A genome-scale metabolic reconstruction of Pseudomonas putida KT2440: iJN746 as a cell factory.
BACKGROUND
Pseudomonas putida is the best studied pollutant degradative bacteria and is harnessed by industrial biotechnology to synthesize fine chemicals. Since the publication of P. putida KT2440's genome, some in silico analyses of its metabolic and biotechnology capacities have been published. However, global understanding of the capabilities of P. putida KT2440 requires the construction of a metabolic model that enables the integration of classical experimental data along with genomic and high-throughput data. The constraint-based reconstruction and analysis (COBRA) approach has been successfully used to build and analyze in silico genome-scale metabolic reconstructions.


RESULTS
We present a genome-scale reconstruction of P. putida KT2440's metabolism, iJN746, which was constructed based on genomic, biochemical, and physiological information. This manually-curated reconstruction accounts for 746 genes, 950 reactions, and 911 metabolites. iJN746 captures biotechnologically relevant pathways, including polyhydroxyalkanoate synthesis and catabolic pathways of aromatic compounds (e.g., toluene, benzoate, phenylacetate, nicotinate), not described in other metabolic reconstructions or biochemical databases. The predictive potential of iJN746 was validated using experimental data including growth performance and gene deletion studies. Furthermore, in silico growth on toluene was found to be oxygen-limited, suggesting the existence of oxygen-efficient pathways not yet annotated in P. putida's genome. Moreover, we evaluated the production efficiency of polyhydroxyalkanoates from various carbon sources and found fatty acids as the most prominent candidates, as expected.


CONCLUSIONS
Here we presented the first genome-scale reconstruction of P. putida, a biotechnologically interesting all-surrounder. Taken together, this work illustrates the utility of iJN746 as i) a knowledge-base, ii) a discovery tool, and iii) an engineering platform to explore P. putida's potential in bioremediation and bioplastic production.
Nogales
Juan
J
Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas-CSIC, Ramiro de Maeztu 9, Madrid, 28040, Spain. jnogales@cib.csic.es
Palsson
Bernhard Ø
BØ
Thiele
Ines
I
D000693:Anaerobiosis; D019295:Computational Biology / Q000379:methods*; D003198:Computer Simulation; D030541:Databases, Genetic; D005227:Fatty Acids / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D016680:Genome, Bacterial; D023281:Genomics / Q000379:methods; D051188:Knowledge Bases; D053858:Metabolic Networks and Pathways / Q000235:genetics; D008954:Models, Biological; D010100:Oxygen / Q000378:metabolism; D054813:Polyhydroxyalkanoates / Q000096:biosynthesis; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism; D014050:Toluene / Q000378:metabolism
D005227:Fatty Acids; D054813:Polyhydroxyalkanoates; D014050:Toluene; D010100:Oxygen
Science. 1987 Jan 30;235(4788):593-6
3468623
J Biol Chem. 2007 Sep 28;282(39):28791-28799
17573341
Appl Environ Microbiol. 1990 Nov;56(11):3360-7
2125185
Genome Biol. 2003;4(9):R54
12952533
Appl Environ Microbiol. 1990 Feb;56(2):569-71
2306096
Biotechnol Bioeng. 2003 Dec 20;84(6):647-57
14595777
Environ Microbiol. 2002 Dec;4(12):799-808
12534463
J Bacteriol. 2007 Apr;189(7):2787-92
17259313
Environ Microbiol. 2002 Dec;4(12):824-41
12534466
Nat Rev Microbiol. 2004 Nov;2(11):886-97
15494745
Appl Environ Microbiol. 1992 Feb;58(2):536-44
1610179
EMBO Rep. 2005 May;6(5):397-9
15864286
Nature. 2004 Feb 26;427(6977):839-43
14985762
Proc Natl Acad Sci U S A. 2007 Feb 6;104(6):1777-82
17267599
Mol Gen Genet. 1977 Jul 20;154(2):203-4
895716
Proteomics. 2006 Feb;6(4):1301-18
16470664
Appl Environ Microbiol. 2001 Nov;67(11):5219-24
