Kubota Takeshi, Watanabe Akira, Suda Masako, Kogure Takahisa, Hiraga Kazumi, Inui Masayuki
Research Institute of Innovative Technology for the Earth, 9-2, Kizugawadai, Kizugawa, Kyoto 619-0292, Japan.
Research Institute of Innovative Technology for the Earth, 9-2, Kizugawadai, Kizugawa, Kyoto 619-0292, Japan; Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0101, Japan.
Metab Eng. 2016 Nov;38:322-330. doi: 10.1016/j.ymben.2016.07.010. Epub 2016 Jul 26.
para-Aminobenzoate (PABA), a valuable chemical raw material, can be synthesized by most microorganisms. This aromatic compound is currently manufactured from petroleum-derived materials by chemical synthesis. To produce PABA from renewable resources, its production by fermentation was investigated. The evaluation of the sensitivity to PABA toxicity revealed that Corynebacterium glutamicum had better tolerance to PABA than several other microorganisms. To produce PABA from glucose, genetically engineered C. glutamicum was constructed by introducing both pabAB and pabC. The generated strain produced 20mM of PABA in a test-tube scale culture; however, during the investigation, an unidentified major byproduct was detected in the culture supernatant. Unexpectedly, the byproduct was also detected after the incubation of PABA with glucose in a buffer solution without bacterial cells. To elucidate the mechanism underlying the formation of this byproduct, PABA analogues and several kinds of sugars were mixed and analyzed. New chemical compounds were detected when incubating aniline with glucose as well as PABA with reducing sugars (mannose, xylose, or arabinose), indicating that an amino group of PABA reacted non-enzymatically with an aldehyde group of glucose. The molecular mass of the byproduct determined by LC-MS suggested that the molecule was generated from PABA and glucose with releasing a water molecule, generally known as a glycation product. Because the glycation reaction was reversible, the byproduct was easily converted to PABA by acid treatment (around pH 2-3) with HCl. Then, pab genes were screened to improve PABA production. The highest PABA concentration was achieved by a strain expressing the pabAB of Corynebacterium callunae and a strain expressing the pabC of Xenorhabdus bovienii, respectively. A plasmid harboring both the pabAB of C. callunae and the pabC of X. bovienii, the best gene combination, was introduced into a strain overexpressing the genes of the shikimate pathway. The resultant strain produced 45mM of PABA in a test-tube scale culture. Under a fermenter-controlled condition, the strain produced up to 314mM (43g/L) of PABA at 48h, with a 20% yield. To our knowledge, this is the highest concentration of PABA produced by a genetically modified microorganism ever reported.
对氨基苯甲酸（PABA）是一种重要的化学原料，大多数微生物都能合成它。这种芳香族化合物目前是通过化学合成从石油衍生材料中制造的。为了从可再生资源生产PABA，对其发酵生产进行了研究。对PABA毒性敏感性的评估表明，谷氨酸棒杆菌对PABA的耐受性比其他几种微生物更好。为了从葡萄糖生产PABA，通过引入pabAB和pabC构建了基因工程改造的谷氨酸棒杆菌。所产生的菌株在试管规模培养中产生了20mM的PABA；然而，在研究过程中，在培养上清液中检测到一种未鉴定的主要副产物。出乎意料的是，在没有细菌细胞的缓冲溶液中，将PABA与葡萄糖一起孵育后也检测到了该副产物。为了阐明该副产物形成的机制，将PABA类似物和几种糖类混合并进行分析。当将苯胺与葡萄糖以及PABA与还原糖（甘露糖、木糖或阿拉伯糖）一起孵育时，检测到了新的化合物，这表明PABA的氨基与葡萄糖的醛基发生了非酶反应。通过液相色谱-质谱法测定的副产物分子量表明，该分子是由PABA和葡萄糖生成并释放出一个水分子，通常称为糖化产物。由于糖化反应是可逆的，通过用HCl进行酸处理（pH约为2-3），副产物很容易转化为PABA。然后，筛选pab基因以提高PABA的产量。分别通过表达卡氏棒杆菌pabAB的菌株和表达博维氏致病杆菌pabC的菌株实现了最高的PABA浓度。将携带卡氏棒杆菌pabAB和博维氏致病杆菌pabC这一最佳基因组合的质粒引入过表达莽草酸途径基因的菌株中。所得菌株在试管规模培养中产生了45mM的PABA。在发酵罐控制条件下，该菌株在48小时时产生了高达314mM（43g/L）的PABA，产率为20%。据我们所知，这是有史以来报道的转基因微生物产生的最高浓度的PABA。
