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Biology As a Basis for Biochemical Engineering

Biology As a Basis for Biochemical Engineering

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Biology as a Basis for Biochemical

Sujata K. Bhatia, M.D., P.E. demonstrates the versatility Harvard Univ. of the living as a miniature reactor. Living cells can be genetically engineered to produce specific , tuned to manufacture novel enzymes, and optimized to guarantee genetic stability and consistent production of desired products.

iochemical engineering harnesses living cells as Major models of molecular and cellular biology miniature chemical reactors, enabling the production A living cell is an elegant reactor, containing the instruc­ Bof designer molecules ranging from pharmaceuticals tions for chemical synthesis and breakdown reactions, pro­ to to . Discoveries in molecular and cellular cess regulation and control, and quality assurance, all within biology have driven the development of biochemical engi­ the core nucleus of the cell. The basic instructions governing neering techniques, and the subsequent rise of the modern all processes within the cell are encoded in deoxyribo­ industry. nucleic acid (DNA), the genetic material of the cell. DNA Live cells possess unique capabilities to manufacture is a long polymer composed of repeating monomer subunits complex chemical entities. Even a relatively simple bacterial called . The subunits are adenine (A), cell performs highly complex synthesis, fueling, and poly­ cytosine (C), guanine (G), and thymine (T), all arranged in merization reactions through native pathways. Such reac­ a specific sequence that codes for all structure and function tions — occurring readily in — would be very within the cell. The living cell operates based on sequences difficult to accomplish via traditional synthetic chemistry or of A, C, G, and T, much the same way that computers oper­ in­organic catalysts. ate based on sequences of 0s and 1s. The structure of DNA Yet living cells also introduce unique challenges and was first presented by Watson and Crick in a now classic tradeoffs for manufacturing and . The work of (1); it is difficult to overestimate demands of large-scale production can place unnatural the impact of this work on biochemical engineering and stresses on cells, affecting both cellular growth and the qual­ industrial biotechnology. ity and reliability of cellular output. In living organisms, DNA typically does not exist as a The biochemical must successfully balance single strand, but rather as two long polymer strands twisted the capacities and limitations of these cellular reactors. around one another to form a double-helix structure. The Designing and optimizing cellular manufacturing processes, two strands are held together by hydrogen bonds, and the therefore, requires an understanding of the biological under­ two strands are completely complementary to one another. pinnings of biochemical engineering. Adenine (A) preferentially binds to thymine (T), and This article describes the fundamental principles of cytosine (C) preferentially binds to guanine (G). Thus the molecular and cellular biology, and explains the flow of structure and nucleotide sequence of a single DNA strand genetic information within biological systems. It then relates completely defines the structure and nucleotide sequence of core biological phenomena to and meta­ its complementary DNA strand (2) (Figure 1). bolic engineering. Finally, it discusses important engineering Every cell within an contains the same DNA considerations in the design of biochemical manufacturing double-helix structure and sequence. Accurate communica­ processes. tion of the information encoded within DNA is absolutely

