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.
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