By Roxanna Pignolet, Biochemistry & Molecular Biology, ‘20

Author’s note: This literature review was originally written as an assignment for my 102B Writing in the Disciplines: Biological Sciences class. At the start of this quarter I was lucky enough to get involved in plant metabolic engineering research in Dr. Patrick Shih’s laboratory, which exposed me to the field of synthetic biology for the first time. I immediately became fascinated with the whole process of engineering plants to produce medically relevant compounds. Through this review I hope to inform others of these surprising and highly relevant applications of plant genetic engineering.

Introduction

Plants produce a variety of secondary metabolites that participate in their natural defense and signaling mechanisms (3). Several of these complex molecules, such as fisetin, benzyl isothiocyanate, taxol, and verazine, have been found to have anticancer properties that have potential for future use in medicines or supplements [1,2,3,4]. The complexity of these molecules makes chemical synthesis in the lab difficult and expensive, so methods utilizing existing plant pathways are of great interest in current research [4]. This manipulation of metabolic pathways through making changes to enzymes, regulatory mechanisms, and using recombinant DNA techniques, is known as metabolic engineering [5].

New applications in plants are driving optimization and innovation of plant metabolic engineering methodology. Current research is focused on biosynthesizing target molecules through microbial cell factories and engineering stable transgenic plant lines. With an estimated 38.4 % of people being diagnosed with cancer in their lifetime and rising costs for treatment in the US, there is an increasing need for implementation of effective treatment [6]. Successful implementation of these methods of biosynthesis could pave the way for cost-efficient and eco-friendly mass production of important pharmaceuticals. This review will cover key advances in anticancer metabolite production and will discuss the outlook of plant bioengineering as a feasible alternative to anticancer molecule chemical synthesis.

Constructing Gene Libraries and Databases

Before the metabolic pathways can be reengineered to favor a specific product, researchers must understand which genes are important for its function. While some studies are working with plants that already have a database of genetic information, others are diving into unexplored territory. cDNA libraries are one way to compile a database of relevant genes. The viral enzyme reverse transcriptase can construct complementary DNA sequences from RNA molecules. This is important because the RNA molecules themselves are too unstable to be sequenced directly. This cDNA can be ligated onto a DNA backbone and introduced into bacteria for translation and detection [7]. The cDNA libraries that this method produces can be used to discover the mRNA sequence of a known gene or to identify unknown genes in a pathway. Future research will rely on this data to further metabolite pathway optimizations.

In a recent study, researchers analyzed Withania somnifera leaf and root tissue cultures to construct a gene database for the withanolide production pathway. Withanolide is a metabolite in W. somnifera that acts as an anticancer drug in the presence of gastric adenocarcinoma cell lines. By isolating RNA from tissue cultures of W. somnifera, the researchers were able to construct a transcript library and categorize it based on gene expression. Using this data, the researchers extracted transcripts coding for enzymes in the withanolide pathway, and mapped expression of these genes in the plant tissues. This database is key to performing further transient expression studies on this species [8].

Microbial Cell Factories

Microbial cell factories are bacteria engineered to produce compounds native to other organisms. This review will focus on their application to plant metabolite production. One benefit of microbial synthesis is the lower cost of growing bacteria compared to growing plants. Synthesising complex molecules in a laboratory is often cost inefficient due to labor intensive multistep reactions and low product yields. Microbial synthesis is an attractive option for utilizing the machinery of many thousands of cells as a means of mass production [9].

One application of this method is for producing benzyl isothiocyanate (BITC), a secondary metabolite found in cruciferous vegetables that is believed to reduce the risk of cancer. Due to the complexity of this compound, research has been conducted to prove the possibility of mass production through bacterial synthesis in E. coli. The scientists modified the first two proteins in the BITC pathway (CYP79A2 and CYP83B1) to function inside the prokaryotic environment. The next five enzymes tended to form nonfunctional aggregates, but analogous genes from A. thaliana were found to produce functional enzymes. Successful expression of the entire BITC pathway was verified by gas chromatography-mass spectrometry (GC-MS), though the identification of the compounds left some room for error [9]. While the researchers ultimately were successful in modifying the pathway, they had to overcome significant hurdles to adapt the system to a bacterial environment. These hurdles are intrinsic to the conversion of plant to bacterial systems in general, so it is important to consider the potential costs associated with the necessary optimisation processes. These successes are highly specific to the BITC pathway; however, this study shows that microbial synthesis is an option for metabolite production.

