Biernacki Mateusz, Riechen Jan, Hähnel Urs, Roick Thomas, Baronian Kim, Bode Rüdiger, Kunze Gotthard
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Correnstr. 3, 06466, Gatersleben, Saxony-Anhalt, Germany.
Jäckering Mühlen- und Nährmittelwerke GmbH, Vorsterhauser Weg 46, 59007, Hamm, Germany.
AMB Express. 2017 Dec;7(1):4. doi: 10.1186/s13568-016-0303-z. Epub 2017 Jan 3.
(R)-3-hydroxybutyric acid can be used in industrial and health applications. The synthesis pathway comprises two enzymes, β-ketothiolase and acetoacetyl-CoA reductase which convert cytoplasmic acetyl-CoA to (R)-3-hydroxybutyric acid [(R)-3-HB] which is released into the culture medium. In the present study we used the non-conventional yeast, Arxula adeninivorans, for the synthesis enantiopure (R)-3-HB. To establish optimal production, we investigated three different endogenous yeast thiolases (Akat1p, Akat2p, Akat4p) and three bacterial thiolases (atoBp, thlp, phaAp) in combination with an enantiospecific reductase (phaBp) from Cupriavidus necator H16 and endogenous yeast reductases (Atpk2p, Afox2p). We found that Arxula is able to release (R)-3-HB used an existing secretion system negating the need to engineer membrane transport. Overexpression of thl and phaB genes in organisms cultured in a shaking flask resulted in 4.84 g L (R)-3-HB, at a rate of 0.023 g L h over 214 h. Fed-batch culturing with glucose as a carbon source did not improve the yield, but a similar level was reached with a shorter incubation period [3.78 g L of (R)-3-HB at 89 h] and the rate of production was doubled to 0.043 g L h which is higher than any levels in yeast reported to date. The secreted (R)-3-HB was 99.9% pure. This is the first evidence of enantiopure (R)-3-HB synthesis using yeast as a production host and glucose as a carbon source.
(R)-3-羟基丁酸可用于工业和健康领域。其合成途径包括两种酶，即β-酮硫解酶和乙酰乙酰辅酶A还原酶，它们将细胞质中的乙酰辅酶A转化为(R)-3-羟基丁酸[(R)-3-HB]，并释放到培养基中。在本研究中，我们使用非常规酵母嗜腺嘌呤节杆菌来合成对映体纯的(R)-3-HB。为了确定最佳产量，我们研究了三种不同的内源性酵母硫解酶（Akat1p、Akat2p、Akat4p）和三种细菌硫解酶（atoBp、thlp、phaAp），并将它们与来自食酸铜绿假单胞菌H16的对映体特异性还原酶（phaBp）和内源性酵母还原酶（Atpk2p、Afox2p）相结合。我们发现嗜腺嘌呤节杆菌能够利用现有的分泌系统释放(R)-3-HB，无需对膜运输进行工程改造。在摇瓶培养的生物体中过表达thl和phaB基因，在214小时内产生了4.84 g/L的(R)-3-HB，速率为0.023 g/(L·h)。以葡萄糖为碳源进行补料分批培养并没有提高产量，但在较短的培养时间（89小时时为3.78 g/L的(R)-3-HB）内达到了类似的水平，生产速率提高了一倍，达到0.043 g/(L·h)，高于迄今为止报道的酵母中的任何水平。分泌的(R)-3-HB纯度为99.9%。这是首次以酵母作为生产宿主、葡萄糖作为碳源合成对映体纯的(R)-3-HB的证据。
