The Outlook for Protein Engineering in Crop Improvement

In the early 1800s the population of the world was estimated to be around 1 billion. It grew to 2 billion in the 1920s and to 6 billion at the close of the last millennium. We are currently adding approximately 80 million more per year and, at this rate, the global population will increase from the current 6 billion to >10 billion people by the mid-2020s (Pimentel and Pimentel, 2000). Simultaneous decline of arable agricultural land will challenge our ability to meet increasing demands for food, feed, fuel, and fiber. These are the grand challenges of the 21st century. Remarkably, almost 300 years ago, the British poet, Jonathan Swift (1667–1745) noted with extraordinary perception that “Whoever could make two ears of corn (Zea mays) or two blades of grass to grow upon a spot of ground where only one grew before, would deserve better of mankind, and do more essential service to his country, than the whole race of politicians put together.” Fast forward to the 21st century, Mr. Swift would be very pleased. Thanks to years of gradual selection through traditional plant breeding techniques, farmers have been able to achieve crop productivity that is orders of magnitude greater than the esteemed poet could have ever anticipated. Yield increases have come from continual improvements in agricultural practices and selections for improved abiotic and biotic stress tolerance and better pest management practices. However, faced with a burgeoning world population, competition for space and climatic changes, classical plant breeding alone is insufficient and not rapid enough for improving the characteristics of crops. Whereas traditional plant breeding is limited to accessing genes from closely related species, genome sequencing efforts of the last decade have yielded a plurality of genes of many different functions from many different species. Together with modern techniques in recombinant DNA methodology and plant transformation protocols, we are now postured to introduce any gene with desired function into a crop of interest. In the ideal case, a gene encoding a protein for a given trait from a given species will behave identically in the transgenic crop and faithfully confer the desired trait or phenotype. Often, owing to compromised expression, folding, and stability, the protein will have to be engineered or redesigned to achieve the end goal. In my opinion, we can attribute our current ability to alter function or impart new function into proteins to three significant observations: (1) the extraordinarily rapid growth in the knowledge base of the disparate disciplines of protein biochemistry, plant science, proteomics, genetics, genomics, molecular biology, mathematics, bioinformatics, and computer science and their synergistic value; (2) technological advances in macromolecular structure analysis methods such as NMR, x-ray crystallography, and mass spectrometry; and (3) the active participation of numerous companies in the development of reagents and tools for advancing the goals of basic and applied research. Thus, in the context of a world of limited and rapidly diminishing natural resources, protein engineering of crop species offers promising solutions to meeting the four Fs, i.e. food, feed, fiber, and fuel of societies in both developed and developing countries. In this narrative, I provide a holistic view of the state-of-the-art in protein engineering and potential applications of these strategies to crop improvement for a variety of societal benefits. A comparison of the steps involved in rational protein engineering (left) and directed protein evolution (right). Similarly, knowledge-based protein engineering of the family of δ-endotoxins from Bacillus thuringiensis has resulted in the development of novel toxins with enhanced insecticidal activity and specificity (Saraswathy and Kumar, 2004; Mandal et al., 2007). It is pertinent to mention here that the widespread use of B. thuringiensis (Bt) crops has raised alarms regarding the possible development of resistance to the insecticidal protein by various pests (Gould, 1998) and at least two crop pests have been reported to have acquired resistance to Bt sprays outside of the laboratory environment (Soberon et al., 2007). In addition to the “high dose/refuge strategy” recommended by the U.S. Environmental Protection Agency (www.epa.gov/pesticides/biopesticides/white_bt.pdf; Gould, 1998), an emerging strategy is the use of protein engineering approaches to counter the adaptive behavior of insects based on a molecular understanding of the toxin and its interaction with proteins in the insect gut. One hypothesis is that upon ingestion the protease-activated toxin binds to the primary receptor cadherin, followed by protease clipping of the N terminus of the toxin that includes helix α-1 in domain 1. These steps facilitate oligomerization and subsequent binding to secondary receptors that result in the formation of pores in cells and eventual cell death (Bravo et al., 2004). On the basis of these observations, it was hypothesized that engineered Bt toxins (Cry1Ab and Cry1Ac), in which the helix α-1 was deleted, could form pore-forming oligomers without the involvement of cadherin. The engineered proteins were indeed found to be toxic to Manduca sexta in which the expression of cadherin was silenced by RNA interference (Soberon et al., 2007). Roesler and Rao (2000) chose barley (Hordeum