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news and views switch NWASP, the SH3 domain from the adaptor protein CRK and the PDZ domain from the adaptor protein syntrophin. Three metabolic enzymes for mevalonate production—E. coli AtoB and Saccharomyces cerevisiae HMGS and HMGR—were targeted to the scaffold using specific ligands corresponding to each docking domain, a strategy reminiscent of the modular domain framework recently used to build ultrasensitive signaling switches7. The advantage of this strategy for enzyme assembly is that it requires only small modifications to the targeted enzymes without the need for structural information, akin to the addition of fusion tags for affinity purification. By noncovalently tethering the enzymes to the synthetic scaffold, Dueber et al.1 showed that mevalonate yields could be increased by as much as 77-fold compared with the unscaffolded pathway, depending on both the number of interaction domain repeats and the domain orientation in the scaffold architecture. This scaffold design represents a considerable advance over the state of the art2, as it permits various enzyme ratios and geometries for systems of two or more enzymes. The ability to configure multiple enzymes both spatially and stoichiometrically is important because it enables balancing of reaction rates in the context of an entire reaction pathway. The exciting observation that the same programmable scaffolds enhanced the production of a structurally unrelated compound, glucaric acid, indicates that this approach might be extended to other metabolic pathways. However, several important issues should be considered when developing synthetic scaffolds for other systems. First, the fact that many metabolic enzymes are multimeric may present an assembly conundrum if enzyme subunit assembly is inhibited by scaffolding, or vice versa8. Similarly, the ability to tag enzymes with ligands without perturbing function is protein dependent, and for some enzymes, it may be necessary to perform more exhaustive searches to identify permissive sites for peptide insertions. Further refinement of scaffold-ligand interactions may also be needed to ensure that the scaffolds assemble more ‘cleanly’ and to prevent the wasteful accumulation of a large pool of scaffold-free enzymes or enzyme-free scaffolds. It should be noted that the concept of metabolic channeling is not without controversy, as direct measurement of this effect can be plagued by experimental artifacts9–11. Dueber et al.1 speculate that the scaffolded enzymes improve pathway efficiency by increasing the effective concentrations of intermediary metabolites. However, there are other possible mechanisms that could contribute to the increased mevalonate yields. These include
improved enzyme folding, reduced enzyme aggregation or degradation, or a combination of these effects. Finally, even if the channeling effect is real, it may be effective only in highly engineered systems where the enzyme fluxes are carefully balanced—as suggested by the finding that the approach was less efficacious for producing glucaric acid than mevalonate1. Regardless, the use of synthetic enzyme scaffolds clearly improves metabolic performance and provides a powerful new method that can be used alongside conventional methods such as modulation of enzyme expression levels by, for instance, tuning intergenic regions12 or evolution of rate-limiting enzymes13. In the long term, the possibility of integrating several of these strategies simultaneously to balance pathway flux, eliminate metabolic bottlenecks, reduce cell stress and prevent accumulation of unwanted intermediates or by-products is limited only by one’s imagination and should clear
the way to produce commercially viable levels of diverse metabolic products such as biofuels, specialty chemicals and therapeutics. 1. Dueber, J.E. et al. Nat. Biotechnol. 27, 753–759 (2009). 2. Conrado, R.J., Varner, J.D. & DeLisa, M.P. Curr. Opin. Biotechnol. 19, 492–499 (2008). 3. Hyde, C.C. et al. J. Biol. Chem. 263, 17857–17871 (1988). 4. An, S., Kumar, R., Sheets, E.D. & Benkovic, S.J. Science 320, 103–106 (2008). 5. Narayanaswamy, R. et al. Proc. Natl. Acad. Sci. USA 106, 10147–10152 (2009). 6. Meynial Salles, I. et al. Metab. Eng. 9, 152–159 (2007). 7. Dueber, J.E., Mirsky, E.A. & Lim, W.A. Nat. Biotechnol. 25, 660–662 (2007). 8. Ljungcrantz, P. et al. Biochemistry 28, 8786–8792 (1989). 9. Cornish-Bowden, A. & Cardenas, M.L. Eur. J. Biochem. 213, 87-92 (1993). 10. Petersson, G. J. Theor. Biol. 152, 65-69 (1991). 11. Wu, X.M., Gutfreund, H., Lakatos, S. & Chock, P.B. Proc. Natl. Acad. Sci. USA 88, 497-501 (1991). 12. Pfleger, B.F., Pitera, D.J., Smolke, C.D. & Keasling, J.D. Nat. Biotechnol. 24, 1027–1032 (2006). 13. Bloom, J.D. & Arnold, F.H. Proc. Natl. Acad. Sci. USA 106, 9995–10000 (2009).
