Manipulating Natural Product Biosynthetic Pathways via DNA Assembler

Abstract

DNA assembler is an efficient synthetic biology method for constructing and manipulating biochemical pathways. The rapidly increasing number of sequenced genomes provides a rich source for discovery of gene clusters involved in synthesizing new natural products. However, both discovery and economical production are hampered by our limited knowledge in manipulating most organisms and the corresponding pathways. By taking advantage of yeast in vivo homologous recombination, DNA assembler synthesizes an entire expression vector containing the target biosynthetic pathway and the genetic elements needed for DNA maintenance and replication. Here we use the spectinabilin clusters originated from two hosts as examples to illustrate the guidelines of using DNA assembler for cluster characterization and silent cluster activation. Such strategies offer unprecedented versatility in cluster manipulation, bypass the traditional laborious strategies to elicit pathway expression, and provide a new platform for de novo cluster assembly and genome mining for discovering new natural products. Curr. Protoc. Chem. Biol. 6:65‐100 © 2014 by John Wiley & Sons, Inc.

A Strategy for Seamless Cloning of Large DNA Fragments from Streptomyces

“…Current methods in the same field (10, 11) are unable to clone large target fragments with repeat sequences, whereas SCLF is able to. In fact, the size of the target fragment could be larger, since conventional plasmids have the ability to harbor DNA fragments ranging from 40 to 100 kb (23), and there is no difference in the procedures for cloning fragments of different sizes.…”

“…However, when used for cloning PKS (polyketide synthase) genes, the assembly efficiency can be low, and most of the constructs undergo severe gene deletions. This failure is due to the repeating PKS domains, which share very similar sequences (10). Similarly, large target fragments with repeats are difficult to clone via such methods.…”

“…The target fragments obtained from the BAC clones could be up to 28 kb, but this approach does not allow direct cloning of DNA regions larger than a certain size, e.g., 10 kb, from genomic DNA. Techniques based on in vivo recombination in yeast (10, 11) allow the cloning of fragments up to 80 kb, but the target large fragments are assembled from a number of relatively small fragments with overlaps. However, when used for cloning PKS (polyketide synthase) genes, the assembly efficiency can be low, and most of the constructs undergo severe gene deletions.…”

Cold Atmospheric Plasma Restores Paclitaxel Sensitivity to Paclitaxel-Resistant Breast Cancer Cells by Reversing Expression of Resistance-Related Genes

“…Paclitaxel (Tx) has been widely used to treat a variety of cancer types, including breast cancer [5]. Tx binds to β-tubulin subunits of the microtubule, preventing them from undergoing the depolymerization process, which is crucial during the course of mitosis [6]. Subsequently, the dissociation of the spindle is inhibited, and therefore, the cell cycle is blocked in the G2/M phase and apoptosis [7].…”

Metabolic engineering strategies for high-level production of aromatic amino acid pathway derivatives in Saccharomyces cerevisiae

“…The strain S. cerevisiae BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was used as the microbial platform for the production of SA and MA, as well as for the assembly of plasmids using the DNA Assembler technique Shao and Zhao, 2014). The yeast cultures were maintained in YPD medium (1% yeast extract, 2% peptone, and 2% dextrose).…”