vulgare) chymotrypsin inhibitor-2 to exemplify the rational mutagenesis approach for nutritional enhancement. On the basis of its 3D structure, other homologous sequences, and a large body of literature on its biochemical and biophysical properties, they engineered a thermodynamically stable protein containing significantly elevated levels of the essential amino acids Lys, Trp, Thr, Ile, and Met. The value and success of sequence-/structure-guided protein engineering for proteins with enhanced nutritional benefits and improved insecticidal activity has been particularly well documented in recent reviews (Saraswathy and Kumar, 2004; Beauregard and Hefford, 2006). I will not dwell further on this subject except to state that despite the investment of millions of dollars by the major seed/plant biotechnology companies, the consumer is yet to see a transgenic cereal or legume crop expressing higher levels of essential amino acids such as Lys, Trp, Met, and Thr. Recently, however, Renessen (a joint venture between Cargill Inc. and Monsanto Company) obtained regulatory approval to produce corn engineered to produce high levels of free Lys. Termed Mavera high-Lys corn, the transgenic crop was to be grown on limited acreages in 2007 to produce grain with enhanced Lys levels for the animal feed industry. The structure-based engineering of strictosidine synthase is an excellent example of redesign of substrate specificity with great potential for changing metabolic pathways and generating novel molecules for health and nutrition. The enzyme catalyzes the condensation of tryptamine and secologanin leading to the synthesis of numerous monoterpenoid indole alkaloids in higher plants. Using the crystallographic structure of strictosidine synthase in complex with strictosidine, Loris et al. (2007) produced and characterized mutants with the capacity to generate novel alkaloid libraries for pharmacological screening. This example augurs well for knowledge-based protein engineering of strategic target molecules to alter pathways and make new products. A particularly exciting rational method to increase protein stability is the consensus engineering approach (Steipe, 2004). In this method, an improved protein is derived on the basis of a consensus sequence that incorporates favorable amino acid substitutions from a family of homologous sequences, or an existing sequence is modified to more closely match the consensus sequence. A remarkably successful example of this approach is the engineering of thermal stability in phytases, enzymes used quite extensively in animal feed technology. A protein with a consensus sequence derived from 13 homologous sequences displayed an increase in protein stability that was 22°C greater than the best parent (Lehmann et al., 2000, 2002). More recently, Dai et al. (2007) have applied consensus engineering to design a novel fluorescent protein using a family of 31 other fluorescent proteins. It is expected that a rapidly growing database of protein sequences in conjunction with algorithms to identify sequence homologies, powerful new structure-prediction programs (Zhang and Skolnick, 2004), and worldwide high-throughput structural genomics initiatives (Bhattacharya et al., 2007) will facilitate more extensive applications of such knowledge-based targeted mutations for modified protein function. However, it is important to bear in mind that an inherent limitation of structure-based rational mutagenesis is its inability to factor in the contributions of side chains that may be distal to the functional site of the molecule. Nevertheless, the structure of a protein is invaluable to a mechanistic understanding of protein function and can provide the template for further engineering by rational or combinatorial genetic approaches. Thus, the recent characterization of the x-ray structure of the glyphosate-insensitive form of the enzyme 5-enolpyruvylshikimate 3-P (ESP) synthase and its interaction with glyphosate (Funke et al., 2006), provides an explanation for the molecular basis of herbicide resistance of Roundup Ready crops. Whereas the ESP synthase was an introduced heterologous protein, acetohydroxyacid synthase (also known as acetolactate synthase) was the first example of a rationally engineered enzyme designed to confer resistance to the sulfonylurea and imidazolinone class of herbicides (Ott et al., 1996). Currently, transgenic soybean (Glycine max) resistant to these herbicides is commercially available. Undoubtedly, the recently derived x-ray structure of acetohydroxyacid synthase (Pang et al., 2003) will facilitate further protein engineering and subsequent identification of other novel herbicides. Phenotypic diversity manifests itself through transcriptional and posttranscriptional regulation of a number of genes. The ability to precisely control gene expression and insert foreign genes in specific sites within the genome has been major quests in molecular biology. In recent years, rapid strides have been made in the design of: (1) zinc finger (ZF) containing artificial transcription factors (TFs) capable of binding to specific DNA sequences (Pabo et al., 2001) and (2) tailored ZF nucleases that can cleave double-stranded DNA at predetermined sites to allow gene insertion by homologous recombination (Durai et al., 2005). The DNA binding modules of the artificial TFs are based on the classic Cys2His2 ZF domains found in many eukaryotic TFs. Importantly, the natural