Growth-coupled evolution of phosphoketolase to improve l-glutamate production by Corynebacterium glutamicum

  • Taiwo Dele-Osibanjo
  • Qinggang Li
  • Xiaoli Zhang
  • Xuan Guo
  • Jinhui Feng
  • Jiao Liu
  • Xue Sun
  • Xiaowei Wang
  • Wenjuan Zhou
  • Ping ZhengEmail author
  • Jibin SunEmail author
  • Yanhe Ma
Biotechnologically relevant enzymes and proteins


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.


Phosphoketolase Growth-coupled evolution Acetyl CoA generation l-glutamate production Corynebacterium glutamicum 



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 information

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).

Compliance with ethical standards

This article does not contain studies with human participants or animals performed by any of the authors. All authors confirm that ethical principles have been followed in the research as well as in manuscript preparation, and approved this submission

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

253_2019_10043_MOESM1_ESM.pdf (752 kb)
ESM 1 (PDF 752 kb)


  1. Atsumi S, Liao JC (2008) Directed evolution of Methanococcus jannaschii citramalate synthase for biosynthesis of 1-propanol and 1-butanol by Escherichia coli. Appl Environ Microbiol 74(24):7802–7808.
  2. Babul J (1978) Phosphofructokinases from Escherichia coli. Purification and characterization of the non-allosteric isozyme. J Biol Chem 253(12):4350–4355Google Scholar
  3. Baneyx F, Mujacic M (2004) Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol 22(11):1399–1408. CrossRefGoogle Scholar
  4. Bergman A, Siewers V, Nielsen J, Chen Y (2016) Functional expression and evaluation of heterologous phosphoketolases in Saccharomyces cerevisiae. AMB Express 6:13. CrossRefGoogle Scholar
  5. Bloom JD, Meyer MM, Meinhold P, Otey CR, MacMillan D, Arnold FH (2005) Evolving strategies for enzyme engineering. Curr Opin Struct Biol 15(4):447–452. CrossRefGoogle Scholar
  6. Bogorad IW, Lin TS, Liao JC (2013) Synthetic non-oxidative glycolysis enables complete carbon conservation. Nature 502(7473):693–698. CrossRefGoogle Scholar
  7. Bradford MM (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal Biochem 72(1-2):248–254. CrossRefGoogle Scholar
  8. Chen RD (2001) Enzyme engineering: rational redesign versus directed evolution. Trends Biotechnol 19(1):13–14. CrossRefGoogle Scholar
  9. Chinen A, Kozlov YI, Hara Y, Izui H, Yasueda H (2007) Innovative metabolic pathway design for efficient L-glutamate production by suppressing CO2 emission. J Biosci Bioeng 103(3):262–269. CrossRefGoogle Scholar
  10. Chwa JW, Kim WJ, Sim SJ, Um Y, Woo HM (2016) Engineering of a modular and synthetic phosphoketolase pathway for photosynthetic production of acetone from CO2 in Synechococcus elongatus PCC 7942 under light and aerobic condition. Plant Biotechnol J 14(8):1768–1776. CrossRefGoogle Scholar
  11. de Jong BW, Shi S, Siewers V, Nielsen J (2014) Improved production of fatty acid ethyl esters in Saccharomyces cerevisiae through up-regulation of the ethanol degradation pathway and expression of the heterologous phosphoketolase pathway. Microb Cell Factories 13:10. CrossRefGoogle Scholar
