A Green Route for High-Yield Production of Tetramethylpyrazine From Non-Food Raw Materials.
A Green Route for High-Yield Production of Tetramethylpyrazine From Non-Food Raw Materials.
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非食品原料高产生产四甲基吡嗪的绿色路线
DOI：
10.3389/fbioe.2021.792023
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发表时间：
2021
影响因子：
5.7
通讯作者：
Xie N
中科院分区：
文献类型：
--
作者：
Li J;Lu J;Ma Z;Li J;Chen X;Diao M;Xie N
2,3,5,6-川芎嗪（tetramethylpyrazine， TMP）是从川芎中分离出来的一种治疗心脑血管疾病的活性药物成分，是食品工业中广泛使用的调味添加剂。因此，人们对开发新的策略以生态和经济的方式生产这种高价值化合物非常感兴趣。在此基础上，提出了一种具有成本竞争力的组合方法来实现TMP的绿色高效生产。首先，通过将生物合成途径与细胞内NAD+再生系统结合引入野生型大肠杆菌，构建微生物细胞工厂，生产乙酰素（3-羟基-2-丁酮，AC），乙酰素是TMP的内源性前体。为了进一步提高(R)-AC的产量，通过基因操作逐步破坏或阻断副产物的代谢途径，在摇瓶中得到40.84 g/L (R)-AC，光学纯度高达99.42%。然后，利用最佳菌株GXASR11将廉价原料的水解产物转化为(R)-AC，在优化的发酵条件下，在5-L发酵罐中，48 h内滴度达到86.04 g/L。据我们所知，这是利用重组大肠杆菌从非食品原料中生产出的最高(R)-AC的光学纯度（≥98%）。将发酵液上清液与磷酸二铵（DAP）混合，使其总积为20 ml，并转移到高压微反应器中。最终，在最佳反应条件下，3 h的缩合反应可制得56.72 g/L的TMP，转化率高达85.30%。这些结果展示了一种绿色和可持续的方法来高效生产高价值的TMP，实现了低成本可再生能源的增值。
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.
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