Spanning high-dimensional expression space using ribosome-binding site combinatorics

Protein levels are a dominant factor shaping natural and synthetic biological systems. Although proper functioning of metabolic pathways relies on precise control of enzyme levels, the experimental ability to balance the levels of many genes in parallel is a major outstanding challenge. Here, we introduce a rapid and modular method to span the expression space of several proteins in parallel. By combinatorially pairing genes with a compact set of ribosome-binding sites, we modulate protein abundance by several orders of magnitude. We demonstrate our strategy by using a synthetic operon containing fluorescent proteins to span a 3D color space. Using the same approach, we modulate a recombinant carotenoid biosynthesis pathway in Escherichia coli to reveal a diversity of phenotypes, each characterized by a distinct carotenoid accumulation profile. In a single combinatorial assembly, we achieve a yield of the industrially valuable compound astaxanthin 4-fold higher than previously reported. The methodology presented here provides an efficient tool for exploring a high-dimensional expression space to locate desirable phenotypes.

[1]  Sang Yup Lee,et al.  Construction and optimization of synthetic pathways in metabolic engineering. , 2010, Current opinion in microbiology.

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

[3]  J. McCarthy,et al.  The mechanism of translational coupling in Escherichia coli. Higher order structure in the atpHA mRNA acts as a conformational switch regulating the access of de novo initiating ribosomes. , 1994, The Journal of biological chemistry.

[4]  M. Pátek,et al.  Metabolic engineering of the L-valine biosynthesis pathway in Corynebacterium glutamicum using promoter activity modulation. , 2009, Journal of biotechnology.

[5]  T. Hwa,et al.  Interdependence of Cell Growth and Gene Expression: Origins and Consequences , 2010, Science.

[6]  M. Kimmel,et al.  Conflict of interest statement. None declared. , 2010 .

[7]  Drew Endy,et al.  Engineering BioBrick vectors from BioBrick parts , 2008, Journal of Biological Engineering.

[8]  M. Oh,et al.  Directed Evolution of Metabolically Engineered Escherichiacoli for Carotenoid Production , 2000, Biotechnology progress.

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

[10]  K. Lemuth,et al.  Engineering of a plasmid-free Escherichia coli strain for improved in vivo biosynthesis of astaxanthin , 2011, Microbial cell factories.

[11]  C. Glover,et al.  Gene expression profiling for hematopoietic cell culture , 2006 .

[12]  Jay D Keasling,et al.  Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli. , 2007, Metabolic engineering.

[13]  Nathan C Shaner,et al.  A guide to choosing fluorescent proteins , 2005, Nature Methods.

[14]  Joseph H. Davis,et al.  Design, construction and characterization of a set of insulated bacterial promoters , 2010, Nucleic acids research.

[15]  Brian F. Pfleger,et al.  Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes , 2006, Nature Biotechnology.

[16]  U. Alon,et al.  Optimality and evolutionary tuning of the expression level of a protein , 2005, Nature.

[17]  D. Schümperli,et al.  Translational coupling at an intercistronic boundary of the Escherichia coli galactose operon , 1982, Cell.

[18]  Christina D Smolke,et al.  A synthetic library of RNA control modules for predictable tuning of gene expression in yeast , 2011, Molecular systems biology.

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

[20]  M. Nomura,et al.  Localization of the target site for translational regulation of the L11 operon and direct evidence for translational coupling in Escherichia coli , 1983, Cell.

[21]  R. Popovic,et al.  Carotenogenesis up-regulation in Scenedesmus sp. using a targeted metabolomics approach by liquid chromatography-high-resolution mass spectrometry. , 2011, Journal of agricultural and food chemistry.

[22]  Farren J. Isaacs,et al.  Programming cells by multiplex genome engineering and accelerated evolution , 2009, Nature.

[23]  Roman Rydzanicz,et al.  Assembly PCR oligo maker: a tool for designing oligodeoxynucleotides for constructing long DNA molecules for RNA production , 2005, Nucleic Acids Res..

[24]  Robert T Sauer,et al.  Engineering controllable protein degradation. , 2006, Molecular cell.

[25]  B. Glick.  Metabolic load and heterologous gene expression. , 1995, Biotechnology advances.

[26]  S. Ho,et al.  Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. , 1989, Gene.

[27]  E. Marcotte,et al.  Absolute protein expression profiling estimates the relative contributions of transcriptional and translational regulation , 2007, Nature Biotechnology.

[28]  Xin-Guang Zhu,et al.  Optimizing the Distribution of Resources between Enzymes of Carbon Metabolism Can Dramatically Increase Photosynthetic Rate: A Numerical Simulation Using an Evolutionary Algorithm1[W][OA] , 2007, Plant Physiology.

[29]  References , 1971 .

[30]  P. R. Jensen,et al.  Synthetic promoter libraries--tuning of gene expression. , 2006, Trends in biotechnology.

[31]  Nikita Vladimirov,et al.  Role of Translational Coupling in Robustness of Bacterial Chemotaxis Pathway , 2009, PLoS biology.

[32]  E. Gantt,et al.  Elucidation of the Pathway to Astaxanthin in the Flowers of Adonis aestivalis[C][W] , 2011, Plant Cell.