Recent Advances in the Physiology and Genetics of Amino Acid-Producing Bacteria

Abstract

Corynebacterium glutamicum and its close relatives, C. flavum and C. lactofennentum, have been used for over 3 decades in the industrial production of amino acids by fermentation. Since 1984, several research groups have started programs to develop metabolic engineering principles for amino acid-producing Corynebacferium strains. Initially, the programs concentrated on the isolation of genes encoding (deregulated) biosynthetic enzymes and the development of general molecular biology tools such as cloning vectors and DNA transfer methods. With most of the genes and tools now available, recombinant DNA technology can be applied in strain improvement. To accomplish these improvements, it is critical and advantageous to understand the mechanisms of gene expression and regulation as well as the biochemistry and physiology of the species being engineered. This review explores the advances made in the understanding and application of amino acid-producing bacteria in the early 1990s.     

Site-directed mutagenesis of Escherichia coli acetylglutamate kinase and aspartokinase III probes the catalytic and substrate-binding mechanisms of these amino acid kinase family enzymes and allows three-dimensional modelling of aspartokinase

We test, using site-directed mutagenesis, predictions based on the X-ray structure of N-acetyl-L-glutamate kinase (NAGK), the paradigm of the amino acid kinase protein family, about the roles of specific residues on substrate binding and catalysis. The mutations K8R and D162E decreased V([sustrate]= infinity ) 100-fold and 1000-fold, respectively, in agreement with the predictions that K8 catalyzes phosphoryl transfer and D162 organizes the catalytic groups. R66K and N158Q increased selectively K(m)(Asp) three to four orders of magnitude, in agreement with the binding of R66 and N158 to the C(alpha) substituents of NAG. Mutagenesis in parallel of aspartokinase III (AKIII phosphorylates aspartate instead of acetylglutamate), another important amino acid kinase family member of unknown 3-D structure, identified in AKIII two residues, K8 and D202, that appear to play roles similar to those of K8 and D162 of NAGK, and supports the involvement of E119 and R198, similarly to R66 and N158 of NAGK, in the binding of the amino acid substrate, apparently interacting, respectively, with the alpha-NH(3)(+) and alpha-COO(-) of aspartate. These results and an improved alignment of the NAGK and AKIII sequences have guided us into 3-D modelling of the amino acid kinase domain of AKIII using NAGK as template. The model has good stereochemistry and validation parameters. It provides insight into substrate binding and catalysis, agreeing with mutagenesis results with another aspartokinase that were not considered when building the model.AKIII is homodimeric and is inhibited by lysine. Lysine may bind to a regulatory region that is C-terminal to the amino acid kinase domain. We make a C-terminally truncated AKIII (AKIIIt) and show that the C-region is involved in intersubunit interactions, since AKIIIt is found to be monomeric. Further, it is inactive, as demanded if dimer formation is essential for activity. Models for AKIII architecture are proposed that account for these findings.     

Identification and expression of a cDNA from Daucus carota encoding a bifunctional aspartokinase-homoserine dehydrogenase

Aspartokinase (EC 2.7.2.4) and homoserine dehydrogenase (EC 1.1.1.3) catalyze steps in the pathway for the synthesis of lysine, threonine, and methionine from aspartate. Homoserine dehydrogenase was purified from carrot (Daucus carota L.) cell cultures and portions of it were subjected to amino acid sequencing. Oligonucleotides deduced from the amino acid sequences were used as primers in a polymerase chain reaction to amplify a DNA fragment using DNA derived from carrot cell culture mRNA as template. The amplification product was radiolabelled and used as a probe to identify cDNA clones from libraries derived from carrot cell culture and root RNA. Two overlapping clones were isolated. Together the cDNA clones delineate a 3089 bp long sequence encompassing an open reading frame encoding 921 amino acids, including the mature protein and a long chloroplast transit peptide. The deduced amino acid sequence has high homology with the Escherichia coli proteins aspartokinase I-homoserine dehydrogenase I and aspartokinase II-homoserine dehydrogenase II. Like the E. coli genes the isolated carrot cDNA appears to encode a bifunctional aspartokinase-homoserine dehydrogenase enzyme.Abbreviations AK aspartokinase - HSDH homoserine dehydrogenase - PCR polymerase chain reaction - SDS sodium dodecyl sulfate The mention of vendor or product does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over vendors of similar products not mentioned.     

Cultural Conditions for l-Leucine Production by Strain No. 218, a Mutant of Brevibacterium lactofermentum 2256

The excellent l-leucine producing mutant No. 218, derived from a biotin requiring glutamic acid producing strain, is methionine and isoleucine auxotrophic. A suboptimum growth condition made by adding a limiting amount of isoleucine was necessary for the maximum production of l-leucine. On the other hand, methionine was indifferent to the productivity if sufficiently supplied for growth.

Biotin of more than 50 μg/liter caused the accumulation of l-leucine; less than 50 μg/liter, however, gave a drastic change in accumulation pattern from l-leucine to l-glutamic acid. Strain No. 218 produced 28 mg/ml of l-leucine after 72 hr cultivation when 13 % glucose was supplied as a carbon source, thus giving the yield of 21.6%.

Effects on l-leucine production of concentrations of inorganic salts, pH, temperature and aeration were also investigated.     

