Transcriptomic analysis of homoserine-evolved MG1655 strain (wild type) and of homoserine -treated wild type, mutant deleted for thrL and mutant bearing thrL* allele
ABSTRACT: The growth of E. coli is inhibited by millimolar concentrations of L-homoserine, which may be problematic for the industrial production of this compound or for products derived from it. In this work, an adapted laboratory evolution (ALE) was applied, which resulted in the isolation of an E. coli strain at least 10-fold more tolerant to L-homoserine than the original (MG1655) strain. Only four genomic modifications were identified after genome sequencing of this evolved strain (designed 4E), including a 49 bp truncation starting from the codon stop of thrL and leading to a modified thrL locus carrying a thrL* allele encoding a 30 amino acids polypeptide, which is 9 amino acids more than the leader peptide encoded by thrL. Replacement of thrL with thrL* enabled the initial strain to be as or slightly more tolerant to L-homoserine than the evolved 4E strain, which is explained by the rapid metabolization of L-homoserine to threonine resulting from ThrL*-dependent transcriptional activation of the threonine thrABC operon. Interestingly, in 4E strain, L-homoserine degradation went beyond threonine degradation, as tdh and kbl encoding threonine degradation pathway II and genes of the glycine cleavage system were strongly upregulated. To infer about the toxicity of L-homoserine, a transcriptomic analysis of wild-type MG1655 in the presence of 10 mM L-homoserine was performed, which identified a potent repression of locomotion-motility-chemotaxis process. Since the magnitude of this repression was reduced in a ΔthrL mutant concomitantly with a twofold lower sensitivity of this mutant to L-homoserine, one could argue that the repression of this biological process contributed to growth inhibition by L-homoserine. Furthermore, in both wild type and thrl mutant, a strong repression of the branched-chain amino acids synthesis and transport, as well as activation of the sulphate assimilation pathway process to cysteine synthesis were observed in the presence of L-homoserine, which may also contribute to toxic effect of this compound. How this non-canonical amino acid triggers these transcriptomic changes is discussed. Overall design: Transcriptomic analysis implicated 4 strains, namely MG1655 (WT), strain deleted of thrl (Dthrl), strain bearing thrL* allele (thrL*) and strain obtained after adapted laboratory evolution on homoserine (4E). Agilent microarray platfprm GPL13359 was used for microarray analysis . The four strains were inoculated at 0D600 of 0.2 in M9 minimal medium containing 10 g/L glucose in the absence or presence of 10 mM L-homoserine. When cultures reached OD600 of around 1.0, samples were taken for RNA was extraction. Notice that strain 4E is referred in the transcriptomic samples as E4
INSTRUMENT(S): Agilent-020097 E. coli Gene Expression Microarray (Probe Name version)
Project description:In addition to threonine auxotrophy, mutation of the Saccharomyces cerevisiae threonine biosynthetic genes THR1 (encoding homoserine kinase) and THR4 (encoding threonine synthase) results in a plethora of other phenotypes. We investigated the basis for these other phenotypes and found that they are dependent on the toxic biosynthetic intermediate homoserine. Moreover, homoserine is also toxic for Candida albicans thr1Delta mutants. Since increasing levels of threonine, but not other amino acids, overcome the homoserine toxicity of thr1Delta mutants, homoserine may act as a toxic threonine analog. Homoserine-mediated lethality of thr1Delta mutants is blocked by cycloheximide, consistent with a role for protein synthesis in this lethality. We identified various proteasome and ubiquitin pathway components that either when mutated or present in high copy numbers suppressed the thr1Delta mutant homoserine toxicity. Since the doa4Delta and proteasome mutants identified have reduced ubiquitin- and/or proteasome-mediated proteolysis, the degradation of a particular protein or subset of proteins likely contributes to homoserine toxicity.
