Utah State University DigitalCommons@USU All Graduate Theses and Dissertations Graduate Studies 12-2017 Investigation and Engineering of Polyketide Biosynthetic Pathways Lei Sun Utah State University Follow this and additional works at: https://digitalcommons.usu.edu/etd Part of the Biological Engineering Commons Recommended Citation Sun, Lei, "Investigation and Engineering of Polyketide Biosynthetic Pathways" (2017). All Graduate Theses and Dissertations. 6903. https://digitalcommons.usu.edu/etd/6903 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. INVESTIGATION AND ENGINEERING OF POLYKETIDE BIOSYNTHETIC PATHWAYS by Lei Sun A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSPHY in Biological Engineering Approved: ______________________ ____________________ Jixun Zhan, Ph.D. David W. Britt, Ph.D. Major Professor Committee Member ______________________ ____________________ Dong Chen, Ph.D. Jon Takemoto, Ph.D. Committee Member Committee Member ______________________ ____________________ Elizabeth Vargis, Ph.D. Mark R. McLellan, Ph.D. Committee Member Vice President for Research and Dean of the School of Graduate Studies UTAH STATE UNIVERSITY Logan, Utah 2017 ii Copyright© Lei Sun 2017 All Rights Reserved iii ABSTRACT Investigation and engineering of polyketide biosynthetic pathways by Lei Sun, Doctor of Philosophy Utah State University, 2017 Major Professor: Jixun Zhan Department: Biological Engineering Polyketides are a large family of natural products widely found in bacteria, fungi and plants, which include many clinically important drugs such as tetracycline, chromomycin, spirolaxine, endocrocin and emodin. They are synthesized from acyl- or malonyl-CoA precursors by polyketide synthases (PKSs). Three types of PKSs are known to date. Type I PKSs are multifunctional enzymes that are organized into modules, each of which harbors a set of distinct, non-iteratively acting domains responsible for the catalysis of one cycle of polyketide chain elongation. Type II PKSs are multienzyme complexes that carry a single set of iteratively acting enzymes. Type III PKSs, also known as chalcone synthase-like PKSs, are homodimeric enzymes that essentially are iteratively acting condensing enzymes. In the first part of my dissertation research, I identified a type I nonreducing PKS (NR-PKS) EmoA and a discrete thioesterase EmoB from the fungus Shiraia sp. strain Slf14 which produces endocrocin and emodin, and heterologously expressed these enzymes in the industrial strains Saachromyces cerevisiae and Pichia pastoris. In order to improve the production of target iv compounds endocrocin and emodin, we co-expressed a more efficient thioesterase ACTE1 with EmoA and introduced an engineered acetyl-CoA carboxylase ACC1 into the biosynthetic pathway, which provides more malonyl-CoA for polyketide biosynthesis. A decarboxylase TpcK was also introduced to further increase the production of emodin. The second part was to characterize a silent type II PKS gene cluster involved in the biosynthetic pathway of chromomycins in Streptomyces reseiscleroticus and optimize the production of chromomycins by engineering two regulatory genes to construct an efficient producing strain. The third part of this research was to identify a type III PKS involved in the biosynthesis of spirolaxine (anti-Helicobacter pylori) in a fungal strain Sporotrichum laxum. Heterologous expression of this PKS gene in Escherichia coli produced alkylresorcinols which serve as the backbone to synthesize spirolaxine. In the fourth part of my research, we manipulated the fermentation broth of E. coli harboring a type III PKS StTS by supplying different nutrients including glucose and sodium pyruvate at different concentrations, from which six novel flaviolin derivatives were produced. It provides a new approach to biosynthesizing new molecules in the widely used heterologous host E. coli. One goal of my dissertation research was to investigate and engineer the biosynthetic pathway of pharmaceutically valuable compounds, such as chromomycin, spirolaxine and emodin, so that we can produce the target compounds more efficiently for potential applications in industry. The other goal was to utilize E. coli as a platform for heterologous expression of PKSs and engineering of particular biosynthetic pathways to generate chemical diversity in natural products, such as novel flaviolin derivatives and alkylresorcinols. (189 pages) v PUBLIC ABSTRACT Investigation and engineering of polyketide biosynthetic pathways Lei Sun This research is focused on investigation and engineering of natural product biosynthetic pathways for efficient production of pharmaceutically important