以L-亮氨酸为底物一步法生物合成α-酮异己酸

One-step biosynthesis ofα-ketoisocaproate using L-leucine as substrate

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、MgCl 2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。

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 inα-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 forα-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 ofα-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 maximalα-ketoisocaproate titter reached 9.23 g/L,with the yield of 0.17 gα-ketoisocaproate/g glucose.However,with the exception of poor yield ofα-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 ofα-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/Lα-ketoisocaproate,but the yield was only 0.014 gα-ketoisocaproate/g glucose.Yet,the production ofα-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 ofα-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 produceα-ketoisocaproate without producing H 2 O 2,thereby reducing the impact on the growth of host cells.It has been widely used in the synthesis of variousα-keto acids,such as phenylpyruvate,α-ketoisovaleric acid,α-ketoglutarate,α-keto-γ-methylthiobutyric acid andα-ketoisocaproate.In a study,α-ketoisocaproate was prepared using the whole-cell transformation technique of Rhodococcus opacus DSM 43250,andα-ketoisocaproate titer reached 1275 mg/L with a yield of 0.254 g/(L·h).In another research,for the development ofα-ketoisocaproate from leucine,an Escherichia coli BL21(DE3)was constructed by whole-cell biocatalyst with membrane-bound L-amino acid deaminase from P.vulgaris.Theα-ketoisocaproate titter was reached 69.1 g/L with the production rate of 3.14 g/(L·h).In another study conducted,an even higherα-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 ofα-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 ofα-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 synthesizeα-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 ofα-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,MgCl 2 concentration 5 mmol/L),after the conversion for 24 h,3.66 g/L ofα-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 ofα-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 ofα-ketoisocaproate with food-safe strain B.subtilis 168 as the host,providing a new strategy for the industrialized and safe synthesis ofα-ketoisocaproate and other importantα-keto acids.