Project description:The nucleotide sequence of the Serratia marcescens threonine operon (thrA1A2BC) was determined. Three long open reading frames were identified; these open reading frames code for aspartokinase I (AKI)-homoserine dehydrogenase I (HDI), homoserine kinase, and threonine synthase, in that order. The predicted amino acid sequences of these enzymes were similar to the amino acid sequences of the corresponding enzymes in Escherichia coli. The AKI-HDI protein is apparently a tetramer composed of monomer polypeptides that are 819 amino acids long. A deletion analysis revealed that the central and C-terminal region was responsible for threonine-resistant HDI activity, a monomeric fragment extending from the N terminus to residue 306 was responsible for threonine-resistant AKI activity, and an N-terminal portion containing 468 residues was responsible for threonine-sensitive AKI activity. The thrA(1)1A(2)1 and thrA(1)5A(2)5 mutations of threonine-excreting strains HNr21 and TLr156, which result in the loss of threonine-mediated feedback inhibition of both AKI activity and HDI activity, cause single amino acid substitutions (Gly to Asp at position 330 and Ser to Phe at position 352, respectively) in the central region of the AKI-HDI protein. The thrA1+A(2)2 mutation of strain HNr59, which results in a threonine-sensitive AKI and a threonine-resistant HDI, also causes a single amino acid substitution (Ala to Thr at position 479).
Project description:Staphylococcus aureus is a Gram-positive nosocomial pathogen. The prevalence of multidrug-resistant S. aureus strains in both hospital and community settings makes it imperative to characterize new drug targets to combat S. aureus infections. In this context, enzymes involved in cell-wall maintenance and essential amino-acid biosynthesis are significant drug targets. Homoserine dehydrogenase (HSD) is an oxidoreductase that is involved in the reversible conversion of L-aspartate semialdehyde to L-homoserine in a dinucleotide cofactor-dependent reduction reaction. HSD is thus a crucial intermediate enzyme linked to the biosynthesis of several essential amino acids such as lysine, methionine, isoleucine and threonine.
Project description:l-Homoserine, which is one of the few amino acids that is not produced on a large scale by microbial fermentation, plays a significant role in the synthesis of a series of valuable chemicals. In this study, systematic metabolic engineering was applied to target Escherichia coli W3110 for the production of l-homoserine. Initially, a basic l-homoserine producer was engineered through the strategies of overexpressing thrA (encoding homoserine dehydrogenase), removing the degradative and competitive pathways by knocking out metA (encoding homoserine O-succinyltransferase) and thrB (encoding homoserine kinase), reinforcing the transport system, and redirecting the carbon flux by deleting iclR (encoding the isocitrate lyase regulator). The resulting strain constructed by these strategies yielded 3.21 g/liter of l-homoserine in batch cultures. Moreover, based on CRISPR-Cas9/dCas9 (nuclease-dead Cas9)-mediated gene repression for 50 genes, the iterative genetic modifications of biosynthesis pathways improved the l-homoserine yield in a stepwise manner. The rational integration of glucose uptake and recovery of l-glutamate increased l-homoserine production to 7.25 g/liter in shake flask cultivation. Furthermore, the intracellular metabolic analysis further provided targets for strain modification by introducing the anaplerotic route afforded by pyruvate carboxylase to oxaloacetate formation, which resulted in accumulating 8.54 g/liter l-homoserine (0.33 g/g glucose, 62.4% of the maximum theoretical yield) in shake flask cultivation. Finally, a rationally designed strain gave 37.57 g/liter l-homoserine under fed-batch fermentation, with a yield of 0.31 g/g glucose.IMPORTANCE In this study, the bottlenecks that sequentially limit l-homoserine biosynthesis were identified and resolved, based on rational and efficient metabolic-engineering strategies, coupled with CRISPR interference (CRISPRi)-based systematic analysis. The metabolomics data largely expanded our understanding of metabolic effects and revealed relevant targets for further modification to achieve better performance. The systematic analysis strategy, as well as metabolomics analysis, can be used to rationally design cell factories for the production of highly valuable chemicals.
