Industrialization of Biology A Roadmap to Accelerate the Advanced Manufacturing of Chemicals (2015) / Chapter Skim
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4 How Do We Get There?
4 How Do We Get There?
Pages 67-100
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From page 67... ...
In order to facilitate biomanufacturing for chemical production, a series of conclusions and roadmap goals are presented and discussed in this chapter. The discussion is organized into three broad categories: feedstocks, enabling transformations, and integrated design toolchain.
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From page 68... ...
In the case of large-volume chemicals, sugar costs can represent the majority of the total product costs. In the extreme case of biofuels, sugar costs represent as much as 65 percent of the total product costs.96 By contrast, for industrial enzyme and specialty chemical production, the overall cost of the carbon source is a small fraction of the total costs.
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From page 69... ...
For the biological production of chemicals to reach its full potential, more abundant, more diverse, and less costly sources of carbon are needed. Cellulosic material derived from agricultural residues, forestry by-products, and even dedicated energy crops are both abundant and diverse.
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From page 70... ...
FIGURE 4-1 The quantity and sources of biofuels mandated by the 2007 Renewable Fuel Standard.
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From page 71... ...
Alternative sources of carbon are needed to realize our full ambitions for the biological production of chemicals. Lignocellulosic Biomass Agricultural residues will be the first source of cellulose used in the biological production of chemicals.
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From page 72... ...
Modifications to the level of lignin and the nature of the hemicellulose content will lead to less recalcitrant biomass, yielding more fermentable sugars per ton, and further reducing the cost of usable cellulosic sugars. Perennial grasses may also be adapted to cultivation on marginal land not currently used for row crops.
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From page 73... ...
Additional process engineering and host organism research are needed to expand the economic viability of C1 feedstocks for the biological production of chemicals. Additional advances in C1s are coming from ICI, INEOS Bio, LanzaTech, and Newlight Technologies.
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From page 74... ...
For such products, both feedstock costs and capital costs are critical considerations. Hence, both product costs and capital costs must be reduced for industrial biology to compete effectively with conventional petrochemical processing.
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From page 75... ...
Such improvement can only come from more productive host organisms, combined with improvements in process engineering. A bioprocessing facility for chemical production consists of a series of operations.
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From page 76... ...
Space-time yield remains low because of constraints of the microorganism and temperature and shear limitations. Historically, host organisms have been selected and engineered to optimize productivity in terms of production rate, fermentation titer, and product yield (per unit feedstock)
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From page 77... ...
This must be done in tandem with the development of host organisms built to this purpose. The ability to build predictive models at the level of individual metabolic pathways, at the level of whole-cell metabolism, and at the level of the overall fermenter operation is a significant need.
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From page 78... ...
Typical commercial uses include a broad range of alcohols, amines, amino acids, and organic acids. Enzyme-mediated reactions can be carried out at high yield.
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From page 79... ...
Key Conclusions Conclusion: Aerobic, fed-batch, monoculture fermentation has been the dominant process for bioproduction of chemicals for many decades. Successful improvement efforts have focused on more pro ductive host organisms.
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From page 80... ...
• Within 10 years, for all bio-based aqueous processes, achieve 95 percent reuse of process water. Organism The core of an expanded industry emerging from the accelerated biological production of chemicals will consist of specialized organisms capable of producing a given compound at titers, productivities, and yields sufficient for economical production.
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From page 81... ...
As proof of concept is established for biological production and the model needs expand to consider the full integration of process design and development, additional specifications may include (5) the quality specification of the finished product (e.g., purity)
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From page 82... ...
Fully Integrated Design Toolchain Across each of the levels of resolution described at the outset, we note a common gap between the scientific design tools available today and the engineering design tools needed to achieve the envisioned future presented in this report. To date, most tools used in organism design are what are colloquially referred to as "pull" tools.
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From page 83... ...
More sophisticated push tools may even be able to prioritize the results based on both estimated confidence in each prediction as well as the likelihood that each result might adversely impact organism performance. Push tools free the biological engineer from needing to query each design against a library of tools and instead rely on software to point out all potential issues in a proposed design.
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From page 84... ...
,106 into the service of synthetic biology. Repositories of information concerning organisms, DNA sequences, and expression systems have begun to emerge, including the iGEM Registry of Standard Biological Parts,107 the ICE repository platform,108 the Virtual Parts Repository,109 the DNASU plasmid repository,110 and AddGene.111 The first three of these specific repositories have established APIs for design tools to access their contents, and efforts are under way to develop a standardized API across these repositories to enable a united "Web of Registries." While these efforts demonstrate that some progress has been and is being made toward the establishment of the standardized APIs, data-exchange standards, and standardized data repositories that will be required to enable a fully integrated design toolchain, it is clear that much work remains (in particular around establishing repositories of experimental measurement and characterization data)
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From page 85... ...
Roadmap Goals • Within 4 years, develop and demonstrate an integrated design toolchain for the design of a biomanufacturing process at and below the level of an individual organism (i.e., everything inside the cell)
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From page 86... ...
