ne w s and vie w s approach to combine molecularly defined antigens with new adjuvants to create fully functional vaccine delivery systems. Certainly, the challenges associated with characterizing and formulating complex and heterogeneous particles make it likely that successful implementation of synthetic nano- to microsized delivery platforms may face regulatory hurdles going forward. On the other hand, adjuvants that assemble in vivo, such as the thermoresponsive polymers described in this report, already suggest a possible path toward translation.
COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Lynn, G.M. et al. Nat. Biotechnol. 33, 1201–1210 (2015). 2. Steinhagen, F., Kinjo, T., Bode, C. & Klinman, D.M. Vaccine 29, 3341–3355 (2011). 3. Vasilakos, J.P. & Tomai, M.A. Expert Rev. Vaccines 12, 809–819 (2013). 4. Kawai, T. & Akira, S. Immunity 34, 637–650 (2011). 5. Lizée, G. et al. Annu. Rev. Med. 64, 71–90 (2013). 6. Wu, T.Y. et al. Sci. Transl. Med. 6, 263ra160 (2014). 7. Hanson, M.C. et al. J. Clin. Invest. 125, 2532–2546 (2015). 8. Kopecˇek, J. & Kopecˇková, P. Adv. Drug Deliv. Rev. 62, 122–149 (2010). 9. Park, H. et al. J. Immunol. 190, 4103–4115 (2013).
Biological synthesis unbounded?
npg
© 2015 Nature America, Inc. All rights reserved.
Brian F Pfleger & Kristala L J Prather Chemical synthesis of specialty and commodity products still reigns supreme but greener, metabolic engineering approaches are gaining ground. Since the emergence of metabolic engineering as a codified discipline some 25 years ago, engineered microbial systems have increasingly been touted as a viable means for the replacement of traditional manufacturing in the synthesis of certain chemicals. With fermentation-derived 1,3-propanediol replacing the chemically synthesized product in 2006 and yeast-derived artemisinin displacing the product extracted from plants in 2013, and with several other products now in advanced stages of commercialization, evidence is mounting that biology can provide alternative paths to useful molecules. A recent report by Smolke and colleagues1 describing the synthesis of opiates from glucose in an engineered yeast represents another such breakthrough. So, have we now reached the point where we are ready to declare biology on par with chemistry for manufacturing? Not quite yet. Opiates are part of a class of natural products known as benzylisoquinoline alkaloids (BIAs). The work by Smolke and colleagues1 to produce opiates from a simple sugar represents a major step forward for the construction of microbial cell factories. The first target for biological synthesis was thebaine, a BIA that can be chemically or biologically converted to semisynthetic opiates of pharmacological importance including hydrocodone Brian F. Pfleger is at the University of Wisconsin-Madison, Madison, Wisconsin, USA, and Kristala L.J. Prather is in the Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. e-mail: [email protected] or [email protected]
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and oxycodone. Using glucose as a feedstock, it was necessary to heterologously express or improve endogenous expression levels of more than 20 individual genes to engineer the pathway. Perhaps unsurprisingly, simply introducing all of the genes into Saccharomyces cerevisiae did not result in production of thebaine. Rather, the authors used a modular strategy, in which the full pathway is segmented into parts so that precursors can be supplied from the culture medium and/or intermediates can be produced and measured, in order to identify and overcome bottlenecks in the pathway. For example, when producing the pathway intermediate (S)-reticuline the authors observed accumulation of two intermediates, dopamine and 3′-hydroxyN-methylcoclaurine. This led to the decision to introduce additional copies of three genes in the pathway, resulting in a fourfold increase in S-reticuline and a twofold decrease in 3′-hydroxy-N-methylcoclaurine. Ultimately, production of BIAs from glucose did not come as a surprise; indeed, it was predicted to occur in the near future once the separate components of the pathway had been identified from nature and were functionally verified2–4, even though commercialization was expected to still be some time away5. Although production of opiates from glucose in yeast is a landmark achievement, the low product titers (6.4 and 0.3 µg/L for thebaine and hydrocodone, respectively) need to be improved 1,000,000-fold or more to enable commercial production. Increasing titers and yields to enable commercial production of chemicals in microorganisms is a