Stahlhut et al. engineered a new biosynthetic pathway into E. coli that produced an anticancer acting plant metabolite known as fisetin. As current understanding of the fisetin production pathway is limited, the researchers hypothesized that they could model the intermediates and steps of the pathway on the known pathway of a similar molecule called quercetin. Gibson assembly, a technique that allows for multiple DNA fragments to be ligated into the same fragment, was used to prepare the plasmids coding for the proposed biosynthetic pathway. The cultures of the complete pathway included the buildup of several intermediates as well as fully formed fisetin. While this method of biosynthesis has the potential for low production costs, the technique would need to be substantially optimized to confront the degradation of product in the bacterial environment.

Transgenic Plant Lines

A current synthetic biology strategy for engineering plant metabolites is heterologous expression of a target pathway in plants. The general procedure includes cloning key genes for the metabolic pathway of interest, constructing a vector with those genes, and testing the expression transiently in the model plant through Agrobacterium mediated genetic transformation. The transient nature of this procedure allows for relatively fast results that can drive the strategic production of stable plant lines. Subsequent testing for expected levels of product in the transgenic plant segment provides information on the success of the transformation [2,10].

Augustin et al. aimed to transform the cost-efficient and fast-growing crop Camelina sativa to produce a metabolite known as verazine. Verazine is the precursor of cyclopamine, which is a chemical that has been shown to inhibit a pathway that plays a role in tumor growth. Four genes known to be necessary for the verazine production pathway were cloned from V. californicum. They prepared three different vectors, the first included the first two enzymes in the pathway, the second vector had the first three, and the last vector coded for all four necessary enzymes. Preliminary research that suggested the gene GAD2 might be necessary for successful verazine biosynthesis, so they included it as part of the last two vectors. GAD2 (glutamate decarboxylase-2) is an enzyme that is required for the production of a co-substrate. C. sativa plants were transformed from each of these vectors and analyzed for evidence of the product. They quantitated verazine amounts in the transgenic lines averaging 54 pg/mg seed. While these levels indicate a successful construction of the pathway, much higher amounts would be needed to make this a viable strategy for mass production. Demands for cyclopamine are predicted to outrun current supply capability and attempts to harvest V. californicum for it or to use cell culture techniques have not proven successful [1]. Augustin et al. provide encouraging results that suggest that optimized transgenic C. sativa plant lines could be the answer to meeting the demand for the medicinally important drug.

While Augustin et al. use standard methods for the cloning of genes and construction of vectors, they chose to produce stable plant lines before analyzing the success of the pathway they constructed. One of the benefits of this method is more accurate results for levels of specific compounds produced, however the time it takes to grow a transgenic plant to seed significantly slows the research down. Transient expression is the temporary expression of genes through the introduction of transformed plasmid DNA to eukaryotic cells. This method allows scientists to efficiently test a pathway at various stages of optimization [10].

In Blanco et al., the production of metabolites known as flavinols was increased through overexpression of a protein known as CcMYB12. Flavinols naturally occur in high quantities in the Globe artichoke and are known to have many medicinal properties including anticancer functions. They targeted a gene believed to code for a transcription factor responsible for regulating the Flavinol synthesis pathway and attempted to integrate this gene into Arabidopsis and tobacco plants to upregulate the production of flavanol. The flavanol production pathway is already present, however medical applications would require higher production levels. In the Arabidopsis and tobacco plant lines transformed with the CcMYB12 gene, higher amounts of flavinols were produced as expected. This confirms the role of CcMYB12 as a regulator of the flavonoid pathway. Due to the understanding of the flavanol production pathway, Blanco et al. were able to focus on regulating the pathway through overexpression. However not all pathways are so well understood, and some are specific to certain plant species. In these cases, different strategies are necessary to express the molecule of interest.  

A related strategy for engineering metabolite production is the manipulation of transcription factors or regulatory mechanisms. Augustine et al. hypothesized that suppressing a gene involved in the conversion of glucoraphanin (an anticancer relevant metabolite), into undesirable metabolites would increase the levels of glucoraphanin accumulated in plants. They created RNA that codes for the downregulation of all homologues, or copies, of the GSL-ALK gene of interest. Agrobacterium was used to transform B. juncea plants, from which stable lines were produced and analyzed. They discovered a varying efficiency of regulation, with an overall 58% decrease in total glucosinolates. This had a pronounced positive effect on the reduction of unwanted side-products and the overall increase in glucoraphanin [11].

