Synergistic Production of Lycopene and β-Alanine Through Engineered Redox Balancing in Escherichia coli

Abstract

The production of β-alanine from fatty acid feedstocks presents a promising synthetic strategy due to its high carbon yield. However, the excessive reducing power generated during fatty acid utilization disrupts cellular redox balance, adversely affecting metabolism and limiting the efficiency and final yield of β-alanine production. To address this challenge, we engineered a co-production system in which excess reducing equivalents generated during fatty acid β-oxidation and β-alanine biosynthesis were consumed by growth-coupled lycopene biosynthesis. The resulting dual-pathway strain, SA01, achieved 44.78 g/L β-alanine and 3.07 g/L lycopene in bioreactor fermentation, representing a 21.45% increase in β-alanine production compared to the β-alanine-producing strain WA01, and a 74.43% increase in lycopene production compared to the lycopene-producing strain LA01. Further optimization in strain SA06, involving cofactor engineering to shift redox flow from NADH to NADPH, enhanced the titers to 52.78 g/L β-alanine and 3.61 g/L lycopene. Metabolite analysis confirmed a decrease in intracellular NADH and FADH₂ levels in SA06, indicating restoration of redox balance during the late fermentation phase. Additional improvements in the fermentation process, including gradual carbon source switching, optimization of the induction strategy, and fine-tuning of conditions during both growth and bioconversion phases, resulted in further increases in product titers, reaching 72 g/L β-alanine and 6.15 g/L lycopene. This study offers valuable insights into the development of microbial co-production systems, highlighting the critical role of dynamic cofactor and redox balance management, as well as process optimization, in improving production efficiency.

Keywords: Escherichia coli; lycopene; metabolic engineering; redox balance; β-alanine.

Figure 1

Comparative analysis of β-alanine production performance in different engineered strains. (A) Shake-flask level evaluation of β-alanine biosynthesis. The bar graph illustrates β-alanine titers achieved through whole-cell bioconversion using palmitic acid as a substrate over 18 h. The line graph represents the corresponding yield, calculated as the mass ratio of β-alanine produced to palmitic acid consumed. (B) Comparative fermentation profiles of strains WA01 and WA05 in 1-L bioreactors.

Figure 2

Reducing equivalent (NAD(P)H/FADH₂) and ATP generation and consumption in engineered metabolic pathways.

Figure 3

Comparative analysis of β-alanine production performance in dual-pathway strains. (A) Shake-flask level evaluation of lycopene and β-alanine production; (B) fermentation profiles of strains SA01 in 1-L bioreactor; (C) fermentation profiles of strains SA05 in 1-L bioreactor; (D) fermentation profiles of strains SA06 in 1-L bioreactor; (E) fermentation profiles of strains SA07 in 1-L bioreactor.

Figure 4

Metabolite analysis of strains WA01 (grey) and SA06 (blue) during fermentation. The stages of fermentation are represented as early (E, 20 h), middle (M, 45 h), and late (L, 60 h).

Figure 5

The optimization of the fermentation process for strain SA06.