Methanol-based acetoin production by genetically engineered Bacillus methanolicus,Green Chemistry

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Methanol-based acetoin production by genetically engineered Bacillus methanolicus
Green Chemistry ( IF 9.3 ) Pub Date : 2019/12/10 , DOI: 10.1039/c9gc03950c
Eivind B. Drejer _{1,

2,

3} , Dennis Tin Chat Chan _{1,

2,

3} , Carsten Haupka _{4,

5,

6,

7} , Volker F. Wendisch _{4,

5,

6,

7} , Trygve Brautaset _{1,

2,

3} , Marta Irla _{1,

2,

3}

Affiliation

Methanol is an attractive alternative non-food feedstock for industrial fermentations that can be used instead of sugar-based raw materials. Here, the thermophilic and methylotrophic bacterium Bacillus methanolicus MGA3 was metabolically engineered to produce the platform chemical (R)-acetoin from methanol at 50 °C. Three different heterologous alsSD/budAB operons, each encoding acetolactate synthase and acetolactate decarboxylase, were functionally expressed under control of an inducible promoter in B. methanolicus MGA3, resulting in up to 0.26 ± 0.04 g L⁻¹ of (R)-acetoin titer in shake flask cultivations. To further improve acetoin production, a total of six different genes or operons were expressed in the acetoin producing strains to increase supply of the acetoin precursor pyruvate. In particular, expression of a gene coding for malic enzyme from Geobacillus stearothermophilus in combination with the isocitrate lyase gene from B. methanolicus MGA3 increased acetoin titers 1.6-fold up to 0.42 ± 0.01 g L⁻¹ which corresponds to 0.07 g g⁻¹ methanol. This resulted in an MGA3 strain overproducing 4 recombinant enzymes in total from two different plasmids with two distinct antibiotics resistance markers, demonstrating the increased complexity of metabolic engineering allowed by newly developed genetic tools for this organism. To our knowledge, this is the first demonstration of microbial production of acetoin from methanol.

中文翻译：

基因改造的甲醇芽孢杆菌生产基于甲醇的乙酰丙酮

甲醇是用于工业发酵的一种有吸引力的非食品原料，可以代替糖基原料使用。在这里，嗜热和甲基营养细菌甲醇芽孢杆菌MGA3经过代谢工程改造，从甲醇在50°C生成平台化学（R）-乙酰丙酮。在甲醇双歧杆菌MGA3中在诱导型启动子的控制下功能性表达三个不同的异源alsSD / budAB操纵子，每个操纵子编码乙酰乳酸合酶和乙酰乳酸脱羧酶，产生高达0.26±0.04g L ^-1的（R摇瓶培养中的）-乙醛滴定度。为了进一步提高乙酰丙酮的产量，在产生乙酰丙酮的菌株中总共表达了六个不同的基因或操纵子，以增加乙酰丙酮前体丙酮酸的供应。特别地，编码来自嗜热热地芽孢杆菌的苹果酸酶的基因与来自甲醇双歧杆菌MGA3的异柠檬酸裂合酶基因的组合表达提高了乙酰化抗体滴度1.6倍，直至0.42±0.01 g L ^-1，相当于0.07 gg ^-1甲醇。这导致MGA3菌株从具有两个不同抗生素抗性标记的两个不同质粒中总共过量生产4种重组酶，证明了新开发的针对该生物的遗传工具允许的代谢工程复杂性增加。据我们所知，这是微生物从甲醇中生产乙缩醛的第一个证明。

更新日期：2020-02-13

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