|
Http://www.abbs.info e-mail:[email protected] ISSN 0582-9879
ACTA BIOCHIMICA et BIOPHYSICA SINICA 2001, 33(6):
600-606
CN 31-1300/Q |
PCR
Based Cloning and Sequence Analysis of the Pichia pastoris Cystathionine
b-Synthase Gene
( Institute of Biochemistry and Cell
Biology, Shanghai Institutes for Biological Sciences,
CBS
is directly involved in the removal of homocysteine from methionine and SAM
cycle and in the biosynthesis of cysteine. Its defectiveness can cause
homocystinuria in human[3]. In S. cerevisiae, disruption of CYS4
(encoding CBS) results in cysteine auxotroph[4]. In this report, we
cloned and sequenced the CBS gene of Pichia pastoris (PpCBS).
Transforming a PpCBS harboring expression vector into a CBS
mutant strain of S. cerevisiae can functionally complement the cysteine
auxotrophy. Disruption of the CBS gene of P. pastoris results in
Cys- phenotype, which can serve as a selectable marker of P.
pastoris expression system.
1 Materials and methods
1.1 Strains,
plasmids, media and reagents
P.
pastoris strains: GS115 (his4) was used as
the source of RNA preparation, JC307 (his4, ura3)[5]
(kindly provided by J.M.Cregg) was used for CBS gene disruption. S.
cerevisiae strain: YPH499[6](MATa, ura3-52, lys2-801amber,
ade2-101ochre, trp1-D1, his3-D200,
leu2-D1) was used to construct a CBS mutant for transformation to
examine function of the cloned gene. E.coli strain TG1 was used for
cloning. pJJ215, pJJ242 and pVT-102U were kindly provided by Professor Shi-Zhou
Ao.
Yeasts
were cultured on YPD medium (1% yeast extract, 2% peptone, 2% glucose, 1.5%
agar for plates) at 30 ℃.
When screening for transformant, synthetic minimal medium was used (SD: 0.67%
yeast nitrogen base without amino acids, 2% glucose, 1.5% agar for plates,
supplemented with nutrients according to strain requirements.) When Cys-
phenotype was screened, 30 mg/L glutathionine was supplemented to SD plate.
Restriction
endonucleases, DNA ligase and Taq DNA polymerase were products of Gibco
BRL. RNase H and terminal deoxynucleotidyl transferase (TdT) were products of
Takara. DEPC and Trizol reagent were purchased from Watson BioTechnology, Inc. Pfu
DNA polymerase, first-strand cDNA synthesis kit and 3S multi-purpose DNAprep
kit were obtained from Biocolor Biological Science & Technology Co., Ltd.
1.2
DNA methods
Recombinant
DNA methods, including genomic DNA preparation, introduction of DNA into yeast
cells and one step gene disruption, were performed as described in Ausubel et
al.[7]. DNA sequence was determined with automated DNA
sequencing method involving PCR (ABI PRISM377 sequencer) using fluoresceined primers.
1.3
Cloning of conservative region of CBS gene of P. pastoris
A
conservative region of P. pastoris CBS gene was obtained by PCR
with a pair of degenerate primers, 5'-GGTGGKWSHRTBAARGAYMG-3', and
5'-CACWGGYRVYTGRTCRAADCC-3' (W＝A＋C,
K=G＋T, S=G＋C,
H=A＋C＋T,
R=A＋G, M=A＋C,
Y=C＋T V=A＋G＋C,
D=A＋G＋T).
To get better amplification, a touch-down PCR was performed using 500 ng GS115
genomic DNA as template. The PCR were carried out as follow: in the first 16
cycles, the reaction mixture was incubated at 94 ℃
for 30 s to denature the template, then annealed for 30 s at a
successive lowering temperature from 55 ℃
to 40 ℃
at intervals of 1 ℃
between adjacent cycles, followed by extension at 72 ℃
for 1 min. In the subsequent 20 cycles, all parameters kept unchanged except
that the annealing step remained at 50 ℃
for 30 s. A control was run using genomic DNA of S. cerevisiae instead
of that of P. pastoris. The PCR products of both S. cerevisiae
and P. pastoris were blunt-end-cloned into SmaI site of pUC18.
1.4
3' RACE of PpCBS
RACE
was carried out basically as described by Frohman[8] with some
modifications. The total RNA of P. pastoris was extracted with Trizol
reagent. The first strand of cDNA was synthesized by MMLV reverse transcriptase
using an anchor primer 5' GGCCTGCAGTCGACTAGTACTTTTTTTTTTTTTTTTT 3' with
approximately 3 mg total P. pastoris RNA, according to the protocol
supplied with the kit, then the remained RNA in the reaction mixture was
removed by digestion of RNase A and RNase H. After purification by a 3S
Multi-Purpose DNAprep column, the product was used as PCR template. First round
of PCR was done with a universal amplification primer (UAP)
5'-GGCCTGCAGTCGACTAGTAC-3' and a gene specific primer 3GSP1,
5'-GGAT-AGAGAAATTGTGGACACTT-3', then nested PCR was performed with UAP primer
and a nested gene specific primer 3GSP2, 5'-CGTCTAACCAAGTT-CGCTGATGA-3'.
