(Optochemical) Control of Synthetic Microbial Coculture Interactions on a Microcolony Level.

Synthetic microbial cocultures carry enormous potential for applied biotechnology and are increasingly the subject of fundamental research. So far, most cocultures have been designed and characterized based on bulk cultivations without considering the potentially highly heterogeneous and diverse single-cell behavior. However, an in-depth understanding of cocultures including their interacting single cells is indispensable for the development of novel cultivation approaches and control of cocultures. We present the development, validation, and experimental characterization of an optochemically controllable bacterial coculture on a microcolony level consisting of two Corynebacterium glutamicum strains. Our coculture combines an l-lysine auxotrophic strain together with a l-lysine-producing variant carrying the genetically IPTG-mediated induction of l-lysine production. We implemented two control approaches utilizing IPTG as inducer molecule. First, unmodified IPTG was supplemented to the culture enabling a medium-based control of the production of l-lysine, which serves as the main interacting component. Second, optochemical control was successfully performed by utilizing photocaged IPTG activated by appropriate illumination. Both control strategies were validated studying cellular growth on a microcolony level. The novel microfluidic single-cell cultivation strategies applied in this work can serve as a blueprint to validate cellular control strategies of synthetic mono- and cocultures with single-cell resolution at defined environmental conditions.

Overexpression of the saccharopine dehydrogenase gene improves lysine biosynthesis in Flammulina velutipes.

Saccharopine dehydrogenase (EC 1.5.1.7) regulates the last step of fungal lysine biosynthesis. The gene (Fvsdh) encoding saccharopine dehydrogenase was identified and cloned from the whole genome of Flammulina velutipes. The genomic DNA of Fvsdh is 1257 bp, comprising three introns and four exons. The full-length complementary DNA of Fvsdh comprises 1107 bp with a deduced amino acid sequence of 368 residues. A 1,000-bp promoter sequence containing the TATA box, CAAT box, and several putative cis-acting elements was also identified. The results of tissue expression analysis showed that the expression level of the Fvsdh gene was higher in the pileus than in the stipe whether in the elongation or maturation stage. Further research showed that the lysine contents were 3.03 and 2.95 mg/g in maturation-pileus and elongation-pileus, respectively. In contrast, the lysine contents were 2.49 and 2.07 mg/g in elongation-stipe and maturation-stipe, respectively. To study the function of Fvsdh, we overexpressed Fvsdh in F. velutipes and found that Fvsdh gene expression was increased from 1.1- to 3-fold in randomly selected transgenic strains. The lysine contents were also increased from 1.12- to 1.3-fold in these five transformants, except for strain T3, in which the lysine contents were the same as the control. These results indicate that the expression of the Fvsdh gene can affect the lysine content of F. velutipes.

Expanding lysine industry: industrial biomanufacturing of lysine and its derivatives.

L-Lysine is widely used as a nutrition supplement in feed, food, and beverage industries as well as a chemical intermediate. At present, great efforts are made to further decrease the cost of lysine to make it more competitive in the markets. Furthermore, lysine also shows potential as a feedstock to produce other high-value chemicals for active pharmaceutical ingredients, drugs, or materials. In this review, the current biomanufacturing of lysine is first presented. Second, the production of novel derivatives from lysine is discussed. Some chemicals like L-pipecolic acid, cadaverine, and 5-aminovalerate already have been obtained at a lab scale. Others like 6-aminocaproic acid, valerolactam, and caprolactam could be produced through a biological and chemical coupling pathway or be synthesized by a hypothetical pathway. This review demonstrates an active and expansive lysine industry, and these green biomanufacturing strategies could also be applied to enhance the competitiveness of other amino acid industry.

