerevisiueISP52 transformants. (Panel A) Ethidium bromidestained agarose gel on which intact chromosomes of yeast transformants were resolved by TAFE (Geneline; Beckman Instruments, Palo Alto, CA). From left to right the lanes were loaded with indicated DNA samples. Standard electrophoretic conditions employed 1.25% agarose, 300 V, 14”C, 1 x TAFE buffer, switching interval of 55 s and running time of 18 h. Gels were stained afterwards with EtdBr to visualize the DNA. Yeast DNA samples were prepared as described by Smith and Cantor (1987). (Panel B) Southern blot probed with the 143%bp N&IPvuII [a-3ZP]DNA fragment. DNA transfer hyb~dization and chromosome identification were carried out as described by Pretorius and Marmur (1988).
tose-4-epimerase, is expressed at a level of approx. 1% of the total polyadenyiated RNA in a yeast cell (St. John and Davis, 1981). The laboratory strain, ISP53, was transformed with pSP5 to Leu+ (SC -Le” agar plates) and GtR (> 600 pg Gt/ml on YPGal -t G418 agar plates). However, efforts to transform industrial strains RSA 1 and RSA2 with pSP5 failed. To increase the transformation efficiency, pSP5 was converted to a centromeric plasmid. The circular m~ic~omosome, pSP6, was successfully transformed into the laboratory strain ISP53 as well as the brewing and distilling strains, RSAl and RSA2, rendering the transformants resistant to 6OOpg Gtjml on YPGal+ Gt agar plates. The presence of pSP6 in these transformants was confirmed by Southe~-blot hyb~dization (results not shown) and the formation of halos on YPP. (c) Synthesis and secretion of GA11 and/or AMY Competent E. coil HBlOl cells were transformed with the recombin~t plasmids and screened for starch hydrolysis. E. coli transformants producing AMY, containing pSP2 through pSP6, formed halos on LBS agar plates (using the iodine staining technique) and LBP agar plates (PAT; Pharmacia Diagnostics, Uppsala, Sweden). PAT starch is a water-insoluble cross-linked glucose polymer coupled to a blue dye and is resistant to the exo-activity of GA (Marciniak and Kula, 1982). Halo formation on LBP agar plates therefore specifically indicates AMY activity. Consistent with the findings of Pretorius et al. (1988), the AMY gene from B. ~~yloi~q~e~~i~ns (present on plasmids pSP2 through pSP6) conferred extracellul~ AMY activity to the E. co& transformants. Due to the inability of E. coli to recognize STA2 regulatory and/or secretory signals, E. coli transformants containing pSP1 did not form halos on LBS and LBP agar plates. S. cerevisiae strain ISP52 was tr~sfo~~ to Ura’ Sta+ with plasmid pSP1, to Ura+ Amy+ with pSP2, to Ura + Sta + Amy + with pSP3 and to Ura + Sta + Amy + and GtR with pSP4, respectively. Strain ISP53 was transformed to Leu + Sta” Amy + and GtR with plasmid pSP5. Laboratory strain ISP53 and industrial strains RSAl and RSA2 were transformed to Leu’ and GtR with pSP6 and exhibited extracellular amylolytic activity. Furthermore, the integrity of pSP6 in the yeast transformants was also confirmed by the transformation of E. coli HBlOl with extrachromosomal DNA from RSAl[pSP6] and RSA2[pSP6]. The E. coli tr~sfo~~ts cont~ning pSP6 formed halos on LBS and LBP agar plates. All the yeast transformants formed halos on YPSB (Pretorius et al., 1986a) and YPS agar plates. All yeast transformants except ISP52(pSPl) formed halos on YPP agar plates (Fig. 3A). Transformants ISP52(pSPl) formed a slightly turbid halo on YPS agar plates, whereas ISP52(pSP2) produced a clearer halo and ISP52(pSP3) a larger and clearer halo (Fig. 3B). These
90
Fig. 3. Secretion of amylolytic enzymes by yeast transformants. (Panel A) yeast transformants containing plasmids pSP1 through pSP3 were spotted on YPP plates. Halos only developed around colonies secreting AMY. (Panel B) The co-operative action of GA11 and AMY visualized on YPS plates after staining with an iodine solution (0.33% 1,/0.66% KI). Agar plates were incubated for live days at 30°C.
results indicate efficient starch degradation by the co-operative action of GA11 and AMY.