Metabolic engineering without plasmids Aashiq H Kachroo, Makkuni Jayaram & Paul A Rowley Tandem gene duplication is harnessed to genetically engineer Escherichia coli, enabling sustained expression of metabolic products. Despite its many successes, metabolic engineering of bacteria remains a work in progress1,2. In this issue, Tyo et al.3 present a clever solution to a major problem in the field: the gradual decline in productivity of cells that express recombinant enzymes from plasmids. Their approach, called chemically inducible chromosomal evolution (CIChE), is shown to enable roughly two- to fourfold increases in the yields of two useful biochemical products, the carotenoid lycopene and the polymer poly-3-hydroxybutyrate (PHB). The prospect of long-term expression of metabolic pathways without the requirement of marker selection has wide-ranging potential applications in metabolic engineering, synthetic biology and industrial biotechnology. Conventional metabolic engineering involves assembling a desired biochemical pathway in a plasmid vector that can autonomously replicate and attain a relatively high copy number when introduced into a host bacterial strain, most Aashiq H. Kachroo, Makkuni Jayaram & Paul A. Rowley are at the University of Texas at Austin, Austin, Texas, USA. e-mail:
[email protected]
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often E. coli K12. However, the metabolic burden shouldered by a plasmid-bearing producer cell puts it at a selective disadvantage relative to a plasmid-free nonproducer. Thus, if cells without the plasmid arise owing to missegregation events, their progeny would eventually dominate the population. In principle, this is not a serious problem, as plasmids can be maintained using antibiotic selection. In practice, however, the situation is more complicated because of the peculiarities of plasmid physiology related to replication and segregation. Two different plasmids that cohabit a host cell will exhibit ‘mutual incompatibility’ if they compete with each other for factors that promote replication or segregation4 (Fig. 1a). Incompatibility can be observed in the absence of external selection pressure, such as from antibiotics, that maintains both plasmids in the cell. Successive cell divisions will eventually give rise to distinct cell populations in which the plasmids have been separated from each other. The degree of incompatibility and the speed with which it occurs depends on the plasmid copy number as well as the mechanisms governing plasmid replication and segregation.
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recA Single-copy genomic integration
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Figure 1 CIChE addresses the productivity loss caused by allele segregation. (a) Plasmid incompatibility and allele segregation. Events leading to plasmid incompatibility and allele segregation are illustrated for a progenitor bacterial cell containing a productive plasmid (green) at a copy number of two. A spontaneous mutation generates a nonproductive plasmid (red) that may compete with its productive counterpart during replication and segregation. As a result, nonproductive cells (harboring only the red plasmid) arise in the population. The sequence of events leading to ‘allele segregation’ is of particular concern to Tyo et al.3 (b) CIChE overcomes allele segregation. Tyo et al.3 drive recA-mediated tandem gene duplication of a recombinant pathway by culturing cells in increasing concentrations of an antibiotic. Deleting recA prevents loss of the recombinant genes in the absence of antibiotic. Blue, recA. Black, recA recombination sites. Green, recombinant pathway and antibiotic-resistance gene.
