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Aims: Disruption of the extracellular Zymomonas mobilis sucrase gene (sacC) to improve levan production. Methods and Results: A PCR-amplified tetracycline ...
Journal of Applied Microbiology 2004, 96, 671–676

doi:10.1111/j.1365-2672.2003.02169.x

Disruption of the Zymomonas mobilis extracellular sucrase gene (sacC) improves levan production V. Senthilkumar1, N. Rameshkumar1, S.J.W. Busby2 and P. Gunasekaran1 1

Department of Microbial Technology, Centre for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai, India, and 2School of Biosciences, The University of Birmingham, Edgbaston, Birmingham, UK 2003/0423: received 22 May 2003, revised 29 October 2003 and accepted 30 October 2003

ABSTRACT V . S E N T H I L K U M A R , N . R A M E S H K U M A R , S . J . W . B U S B Y A N D P . G U N A S E K A R A N . 2004.

Aims: Disruption of the extracellular Zymomonas mobilis sucrase gene (sacC) to improve levan production. Methods and Results: A PCR-amplified tetracycline resistance cassette was inserted within the cloned sacC gene in pZS2811. The recombinant construct was transferred to Z. mobilis by electroporation. The Z. mobilis sacC gene, encoding an efficient extracellular sucrase, was inactivated. A sacC defective mutant of Z. mobilis, which resulted from homologous recombination, was selected and the sacC gene disruption was confirmed by PCR. Fermentation trials with this mutant were conducted, and levansucrase activity and levan production were measured. In sucrose medium, the sacC mutant strain produced threefold higher levansucrase (SacB) than the parent strain. This resulted in higher levels of levan production, whilst ethanol production was considerably decreased. Conclusions: Zymomonas mobilis sacC gene encoding an extracellular sucrase was inactivated by gene disruption. This sacC mutant strain produced higher level of levan in sucrose medium because of the improved levansucrase (SacB) than the parent strain. Significance and Impact of the Study: The Z. mobilis CT2, sacC mutant produces high level of levansucrase (SacB) and can be used for the production of levan. Keywords: cassette mutagenesis, extracellular sucrase, levan, Zymomonas mobilis.

INTRODUCTION Zymomonas mobilis is an anaerobic, Gram-negative bacterium that produces ethanol from glucose via the Entner– Doudoroff pathway. It can ferment glucose and fructose efficiently to produce ethanol and carbon dioxide as the sole fermentation products. Three different sucrases, namely intracellular sucrase (SacA), extracellular levansucrase (SacB) and extracellular sucrase (SacC), contribute to sucrose hydrolysis by this organism. In addition, when growing in sucrose medium, the SacB levansucrase polymerizes fructose units to form levan, an industrially important fructan. Due to its high activity, the SacC sucrase Correspondence to: P. Gunasekaran, Department of Microbial Technology, Center for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai 625021, India (e-mail: [email protected]).

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is responsible for >70% of total sucrase activity (Preziosi et al. 1990; O’Mullan et al. 1992). In our previous work, the genes coding for levansucrase (sacB) and extracellular sucrase (sacC) have been cloned, sequenced and characterized. They have a high degree of identity (70% at the base sequence level) and they are organized in an operon (Gunasekaran et al. 1995; Kannan et al. 1995). It is known that transcription of sacB is upregulated by the presence of sucrose in growth medium (Song et al. 1999). SacB levansucrase mutants are known to grow on sucrose medium without levan production and produce higher levels of ethanol (Kannan et al. 1993). However, no information is currently available about the effects of mutations that disrupt the SacC sucrase. Thus, in this work, we disrupted the Z. mobilis sacC gene. We report that this results in higher levels of levansucrase activity, and consequent levan production, during sucrose fermentation.

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M A T E R I A LS A N D M E T H O D S

Table 1 Oligonucleotide primers used in this study

Bacterial strains, plasmids and culture conditions )

