Overexpression of wild-type aspartokinase increases L-lysine production in the thermotolerant methylotrophic bacterium Bacillus methanolicus

Appl Environ Microbiol. 2009 Feb;75(3):652-61. doi: 10.1128/AEM.01176-08. Epub 2008 Dec 5.

Abstract

Aspartokinase (AK) controls the carbon flow into the aspartate pathway for the biosynthesis of the amino acids l-methionine, l-threonine, l-isoleucine, and l-lysine. We report here the cloning of four genes (asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; dapG, encoding AKI; and yclM, encoding AKIII) of the aspartate pathway in Bacillus methanolicus MGA3. Together with the known AKII gene lysC, dapG and yclM form a set of three AK genes in this organism. Overexpression of dapG, lysC, and yclM increased l-lysine production in wild-type B. methanolicus strain MGA3 2-, 10-, and 60-fold (corresponding to 11 g/liter), respectively, without negatively affecting the specific growth rate. The production levels of l-methionine (less than 0.5 g/liter) and l-threonine (less than 0.1 g/liter) were low in all recombinant strains. The AK proteins were purified, and biochemical analyses demonstrated that they have similar V(max) values (between 47 and 58 micromol/min/mg protein) and K(m) values for l-aspartate (between 1.9 and 5.0 mM). AKI and AKII were allosterically inhibited by meso-diaminopimelate (50% inhibitory concentration [IC(50)], 0.1 mM) and by l-lysine (IC(50), 0.3 mM), respectively. AKIII was inhibited by l-threonine (IC(50), 4 mM) and by l-lysine (IC(50), 5 mM), and this enzyme was synergistically inhibited in the presence of both of these amino acids at low concentrations. The correlation between the impact on l-lysine production in vivo and the biochemical properties in vitro of the individual AK proteins is discussed. This is the first example of improving l-lysine production by metabolic engineering of B. methanolicus and also the first documentation of considerably increasing l-lysine production by overexpression of a wild-type AK.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Aspartate Kinase / genetics*
  • Aspartate Kinase / isolation & purification
  • Aspartate Kinase / metabolism*
  • Aspartate-Semialdehyde Dehydrogenase / genetics
  • Aspartate-Semialdehyde Dehydrogenase / metabolism
  • Aspartic Acid / metabolism
  • Bacillus / enzymology*
  • Bacillus / genetics
  • Bacillus / metabolism*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics
  • Diaminopimelic Acid / pharmacology
  • Enzyme Inhibitors / pharmacology
  • Gene Dosage*
  • Gene Expression*
  • Hydro-Lyases / genetics
  • Hydro-Lyases / metabolism
  • Inhibitory Concentration 50
  • Kinetics
  • Lysine / biosynthesis*
  • Methionine / biosynthesis
  • Molecular Sequence Data
  • Sequence Analysis, DNA
  • Threonine / pharmacology

Substances

  • Bacterial Proteins
  • DNA, Bacterial
  • Enzyme Inhibitors
  • Threonine
  • Aspartic Acid
  • Diaminopimelic Acid
  • Methionine
  • Aspartate-Semialdehyde Dehydrogenase
  • Aspartate Kinase
  • Hydro-Lyases
  • 4-hydroxy-tetrahydrodipicolinate synthase
  • Lysine

Associated data

  • GENBANK/FJ485942
  • GENBANK/FJ485943