Construction of lycopene-overproducing E. coli strains by combining systematic and combinatorial gene knockout targets

Identification of genes that affect the product accumulation phenotype of recombinant strains is an important problem in industrial strain construction and a central tenet of metabolic engineering. We have used systematic (model-based) and combinatorial (transposon-based) methods to identify gene knockout targets that increase lycopene biosynthesis in strains of Escherichia coli. We show that these two search strategies yield two distinct gene sets, which affect product synthesis either through an increase in precursor availability or through (largely unknown) kinetic or regulatory mechanisms, respectively. Exhaustive exploration of all possible combinations of the above gene sets yielded a unique set of 64 knockout strains spanning the metabolic landscape of systematic and combinatorial gene knockout targets. This included a global maximum strain exhibiting an 8.5-fold product increase over recombinant K12 wild type and a twofold increase over the engineered parental strain. These results were further validated in controlled culture conditions.

[1]  G. Storz,et al.  The response regulator RssB controls stability of the sigma(S) subunit of RNA polymerase in Escherichia coli. , 1996, The EMBO journal.

[2]  G. Stephanopoulos,et al.  Identifying gene targets for the metabolic engineering of lycopene biosynthesis in Escherichia coli. , 2005, Metabolic engineering.

[3]  J. Keasling,et al.  Low-copy plasmids can perform as well as or better than high-copy plasmids for metabolic engineering of bacteria. , 2000, Metabolic engineering.

[4]  P. Matthews,et al.  Metabolic engineering of carotenoid accumulation in Escherichia coli by modulation of the isoprenoid precursor pool with expression of deoxyxylulose phosphate synthase , 2000, Applied Microbiology and Biotechnology.

[5]  Jeffrey H. Miller,et al.  A short course in bacterial genetics , 1992 .

[6]  B. Wanner,et al.  One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Yaoguang Liu,et al.  Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. , 1995, Genomics.

[8]  D. Botstein,et al.  Cluster analysis and display of genome-wide expression patterns. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[9]  C. Schmidt-Dannert,et al.  Metabolic engineering towards biotechnological production of carotenoids in microorganisms , 2002, Applied Microbiology and Biotechnology.

[10]  Monica Riley,et al.  A functional update of the Escherichia coli K-12 genome , 2001, Genome Biology.

[11]  G. Sandmann,et al.  Identification of carotenoids in Erwinia herbicola and in a transformed Escherichia coli strain. , 1990, FEMS microbiology letters.

[12]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[13]  F X Cunningham,et al.  Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC7942. , 1994, The Plant cell.

[14]  D. Hannaway,et al.  Nodulation of Lupinus albus and Vicia faba by Rhizobium spp. as influenced by Agrobacterium rhizogenes , 1989 .

[15]  N. Misawa,et al.  Metabolic engineering for the production of carotenoids in non-carotenogenic bacteria and yeasts. , 1998, Journal of biotechnology.

[16]  W. R. Farmer,et al.  Improving lycopene production in Escherichia coli by engineering metabolic control , 2000, Nature Biotechnology.

[17]  J. Keasling,et al.  Metabolic engineering of the nonmevalonate isopentenyl diphosphate synthesis pathway in Escherichia coli enhances lycopene production. , 2001, Biotechnology and bioengineering.

[18]  J. Shendure,et al.  Selection analyses of insertional mutants using subgenic-resolution arrays , 2001, Nature Biotechnology.

[19]  M. Becker-Hapak,et al.  RpoS dependent overexpression of carotenoids from Erwinia herbicola in OXYR deficient Escherichia coli. , 1997, Biochemical and biophysical research communications.

[20]  Gregory Stephanopoulos,et al.  Optimizing bioconversion pathways through systems analysis and metabolic engineering , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[21]  W. Eisenreich,et al.  Biosynthesis of terpenes: Studies on 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[22]  Gregory Stephanopoulos,et al.  Engineering metabolism and product formation in Corynebacterium glutamicum by coordinated gene overexpression. , 2003, Metabolic Engineering.

[23]  J. Keasling,et al.  Controlling the metabolic flux through the carotenoid pathway using directed mRNA processing and stabilization. , 2001, Metabolic engineering.

[24]  Jens Nielsen,et al.  Increasing galactose consumption by Saccharomyces cerevisiae through metabolic engineering of the GAL gene regulatory network , 2000, Nature Biotechnology.