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Previous Article | Next Article 
Applied and Environmental Microbiology, December 2003, p. 7563-7566, Vol. 69, No. 12
0099-2240/03/$08.00+0 DOI: 10.1128/AEM.69.12.7563-7566.2003
Copyright © 2003, American
Society for
Microbiology. All Rights Reserved.
Genetic Characterization of the Carotenoid Biosynthetic Pathway in Methylobacterium extorquens AM1 and Isolation of a Colorless Mutant
Stephen J. Van Dien,1 Christopher J. Marx,2,
Brooke N. O'Brien,2 and Mary E. Lidstrom1,2*
Department
of Chemical Engineering,1
Department of
Microbiology, University of Washington,
Seattle, Washington 98195-21802
Received 30 June 2003/
Accepted 22 September 2003
 |
ABSTRACT
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Genomic
searches were used to reconstruct the putative carotenoid biosynthesis
pathway in the pink-pigmented facultative methylotroph
Methylobacterium extorquens AM1. Four genes for putative
phytoene desaturases were identified. A colorless mutant was obtained
by transposon mutagenesis, and the insertion was shown to be in one of
the putative phytoene desaturase genes. Mutations in the other three
did not affect color. The tetracycline marker was removed from the
original transposon mutant, resulting in a pigment-free strain with
wild-type growth properties useful as a tool for future
experiments.
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INTRODUCTION
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Methylotrophic bacteria are capable of growth using C1
compounds such as methanol as their only carbon and energy source, and
therefore they could potentially serve as biocatalysts for the
conversion of methanol to useful products. Methanol is a good candidate
for an alternative feedstock, as it is abundant, soluble, and low in
cost. The pink-pigmented facultative methylotroph Methylobacterium
extorquens AM1 is known to generate a pink carotenoid
(6,
11). Since carotenoids
have a variety of industrial uses
(4), the carotenoid
biosynthesis pathway is a potential target for metabolic engineering of
this bacterium. In this work, genomic reconstruction and mutagenesis
were used to characterize the carotenoid biosynthetic pathway in M.
extorquens AM1.
|
Reconstruction of the
carotenoid biosynthetic pathway from the genome sequence.
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The biosynthetic pathway for
carotenoids has been elucidated through extensive study of various
plants and bacteria (3)
and is outlined in Fig.
1. The first part of the pathway, common to all isoprenoids, is the
production of isopentenyl pyrophosphate (IPP). In most bacteria, this
occurs through a pathway that begins with glyceraldehyde-3-phosphate
and proceeds through 1-deoxy-D-xylulose-5-phosphate (DXP),
as shown in Fig. 1
(13). This is referred to
as the mevalonate-independent pathway. Although the details of the
conversion to IPP are not fully understood, some of the genes in these
intermediate steps have recently been identified
(3,
12). Protein sequences of
the enzymes involved in the upper pathway were obtained from the
GenBank
(http://www.ncbi.nlm.nih.gov)
database and used to search the M. extorquens AM1
partial genome sequence
(http://www.integratedgenomics.com/genomereleases.html#list6).
Putative gene function was further evaluated by a BLAST search of the
corresponding translated sequence against the National Center for
Biotechnology Information database (http://www.ncbi.nlm.nih.gov/BLAST).
The results of this search are listed in Table
1. Genes potentially encoding all of the steps of the pathway were found,
with the exception of the IPP delta isomerase (idi gene).
Since the production of IPP is essential for all organisms, it is
likely that this gene is either divergent or located in the unsequenced
portion of the genome. The other pathway for IPP synthesis, uncommon in
bacteria, proceeds through mevalonic acid as an intermediate
(3). No genes predicted to
encode enzymes of this pathway were identified in the M.
extorquens AM1 genome sequence.
IPP and its isomer
dimethylallyl pyrophosphate are condensed to various degrees to form a
wide variety of isoprenoid compounds, some of which are essential for
growth. The first step unique to carotenoid synthesis is the
condensation of four such units to make geranylgeranyl diphosphate
(GGPP) via the enzyme GGPP synthase. Two of these molecules are then
condensed by phytoene synthase to form phytoene, a precursor of all
carotenoids (3). Phytoene
desaturases then introduce a variable number of desaturations to create
molecular diversity. The Rhodobacter capsulatus phytoene
desaturase introduces three desaturations to produce neurosporene,
which is yellow, while phytoene desaturases from many other bacteria
introduce four desaturations to produce the red compound lycopene
(7). Beginning with these
compounds, further diversity in carotenoids is introduced by
modifications such as desaturation, methylation, and hydroxylation.
Examples include ß-carotene, zeaxanthin, and astaxanthan
(3).
Identitysearches were performed by using translated sequences of known R.
capsulatus (1) and
Erwinia herbicola
(8,
15) genes to locate
putative carotenoid biosynthetic genes in the M. extorquens
AM1 partial genome sequence (Table 2), which were further
evaluated as described above by using BLAST. The predicted carotenoid
biosynthesis genes are not tightly clustered, as they are located on
seven different contigs in the genome sequence, separated by
intervening DNA. Genes predicted to encode all steps for the formation
of the first colored carotenoid (e.g., lycopene) from IPP were
identified in the genome. In addition, three candidates for genes
involved in further modification were found, with the highest identity
to the hydroxyspheroidene pathway of R. sphaeroides
(1). No candidates for the
Erwinia ß-carotene-zeaxanthan pathway
(8) were found. Multiple
candidates were identified for some carotenoid biosynthesis genes: two
genes for phytoene synthase (crtB) and four for phytoene
desaturase (crtI).
|
Isolation and
genetic characterization of a colorless mutant of M.
extorquens AM1 and generation of unmarked strain AM1-W.