1.5
5' RACE of PpCBS
Two
gene specific primers were designed in the 5' region of the conservative
fragment, one was used as 5' RT primer 5'-CCATACTGGTCTAA-3', and the other 5'
GSP2, 5'-CGAATGTGAGACTCTGGGG-AAT-3', was used for amplification.
Reverse-transcribed P. pastoris total RNA with 5' RT primer according to
the protocol supplied with the cDNA synthesis kit. RNA in the reaction mixture
was digested with RNase A and RNase H, then the first strand cDNA for 5' RACE
was purified using 3S Multi-Purpose DNAprep column. After purification, a
poly(dA) tail was appended to the 5' end of the first strand cDNA using TdT
(terminal deoxynucleotidyl transferase) at 37 ℃
for 10-15
min. The poly(dA) tailed cDNA was initially amplified with anchor primer and
5GSP2, then reamplified the primary PCR product with primer UAP and 5GSP2.
2.1
Cloning of conservative fragment of P. pastoris CBS gene
In
order to clone CBS gene of P. pastoris, database searching was
carried out. 23 sequences of CBS from different sources were retrieved.
Selected 4 representative sequences to perform alignment, both of DNA sequences
and deduced amino acids sequences. Then based on two conservative regions, two
PCR degenerate primers were designed separately (Fig.1). Using P. pastoris
genomic DNA as template, a touch-down PCR was run to elevate specificity. After
36 cycles a single 1.0 kb fragment was amplified from P. pastoris
genomic DNA [Fig.2(A)], this DNA fragment was cloned into a T-vector, and
sequenced. This conservative DNA fragment showed 64% identity of nucleotide
sequences to that of CBS gene from S. cerevisiae.
Fig.1 Nucleotide sequence of the Pichia
pastoris CBS gene and deduced amino acids sequence
Two
conservative region is shown in bold type, the RACE primer sequence is shown
underlined.
The
reverse transcribed cDNA with anchor primer, was used as template of 3' RACE.
The first run of PCR with primer UAP and 3GSP1 amplified a vague band around
800 bp, then dilute the PCR product for 1 000 fold as the next round PCR
template. A second set of PCR cycles using nested PCR primer 3' GSP2 and UAP
amplified a single band of 610 bp [Fig.2(B)]. This 3'RACE product was purified
by agarose gel then directly sequenced with 3' GSP2 primer.
We
used the same set of universal amplification primer and adaptor primer for both
3' RACE and 5' RACE, in this way, we can decrease both variability in the 5'
RACE protocol and cost. The reverse-transcribed cDNA with 5' RT primer was used
for 5' RACE. After appending poly(dA) tail, the cDNA was amplified using anchor
primer and 5′GSP2,
there is no visible band of the PCR product in EB stain agrose gel. A second
round of PCR using primer UAP and GSP2 was run with the dilution of previous
PCR product as template, and a fragment around 500 bp was amplified [Fig.2(C)].
After purification, it was directly sequenced with 5' GSP2.
Fig.2 Agarose gel analysis of PCR
products
(A) Touch-down PCR amplification of the
conservative region of CBS gene. 1, negative control without template;
2, P. pastoris genomic DNA as template; 3, S. cerevisiae genomic
DNA as template; M, DL 2000 DNA size marker. (B) Gel analysis of 3' RACE product. 1, the primary PCR
product of 3' RACE with primer
anchor and GSP1; 2, nested PCR product of two in a thousand primary PCR product
as template with primer UAP and 3GSP2; M, size marker lDNA EcoRI/HindIII.
(C) Gel analysis of 5' RACE product. The first strand cDNA reverse-transcribed
using 5' RACE RT primer was
initially PCR-amplified with anchor prime and 5GSP2, then reamplified with UAP
primer and 5GSP2. 1, a negative control without TdT tailing of the cDNA; 2, TdT
tailing for 10 min; 3, TdT tailing for 15 min; M, size marker lDNA EcoRI/HindIII.
The
sequences of both RACE fragments were assembled with that of the conservative
fragment of PpCBS to reveal the whole sequence of PpCBS gene
which contains an ORF of 1 503 bp coding for a polypeptide of 501 residues
(Fig.1). The deduced amino acid sequence showed 55% identity to that of S.
cerevisiae CBS (ScCBS), 41% identity to rat CBS; 40% identity to human CBS
as shown in Fig.3. The similarity of protein sequences between PpCBS and ScCBS
is lower than that between human CBS and rat CBS (approximately 90% identity)[5],
but higher than that between human CBS and ScCBS (36% identity).
