Evaluation of thrB and thrC gene expression in Corynebacterium glutamicum ATCC 13032 during in vitro L-threonine production

Daryaei Ghazani, Nastaran; Abbasi Yusefabad, Nadia; Rahbarnia, Leila; Dehnad, Alireza; Ebrahimpour, Aria; Shokrollahi, Amir Ardalan; Benyamin, Pollet; Farid, Ali

doi:10.48307/bcbn.2025.561212.1042

Evaluation of thrB and thrC gene expression in Corynebacterium glutamicum ATCC 13032 during in vitro L-threonine production

Document Type : Original Article

Authors

Nastaran Daryaei Ghazani ¹

Nadia Abbasi Yusefabad ¹

Leila Rahbarnia ²

Alireza Dehnad ³

Aria Ebrahimpour ¹

Amir Ardalan Shokrollahi ⁴

Pollet Benyamin ¹

Ali Farid ¹

¹ Rabe Rashid Institute of Higher Education, Tabriz, Iran

² Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

³ Department of Biotechnology and Microbiology, Center for Research and Education of Agriculture and Natural Resources of East Azarbaijan Province, Department of Bacterial Disease Research, Tabriz, Iran

⁴ Department of Food Science, College of Agriculture, University of Tabriz, Tabriz, Iran

10.48307/bcbn.2025.561212.1042

Abstract

Background: L-threonine is an essential amino acid required for normal growth and tissue synthesis in humans and animals and is widely used in the food and pharmaceutical industries.
Objectives: The present study aimed to enhance laboratory-scale threonine production by optimizing the culture medium composition and physicochemical conditions of Corynebacterium glutamicum as a suitable production host. In parallel, the expression patterns of the key threonine biosynthetic genes, thrB and thrC, were evaluated using real-time polymerase chain reaction (PCR).
Methods: C. glutamicum was initially cultivated in ISP2 medium, after which different temperature ranges, pH values, and alternative carbon sources, including cheesewater, bagasse, molasses, and corn syrup, were assessed to optimize the medium formulation. Threonine production under different sugar sources and pH conditions was quantified using spectrophotometric and chromatographic methods. The optimal physicochemical conditions for threonine biosynthesis were selected, and the expression levels of the key biosynthetic genes thrB and thrC were analyzed by real-time PCR.
Results: Chromatographic analysis indicated that supplementation with 6 g/L cheesewater and adjustment of the pH to 7.3 provided the most favorable conditions for L-threonine production. The highest threonine concentrations obtained in the tested culture media were 18,263 mg/L for molasses, 16,219.7 mg/L for bagasse, 6,843.2 mg/L for corn syrup, and 39,393.8 mg/L for cheesewater. Among the evaluated substrates, the cheesewater-containing medium yielded the highest threonine concentration. Maximum threonine production was consistently observed at pH 7.3. Real-time PCR analysis demonstrated that the cheesewater-based medium induced a significant upregulation of threonine biosynthetic genes. Specifically, thrB expression increased from 1.09-fold to 1.10-fold (p<0.0001), while thrC expression increased from 1.08-fold to 1.09-fold relative to the standard medium (p<0.00017).
Conclusion: These findings indicate that natural substrates such as cheesewater can serve as safe, economical, and effective carbon sources for improving threonine production. Moreover, the results underscore the substantial influence of physicochemical parameters of the culture medium on L-threonine biosynthesis by C. glutamicum, as reflected at both the metabolic and transcriptional levels.

Keywords

Threonine

Corynebacterium glutamicum

Real-time PCR

thrB and thrC gene expression

Subjects