molecules or generation of new bioactive molecules for drug development. Natural products are an important source of therapeutics, such as chromomycin (anti-cancer), emodin (anti-inflammatory and anti-tumor) and sprolaxine (anti-Helicobacter pylori). Metabolic engineering of natural product biosynthetic pathways shows its promise for creating and producing valuable compounds with chemical diversity for drug discovery. One goal of this research is to create highly efficient strains to biosynthesize valuable natural products. The engineered Streptomyces roseiscleroticus strain constructed in this work showed higher titers of chromomycins than previously reported, which was achieved by characterizing and engineering the chromomycin biosynthetic gene cluster. I activated the polyketide biosynthetic pathway by engineering two regulatory genes, and optimized the culture conditions to increase the titer of chromomycins. The production of emodin nowadays mostly relies on conventional plant cultivation and organic solvent extraction, which is time-consuming and cost-ineffective. This work built a biosynthetic platform using industrial strains Saccharomyces cerevisiae and Pichia pastoris with eight genes from fungi and yeast, which affords a more efficient biosynthetic process of emodin. On the other hand, we used Escherichia coli as a platform for heterologous expression of PKSs and engineering of particular biosynthetic pathways to generate chemical diversity in natural vi products. The type III polyketide synthase (PKS) involved in the biosynthesis of spirolaxine was identified in this research, which is important for complete elucidation of the biosynthetic pathway of this anti-Helicobacter pylori natural product. Heterologous expression of this PKS in E. coli generated five new pharmaceutically valuable alkylresorcinols. Addition of glucose or pyruvate into the fermentation broths of E. coli expressing another type III PKS StTS resulted in a significant change in the product profiles. Five new products are produced and structurally characterized. Therefore, this work provides a new approach to generating new bioactive molecules in E. coli, the most widely used heterologous expression host. vii DEDICATION I dedicate this doctoral dissertation to my parents, Yulong Sun and Yifeng Tang, for nursing me with affections and love and their dedicated partnership for success in my life. To my wonderful wife, Jing Gao, and beloved daughter, Olivia Sun, for encouragement and support along the way. Lei Sun viii ACKNOWLEDGMENTS I would like to express my gratitude to the American Heart Association grant (16GRNT26430067) and the National Institutes of Health grants (AI065357 RM DP 008 and AI089347) for supporting my research. My deepest gratitude goes first and foremost to my major professor, Dr. Jixun Zhan, for his outstanding guidance, generous support and constant encouragement. He leads me an access to the academic world, and his mentoring is invaluable in the development of my future career. Without his consistent and illuminating instructions, it would have been impossible for me to get so much work done in four and half years and finish this dissertation. I would also like to thank my committee members, Dr. David W. Britt, Dr. Jon Takemoto, Dr. Dong Chen and Dr. Elizabeth Vargis for their suggestions and help, which helped me to grow professionally. I am also deeply indebted to all my friends and colleagues, in the Metabolic Engineering Laboratory, for the inspiring moments we shared and positive scientific atmosphere we created. Last but not least, I would like to extend my thanks to all the staff in the Department of Biological Engineering, especially Anne Martin, Kami McNeil, Levi Sanchez and Jed Moss for their help. To all, thank you for being part of these past years and for those to come. Lei Sun ix CONTENTS ABSTRACT ................................................................................................................................... iii PUBLIC ABSTRACT .....................................................................................................................v DEDICATION .............................................................................................................................. vii ACKNOWLEDGMENTS ........................................................................................................... viii CONTENTS ................................................................................................................................... ix LIST OF TABLES .........................................................................................................................xv