Editorial pubs.acs.org/synthbio
Special Issue on Genome Engineering ngineering microbial cells for high-yield production of fine chemicals, feedstocks and fuels requires sophisticated molecular tools. The field of synthetic biology has enabled us to fine-tune production strains by rewriting and editing genomes. Advances in DNA sequencing and synthesis have streamlined our ability to identify genes of interest, create synthetic DNA constructs and perform genome-wide searches. However, the time required to implement genetic engineering strategies is limiting. One of the most successful commercial applications of metabolic engineering to date is the development of Sorona by Dupont. Sorona is a copolymer made from 1,3-propanediol produced by Escherichia coli fermentation and petroleum derived terephthalic acid. The development of the E. coli strain capable of converting corn starch into 1,3propanediol was a major research effort, requiring 12 years, 26 genetic manipulations and millions of dollars of investment. This highlights the need to develop genome engineering tools that allow not only the rapid identification of genotype/ phenotype relationships, especially for complex phenotypes, but also multiplexing the desired genetic mutations so that strain development time can be cut drastically. This special issue of ACS Synthetic Biology is focused on genome engineering tools and their application to engineering novel phenotypes into microbes. One of the most significant advances in engineering E. coli is the identification of phagebased recombineering techniques that have allowed much higher rates of recombination. Pines et al. is a comprehensive review of the use of bacteriophages to enable high rates of recombination in E. coli as well as multiplexing. Further optimization of phase-based recombineering has been achieved by identification of the mechanism by which it occurs, which is described in detail. The authors also discuss the challenges associated with phage-based recombineering and approaches that have (or may) lead to increased efficiency of this tool. Freed et al. report the construction of a genome-wide gene expression library that varies each gene over a 104 range. This tool is based on the previous genome-wide tool developed by the Gill Laboratory, TRMR.1 The original TRMR library contained two levels of expression: one under the control of a strong promoter (up regulated) and one in which translation was decreased by replacing the native ribosome binding site (RBS) with an inert one. The new tool, coined Tunable TRMR (or T2RMR), used an inducible promoter so that the expression level of the library could be controlled over a 104 range. The new library also includes dual RBS sequences to remove context-specific effects on expression. Using this method, a single researcher can screen the library of ∼20 000 mutants in less than 1 week, and the mutations are barcoded so they are easily identified postselection. T2RMR allows fast and efficient identification of genes that confer desired phenotypes, but the major advance in this method is the ability to easily fine-tune gene expression in the library, thus enabling the engineering of more balanced metabolic pathways. CRISPR/Cas, a prokaryotic immunity system, has recently received a lot of attention due to the ability of researchers to re-
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© 2015 American Chemical Society
engineer it to allow very efficient and highly specific engineering of genomes. In this issue, two new CRISPR/Cas based technology are described. Standage-Beier et al. show that nicking versions of Cas9 are nonlethal and can be used to specifically delete single genes all the way up to 97 kb of the genome. Mulitplexing this technique allows for deletion of up to 3% of the E. coli genome. Jakočiu̅nas et al. report the development of a multiplex and marker-free genome engineering tool (CasEMBLR) in Saccharomyces cerevisiae by combining in vivo DNA assembly with CRISPR/Cas9. The authors used CasEMBLR to engineer a yeast strain with increased aromatic amino acid production and report an efficiency of 58% without any selection, which is on par or better than current strain building tools for multilocus DNA integration. CasEMBLR is an invaluable tool for strain engineering of yeast and will further enable widespread use of this organism as chassis strain for biological production of fuels, fine chemicals and feedstocks. Photosynthetic microbes have received a lot of attention recently as potential platform strains for sustainable production of biofuels and bioproducts. Unfortunately, our ability to reengineer the genome is hampered by a lack of sophisticated molecular tools. Ramey