repertoire of ZF domains has been extended by mutant libraries and novel ZF domains binding to specific DNA sequences have been selected by phage display (Dreier et al., 2001; Blancafort et al., 2004). The regulation of plant gene expression by TFs to effect complex phenotypic properties has been successfully demonstrated in plants (for review, see Segal et al., 2003). It is particularly exciting to speculate on the opportunities for controlling metabolic pathways to make novel pharmacological products by turning on or turning off specific genes with designer TFs (Gandet and Memelink, 2002). It is evident, therefore, that a variety of improved protein functions can be achieved through experimental protein engineering. However, a major limitation, even for directed evolution, is the highly restricted coverage of sequence space (103–106 sequences) for targeted mutagenesis (Voigt et al., 2001). Following the pioneering work of DeGrado and colleagues in the de novo design of proteins with predefined structure, i.e. helical proteins adopting the four-helix bundle (DeGrado, 1988; DeGrado et al., 1989; Hecht et al., 1990), more recent advances in computational protein design have allowed for a greater exploration of the protein sequence space and ushered in the era of designer synthetic proteins with novel functionalities. Thus, Mayo and colleagues at California Institute of Technology have developed sophisticated computational tools for the design of enzyme-like proteins from protein scaffolds devoid of catalytic activity (Bolon and Mayo, 2001). Although such enzymes have poor kinetic properties, they nevertheless provide the template for adoption of iterative combinatorial mutagenesis strategies and selection schemes for optimizing the desired function. Hellinga and colleagues at the Duke University Medical Center have also used computational tools to specifically focus on redesigning the signaling properties of receptor molecules via changes in ligand recognition sites (Benson et al., 2002). Typically, approximately 12 to 18 amino acid residues on the receptor surface are in direct contact with the wild-type ligand and there is a major challenge associated with experimentally producing and testing libraries for novel ligand-binding functions resulting from mutations of these residues. To overcome this combinatorial challenge, the Hellinga group developed an algorithm that is essentially an in silico evolution and screening program (Looger et al., 2003). In an exciting application, Hellinga's group redesigned the binding specificity of Escherichia coli periplasmic binding protein from native binding to the sugar Ara to high-affinity binding of molecules such as trinitrotoluene, l-lactate, and the neurotransmitter, serotonin. Furthermore, they provided practical evidence for the biosensor properties of the engineered receptors. This pioneering approach to the computational redesign of biomolecular recognition sites has profound implications for the development of the next generation of crop plants that can be specifically regulated by engineered receptor-ligand interactions. A broader concept of protein engineering is found in the exciting work of Lim and at the University of are biochemical in eukaryotic cells to new in cell behavior (Bhattacharya et al., 2006). approach is on the extensive biochemical and structural of in that have demonstrated the highly of proteins. in the domain is the ligand-binding domain and the domain has of the domain in specific with proteins that the for the proteins of can be designed to control the specificity of the leading to of the use of designer proteins to cell and is in two recent by the group et al., et al., 2007). It is to speculate on the of to control plant The genome the of which are and 2001). identification and direct receptor-ligand binding have been demonstrated only for a with the the signaling and 2006). A more understanding of the molecular of signaling and the of their will the development of novel crops expressing engineered proteins designed to and to specific and In addition to turning on engineered pathways in crop plants for resistance and stress one can the of designer plants with molecules to a variety of such as or toxic The of proteins and their ability to new functions selection is well documented by numerous in the the example of in evolution is that of the coli protein, with or activity to one with activity et al., This concept of proteins in the laboratory by the group at California Institute of Technology and by and colleagues at the in the has the of protein engineering both from the of tools for in protein structure and function as well as the development of products for the biotechnology industry. to as directed evolution, molecular or DNA the method not a any structural or mechanistic understanding of the target and has the used for protein the on two (1) of a of mutants and (2) a screening for the selection of the protein with the best for the desired from of of and In the libraries of mutants are made by and of proteins from a gene that a protein at least of the desired biochemical properties or using et al., to introduce mutations The genes are and the proteins for improved or modified Typically, the iterative of in recombination and screening is to the end is mutations are of mutagenesis and screening are to the of favorable mutants and the of In the the of in evolution is related to the of the and 2004). The greater