Combinatorial pathway engineering for optimized production of the anti‐malarial FR900098. Issue 2 (3rd September 2015)

• Record Type: Journal Article
• Authors: Freestone, Todd S.; Zhao, Huimin
• Is Part Of: Biotechnology and bioengineering. Volume 113:Issue 2(2016)
• Page Start: 384
• Page End: 392
• Publication Date: 2015-09-03
• Subjects: FR900098 -- pathway engineering -- metabolic engineering -- anti‐malarial; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.25719
• Languages: English
• ISSNs: 0006-3592

Abstract

ABSTRACT: As resistance to current anti‐malarial therapeutics spreads, new compounds to treat malaria are increasingly needed. One promising compound is FR900098, a naturally occurring phosphonate. Due to limitations in both chemical synthesis and biosynthetic methods for FR900098 production, this potential therapeutic has yet to see widespread implementation. Here we applied a combinatorial pathway engineering strategy to improve the production of FR900098 in Escherichia coli by modulating each of the pathway's nine genes with four promoters of different strengths. Due to the large size of the library and the low screening throughput, it was necessary to develop a novel screening strategy that significantly reduced the sample size needed to find an optimal strain. This was done by using biased libraries that localize searching around top hits and home in on high‐producing strains. By incorporating this strategy, a significantly improved strain was found after screening less than 3% of the entire library. When coupled with culturing optimization, a strain was found to produce 96 mg/L, a 16‐fold improvement over the original strain. We believe the enriched library method developed here can be used on other large pathways that may be difficult to engineer by combinatorial methods due to low screening throughput. Biotechnol. Bioeng. 2016;113: 384–392. © 2015 Wiley Periodicals, Inc. Abstract : An enriched library screening strategy was developed that uses iterative rounds of library building and screening to drastically reduce the search space of metabolic pathways constructed using combinatorial methods. This strategy is most beneficial for large pathways that require low‐throughput screening, as exemplified by the implementation of this strategy on the pathway of the anti‐malarial FR900098 in this study.