Structural basis for substrate specificity of meso-diaminopimelic acid decarboxylase from Corynebacterium glutamicum.

l-lysine is an essential amino acid that is widely used as a food supplement for humans and animals. meso-Diaminopimelic acid decarboxylase (DAPDC) catalyzes the final step in the de novol-lysine biosynthetic pathway by converting meso-diaminopimelic acid (meso-DAP) into l-lysine by decarboxylation reaction. To elucidate its molecular mechanisms, we determined the crystal structure of DAPDC from Corynebacterium glutamicum (CgDAPDC). The PLP cofactor is bound at the center of the barrel domain and forms a Schiff base with the catalytic Lys75 residue. We also determined the CgDAPDC structure in complex with both pyridoxal 5'-phosphate (PLP) and the l-lysine product and revealed that the protein has an optimal substrate binding pocket to accommodate meso-DAP as a substrate. Structural comparison of CgDAPDC with other amino acid decarboxylases with different substrate specificities revealed that the position of the α15 helix in CgDAPDC and the residues located on the helix are crucial for determining the substrate specificities of the amino acid decarboxylases.

Improved growth performance, food efficiency, and lysine availability in growing rats fed with lysine-biofortified rice.

Rice is an excellent source of protein, and has an adequate balance of amino acids with the exception of the essential amino acid lysine. By using a combined enhancement of lysine synthesis and suppression of its catabolism, we had produced two transgenic rice lines HFL1 and HFL2 (High Free Lysine) containing high concentration of free lysine. In this study, a 70-day rat feeding study was conducted to assess the nutritional value of two transgenic lines as compared with either their wild type (WT) or the WT rice supplemented with different concentrations of L-lysine. The results revealed that animal performance, including body weight, food intake, and food efficiency, was greater in the HFL groups than in the WT group. Moreover, the HFL diets had increased protein apparent digestibility, protein efficiency ratio, and lysine availability than the WT diet. Based on the linear relationship between dietary L-lysine concentrations and animal performance, it indicated that the biological indexes of the HFL groups were similar or better than that of the WT20 group, which was supplemented with L-lysine concentrations similar to those present in the HFL diets. Therefore, lysine-biofortified rice contributed to improved growth performance, food efficiency, and lysine availability in growing rats.

Disruptions of the genes involved in lysine biosynthesis, iron acquisition, and secondary metabolisms affect virulence and fitness in Metarhizium robertsii.

Based on genomic analysis, polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) pathways account for biosynthesis of the majority of the secondary metabolites produced by the entomopathogenic fungus Metarhizium robertsii. To evaluate the contribution of these pathways to M. robertsii fitness and/or virulence, mutants deleted for mrpptA, the Sfp-type 4' phosphopantetheinyl transferase gene required for their activation were generated. ΔmrpptA strains were deficient in PKS and NRPS activity resulting in colonies that lacked the typical green pigment and failed to produce the nonribosomal peptides (destruxins, serinocylins, and the siderophores ferricrocin and metachelins) as well as the hybrid polyketide-peptides (NG-39x) that are all produced by the wild type (WT) M. robertsii. The ΔmrpptA colonies were also auxotrophic for lysine. Two other mutant strains were generated: ΔmraarA, in which the α-aminoadipate reductase gene critical for lysine biosynthesis was disrupted, and ΔmrsidA, in which the L-ornithine N5-oxygenase gene that is critical for hydroxamate siderophore biosynthesis was disrupted. The phenotypes of these mutants were compared to those of ΔmrpptA to separate effects of the loss of lysine or siderophore production from the overall effect of losing all polyketide and non-ribosomal peptide production. Loss of lysine biosynthesis marginally increased resistance to H2O2 while it had little effect on the sensitivity to the cell wall disruptor sodium dodecyl sulfate (SDS) and no effect on sensitivity to iron deprivation. In contrast, combined loss of metachelin and ferricrocin through the inactivation of mrsidA resulted in mutants that were as hypersensitive or slightly more sensitive to H2O2, iron deprivation, and SDS, and were either identical or marginally higher in ΔmrpptA strains. In contrast to ΔmrpptA, loss of mrsidA did not completely abolish siderophore activity, which suggests the production of one or more non-hydroxamate iron-chelating compounds. Deletion of mrpptA, mrsidA, and mraarA reduced conidium production and conidia of a GFP-tagged ΔmrpptA strain displayed a longer germination delay than WT on insect cuticles, a deficiency that was rescued by lysine supplementation. Compared with WT, ΔmrpptA strains displayed ∼19-fold reduction in virulence against Drosophila suzukii. In contrast, lysine auxotrophy and loss of siderophores accounted for ∼2 and ∼6-fold decreases in virulence, respectively. Deletion of mrpptA had no significant effect on growth inhibition of Bacillus cereus. Our results suggest that PKS and NRPS metabolism plays a significant role in M. robertsii virulence, depresses conidium production, and contributes marginally to resistance to oxidative stress and iron homeostasis, but has no significant antibacterial effect.

l-Lysine production independent of the oxidative pentose phosphate pathway by Corynebacterium glutamicum with the Streptococcus mutans gapN gene.