For a fixed steady-state copy number of the plasmid, incompatibility can arise if ‘random replication’ occurs—that is, if the machinery for plasmid replication randomly chooses which plasmid to use as a template. As a result, a single cell may contain unequal numbers of the two plasmid types before plasmid segregation and cell division (Fig. 1a). Alternatively, if each plasmid molecule is duplicated once—so-called ‘ordered replication’—the two plasmid types are maintained at equal numbers. Regardless, in both cases the effect of incompatibility takes hold when cells containing only mutant plasmids are generated by random segregation of the plasmids between the two daughter cells. In a cell that receives more or fewer plasmid molecules than normal due to unequal segregation, the steady-state copy number can be restored by under- or overreplication, as required. For a homogenous starting population of plasmids, the events leading to incompatibility are set into motion by spontaneous mutation. Tyo et al.3 investigate a scenario that is particularly relevant to metabolic engineering: mutations that impair the engineered metabolic pathway (denoted by the change in plasmid color from green to red in Fig. 1a). In this situation, the desired output from the population falls off quickly once nonproducer cells are formed by incompatibility and begin to multiply faster than the producers. The authors quantify this effect for a particular case called ‘allele segregation’, which results
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from ordered plasmid replication followed by equal but random segregation (Fig. 1a). They calculate that for a plasmid copy number of 40 and a mutation frequency that introduces one nonproducer plasmid in ~10 generations, allele segregation leads to roughly 80% loss of active plasmids in ~40 generations—a result borne out by experimental observation that product yield drops dramatically within this period. By contrast, if alleles cannot segregate (that is, if replication and segregation are ‘ordered’ as is the case for genes on the chromosome), spontaneous mutation alone would be expected to take >500 generations to abolish productivity. To avoid the problems of allele segregation, Tyo et al.3 therefore abandon plasmids and turn to modification of the E. coli chromosome itself using CIChE. In the first step of CIChE, the E. coli chromosome is modified by inserting a DNA construct containing recA homologous recombination sites that flank a recombinant-pathway expression cassette and an antibiotic-resistance marker. This strain is then cultured in increasing concentrations of the antibiotic. Such conditions select for bacteria that contain higher copy numbers of the DNA construct, which are generated by recA-mediated tandem gene duplication (Fig. 1b). Once the construct is present at sufficiently high copy number, the amplified pathway is permanently cemented into the chromosome by deleting recA, preventing loss of any copies of the pathway by recombination when antibiotic selection is discontinued. Such selection
marker–free culture conditions are often highly desirable, as the markers may not be practical in conditions required for optimal industrial production. Tyo et al.3 demonstrate that CIChEengineered cells sustain production levels for >70 generations compared with 20 generations or less without CIChE. The yields of lycopene and PHB are increased 1.6- and 4=fold, respectively, over those from corresponding plasmidbased systems. Chromosomal integration accounts for the sustained production by CIChE-engineered cells. Even though spontaneous mutations may inactivate genes located on the chromosome or on plasmids at the same frequency, mutated genes on the plasmid can be separated from normal genes by allele segregation, but such separation is prevented if mutant and normal genes are located on the same chromosome. It is this critical difference that delays the spread of nonproductive cells in the chromosomally engineered strain. In principle, a logical alternative to the CIChE approach would be to house the recombinant genes in a unit copy plasmid, such as the E. coli F episome, that segregates equally to daughter cells with the help of an active partitioning system5,6. Low-copy bacterial plasmids (such as P1 and R1) that also encode active partitioning systems would seem potentially useful as well. Such plasmids replicate, on average, once per cell cycle. Even so, it is known that such active systems do not always replicate every plasmid mol-
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news and views ecule exactly once, and they are not completely unbiased in their partitioning of the resulting sister plasmids. Hence it is possible that allele segregation would still occur if these alternative plasmids were used, and owing to their low copy numbers, these plasmids may not be able to express the recombinant pathway at sufficiently high levels. Moreover, the introduction of a large block of amplified foreign DNA containing multiple copies of a metabolic pathway might also be deleterious to plasmid replication and/or maintenance. Nevertheless, the option of expressing recombinant pathways in an extrachromosomal context could still be valuable in certain cases, provided the disadvantages of allele segregation can be overcome. This would require a multi-copy plasmid system in which every molecule replicates once during a cell cycle. Additionally, sister plasmids resulting from each replication event must be segregated equally, one each to the two daughter cells.
