Biosensor Innovation Enhances Itaconic Acid Yields

Abstract

Acetate is a cost-effective and sustainable carbon source that, despite its potential, remains underutilized. This study employed biosensor-assisted adaptive laboratory evolution (ALE) to enhance itaconic acid production and acetate metabolism in Escherichia coli. The evolved E. coli W strains exhibited 65% increase in itaconic acid production and 71% increase in growth rate, and 45% increase in itaconic acid yield. A common 31-kb genomic deletion was identified in the evolved strains, with two genes, ecw_m2276 and ecw_m2277, driving the observed phenotypic changes. The evolved strains exhibited an intensified stringent response, which enhanced the acetate-utilizing pathway and resulted in over a 5,000% increase in the expression of the glyoxylate shunt, thereby boosting microbial growth. Overexpression of relA further replicated these enhanced phenotypes. Our findings highlight not only significant physiological improvements but also present a novel strategy for enhancing microbial growth and bioproduction from acetate, offering valuable insights for industrial biotechnology applications.

A novel evolutionary technique, designed to select E. coli strains capable of more efficiently metabolizing acetate-a sustainable, cost-effective carbon source-has been developed. Strains evolved through this method demonstrate a 1.7-fold increase in their ability to convert acetate into itaconic acid, a key raw material for eco-friendly adhesives and bioplastics. The research team anticipates that this advancement paves the way for transforming microbial cell factories into stable, efficient producers of chemical raw materials.

A research team, led by Professor Donghyuk Kim from the School of Energy and Chemical Engineering at UNIST, in collaboration with Professor Gyoo Yeol Jung from POSTECH and Dr. Myung Hyun Noh from the Korea Research Institute of Chemical Technology (KRICT), announced that they successfully developed E. coli strains with an average 1.7-fold improvement in converting acetate to itaconic acid.

Itaconic acid is a biodegradable polymer and a key component in medical adhesives. Currently, it is predominantly produced via fermentation of starches by fungi, a process that consumes significant food resources and incurs high production costs. As an alternative, acetate-mainly derived from vinegar-offers a cheap and abundant substrate. Moreover, utilizing acetate coupled with carbon dioxide capture can contribute to carbon reduction efforts. However, microbes typically struggle to efficiently metabolize acetate due to toxicity and metabolic burden, resulting in limited itaconic acid yields.

To overcome this challenge, the research team employed an adaptive laboratory evolution (ALE) approach, selecting for strains that thrive on high itaconic acid levels. They integrated biosensors-designed to express antibiotic resistance genes in response to acetate-derived itaconic acid concentrations-into *E. coli*. By gradually increasing antibiotic levels during repeated cultivation cycles, only those strains capable of producing high levels of itaconic acid survived and thrived.

After approximately 50 generations of laboratory evolution, the team isolated strains exhibiting 1.7 times higher itaconic acid production and faster cell division rates compared to the original strains.

To elucidate the genetic basis of these improvements, whole-genome and transcriptome analyses were performed. Remarkably, the evolved strains possessed a large genomic deletion spanning approximately 31,000 base pairs, including two genes involved in acetate metabolism and growth efficiency. The deletion was found to induce the stringent response, a stress adaptation mechanism, altering cellular physiology to favor enhanced acetate utilization and growth.

Jihoon Woo, the first author of the study, explained, "While the stringent response is typically known to suppress growth and conserve resources under stress, in this case, it surprisingly facilitated the more efficient metabolism of acetate and improved both growth and production."

Further experiments demonstrated that overexpressing relA, a gene responsible for synthesizing the signaling molecule ppGpp that triggers the stringent response, recapitulated the enhanced phenotypes observed in the evolved strains.

Professor Kim commented, "Through this evolutionary and systems biology approach, we reinterpreted microbial physiological responses and turned what was previously considered a disadvantage-the stringent response-into an advantage for bioproduction." He further noted, "This strategy offers promising insights for developing sustainable chemical manufacturing technologies in the post-fossil fuel era."

This research was supported by the Ministry of Science and ICT (MSIT), the National Research Foundation (NRF) of Korea, and the Ministry of Oceans and Fisheries' Marine Biotechnology Program. It was published in the June 2025 of Bioresource Technology, an international peer-reviewed journal.

Journal Reference

Jo Hyun Moon, Jihoon Woo, Joon Young Park, et al., "Biosensor-guided evolution boosts itaconic acid production, unveiling unique insights into the stringent response," Bioresour. Technol., (2025).