Project description:De novo synthesis of threonine from aspartate occurs via the ?-aspartyl phosphate pathway in plants, bacteria and fungi. However, the Trypanosoma brucei genome encodes only the last two steps in this pathway: homoserine kinase (HSK) and threonine synthase. Here, we investigated the possible roles for this incomplete pathway through biochemical, genetic and nutritional studies. Purified recombinant TbHSK specifically phosphorylates L-homoserine and displays kinetic properties similar to other HSKs. HSK null mutants generated in bloodstream forms displayed no growth phenotype in vitro or loss of virulence in vivo. However, following transformation into procyclic forms, homoserine, homoserine lactone and certain acyl homoserine lactones (AHLs) were found to substitute for threonine in growth media for wild-type procyclics, but not HSK null mutants. The tsetse fly is considered to be an unlikely source of these nutrients as it feeds exclusively on mammalian blood. Bioinformatic studies predict that tsetse endosymbionts possess part (up to homoserine in Wigglesworthia glossinidia) or all of the ?-aspartyl phosphate pathway (Sodalis glossinidius). In addition S.?glossinidius is known to produce 3-oxohexanoylhomoserine lactone which also supports trypanosome growth. We propose that T. brucei has retained HSK and threonine synthase in order to salvage these nutrients when threonine availability is limiting.
Project description:Aspartate kinase (AK) and homoserine dehydrogenase (HSD) function as key regulatory enzymes at branch points in the aspartate amino acid pathway and are feedback-inhibited by threonine. In plants the biochemical features of AK and bifunctional AK-HSD enzymes have been characterized, but the molecular properties of the monofunctional HSD remain unexamined. To investigate the role of HSD, we have cloned the cDNA and gene encoding the monofunctional HSD (GmHSD) from soybean. Using heterologously expressed and purified GmHSD, initial velocity and product inhibition studies support an ordered bi bi kinetic mechanism in which nicotinamide cofactor binds first and leaves last in the reaction sequence. Threonine inhibition of GmHSD occurs at concentrations (K(i) = 160-240 mM) more than 1000-fold above physiological levels. This is in contrast to the two AK-HSD isoforms in soybean that are sensitive to threonine inhibition (K(i) approximately 150 microM). In addition, GmHSD is not inhibited by other aspartate-derived amino acids. The ratio of threonine-resistant to threonine-sensitive HSD activity in soybean tissues varies and likely reflects different demands for amino acid biosynthesis. This is the first cloning and detailed biochemical characterization of a monofunctional feedback-insensitive HSD from any plant. Threonine-resistant HSD offers a useful biotechnology tool for manipulating the aspartate amino acid pathway to increase threonine and methionine production in plants for improved nutritional content.
Project description:We have cloned the homoserine dehydrogenase genes (hom) from the gram-negative obligate methylotrophs Methylobacillus glycogenes ATCC 21276 and ATCC 21371 by complementation of an Escherichia coli homoserine dehydrogenase-deficient mutant. The 4.15-kb DNA fragment cloned from M. glycogenes ATCC 21371 also complemented an E. coli threonine synthase-deficient mutant, suggesting the DNA fragment contained the thrC gene in addition to the hom gene. The homoserine dehydrogenases expressed in the E. coli recombinants were hardly inhibited by L-threonine, L-phenylalanine, or L-methionine. However, they became sensitive to the amino acids after storage at 4 degrees C for 4 days as in M. glycogenes. The structures of the homoserine dehydrogenases overexpressed in E. coli were thought to be different from those in M. glycogenes, probably in subunit numbers of the enzyme, and were thought to have converted to the correct structures during the storage. The nucleotide sequences of the hom and thrC genes were determined. The hom genes of M. glycogenes ATCC 21276 and ATCC 21371 encode peptides with M(r)s of 48,225 and 44,815, respectively. The thrC genes were located 50 bp downstream of the hom genes. The thrC gene of ATCC 21371 encodes a peptide with an M(r) of 52,111, and the gene product of ATCC 21276 was truncated. Northern (RNA) blot analysis suggests that the hom and thrC genes are organized in an operon. Significant homology between the predicted amino acid sequences of the hom and thrC genes and those from other microorganisms was found.