widespread problem that must be tackled if the metabolic engineering field is to fulfill its potential. Engineering a microorganism to produce chemicals requires the following: first, expression of the various enzymes needed to catalyze each reaction step; second, sufficient activities of those enzymes to meet productivity targets; and third, adequate metabolite pools to support the desired metabolic fluxes. Although this might seem straightforward, finding enzymes with the requisite activities—and specificities—is often a limiting factor, especially when designing pathways not found in nature6. Even when engineering a natural product pathway, key enzymatic steps may need to be characterized, as was demonstrated by Smolke and colleagues1 for the isomerization of (S)-reticuline to (R)-reticuline. Although a treasure trove of enzymatic activities are available in nature, identifying the best candidate enzyme, with specific and high activity, and then achieving functional expression in different species can be challenging. A rapidly growing number of genes that encode enzymes have been identified through automated genome annotation, but functional validation and confirmation of putative activities have lagged behind. Once a suitable candidate enzyme has been identified, specificity and activity may be altered by rational design or directed evolution. Rational design requires structural information but can result in a small number of variants that need to be screened; on the other hand, directed evolution can identify properties not easily predicted from structure but requires screening methods that can match the large library sizes generated (Fig. 1). Metabolite pool sizes can be modulated by enzymes in a multistep pathway, by unrelated enzymes that affect overall fluxes and by substrate transport. Balancing metabolic fluxes, by altering both absolute and relative levels of pathway enzymes, increasing or decreasing endogenous enzymes, and up- or downregulating transporters, is crucial for achieving highlevel production of target molecules. Advances in DNA synthesis mean that we can now construct any DNA sequence. We are no longer restricted to sequences that we can clone from source material. Furthermore, we can apply synthetic biology methods to assemble diverse libraries of genes and regulatory elements in a combinatorial way. This approach has dramatically increased the sequence space (upwards of 1010 sequences per library) that can be searched for optimal DNA constructs encoding the best enzyme, the optimal regulatory strategy or novel enzyme activity. However, screening capacity in general lags
volume 33 number 11 November 2015 nature biotechnology
ne w s and vie w s Screening platform capacity Can you develop a growth-linked selection to detect your product? Yes No 108–1010 per day
Is the product easily detected (i.e., by fluorescent, spectroscopic techniques)?
Yes No 9
COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Lynn, G.M. et al. Nat. Biotechnol. 33, 1201–1210 (2015). 2. Steinhagen, F., Kinjo, T., Bode, C. & Klinman, D.M. Vaccine 29, 3341–3355 (2011). 3. Vasilakos, J.P. & Tomai, M.A. Expert Rev. Vaccines 12, 809–819 (2013). 4. Kawai, T. & Akira, S. Immunity 34, 637–650 (2011). 5. Lizée, G. et al. Annu. Rev. Med. 64, 71–90 (2013). 6. Wu, T.Y. et al. Sci. Transl. Med. 6, 263ra160 (2014). 7. Hanson, M.C. et al. J. Clin. Invest. 125, 2532–2546 (2015). 8. Kopecˇek, J. & Kopecˇková, P. Adv. Drug Deliv. Rev. 62, 122–149 (2010). 9. Park, H. et al. J. Immunol. 190, 4103–4115 (2013).
Biological synthesis unbounded?
npg
© 2015 Nature America, Inc. All rights reserved.