  12. García-Fruitós E, González-Montalbán N, Morell M, Vera A, Ferraz RM, Arís A, Ventura S, Villaverde A (2005) Aggregation as bacterial inclusion bodies does not imply inactivation of enzymes and fluorescent proteins. Microb Cell Factories 4:27. CrossRefGoogle Scholar
  13. Glenn K, Smith KS (2015) Allosteric Regulation of Lactobacillus plantarum Xylulose 5-Phosphate/Fructose 6-Phosphate Phosphoketolase (Xfp). J Bacteriol 197(7):1157–1163. CrossRefGoogle Scholar
  14. Glenn K, Ingram-Smith C, Smith KS (2014) Biochemical and kinetic characterization of xylulose 5-phosphate/fructose 6-phosphate phosphoketolase 2 (Xfp2) from Cryptococcus neoformans. Eukaryot Cell 13(5):657–663. CrossRefGoogle Scholar
  15. Gonzalez-Montalban N, Carrio MM, Cuatrecasas S, Aris A, Villaverde A (2005) Bacterial inclusion bodies are cytotoxic in vivo in absence of functional chaperones DnaK or GroEL. J Biotechnol 118(4):406–412.
  16. Grill JP, Crociani J, Ballongue J (1995) Characterization of fructose 6 phosphate phosphoketolases purified from Bifidobacterium species. Curr Microbiol 31(1):49–54CrossRefGoogle Scholar
  17. Henard CA, Freed EF, Guarnieri MT (2015) Phosphoketolase pathway engineering for carbon-efficient biocatalysis. Curr Opin Biotechnol 36:183–188. CrossRefGoogle Scholar
  18. Henard CA, Smith HK, Guarnieri MT (2017) Phosphoketolase overexpression increases biomass and lipid yield from methane in an obligate methanotrophic biocatalyst. Metab Eng 41:152–158. CrossRefGoogle Scholar
  19. Hofmann E, Kopperschlager G (1982) Phosphofructokinase from yeast. Methods Enzymol 90(Pt E):49–60CrossRefGoogle Scholar
  20. Keasling JD (2010) Manufacturing molecules through metabolic engineering. Science 330(6009):1355–1358. CrossRefGoogle Scholar
  21. Keilhauer C, Eggeling L, Sahm H (1993) Isoleucine synthesis in Corynebacterium-glutamicum - Molecular analysis of the ilvB-ilvN-ilvC operon. J Bacteriol 175(17):5595–5603CrossRefGoogle Scholar
  22. Kocharin K, Siewers V, Nielsen J (2013) Improved polyhydroxybutyrate production by Saccharomyces cerevisiae through the use of the phosphoketolase pathway. Biotechnol Bioeng 110(8):2216–2224. CrossRefGoogle Scholar
  23. Kotlarz D, Buc H (1982) Phosphofructokinases from Escherichia coli. Methods Enzymol 90(Pt E):60–70CrossRefGoogle Scholar
  24. Kozlov YI, Chinen A, Izui H, Hara Y, Yasueda H, Rybak KV, Slivinskaya EA, Katashkina JY, Kozlov Y, Katashikina JY, Hisashi Y, Chinen AAC, Izui HAC, Hara YAC, Yasueda HAC (2006) New bacterium, which is modified to have an increased activity of D-xylulose-5-phosphate phosphoketolase and/or fructose-6-phosphate phosphoketolase, useful for producing useful metabolites, e.g. L-glutamic acid or L-proline. WO2006016705-A1Google Scholar
  25. Krusemann JL, Lindner SN, Dempfle M, Widmer J, Arrivault S, Debacker M, He H, Kubis A, Chayot R, Anissimova M, Marliere P, Cotton CAR, Bar-Even A (2018) Artificial pathway emergence in central metabolism from three recursive phosphoketolase reactions. FEBS J 285:4367–4377. CrossRefGoogle Scholar
  26. Lee SM, Jellison T, Alper HS (2012) Directed evolution of xylose isomerase for improved xylose catabolism and fermentation in the yeast Saccharomyces cerevisiae. Appl Environ Microbiol 78(16):5708–5716. CrossRefGoogle Scholar
  27. Liu Q, Ouyang SP, Kim J, Chen GQ (2007) The impact of PHB accumulation on L-glutamate production by recombinant Corynebacterium glutamicum. J Biotechnol 132(3):273–279. CrossRefGoogle Scholar
