Efficient whole-cell catalysis for 5-aminovalerate production from L-lysine by using engineered Escherichia coli with ethanol pretreatment

Abstract

Microorganisms can utilize biomass to produce valuable chemicals, showing sustainable, renewable and economic advantages compared with traditional chemical synthesis. As a potential five-carbon platform polymer monomer, 5-aminovalerate has been widely used in industrial fields such as clothes and disposable goods. Here we establish an efficient whole-cell catalysis for 5-aminovalerate production with ethanol pretreatment. In this study, the metabolic pathway from L-lysine to 5-aminovalerate was constructed at the cellular level by introducing L-lysine α-oxidase. The newly produced H₂O₂ and added ethanol both are toxic to the cells, obviously inhibiting their growth. Here, a promising strategy of whole-cell catalysis with ethanol pretreatment is proposed, which greatly improves the yield of 5-aminovalerate. Subsequently, the effects of ethanol pretreatment, substrate concentration, reaction temperature, pH value, metal ion additions and hydrogen peroxide addition on the whole-cell biocatalytic efficiency were investigated. Using 100 g/L of L-lysine hydrochloride as raw material, 50.62 g/L of 5-aminovalerate could be excellently produced via fed-batch bioconversion with the yield of 0.84 mol/mol. The results show that a fast, environmentally friendly and efficient production of 5-aminovalerate was established after introducing the engineered whole-cell biocatalysts. This strategy, combined with ethanol pretreatment, can not only greatly enhance the yield of 5-aminovalerate but also be applied to the biosynthesis of other valuable chemicals.

Figure 1

E. coli whole-cell overexpressing RaiP system with ethanol pretreatment converting L-lysine to 5-aminovalerate.

Figure 2

5AVA titer by whole-cell biocatalysts CJ02RaiP with ethanol pretreatment of different concentration at 37 °C, 250 rpm and pH 8.0 after 12 h. 5 g/L of L-lysine HCl was as substrate and the OD₆₀₀ was 40. Various ethanol concentration of 0%, 1%, 2%, 3%, 4% and 5% were investigated. Data are means ± SD (n = 3). Statistics were performed by the two-tailed Student’s t-test. *p < 0.05; ns, not significant.

Figure 3

Expression analysis of RaiP. Protein samples were separated by 12% SDS-PAGE and stained with coomassie brilliant blue. Lane 1, molecular weight markers (kDa); Lane 2, RaiP noninduced control; Lane 3, RaiP induced in the absence of ethanol; Lane 4, RaiP induced in the presence of 4% ethanol. The expression was induced with 0.5 mM IPTG.

Figure 4

(A) Biosynthesis of 5AVA achieved via whole-cell biocatalysts CJ02RaiP with 4% ethanol pretreatment at different temperature, 5 g/L L-lysine HCl, 250 rpm and pH 8.0 for 12 h. Various temperature of 16 °C, 23 °C, 30 °C, 37 °C, 44 °C, 51 °C and 58 °C were investigated. (B) Biosynthesis of 5AVA achieved via whole-cell biocatalysts CJ02RaiP with 4% ethanol pretreatment at different rotational speed, 5 g/L L-lysine HCl, 37 °C and pH 8.0 for 12 h. Various rotational speed of 100 rpm, 150 rpm, 200 rpm, 250 rpm and 300 rpm were investigated. Data are means ± SD (n = 3). Statistics were performed by the two-tailed student t-test. *p < 0.05; ns, not significant. C: Biosynthesis of 5AVA achieved via whole-cell biocatalysts CJ02RaiP with 4% ethanol pretreatment at different pH, 5 g/L L-lysine HCl, 37 °C and 200 rpm for 12 h. Various pH of 6, 7, 8, 9 and 10 were investigated. D: The effect of metal ions on 5AVA production were investigated with 4% ethanol pretreatment at 5 g/L L-lysine HCl, 37 °C, pH 8.0 and 200 rpm for 12 h. Various metal ions of Mg²⁺, Mo²⁺, Ca²⁺, Zn²⁺, Fe²⁺, Fe³⁺, Cu²⁺ and Co²⁺ (3 mM) were investigated. Data are means ± SD (n = 3). Statistics were performed by the two-tailed Student’s t-test. *p < 0.05; ns, not significant.

Figure 5

5AVA production achieved by engineered whole-cell biocatalysts CJ02RaiP in the presence of different H₂O₂ concentrations. The experiments were conducted at 5 g/L L-lysine HCl, 4% ethanol pretreatment, 37 °C, 3 mM Mg²⁺, pH 8.0 and 200 rpm for 12 h. Various H₂O₂ concentrations (0, 5, 10 and 15 mM) were added after reaction 8 h. Values and error bars represent the mean and the standard deviation of triplicate cultivations. Statistics were performed by the two-tailed Student’s t-test. *p < 0.05; ns, not significant.

Figure 6

(A) Time profiles of 5AVA production were investigated by engineered whole-cell biocatalysts CJ02RaiP. The experiments were conducted at 5 g/L L-lysine HCl, 4% ethanol pretreatment, 37 °C, 3 mM Mg²⁺, pH 8.0 and 200 rpm. 10 mM H₂O₂ was added after reaction 8 h. Values and error bars represent the mean and the standard deviation of triplicate cultivations. (B) Fed-batch strategy for the production of 5AVA by engineered whole-cell biocatalysts CJ02RaiP in a 400 mL reactor. The experiments were conducted at 100 g/L L-lysine HCl, 4% ethanol pretreatment, 37 °C, 3 mM Mg²⁺, pH 8.0 and 200 rpm. 10 mM H₂O₂ was added after reaction 8 h. Data are means ± SD (n = 3).

Figure 7

LC-MS confirmation of 5AVA and 2K6AC biosynthesis by whole-cell biocatalysts CJ02RaiP. (A) HPLC results of 5AVA and 2K6AC. (B) Mass spectrum of 5AVA. (C) Mass spectrum of 2K6AC. Samples were derived with phenyl isothiocyanate (PITC) for LC-MS analysis. 5AVA, 5-Aminovalerate. 2K6AC, 2-Keto-6-aminocaproate.