Abstract
The introduction of the key non-oxidative glycolytic (NOG) pathway enzyme, phosphoketolases (PKTs), into heterologous hosts can improve the yield of a variety of acetyl CoA-derived products of interest. However, the low specific activity of existing PKTs compared with that of 6-phosphofructokinase (PFK), the key EMP pathway enzyme, largely limits their potential applications. To improve PKT activity, previous attempts have focused on increasing intracellular PKT concentration via the use of strong promoters. Herein, we report the establishment of a growth-coupled evolution strategy for the enrichment and selection of PKT mutants with improved specific activity in Corynebacterium glutamicum hosts with defective PFK. Five mutants from 9 Bifidobacterium adolescentis-source PKT (BA-PKT) mutant libraries were obtained. Site-directed mutagenesis analysis revealed 11 mutant sites which contributed to improved BA-PKT specific activity. Further structural analysis revealed that the mutant sites were located far away from the enzyme active site, which makes them almost unpredictable using a rational design approach. Mutant site recombination led to the construction of a novel mutant, PKTT2A/I6T/H260Y, with Vmax 29.77 ± 1.58 U/mg and Kcat/Km 0.32 ± 0.01 s−1/mM, which corresponds to 73.27 ± 3.25% and 80.16 ± 3.38% improvements, respectively, compared with the wildtype (Vmax; 17.17 ± 0.59 U/mg, Kcat/Km; 0.17 ± 0.01 s−1/mM). Expression of PKTT2A/I6T/H260 in C. glutamicum Z188 resulted in 16.67 ± 2.24% and 18.19 ± 0.53% improvement in l-glutamate titer and yield, respectively, compared with the wildtype BA-PKT. Our findings provide an efficient approach for improving the activity of PKTs. Furthermore, the novel mutants could serve as useful tools in improving the yield of l-glutamate and other acetyl CoA-associated products.
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Acknowledgments
We thank Guoqiang Cao and Zijian Tan (both from Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences) for technical support in Z188Δpfk strain construction and BA-PKT 3-D structure analysis, respectively.
Funding
This research was supported by grants from the National Natural Science Foundation of China (31870081), the National Key R&D Program of China (2018YFA0901403), the Special Program of Talents Development for Excellent Youth Scholars in Tianjin (TJTZJH-QNBJRC-2-10), the Youth Innovation Promotion Association of Chinese Academy of Sciences (2016164), and the Science and Technology Project of Tianjin (15PTCYSY00020 and 14ZCZDSY00157).
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Dele-Osibanjo, T., Li, Q., Zhang, X. et al. Growth-coupled evolution of phosphoketolase to improve l-glutamate production by Corynebacterium glutamicum. Appl Microbiol Biotechnol 103, 8413–8425 (2019). https://doi.org/10.1007/s00253-019-10043-6
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DOI: https://doi.org/10.1007/s00253-019-10043-6