(3dv) Refactoring Biosynthetic Pathways Via Synthetic Biology

The overall theme of my postdoctoral research is to develop synthetic biology strategies to address the challenges associated with engineering natural product gene clusters for both new drug discovery and product yield optimization.  As transiting from a PhD candidate to a more independent researcher, my research focus was correspondingly shifted from engineering and characterization of single enzymes to constructing biological systems composed of multiple components.  

Projects in My Postdoctoral Study

DNA Assembler, an in vivo Genetic Method for Rapid Construction of Biochemical Pathways.  A highly efficiently approach, named “DNA assembler”, was developed, which allows the rapid assembly of an entire biochemical pathway in a single step via in vivo homologous recombination in Saccharomyces cerevisiae.  Three distinct functional biochemical pathways with sizes ranging from 9 to 19 kb were assembled on both a plasmid and a chromosome with a high efficiency.  As this method only requires simple DNA preparation and one-step yeast transformation, and circumvents the potential problems associated with the existing cloning methods, it represents a versatile approach in the construction of biochemical pathways for synthetic biology, metabolic engineering, and functional genomics studies.

Rapid Characterization and Engineering of Natural Product Biosynthetic Pathways via DNA Assembler.   Sequenced genomes and metagenomes provide a tremendously rich source for discovery of new genes and pathways.  However, the discovery and sustainable production of valuable natural products are often hampered owing to our limited ability to manipulate the corresponding biosynthetic pathways.  We redesigned the DNA assembler as a genomics-driven, synthetic biology-based method, for discovery, characterization, and engineering of natural product biosynthetic pathways.  This method synthesizes an entire expression vector containing the target biosynthetic pathway and the genetic elements needed for DNA maintenance and replication in S. cerevisiae, Escherichia coli, and a target heterologous expression host in a single-step manner.  Two important biosynthetic pathways from Streptomyces including the aureothin and the spectinabilin biosynthetic pathways were chosen for study.  It was demonstrated that since all the fragments are prepared by PCR, various manipulations including site-directed mutagenesis, scar-less gene deletion and artificial gene cluster construction can be easily achieved.  Such a method with unprecedented flexibility and versatility has many applications in heterologous pathway expression, pathway functional studies, and combinatorial biosynthesis. 

Refactored Biosynthetic Pathways: Delineating Pathway Expression from Sophisticated Regulation Cascades via Synthetic Biology.  The production of natural product is subjected to sophisticated regulation cascades in which target gene and multiple regulators interact with each other, resulting in a complex regulatory network that responds to a variety of physiological and environmental signals.  This highly regulated process complicates the discovery and characterization of new natural products with important biological activities and hampers sustainable production of the high-value ones.  We developed a general synthetic biology-based strategy to resolve the complexity and delineate the pathway expression from sophisticated regulation cascades.  As proof of concept, three important biosynthetic pathways from Streptomyces including the spectinabilin gene cluster, the fosfomycin gene cluster, and the FR900098 gene cluster were chosen for study.  We propose to construct refactored pathways by inserting a constitutive promoter in front of each pathway gene.  Approximately 40 constitutive promoter candidates in front of the house-keeping genes were cloned from the genomes of Streptomyces and other related actinobacteria and fused with reporter genes.  Currently, they are being evaluated in different growth conditions.  The strong constitutive promoters will be inserted in front of the genes in the spectinabilin gene cluster in order to activate the silent spectinabilin biosynthesis in S. lividans.  If successful, it will be very useful in activating numerous uncharacterized cryptic pathways for discovering new potential natural products with biological and pharmaceutical importance.  In addition, the fosfomycin biosynthetic pathway and the FR900098 biosynthetic pathway, both having important biomedical applications are chosen and will be reconstructed with strong and constitutive promoters for higher production levels.

Selected Projects in My PhD Study

Biosynthesis of Triacetic Acid Lactone (TAL) from D-Glucose.  Rational design was applied to establish the biosynthesis of TAL from a renewable feedstock D-glucose in vivo.  Based on the catalytic mechanism of 6-methylsalycylic acid synthase (MSAS), we hypothesized that inactivating the ketoreductase (KR) domain would lead to TAL formation in vivo.  By using bioinformatics tools for BLAST search and sequence alignment, we identified the key catalytic residue of the KR domain.  Heterologous expression of a variant of MSAS, Y1572F, in S. cerevisaie, enabled the synthesis of TAL at a maximal titer of 0.28 g/L in flask.  TAL production was further optimized to 1.7 g/L by fed-batch fermentation.  This study demonstrates the power of rational design in engineering of multifunctional proteins.

Characterization of Alcohol Dehydrogenases in the Biosynthesis of Phosphonic Acid Antibiotics.  Phosphonates represent a potent, yet underexploited, group of bioactive compounds, mainly produced by Streptomyces.  Despite the significant structural differences among many of phosphonic acids, their biosynthetic routes contain an unexpected common intermediate, 2-hydroxyethyl-phosphonate (HEP), which is synthesized from phosphonoacetaldeyhyde (PnAA) by a distinct family of iron-dependent alcohol dehydrogenases (ADHs).  Although the sequence identity of the ADH family members is relatively low (34–37%), in vitro biochemical characterization of the homologs involved in biosynthesis of the antibiotics fosfomycin, bialaphos, and dehydrophos unequivocally confirms their enzymatic activities.  These unique ADHs have exquisite substrate specificity, unusual metal requirements, and an unprecedented monomeric quaternary structure.  Further, sequence analysis shows that these ADHs form a monophyletic group along with additional family members encoded by putative phosphonate biosynthetic gene clusters.  Thus, the reduction of PnAA to HEP may represent a common step in the biosynthesis of many phosphonate natural products, a finding that lends insight into the evolution of phosphonate biosynthetic pathways and the chemical structures of new phosphonic acids containing secondary metabolites.