Bacteria appear to be devoid of such a system. However, the 2-µm plasmid of Saccharomyces cerevisiae, present in the nucleus at roughly 60 copies per cell, satisfies these demands7,8. This plasmid could be potentially useful in the design and construction of yeast-based biofactories with sustained productivity. But for now, the most effective safeguard against the effects of allele segregation in bacterial systems appears to be CIChE. 1. Keasling, J.D. Trends Biotechnol. 17, 452–460 (1999). 2. Keasling, J.D. ACS Chem. Biol. 3, 64–76 (2008). 3. Tyo, K.E.J. et al. Nat. Biotechnol. 27, 760–765 (2009) 4. Novick, R.P. Microbiol. Rev. 51, 381–395 (1987). 5. Funnell, B.E. & Slacev, R.A. Partition systems of bacterial plasmids, in Plasmid Biology (ed. Funnell, B.E. & Phillips, G.J.) 81–103 (ASM Press, Washington, DC, 2004). 6. Ebersbach, G. & Gerdes, K. Annu. Rev. Genet. 39, 453– 479 (2005). 7. Ghosh, S.K., Hajra, S., Paek, A. & Jayaram, M. Annu. Rev. Biochem. 75, 211–241 (2006). 8. Ghosh, S.K., Hajra, S. & Jayaram, M. Proc. Natl. Acad. Sci. USA 104, 13034–13039 (2007).
Universal cell-free protein synthesis James R Swartz Unstructured translation-initiation sequences enable protein synthesis in cell extracts from multiple organisms.
initiation is often inefficient, again because of secondary and tertiary mRNA structure. Mureev et al.1 address this hurdle with a remarkable new development. Taking their cue from the upstream sequences of late poxvirus genes5, they reason that 5′ untranslated regions enriched with poly(A) sequences could bypass the need for early translation-initiation steps. They also insert weakly stable hairpin structures after the initiating AUG codon to facilitate AUG recognition by the small ribosomal subunit and to provide more time for recruitment of the large subunit (Fig. 1). This approach proves to be very effective in activating protein production using cell extracts from L. tarantolae, a eukaryotic organism amenable to large-scale, inexpensive extract production. The new sequences enable much higher protein yields than the 5′ omega6 and 5′ obelin7 sequences previously optimized for cell-free expression. Most importantly, the strategy is generally effective for cell extracts from yeast, wheat germ, insect cells, rabbit reticulocytes and even E. coli—leading the authors to name the new sequences “species-independent translational sequences” (SITS). Using SITS-activated genes, Mureev et al.1 further develop the L. tarantolae cell-free system by using upstream interfering RNAs to block the production of native proteins other than the desired product. They also establish combined transcription and translation and demonstrate useful expression levels using Promoter
The exponential increase in the number of sequenced genomes has focused attention on how best to produce and study the encoded gene products. Cell-free protein synthesis offers the promise of rapid, multiplexed production of these proteins, but it often takes years of work to develop such systems for a new organism. As described in this issue by Mureev et al.1, this impediment has now been overcome with an apparently generic approach to cell-free protein synthesis. Using optimized translational leaders and rapid preparation procedures, the authors achieve effective protein synthesis in cell extracts from the protozoan Leishmania tarentolae as well as from various eukaryotes and prokaryotes. Cell-free protein synthesis is not a new technique—in fact, it was used to help decipher the genetic code2. After decades of development, we now have commercially available systems based on Escherichia coli, wheat germ, rabbit reticulocyte and insect cell extracts, as James R. Swartz is in the Department of Chemical Engineering and the Department of Bioengineering, Stanford University, California, USA. e-mail:
[email protected]
well as a system using purified components3. In addition, although cell extracts are usually considerably diluted relative to intracellular concentrations, even complex aspects of ancillary metabolism, such as oxidative phosphorylation4, can be activated in cell-free reactions. This underscores the additional potential of these systems for studying intracellular metabolism and regulation as well as protein synthesis and modification. The open nature of the systems also enables precise manipulation of substrate and catalyst concentrations as well as rapid sampling. A cell-free system using the native expression apparatus (i.e., cell extract) is most likely to ensure that the protein products of newly sequenced genomes possess their authentic activities and post-translational modifications. Initiating translation is often the major barrier in activating a new cell-free protein synthesis system. For prokaryotes, the lower concentrations of initiating factors in the diluted extract are ineffective if the mRNA is blocked by mRNA secondary or tertiary structure. For eukaryotes, the native mRNA is usually modified by capping and polyadenylation before it can be recognized for translation, and translation
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SITS
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Folded protein Figure 1 Tagging a gene of interest with a species-independent translation sequence (SITS) enables cell-free synthesis of the gene product. The method works with cell extracts of the protozoan Leishmania tarantolae, of the bacterium Escherichia coli and of four eukaryotes, suggesting that it is applicable for a broad variety of organisms. Potential applications of this cellfree protein synthesis approach include genomewide expression of proteins in newly sequenced organisms.
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