40 www.aiche.org/cep July 2013 CEP Copyright © 2013 American Institute of Chemical (AIChE) essential for the organism to survive. Cells have evolved an some), amino acids line up along the single-stranded RNA ingenious mechanism for ensuring consistent and error-free with corresponding codon triplets, and the amino acids bind transmission of DNA-encoded information. to one another to form a . The sequence of the RNA . The first step of the information transmis­ strand completely defines the sequence of amino acids in the sion process is known as transcription. During transcrip­ protein (Figure 3). Just as DNA completely defines RNA, tion, the unzips into two single DNA strands. RNA completely defines a protein. A protein’s composi­ Nucleotides line up along one of the strands to form a new tion and sequence can be predicted from its corresponding single-stranded polymeric molecule called ribonucleic acid DNA sequence, and the reverse is also true, so that a DNA (RNA). RNA is composed of a sequence of the nucleotide sequence can be derived from a known protein sequence. subunits adenine (A), cytosine (C), guanine (G), and uracil The main sources of information in the cell — DNA and (U) — uracil in RNA fulfills an analogous role to that of RNA — as well as the main sources of structure and func­ thymine in DNA. Because the sequence of nucleotides in tion in the cell — proteins — are all polymers. a single DNA strand completely defines the sequence of nucleotides in its complementary DNA strand, it is also true Genetic engineering: Cells as reactors that the nucleotide sequence of a single DNA strand defines Genetic engineering, the foundation of the modern bio­ the sequence of nucleotides in its complementary RNA industry, is based on the following key insight: strand (Figure 2). Since a DNA sequence completely defines an RNA sequence Translation. Just as DNA codes for RNA during tran­ through transcription, and an RNA sequence completely scription, RNA in turn codes for the synthesis of proteins, defines a protein sequence through translation, then any as well as the structural and functional molecules of the cell desired protein can be manufactured by cellular machinery, and the entire organism. RNA is an intermediary between the provided that the cell possesses the DNA template corre­ DNA of the cellular nucleus, and the synthesis of sponding to the protein. proteins in the cytoplasm outside the cellular nucleus. While Cells containing the DNA template for a desired protein DNA and RNA are macromolecules composed of nucleotide can be cultured in large batches and the protein subunits, proteins are macromolecules composed of amino can be produced at a large scale. For instance, suppose that acid subunits. Each three-nucleotide sequence of RNA (for you want to produce human protein at a large scale instance, ATC, CUG, GGG, etc.) is known as a codon and for therapeutic purposes. If the DNA coding for human insu­ codes for a specific amino acid(3) . The process of protein lin can be inserted into a robust cellular host such as a synthesis based on the RNA code is called translation. cell, and yeast cells subsequently grown in large fermenta­ During translation (which takes place inside the ribo­ tion batches (much the same way as beer is brewed), then large amounts of human insulin can be readily produced. The biggest challenge of this process is inserting the human Base Pairs Structure of DNA Deoxyribonucleic Acid insulin into the yeast cellular host. Article continues on page

C = Cytosine G = Guanine Nitrogenous A = Adenine Base T = Thymine

