Zhou Hai-Yan, Wu Wang-Jie, Niu Kun, Xu Yue-Ying, Liu Zhi-Qiang, Zheng Yu-Guo
1Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, No. 18 Chaowang Road, Hangzhou, 310014 Zhejiang Province People's Republic of China.
2Engineering Research Center of Bioconversion and Biopurification, Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014 People's Republic of China.
3 Biotech. 2019 Mar;9(3):96. doi: 10.1007/s13205-019-1609-8. Epub 2019 Feb 19.
Microbial fermentation for L-methionine (L-Met) production based on natural renewable resources is attractive and challenging. In this work, the effects of medium composition and fermentation conditions were investigated to improve L-Met production by genetically engineered MET-3. Statistical optimization techniques including Plackett-Burman (PB) design and Box-Behnken design (BBD) were adopted first to optimize the culture medium. Results of PB-designed experiments indicated that the culture medium components including glucose, yeast extract, KHPO, and MgSO.7HO had significant effects on L-Met biosynthesis. With their best-predicted concentration established by BBD (glucose 37.43 g/L, yeast extract 0.95 g/L, KHPO 1.82 g/L, and MgSO.7HO 4.51 g/L), L-Met titer was increased to 3.04 g/L from less than 2.0 g/L. For further enhancement of L-Met biosynthesis, the fermentation conditions of batch cultivation carried out in a 5-L fermentor were optimized, and the optimum results were obtained at an agitation rate of 300 rpm, medium pH of 7.0, and induction temperature of 28 °C. Based on the optimization parameters, fed-batch fermentation with the modified medium was conducted. As a result, great improvement of L-Met titer (12.80 g/L) and yield (0.13 mol/mol) were achieved, with an increase of 38.53% and 30.0% compared with those of the basal medium, respectively. Furthermore, higher L-Met productivity of 0.261 g/L/h was obtained, representing 2.13-fold higher in comparison to the original medium. The results may provide a helpful reference for further study on strain improvement and fermentation control.
基于天然可再生资源的微生物发酵生产L-蛋氨酸（L-Met）具有吸引力且具有挑战性。在本研究中，研究了培养基组成和发酵条件对基因工程菌MET-3生产L-Met的影响。首先采用包括Plackett-Burman（PB）设计和Box-Behnken设计（BBD）在内的统计优化技术对培养基进行优化。PB设计实验结果表明，葡萄糖、酵母提取物、KHPO和MgSO₄·7H₂O等培养基成分对L-Met生物合成有显著影响。通过BBD确定其最佳预测浓度（葡萄糖37.43 g/L、酵母提取物0.95 g/L、KHPO 1.82 g/L和MgSO₄·7H₂O 4.51 g/L）后，L-Met产量从低于2.0 g/L提高到3.04 g/L。为进一步提高L-Met生物合成，对5-L发酵罐中分批培养的发酵条件进行了优化，在搅拌速度300 rpm、培养基pH 7.0和诱导温度28°C时获得了最佳结果。基于优化参数，使用改良培养基进行补料分批发酵。结果，L-Met产量（12.80 g/L）和产率（0.13 mol/mol）得到了显著提高，分别比基础培养基提高了38.53%和30.0%。此外，还获得了更高的L-Met生产率0.261 g/L/h，比原始培养基高2.13倍。这些结果可为进一步研究菌株改良和发酵控制提供有益参考。
