Effective 5‐aminolevulinic acid production via T7 RNA polymerase and RuBisCO equipped Escherichia coli W3110

Abstract

Chromosome-based engineering is a superior approach for gene integration generating a stable and robust chassis. Therefore, an effective amplifier, T7 RNA polymerase (T7RNAP) from bacteriophage, has been incorporated into Escherichia coli W3110 by site-specific integration. Herein, we performed the 5-aminolevulinic acid (5-ALA) production in four T7RNAP-equipped W3110 strains using recombinant 5-aminolevulinic synthase and further explored the metabolic difference in best strain. The fastest glucose consumption resulted in the highest biomass and the 5-ALA production reached to 5.5 g/L; thus, the least by-product of acetate was shown in RH strain in which T7RNAP was inserted at HK022 phage attack site.Overexpression of phosphoenolpyruvate (PEP) carboxylase would pull PEP to oxaloacetic acid in tricarboxylic acid cycle, leading to energy conservation and even no acetate production, thus, 6.53 g/L of 5-ALA was achieved. Amino acid utilization in RH deciphered the major metabolic flux in α-ketoglutaric acid dominating 5-ALA production. Finally, the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulokinase were expressed for carbon dioxide recycling; a robust and efficient chassis toward low-carbon assimilation and high-level of 5-ALA production up to 11.2 g/L in fed-batch fermentation was established.

Non-native Pathway Engineering with CRISPRi for Carbon Dioxide Assimilation and Valued 5-Aminolevulinic Acid Synthesis in Escherichia coli Nissle

“…An artificial CO 2 -fixing pathway in EcN was reconstructed at the downstream of the ribulose route to provide an additional carboxylation step, as in former works. , Two different routes, R15P and Ru5P, were added to connect the pentose phosphate (PP) pathway to 3-phosphoglycerate (3PG), which is an intermediate in glycolysis. The first and second schemes were designated as RR and PR, respectively (Figure A).…”

“…E. coli DH5α was used for regular cloning, while T7RNAP-equipped MG1655 and EcN (MT7 and ET7) were used for functional tests. Luria–Bertani (LB) medium was used for preculture, while the MM9 medium (12.8 g/L Na 2 HPO 4 , 3 g/L KH 2 PO 4 , 0.5 g/L NaCl, 1 g/L NH 4 Cl, 0.24 g/L MgSO 4 , 0.011 g/L CaCl 2 ) with 10 g/L glucose or xylose was used in the CO 2 assimilation and 5-ALA production . The antibiotics at concentrations of 100 mg/L ampicillin, 50 mg/L kanamycin, and 25 mg/L chloramphenicol were added, while 0.1 mM IPTG was used for induction.…”

“…The recombinant cells were cultured aerobically in a 250 mL baffled flask with 30 mL of glucose-based MM9 medium without CO 2 supply . The whole culturing process was placed at 37 °C with shaking at 200 rpm.…”

“…Recently, EcN studies have been broadened as a safe chemical producer, thus leveraging its exploration for MCF. For instance, EcN has effectively produced a high titer bulk chemical of itaconic acid and high-value chemicals of p -coumaric acid, heparosan, aminobutyric acid, and 5-aminolevulinic acid (5-ALA). − Of valuable chemicals and drugs, 5-ALA is a metabolic hub crucial for heme precursor and also has received approval from the FDA as a second-generation photosensitizer and photodynamic drug for the treatment of glioma. − Biosynthesis of 5-ALA is majorly accomplished via the C4 pathway by overexpressing heterologous ALA synthase (ALAS), and CO 2 is a definitive side product. , Previous works of 5-ALA production from glucose have been integrated with the CO 2 biomitigation system by co-overexpressing RuBisCO and PRK. , However, rerouting flux from glucose into Ru5P would release additional CO 2 , indicating that CO 2 assimilation from chemical biosynthesis has not been effectively executed. , Moreover, as the strain used was E. coli BL21 or K-12, manipulating the acetate pathway was necessary to hinder a high acetate accumulation and maintain strain durability. , Hence, harnessing the acidophilic nature of EcN to express the CO 2 -fixing system and fine-tuning its artificial pathway are outright solutions for assimilating CO 2 emission during 5-ALA production.…”

ASIA: An automated stress‐inducible adaptor for enhanced stress protein expression in engineered Escherichia coli

“…The orthogonality of T7 RNA polymerase (T7RNAP) with the T7 promoter is highly effective for initiating protein expression in Escherichia coli, and it is widely applicable across diverse organisms, including Bacillus subtilis, yeast, cyanobacteria-spanning from prokaryotic to eukaryotic systems (Wang et al, 2018), and even in cellfree systems recently (Wang et al, 2023). However, the substantial energy demands associated with T7 system (Landberg et al, 2020;Tan & Ng, 2020), particularly when expressing toxic and stressinducing proteins, have been known to hinder cell growth and lead to cell lysis during cultivation occasionally (Stargardt et al, 2020(Stargardt et al, , 2021Ting & Ng, 2023). On the other hand, the leakage expression of T7RNAP results in the rapid production of target proteins during the noninducing period and depletes resources for essential cellular growth.…”