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Transposon mutagenesis using the
mini-Tn5 derivative ISphoA/hah-Tc
(5) was performed as
previously described (10)
in order to determine whether a single insertion mutation could result
in loss of carotenoid production. Three white transposon mutants were
isolated out of approximately 7,000 screened visually. The chromosomal
site of insertion in each of these strains was determined as previously
described (10). The
sequences obtained were all mapped to the same open reading frame (ORF)
in the M. extorquens AM1 genome sequence: one of the four
genes predicted to encode phytoene desaturase (phytoene dehydrogenase).
This gene is shown in bold in Table
2 and will be referred to as crtI.
For future genetic
screening of this strain with colorimetric substrates, it would be
useful to have a colorless, unmarked mutant. In order to generate such
a pigment-free mutant, the tetracycline resistance gene of
crtI::ISphoA/hah-Tc mutant
S234-13 was removed by using cre expression plasmid pCM158
(9). The resulting
unmarked, white mutant of M. extorquens AM1, AM1-W, was
confirmed by analytical PCR assay. Under all standard laboratory
conditions, growth of AM1-W was indistinguishable from that of the
pigmented wild-type strain (data not shown).
To verify that
crtI is responsible for the loss of color in the mutants, as
opposed to a polar effect, gene complementation was performed. A 1.5-kb
region containing crtI was amplified by PCR, cloned directly
into pCR2.1 (Invitrogen), and subcloned as a
BamHI-EcoRI fragment into pCM66 and pCM160
(10) to produce pCAR12
and pCAR14. The resulting plasmids contain crtI expressed from
Escherichia coli Plac or M.
extorquens AM1 PmxaF, respectively. Both
plasmids were introduced by conjugation with E. coli S17-1
(16) into AM1-W,
resulting in pink colonies. In contrast, colonies of AM1-W containing
either pCM66 or pCM160 remained white. These results demonstrate that
the M. extorquens crtI gene is essential for formation of the
pink pigment and confirm previous suggestions
(6,
11) that the pigment was
due to the presence of a carotenoid.
Surprisingly, crtI
was not the most likely candidate to encode an active phytoene
desaturase, on the basis of the information in Table
2. The gene labeled
orf1 is located immediately downstream of a putative phytoene
synthase gene and has greater identity to a known phytoene desaturase.
In contrast, crtI is surrounded by putative phospholipid and
cell wall biosynthesis genes and a GMP synthase. Deletion mutants with
changes in two of the remaining phytoene desaturase gene candidates,
orf2 and orf3, were prepared by using PCR products of
these ORFs and the allelic exchange vector pCM184
(9). These mutants were
confirmed to be double crossovers by PCR analysis, indicating that they
have null mutations in the corresponding gene. Both mutants remained
pink, suggesting that these genes do not play a major role in the
synthesis of colored carotenoids in M. extorquens
AM1.
|
Isolation of carotenoid from
wild-type M. extorquens AM1.
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Carotenoids were extracted from M.
extorquens AM1 by using a modification of the procedure commonly
used for photosynthetic bacteria
(17). Cell pellets
obtained from approximately 100 ml of cell culture grown in mineral
salts medium (2)
containing 125 mM methanol were resuspended in 1 ml of methanol at
65°C and vortexed for 1 min. A 0.4-ml volume of water and 0.3
ml of chloroform were added, and the sample was vortexed for an
additional 2 min. The bottom organic layer, containing the carotenoid,
was extracted into clean tubes. A 1-ml volume of methanol and 0.4 ml of
water were added, and the organic layer was extracted again. The
extract from wild-type cells was pink, whereas that from mutant strain
AM1-W was colorless. A 2-ml volume of acetone was added to the extract
(about 0.15 ml) in order to remove phospholipids and glycolipids
(14), and the sample was
placed at -20°C overnight. The sample was centrifuged,
and the supernatant was evaporated to dryness and redissolved in 0.1 ml
of chloroform. The visible absorption spectrum of the sample, with
maxima at 480, 510, and 540 nm, matched that of the extract from a
related pink-pigmented methylotroph, previously referred to as
Protaminobacter ruber(14). Furthermore, as
observed with the carotenoid from P. ruber
(14), a 9-nm
red shift in the absorption spectra was observed following hydrolysis
of the carotenoid in 0.2 M HCl-methanol. These results suggest
that the pink carotenoid in M. extorquens AM1 is similar to
that in P. ruber, which has been shown to be a carotenoid with
an attached sugar moiety that is lost during hydrolysis. The
1H nuclear magnetic resonance spectrum of the P.
ruber extract indicated the presence of a carbonyl group and about
13 conjugated double bonds, and thin-layer chromatography data
suggested that the carotenoid has a bacterioruberin- or
oscillaxanthin-like structure
(14).
|
Nucleotide
sequence accession number.
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The sequence of M.
extorquens AM1 crtI has been deposited in the GenBank
database (accession number
AY331188).
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ACKNOWLEDGMENTS
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This
work was supported by a grant from the National Institutes of Health
(GM58933).
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FOOTNOTES
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* Corresponding
author. Mailing address: Department of Chemical Engineering, University
of Washington, Box 352180, Seattle, WA 98195-2180. Phone: (206)
616-5282. Fax: (206) 616-5721. E-mail:
lidstrom{at}u.washington.edu. 
Present
address: 2215 Biomedical Physical Sciences, Michigan State University,
East Lansing, MI 48824-4320.
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Applied and Environmental Microbiology, December 2003, p. 7563-7566, Vol. 69, No. 12
0099-2240/03/$08.00+0 DOI: 10.1128/AEM.69.12.7563-7566.2003
Copyright © 2003, American
Society for
Microbiology. All Rights Reserved.
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Abstract
Introduction