Fig.3 Alignment of amino acid
sequences of CBS proteins from different resourses
Pichia
pastoris(Pp), Saccharomyces cerevisiae(Sc),
Rattus norvegicus(Rn), Homo sapiens(Hs).
To
corroborate the cloned gene functional, a S. cerevisiae strain with a CBS
mutation was constructed. The PCR product of S. cerevisiae CBS
conservative region were blunt-end-cloned into SmaI site of pUC18, the
resulting plasmid was designated as pSCBS. A BamHI fragment of pJJ215,
containing S. cerevisiae HIS3 gene, was end-blunted by Klenow
enzyme then ligated into EcoRV site of pSCBSc (Fig.4). This
construct was used to disrupt CBS gene of S. cerevisiae strain
YPH499. The transformants were selected on the SD plate supplemented with
uracil, leucine, lysine, adenosine, tryptophan and glutathionine but no
histidine. Then the Cys- phenotype of the transformant was
demonstrated on SD plate without glutathionine. The successful disruption of CBS
gene was verified by PCR. This strain, designated as YC12, has a Cys-
phenotype which needs supplementation of cysteine or glutathionine to grow.
Fig.4 Construction of three plasmids
for functional analysis of PpCBS
The
plasmid pVT102-PCBS which contains the entire P. pastoris CBS
cDNA was used to transform Cys– strain YC12. The plasmid expressing
the P. pastoris CBS, allows this Cys- strain to form colonies
in the absence of exogenous cysteine, whilethe same strain containing the
expression vector alone, pVT-102U, does not (Fig.5).
Fig.5 Growth of yeast strains on SD
media supplemented with histidine, leucine, uracil, adenine, lysine and
tryptophan except glutathionine for 3 days
(A)
YHP499; (B) YC12 transformed with pVT-102U; (C) YC12 Transformed with pVT-PCBS.
The
methylotrophic yeast, P. pastoris, has been developed as highly
successful expression systems for recombinant proteins. The favorable and most
advantageous characteristics of these species resulted in an increasing number
of biotechnological applications[9,10]. Two of these merits are: (1)
Strong and tightly regulated promoters, such as alcohol oxidase I promoter that
is uniquely suited for the controlled expression of foreign genes; (2) The
ability to grow to very high cell densities in simple defined minimal salt
media. Besides expression system, methylotrophic yeasts are also exploited as a
model organism for peroxisome assembly[11,12] or industrial production
of metabolites[13]. Its high cell density fermentation makes it a
potential to produce fine chemicals, such as SAM, at low cost. However all the
genetic study on P. pastoris focus on methanol metabolism and peroxisome
biogenesis related gene, there is less knowledge of the sulfur-containing amino
acids metabolism in P. pastoris.
The
researches in molecular biology of P. pastoris benefit from S.cerevisiae
genetic and molecular information. Several genes of P. pastoris were
cloned based on the homology of the genes between these two yeasts[14,15].
We reported here the cloning and sequencing of the P. pastoris CBS
mainly based on homology alignment and PCR technique. First, based on the
homology alignment of different source of CBS, a set of degenerate PCR
primers was designed to amplify a conservative fragment of P. pastoris CBS,
after the sequence was revealed, 5' RACE and 3' RACE were performed separately
to get the 5' and 3' end sequence. The whole sequence of P. pastoris CBS
was assembled through these three sequences. In this way, we avoid tedious
screening of genomic DNA or cDNA library.
Although
classic and molecular genetic techniques are generally well-developed for P.
pastoris, few selectable marker genes have been described for the molecular
genetic manipulation of the yeast. Existing markers are limited to the
biosynthetic pathway genes HIS4 from either P.pastoris or S.cerevisiae,
ARG4 from S. cerevisiae, until recently Lin et al.[5]
isolate a new set of biosynthetic markers: the P. pastoris ADE1
(PR-amidoimidazolesuccinocarboxamide synthase), ARG4 (argininosuccinate
lyase) and URA3 (orotidine 5'-phosphate decarboxylase) genes. Now, a new
biosynthetic marker was added to this family. Disruption of CBS results
in a cysteine auxotrophy in P. pastoris, this trait make it can server
as a selectable marker.
CBS
gene mutations in human cause homocystinuria, a recessive disorder
characterized by excessive levels of total homocysteine (tHcy) in plasma. The
primary cause of mortality is thromboembolism induced by the excessive tHcy
levels. Mild increases in tHcy levels are a significant risk factor in the
development of vascular disease in the general population. Shan et al[16]
found that CBS protein has two separate domain, one of which in N-terminus is
responsible for the catalytic activity, the other in C-terminus for a negative
regulator of enzyme activity. The identification of the C-terminus as a
negative regulatory domain suggests it would be a good target for pharmacological
intervention to lower tHcy levels in both homocystinuria and individuals with
normal CBS function. Potentially, drugs could be identified which disrupt the
ability of the C-terminal fragment to inhibit catalytic activity. Such
molecules could be useful for reducing the risk of homocysteine-related
vascular disease.
