Keasling recently was inducted into the National Academy of Engineering. He is also co-founder of Amyris Biotechnologies, a bioenergy startup which recently went public.

Metabolic engineering is the practice of altering genes and metabolic pathways within a cell or microorganism to increase its production of a specific substance. In the review in Science, Keasling notes that even with the substantial development of tools for metabolic engineering, and that metabolic engineers must weigh many trade-offs in the development of microbial catalysts:

1. cost and availability of starting materials (e.g., carbon substrates);
2. metabolic route and corresponding genes encoding the enzymes in the pathway to produce the desired product;
3. most appropriate microbial host;
4. robust and responsive genetic control system for the desired pathways and chosen host;
5. methods for debugging and debottlenecking the constructed pathway; and
6. ways to maximize yields, titers, and productivities.

Unfortunately, these design decisions cannot be made independently of each other: Genes cannot be expressed, nor will the resulting enzymes function, in every host; products or metabolic intermediates may be toxic to one host but not another host; different hosts have different levels of sophistication of genetic tools available; and processing conditions (e.g., growth, production, product separation and purification) are not compatible with all hosts. Even with these many challenges, metabolic engineering has been successful for many applications, and with continued developments more applications will be possible.

One area where metabolic engineering has a sizable advantage over synthetic organic chemistry is in the production of natural products, particularly active pharmaceutical ingredients (APIs), some of which are too complex to be chemically synthesized and yet have a value that justifies the cost of developing a genetically engineered microorganism. The cost of starting materials is generally a small fraction of their cost, and relatively little starting material is necessary so availability is not an issue. Most APIs fall into three classes of natural products, and many of the biosynthetic pathways for their precursors have been reconstituted in heterologous hosts.

—Jay Keasling

These three classes are: alkaloids; polyketides and nonribosomal peptides (NRPs); and isoprenoids. Keasling notes that bulk chemicals such as solvents and polymer precursors currently are rarely produced from microorganisms, because they can be produced inexpensively from petroleum by chemical catalysis. However, he adds, due to fluctuations in petroleum prices and recognition of dwindling reserves, trade imbalances, and political considerations, “it is now possible to consider production of these inexpensive chemicals from low-cost starting materials such as starch, sucrose, or cellulosic biomass (e.g., agricultural and forest waste, dedicated energy crops, etc.) with a microbial catalyst”. The key to producing bulk chemicals (e.g., polymer precursors) by using metabolically engineered cells will be to produce the exact molecule needed for existing products rather than something “similar but green” that will require extensive product testing before it can be used.

By far the highest-volume (and lowest-margin) application for engineered metabolism is the production of transportation fuels...Recent advances in metabolic pathway and protein engineering have made it possible to engineer microorganisms to produce hydrocarbons with properties similar or identical to those of petroleum-derived fuels and thus compatible with our existing transportation infrastructure. Linear hydrocarbons (alkanes, alkenes, and esters) typical of diesel and jet fuel can be produced by way of the fatty acid biosynthetic pathway. For diesel in cold weather and jet fuel at high altitudes, branches in the chain are beneficial—regularly branched and cyclic hydrocarbons of different sizes with diverse structural and chemical properties can be produced via the isoprenoid biosynthetic pathway. Both the fatty acid–derived and the isoprenoid-derived fuels diffuse (or are pumped) out of the engineered cells and phase separate in the fermentation, making purification simple and reducing fuel cost.

Although the pathways described above produce a wide range of fuel-like molecules, there are many other molecules that one might want to produce, such as short, highly branched hydrocarbons (e.g., 2,2,4-trimethyl pentane or isooctane) that would be excellent substitutes for petroleum-derived gasoline. Additionally, most petroleum fuels are mixtures of large numbers of components that together create the many important properties of the fuels. It should be possible to engineer single microbes or microbial consortia to produce a mixture of fuels from one of the biosynthetic pathways or from multiple biosynthetic pathways. Indeed, some enzymes produce mixtures of products from a single precursor—maybe these enzymes could be tuned to produce a fuel mixture ideal for a particular engine type or climate.

To make these new fuels economically viable, we must tap into inexpensive carbon sources (namely, sugars from cellulosic biomass). Given the variety of sugars in cellulosic biomass, the fuel producer must be able to consume both five- and six-carbon sugars. Because many yeasts do not consume five-carbon sugars, recent developments in engineering yeast to catabolize these sugars will make production of these fuels more economically viable. Engineering fuel-producing microorganisms to secrete cellulases and hemicellulases to depolymerize these sugar polymers into sugars before uptake and conversion into fuels has the potential to substantially reduce the cost of producing the fuel.

—Jay Keasling

In the paper, Keasling discusses the roadblocks that stand in the way of a future in which microorganisms and molecules can be tailor-made through metabolic engineering, including the need for debugging routines that can find and fix errors in engineered cells. However, he is convinced these roadblocks can and will be overcome.

One can even envision a day when cell manufacturing is done by different companies, each specializing in certain aspects of the synthesis—one company constructs the chromosome, one company builds the membrane and cell wall (the “bag”), one company fills the bag with the basic molecules needed to boot up the cell.

Until this future arrives, manufacturing of molecules will be done with well-known, safe, industrial microorganisms that have tractable genetic systems. Continued development of tools for existing, safe, industrial hosts, cloning and expressing genes encoding precursor production pathways, and the creation of novel enzymes that catalyze unnatural reactions will be necessary to expand the range of products that can be produced from biological systems. When more of these tools are available, metabolic engineering should be just as powerful as synthetic chemistry, and together the two disciplines can greatly expand the number of products available from renewable resources.

—Jay Keasling

Resources

• Jay D. Keasling (2010) Manufacturing Molecules Through Metabolic Engineering. Science Vol. 330 no. 6009 pp. 1355-1358 doi: 10.1126/science.1193990