Sustainable production of 2,3,5,6-tetramethylpyrazine at high titer in engineered Corynebacterium glutamicum.

The industrial amino acid production workhorse, Corynebacterium glutamicum naturally produces low levels of 2,3,5,6-tetramethylpyrazine (TMP), a valuable flavor, fragrance and commodity chemical. Here we demonstrate TMP production (∼0.8 ​g L-1) in C. glutamicum type strain ATCC13032 via overexpression of acetolactate synthase and/or alpha-acetolactate decarboxylase from Lactococcus lactis in CGXII minimal medium supplemented with 40 g L-1 glucose. This engineered strain also demonstrated growth and TMP production when the minimal medium was supplemented with up to 40% (v v-1) hydrolysates derived from ionic liquid pretreated sorghum biomass. A key objective was to take the fully engineered strain developed in this study and interrogate media parameters that influence the production of TMP, a critical post strain engineering optimization. Design of experiments in a high throughput plate format identified glucose, urea and their ratio as significant components affecting TMP production. These two components were further optimized using response surface methodology. In the optimized CGXII medium, the engineered strain could produce up to 3.56 g L-1 TMP (4-fold enhancement in titers and 2-fold enhancement in yield, mol mol-1) from 80 g L-1 glucose and 11.9 g L-1 urea in shake flask batch cultivation.