Cloning, sequencing and expression in Escherichia coli of a Streptomyces aureofaciens gene encoding glyceraldehyde-3-phosphate dehydrogenase

Gene, 165 ( 1995) 77-80 0 1995 Elsevier Science B.V. All rights reserved.

77

0378-l 119/95/$09.50

GENE 09225

Cloning, sequencing and expression in Escherichia coli of a Streptomyces aureofaciens gene encoding glyceraldehyde-3-phosphate dehydrogenase (Recombinant DNA; overexpression; sequence comparison; AraC-like transcriptional isomerase)

J&n Kormanec,

Aurelia Lempelovh,

Marian FarkaSovskji

and Dagmar

regulator; A5-3-ketosteroid

HomerovA

Institute ofMolecular Biology, Slovak Academy oj‘Sciences. 842 51 Bratislara. Slovak Republic Received by K.F. Chater:

18 April 1995; Revised/Accepted:

2 June/5 June 1995; Received at publishers:

24 July 1995

SUMMARY

The structural

gene (gap) encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) from Streptomyces aureofaciens (Sa) has been cloned and sequenced. The predicted gap product consists of 332 amino acids (aa) (35 312 Da), and has considerable homology (up to 52% aa identity) with other bacterial and eukaryotic gap genes. Sequence analysis of the regions flanking gap revealed two incomplete open reading frames encoding proteins similar to the AraC family of bacterial transcriptional regulators and A5-3-ketosteroid isomerase. The Sa gap gene was expressed at a high level in Escherichia coli (EC). Transformation of the EC strain resulted in an up to eightfold increase in specific GAPDH activity.

INTRODUCTION

Streptomyces are mycelial, sporulating Gram+ bacteria which produce a wide variety of antibiotics and bioactive molecules as secondary metabolites (Chater, 1989). Although genes for secondary metabolism have been extensively studied, molecular studies of primary metabolism have received comparatively little attention in these Correspondence Slovak Slovak

to: Dr. J. Kormanec,

Institute

of Molecular

Biology,

Academy of Sciences. Dubravska cesta 21, 842 51 Bratislava. Republic. Tel. (42-7) 378-2432; Fax (42-7) 372-316;

e-mail: [email protected] Abbreviations:

A, absorbance

I1 cm); aa, amino acid(s); Ap, ampicillin;

B., Bacillus; bp, base pair(s); dCTP, GAPDH, glyceraldehyde-3-phosphate

deoxyCTP; EC. Escherichia coli; dehydrogenase; gap, gene encod-

ing GAPDH; IPTG, isopropyl-B-D-thiogalactopyranoside; kb, kilobase(s) or 1000 bp; NAD. nicotinamide-adenine dinucleotide; nt, nucleotide(s); frame; (large)

PAGE, fragment

oligo,

oligodeoxyribonucleotide;

ORF,

open

reading

polyacrylamide-gel electrophoresis; PolIk. Klenow of Ec DNA polymerase I; RBS, ribosome-binding

site(s); S.. Streptomyces; Sa. S. auwfaciens; [I, denotes plasmid-carrier state. SSDI 0378-1119(95)00510-2

SDS. sodium dodecyl sulfate:

organisms. Studies with several examples of primary metabolism genes, including the glycerol utilization operon (Smith and Chater, 1988) and the glucose-kinaseencoding gene (Angel1 et al., 1992) have indicated different mechanisms from those of regulatory Escherichia coli (EC). Although many Streptomyces fermentations are glucose based, to date, only one Streptomyces gene encoding a glycolytic enzyme, 3-phosphoglycerate mutase, has been cloned and characterized (White et al., 1992). While searching for genes encoding o factor, we identified a Streptomyces aureofaciens (Sa) chromosomal fragment encoding a GAPDH-like protein. GAPDH, a key enzyme of glycolysis, reversibly catalyzes the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate using NAD+ as the coenzyme (Harris and Waters, 1976). The primary structure of GAPDH is very similar in many organisms (Branlant and Branlant, 1985). The aim of this report was to sequence and analyze the Sa gap gene. High-level expression of gap in EC was also investigated.