Escherichia coli DH5a[F / 80 d lacZ M15, D (lacZYA-argF) U169, endA1, hsdR17, supE44, k), gyrA96, relA1] and Z. mobilis B14023 strains were used. Plasmids pBR322 and pZS2811 harbouring the cloned sacC gene (Gunasekaran et al. 1995) were used. Zymomonas mobilis was grown on RMG medium (glucose, 20 g l)1; yeast extract, 10 g l)1; KH2PO4, 2 g l)1; pH 6Æ0) at 30C in stationary flasks. Escherichia coli cultures were grown at 37C with agitation in LB medium (tryptone, 10 g l)1; yeast extract, 5 g l)1; sodium chloride, 10 g l)1; pH 7Æ2). When necessary, media were supplemented with ampicillin (100 lg ml)1) and tetracycline (30 lg ml)1 for Z. mobilis and 12Æ5 lg ml)1 for E. coli). For solid media, 15 g l)1 agar was added. DNA manipulations Plasmid DNA was extracted using miniprep kits (Qiagen, Hilden, Germany). Genomic DNA was extracted according to Byun et al. (1986). Restriction digestions and ligations were performed according to manufacturer’s instructions (Fermentas, Opelstrasse, Germany). Standard protocols were used for recovery of DNA from agarose gels (Suzuki et al. 1991). The insertion of the tetracycline cassette into the sacC ORF was confirmed by PCR with gene specific primers, synthesized by Alta Bioscience (Birmingham University, Birmingham, UK). DNA sequencing was performed using the dideoxy chain-termination method in an automated DNA sequencer (model 310A; Applied Biosystems, Foster City, CA, USA). The sequencing reaction was carried out using ABI PRISMTM dye terminator cycle sequencing kit (Applied Biosystems) according to the manufacturer’s instructions. Cassette mutagenesis of sacC A 1Æ5-kbp fragment carrying the pBR322 tetracycline resistance gene (tetR) was amplified by PCR with oligonucleotide primers (Table 1). The fragment was cloned by ligation into EcoRV linearized pZS2811 and transformation into E. coli DH5a. Zymomonas mobilis B14023 was transformed with pZST2 by electroporation. To prepare electrocompetent cells, Z. mobilis B14023 cells were grown in RMG medium to an O.D.600 of 0Æ3–0Æ4. Cells were harvested by centrifugation, washed twice in ice-cold water, and once in 10% glycerol. Finally, the cell pellet was suspended in 2 ml of 10% glycerol and electroporated with 150–200 ng of plasmid DNA at 1Æ8 kV in a 0Æ2 mm cuvette (BTX-PEP, San Diego, CA, USA). Then, the cells in RMG medium were incubated without shaking for 16–18 h at 30C (for expression) and plated on RMG containing tetracycline (30 lg ml)1).

Primer

Corresponding gene Sequence (5¢ fi 3¢) of primers

Tet5 F tetR Tet3 R tetR Sb F SacB Sb R

SacB

Sc F Sc R

SacC SacC

TCAAGAATTCTCATGTTTGACAGC GTATCGGTGATTCATTCTTGC GTCAAAGCTTATTAGGATAGTT CTTATGTT GTCAAAGCTTCGTCATTTACAA TGAATAATC TGATGAACACCCTTCCTATTC GGTATGATTGGCGTTATTTCAGCG

Fermentation Seed cultures were grown in RMG medium for 18 h at 30C under static conditions and these were used as inoculum. For fermentation, the 20 g l)1 glucose in the growth medium was replaced with 100 g l)1 of glucose or fructose or sucrose. Fermentation experiments were carried out with 100 ml of medium in Erlenmeyer flasks. At 8-h intervals, samples were withdrawn and the biomass, reducing sugar, ethanol and levan production were estimated. Analytical methods For biomass estimation, cells were washed twice and suspended in 0Æ85% (w/v) NaCl. After measuring the absorbance at 550 nm, the corresponding dry weight was obtained from established calibration curves as described by Doelle and Greenfield (1985). Ethanol concentrations were determined by the method of Caputi et al. (1968). Residual sugar was estimated by the Somogyi (1952) method when glucose or fructose was used as substrate. The phenol– sulphuric acid method (Dubois et al. 1956) was used when the substrate was sucrose. Levan from the culture supernatants was separated according to Viikari and Gisler (1986) and estimated as fructose units after hydrolysing the levan in 0Æ1 N HCl at 100C for 1 h. Enzyme extraction and assay Cells were harvested by centrifugation at 1000 g at 4C for 15 min. The culture filtrate was used as the source of extracellular enzyme. Cell pellets were washed and suspended in 50 mM acetate buffer (pH 5) containing 1 mM phenyl methyl sulphonyl fluoride (PMSF). Cells were sonicated (three 20-s periods at 40 W, with 45-s intervals between the periods) using a Braun sonifier (Labsonic 2000, Melsungen, Germany), and clarified by centrifugation at 4500 g for 30 min at 4C. The supernatant was used as a source of intracellular enzyme. Sucrose hydrolysing and levan forming activities were assayed as described earlier (Kannan et al.

ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 671–676, doi:10.1111/j.1365-2672.2003.02169.x

DISRUPTION OF Z. MOBILIS sacC

1993). Extracellular proteins of Z. mobilis B14023 and CT2 were separated by PAGE in the absence of denaturing agents and gels were incubated at 30C for 12 h in 5% (w/v) sucrose. Appearance of a slimy white band indicates levanforming activity.

