# Optimization of Fermentation Conditions for the Production of 2,3,5-Trimethylpyrazine by Recombinant Bacillus licheniformis

## TL;DR

This study improves the production of 2,3,5-trimethylpyrazine using a genetically modified Bacillus licheniformis strain and optimized fermentation conditions.

## Contribution

A novel recombinant strain and optimized fermentation parameters significantly increased 2,3,5-trimethylpyrazine yield.

## Key findings

- Recombinant strain YC7/pHT01-BlTDH (N157A) achieved a TMP yield of 15.35 mg/L.
- Optimized conditions (substrate ratio 1:2, 1.0 mM IPTG, 4 d fermentation) increased TMP yield to 44.52 mg/L.
- The yield improvement was 29.17 mg/L compared to initial conditions.

## Abstract

2,3,5-Trimethylpyrazine (TMP) is an alkyl pyrazine with broad application prospects in the fields of food additives and medicine. L-threonine-3-dehydrogenase (TDH) is a key enzyme in the biosynthesis pathway of TMP. To explore the efficient and environmentally friendly production method of TMP, we constructed recombinant strains overexpressing the BlTDH gene and its mutant BlTDH (N157A) using Bacillus licheniformis YC7. The TMP yield of recombinant strains with pHT01-BlTDH (N157A) reached 15.35 ± 1.51 mg/L, which was significantly higher than that of strains with pHT01-BlTDH (9.86 ± 1.24 mg/L) and strains with vector pHT01 (2.35 ± 0.58 mg/L). To further increase the TMP yield of strain YC7/pHT01-BlTDH (N157A), the fermentation process was optimized by single-factor experiments, and the response surface test was conducted using the Box–Behnken design. The results revealed that the substrate ratio, IPTG concentration and fermentation time had significant effects on the yield of TMP, and the degree of influence was substrate ratio > fermentation time > IPTG concentration. The optimization results of response surface indicated that the optimal fermentation conditions were as follows: substrate ratio of 1:2, IPTG concentration of 1.0 mM, and fermentation time of 4 d. Under these conditions, the TMP yield reached 44.52 ± 0.21 mg/L, which was around 0.005 mg/L different from the predicted value (45.515 mg/L), and increased by 29.17 mg/L compared with the initial condition. The optimization of fermentation conditions significantly increased the yield of TMP produced by recombinant strains, which provided a theoretical basis and strain resources for industrial production of TMP.