the expected in the desired or function novel substrate the greater the sequence space to be to the sequence with the best activity 2004). gene is to generate the sequence diversity the On the other in or family recombination of protein a homologous family of proteins that are related in sequence more have a of the family has to a functional libraries through family are also expected to a significantly high of A classic of is found in the evolution of activity from a family of genes encoding et al., Currently, a number of different methods have been developed for libraries in which sequence are or are not These whole gene and I the to many excellent reviews and for more coverage et al., 2001; et al., 2001; and 2004; 2004; et al., 2005). The success of DNA the of the method is the high of methods that overcome this to a great have been One is the known as et al., 2003) and the other is Using known as and 2004). In an excellent recent et al. (2007) the of these recombination with that of DNA and the significant improvements they to the of directed of the method, however, there are two other that are to the success of directed (1) screening methods such as phage display (for review, see and and other emerging techniques using synthetic cells and and et al., to the population of from to a number and (2) the of high-throughput to the function of the protein and the best ligand binding and The increase in crop productivity in the last of can be in large to the development of transgenic crops expressing proteins that resistant to herbicides such as and In existing tolerance is derived from the expression of an ESP synthase gene from that is to In recent other successful have been made to identify novel mutants of ESP synthase by protein engineering. et al. demonstrated that even a of directed evolution yielded with kinetic properties to the parent ESP synthase enzymes from coli and More recently, et al. a mutant ESP synthase by mutagenesis of the The a in the protein kinetic properties, and was to confer glyphosate tolerance in transgenic an to an enzyme for tolerance to et al. a strategy using directed evolution to design a glyphosate that the herbicide by glyphosate to a that is a poor of ESP genes encoding related enzymes with very poor activity glyphosate were from the Bacillus were to of DNA to an active enzyme that was more active than the enzymes and was approximately in amino acid sequence to the proteins. The inability to active enzyme by amino acids from the in the wild-type enzyme or the active was an of the of combinatorial The recently x-ray structure of in complex with A and the offers into the molecular basis of glyphosate resistance et al., 2007) and possible further of activity by directed the corn, and other crops containing the enzyme are to be the next years the site at is example of a herbicide to the class that is rapidly by in in et al. applied a evolution strategy to two and mutants with enhanced the best of which a and higher activity than the parent targeted mutagenesis of the mutant yielded an enzyme that higher are subject to of the environment and can be by and have that this in be to from a population of at high and It is also known that the active state of is regulated by an enzyme that is at and recent by et al. in transgenic plants a between expression levels and to To the hypothesis that increasing the thermal stability of can to activity at elevated et al. (2007) used two of directed evolution of the gene to produce that were first extensively characterized in and in This provides evidence for higher activity in transgenic plants expressing mutants and that protein engineering of and other proteins in the may be more than et al., 2004). The family of insecticidal proteins from B. thuringiensis has been used for plant resistance to insect the that insects will their to the biochemical of and has been by laboratory and The diversity of sequences and 3D of toxins et al., as well as the identification of receptor molecules such as and in the insect and 2001) will only the use of both rational and directed evolution methods to novel solutions for plant resistance and of the adaptive evolution by the insect species. The development of as of the is a A and understanding of the of and via rational and molecular evolution methods exciting opportunities for plant cell to transgenic crops more to rapid to I will stress that in this is and we are years from engineering crop plants for the industry. Nevertheless, of a number of different with improved thermal stability and modified for use in (for review, see et al., 2006), provide a of the potential opportunities in plant In an the engineering of strictosidine synthase was as an example of the potential of plant metabolic engineering for changing the natural repertoire of pharmacological The diversity of sequences in plants and their involvement in complex metabolic pathways a of genes that could be further engineered for advancing the benefits of plant biotechnology for and producing and in et al., et al., 2007). The synergistic of rational and directed evolution on the one and advances in plant and the on the opportunities for genetic engineering of novel into the next generation of crop plants to benefits that the four However, the of agricultural biotechnology for in both developing and developed will be only the developed can put their the and benefits of modified and on regulatory for rapidly products to the 2000).