Engineering of a microbial coculture of Escherichia coli strains for the biosynthesis of resveratrol.
BACKGROUND
Resveratrol is a plant natural product with many health-protecting effects which makes it an attractive chemical both for academic studies and industrial purposes. However, the low quantities naturally produced by plants as well as the unsustainable procedures of extraction, purification and concentration have prompted many biotechnological approaches to produce this chemical in large quantities from renewable sources. None of these approaches have considered a microbial coculture strategy to produce this compound. The aim of this study was to prove the functionality of a microbial coculture for the biosynthesis of resveratrol.


RESULTS
In this work, we have successfully applied a coculture system strategy comprised of two populations of Escherichia coli strains, each with a partial and complementary section of the pathway leading to the biosynthesis of the stilbene resveratrol. The first strain is a pheA knockout mutant previously engineered to excrete p-coumaric acid into the medium through the overexpression of genes encoding a tyrosine ammonia lyase from Rhodothorula glutinis, a feedback resistant 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and a transketolase. The second strain in the coculture was engineered to express the second part of the resveratrol biosynthetic pathway through the introduction of synthetic genes encoding the 4-coumaroyl-CoA ligase from Streptomyces coelicolor A2 and the stilbene synthase either from the peanut Arachis hypogaea or the grapevine Vitis vinifera, the latter synthesized employing a gene harmonization strategy and showing better resveratrol production performance. Batch cultures were performed in mineral medium with glycerol as the sole carbon source, where a final titer of 22.6 mg/L of resveratrol was produced in 30 h.


CONCLUSIONS
To our knowledge, this is the first time that a coculture of bacterial strains is used for the biosynthesis of resveratrol from glycerol, having the potential for a greater improvement in the product yield and avoiding the use of precursors such as p-coumaric acid, yeast extract or an expensive inhibitor such as cerulenin.
Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. Postal 510-3, 62210, Cuernavaca, Morelos, Mexico.
José M
JM
Camacho-Zaragoza
Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. Postal 510-3, 62210, Cuernavaca, Morelos, Mexico.
Georgina
G
Hernández-Chávez
Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. Postal 510-3, 62210, Cuernavaca, Morelos, Mexico.
Fabian
F
Moreno-Avitia
Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. Postal 510-3, 62210, Cuernavaca, Morelos, Mexico.
René
R
Ramírez-Iñiguez
Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. Postal 510-3, 62210, Cuernavaca, Morelos, Mexico.
Alfredo
A
Martínez
Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. Postal 510-3, 62210, Cuernavaca, Morelos, Mexico.
Francisco
F
Bolívar
Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. Postal 510-3, 62210, Cuernavaca, Morelos, Mexico. gosset@ibt.unam.mx.
Guillermo
http://orcid.org/0000-0003-3127-0911
G
Gosset
Escherichia coli; Glycerol; Metabolic engineering; Microbial coculture; Resveratrol
Annu Rev Microbiol. 2007;61:51-69
17456010
J Biotechnol. 2013 Sep 20;167(4):404-11
23916948
Appl Environ Microbiol. 2006 Aug;72(8):5670-2
16885328
Biomed Res Int. 2013;2013:780145
23710459
Microb Cell Fact. 2015 Jan 16;14:6
25592545
Anticancer Res. 2004 Sep-Oct;24(5A):2783-840
15517885
PLoS One. 2008 May 14;3(5):e2189
18478103
Proc Natl Acad Sci U S A. 2015 Jul 7;112(27):8266-71
26111796
J Bacteriol. 2003 Jan;185(1):20-7
12486036
J Gen Microbiol. 1988 Aug;134(8):2249-53
3075658
Am J Physiol Endocrinol Metab. 2006 Jun;290(6):E1339-46
16434553
Lancet. 1992 Jun 20;339(8808):1523-6
1351198
Curr Opin Biotechnol. 2015 Aug;34:73-81
25499800
Cardiovasc Drug Rev. 2004 Fall;22(3):169-88
15492766
Mol Pharm. 2008 Mar-Apr;5(2):257-65
18333619
J Bacteriol. 2004 Nov;186(21):7196-204
15489430
Chem Biol. 2007 Jun;14(6):613-21
17584609
J Biomed Biotechnol. 2012;2012:579089
22654481
Nat Biotechnol. 2009 Oct;27(10):946-50
19801975
Metab Eng. 2015 Nov;32:1-11
26344106
Nat Biotechnol. 2013 Apr;31(4):335-41
23417095
J Antimicrob Chemother. 2001 Feb;47(2):243-4
11157919
Bacteriol Rev. 1972 Dec;36(4):525-57
4568763
Appl Environ Microbiol. 2011 May;77(10):3451-60
21441338
Microb Cell Fact. 2015 Sep 15;14:134
26369810
BMC Biotechnol. 2006 Mar 21;6:22
16551366
Metab Eng. 2016 May;35:55-63
26860871
Clin Cancer Res. 2002 Mar;8(3):893-903
11895924
Gene. 1988 Sep 30;69(2):301-15
3069586
Metab Eng. 2011 Jul;13(4):392-400
21320631
Plant Mol Biol. 2004 Aug;55(6):869-81
15604722
Microb Cell Fact. 2009 Apr 02;8:19
19341482