The synthetic xylulose-1 phosphate pathway increases production of glycolic acid from xylose-rich sugar mixtures.

BACKGROUND: Glycolic acid (GA) is a two-carbon hydroxyacid with applications in the cosmetic, textile, and medical industry. Microbial GA production from all sugars can be achieved by engineering the natural glyoxylate shunt. The synthetic (d)-xylulose-1 phosphate (X1P) pathway provides a complementary route to produce GA from (d)-xylose. The simultaneous operation of the X1P and glyoxylate pathways increases the theoretical GA yield from xylose by 20 %, which may strongly improve GA production from hemicellulosic hydrolysates. RESULTS: We herein describe the construction of an E. coli strain that produces GA via the glyoxylate pathway at a yield of 0.31 , 0.29 , and 0.37 g/g from glucose, xylose, or a mixture of glucose and xylose (mass ratio: 33:66 %), respectively. When the X1P pathway operates in addition to the glyoxylate pathway, the GA yields on the three substrates are, respectively, 0.39 , 0.43 , and 0.47 g/g. Upon constitutive expression of the sugar permease GalP, the GA yield of the strain which simultaneously operates the glyoxylate and X1P pathways further increases to 0.63 g/g when growing on the glucose/xylose mixture. Under these conditions, the GA yield on the xylose fraction of the sugar mixture reaches 0.75 g/g, which is the highest yield reported to date. CONCLUSIONS: These results demonstrate that the synthetic X1P pathway has a very strong potential to improve GA production from xylose-rich hemicellulosic hydrolysates.

Engineering of a Synthetic Metabolic Pathway for the Assimilation of (d)-Xylose into Value-Added Chemicals.

A synthetic pathway for (d)-xylose assimilation was stoichiometrically evaluated and implemented in Escherichia coli strains. The pathway proceeds via isomerization of (d)-xylose to (d)-xylulose, phosphorylation of (d)-xylulose to obtain (d)-xylulose-1-phosphate (X1P), and aldolytic cleavage of the latter to yield glycolaldehyde and DHAP. Stoichiometric analyses showed that this pathway provides access to ethylene glycol with a theoretical molar yield of 1. Alternatively, both glycolaldehyde and DHAP can be converted to glycolic acid with a theoretical yield that is 20% higher than for the exclusive production of this acid via the glyoxylate shunt. Simultaneous expression of xylulose-1 kinase and X1P aldolase activities, provided by human ketohexokinase-C and human aldolase-B, respectively, restored growth of a (d)-xylulose-5-kinase mutant on xylose. This strain produced ethylene glycol as the major metabolic endproduct. Metabolic engineering provided strains that assimilated the entire C2 fraction into the central metabolism or that produced 4.3 g/L glycolic acid at a molar yield of 0.9 in shake flasks.

Optimization of ethylene glycol production from (D)-xylose via a synthetic pathway implemented in Escherichia coli.

BACKGROUND: Ethylene glycol (EG) is a bulk chemical that is mainly used as an anti-freezing agent and a raw material in the synthesis of plastics. Production of commercial EG currently exclusively relies on chemical synthesis using fossil resources. Biochemical production of ethylene glycol from renewable resources may be more sustainable. RESULTS: Herein, a synthetic pathway is described that produces EG in Escherichia coli through the action of (D)-xylose isomerase, (D)-xylulose-1-kinase, (D)-xylulose-1-phosphate aldolase, and glycolaldehyde reductase. These reactions were successively catalyzed by the endogenous xylose isomerase (XylA), the heterologously expressed human hexokinase (Khk-C) and aldolase (Aldo-B), and an endogenous glycolaldehyde reductase activity, respectively, which we showed to be encoded by yqhD. The production strain was optimized by deleting the genes encoding for (D)-xylulose-5 kinase (xylB) and glycolaldehyde dehydrogenase (aldA), and by overexpressing the candidate glycolaldehyde reductases YqhD, GldA, and FucO. The strain overproducing FucO was the best EG producer reaching a molar yield of 0.94 in shake flasks, and accumulating 20 g/L EG with a molar yield and productivity of 0.91 and 0.37 g/(L.h), respectively, in a controlled bioreactor under aerobic conditions. CONCLUSIONS: We have demonstrated the feasibility to produce EG from (D)-xylose via a synthetic pathway in E. coli at approximately 90 % of the theoretical yield.

