Efficient Production Of Glutarate From L-lysine Via A Multi-enzyme Cascade Pathway

Glutarate is an important five-carbon compound,which is widely applied in plastic chemical,medicine,and agriculture fields.In comparision with the traditional chemical synthesis methods,the enzymatic transformation using 5-aminovalerate as the substrate is one of the most concerned glutarate biosynthesis methods.Unfortunately,high cost of substrate and low titer,yield,and productivity can not satisfy the demand of industrialized production.In this study,we designed a five-enzyme cascade for one-pot transformation of cheap L-lysine to glutarate.Combined with RBS regulation,protein engineering,and transporter engineering strategies,the catalytic efficiency of glutarate production from L-lysine was significantly improved.The major results are described as follows:（1）Design and construction of glutarate synthesis cascade pathway.First,L-lysine decarboxylase from E.coli（Ec CA）,putrescine aminotransferase andγ-aminovaleraldehyde dehydrogenase from K.pneumoniae（Kpc PA and Kpc PD）,4-aminobutyrate aminotransferase and succinate-semialdehyde dehydrogenase from P.fluorescens（Pfe GT and Pfe GD）were selected via the comparison of specific enzyme activity and initial reaction rate.Then,the glutarate synthesis cascade reaction was reconstructed in vitro,and the generation of glutarate was determined by mass spectrometry,suggesting that the five-enzyme cascade pathway for glutarate production was successfully achieved.（2）Reconstruction and optimization of upstream synthesis pathway of glutarate.First,the contribution of individual enzymes on the initial reaction rate was examined by varying the concentrations of target enzymes,and Kpc PA was identified as the rate-limiting enzyme.Based on this,the orthogonal optimization experiment was performed to determine that there was a synergistic effect among the three enzymes of the upstream synthesis pathway.When Ec CA:Kpc PA:Kpc PD=4:8:7,the initial reaction rate reached the maximum of 179μM NADH·min^-1,which was 7.2-fold higher than that of the control.Secondly,RBS regulation was applied to fine-tune the expression levels of Ec CA,Kpc PA,and Kpc PD,and the recombinant strain AVA-02 was constructed,which produced 21.6 g·L^-1 5-aminovalerate,an increase of 84.6%from level in control strain AVA-01(11.7 g·L^-1).Third,combined with volume scanning and hydrophobicity scanning strategies,12 potential mutation sites that may be beneficial to enhance the catalytic activity of Kpc PA towards cadaverine were screened out,leading to two positive mutation sites E120G and T332A.The specific enzyme activity of the double mutant Kpc PA^E120G/T332A was increased to 75.87±1.51 U·mg^-1,which was 4.77-fold higher than that of the wild-type Kpc PA.By introducing the best variant Kpc PA^E120G/T332Ainto strain AVA-02,we constructed the recombinant strain AVA-03,which produced 29.5 g·L^-15-aminovalerate,an increase of 36.6%from level in strain AVA-02.（3）Reconstruction and optimization of downstream synthesis pathway of glutarate.First,the contribution of individual enzymes on the initial reaction rate was examined by varying the concentrations of target enzymes,and the results showed that Pfe GT and Pfe GD had no significant effect on glutarate production.Furthermore,the initial reaction rate of downstream pathway(282μM NADH·min^-1)was much higher than that of upstream pathway(25μM NADH·min^-1),which further confirmed that there was no rate-limiting enzyme in the downstream synthesis pathway.Secondly,based on the evaluation of substrate utilization ability,the low transport efficiency of 5-aminovalerate from extracellular to intracellular was identified as the major bottleneck.By overexpressing the 4-aminobutyrate transporter（Gab P）in strain Glu-01,the recombinant strain Glu-02 was generated.Finally,the accumulation of5-aminovalerate decreased from 8.9 g·L^-1 to 1.2 g·L^-1,while the titer of glutarate increased from 32.5 g·L^-1 to 41.2 g·L^-1,an increase of 26.8%from level in strain Glu-01.（4）Assembly and application of glutarate synthesis cascade pathway.First,the optimum upstream and downstream synthetic pathways of glutarate were assembled into the strain E.coli F0723 to construct the recombinant strain Glu-03.Then,the conditions for enzyme production and whole-cell conversion system were optimized with the strain Glu-03.The optimum enzyme production conditions were determined as follows:0.4 m M IPTG was added into the culture when the OD₆₀₀ of cells reaching 0.8,and enzymes were induced at25^oC for 14 h.Furthermore,the optimum whole-cell conversion conditions were determined as follows:30^oC,p H 8.5,0.6 m M NAD⁺,50 m Mα-KG,1 g·L^-1 triton（X-100）,and 30 g·L^-1wet-cells were added to convert 100 g·L^-1 L-lysine to glutarate.Finally,the whole-cell transformation system was tested in a 5-L scale bioreactor,resulting in 77.6 g·L^-1 glutarate in42 h,with the conversion rate,yield,and productivity of 85.9%,0.78 g·g^-1 L-lysine,and 1.85g·L^-1·h^-1,respectively.