A Green Route for High-Yield Production of Tetramethylpyrazine From Non-Food Raw Materials

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.

Metabolic Engineering of Escherichia coli for High-Level Production of (R)-Acetoin from Low-Cost Raw Materials

“…Cassava powder and cottonseed meal were respectively obtained from Guangxi State Farms Mingyang Biochemical Group, Inc. (Nanning, China) and Jinan Oumi Biotechnology Co., Ltd. (Jinan, China). Thermostable α-amylase and glucoamylase obtained from Novozymes (Novozymes China Biotechnology Co., Ltd., Tianjin, China) were used for liquefaction and saccharification of raw starch as previously described [ 7 ]. The acid protease was purchased from Imperial Jade Biotechnology Co., Ltd. (Yinchuan, China) for protein hydrolysis [ 7 ].…”

“…Thermostable α-amylase and glucoamylase obtained from Novozymes (Novozymes China Biotechnology Co., Ltd., Tianjin, China) were used for liquefaction and saccharification of raw starch as previously described [ 7 ]. The acid protease was purchased from Imperial Jade Biotechnology Co., Ltd. (Yinchuan, China) for protein hydrolysis [ 7 ].…”

“…The composition of the initial fermentation medium (IFM) was as follows: 100 g/L glucose, 10 g/L peptone, 7 g/L yeast extract, 0.5 g/L NaCl, 0.2 g/L MgSO 4 , 0.5 g/L betaine monohydrate and 0.1 g/L thiamine. The cassava powder and cottonseed meal were enzymatically hydrolyzed as described previously to make the hydrolysate medium [ 7 ]. Briefly, 18.8 and 6.7% ( w/v ) cassava powder and cottonseed meal (pH 6.3) were pretreated by autoclaving with 10 mL of α-amylase solution (0.5 g CaCl 2 was added to 4 mL α-amylase and diluted with distilled water to a total volume of 100 mL) at 121 °C for 15 min.…”

“…As a popular flavoring agent used in foods and cosmetics industries, due to its pleasant aroma, acetoin is recognized as safe (GRAS) by JECFA and US FDA [ 5 ]. Additionally, acetoin is also an important precursor of tetramethylpyrazine, an active pharmaceutical ingredient originally isolated from Ligusticum wallichii for curing cardiovascular and cerebrovascular diseases [ 6 , 7 ]. Acetoin has one chiral center in the molecule and, therefore, exists as two stereoisomers, ( R )-acetoin and ( S )-acetoin.…”

Prospective bacterial and fungal sources of hyaluronic acid: A review

“…Corynebacterium glutamicum is a non-pathogenic and non-sporulating bacteria classified as GRAS. It is widely used for large-scale production of various organic compounds, such as organic acids [262] , [263] , alcohols [264] , [265] , [266] and pyrazine [267] , [268] , [269] , and has a special significance in the industrial production of amino acids [270] , [271] , [272] , [273] , [274] . In a detailed survey, the variety of applications of this organism in industry was analyzed by Becker [275] .…”