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scored, after growing the cells overnight in RMG broth without an antibiotic, to provide sufficient time for a double crossover event. The disruption of the sacC gene was confirmed by PCR, using both the parent and the mutant that was designated as Z. mobilis CT2. Figure 2 shows the result of PCR amplification of the sacC chromosomal region of both strains. The parent strain shows a 1Æ3-kbp amplicon, whilst the CT2 mutant shows a 2Æ8-kbp amplicon, because of the insertion. Further PCR amplification with tetR specific primers (Table. 1) suggested that the mutant CT2 strain only had a tetR insertion within the sacC gene. The precise site of the insertion was confirmed by sequencing (data not shown). PCR amplification of sacB gene (encoding levansucrase), with sacB specific primers, showed that the sacC disruption had not affected sacB. Zymogram analysis confirmed the absence of extracellular SacC sucrase activity in the culture filtrate of the mutant CT2 strain, whilst the SacB band was present in both parent and mutant (Fig. 3).

RESULTS Construction of a Z. mobilis sacC sucrase mutant A tetR cassette was inserted at the EcoRV site of the sacC gene cloned in pZS2811 as described in materials and methods. The resultant recombinant plasmid was designated as pZST2 (Fig. 1) and transferred to Z. mobilis by electroporation. As the plasmid pZST2 does not replicate in Z. mobilis but can be maintained in cells only after homologous recombination into chromosome, resulting in insertional inactivation of the sacC gene (Fig. 1). Thus, transformants, in which the sacC gene was defective were

R

p

am

C

PCR amplified tet R gene from pBR322 sac

pZS 2811 4·5 kbp

EcoRV

amp

R

sacC

sa cC

pZST2 6·0 kbp R

tet

cC

sa

Homologous recombination

Zymomonas mobilis chromosome

cB

sa

Fig. 1 Construction of a sacC defective Zymomonas mobilis strain by homologous recombination. pZST2 was transformed into Z. mobilis B14023. Homologous recombination between the sacC gene flanking sequences in the plasmid and chromosomal DNA of B14023 occurs

1·3 kbp

sacB

2·8 kbp

sacC

tet R gene

sacC

Zymomonas mobilis CT2 chromosome

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1

M

2

3

4

Fermentation characteristics of sacC defective Z. mobilis CT2

5

3 kbp

1·5 kbp

Fig. 2 Confirmation of tetR insertion by PCR. M- 1 kbp ladder. Lane 1 – amplification of sacC gene in strain CT2; lane 2 – amplification of sacC gene in strain B14023; lane 3 – amplification of internal region of 2Æ8 kbp PCR product with tetR gene specific primers; lanes 4 and 5 – amplification of sacB gene in Strain CT2 and B14023, respectively

(a) 1

(b) 2

3

Fermentation kinetics of the parent and the CT2 mutant strain were examined during growth on different carbon sources. Biomass production by CT2 was relatively low in glucose and fructose fermentations whereas, with sucrose, it was higher (equal to that of the parent strain: Fig. 4a). Comparison of the parent and CT2 strains showed that there was a significant difference between them with respect to substrate consumption during sucrose fermentation (Fig. 4b). Thus, ethanol production by CT2 was high (41 g l)1 at 48 h) in glucose and fructose media but lower (22 g l)1) in sucrose medium (Fig. 4c). The decreased ethanol production in sucrose medium could be attributed to the increased level of production of levan (21Æ1 g l)1 in the CT2 strain at 24 h compared with 15Æ5 g l)1 in the parent strain; Fig. 4d). As the increased level of levan production could be attributed to the higher level of levansucrase, this activity was compared in the mutant and parent strains. Data in Table 2 show that there is a significant reduction in the sucrose hydrolysing activity of Z. mobilis CT2 grown in glucose and fructose. However, the strain CT2 when grown in sucrose medium produced threefold (100 U ml)1) higher level of levansucrase activity compared to that of parent strain. This increase was twofold (30Æ9 and 22 U ml)1) in the cells grown in glucose and fructose media. After 24 h, the levan production decreased, perhaps due to the reversible action of levansucrase. DISCUSSION

4

SacC SacB

Fig. 3 Zymogram analysis of sucrase and levanforming activities of the parent B14023 and strain CT2. Cellular extracts (150 lg) of Zymomonas mobilis B14023 (lanes 1 and 3) and CT2 (lanes 2 and 4) were resolved on native PAGE and zymogram stained for levansucrase activity. (a) Sucrose hydrolysing activity, (b) Levanforming activity