We have recently developed a Corynebacterium glutamicum strain that generates NADPH via the glycolytic pathway by replacing endogenous NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GapA) with a nonphosphorylating NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) from Streptococcus mutans. Strain RE2, a suppressor mutant spontaneously isolated for its improved growth on glucose from the engineered strain, was proven to be a high-potential host for l-lysine production (Takeno et al., 2010). In this study, the suppressor mutation was identified to be a point mutation in rho encoding the transcription termination factor Rho. Strain RE2 still showed retarded growth despite the mutation rho696. Our strategy for reconciling improved growth with a high level of l-lysine production was to use GapA together with GapN only in the early growth phase, and subsequently shift this combination-type glycolysis to one that depends only on GapN in the rest of the growth phase. To achieve this, we expressed gapA under the myo-inositol-inducible promoter of iolT1 encoding a myo-inositol transporter in strain RE2. The resulting strain RE2A(iol) was engineered into an l-lysine producer by introduction of a plasmid carrying the desensitized lysC, followed by examination for culture conditions with myo-inositol supplementation. We found that as a higher concentration of myo-inositol was added to the seed culture, the following fermentation period became shorter while maintaining a high level of l-lysine production. This finally reached a fermentation period comparable to that of the control GapA strain, and yielded a 1.5-fold higher production rate compared with strain RE2. The transcript level of gapA, as well as the GapA activity, in the early growth phase increased in proportion to the myo-inositol concentration and then fell to low levels in the subsequent growth phase, indicating that improved growth was a result of increased GapA activity, especially in the early growth phase. Moreover, blockade of the pentose phosphate pathway through a defect in glucose 6-phosphate dehydrogenase did not significantly affect l-lysine production in the engineered GapN strains, while a drastic decrease in l-lysine production was observed for the control GapA strain. Determination of the intracellular NADPH/NADP(+) ratios revealed that the ratios in the engineered strains were significantly higher than the ratio of the control GapA strain irrespective of the pentose phosphate pathway. These results demonstrate that our strain engineering strategy allows efficient l-lysine production independent of the oxidative pentose phosphate pathway.

Enhancing L-Lysine Production of Beet Molasses by Engineered Escherichia coli Using an In Situ Pretreatment Method.

Reducing the viscosity of molasses environmentally and selectively removing the harmful ingredients for microbes are the keys to promoting the bioavailability of molasses. A simple and environmental in situ pretreatment method integrating surfactants and alkali was developed to reduce the viscosity of molasses prior to L-lysine production using Escherichia coli ZY0217. Adding activated carbon and modified orange peel based on the in situ pretreatment process effectively removed pigments and excessive zinc in the molasses and also significantly increased the cell growth and L-lysine yield from E. coli ZY0217. The experimental results showed that a mixture of secondary alkane sulfonate, an anionic surfactant, and HodagCB-6, a non-ionic surfactant, effectively reduced the viscosity of the molasses more so than any single surfactant. When the surfactant mixture was added at a concentration of 0.04 g/L to the molasses, the ω value was 0.4, and when ammonia was added at 0.6 %, the lowest viscosity of 705 mPa · s was obtained. Further, 91.5 % of the color and 86.68 % of the original levels of zinc were removed using an activated carbon and modified orange peel treatment on the molasses with the lowest viscosity, which further promoted cell growth and L-lysine production. In the fed-batch cultivation process, the L-lysine concentration achieved using a constant-speed feeding strategy was 45.89 g/L, with an L-lysine yield of 27.18 %, whereas the L-lysine yield from untreated molasses was only 10.13 %. The increase in L-lysine yield was related to the reduced viscosity and the detoxification of the molasses. Lastly, the pretreatment was found to significantly enhance the conversion of sugars in the molasses to L-lysine.