Construction of Lactobacillus plantarum strain with enhanced L-lysine yield

AIM: To enhance L-lysine secretion in Lactobacillus plantarum. METHODS AND RESULTS: An S-2-aminoethyl-L-cystein (AEC)-resistant mutant of L. plantarum was isolated, and it produced L-lysine at considerably higher level than the parent strain. Aspartokinase in the mutant has been desensitized to feedback inhibition by L-lysine. The nucleotide sequence analysis of thrA2 that codes for aspartokinase in the mutant predicted a substitution of glutamine to histidine at position 421. L-Lysine-insensitive aspartokinase, together with aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase, and dihydrodipicolinate reductase genes, was cloned from L. plantarum DNA to a shuttle vector, pRN14, and the genes were then transformed individually into the AEC-resistant mutant and the parent strain. The overexpression of the genes led to the increase in the activity of enzymes they encode in vitro. However, only the strain overexpressing aspartokinase or dihydrodipicolinate synthase produced more L-lysine. CONCLUSIONS: The desensitization of aspartokinase to L-lysine in L. plantarum led to the overproduction of L-lysine. The overexpression of L-lysine-insensitive aspartokinase or dihydrodipicolinate synthase enhanced L-lysine secretion in L. plantarum. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of the L-lysine-overproducing strain of L. plantarum in food or feed fermentation may increase the L-lysine content of fermented products.     

207 Nanopore immobilization of DNA polymerase enhances single-molecule sequencing

A zero-mode waveguide (ZMW) is a nanoscale optical waveguide driven at a frequency below its cut-off. In this mode, the electric field, instead of traveling down the axis of the conducting cavity, decays exponentially. By fabricating waveguides with sub-wavelength diameters and illuminating them with laser light, the electric field in the waveguide is confined enough to enable single-molecule optical detection at micromolar concentration [1]. Immobilizing single DNA polymerases in ZMWs and using special phosphate-fluorescently labeled dNTPs form the basis for single-molecule real-time DNA sequencing, one of the most promising next-generation sequencing platforms [2]. In this method, the polymerase replicates the sample DNA, and as it incorporates new bases into the product strand, the labeled dNTPs emit a burst of light before the phosphate is cleaved off. The sequence of colors corresponds to the DNA sequence (see Figure 1 below from Eid et al., 2009). Because the ZMW aperture’s diameter is sub-diffraction-limit, it is impossible to optically distinguish one polymerase in a ZMW from two. Having only one polymerase in each waveguide is critical to sequencing accuracy. In its present state, experimenters use diffusion to fill ZMWs with polymerases, resulting in a Poisson distribution for filling ZMWs, and consequently a theoretical limit of 36.8% of ZMWs having only one polymerase [2]. We achieve full polymerase occupancy of ZMWs by fabricating the structures on an ultrathin silicon nitride membrane and drilling a nanopore at the base of each waveguide with an ion beam. A short DNA fragment with biotin on either end is conjugated to a streptavidin and then drawn into the nanopore with a voltage bias. There is then a free biotin at the base of the ZMW. A polymerase–streptavidin complex can diffuse into the ZMW and bind to the exposed biotin. Because the nanopore is too small to fit more than one molecule, only one ZMW will bind to a biotin in the nanopore. Upon flushing the ZMW chamber, the biotin-bound polymerase will remain trapped in the pore, and only a single polymerase will remain at the base of each waveguide.      

Effects of a Feedback-Resistant Aspartokinase III Gene on L-Isoleucine Production in Escherichia coli K-12

An L-isoleucine-overproducing recombinant strain of E. coli, TVD5, was also found to overproduce L-valine. The L-isoleucine productivity of TVD5 was markedly decreased by addition of L-lysine to the medium. Introduction of a gene encoding feedback-resistant aspartokinase III increased L-isoleucine productivity and decreased L-valine by-production. The resulting strain accumulated 12 g/l L-isoleucine from 40 g/l glucose, and suppression of L-isoleucine productivity by L-lysine was relieved.     

Allosteric transition of aspartokinase I-homoserine dehydrogenase I studied by time-resolved fluorescence

The allosteric transition of threonine-sensitive aspartokinase I-homoserine dehydrogenase I from Escherichia coli has been studied by time-resolved fluorescence spectroscopy. Fluorescence decay can be resolved into 2 distinct classes of tryptophan emitters: a fast component, with a lifetime of about 1.5 ns; and a slow component, with a lifetime of about 4.5 ns. The fluorescence properties of the slow component are modified by the allosteric transition. In the T-form of the enzyme stabilized by threonine, the lifetime of the slow component is longer, with a red-shifted spectrum; its accessibility to quenching by acrylamide becomes slightly higher without any decrease of fluorescence anisotropy. These results indicate a change in polarity of the slow component environment. The quaternary structure change associated with the allosteric transition probably involves global movements of structural domains without leading to any local mobility on the nanosecond time-scale. We suggest that the slow component corresponds to the unique tryptophan of the buried kinase domain.     

Dynamic energy correlation analysis of E. coli aspartokinase III and alteration of allosteric regulation by manipulating energy transduction pathways

Conformational change associated with allosteric regulation in a protein is ultimately driven by energy transformation. However, little is known about the latter process. In this work, we combined steered molecular dynamics simulations and sequence conservation analysis to investigate the conformational changes and energy transformation in the allosteric enzyme aspartokinase III (AK III) from Escherichia coli. Correlation analysis of energy change at residue level indicated significant transformation between electrostatic energy and dihedral angle energy during the allosteric regulation. Key amino acid residues located in the corresponding energy transduction pathways were identified by dynamic energy correlation analysis. To verify their functions, residues with a high energy correlation in the pathways were altered and their effects on allosteric regulation of AKIII were determined. This study sheds new insights into energy transformation during allosteric regulation of AK III and proposes a strategy to identify key residues that are involved in intramolecular energy transduction and thus in driving the allosteric process.