One-step biosynthesis of α-ketoisocaproate from L-leucine by an Escherichia coli whole-cell biocatalyst expressing an L-amino acid deaminase from Proteus vulgaris

Abstract

This work aimed to develop a whole-cell biotransformation process for the production of α-ketoisocaproate from L-leucine. A recombinant Escherichia coli strain was constructed by expressing an L-amino acid deaminase from Proteus vulgaris. To enhance α-ketoisocaproate production, the reaction conditions were optimized as follows: whole-cell biocatalyst 0.8 g/L, leucine concentration 13.1 g/L, temperature 35 °C, pH 7.5, and reaction time 20 h. Under the above conditions, the α-ketoisocaproate titer reached 12.7 g/L with a leucine conversion rate of 97.8%. In addition, different leucine feeding strategies were examined to increase the α-ketoisocaproate titer. When 13.1 g/L leucine was added at 2-h intervals (from 0 to 22 h, 12 addition times), the α-ketoisocaproate titer reached 69.1 g/L, while the leucine conversion rate decreased to 50.3%. We have developed an effective process for the biotechnological production of α-ketoisocaproate that is more environmentally friendly than the traditional petrochemical synthesis approach.

Figure 1. The deamination reaction of l-amino acid deaminase (l-AAD) from P. vulgaris.

Figure 2

(A) Specific growth rate of recombinant strain at different lactose-induction concentration. E. coli-pET-28a (+) (black bars), E. coli-pET-28a (+)-lad (white bars); (B) Effect of biocatalyst activity (black bars), α-ketoisocaperate (grey bars) and pH (white bars) at different induction intensities.

Figure 3. The effect of KIC, substrate, pH, temperature, DCW, and time on whole-cell biotransformation.

(A) Influence of KIC concentration; (B) effect of substrate concentration; (C) Influence of pH; (D) Influence of temperature; (E) Influence of biocatalyst; (F) KIC production at 0.8 g/L DCW over time.

Figure 4. The comparison of biocatalyst activity by whole-cell biocatalyst and production at different concentration of CCCP.

(A) biocatalyst activity at different CCCP concentration; (B) α-ketoisocaporate accumulation during bioconversion in 52.47 g/L substrate. CCCP concentration: : 0 μM, : 5 μM, : 10 μM, : 15 μM, : 20 μM, : 25 μM.

Figure 5. The effect of different feeding strategies on production of α-ketoisocaperate and biocatalyst activity.

(A) The production of α-ketoisocaperate (black bars) and biocatalyst activity (grey bars) with different initial substrate concentrations; (B) The α-ketoisocaperateproductionat different feeding interval time. : 2 h, : 4 h, : 6 h, : 8 h; : 10 h.

Figure 6. Immobilization and recyclablilty of the whole-cell biocatalyst.

(A) Comparision of immobilization on whole-cell biocatalyst activity. (B) The comparision of the recycle ability between the free cells and immoblized cells. [straight line with square mark () for free cell and dot line with triangle mark () for immobilized cell]; Comparison of the pH (C), and thermal (D) (black bar for free and light grey bar for immobilized recombinant whole cell biocatalyst).