78 EXPERIMENTAL

AND DISCUSSION

(a) Cloning and sequencing of the gap gene

To identify the gene encoding 04’ factor of RNA polymerase recognizing the AI promoter of the Sa plasmid pSA2201 (FarkaSovskg et al., 1991), a Sa genomic library (2-4-kb TuqI partially digested chromosomal fragments cloned into the C/a1 site of pBR322) was hybridized with a mixed reverse oligo probe, S-TCSAGRTTVGGCAC (where R=A or G; S=G or C; V=A or G or C), prepared according to the first five aa of the N-terminal sequence, VPNLDXFAEXLQLI (where X denotes unidentified aa), of the purified 04’ protein. Sequence analysis of the hybridizing region in one of the two representative clones revealed a sequence identical to the oligo probe used, but the aa in the ORF following this sequence did not correspond to the further 049 aa sequence. Comparison of the aa sequence with databases by the BLAST Network Service, NCBI (Altschul et al., 1990) revealed strong similarity to GAPDHs from various organisms. The full sequence of the gap-like gene is given in Fig. 1. The gene would encode a protein of 332 aa (35 312 Da). The most likely start codon ATG at nt 527 is preceded by an RBS (AAAGGA). The ORF terminates at a TGA

stop codon at nt 1523, followed by an inverted repeat (nt 1541-1568). The repeat may function as a Rhoindependent terminator, which could form a stable RNA stem-loop structure (AG = - 39.2 kcal/mol) (Tinoco et al., 1973). This ORF shows a codon usage typical for Streptomyces genes, with a marked preference for G or C in the third position (95.4%; Bibb et al., 1984). Upstream and downstream from gap, two truncated coding ORFs, fulfilling the criteria for Streptomyces regions, were identified. Neither of them appeared to encode a glycolytic enzyme. The deduced product of the upstream ORFl resembles a group of bacterial transcription regulators of the AraC family (Ramos et al., 1990), the strongest similarity being in the C-terminal region containing a potential helix-turn-helix DNA-binding domain. The downstream 0RF2 encodes a product with significant similarity (25% identical matches) to A5-3ketosteroid isomerases from Pseudomonas putida, catalyzing the allylic isomerization of the 5.6 double bond of A5-3-ketosteroids to the 4,5 position by stereospecific intramolecular transfer of a proton (Kim et al., 1994). Thus, the gup gene of Su is not part of a glycolytic or gluconeogenetic operon as recently described for several eubacteria (Alefounder and Perham, 1989; Eikmanns. 1992; Martin et al., 1993).

100 IS0 137 600 25 750 75 900

Fig. 1. The nt sequence of the Sa gap gene and flanking DNA. The deduced gap gene product, and the products of the upstream the downstream 3’-truncated ORFs are given in single-letter aa code in the second position of each codon. Start and stop codons,

5’-truncated and and presumptive

RBS are underlined. A putative terminator structure is underlined with convergent arrows. The asterisks indicate aa that are identical in all aligned six sequences (see section b); the crosses indicate aa that are similar in all six sequences (similar aa were: I,V,L,M; F,Y; N,Q; S,T,A; D,E; R,K). The aa, which have been identified as being important in the NAD-binding and catalytic mechanism (Skarzynski et al., 1987) are in boldface and underlined. This sequence has been deposited in GenBank/EMBL/DDBJ databases under accession No. U21191. Methods: The nt sequence was determined in both strands after 3’-end labelling with PolIk and [cc-~~P]~CTP according to Maxam and Gilbert (1980). Compressions caused by a high percentage of G-C pairs were removed by running equilibrated gels at 75°C. DNA manipulations in EC were performed as described in Ausubel et al. (1987). EC SURE (Stratagene, LaJolla. CA, USA) was used in cloning experiments.

79 EC and cyanobacterium

Anabaena

0

variabilis

contain additional gap genes (Alefounder and Perham, 1989; Martin et al., 1993). Low-stringency hybridization of Sa chromosomal DNA with a DNA fragment internal to the identified gap gene revealed, in addition to the stronger signals confirming colinearity of the cloned gap gene with chromosome, a weak signal, possibly corresponding to a second gap gene (data not shown). Cloning of the potential second gene is in progress.