Two new prenylflavonoids from Epimedii Herba and their inhibitory effects on advanced glycation end-products.

Because inhibitors of advanced glycation end-products (AGEs), for example pyridoxamine, significantly inhibit the development of retinopathy and neuropathy in rats with streptozotocin-induced diabetes, treatment with AGE inhibitors is believed to be a potential strategy for the prevention of lifestyle-related diseases such as diabetic complications. In the present study, the MeOH extract of Epimedii Herba (EH; aerial parts of Epimedium spp.) was found to inhibit the formation of N (ε) -(carboxymethyl)lysine (CML) and N (ω) -(carboxymethyl)arginine (CMA) during incubation of collagen-derived gelatin with ribose. Furthermore, compounds with inhibitory effects against CML and CMA formation were isolated from EH. Two new prenylflavonoids (compounds 1 and 2) and two known compounds (3 and 4) were found to significantly inhibit the formation of both CML and CMA; compound 4 (epimedokoreanin B) had the strongest inhibitory effect of the isolated compounds. These data suggest that epimedokoreanin B could prevent clinical complications of diabetes by inhibiting AGEs.

Structures of two bacterial resistance factors mediating tRNA-dependent aminoacylation of phosphatidylglycerol with lysine or alanine.

The cytoplasmic membrane is probably the most important physical barrier between microbes and the surrounding habitat. Aminoacylation of the polar head group of the phospholipid phosphatidylglycerol (PG) catalyzed by Ala-tRNA(Ala)-dependent alanyl-phosphatidylglycerol synthase (A-PGS) or by Lys-tRNA(Lys)-dependent lysyl-phosphatidylglycerol synthase (L-PGS) enables bacteria to cope with cationic peptides that are harmful to the integrity of the cell membrane. Accordingly, these synthases also have been designated as multiple peptide resistance factors (MprF). They consist of a separable C-terminal catalytic domain and an N-terminal transmembrane flippase domain. Here we present the X-ray crystallographic structure of the catalytic domain of A-PGS from the opportunistic human pathogen Pseudomonas aeruginosa. In parallel, the structure of the related lysyl-phosphatidylglycerol-specific L-PGS domain from Bacillus licheniformis in complex with the substrate analog L-lysine amide is presented. Both proteins reveal a continuous tunnel that allows the hydrophobic lipid substrate PG and the polar aminoacyl-tRNA substrate to access the catalytic site from opposite directions. Substrate recognition of A-PGS versus L-PGS was investigated using misacylated tRNA variants. The structural work presented here in combination with biochemical experiments using artificial tRNA or artificial lipid substrates reveals the tRNA acceptor stem, the aminoacyl moiety, and the polar head group of PG as the main determinants for substrate recognition. A mutagenesis approach yielded the complementary amino acid determinants of tRNA interaction. These results have broad implications for the design of L-PGS and A-PGS inhibitors that could render microbial pathogens more susceptible to antimicrobial compounds.

Report: Bioconversion of agriculture waste to lysine with UV mutated strain of brevibacterium flavum and its biological evaluation in broiler chicks.

Lysine executes imperative structural and functional roles in body and its supplementation in diet beneficial to prevent the escalating threat of protein deficiency. The physical mutagenesis offers new fascinating avenues of research for overproduction of lysine through surplus carbohydrate containing agriculture waste especially in developing countries. The current study was aimed to investigate the potential of UV mutated strain of Brevibacterium flavum at 254 nm for lysine production. The physical and nutritional parameters were optimized and maximum lysine production was observed with molasses (4% substrate water ratio). Moreover, supplementation of culture medium with metal cations (i.e. 0.4% CaSO4, 0.3% NaCl, 0.3% KH2PO4, 0.4% MgSO4, and 0.2% (NH4) 2SO4w/v) together with 0.75% v/v corn steep liquor significantly enhanced the lysine production up to 26.71 ± 0.31 g/L. Though, concentrations of urea, ammonium nitrate and yeast sludge did not exhibit any profound effect on lysine production. Biological evaluation of lysine enriched biomass in terms of weight gain and feed conversion ratio reflected non-significant difference for experimental and control (+ve) groups. Conclusively, lysine produced in the form of biomass was compatible to market lysine in its effectiveness and have potential to utilize at commercial scale.