Brian F Pfleger & Kristala L J Prather Chemical synthesis of specialty and commodity products still reigns supreme but greener, metabolic engineering approaches are gaining ground. Since the emergence of metabolic engineering as a codified discipline some 25 years ago, engineered microbial systems have increasingly been touted as a viable means for the replacement of traditional manufacturing in the synthesis of certain chemicals. With fermentation-derived 1,3-propanediol replacing the chemically synthesized product in 2006 and yeast-derived artemisinin displacing the product extracted from plants in 2013, and with several other products now in advanced stages of commercialization, evidence is mounting that biology can provide alternative paths to useful molecules. A recent report by Smolke and colleagues1 describing the synthesis of opiates from glucose in an engineered yeast represents another such breakthrough. So, have we now reached the point where we are ready to declare biology on par with chemistry for manufacturing? Not quite yet. Opiates are part of a class of natural products known as benzylisoquinoline alkaloids (BIAs). The work by Smolke and colleagues1 to produce opiates from a simple sugar represents a major step forward for the construction of microbial cell factories. The first target for biological synthesis was thebaine, a BIA that can be chemically or biologically converted to semisynthetic opiates of pharmacological importance including hydrocodone Brian F. Pfleger is at the University of Wisconsin-Madison, Madison, Wisconsin, USA, and Kristala L.J. Prather is in the Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. e-mail: [email protected] or [email protected]
1148
and oxycodone. Using glucose as a feedstock, it was necessary to heterologously express or improve endogenous expression levels of more than 20 individual genes to engineer the pathway. Perhaps unsurprisingly, simply introducing all of the genes into Saccharomyces cerevisiae did not result in production of thebaine. Rather, the authors used a modular strategy, in which the full pathway is segmented into parts so that precursors can be supplied from the culture medium and/or intermediates can be produced and measured, in order to identify and overcome bottlenecks in the pathway. For example, when producing the pathway intermediate (S)-reticuline the authors observed accumulation of two intermediates, dopamine and 3′-hydroxyN-methylcoclaurine. This led to the decision to introduce additional copies of three genes in the pathway, resulting in a fourfold increase in S-reticuline and a twofold decrease in 3′-hydroxy-N-methylcoclaurine. Ultimately, production of BIAs from glucose did not come as a surprise; indeed, it was predicted to occur in the near future once the separate components of the pathway had been identified from nature and were functionally verified2–4, even though commercialization was expected to still be some time away5. Although production of opiates from glucose in yeast is a landmark achievement, the low product titers (6.4 and 0.3 µg/L for thebaine and hydrocodone, respectively) need to be improved 1,000,000-fold or more to enable commercial production. Increasing titers and yields to enable commercial production of chemicals in microorganisms is a
widespread problem that must be tackled if the metabolic engineering field is to fulfill its potential. Engineering a microorganism to produce chemicals requires the following: first, expression of the various enzymes needed to catalyze each reaction step; second, sufficient activities of those enzymes to meet productivity targets; and third, adequate metabolite pools to support the desired metabolic fluxes. Although this might seem straightforward, finding enzymes with the requisite activities—and specificities—is often a limiting factor, especially when designing pathways not found in nature6. Even when engineering a natural product pathway, key enzymatic steps may need to be characterized, as was demonstrated by Smolke and colleagues1 for the isomerization of (S)-reticuline to (R)-reticuline. Although a treasure trove of enzymatic activities are available in nature, identifying the best candidate enzyme, with specific and high activity, and then achieving functional expression in different species can be challenging. A rapidly growing number of genes that encode enzymes have been identified through automated genome annotation, but functional validation and confirmation of putative activities have lagged behind. Once a suitable candidate enzyme has been identified, specificity and activity may be altered by rational design or directed evolution. Rational design requires structural information but can result in a small number of variants that need to be screened; on the other hand, directed evolution can identify properties not easily predicted from structure but requires screening methods that can match the large library sizes generated (Fig. 1). Metabolite pool sizes can be modulated by enzymes in a multistep pathway, by unrelated enzymes that affect overall fluxes and by substrate transport. Balancing metabolic fluxes, by altering both absolute and relative levels of pathway enzymes, increasing or decreasing endogenous enzymes, and up- or downregulating transporters, is crucial for achieving highlevel production of target molecules. Advances in DNA synthesis mean that we can now construct any DNA sequence. We are no longer restricted to sequences that we can clone from source material. Furthermore, we can apply synthetic biology methods to assemble diverse libraries of genes and regulatory elements in a combinatorial way. This approach has dramatically increased the sequence space (upwards of 1010 sequences per library) that can be searched for optimal DNA constructs encoding the best enzyme, the optimal regulatory strategy or novel enzyme activity. However, screening capacity in general lags
volume 33 number 11 November 2015 nature biotechnology
ne w s and vie w s Screening platform capacity Can you develop a growth-linked selection to detect your product? Yes No 108–1010 per day
Is the product easily detected (i.e., by fluorescent, spectroscopic techniques)?
Yes No 9