180

0

30

pTrc99A

60 P=-wPl

180

-

min

7 marker

kDa

- 94.0 - 67.0

(b) Comparison of deduced aa sequences of GAPDH from Sa and from other organisms

Alignment of the predicted aa sequence of the Sa gap gene product with GAPDHs of B. stearothermophilus (Branlant et al., 1989) Corynebacterium glutamicum (Eikmanns, 1992) Zymomonas mobilis (Conway et al., 1987) EC (Branlant and Branlant, 1985) and human muscle (Nowak et al., 1981) revealed identities of between 41.6 and 57.8% (examples of Gram+ and Gram- prokaryotes, and eukaryotes were chosen). Although similarities of the Sa GAPDH with aligned GAPDHs are dispersed throughout the whole sequence, there are several highly conserved regions which are identical in all tested GAPDHs (Fig. 1). The aa positions of these regions (underlined and in boldface in Fig. 1), which have been identified as being important in the NAD-binding and catalytic mechanism (Skarzynski et al., 1987), were all conserved in SLI GAPDH. The comparison strongly suggests that the gap gene, identified in Sa, encodes GAPDH.

1.4

5.1

6.9

11.8

Fig. 2. The 0.1% SDS-IO%

PAGE

29.2

59.5

showing

expression

gene in EC. The gel was stained

with Coomassie

of GAPDH

extract

Methods:

in particular The 1800-bp

crude

BspHI-X/r01

- GAPDH (u/w)

is indicated

(blunted

of the Sa gap

blue. Specific activity below each lane.

with PolIk) fragment

of

Su DNA containing gup from the start Met (nt 527) was cloned into pTrc99A (Amann et al., 1988) digested with Hind111 (blunted with PolIk)+

NcoI, resulting

corresponding

in plasmid

plasmid

Ap/ml (Ausubel

was grown

by the addition

fraction

of the cells was collected

as described GAPDH

in Laemmli

(Byers.

(1970),

was determined

was assayed

as the amount A,,,

of 1 mM IPTG.

At the corresponding

by centrifugation

Each sample containing

concentration

containing

containing

0.1 mg

et al., 1987) at 37°C till Asoo = 0.7. and expression

induced

sonication.

EC SURE

pKK-gapl.

in M9 medium

and disrupted

10 ug of total proteins and loaded

of enzyme which reduces

by

was treated

on the gel. The protein

by the method

by the arsenolysis

was

time, a

procedure;

of Bradford

(1976).

1 unit (u) is defined

1 umol of NAD+ per min at

1982).

(d) Conclusions

(c) Expression of the Sa gap gene in EC In order to confirm that the Sa gap gene encodes functional GAPDH, an attempt was made to express the gene in EC. The gap coding region was placed under the control of strong trc promoter in EC expression plasmid pTrc99A (Amann et al., 1988). Soluble protein extracts of EC transformed with the plasmid pKK-gap1 and pTrc99A, before and after induction with IPTG, were examined by SDS-PAGE and GAPDH enzyme assay. As shown in Fig. 2, a prominent band with increasing intensity was clearly visible after induction with IPTG in the region corresponding to a molecular mass of 39.4 kDa. This value was little higher than the calculated molecular mass (35 312 Da) of the gap gene product. The same discrepancy in the mobility of GAPDH in SDS-PAGE was also shown in Zymomonas mobilis (Conway et al., 1987). The about eightfold higher specific activity of GAPDH in crude extracts of Ec[pKK-gapl] after 3 h of IPTG induction, compared to Ec [ pTrc99Al (Fig. 2), clearly demonstrates that the overproduced protein corresponds to GAPDH.

(I ) The gap gene of Sa encoding GAPDH was cloned and sequenced. (2) The Sa gap gene seems to be not organized in a glycolytic or gluconeogenetic operon. (3) Two incomplete ORFs, with similarity to AraC transcriptional regulators and A5-3-ketosteroid isomerases, were identified in the upstream and downstream region of gap, respectively. (4) Hybridization data suggest the presence of a second gap gene in Sa. (5) The gap gene of Sa was expressed in EC confirming that it encodes functional GAPDH.

ACKNOWLEDGEMENTS

We would like to thank Mrs. Renata Knirschova for excellent technical assistance. This work was supported in part by the Slovak Grant Agency for Science (grant No. GA999135).

80 glycolytic enzymes, 3-phosphoglycerate

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