The overall goal of the project was to develop a cell factory to produce 1,5-Pentanediol (1,5-PDO) from glucose in Escherichia coli through the synthetic biology tools. Firstly, we designed an artificial 1,5-PDO biosynthetic pathway, and assembled the pathway in Escherichia coli NT1003 after the screening of the functional enzymes. Subsequently, we improved strain production ability by optimizing gene expression plasmid. In addition, we knock out the genes of branched pathways which catalyze the synthesis of acetic acid from pyruvate and 1,5-PDO degradation. Meanwhile, we further optimized pathway by engineering limiting enzyme of MmCAR through the mutation and protein scaffold assembly strategies. Figure 1a shows the whole design of our project, and Figure 1b shows the results of 1,5-PDO production obtained after each genetic manipulation.
Figure 1 (a) Metabolic engineering strategies. (b) Results involved 1,5-PDO content obtained at each step
For our final engineered strain of NT1003-P4-ΔYcjQ, we first detected its 1,5-PDO production activity in a 500 ml shake flask. The fermentation medium contains 20 g/L glucose, 10 g/L (NH4)2SO4, tryptone 5 g/L, 2 g/L yeast extract, and trace element. After cultivation of 96 h, the 1,5-PDO titer could reach 2.4 g/L.
Figure 2 The fermentation of E. coli NT1003-P4-ΔYcjQ in a 500 ml shake flask.
To further identify the performance of the E. coli NT1003-P4-ΔYcjQ, we performed a fed-batch fermentation using a 5-liter bioreactor. The strain was inoculated in the fermentation medium that containing 50 g/L glucose, 10 g/L (NH4)2SO4, tryptone 5 g/L, 2 g/L yeast extract, and trace element. When the glucose was exhausted, the medium that containing 400 g/L ammonium sulfate and 600 g/L glucose were supplemented during fermentation. After fermentation of 80 h, the 1,5-PDO production was reached to 48 mM (5.0 g/L) (Figure 3), which was higher than that of flask fermentation. In future, the fermentation medium and feeding strategy are required to be further optimized to obtain a higher 1,5-PDO production.
Figure 3 The fermentation of E. coli NT1003-P4-ΔYcjQ in 5 L bioreactor.
In future, in addition to further engineer the strain to improve 1,5-PDO production by cofactor supply engineering strategy, and so on, fermentation medium and feeding strategy in 5 L fermentor are also required to be further optimized to obtain a higher 1,5-PDO production. What is more, we also designed a separation process for obtaining purified 1,5-PDO products from fermentation broth, hopping to examine the property of 1,5-PDO in the synthesize polyurethane and its derived polyurethane coating.
Figure 4 The proposed separation process to obtain purified 1,5-PDO product