Liu Huaiwei, Wang Yang, Tang Qiang, Kong Wentao, Chung Wook-Jin, Lu Ting
Microb Cell Fact. 2014 Sep 12;13:135. doi: 10.1186/s12934-014-0135-y.
Isopentenols, such as prenol and isoprenol, are promising advanced biofuels because of their higher energy densities and better combustion efficiencies compared with ethanol. Microbial production of isopentenols has been developed recently via metabolically engineered E. coli. However, current yields remain low and the underlying pathways require systematic optimization.
In this study, we targeted the E. coli native 2-methyl-(D)-erythritol-4-phosphate (MEP) pathway and its upstream glycolysis pathway for the optimization of isopentenol production. Two codon optimized genes, nudF and yhfR from Bacillus subtilis, were synthesized and expressed in E. coli W3110 to confer the isopentenol production of the strain. Two key enzymes (IspG and Dxs) were then overexpressed to optimize the E. coli native MEP pathway, which led to a significant increase (3.3-fold) in isopentenol production. Subsequently, the glycolysis pathway was tuned to enhance the precursor and NADPH supplies for the MEP pathway by activating the pentose phosphate pathway (PPP) and Entner-Doudoroff pathway (ED), which resulted in additional 1.9 folds of increase in isopentenol production. A 5 L-scale batch cultivation experiment was finally implemented, showing a total of 61.9 mg L(-1) isopentenol production from 20 g L(-1) of glucose.
The isopentenol production was successfully increased through multi-step optimization of the MEP and its upstream glycolysis pathways. It demonstrated that the total fluxes and their balance of the precursors of the MEP pathway are of critical importance in isopentenol production. In the future, an elucidation of the contribution of PPP and ED to MEP is needed for further optimization of isopentenol production.
异戊烯醇，如prenol和异戊二烯醇，由于其能量密度高于乙醇且燃烧效率更高，是很有前景的先进生物燃料。最近通过代谢工程改造的大肠杆菌实现了异戊烯醇的微生物生产。然而，目前的产量仍然很低，其潜在途径需要系统优化。
在本研究中，我们针对大肠杆菌天然的2-甲基-（D）-赤藓糖醇-4-磷酸（MEP）途径及其上游糖酵解途径来优化异戊烯醇的生产。合成了来自枯草芽孢杆菌的两个密码子优化基因nudF和yhfR，并在大肠杆菌W3110中表达，以赋予该菌株生产异戊烯醇的能力。然后过表达两种关键酶（IspG和Dxs）以优化大肠杆菌天然的MEP途径，这导致异戊烯醇产量显著增加（3.3倍）。随后，通过激活磷酸戊糖途径（PPP）和Entner-Doudoroff途径（ED）来调节糖酵解途径，以增强MEP途径的前体和NADPH供应，这使得异戊烯醇产量又增加了1.9倍。最后进行了5 L规模的分批培养实验，结果表明以20 g L(-1)葡萄糖为原料可生产总共61.9 mg L(-1)的异戊烯醇。
通过对MEP及其上游糖酵解途径进行多步优化，成功提高了异戊烯醇的产量。这表明MEP途径前体的总通量及其平衡在异戊烯醇生产中至关重要。未来，为了进一步优化异戊烯醇生产，需要阐明PPP和ED对MEP的贡献。