Conclusion

Plant metabolic engineering has become an important topic due to the variety of anticancer relevant compounds that are only produced in certain plants. The goal of current research is to manipulate the metabolic pathways through microbial synthesis or transgenic expression in plants in order to produce high levels of these target compounds. Microbial expression comes with the challenges associated with transferring a plant based metabolic process into a completely foreign environment. However, microbial cell factories may be more cost-efficient than transgenic plants when considering the minimal maintenance requirements of bacterial colonies. In many cases, preliminary research must still be done to acquire a basic understanding of the main enzymes and genes necessary for each pathway’s function. As researchers begin to compile results and build transcript libraries, more precise optimization of metabolic pathways will become possible. Rather than focusing on recreating the natural production pipeline, future studies will have the opportunity to significantly manipulate the amount of an anticancer molecule in a plant. In a larger sense, this technology has the potential to revolutionize the pharmaceutical industry by shifting from chemical factories to eco-friendly plant based drug production.

References

1. Augustin MM, Shukla AK, Starks CM, O’Neil-Johnson M, Han L, Holland CK, Kutchan TM. Biosynthesis of Veratrum californicum specialty chemicals in Camelina sativa seed. Plant Biotechnology Reports. 2017 [accessed 2019 Jan 23];11(1):29–41. https://search.proquest.com/docview/1879495789?accountid=14505. doi:10.1007/s11816-017-0427-x
2. Lu X, Tang K, Li P. Plant Metabolic Engineering Strategies for the Production of Pharmaceutical Terpenoids. Frontiers in Plant Science. 2016 [accessed 2019 Jan 28];7. doi:10.3389/fpls.2016.01647
3. Marasco EK, Schmidt-Dannert C. Biosynthesis of Plant Natural Products and Characterization of Plant Biosynthetic Pathways in Recombinant Microorganisms. Applications of Plant Metabolic Engineering. 2012 Jun 9:1–43. doi:10.1007/978-1-4020-6031-1_1
4. Stahlhut SG, Siedler S, Malla S, Harrison SJ, Maury J, Neves AR, Forster J. Assembly of a novel biosynthetic pathway for production of the plant flavonoid fisetin in Escherichia coli. Metabolic Engineering. 2015 [accessed 2019 Jan 29];31:84–93. doi:10.1016/j.ymben.2015.07.002
5. Yang, Shang-Tian & Liu, Margaret & Zhang, Yali. (2007). Chapter 4. Metabolic Engineering—Applications, Methods, and Challenges. Bioprocessing for Value-Added Products from Renewable Resources. 73-118. 10.1016/B978-044452114-9/50005-0.
6. Cancer Statistics. National Cancer Institute. 2018 Apr 27 [accessed 2019 Mar 25]. https://www.cancer.gov/about-cancer/understanding/statistics
7. Polyak K, Meyerson M. Gene Expression: MRNA Transcript Analysis. In: Holland-Frei Cancer Medicine. 6th ed. https://www.ncbi.nlm.nih.gov/books/NBK12777/
8. Senthil K, Jayakodi M, Thirugnanasambantham P, Lee S, Duraisamy P, Purushotham PM, Rajasekaran K, Charles SN, Roy IM, Nagappan A, et al. Transcriptome analysis reveals in vitro cultured Withania somnifera leaf and root tissues as a promising source for targeted withanolide biosynthesis. BMC Genomics. 2015 [accessed 2019 Jan 23];16(1):14.https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-015-1214-0. doi:10.1186/s12864-015-1214-0
9. Liu F, Yang H, Wang L, Yu B. Biosynthesis of the High-Value Plant Secondary Product Benzyl Isothiocyanate via Functional Expression of Multiple Heterologous Enzymes in Escherichia coli. ACS Synthetic Biology. 2016 [accessed 2019 Jan 29];5(12):1557–1565. https://www.sciencedirect.com/science/article/pii/S1096717615000828?via=ihub. doi:10.1021/acssynbio.6b00143
10. Blanco E, Sabetta W, Danzi D, Negro D, Passeri V, Lisi AD, Paolocci F, Sonnante G. Isolation and Characterization of the Flavonol Regulator CcMYB12 From the Globe Artichoke [Cynara cardunculus var. scolymus (L.) Fiori]. Frontiers in Plant Science. 2018 [accessed 2019 Jan 29];9. doi:10.3389/fpls.2018.00941
11. Augustine R, Bisht NC. Biofortification of oilseed Brassica juncea with the anti-cancer compound glucoraphanin by suppressing GSL-ALK gene family. Scientific Reports. 2015 [accessed 2019 Jan 23];5(1). https://www.nature.com/articles/srep18005. doi:10.1038/srep18005
12. Sun J, Manmathan H, Sun C, Peebles CAM. Examining the transcriptional response of overexpressing anthranilate synthase in the hairy roots of an important medicinal plant Catharanthus roseus by RNA-seq. BMC Plant Biology. 2016 [accessed 2019 Jan 23];16(1). https://bmcplantbiol.biomedcentral.com/articles/10.1186/s12870-016-0794-4#Abs1. doi:10.1186/s12870-016-0794-4