image: A: Optimization of the inorganic nitrogen source; B: Optimization of the supplementation of organic nitrogen sources; C: Fermentation curve of strain E. coli H25 after nitrogen source optimization in a 5 L fermenter; D: Single-factor optimization of fermentation parameters in a 5 L fermenter; E: Fermentation curve of strain E. coli H25 under the optimized fermentation conditions in a 5 L fermenter; F: The impact of process optimization on key gene expression; G: The impact of process optimization on the content of key intracellular metabolites
Credit: Acta Microbiologica Sinica
L-Histidine is a high-value, imidazole-containing amino acid with broad applications in the pharmaceutical, food, and feed-additive industries, and its functional derivatives such as carnosine and ergothioneine play important roles in antioxidant defense. However, fermentative production of L-histidine has long been hampered by three interconnected bottlenecks: low carbon flux through the core biosynthetic route, insufficient supply of the precursor 5-phosphoribosyl pyrophosphate (PRPP), and heavy accumulation of organic acid by-products such as formate and acetate. Now, a research team led by Prof. Jing Wu at Jiangnan University in Wuxi, China, has systematically re-engineered Escherichia coli BL21 to overcome these limitations.
“L-histidine is a vital amino acid for human health and nutrition, yet its sustainable production by fermentation has lagged behind other amino acids because the biosynthetic pathway is tightly regulated by both transcriptional attenuation and end-product feedback inhibition, and synthesizing one molecule consumes about 9.4 molecules of ATP,” said Jing Wu, corresponding author of the study and a professor at Jiangnan University. “We set out to provide a more efficient and greener route for producing this high-value amino acid.”
The team applied an integrated four-part engineering strategy. First, to strengthen the core biosynthetic flux, they removed the transcriptional attenuation of the histidine operon (hisL), introduced a feedback-resistant ATP phosphoribosyltransferase mutant (CgHisGG233H/T235Q) from Corynebacterium glutamicum, and overexpressed the hisDCBHAFI gene cluster on a plasmid. Second, to reinforce precursor supply, they deleted the purine repressor purR and introduced the PRPP synthase SmPrs from Serratia marcescens and the allosterically deregulated 6-phosphogluconate dehydrogenase CgGndS361F, while overexpressing glucose-6-phosphate dehydrogenase Zwf, thereby reconstructing the pentose phosphate pathway to increase PRPP availability. Third, to reduce carbon loss, they eliminated key formate- and acetate-generating genes (pflB-focA, pta, tdcD, and tdcE). Finally, they optimized the nitrogen-source composition and fermentation parameters.
Under the optimized conditions in a 5-L fermenter, the optimal combination of 7 g/L ammonium sulfate, 7 g/L yeast extract, and 21 g/L corn steep liquor powder, together with a temperature of 34 °C, pH 6.7, and 35% dissolved oxygen, enabled the final strain E. coli H25 to reach an L-histidine titer of 34.84 g/L, a yield of 0.13 g/g, and a productivity of 0.73 g/(L·h) — an increase of about 127% in titer over the pathway-optimized strain H10. Formate and acetate were reduced to only 0.2 g/L and 0.3 g/L, respectively, markedly lowering carbon loss.
Transcript and intracellular metabolite analyses further revealed the drivers of these improvements. Process optimization enhanced the expression of the core-pathway operon hisGDCBHFI and up-regulated the ammonia-donor genes gdhA and glnA by 32.34% and 46.21%, respectively, while intracellular levels of ATP, NAD⁺, L-glutamate, and L-glutamine rose by 71.59%, 19.19%, 25.77%, and 24.00%. The synergy of improved energy status, redox balance, and nitrogen-precursor supply created a favorable intracellular environment that drives efficient L-histidine accumulation.
The team is now working to push the strain further, planning to strengthen substrate uptake and nitrogen assimilation networks and to integrate transcriptomics, metabolomics, and 13C flux analysis to uncover new targets for even higher yields and process stability, with the goal of providing a practical, sustainable route for the industrial biosynthesis of L-histidine and other high-nitrogen amino acids.
The study was conducted by Zhaoyang Chen, Jia Liu, Xin Dong, Guipeng Hu, Yiwen Zhou, and Jing Wu, affiliated with the School of Life Sciences and Health Engineering and the School of Biotechnology (Key Laboratory of Industrial Biotechnology of Ministry of Education) at Jiangnan University. The research was supported by the Key Program of the National Natural Science Foundation of China (22538006). The paper was published in Acta Microbiologica Sinica (DOI: 10.13343/j.cnki.wsxb.20260208).
Article Title
Construction and fermentation optimization of an Escherichia coli cell factory for L-histidine production
Article Publication Date
4-Oct-2026