Control of NO level in rhizobium-legume root nodules: not only a plant globin story.

Nitric oxide (NO ) is a gaseous signaling molecule which plays both regulatory and defense roles in animals and plants. In the symbiosis between legumes and rhizobia, NO has been shown to be involved in bacterial infection and nodule development steps as well as in mature nodule functioning. We recently showed that an increase in NO level inside Medicago truncatula root nodules also could trigger premature nodule senescence. Here we discuss the importance of the bacterial Sinorhizobium meliloti flavohemoglobin to finely tune the NO level inside nodules and further, we demonstrate that S. meliloti possesses at least two non redundant ways to control NO and that both systems are necessary to maintain efficient nitrogen fixing activity.

Nitric oxide (NO): a key player in the senescence of Medicago truncatula root nodules.

Nitric oxide (NO) is a signalling and defence molecule involved in diverse plant developmental processes, as well as in the plant response to pathogens. NO has also been detected at different steps of the symbiosis between legumes and rhizobia. NO is required for an optimal establishment of the Medicago truncatula-Sinorhizobium meliloti symbiotic interaction, but little is known about the role of NO in mature nodules. Here, we investigate the role of NO in the late steps of symbiosis. Genetic and pharmacological approaches were conducted to modulate the NO level inside root nodules, and their effects on nitrogen fixation and root nodule senescence were monitored. An increase in endogenous NO levels led to a decrease in nitrogen fixation and early nodule senescence, characterized by cytological modifications of the nodule structure and the early expression of a specific senescence marker. By contrast, a decrease in NO levels led to a delay in nodule senescence. Together, our results strongly suggest that NO is a signal in developmental as well as stress-induced nodule senescence. In addition, this work demonstrates the pivotal role of the bacterial NO detoxification response in the prevention of early nodule senescence, and hence the maintenance of efficient symbiosis.

Nitric oxide is required for an optimal establishment of the Medicago truncatula-Sinorhizobium meliloti symbiosis.

Nitric oxide (NO) is a gaseous molecule that participates in numerous plant signalling pathways. It is involved in plant responses to pathogens and development processes such as seed germination, flowering and stomatal closure. Using a permeable NO-specific fluorescent probe and a bacterial reporter strain expressing the lacZ gene under the control of a NO-responsive promoter, we detected NO production in the first steps, during infection threads growth, of the Medicago truncatula-Sinorhizobium meliloti symbiotic interaction. Nitric oxide was also detected, by confocal microscopy, in nodule primordia. Depletion of NO caused by cPTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide), an NO scavenger, resulted in a significant delay in nodule appearance. The overexpression of a bacterial hmp gene, encoding a flavohaemoglobin able to scavenge NO, under the control of a nodule-specific promoter (pENOD20) in transgenic roots, led to the same phenotype. The NO scavenging resulting from these approaches provoked the downregulation of plant genes involved in nodule development, such as MtCRE1 and MtCCS52A. Furthermore, an Hmp-overexpressing S. meliloti mutant strain was found to be less competitive than the wild type in the nodulation process. Taken together, these results indicate that NO is required for an optimal establishment of the M. truncatula-S. meliloti symbiotic interaction.

The response to nitric oxide of the nitrogen-fixing symbiont Sinorhizobium meliloti.

Nitric oxide (NO) is crucial in animal- and plant-pathogen interactions, during which it participates in host defense response and resistance. Indications for the presence of NO during the symbiotic interaction between the model legume Medicago truncatula and its symbiont Sinorhizobium meliloti have been reported but the role of NO in symbiosis is far from being elucidated. Our objective was to understand the role or roles played by NO in symbiosis. As a first step toward this goal, we analyzed the bacterial response to NO in culture, using a transcriptomic approach. We identified approximately 100 bacterial genes whose expression is upregulated in the presence of NO. Surprisingly, most of these genes are regulated by the two-component system FixLJ, known to control the majority of rhizobial genes expressed in planta in mature nodules, or the NO-dedicated regulator NnrR. Among the genes responding to NO is hmp, encoding a putative flavohemoglobin. We report that an hmp mutant displays a higher sensitivity toward NO in culture and leads to a reduced nitrogen fixation efficiency in planta. Because flavohemoglobins are known to detoxify NO in numerous bacterial species, this result is the first indication of the importance of the bacterial NO response in symbiosis.