  28. Liu B, Peng Q, Sheng M, Hu S, Qian M, Fan B, He J (2018) Directed evolution of sulfonylurea esterase and characterization of a variant with improved activity. J Agric Food Chem 67:836–843. CrossRefGoogle Scholar
  29. Meile L, Rohr LM, Geissman TA, Herensperger M, Teuber M (2001) Characterization of the D-xylulose 5-phosphate/D-Fructose 6-phosphate phosphoketolase gene (xfp) from Bifidobacterium lactis. J Bacteriol 183(9):2929–2936. CrossRefGoogle Scholar
  30. Miyazaki K, Takenouchi M (2002) Creating random mutagenesis libraries using megaprimer PCR of whole plasmid. Biotechniques 33(5):1033–1038CrossRefGoogle Scholar
  31. Niebisch A, Bott M (2001) Molecular analysis of the cytochrome bc1-aa3 branch of the Corynebacterium glutamicum respiratory chain containing an unusual diheme cytochrome c1. Arch Microbiol 175(4):282–294CrossRefGoogle Scholar
  32. Nissler K, Otto A, Schellenberger W, Hofmann E (1983) Similarity of activation of yeast phosphofructokinase by AMP and fructose-2,6-bisphosphate. Biochem Biophys Res Commun 111(1):294–300CrossRefGoogle Scholar
  33. Orban JI, Patterson JA (2000) Modification of the phosphoketolase assay for rapid identification of Bifidobacteria. J Microbiol Methods 40(3):221–224. CrossRefGoogle Scholar
  34. Pan J, Wu F, Wang J, Yu LQ, Khayyat NH, Stark BC, Kilbane JJ (2013) Enhancement of desulfurization activity by enzymes of the Rhodococcus dsz operon through coexpression of a high sulfur peptide and directed evolution. Fuel 112:385–390. CrossRefGoogle Scholar
  35. Peyret JL, Bayan N, Joliff G, Gulik-Krzywicki T, Mathieu L, Schechter E, Leblon G (1993) Characterization of the cspB gene encoding PS2, an ordered surface-layer protein in Corynebacterium glutamicum. Mol Microbiol 9(1):97–109Google Scholar
  36. Posthuma CC, Bader R, Engelmann R, Postma PW, Hengstenberg W, Pouwels PH (2002) Expression of the xylulose 5-phosphate phosphoketolase gene, xpkA, from Lactobacillus pentosus MD363 is induced by sugars that are fermented via the phosphoketolase pathway and is repressed by glucose mediated by CcpA and the mannose phosphoenolpyruvate phosphotransferase system. Appl Environ Microbiol 68(2):831–837.
  37. Ruan YL, Zhu LJ, Li Q (2015) Improving the electro-transformation efficiency of Corynebacterium glutamicum by weakening its cell wall and increasing the cytoplasmic membrane fluidity. Biotechnol Lett 37(12):2445–2452. CrossRefGoogle Scholar
  38. Schafer A, Tauch A, Jager W, Kalinowski J, Thierbach G, Puhler A (1994) Small mobilizable multipurpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19 - selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145(1):69–73. CrossRefGoogle Scholar
  39. Servinsky MD, Germane KL, Liu S, Kiel JT, Clark AM, Shankar J, Sund CJ (2012) Arabinose is metabolized via a phosphoketolase pathway in Clostridium acetobutylicum ATCC 824. J Ind Microbiol Biotechnol 39(12):1859–1867. CrossRefGoogle Scholar
  40. Sgorbati B, Lenaz G, Casalicchio F (1976) Purification and properties of two fructose-6-phosphate phosphoketolases in Bifidobacterium. Antonie Van Leeuwenhoek 42(1-2):49–57CrossRefGoogle Scholar