Enhanced L-lysine production from pretreated beet molasses by engineered Escherichia coli in fed-batch fermentation.

Faster sugar consumption rate and low-cost nitrogen source are required for the chemical biosynthesis using molasses. Five pretreatment methods were applied to beet molasses prior to fermentation through engineered Escherichia coli, respectively, and corn steep liquid was used as an organic nitrogen source to replace expensive yeast extract. Furthermore, the effects of different feeding strategy in fed-batch fermentation on L-lysine production were investigated. The experimental results showed that combined tricalcium phosphate, sulfuric acid, and activated carbon pretreatment method (TPSA) pretreatment could improve the sugar consumption rate most greatly, and the initial total sugar concentration of 35 g/L from TPSA-pretreated beet molasses gave the best results with respect to L-lysine production, dry cell weight concentration, and L-lysine yield in batch fermentation. Moreover, a mixture of low-cost corn steep liquid and yeast extract containing equal amount of nitrogen could be used as the organic nitrogen source for effective L-lysine fermentation, and constant speed feeding strategy of TPSA-pretreated beet molasses promoted L-lysine production by engineered E. coli. The TPSA-pretreated beet molasses had a sugar consumption rate of 1.75 g/(L h), and a L-lysine yield of 27.81% was achieved, compared with the theoretical yield of 62% by glucose. It was clarified that the pretreatment significantly enhanced the conversion of sugars in beet molasses to L-lysine.

Glycation of human cortical and cancellous bone captures differences in the formation of Maillard reaction products between glucose and ribose.

To better understand some aspects of bone matrix glycation, we used an in vitro glycation approach. Within two weeks, our glycation procedures led to the formation of advanced glycation end products (AGEs) at the levels that corresponded to approx. 25-30 years of the natural in vivo glycation. Cortical and cancellous bones from human tibias were glycated in vitro using either glucose (glucosylation) or ribose (ribosylation). Both glucosylation and ribosylation led to the formation of higher levels of AGEs and pentosidine (PEN) in cancellous than cortical bone dissected from all tested donors (young, middle-age and elderly men and women). More efficient glycation of bone matrix proteins in cancellous bone most likely depended on the higher porosity of this tissue, which facilitated better accessibility of the sugars to the matrix proteins. Notably, glycation of cortical bone from older donors led to much higher AGEs levels as compared to young donors. Such efficient in vitro glycation of older cortical bone could result from aging-related increase in porosity caused by the loss of mineral content. In addition, more pronounced glycation in vivo would be driven by elevated oxidation processes. Interestingly, the levels of PEN formation differed pronouncedly between glucosylation and ribosylation. Ribosylation generated very high levels of PEN (approx. 6- vs. 2.5-fold higher PEN level than in glucosylated samples). Kinetic studies of AGEs and PEN formation in human cortical and cancellous bone matrix confirmed higher accumulation of fluorescent crosslinks for ribosylation. Our results suggest that in vitro glycation of bone using glucose leads to the formation of lower levels of AGEs including PEN, whereas ribosylation appears to support a pathway toward PEN formation. Our studies may help to understand differences in the progression of bone pathologies related to protein glycation by different sugars, and raise awareness for excessive sugar supplementation in food and drinks.

Optimization of culture conditions for enhanced lysine production using engineered Escherichia coli.

In this study, culture conditions, including dissolved oxygen (DO) content, presence of osmoprotectants, residual glucose concentration, and ammonium sulfate-feeding strategies, were investigated for decreasing the inhibition effects of acetic acid, ammonium, and osmotic stress on L-lysine fermentation by Escherichia coli. The results revealed that higher DO content and lower residual glucose concentration could decrease acetic acid accumulation, betaine supplementation could enhance osmotic stress tolerance, and variable speed ammonium sulfate-feeding strategy could decrease ammonium inhibition. Thus, with 25 % DO content, 0-5.0 g/L of residual glucose concentration, and 1.5 g/L of betaine supplementation, 134.9 g/L of L-lysine was obtained after 72 h of culture, with L-lysine yield and productivity of 45.4 % and 1.9 g/(L · h), respectively.

