Gene, 118 (1992) 65-72 0 1992 Elsevicr Science

GENE

Publishers

B.V. All rights reserved.

65

0378-l 119,‘92~%05.00

06600

The isolation and characterization from the yeast Yarrowia lipolytica

of the pyruvate

(Lower eukaryote;

sequence

glycolytic

enzyme;

C.A. Strick, L.C. James,

recombinant

DNA;

M.M. O’Donnell,

analysis;

M.G. Gollaher

kinase-encoding

gene

intron)

and A.E. Franke

Molecular Genetics ctnd Protein Chemistry Depcrrtrnent. Central Re.yeurch Division, Pfizer Inc. Groton, c7‘ 06340. USA Received

by J. Marmur:

5 March

1992; RcvisedlAccepted:

19 April,‘20 .4pril 1992; Rcccived

at publishers:

18 May 1992

SUMMARY

The dimorphic yeast, Yurvowia lipolytica, has been developed as a useful expression/secretion system for heterologous proteins such as chymosin and tissue plasminogen activator. To further develop this expression system, we have cloned the gene (PYK) encoding the highly expressed glycolytic enzyme, pyruvate kinase (PYK). Genomic clones were selected by their specific hybridization to synthetic oligodeoxyribonucleotide probes based on regions of the enzyme that were conserved through evolution. The clones identified by hybridization contained overlapping DNA inserts. We have confirmed the identity of the cloned gene based on two criteria: (I) the nucleotide sequence of the proposed PYK gene predicts a protein activity was that is highly homologous to the corresponding Saccharomyces cerevisiue enzyme, and (2) PYK-specific increased twofold when wild-type Y. lipolytica strains were transformed with the isolated DNA. Interestingly, we found that the open reading frame of the Y. lipolytica PYK gene was interrupted by an intron. This represents the first report of an intron in a Y. l@olytica gene.

INTRODUCTION

The dimorphic yeast Yurrowia lipolytica has a number of characteristics that make it amenable for development as an expression system for heterologous proteins. Foremost among these is its ability to secrete high levels of large proteins such as an alkaline protease and several acid proteases (Tobe et al., 1976; Ogrydziak and Schraf, 1982; Ya-

Correspondence Chemistry

to: Dr. C.A.

Department,

Point Rd., Groton. Fax (203)441-3783. Abbreviations:

Strick,

Central

Molecular

Research

Genetics

Division,

and

Protein

Pfizer Inc, Eastern

CT 06340, USA. Tel. (203)441-4350;

aa, amino acid(s); bp, base pair(s): ExoIII, exonuclease

from E. co/i; kb, kilobase

or 1000 bp; nt, nucleotide(s);

oxyribonucleotidc;

ORF,

PYK. gene (DNA,

probe) encoding

open

M NaCI/O.OlS M Na,.citrate wild type: Y.. Ycrrrowim.

reading

frame;

PYK,

pyruvate

PYK; S., Saccharomyces;

pH 7.6;

tsp,transcription

III

oligo, oligodekinase;

SSC, 0.15

start point(s):

wt,

mada and Ogrydziak, 1983) and an RNase (Cheng and Ogrydziak, 1986). A DNA-mediated integrative transformation system has been developed for Y. lipolrticu (Davidow et al., 1985; Gaillardin et al., 1985), and several biosynthetic genes have been cloned and sequenced for use as selectable markers (Davidow et al., 1987a; A.E.F., unpublished). The XPR2 gene, which encodes a highly expressed alkaline extracellular protease, has also been cloned and sequenced (Davidow et al., 1987b; Matoba et al., 1988; Heslot et al., 1990) and its promoter, signal sequence and protease processing sites have been adapted to form the basis of a system for the expression and secretion of heterologous proteins. A number of foreign proteins such as prochymosin (Franke et al., 1988), invertase, interferon (Heslot et al., 1990) and human tissue plasminogen activator (A.E.F., unpublished) have been successfully secreted by Y. lipolytica using this system. To further develop the potential of this expression sys-

