Abstract
2,3,5,6-Tetramethylpyrazine (TMP) is an active pharmaceutical ingredient originally isolated from Ligusticum wallichii for curing cardiovascular and cerebrovascular diseases and is widely used as a popular flavoring additive in the food industry. Hence, there is a great interest in developing new strategies to produce this high-value compound in an ecological and economical way. Herein, a cost-competitive combinational approach was proposed to accomplish green and high-efficiency production of TMP. First, microbial cell factories were constructed to produce acetoin (3-hydroxy-2-butanone, AC), an endogenous precursor of TMP, by introducing a biosynthesis pathway coupled with an intracellular NAD+ regeneration system to the wild-type Escherichia coli. To further improve the production of (R)-AC, the metabolic pathways of by-products were impaired or blocked stepwise by gene manipulation, resulting in 40.84 g/L (R)-AC with a high optical purity of 99.42% in shake flasks. Thereafter, an optimal strain designated GXASR11 was used to convert the hydrolysates of inexpensive feedstocks into (R)-AC and achieved a titer of 86.04 g/L within 48 h in a 5-L fermenter under optimized fermentation conditions. To the best of our knowledge, this is the highest (R)-AC production with high optical purity (≥98%) produced from non-food raw materials using recombinant E. coli. The supernatant of fermentation broth was mixed with diammonium phosphate (DAP) to make a total volume of 20 ml and transferred to a high-pressure microreactor. Finally, 56.72 g/L TMP was obtained in 3 h via the condensation reaction with a high conversion rate (85.30%) under optimal reaction conditions. These results demonstrated a green and sustainable approach to efficiently produce high-valued TMP, which realized value addition of low-cost renewables.
Highlights
B. licheniformis BL1 (Engineered) B. licheniformis BLC (Engineered) B. subtilis BJ3-2 B. subtilis BS2 B. subtilis CICC 10211 B. subtilis CICC 20030
The pH was adjusted to 6.3 using 20% NaOH solution and 7.5 ml α-amylase solution (0.5 g CaCl2 was added to 2 ml α-amylase, the final volume was adjusted to 100 ml with distilled water) was added before autoclaving at 121°C for 15 min to degrade the structure of starch grains for better hydrolysis performance
Compared with a standpoint of an enzymatic condensation process, the predominant experimental results tend to support the viewpoint that the precursor of TMP was biosynthesized in vivo whereas the subsequent condensation was an in vitro nonenzymatic process (Zhu et al, 2010; Xiao et al, 2014)
Summary
2,3,5,6-Tetramethylpyrazine (TMP), known as ligustrazine, is a biologically active alkaloid, which occurs naturally in the rhizome of a Chinese traditional medicinal herb, Chuanxiong (Ligusticum wallichii) (Xiao et al, 2006; Chen et al, 2018). TMP is a colorless compound with pleasant roasted, nutty flavor characteristics, and is usually used as a flavor additive in the food and. B. licheniformis BL1 (Engineered) B. licheniformis BLC (Engineered) B. subtilis BJ3-2 B. subtilis BS2 B. subtilis CICC 10211 B. subtilis CICC 20030. Glucose Glucose, acetaldehyde Adlay Glucose Glucose Rapeseed meal, glucose, wheat bran Glucose Glucose. Yeast extract Tryptone, yeast extract Adlay Tryptone, yeast extract Yeast extract, tryptone Rapeseed meal, wheat bran. B. subtilis LB5 B. subtilis XZ1124 Bacillus sp. Diacetylactis FC1 Monacus strain M-3 Paenibacillus polymyxa CICC 23617 P. polymyxa CICC 23617 Serratia marcescens CICC 10187. Starch Glucose, acetoin Glucose Sucrose, corn steep liquor powder, acetoin Corn flour
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