A 1
z 100
_.-•-*~st'52[psP3]
(d) Starch utilization and hydrolysis
Because secreted amylolytic enzymes are present continuously during growth of yeast cells, the sugar uptake and carbohydrate content of the culture broth at a given time will reflect both the types and activities of the amylolytic enzymes present as well as the ability of the cells to assimilate various starch hydrolysis products. The utilization of starch by the different yeast strains and transformants were compared with each other (Fig. 4A). As expected, the control strain ISP52, secreting neither GA11 nor AMY, consumed almost no starch. Transformant ISP52(pSPl), secreting GAII, consumed 69% of the available starch, indicating the ineffectiveness of this strain to utilize starch efficiently. Transformant ISP52(pSP2), secreting AMY, utilized 84% of the available starch. The relatively low concentration of residual sugar left in the culture medium indicates rapid sugar hydrolysis and utilization. Transformant ISP52(pSP3), secreting both GA11 and AMY, assimilated more than 93% of the starch within 120 h. In fact, 80% of the starch was assimilated within 48 h. The yeast strains and transformants were further analyzed with respect to their ability to hydrolyze residual starch (based on the visual disappearance of the purple colour of the starch-iodine complex) during a five-day growth period. As shown in Fig. 4B, a significant fraction of the starch was not hydrolyzed by S. diastaticus strain ISP55 and S. cerevisiae transformant ISP52(pSPl). This illustrates the inability of the secreted GA11 to hydrolyze starch completely. The AMY-containing yeast transforquite differently. Transformants mants behaved
.e---~-~-.~sP52[psP2]
0
12 24 36 46 60 72 64 96 106120
TIME (hours)
B
0
ii 24 i6 4'6 sb i2 Sk 96 106120
TIME (hours)
Fig. 4. Starch utilization and hydrolysis. Time course of starch utilization (part A) and residual starch hydrolysis (part B) by yeast strains and transformants. To analyze residual starch in the culture medium, 1 ml of supernatants ofeach ofthe different strains grown in YPS was mixed with 1 ml of 2 M HCl and complete hydrolysis was accomplished by heating the mixture in a boiling water bath for 30 min. After neutralization of the hydrolysate with 1 ml 2 M NaOH, the reducing sugars released from starch was determined, using the DNS calorimetric method (Bergmeyer, 1974).
91 TABLE II
ISP52(pSP2) hydrolyzed 98% of the starch after five days. In the culture supernatant of transformant ISP52(pSP3), containing both STA2 and AMY, starch hydrolysis was complete after 72 h. Thus, ISP52(pSP3) hydrolyzes starch much faster than any of the other transformants, indicating a co-operative action of GA11 and AMY. To quantify the levels of extracellular amylolytic activity produced by the various yeast strains and transformants, cell-free culture fluids were assayed for AMY and GA11 activity. The PAT assay was used to specifically quantify AMY activity, the DNS method was used to measure the amount of reducing sugars released from starch and the glucose-peroxidase test was used to determine the amount of glucose released from starch (Table II). The recipient strain, ISP52, exhibited no amylolytic activity as measured by any of the mentioned techniques. AMY activity was only present in the culture supernatant of the AMY containing transformants, ISP52(pSP2) (860 u/liter) and ISP52(pSP3) (850 u/liter). The effect of the STA2 and AMY genes on the production of amylolytic activity by ISP52(pSP3) appears to be more or less additive when reducing sugar was assayed. The sum of the GA11 activity (48 u/ml/min) produced by ISP52(pSPl) and the AMY activity (20 u/ml/min) produced by ISP52(pSP2) was 68 u/ml/min, similar to the value of 61 u/mI/min obtained with ISP52(pSP3). However, when amylolytic activity was assayed by the glucose-peroxidase test, important differences were observed. Low amounts of glucose were liberated from starch by the action of AMY (10.5 u/ml/mm) present in the culture supernatant of ISP52(pSP2), whereas amylolytic enzymes in the culture supernatants of both ISP52(pSPl) and ISP52(pSP3) released high amounts of
A
Amylolytic activities of yeast strains and transformants Yeast strains*
Phadebas b (u/liter)
Reducing sugars c (u/ml/min)
Glucose d (u/ml/mm)
