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食品与发酵工业  2021, Vol. 47 Issue (13): 1-8    DOI: 10.13995/j.cnki.11-1802/ts.027150
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
L-亮氨酸为底物一步法生物合成α-酮异己酸
AL-ADEEB Abdulqader, 乔郅钠, 徐美娟, 杨套伟, 张显, 邵明龙, 饶志明*
(江南大学 生物工程学院,江苏 无锡,214122)
One-step biosynthesis of α-ketoisocaproate using L-leucine as substrate
Al-ADEEB Abdulqader, QIAO Zhina, XU Meijuan, YANG Taowei, ZHANG Xian, SHAO Minglong, RAO Zhiming*
(School of Biotechnology,Jiangnan University,Wuxi 214122,China)
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摘要 α-酮异己酸是重要的有机合成和药物合成中间体,在食品、医药和化工行业中应用广泛。目前,α-酮异己酸的合成以化学法为主,需要高成本的催化剂或特殊的起始结构,导致α-酮异己酸生产成本较高。首次在食品安全性菌株枯草芽孢杆菌(Bacillus subtilis)168中异源表达了普通变形杆菌(Proteus vulgaris)来源的L-氨基酸脱氨酶,以重组枯草芽孢杆菌作为全细胞催化剂、L-亮氨酸为底物实现了α-酮异己酸的一步法生物合成。其次,针对全细胞催化条件进行优化,最优条件(全细胞催化剂20 g/L、L-亮氨酸浓度100 mmol/L、反应温度45 ℃、pH 10.0、MgCl2浓度5 mmol/L)下,转化24 h,可获得3.66 g/L的α-酮异己酸,且重复转化3次后,固定化细胞比游离细胞的再利用率提高了37.3%。该研究成功实现了以食品安全菌株B.subtilis 168为宿主一步法生物合成α-酮异己酸,为α-酮异己酸以及其他重要α-酮酸的工业化安全合成提供了新策略。
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AL-ADEEB Abdulqader
乔郅钠
徐美娟
杨套伟
张显
邵明龙
饶志明
关键词:  普通变形杆菌  L-氨基酸脱氨酶  枯草芽孢杆菌  L-亮氨酸  全细胞催化  固定化细胞  α-酮异己酸    
Abstract: α-ketoisocaproate is an important intermediate in organic and pharmaceutical synthesis, and is widely used in food, medicine and chemical industries. Chemical synthesis is the most commonly used method in α-ketoisocaproate production, including the Grignard reagents with diethyloxamates, the dual carbonylation and the hydantoin process. All these methods require addition of high-cost catalysts or a special starting structure, resulting in higher expenses for α-ketoisocaproate manufacturing. Secondly, all these procedures require the use of toxic reactants that can cause environmental harm. There are more and more studies on biosynthesis of α-ketoisocaproate, mainly through fermentation and whole cell transformation. In previous study, in order to establish a successful fermentation mechanism, a recombinant Corynebacterium glutamicum strain was designed by metabolic engineering, and the maximal α-ketoisocaproate titter reached 9.23 g/L, with the yield of 0.17 g α-ketoisocaproate/g glucose. However, with the exception of poor yield of α-ketoisocaproate, an auxotroph for branch-chained amino acids is still a barrier to industrial development owing to the deletion of ilvE. It can be seen that the synthesis method of α-ketoisocaproate based on metabolic engineering has low yield, many by-products, and needs to add a variety of expensive amino acids, which is not suitable for industrial production. Another study has fabricated a plasmid-free C. glutamicum to produce 6.1 g/L α-ketoisocaproate, but the yield was only 0.014 g α-ketoisocaproate/g glucose. Yet, the production of α-ketoisocaproate of C. glutamicum by metabolic engineering is still narrow by the growth reliant on the L-isoleucine. The whole-cell biosynthesis mechanism offers a bright path to the low-cost development process of α-ketoisocaproate. The whole-cell transformation method has many benefits such as fewer by-products, simple operation, few synthesis steps, no need to add toxic chemical raw materials during the reaction process, product with high purity, easy to separate and purify, and is more suitable for industrial production. The membrane-bound L-amino acid deaminase derived from Proteus vulgaris can catalyze the deamination of L-leucine to produce α-ketoisocaproate without producing H2O2, thereby reducing the impact on the growth of host cells. It has been widely used in the synthesis of various α-keto acids, such as phenylpyruvate, α-ketoisovaleric