11679348
Microbiol Mol Biol Rev. 1999 Mar;63(1):21-53
10066830
J Bacteriol. 1973 Nov;116(2):908-14
4745434
J Biol Chem. 2005 Mar 25;280(12):11683-95
15572364
Curr Opin Biotechnol. 2004 Feb;15(1):50-1
15102466
Microbiol Rev. 1992 Dec;56(4):662-76
1480114
Proc Natl Acad Sci U S A. 2006 Feb 21;103(8):2833-8
16477005
Proc Natl Acad Sci U S A. 1981 Dec;78(12):7458-62
6950388
Nat Genet. 2004 Oct;36(10):1056-8
15448692
Proc Natl Acad Sci U S A. 2007 May 8;104(19):7797-802
17463081
J Bacteriol. 1975 Oct;124(1):7-13
1176436
Nat Rev Genet. 2006 Feb;7(2):130-41
16418748
Adv Microb Physiol. 1990;31:1-69
2264522
Nucleic Acids Res. 2006 Jan 1;34(Database issue):D354-7
16381885
Appl Environ Microbiol. 2006 Jan;72(1):585-95
16391095
Nat Rev Genet. 2004 Feb;5(2):101-13
14735121
Mol Syst Biol. 2007;3:121
17593909
Nat Protoc. 2007;2(3):727-38
17406635
Nature. 2002 Nov 14;420(6912):186-9
12432395
Proc Natl Acad Sci U S A. 2006 Nov 14;103(46):17480-4
17088549
Genome Biol. 2007;8(5):R89
17521419
J Bacteriol. 1989 Sep;171(9):5048-55
2549010
Curr Opin Biotechnol. 2001 Jun;12(3):289-95
11404107
Anal Biochem. 2004 Feb 15;325(2):308-16
14751266
BMC Microbiol. 2005 Mar 07;5:8
15752426
Environ Microbiol. 2001 Oct;3(10):612-8
11722541
Biotechnol Bioeng. 2005 Dec 30;92(7):850-64
16155945
Biotechnol Bioeng. 2006 Dec 5;95(5):992-1002
16807925
Photochem Photobiol. 1978 Oct-Nov;28(4-5):733-41
216032
Mol Syst Biol. 2006;2:2006.0004
16738551
Curr Opin Microbiol. 1998 Jun;1(3):303-10
10066491
Appl Environ Microbiol. 2005 Apr;71(4):2046-52
15812037
Nat Biotechnol. 2001 Feb;19(2):125-30
11175725
Nucleic Acids Res. 2007 Jan;35(Database issue):D533-7
17202169
Annu Rev Plant Physiol Plant Mol Biol. 2001 Jun;52:527-560
11337408
Antonie Van Leeuwenhoek. 1982 May;48(2):105-30
6808915
BMC Microbiol. 2005 Jun 27;5:39
15982422
Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):14339-44
14617778
Trends Biotechnol. 2005 Jul;23(7):349-58
15923052
J Bacteriol. 2007 Jul;189(14):5142-52
17483213
Nat Biotechnol. 2008 Jun;26(6):659-67
18536691
J Biol Chem. 2006 Apr 28;281(17):11981-91
16495222
Science. 2002 Aug 30;297(5586):1551-5
12202830
Am J Physiol Regul Integr Comp Physiol. 2001 Mar;280(3):R695-704
11171647
J Mol Microbiol Biotechnol. 2003;5(4):230-9
12867747
J Bacteriol. 2008 Apr;190(8):2790-803
18192387
J Bacteriol. 1994 Mar;176(6):1661-6
8132461
Mol Gen Genet. 1990 Mar;221(1):113-20
2183008
J Bacteriol. 2005 Aug;187(16):5818-30
16077130
Appl Environ Microbiol. 1994 Oct;60(10):3724-31
7986045
Chemosphere. 2004 Feb;54(8):1255-65
14664855
J Bacteriol. 2005 Mar;187(5):1581-90
15716428
Environ Microbiol. 2004 Dec;6(12):1264-86
15560824
Appl Environ Microbiol. 1989 Aug;55(8):1949-54
2506811
Appl Microbiol Biotechnol. 2003 Mar;61(1):77-81
12658518
J Biol Chem. 1972 Apr 25;247(8):2283-8
5019949
Mol Microbiol. 2006 Dec;62(6):1643-54
17116241
Nat Biotechnol. 2004 Oct;22(10):1218-9
15470454
Appl Microbiol Biotechnol. 2007 Feb;74(1):1-12
17146652
Environ Microbiol. 2006 Jan;8(1):165-77
16343331
Annu Rev Microbiol. 1994;48:525-57
7826017
J Bacteriol. 2007 Sep;189(18):6602-10
17616587
J Bacteriol. 2000 Jan;182(1):91-9
10613867
BMC Syst Biol. 2007 Jun 08;1:26
17555602
Nucleic Acids Res. 2006 Jan 05;34(1):1-9
16397293
Appl Environ Microbiol. 1985 Dec;50(6):1409-13
3911905
J Biol Chem. 2005 Oct 21;280(42):35382-90
16030014
Environ Microbiol. 2001 Oct;3(10):649-57
11722545
J Bacteriol. 1989 Dec;171(12):6782-90
2687253
Annu Rev Microbiol. 1997;51:341-73
9343354
J Gen Microbiol. 1966 May;43(2):159-271
5963505
Genome Res. 2004 Nov;14(11):2367-76
15520298
Biomacromolecules. 2007 Aug;8(8):2504-11
17661516
Appl Environ Microbiol. 1990 Nov;56(11):3354-9
16348341
Environ Microbiol. 2000 Dec;2(6):687-94
11214801
Nature. 2001 Jan 11;409(6817):258-68
11196655
Biotechnol Bioeng. 2000-2001;71(4):286-306
11291038