ISP52 ISP55 ISP52(pSPl) ISP52(pSP2) ISP52(pSP3)
0 0 0 860 850
0 36.80 48.00 20.00 61.00
0 36.40 50.30 10.50 53.30
a Yeast cultures (see Table I) were grown in YPGE medium for 72 h and the cells harvested by centrifugation. The culture supematants were concentrated 60-fold by ultrafiltration using the Millipore Minitan system, the Millipore filter type 1000 NMWL and the Millipore Durapore membrane PTGC 00001 (Millipore Corporation, Bedford, MA). Reactions for ISP55 and ISP52(pSPl) were carried out at pH 5.0 for ISP52(pSP2) at pH 6.0 and for ISP52(pSP3) at pH 5.5. b AMY activity in the culture supernatants was quantified with the PAT assay (Pharmacia Diagnostics, Uppsala, Sweden) using a 20 mM sodium acetate buffer (pH 5.0, pH 5.5 or pH 6.0) and incubation at 60°C for 10 min. Starch hydrolysis in the culture supernatant was assayed by the iodine reaction as described by Filho et al. (1986). ’ Amylolytic activity in the culture supernatants was quantified using the DNS calorimetric method that detects reducing sugars released from starch (Pretorius et al., 1988). Reaction tubes contained 500 nl of culture supematant and 500 ~1 of a 1y0 Lintner starch solution in a 20 mM Na . acetate buffer (pH 5.0, pH 5.5 or pH 6.0). Tubes were incubated at 60°C for 10 min. The reaction was stopped by adding 1 ml DNS solution, boiled for 10 min and chilled on ice. Absorbance was measured at 546 nm (A&. d Amylolytic activity in culture supernatants was also quantified with the PGO/ODAD assay (Sigma kit No. 510; Sigma Chemical Company, St. Louis, MO) that detects glucose released from starch. Standard curves were constructed using glucose and maltose (Sigma CAR-1 1). One unit of amylolytic activity was defined as the amount of enzyme that liberated 1 pmol of reducing sugar or glucose/min/ml at 60°C.
B
C
$ $4 8 18
1
ol
b t
c H L
&
0)
E
a
:!__JI_ b: ‘n ,
5
7
5
Time (min)
7
18
Time (min)
5
7
18
T!me (mm)
Fig. 5. Gas-chromatographic analysis of the starch hydrolysates from the supematants of ISP52(pSPl) (part A), ISP52(pSP2) (part B) and ISP52(pSP3) (part C). No glucose or maltose were detected in the supematant of ISP52. Samples were analyzed with a Varian Vista 6000 gas chromatograph equipped with FID. A 12 m x 0.32 mm ID SE30 fused silica capillary column was used. The following operating conditions were used: column temperature was increased from 130” to 230°C at S”C/min intervals; injector temperature 220°C; FID temperature 250°C; helium carrier linear flow velocity 100 cm/s. Maltotriose, maltotetraose and carbohydrate standard solutions (CAR-11; Sigma Chemical Company, St. Louis, MO) were used as standards. Results were analyzed with a Varian Vista 402 data system. The supematants of strains grown in YPS were incubated at 60°C for 3 h, using the appropriate buffer. The samples were then frozen and freeze-dried. The carbohydrates were subsequently silylated with Tri-sil Z (500 ~1) at 60-65°C for 15 min.
92 glucose from starch (50.3 and 53.3 u/ml/min, respectively). These results can be explained together with the gas chromatographic analyses of the starch hydrolysates (Fig. 5). The STA2-encoded GAII, secreted by ISP52(pSPl), is an a-1,4 glucanglucohydrolase, releasing glucose (Fig. 5A) from the nonreducing end of the starch molecule (Modena et al., 1986). AMY is an a-1,4 glucan 4-glucanohydrolase, hydrolyzing the linkages of starch in a random fashion, producing primarily glucose and maltose (1: 1) (Fig. 5B). The GA11 and AMY activities, secreted by ISP52(pSP3), cooperatively liberated glucose and maltose (3 : 1) from starch (Fig. 5C). Since glucoamylases have a low affinity for low-M, substrates (Modena et al., 1986; Tucker et al., 1984; Whitaker, 1972), only part of the maltose and oligomeric products of AMY action served as substrate for the STAZ-encoded enzyme. In conclusion, laboratory and industrial strains of 5’. cerevisiue transformed with recombinant plasmids carrying both the STA2 and AMY genes, were capable of secreting glucoamylase and a-amylase simultaneously, thereby mediating efficient one-step starch utilization.
ACKNOWLEDGEMENTS
The authors thank Desmond van Jaarsveld for helping us to ultraliltrate culture supernatants and Dr. Marita le Roux for help with the gas-chromatographic analysis of starch hydrolysates.
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