Development of a growth coupled dynamic regulation network balancing malonyl-CoA node to enhance (2S)-naringenin synthesis inE. coli

Abstract

<jats:title>Abstract</jats:title><jats:p>Generally, high- and low-performance nongenetic variants and young and aged cells co-existed in culture at all growth phases. In this regard, individually and dynamically regulating the metabolic flux of single cells based on their cellular state is highly useful for improving the performance of populations. However, balancing the trade-offs between biomass formation and compound over-production requires sophisticated dynamic regulation networks (DRNs) which can be challenging. Here, we developed a growth coupled NCOMB (Naringenin-Coumaric acid-Malonyl-CoA-Balanced) DRN with systematic optimization of (2<jats:italic>S</jats:italic>)-naringenin and <jats:italic>p</jats:italic>-coumaric acid-responsive regulation pathways for real-time control of intracellular supply of malonyl-CoA. In doing so, the acyl carrier protein was used as a new critical node for fine-tuning malonyl-CoA consumption instead of fatty acid synthase. Following directed evolution of the NCOMB DRN, we obtained a strain with cumulative 8.4-fold improvement in (2<jats:italic>S</jats:italic>)-naringenin production with balanced cell growth, demonstrating the high efficiency of this system for improving pathway production.</jats:p>

Fermentation and Metabolic Pathway Optimization to De Novo Synthesize (2S)-Naringenin in Escherichia coli

“…4A). This (2S)-naringenin titer was 1.6-fold (391 mg/l vs. 251 mg/l) higher than the un-optimized titer in our previous report [19].…”

“…Furthermore, an expensive inducer (isopropyl-β-d-thiogalactoside) was used in the fermentation process, which elevated the biosynthesis cost. To overcome these challenges, using our previously constructed dynamic regulation strain (Mut-17) [ 19 ], we systemically optimized the components of the fermentation medium and fermentation conditions, obtaining 391 mg/l (2 S )-naringenin from 3 mM L-tyrosine. Furthermore, we released the tyrosine feedback inhibition by deleting tyrR and overexpressing aroG fbr and tyrA fbr .…”

“…The thin arrows represent genes that were natively expressed. ( B ) The dynamic regulation network of Mut-17, which was constructed in our previous study [ 19 ]. (2 S )-Naringenin synthesis pathway was constitutively overexpressed [ 7 ].…”

“…In this study, we established an induction-free system in our previously constructed dynamic regulated E. coli strain, which can achieve automatic balancing of (2 S )-naringenin production, p -coumaric acid accumulation, and cell growth ( Figs. 1A and 1B ) [ 19 ]. To do this, we first optimized the fermentation conditions (pH and temperature) and medium contents (carbon sources, nitrogen source, and MnCl 2 ).…”

Design and Assembly of a Biofactory for (2S)-Naringenin Production in Escherichia coli: Effects of Oxygen Transfer on Yield and Gene Expression

“…These are the highest titers that have been reported [ 16 , 17 ]. Nonetheless, several strategies have been used to optimize (2 S )-naringenin production, such as the used a cipher of evolutionary design (CiED) to predict gene deletions, and the development a biosensor to respond to dynamical environmental changes [ 16 , 18 , 19 ]. In order to obtain commercially viable microbial factories, it is necessary to study variables such as biosynthetic pathways and gene selection, fermentation conditions, and microorganism type using a systematic approach.…”