Pathway optimization and key enzyme evolution of N-acetylneuraminate biosynthesis using an in vivo aptazyme-based biosensor.
N-acetylneuraminate (NeuAc) biosynthesis has drawn much attention owing to its wide applications in many aspects. Previously, we engineered for the first time an artificial NeuAc biosynthetic pathway in Escherichia coli using glucose as sole substrate. However, rigorous requirements for the flux and cofactor balance make subsequent strain improvement rather difficult. In this study, an in vivo NeuAc biosensor was designed and applied for genetic screening the mutant library of NeuAc producer. Its NeuAc responsive manner was demonstrated using sfgfp as a reporter and a Ni2+-based selection system was developed to couple the cell growth with in vivo NeuAc concentration. Employing this selection system, the NeuAc biosynthesis pathway was optimized and the key enzyme NeuAc synthase was evolved, which improved the titer by 34% and 23%, respectively. The final strain produced up to 8.31g/L NeuAc in minimal medium using glucose as sole carbon source. This work demonstrated the effectiveness of NeuAc biosensor in genetic screening and great potential in metabolic engineering of other organisms.
['Yang|Peng|P|', 'Wang|Jing|J|', 'Pang|Qingxiao|Q|', 'Zhang|Fengyu|F|', 'Wang|Junshu|J|', 'Wang|Qian|Q|', 'Qi|Qingsheng|Q|']
D052157:Aptamers, Nucleotide D015374:Biosensing Techniques D019020:Directed Molecular Evolution D004926:Escherichia coli D029968:Escherichia coli Proteins D019158:N-Acetylneuraminic Acid D007652:Oxo-Acid-Lyases
Pathway optimization N-acetylneuraminate Biosensor NeuAc synthase Genetic selection