RNA Polymerase

Sugar-Phosphate Backbone RNA Transcript

p Figure 2. During transcription, the DNA double-helix unwinds. Nucleotides line up along one of the strands to form an RNA transcript. The nucleotide sequence of a single DNA strand completely defines the p Figure 1. In a DNA strand, adenine (A) preferentially binds to thymine sequence of nucleotides in its complementary RNA strand. Source: National (T), and cytosine (C) preferentially binds to guanine (G). Human Genome Research Institute.

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Cohen and Boyer devised such a method for reliably genetically engineered human therapeutic to gain U.S. Food introducing any given DNA sequence into a chosen cellular and Drug Administration (FDA) approval — ushering in the host (4). The method relies on catalysts known as restric­ age of biotechnology. tion enzymes or restriction , which precisely cut DNA sequences at specific sites based on the nucleotide : sequence. Different restriction enzymes correspond to differ­ Cells as highly tunable reactors ent sequences of DNA. For example, the EcoRI (eco-R-one) Biochemical engineering views the cell as the enzyme cuts DNA only at the sequence GAATTC. for producing a desired compound, and its subdiscipline of If two different pieces of DNA are cut with the same metabolic engineering aims to make the cell more produc­ , then the ends of these two pieces of tive and more versatile. In essence, metabolic engineering DNA will be complementary to one another. The two pieces modifies and exploits the cell’s innate pathways to manufac­ of DNA can join together in a ligation reaction catalyzed by ture useful compounds. Metabolic engineering is a powerful DNA ligase enzyme to form a recombinant DNA molecule. technique that takes advantage of the fact that virtually all Figure 4 illustrates a common technique for creating enzymes, the natural catalysts of biochemical reactions, are recombinant DNA. A desired DNA fragment is inserted into proteins. a vector (i.e., a circular piece of DNA that replicates While genetic engineering leverages the catalytic in the cellular cytoplasm, independently of the chromosomal machinery of the cell to manufacture proteins, metabolic DNA in the ). The recombinant DNA molecule engineering goes one step further, enabling the cell to manu­ can then be inserted into a host cell, such as a bacterium, facture enzymes to tune the cell’s own catalytic machinery. yeast, or mammalian cell, through a process known as trans­ Furthermore, metabolic engineering empowers the cell to formation. A typical method for cellular transformation is to carry out a wider variety of chemical reactions. Whereas apply an electrical field, which causes the cell membrane to genetic engineering enables the cell to make proteins, become more porous to allow the recombinant DNA vector to enter the cell. So, to make human insulin, start by choosing an Vector DNA Chromosomal DNA Fragment appropriate restriction enzyme to cut both a plasmid DNA To Be Cloned vector and the ends of a DNA fragment coding for human insulin. Then join together these two pieces of DNA using DNA ligase, and insert the resulting recombinant DNA Cut DNA molecules plasmid into a host cell. If the host cells replicate in a with restriction enzyme to generate fermentation , human insulin will be produced at complementary a large scale. In fact, in 1982, human insulin was the first Join vector and chromosomal sequences on the DNA fragment using the vector and fragment enzyme DNA ligase Growing Amino Acid Chain Amino Acid