acid, α-ketoglutarate, α-keto-γ-methylthiobutyric acid and α-ketoisocaproate. In a study, α-ketoisocaproate was prepared using the whole-cell transformation technique of Rhodococcus opacus DSM 43250, and α-ketoisocaproate titer reached 1 275 mg/L with a yield of 0.254 g/(L·h). In another research, for the development of α-ketoisocaproate from leucine, an Escherichia coli BL21 (DE3) was constructed by whole-cell biocatalyst with membrane-bound L-amino acid deaminase from P. vulgaris. The α-ketoisocaproate titter was reached 69.1 g/L with the production rate of 3.14 g/(L·h). In another study conducted, an even higher α-ketoisocaproate production of 86.55 g/L and yield of 3.6 g/(L·h) were achieved via three engineering strategies; altering the plasmid origin with various copy numbers, modulating the mRNA composition downstream of the initiation codon, and designing the ribosome binding-site synthesis sequences, which was at a relatively high level. However, E. coli may bring harmful substances that do not meet the food hygiene requirements into the product, restricting the application of α-ketoisocaproate in the food and pharmaceutical industries. Therefore, it is necessary to construct a food-grade expression system with a clear background to realize the safe production of α-ketoisocaproate. Bacillus subtilis is a non-pathogenic bacteria and is generally considered as a GRAS (Generally Recognized As Safe) strain by the US Food and Drug Administration. In addition, there is currently no report on the heterologous expression of L-amino acid deaminase derived from P. vulgaris in B. subtilis to synthesize α-ketoisocaproate. Therefore, in this study, for the first time, the GRAS strain B. subtilis 168 heterologously expressed the L-amino acid deaminase derived from P. vulgaris, using recombinant B. subtilis 168 as the whole cell catalyst and L-Leucine as the substrate to realize the one-step biosynthesis of α-ketoisocaproate. Secondly, the conditions of whole-cell catalysis were optimized. Under the optimal conditions (whole-cell catalyst 20 g/L, L-leucine concentration 100 mmol/L, reaction temperature 45°C, pH 10.0, MgCl2 concentration 5 mmol/L), after the conversion for 24 h, 3.66 g/L of α-ketoisocaproate could be obtained, and the yield was 0.15 g/(L·h). Although the production and yield were not high, it provided a new strategy for the industrialized and safe production of α-ketoisocaproate. After three repeated transformations, the reutilization rate of immobilized cells was 37.3% higher than that of free cells. This study successfully realized the one-step biosynthesis of α-ketoisocaproate with food-safe strain B. subtilis 168 as the host, providing a new strategy for the industrialized and safe synthesis of α-ketoisocaproate and other important α-keto acids.
Key words:  Proteus vulgaris    L-amino acid deaminase    Bacillus subtilis    L-leucine    whole-cell catalysis    immobilized cell    α-ketoisocaproate
收稿日期:  2021-03-01      修回日期:  2021-03-12           出版日期:  2021-07-15      发布日期:  2021-08-02      期的出版日期:  2021-07-15
基金资助: 国家自然科学基金项目(31770058);江苏省自然科学基金项目(BK20181205)
作者简介:  硕士研究生(饶志明教授为通讯作者,E-mail:raozhm@jiangnan.edu.cn)
引用本文:    
AL-ADEEB Abdulqader,乔郅钠,徐美娟,等. 以L-亮氨酸为底物一步法生物合成α-酮异己酸[J]. 食品与发酵工业, 2021, 47(13): 1-8.
Al-ADEEB Abdulqader,QIAO Zhina,XU Meijuan,et al. One-step biosynthesis of α-ketoisocaproate using L-leucine as substrate[J]. Food and Fermentation Industries, 2021, 47(13): 1-8.
链接本文:  
http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.027150  或          http://sf1970.cnif.cn/CN/Y2021/V47/I13/1
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