66

PYK probe

Ala

Ile

Ala

1

Leu Asp Thr Lys Gly Pro Glu Ile Arg Thr Gly

5'.GCC ATC GCC CTG GAC ACC AAG GGA CCC GAG ATC CGA ACC GGA T I: T T T C T T C T T T 1

PYK probe 2

Met Val Ala Arg Gly Asp Leu Gly Ile Glu Ile Pro Ala 5'.ATG GTC GCC CGA GGA GAC CTG GGA ATC GAG ATC CCC GCC T C T C C T T T T : T T

Fig. I. Oligo probus dcslgncd lo isolate P>.K. Oligos wcrc designed to correspond to regions of conscr\ cd xi dcmmincd

h> ahgnrncn~ of knov n

PYK ;):I scqucnws from chlckcn (I.onhcrg and Gilbert.

et al.. I’M) md S. wwu.~i~w(Burke

ticrlincd lrith dashed 1111~sin Fig. 4. P)‘k underlined regmn tuwwd lhc undcrlmcd

the N tcrminu:

6500 DNA

(Inouc

probe I correspond!,

to rhc

PYK probe 1 corresponds

region tov 3rd the C terminus.

using 3 MilliGen

10X5). rat

CI ;II . 1983). Thcac regmn\ xc un-

Oligos wre

to

s>nthcs~rcd

S>nthcsizcr.

tern, \ve have cloned and sequenced the Y. lipol_~~ticrt gene encoding the highly expressed glycolytic enzyme, PYK, a pivotal enzyme in intermediary carbohydrate metabolism which catalyzes the essentially irreversible conversion of phosphocnolpyruvate to pyruvate with concomitant con version of ADP to ATP. PYK appears to pla), a kc! role in the regulation of glycolysis. In this paper, \ve describe the cloning of the Y. Ijl)o(lku PYK gcnc, and present data on gene structure and organization. transcript analysis. and protein product homology.

RLSIILTS

.-\ND

sequcncc of this enzyme has been quite conserved acrosS spccics. At the time we undertook this project, the scquences of the PYK genes from scvcral organisms wcrc known. We used Intelligcnetics DNA sequence analysis programs (Mountain View, CA) to align PYK aa sequences from chicken (Lonberg and Gilbert, 1985), rat (Inouc et al., 1986) and S. cerevislrre (Burke et al., 1983), and determined the longest stretches of aa identity among the scqucnces.

HAA

B

APA

XS I

I

APA

II

HS

III

BgT Sa StX A

St

I

Av Av Av A x

11

lllllIllliI

II

DISCUSSION

(a) Probe design To isolate the gcnc encoding the glycolytic enzyme PYK from Y. lipo(uiccr. \ve took advantage of the fact that the aa

Fig. 3. Scqucncing strateg! for Y. li&~r;~rr PY6.

DNA

frnqcnt\

con-

taining the rcgiona that hqhridiLcd to the oligo prohcs (shown :I\ black bows m Fig. 2) plus additional pN I3 I7

I

scqucnccs upstrcnm from this regmn in

wcrc aubcloncd Into cithcr pBR312

or pBlucact-ipt vector\

11)1

xqucncing.

,\ comhmatl~u~ of the chemical dcgrndatlnn nlcthod (hlaxam

and Gilbcrt.

1980) and the chain-termination

method (Sangcr CI ;d.. 1977:

Scqucn~w. US Biochemical, Clcvcland, OH) wx lng. Due to :I Ixk

used for DNA

clone, dclction clones wcrc gcncratcd using ExoIIl cluscs

(Stutugenc.

tcrmmation tcrminatlon:

open circles.

open squnres. DNA

hew nuclusu uitcs ,ho\rn

LaJolla. Ci-\) to l’xtlitntc

method. Filled wclcs.

Ic;lJ dcgradauon:

;md mung bwn nu-

scqucncing \ i;L the ch;litl-

DN 4 fragments sequcnccd hq chcnDNA

fragments

xqucnccd

1. A,,