A new anaplerotic respiratory pathway involving lysine biosynthesis in isocitrate dehydrogenase-deficient Arabidopsis mutants.

The cornerstone of carbon (C) and nitrogen (N) metabolic interactions - respiration - is presently not well understood in plant cells: the source of the key intermediate 2-oxoglutarate (2OG), to which reduced N is combined to yield glutamate and glutamine, remains somewhat unclear. We took advantage of combined mutations of NAD- and NADP-dependent isocitrate dehydrogenase activity and investigated the associated metabolic effects in Arabidopsis leaves (the major site of N assimilation in this genus), using metabolomics and (13)C-labelling techniques. We show that a substantial reduction in leaf isocitrate dehydrogenase activity did not lead to changes in the respiration efflux rate but respiratory metabolism was reorchestrated: 2OG production was supplemented by a metabolic bypass involving both lysine synthesis and degradation. Although the recycling of lysine has long been considered important in sustaining respiration, we show here that lysine neosynthesis itself participates in an alternative respiratory pathway. Lys metabolism thus contributes to explaining the metabolic flexibility of plant leaves and the effect (or the lack thereof) of respiratory mutations.

Fortifying plants with the essential amino acids lysine and methionine to improve nutritional quality.

Humans, as well as farm animals, cannot synthesize a number of essential amino acids, which are critical for their survival. Hence, these organisms must obtain these essential amino acids from their diets. Cereal and legume crops, which represent the major food and feed sources for humans and livestock worldwide, possess limiting levels of some of these essential amino acids, particularly Lys and Met. Extensive efforts were made to fortify crop plants with these essential amino acids using traditional breeding and mutagenesis. However, aside from some results obtained with maize, none of these approaches was successful. Therefore, additional efforts using genetic engineering approaches concentrated on increasing the synthesis and reducing the catabolism of these essential amino acids and also on the expression of recombinant proteins enriched in them. In the present review, we discuss the basic biological aspects associated with the synthesis and accumulation of these amino acids in plants and also describe recent developments associated with the fortification of crop plants with essential amino acids by genetic engineering approaches.

Genome sequence of thermotolerant Bacillus methanolicus: features and regulation related to methylotrophy and production of L-lysine and L-glutamate from methanol.

Bacillus methanolicus can utilize methanol as its sole carbon and energy source, and the scientific interest in this thermotolerant bacterium has focused largely on exploring its potential as a biocatalyst for the conversion of methanol into L-lysine and L-glutamate. We present here the genome sequences of the important B. methanolicus model strain MGA3 (ATCC 53907) and the alternative wild-type strain PB1 (NCIMB13113). The physiological diversity of these two strains was demonstrated by a comparative fed-batch methanol cultivation displaying highly different methanol consumption and respiration profiles, as well as major differences in their L-glutamate production levels (406 mmol liter(-1) and 11 mmol liter(-1), respectively). Both genomes are small (ca 3.4 Mbp) compared to those of other related bacilli, and MGA3 has two plasmids (pBM19 and pBM69), while PB1 has only one (pBM20). In particular, we focus here on genes representing biochemical pathways for methanol oxidation and concomitant formaldehyde assimilation and dissimilation, the important phosphoenol pyruvate/pyruvate anaplerotic node, the tricarboxylic acid cycle including the glyoxylate pathway, and the biosynthetic pathways for L-lysine and L-glutamate. Several unique findings were made, including the discovery of three different methanol dehydrogenase genes in each of the two B. methanolicus strains, and the genomic analyses were accompanied by gene expression studies. Our results provide new insight into a number of peculiar physiological and metabolic traits of B. methanolicus and open up possibilities for system-level metabolic engineering of this bacterium for the production of amino acids and other useful compounds from methanol.

Sfp-type 4'-phosphopantetheinyl transferase is required for lysine synthesis, tolerance to oxidative stress and virulence in the plant pathogenic fungus Cochliobolus sativus.

Polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) are the major enzymes involved in the biosynthesis of secondary metabolites, which have diverse activities, including roles as pathogenicity/virulence factors in plant pathogenic fungi. These enzymes are activated by 4'-phosphopantetheinylation at the conserved serine residues, which is catalysed by 4'-phosphopantetheinyl transferase (PPTase). PPTase is also required for primary metabolism (α-aminoadipate reductase, AAR). In the genome sequence of the cereal fungal pathogen Cochliobolus sativus, we identified a gene (PPT1) orthologous to the PPTase-encoding genes found in other filamentous ascomycetes. The deletion of PPT1 in C. sativus generated mutants (Δppt1) that were auxotrophic for lysine, unable to synthesize melanin, hypersensitive to oxidative stress and significantly reduced in virulence to barley cv. Bowman. To analyse the pleiotropic effects of PPT1, we also characterized deletion mutants for PKS1 (involved in melanin synthesis), AAR1 (for AAR) and NPS6 (involved in siderophore-mediated iron metabolism). The melanin-deficient strain (Δpks1) showed no differences in pathogenicity and virulence compared with the wild-type strain. Lysine-auxotrophic mutants (Δaar1) induced spot blotch symptoms, as produced by the wild-type strain, when inoculated on wounded barley leaves or when lysine was supplemented. The Δnps6 strain showed a slightly reduced virulence compared with the wild-type strain, but exhibited significantly higher virulence than the Δppt1 strain. Our results suggest that an unknown virulence factor, presumably synthesized by PKSs or NRPSs which are activated by PPTase, is directly responsible for high virulence of C. sativus on barley cv. Bowman.

Coexisting/Coexpressing Genomic Libraries (CoGeL) identify interactions among distantly located genetic loci for developing complex microbial phenotypes.

In engineering novel microbial strains for biotechnological applications, beyond a priori identifiable pathways to be engineered, it is becoming increasingly important to develop complex, ill-defined cellular phenotypes. One approach is to screen genomic or metagenomic libraries to identify genes imparting desirable phenotypes, such as tolerance to stressors or novel catabolic programs. Such libraries are limited by their inability to identify interactions among distant genetic loci. To solve this problem, we constructed plasmid- and fosmid-based Escherichia coli Coexisting/Coexpressing Genomic Libraries (CoGeLs). As a proof of principle, four sets of two genes of the l-lysine biosynthesis pathway distantly located on the E. coli chromosome were knocked out. Upon transformation of these auxotrophs with CoGeLs, cells growing without supplementation were found to harbor library inserts containing the knocked-out genes demonstrating the interaction between the two libraries. CoGeLs were also screened to identify genetic loci that work synergistically to create the considerably more complex acid-tolerance phenotype. CoGeL screening identified combination of genes known to enhance acid tolerance (gadBC operon and adiC), but also identified the novel combination of arcZ and recA that greatly enhanced acid tolerance by 9000-fold. arcZ is a small RNA that we show increases pH tolerance alone and together with recA.

The aspartate-family pathway of plants: linking production of essential amino acids with energy and stress regulation.

The Asp family pathway of plants is highly important from a nutritional standpoint because it leads to the synthesis of the four essential amino acids Lys, Thr, Met and Ile. These amino acids are not synthesized by human and its monogastric livestock and should be supplemented in their diets. Among the Asp-family amino acids, Lys is considered as the nutritionally most important essential amino acid because its level is most limiting in cereal grains, representing the largest source of plant foods and feeds worldwide. Metabolic engineering approaches led to significant increase in Lys level in seeds by enhancing its synthesis and reducing its catabolism. However, results from the model plant Arabidopsis showed that this approach may retard seed germination due to a major negative effect on the levels of a number of TCA cycle metabolites that associate with cellular energy. In the present review, we discuss the regulatory metabolic link of the Asp-family pathway with the TCA cycle and its biological significance upon exposure to stress conditions that cause energy deprivation. In addition, we also discuss how deep understanding of the regulatory metabolic link of the Asp-family pathway with energy and stress regulation can be used to improve Lys level in seeds of important crop species, minimizing the interference with the cellular energy status and plant-stress interaction. This review thus provides an example showing how deep understanding the inter-regulation of metabolism with plant stress physiology can lead to successful nutritional improvements with minimal negative effect on plant growth and response to stressful environments.