To
gain such an objective, A suitable assay system is needed. The resultant P.
pastoris strain in this work can be used as an expression system for the
human CBS protein. No background enzyme activity combined with outstanding
expression level make it an ideal system for expression of human CBS and drug
screening. Meanwhile, the high expression level of P. pastoris make it a
potential to get enough amount of protein to crystallize. Resolving CBS protein
structure will help to design drugs to curb the homocystinuria.
Further
studies on this CBS mutant will shed more light on the sulphur amino
acid metabolism in P. pastoris, this will be conducive to exploiting P.
pastoris to produce sulphur amino acids and SAM, which is hot new dietary
supplement that can ease depression, restore arthritic joints and combat
chronic liver disease. The main production of SAM is from fermentation of S.cerevisiae.
With the merit of high cell density fermentation in defined salts medium, Pichia
pastoris has a potential of producing SAM at low cost.
1 Thomas D, Surdin-Kerjan
Y. Metabolism of sulfur amino acids in S.cerevisiae. Microbiol and
Mol Biol Rev, 1997, 61: 503-532
2 Hansen J, Johannesen
PF. Cysteine is essential for transcriptional regulation of the sulfur
assimilation genes in Saccharomyces cerevisiae. Mol Gen Genet,
2000, 263(3): 535-542
3 Mudd SH, Levy HL,
Skovby F. Disorders in transsulfuration. In: Scriver CR, Beaudet A, Sly W,
Valle D ed. The Metabolic Basis of Inherited Disease, New York:
McCraw-Hill, 1995, 693-734
4 Kruger WD, Cox DR. A
yeast system for expression of human cystathionine b-synthase: Structural and
functional conservation of the human and yeast genes. Proc Natl Acad Sci USA,
1994, 91: 6614-6618
5 Lin Cereghino GP, Lin
Cereghino J, Sunga AJ, Johnson M A, Lim M, Gleeson M, Cregg J M. New selectable
marker/auxotrophic host strain combinations for molecular genetic manipulation
of Pichia pastoris. Gene, 2001, 263(1-2): 159-169
6 Sikorski RS, Hieter P.
A system of shuttle vectors and yeast host strains designed for efficient
manipulation of DNA in Saccharomyces cerevisiae. Genetics, 1989, 122:
19-27
7 Ausubel FM, Brent R,
Kingston RE, Moore DD, Seidman JG, Simth JA, Struhl K. Short Protocols in
Molecular Biology, 2nd ed, New York: John Wiley & Sons, 1995
8 Frohman MA. PCR Primer:
A Laboratory Manual, 2nd ed, New York: Cold Spring Harbor Laboratory
Press, 1995, 381-409
9 Sudbery PE. The
expression of recombinant proteins in yeasts. Curr Opin Biotechnol,
1996, 7(5): 517-524
10 Cereghino JL, Cregg JM.
Heterologous protein expression in the methylotrophic yeast Pichia pastoris.
FEMS Microbiol Rev, 2000, 24(1): 45-66
11 Faber K N, Elgersma Y, Heyman
JA, Koller A, Luers GH, Nuttley W M, Terlecky S R et al. Use of Pichia
pastoris as a model eukaryotic system. Peroxisome biogenesis. Methods
Mol Biol, 1998, 103: 121-147
12 Gould SJ, McCollum D, Spong
AP, Heyman J A, Subramani S. Development of the yeast Pichia pastoris as
a model organism for a genetic and molecular analysis of peroxisome assembly. Yeast,
1992, 8(8): 613-628
13 Wegner GH. Emerging
applications of the methylotrophic yeasts. FEMS Microbiol Rev, 1990, 87:
279-284
14 Cosano IC, Alvarez P, Molina
M, Nombela C. Cloning and sequence analysis of the Pichia pastoris TRP1,
IPP1 and HIS3 genes. Yeast, 1998, 14(9): 861-867
15 Ohi H, Ohtani W, Okazaki N,
Furuhata N, Ohmura T. Cloning and characterization of the Pichia pastoris
PRC1 gene encoding carboxypeptidase Y. Yeast, 1996, 12(1): 31-40
16 Shan X, Kruger WD. Correction
of disease-causing CBS mutations in yeast. Nat Genet, 1998, 19(1):
91-93
Received: June 22, 2001 Accepted: July 31, 2001
*Corresponding author: Tel,
86-21-64374430-5289; Fax, 86-21-64338357; e-mail, [email protected]