Project description:In Escherichia coli, growth is limited at elevated temperatures mainly because of the instability of a single enzyme, homoserine o-succinyltransferase (MetA), the first enzyme in the methionine biosynthesis pathway. The metA gene from the thermophile Geobacillus kaustophilus cloned into the E. coli chromosome was found to enhance the growth of the host strain at elevated temperature (44 degrees C), thus confirming the limited growth of E. coli due to MetA instability. In order to improve E. coli growth at higher temperatures, we used random mutagenesis to obtain a thermostable MetA(E. coli) protein. Sequencing of the thermotolerant mutant showed five amino acid substitutions: S61T, E213V, I229T, N267D, and N271K. An E. coli strain with the mutated metA gene chromosomally inserted showed accelerated growth over a temperature range of 34 to 44 degrees C. We used the site-directed metA mutants to identify two amino acid residues responsible for the sensitivity of MetA(E. coli) to both heat and acids. Replacement of isoleucine 229 with threonine and asparagine 267 with aspartic acid stabilized the protein. The thermostable MetA(E. coli) enzymes showed less aggregation in vivo at higher temperature, as well as upon acetic acid treatment. The data presented here are the first to show improved E. coli growth at higher temperatures solely due to MetA stabilization and provide new knowledge for designing E. coli strains that grow at higher temperatures, thus reducing the cooling cost of bioprocesses.
Project description:The fungally conserved subset of amino acid biosynthetic enzymes not present in humans offer exciting potential as an unexploited class of antifungal drug targets. Since threonine biosynthesis is essential in Cryptococcus neoformans, we further explored the potential of threonine biosynthetic enzymes as antifungal drug targets by determining the survival in mice of Saccharomyces cerevisiae homoserine kinase (thr1Delta) and threonine synthase (thr4Delta) mutants. In striking contrast to aspartate kinase (hom3Delta) mutants, S. cerevisiae thr1Delta and thr4Delta mutants were severely depleted after only 4 h in vivo. Similarly, Candida albicans thr1Delta mutants, but not hom3Delta mutants, were significantly attenuated in virulence. Consistent with the in vivo phenotypes, S. cerevisiae thr1Delta and thr4Delta mutants as well as C. albicans thr1Delta mutants were extremely serum sensitive. In both species, serum sensitivity was suppressed by the addition of threonine, a feedback inhibitor of Hom3p. Because mutation of the HOM3 and HOM6 genes, required for the production of the toxic pathway intermediate homoserine, also suppressed serum sensitivity, we hypothesize that serum sensitivity is a consequence of homoserine accumulation. Serum survival is critical for dissemination, an important virulence determinant: thus, together with the essential nature of C. neoformans threonine synthesis, the cross-species serum sensitivity of thr1Delta mutants makes the fungus-specific Thr1p, and likely Thr4p, ideal antifungal drug targets.
Project description:We have subcloned the coding sequence for the Escherichia coli threonine synthase gene into a eukaryotic expression vector based on the simian-virus-40 early promoter. When mouse 3T3 cells which already expressed homoserine kinase were transfected with the new plasmid, the cells were able to incorporate radioactivity from [14C]homoserine into their cell proteins. Stable cell lines were established by co-transfecting 3T3 cells with the plasmid coding for threonine synthase and another coding for homoserine kinase and G-418 (Geneticin) resistance. Cells were selected for G-418 resistance and then screened for an ability to synthesize threonine from homoserine and incorporate it into the cell protein. A cell line which expressed both the homoserine kinase and threonine synthase genes was capable of growth in a threonine-deficient medium containing homoserine.