Production of tetra-methylpyrazine using engineered Corynebacterium glutamicum

Abstract

Corynebacterium glutamicum ATCC 13032 is an established and industrially-relevant microbial host that has been utilized for the expression of many desirable bioproducts. Tetra-methylpyrazine (TMP) is a naturally occurring alkylpyrazine with broad applications spanning fragrances to resins. We identified an engineered strain of C. glutamicum which produces 5 ​g/L TMP and separately, a strain which can co-produce both TMP and the biofuel compound isopentenol. Ionic liquids also stimulate TMP production in engineered strains. Using a fed batch-mode feeding strategy, ionic liquid stimulated strains produced 2.2 ​g/L of tetra-methylpyrazine. We show that feedback from a specific heterologous gene pathway on host physiology leads to acetoin accumulation and the production of TMP.

Advances in metabolic engineering of Corynebacterium glutamicum to produce high-value active ingredients for food, feed, human health, and well-being

“…Together with specifically created C. glutamicum deletion mutants these experiments allowed elucidation of the biosynthetic pathways that produce pyrazines in detail. More recently, heterologous strains of C. glutamicum, which expressed mevalonate kinase from S. aureus and C. kroppenstedtii and 3-hydroxy-3-methylglutaryl-CoA reductase from S. aureus, allowed the synthesis of up to 5 g.l −1 tetra-methylpyrazine [115].…”

Genomics Characterization of an engineered Corynebacterium glutamicum in Bioreactor Cultivation under Ionic Liquid Stress

“…These included NADH dehydrogenase (Cgl1465), succinate dehydrogenase, sdhABC genes at 24 h and 41 h; cytochrome oxidase, ctaDCEF, cytochrome reductase, qcrCAB and the ATP synthase complex (Cgl1206 to Cgl1213) genes at 24 h, 41 h and 65 h. Amino acid biosynthetic genes upregulated at the later time points included the arginine biosynthetic genes argC argJ, argB and argH at 65 h and argG and argD at mid cultivation phase (41 h). ArgJ protein was also enriched in the acetoin/TMP producing C. glutamicum strain (Eng et al, 2020). Genes encoding for other amino acid biosynthesis included Cgl1139/metE, Cgl2446/metB and Cgl0653/metY at 24 h and 41 h from the methionine/homocysteine pathway; Cgl2204/ilvE at 24 h and Cgl1273/ilvC at 24 h and 41 h in the branched amino acid pathway.…”

“…Several genes encoding for metabolic reactions related to acetoin and TMP accumulation were specifically upregulated in the presence of 50 mM of [Ch][Lys] at the 24 h or 41 h time points (Supplementary Data, Dataset S3, Supplementary Figure S4) when compared to the control samples at the same time points. Of the two subunits of the acetolactate synthase (ALS) ilvB and ilvN, the smaller regulatory subunit, Cgl1272/ilvN was upregulated in the presence of IL fed-batch cultivation when compared to the absence of IL at 24 h. Acetolactate synthase in C. glutamicum takes part in diverting pyruvate flux towards branched chain amino acids biosynthesis and acetoin biosynthesis and could be a precursor to TMP (Eng et al, 2020) (Figure 5). Although branched chain amino acid biosynthesis has been extensively researched for engineering branched chain alcohol (e.g.…”

“…For RNAseq extraction, 5mL culture samples were harvested in 1 mL aliquots, collected by centrifugation at 14,000xg for 3 minutes, and stored at -80 ˚C until subsequent RNA extraction. The supernatant from one of the appropriate time point aliquots was processed for organic acid analysis as described previously (Eng et al, 2020). Lab-scale IP production runs in deep well plates or 5 mL culture tubes were conducted as previously described (Eng et al, 2020).…”

“…The supernatant from one of the appropriate time point aliquots was processed for organic acid analysis as described previously (Eng et al, 2020). Lab-scale IP production runs in deep well plates or 5 mL culture tubes were conducted as previously described (Eng et al, 2020). Isopentenol titers reported for the deep well plate format were corrected for evaporation at the 48 h time point as conducted previously (Sasaki et al, 2019).…”