Two sucrases, namely the SacB levansucrase and the extracellular SacC sucrase, contribute 30 and 70%, respectively, to the extracellular sucrose hydrolysing activity of Z mobilis. Due to its higher specific activity, the SacC sucrase plays a major role in the growth of Z. mobilis in sucrose medium (Ananthalakshmi and Gunasekaran 1999). However, levan production because of SacB levansucrase decreases the ethanol fermentation of Z. mobilis and levansucrase mutants were found to produce more ethanol, because of loss of levan formation (Kannan et al. 1993). In this work, we have focused on the study of Z. mobilis that lacks SacC and hence is solely dependent on SacB for extracellular sucrase activity. As far as we know, this is the first report of the construction of a Z. mobilis sacC mutant. We found that, as expected, our mutant strain expressed only SacB and that the extracellular levansucrase activity was more than 50% of the sucrase activity of the parent strain. This resulted in a mutant strain that, in sucrosecontaining medium, produces less ethanol and more levan, because of the levansucrase activity of SacB protein. This strain, with its high fermentation rates may have advantages in continuous cultivation for levan production.

ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 671–676, doi:10.1111/j.1365-2672.2003.02169.x

DISRUPTION OF Z. MOBILIS sacC

(a) 2·5

675

(b) 100

Residual sugar (g l–1)

Biomass (g l–1)

2 1·5 1 0·5

0

8

16

24

32

40

50

0

48

8

16

24

32

40

48

40

48

Time (h)

Time (h) (d)

(c)

25

60

Levan (g l–1)

Ethanol (g l–1)

20 40

20

15 10 5

0

8

16

24

32

40

0

48

8

16

24

32

Time (h)

Time (h)

Fig. 4 Kinetics of fermentation by Zymomonas mobilis B14023 and CT2. Biomass (a), Residual sugar (b), ethanol (c), and levan formation (d) in glucose ((, j), fructose (D, m) and sucrose (s, d) fermentation by B14023 and strain CT2 was examined. Open symbols corresponding to wild type and closed symbols corresponding to the mutant CT2 strain Table 2 Comparison of sucrase and levansucrase activities of Zymomonas mobilis B 14023 and strain CT2 in glucose, fructose and sucrose Levansucrase activity (U ml)1)

Sucrase activity (U ml)1) Substrate

B14023

CT2

B14023

CT2

Glucose Fructose Sucrose

2Æ65 2Æ53 3Æ82

1Æ51 1Æ24 3Æ20

19Æ3 14Æ3 32Æ0

30Æ9 22Æ0 100Æ0

Fermentations were carried out for12 h with 100 g l)1 of carbon source.

(SacC) could increase the SacB levansucrase activity (Ananthalakshmi and Gunasekaran 1999). Another possibility, that results from sacB and sacC being adjacent cistrons in an operon, is that disruption of sacC leads to increased stability of the sacB transcript. This may be due to an intercistronic transcription terminator that is responsible for the partial generation of monocistronic mRNA for sacB (Song et al. 1999). Thus, disruption of the sacC gene in strain CT2 alters the transcript length of the bi-cistronic messenger and this might increase stability of the sacB transcript. ACKNOWLEDGEMENTS

The reasons for the increased SacB levels in the CT2 mutant strain are unclear. For example, it has been suggested that the loss of extracellular sucrase activity

We thank Department of Science and Technology (New Delhi, India) for financial support through an Indo-UK project (INT/UK/P-8/99). VS thanks Madurai Kamaraj

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University (Madurai, India) for financial assistance as USRF. REFERENCES Ananthalakshmi, V.K. and Gunasekaran, P. (1999) Overproduction of levan in Zymomonas mobilis using cloned sacB gene. Enzyme and Microbial Technology 25, 109–115. Byun, M.O.K., Kaper, J.B. and Ingram, L.O. (1986) Construction of new vector for the expression of foreign genes in Zymomonas mobilis. Journal of Industrial Microbiology 1, 9–15. Caputi, A., Ueda, M. and Brown, T. (1968) Spectrophotometric determination of ethanol in wine. American Journal of Enology and Viticulture 19, 160–165. Doelle, H.W. and Greenfield, P.F. (1985) The production of ethanol from sugar using Zymomonas mobilis. Applied Microbiology and Biotechnology 22, 405–410. Dubois, M., Gilles, K.A., Roberts, P.A. and Smith, F. (1956) Colorimetric method for determination of sugars and related substances. Analytical Chemistry 28, 350–356. Gunasekaran, P., Mukundan, G., Kannan, R., Velmurugan, S., AitAbdelkadhar, N., Alvarez-Macarie, E. and Baratti, J. (1995) The sacB and sacC genes encoding levansucrase and sucrase from a gene cluster in Zymomonas mobilis. Biotechnology Letters 17, 635–42. Kannan, R., Mukundan, G., Ait-Abdelkader, N., Augier-Magro, V., Baratti, J. and Gunasekaran, P. (1995) Molecular cloning and

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ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 671–676, doi:10.1111/j.1365-2672.2003.02169.x