# Enhancing Synthesis Efficiency in Microbial 1,5-Pentanediol Production Through Transcriptomics-Informed Metabolic Engineering of Escherichia coli

## TL;DR

This study uses transcriptomics to improve 1,5-pentanediol production in E. coli by identifying and modifying key genes, leading to higher yields and efficiency.

## Contribution

The study introduces a transcriptomics-guided approach to identify novel genetic targets for enhancing 1,5-PDO production in E. coli.

## Key findings

- Overexpression of fecA and deletion of gadA significantly increased 1,5-PDO production in shake flask cultures.
- Strain S7 achieved a 12.45 g/L titer and improved glucose yield in 5 L bioreactor fed-batch fermentation.
- The optimized strain showed a 15.6% improvement in carbon conversion efficiency and 7.6% better biomass accumulation.

## Abstract

The microbial production of 1,5-pentanediol (1,5-PDO), a versatile platform chemical with extensive industrial applications, remains limited by suboptimal fermentation titers and incomplete understanding of metabolic bottlenecks. To address these challenges, this study employed comparative transcriptomics to systematically identify novel genetic targets capable of enhancing 1,5-PDO biosynthesis in engineered Escherichia coli. Transcriptomic profiling of the 1,5-PDO-producing strain relative to the parental E. coli W3110, conducted at both exponential (24 h) and stationary (96 h) growth phases, revealed 1384 significantly differentially expressed genes, including 851 upregulated and 533 downregulated genes. From these, 20 candidate metabolic genes associated with 1,5-PDO synthesis were selected for functional validation through plasmid-based overexpression or CRISPR interference (CRISPRi)-mediated repression. Reverse engineering confirmed that overexpression of fecA (encoding an iron(III)-citrate transporter) and deletion of gadA (encoding glutamate decarboxylase) significantly enhanced 1,5-PDO production. Subsequent chromosomal integration of fecA at the neutral ilvG locus and deletion of gadA generated the optimized strain S7, which achieved a 1,5-PDO titer of 1.7 g/L in shake flask cultures, representing a 13.3% increase over the parental strain, with a concomitant 50% improvement in glucose yield (0.18 mol/mol). In fed-batch fermentation at the 5 L bioreactor scale, strain S7 attained a titer of 12.45 g/L and a glucose yield of 0.26 mol/mol, marking a 15.6% enhancement in carbon conversion efficiency relative to the parental strain (0.225 mol/mol), while concurrently improving biomass accumulation by 7.6%. These findings demonstrate that transcriptomics-guided reverse engineering constitutes an effective strategy for elucidating nonobvious metabolic determinants and optimizing microbial cell factories for efficient 1,5-PDO production. The identification of fecA and gadA as beneficial targets provides valuable insights into the metabolic rewiring underlying enhanced 1,5-PDO biosynthesis and establishes a foundation for further strain improvement through systems metabolic engineering.