G U U

Anticodon Introduce the vector C into bacterium C G C A A C G C U U G C A C C C A A U U U G C G A G U Recombinant G G U C G DNA Molecule Codon RNA Strand Bacterial p Figure 3. During translation, amino acids line up along the single-­ stranded RNA with corresponding codon triplets, and the amino acids p Figure 4. During the production of recombinant DNA, both a DNA vector bind to one another to form a protein. The sequence of the RNA strand (such as a plasmid) and a desired DNA fragment are cut with the same completely defines the sequence of amino acids in the protein. Genetic restriction enzyme, creating complementary sticky ends. The DNA vector engineering leverages this relationship to manufacture desired proteins in and the DNA fragment are joined using DNA ligase enzyme. The recom- live cells. Metabolic engineering further leverages the fact that all enzymes binant DNA sequence can then be introduced into a host cell such as a are proteins. bacterium. Source: U.S. Dept. of Genome Program.

42 www.aiche.org/cep July 2013 CEP Copyright © 2013 American Institute of Chemical Engineers (AIChE) metabolic engineering enables the cell to make vitamins (5), In addition, cellular systems have innate mechanisms antibiotics, chemotherapeutics (6), other small­molecule for maintaining homeostasis via feedback control loops. As drugs, industrial polymers, dyes, fuels (7), and other spe­ a result, an engineer may design an up­regulated pathway cialty chemicals. All that is required is a host cell with the within the cell, only to find later that the pathway has been necessary starting materials and the necessary DNA genetic down­regulated by cellular feedback­control mechanisms. material coding for the pathway enzymes. Toxicity of reaction intermediates and final products can For example, suppose that you want to manufacture also limit throughput of metabolically engineered pathways, indigo dye from the starting material , a natural and must be considered. Detailed insights and mathematical amino acid. The enzymes tryptophanase and naphthalene models of metabolic networks are necessary to overcome dioxygenase catalyze the pathway from these challenges; computational simulations and analyses of tryptophan to indigo. You can first introduce a recombinant metabolic fluxes are critical for metabolic engineering. plasmid DNA vector coding for the tryptophanase and naph­ The interrelated biological phenomena of metabolic flux, thalene dioxygenase enzymes into a bacterial host cell using metabolic burden, and genetic instability also complicate the methods of genetic engineering described in the previ­ and frustrate metabolic engineering efforts. ous section. Then grow the transformed bacterial cells in a Metabolic flux. Metabolic flux is the rate of turnover of fermentation bioreactor and supply the cells with tryptophan molecules through a metabolic pathway. Engineered meta­ as a starting material. Indigo dye will be manufactured in the bolic pathways can suffer from flux imbalances, as these bioreactor. pathways lack the regulatory mechanisms present in innate Metabolic engineering turns the cell into a powerhouse metabolic pathways. Metabolically engineered pathways chemical reactor with tunable control systems. If a reaction often exhibit bottlenecks as a result of flux imbalances, is deemed productive, then the DNA that encodes its enzyme leading to the diversion of molecules away from the desired catalyst can be introduced (i.e., up­regulated) within the product, as well as the accumulation of toxic intermedi­ cell. Conversely, if a reaction is deemed counter­productive, ates. This is particularly problematic when several different the DNA that encodes its enzyme catalyst can be deleted, enzymes from different organisms are introduced into one interrupted, or down­regulated. The intracellular concentra­ cellular host. tion of any targeted enzyme can be increased or decreased Flux balancing can address rate­limiting steps in produc­ by changing either the number of copies of DNA encoding tion to solve these metabolic flux issues. Flux balancing the enzyme, or the rate at which the DNA is transcribed increases product titers (i.e., concentrations) and decreases and translated into the enzyme. The rates of transcription the buildup of intermediates. and translation can be modified by altering the balance of A major strategy for balancing metabolic flux is to molecular activators and repressors in the cell; these regula­ modulate the expression levels of individual enzymes (i.e., tory molecules are generally proteins. modulating the processing of DNA into RNA, and RNA into There is no doubt that the ready production of therapeu­ protein). Another strategy for addressing flux imbalance is to tic proteins and enzymes via genetic engineering has made a improve the activities of rate­limiting enzymes by directed dramatic impact on . Yet metabolic engineering has . An emerging strategy for improving metabolic the potential to impact all industries by enabling the ready flux is to control the spatial organization of successive production of any chemical species. enzymes in a given metabolic pathway; this approach mimics natural cellular mechanisms and places sequential enzymes Reactor optimization: close to one another within the cellular cytoplasm (8). Complexities of cellular manufacturing Metabolic burden. Problems arise because expression The ability of living cells to adapt to environmental levels of enzymes are often dramatically higher in a meta­ changes in real time makes these cellular reactors highly bolically engineered pathway than would be typically found desirable for manufacturing complex molecules. At the same in any natural metabolic pathway. The metabolic burden of time, the natural adaptations of cells introduce real chal­ an engineered pathway robs the cell of its building blocks, lenges for biochemical engineering. including amino acids and nucleotide monomers; these First and foremost, cellular systems have evolved a high building blocks are necessary for the cell to maintain func­ degree of redundancy in pathways. Any given intracellular tion and remain viable. When novel pathways are introduced reaction may be catalyzed by more than one enzyme. into a cell, essential building blocks can be diverted into The reverse is also true: Any given enzyme may catalyze those nonessential pathways as the cell ramps up production multiple intracellular reactions that share identical reaction of the engineered protein. The overproduction of non­ mechanisms. This redundancy makes it difficult for the bio­ essential proteins triggers stress responses within the cell and to target a pathway enzyme in isolation. slows its growth. Article continues on next page