Design tools that improve the accuracy of functional prediction -- and, ultimately, the ability to predict not just whether an enzyme will be active but how active it will be -- can greatly accelerate the initial steps of establishing proof of concept for biosynthesis. The integration of pathway design and enzyme specification tools, resulting in exquisite computational tools that can reliably present a feasible de novo pathway toward a target compound, would herald a revolution in industrial iology as these tools would immediately b and dramatically expand the scope of chemical compounds that would be candidates for biomanufacturing.
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From page 87... ...
The fully integrated design toolchain should be able to satisfy these layered objectives, accounting for endogenous metabolism, heterologous product formation, and redox and energy balances to predict the optimal combination of genetic manipulations. To this end, it would be desirable to also have registries containing the characteristics of hundreds of host organisms and their phenotypes under a wide range of conditions, such as different temperatures, pressures, salinities, and carbon sources.
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From page 88... ...
These performance specifications can then be translated into well-established parameters, including, for example, observable product yield on substrate, product yield on biomass, and specific productivity, that have been successfully used for decades to model and design bioprocesses. As cellular behavior is more complex, for example, exhibiting dynamic behaviors through the incorporation of feedback control mechanisms, these behaviors can be modeled at the bioreactor scale to predict overall process performance, ultimately generating the predictions in titer, yield, and productivity that are necessary to evaluate the commercial viability of a process.
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From page 89... ...
There have been several enabling improvements in protein design tools, most notably the widespread use of the Rosetta suite. In concert with the improvements in DNA synthesis that have been noted elsewhere, this has meant that it is frequently possible to redesign a given protein scaffold for novel structure, synthesize tens to hundreds of predicted variants, and quickly assay for those that have the required capabilities.
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From page 90... ...
Between improvements in computational design and directed evolution, the prospect exists for taking a relatively small list of parts and endlessly morphing their function to suit the needs of industry. This in turn suggests that there will likely be productive niches within the corporate ecosystem devoted to parts improvement.
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From page 91... ...
cerevisiae, and other model organisms are so highly used is the extensive repertoire of genetic tools available for these hosts. As a result, the correlation between genomic, proteomic, metabolic, and other information is relatively complete and is already laid down into systems biology models that are increasingly being quantified (as apparent from the Design Toolchain described above)
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From page 92... ...
Indeed, this may be an area where synthetic biology can provide modules that go well beyond regulation or metabolism. Into the future, it should be possible to take a toolbox of standardized and orthogonal origins, polymerases, promoters, ribosomes, and encoded amino acid biosynthetic and charging capacities and create made-to-order episomes for any of a variety of industrially relevant bacteria.
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From page 93... ...
Paradoxically, before a chassis is fixed into an evolutionarily stable trajectory, directed evolution methods applicable to whole organisms will be of increasing importance. As systems biology approaches provide increasingly excellent "roadmaps" for metabolic and regulatory engineering in a wide variety of organisms, it should be possible to delimit what pathways, loci, or regulatory networks should be the focus of directed evolution.
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From page 94... ...
Conclusion: Expanding the palette of domesticated microbial and cell-free platforms for biomanufacturing is critical to expanding the repertoire of feedstocks and chemicals accessible via bio-based manufacturing. Conclusion: The design, creation, and cultivation of robust strains that remain genetically stable and retain performance stability over time in the presence of diverse feedstocks and products will reduce the costs involved in the use and scaling of biological production.
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From page 95... ...
Design and evolution can provide basal circuitry that frequently requires additional optimization. Improvements in design tools can reduce the number of circuits that need to be tested and can improve the overall quality of those circuits, while facile directed evolution methods allow an ever larger number of variants to be screened and selected for improved function.
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From page 96... ...
makes it an ideal technology for many different types of measurements, beyond just sequencing genomes, constructs, and RNA expression levels. To the extent that protein and other analytes can be transduced into nucleic acids it may be possible to deconvolute extremely complex mixtures using NGS.
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From page 97... ...
At the same time broadly useful measurement platforms are extended for use by biological engineers, synthetic biology is enabling and introducing new measurement paradigms particularly well suited for the needs of engineering biology. Circuits can be easily linked to readily observed reporters, such as green fluorescent protein, and high-throughput devices and methods that have already been developed, such as plate readers or fluorescence-activated cell sorting, can be used to parse performance.
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From page 98... ...
Engineering biology brings a perspective of altering organisms for utilitarian purposes including the development of bioensor measure s ment devices at both the molecular and the organism levels for readout, feedback, and control. In parallel, as circuits become increasingly complex there will be a need to increase the number of different parameters that can be measured in parallel, such as the expression of multiple genes or the production of multiple metabolites.
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From page 99... ...
Conclusion: The fall in cost and increase in throughput of measure ment technologies should track that of strain engineering technolo gies and vice versa. Roadmap Goals • Within 4 years, develop the ability to routinely and reproduc ibly measure nucleic acids, proteins, and metabolites targeted to characterize 50 or more high-priority, selected model parameters for 2,000 strains and measure 1,000 or more parameters for 200 strains within 1 week at a cost no higher than the full cost of designing and building those strains.
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