  41. Sonderegger M, Schumperli M, Sauer U (2004) Metabolic engineering of a phosphoketolase pathway for pentose catabolism in Saccharomyces cerevisiae. Appl Environ Microbiol 70(5):2892–2897. CrossRefGoogle Scholar
  42. Strandberg L, Enfors SO (1991) Factors influencing inclusion body formation in the production of a fused protein in Escherichia coli. Appl Environ Microbiol 57(6):1669–1674Google Scholar
  43. Suzuki R, Katayama T, Kim BJ, Wakagi T, Shoun H, Ashida H, Yamamoto K, Fushinobu S (2010a) Crystal structures of phosphoketolase: thiamine diphosphate-dependent dehydration mechanism. J Biol Chem 285(44):34279–34287. CrossRefGoogle Scholar
  44. Suzuki R, Kim BJ, Shibata T, Iwamoto Y, Katayama T, Ashida H, Wakagi T, Shoun H, Fushinobu S, Yamamoto K (2010b) Overexpression, crystallization and preliminary X-ray analysis of xylulose-5-phosphate/fructose-6-phosphate phosphoketolase from Bifidobacterium breve. Acta Crystallogr Sect F Struct Biol Cryst Commun 66(Pt 8):941–943. CrossRefGoogle Scholar
  45. Takahashi K, Tagami U, Shimba N, Kashiwagi T, Ishikawa K, Suzuki E (2010) Crystal structure of Bifidobacterium Longum phosphoketolase; key enzyme for glucose metabolism in Bifidobacterium. FEBS Lett 584(18):3855–3861. CrossRefGoogle Scholar
  46. van der Rest ME, Lange C, Molenaar D (1999) A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA. Appl Microbiol Biotechnol 52(4):541–545. CrossRefGoogle Scholar
  47. Wang CY, Li Y, Gao ZW, Liu LC, Zhang MY, Zhang TY, Wu CF, Zhang YX (2018a) Establishing an innovative carbohydrate metabolic pathway for efficient production of 2-keto-L-gulonic acid in Ketogulonicigenium robustum initiated by intronic promoters. Microb Cell Factories 17(1):81. CrossRefGoogle Scholar
  48. Wang Q, Xu J, Sun Z, Luan Y, Li Y, Wang J, Liang Q, Qi Q (2018b) Engineering an in vivo EP-bifido pathway in Escherichia coli for high-yield acetyl-CoA generation with low CO2 emission. Metab Eng 51:79–87.
  49. Wang Y, Cao GQ, Xu DY, Fan LW, Wu XY, Ni XM, Zhao SX, Zheng P, Sun JB, Ma YH (2018c) A Novel Corynebacterium glutamicum L-Glutamate Exporter. Appl Environ Microbiol 84(6):15. Google Scholar
  50. Welch P, Scopes RK (1981) Rapid purification and crystallization of yeast phosphofructokinase. Anal Biochem 112(1):154–157CrossRefGoogle Scholar
  51. Whitworth DA, Ratledge C (1977) Phosphoketolase in Rhodotorula graminis and Other Yeasts. J Gen Microbiol 102:397–401CrossRefGoogle Scholar
  52. Yang XY, Yuan QQ, Zheng YY, Ma HW, Chen T, Zhao XM (2016) An engineered non-oxidative glycolysis pathway for acetone production in Escherichia coli. Biotechnol Lett 38(8):1359–1365. CrossRefGoogle Scholar
  53. Yevenes A, Frey PA (2008) Cloning, expression, purification, cofactor requirements, and steady state kinetics of phosphoketolase-2 from Lactobacillus plantarum. Bioorg Chem 36(1-3):121–127. CrossRefGoogle Scholar
  54. Zhang J, Liu YJ (2013) Computational studies on the catalytic mechanism of phosphoketolase. Comput Theor Chem 1025:1–7. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Tianjin Institute of Industrial BiotechnologyTianjinChina
  2. 2.Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of SciencesTianjinChina
  3. 3.University of Chinese Academy of SciencesBeijingChina
  4. 4.College of BiotechnologyTianjin University of Science and TechnologyTianjinChina

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