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Ultimately, metabolic burden can limit product con­ In 2010, the trailblazing bioengineer J. Craig Venter cre­ centrations. Thus, the engineer must consider the tradeoff ated the first cell with a synthetic genome, thereby creating between cellular growth rate and engineered protein expres­ synthetic life (11). Venter’s team demonstrated that a com­ sion level, and determine the optimum plete genetic system can be reproduced by chemical synthe­ level for a sustained process. It is necessary to weigh the sis, starting with only the digitized DNA sequence contained cell’s basic need for survival and growth against efficient in a computer (11). If DNA is viewed as the software of life production of the desired product. as Venter has suggested, then biochemical engineers can Genetic instability. Because the metabolic burden program and build cells from scratch to carry out multiple shouldered by a plasmid­bearing producer cell places it at complex functions. For instance, engineered cells may be a dis advantage relative to a plasmid­free nonproducer cell, designed for water purification, environmental remediation, genetic instability can occur in engineered cells. Genetic food production, and cost­effective pharmaceutical manu­ instabilities may be present if product titers decrease while facturing. Synthetic cells may even serve as novel sources of cell growth rates increase. in plasmid DNA vec­ energy. tors (9) as well as losses of plasmid DNA vectors (10) can Biochemical engineering of synthetic life will thus create genetic instability. enable engineers to address global challenges in healthcare, Recognizing the tradeoff between engineered protein energy, and . This evolving area of cellular expression levels and genetic stability is the first step toward engineering will require a deep understanding of cellular managing genetic instability. Then, the engineer must find pathways and control mechanisms, mathematical models of the optimal protein production level that guarantees genetic regulatory networks, and optimization of cellular reactors. stability and reproducible production of the desired product Chemical engineers, already experts in reactor design and in the cellular reactor. process control, are uniquely poised to contribute to the new wave of synthetic cellular bioengineering. CEP The future of biochemical engineering Biochemical engineering demonstrates the versatil­ Literature Cited ity of the living cell as a mini­reactor, and leverages the 1. Watson, J. D., and F. H. C. Crick, “A Structure for Deoxyribose innate information pathway of the cell: DNA is transcribed Nucleic Acid,” Nature, 171, pp. 737–738 (Apr. 25, 1953). to RNA, and RNA is translated into protein. While genetic 2. Watson, J. D., and F. H. C. Crick, “Genetical Implications of engineering has established that individual proteins can be the Structure of Deoxyribonucleic Acid,” Nature, 171, produced in cells, and metabolic engineering has now estab­ pp. 964–967 (May 30, 1953). lished that individual metabolic pathways can be introduced 3. Crick, F. H. C., et al., “General Nature of the for in cells, an important step in the future of biochemical engi­ Proteins,” Nature, 192, pp. 1227–1232 (Dec. 30, 1961). 4. Cohen, S. N., et al., “Construction of Biologically Functional neering will be to engineer entirely new cells. This emerging Bacterial Plastids in Vitro,” Proceedings of the National Academy field is known as . of Sciences USA, 70, pp. 3240–3244 (Nov. 1973). 5. Ye, V. M., and S. K. Bhatia, “Pathway Engineering Strategies SujaTa K. BhaTia, M.D., P.E., is a and bioengineer who serves on for Production of Beneficial Carotenoids in Microbial Hosts,” the teaching faculty at Harvard Univ. (Phone: Biotechnology Letters, 34, pp. 1405–1414 (Aug. 2012). (617) 496-2840; Email: [email protected]). She is the Assistant 6. Ye, V. M., and S. K. Bhatia, “Metabolic Engineering for the Director for Undergraduate Studies in Biomedical Engineering at Production of Clinically Important Molecules: Omega­3 Fatty Harvard, and an Assistant Dean for Harvard Summer School. She is also an Associate of the Harvard Kennedy School of Government, for Acids, , and Taxol,” Biotechnology Journal, 7, the Science, Technology, and Globalization Project, as well as a faculty pp. 20–33 (Jan. 2012). member in the Harvard Kennedy School Executive Education program 7. Keasling, J. D., and H. Chou, “Metabolic Engineering Delivers on Innovation for Economic Development. She received bachelor’s Next­Generation Biofuels,” Nature Biotechnology, 26, degrees in biology, , and , and a mas- ter’s degree in chemical engineering, from the Univ. of Delaware, and pp. 298–299 (Mar. 2008). she received an M.D. and a PhD in bioengineering, both from the Univ. 8. Dueber, J. E., et al., “Synthetic Protein Scaffolds Provide Modu­ of Pennsylvania. Prior to joining Harvard, she was a principal investiga- lar Control over Metabolic Flux,” Nature Biotechnology, 27, tor at the DuPont Co., where her projects included the development of for wound closure and the development of minimally pp. 753–759 (Aug. 2009). invasive medical devices. She has written two books, for 9. Kachroo, A. H., et al., “Metabolic Engineering without Plas­ Clinical Applications (a textbook that discusses opportunities for both mids,” Nature Biotechnology, 27, pp. 729–731 (Aug. 2009). biomaterials scientists and to alleviate diseases worldwide) and Engineering Biomaterials for . She received 10. Tyo, K. E. J., et al., “Stabilized Gene Duplication Enables Long­ an award from the Harvard Univ. President’s Innovation Fund for Faculty Term Selection-Free Heterologous Pathway Expression,” Nature in recognition of her innovative approaches to biomedical engineering Biotechnology, 27, pp. 760–765 (Aug. 2009). education, the John R. Marquand Award for Exceptional Advising and 11. Gibson, D. G., et al., “Creation of a Bacterial Cell Controlled Counseling of Harvard Students, and the Capers and Marion McDonald Award for Excellence in Mentoring and Advising. She is a member of by a Chemically Synthesized Genome,” Science, 329, pp. 52–56 AIChE and is a registered P.E. in the state of Massachusetts. (July 2, 2010).

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