et al. provides a valuable discussion of the current state of genome engineering tools for cyanobacteria and highlights the particular tools that need further development in order to allow advanced synthetic biology methods to be applied to these systems. A particular challenge of working with nonmodel organisms is that many of the genetic control elements that are available were developed and characterized in model organisms. The use of these systems in new hosts do not always result in predictable outcomes. Previous efforts to engineer the 2,3-butanediol pathway into Synechococcus elongatus PCC 7942 under the control of the PLlacO1promoter from E. coli reported expression of genes even in the absence of the inducer molecule, IPTG.2 In this issue, Nozzi and Atsumi characterize the PLlacO1 promoter from E. coli in Synechococcus using superfolder GFP as a reporter. They report that the promoter has similar ranges of induction compared to E. coli and tight repression without the addition of IPTG. Further investigation of single gene constructs allowed the authors to identify previously unknown hidden promoter upstream of the alsS and adh genes. This highlights the need to develop more standard parts that can easily be used in different organisms with similar outcomes; until that can be achieved, genetic constructs need to be fully characterized in their intended host strains to identify off target effects. One of the more challenging aspects of genome engineering is identifying clones with the desired genotype, especially if the mutations introduced do not lead to a distinct phenotype. Chen et al. developed a novel microscopy bar-coding method, called MiCodes, to enable high throughput identification of phenotypes. Each miCode is made up of a series of spectrally Special Issue: Genome Engineering Received: November 3, 2015 Published: November 20, 2015 1165
DOI: 10.1021/acssynbio.5b00220 ACS Synth. Biol. 2015, 4, 1165−1166
ACS Synthetic Biology
Editorial
distinct fluorescent proteins targeted to visually discernible organelles. Using just four fluorescent proteins targeted to 4 organelles allows up to 65 536 unique barcodes to be used to identify genotype/phenotype relationships. Quantifying metabolic fluxes is yet another way to identify phenotypic changes. Klesmith et al. developed a new method, FluxScan, which is capable of identifying point mutations that lead to improved flux through the desired enzymatic reaction by coupled it to growth. 8000 single point mutations were screened for improved flux through levoglucosan kinase; 15 beneficial mutations were combined, which resulted in 15-fold improvement in growth and 24-fold improvement in enzyme activity. This is an impressive result, especially because combining the beneficial mutations appear to be synergistic and not destructive. FluxScan is an incredibly useful tool for protein engineering of enzymes that can be linked to growth; however many bioproducts are fermentative, thus this tool should be extended by developing an easily identifiable selection for nongrowth associated products. Our ability to rewrite entire genomes has drastically increased in recent years. Technologies that allow fast and inexpensive DNA sequencing and synthesis have also opened the field to a much larger audience. This issue of ACS Synthetic Biology is just a small sampling of the exciting new tools being developed to aide our efforts to engineer microbes. This is an especially exciting time for metabolic engineers and synthetic biologist due to the wide applicability and specificity of CRISPR/Cas, which allows genome engineering to be performed in a much wider variety of organisms. At the rate which new, more sophisticated tools are being developed (and for a much more diverse set or systems) we will soon only be limited by our creativity and not by our technology.
Nanette Boyle
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Chemical And Biological Engineering, Colorado School of Mines, Golden, Colorado 80401, United States
AUTHOR INFORMATION
Notes
Views expressed in this editorial are those of the author and not necessarily the views of the ACS.
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REFERENCES
(1) Warner, J. R., Reeder, P. J., Karimpour-Fard, A., Woodruff, L. B. A., and Gill, R. T. (2010) Rapid profiling of a microbial genome using mixtures of barcoded oligonucleotides. Nat. Biotechnol. 28, 856−862. (2) Oliver, J. W. K., Machado, I. M. P., Yoneda, H., and Atsumi, S. (2013) Cyanobacterial conversion of carbon dioxide to 2,3-butanediol. Proc. Natl. Acad. Sci. U. S. A. 110, 1249.
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DOI: 10.1021/acssynbio.5b00220 ACS Synth. Biol. 2015, 4, 1165−1166
