Rational Engineering Of L-Glutimine Producing Strain

L-glutamine plays important roles in people’s life and has been applied to food, medicine and many other domains.With increasing demand of L-glutamine market, it is very crucial to improve glutamine productivity effectively. It seems reasonable to suggest that an efficient biosynthetic pathway would provide the conditions necessary for L-glutamine overproduction.A series of molecular modification were carried out to improve L-glutamine yield, decrease byproduct glutamate accumulation and construct temperature-sensitive glutamine-producing strain.The main research contents and results were as follows:(1) Previous studies showed that protein encoded by NCg11221was an important glutamate exporter.In this study NCg11221gene of the L-glutamine producing strain C. glutamicum GM34was disrupted using homologous recombination technology and NCg11221mutant GM34△NCg11221was constructed.Fed-batch fermentation of GM34△NCg11221was carried out in5L fermentor to investigate the byproduct glutamate production. The final glutamate accumulation of the NCg11221disruption strain was0.17(g/g DCW), which was80.9%of the original strain, while the glutamine accumulation did not chang much.(2) Pyruvate carboxylase(Pc, encoded by pyc) played important roles in in supplying oxaloacetate to the tricarboxylic acid cycle.The pyc gene was overexpressed in GM34using the shuttle vector pXMJ19and pyc overexpress strian GM34pXMJ19pyc was constructed.Fed-batch fermentation of recombinant strain was carried out in5L fermentor.Results showed that the growth rate of the pyc overexpressed strain was increased and the final glutamine accumulation was3.3(g/gDCW),which was5.5%higher than control.(3) Compared with biotin limitation strains,temperature-sensitive strains have great advantages.Previous studies showed that ItsA mutant could turn some C. glutamicum strians into temperature-sensitive strains.The ItsA gene of C. glutamicum GM34was disrupted using homologous recombination technology and ItsA mutant GM34△ltsA was constructed.The results showed that GM34△ltsA could still grow at38℃.Fed-batch fermentation of GM34℃ltsA was carried out in5L fermentor,glutamine could not accumulate after temperature shock. The results have valuable reference to further further research.(4) PH has great influence on L-glutamine fermentation.After20h of glutamine fermentation,the precursor glutamate did not increase any more.we conjecture that the glutamate was not sufficient enough for L-glutamine synthesis then.We adjust pH value to5.8,6.0and6.2respectively then to investagate its influce on L-glutamine fermentation.It turned out that at pH5.8and6.0,the glutamate did not increase and L-glutamine production was54.3g/L and57.2g/L respectively.At pH6.2,L-glutamate accumulation increased to12.4g/L, L-glutamine production was50.6g/L.