Arthrospira promotes plant growth and soil properties under high salinity environments.

Salt stress detrimentally impacts plant growth, imperiling crop yield and food quality. Ameliorating plant resilience and productivity in saline environments is critical for global food security. Here, we report the positive effect of Arthrospira (Spirulina) on plant growth and salt tolerance in Arabidopsis and sweet sorghum. Arthrospira application greatly promotes seed germination and seedling growth in both species under salt stress conditions in a dosage-dependent manner. Application of 6 mg Arthrospira per plate significantly enhances K+/Na+ equilibrium and reactive oxygen species (ROS) scavenging in Arabidopsis, reducing salt-induced toxicity. The primary root length, survival rate, chlorophyll content, photosynthesis, plant height, biomass and yield were all improved in both species. Concurrently, Arthrospira demonstrated the synthesis of compatible solutes, such as trehalose (Tre) and glucosylglycerol (GG), contributing to heightened stress tolerance when co-cultivated with Arabidopsis on plates. Transcriptome analysis revealed dramatic up-/down- regulation of genes involved in phytohormone signal transduction, chlorophyll and photosynthesis metabolism, and phenylpropanoid metabolism in Arabidopsis. Furthermore, the application of Arthrospira exerted a positive influence on the rhizosphere bacteriome structure in sweet sorghum, crucial for nutrient cycling and soil health enhancement. Our findings uncovered the underlying mechanisms of algae-plants interaction in saline soil, proposing strategies to enhance crop productivity and soil quality, thereby addressing the urgent need for sustainable agriculture practices to mitigate salinity's repercussions amidst climate change challenges.

Biological sources, metabolism, and production of glucosylglycerols, a group of natural glucosides of biotechnological interest.

Glucosylglycerols (GGs) are a group of functional heterosides comprising glycerol and glucose. In nature, they are mainly produced by many moderately salt-tolerant cyanobacteria as compatible solutes in a salt-dependent manner and synthesized in a few higher plants and fermentation processes. Because of their many interesting physicochemical properties and biological activities, such as low sweetness, low hygroscopicity, high water-holding capacity, excellent biocompatibility, favorable performance in protecting macromolecules, and antitumor activity, GGs exhibit large application potential in the fields of cosmetics, health care, food service, enzyme production, and pharmaceuticals. Many in vitro systems using different members of the GH (glycoside hydrolase) family have been established for the enzymatic synthesis of GGs, and a few of them are in use for commercial production. Based on a good understanding of the genetic bases, biochemical processes, and regulatory mechanisms of GG metabolism in microorganisms (mainly cyanobacteria), in recent years GG production technologies with in vivo systems have also been developed by applying metabolic and bioprocess engineering to a few native or heterologous microbial cell factories. This successfully provides the market GG products with an alternative source. With the further elucidation of details about the biological functions of GGs and related mechanisms, the application scope of GGs will be greatly expanded. In the present review, the biological sources and physiological roles of GGs, the molecular bases and regulation of GG metabolism, and the recent progress in GG production and application are systematically summarized. A few new questions that have arisen in the basic research of GGs and perspectives on GG application are also discussed.

Rapidly Improving High Light and High Temperature Tolerances of Cyanobacterial Cell Factories Through the Convenient Introduction of an AtpA-C252F Mutation.

Photosynthetic biomanufacturing is a promising route for green production of biofuels and biochemicals utilizing carbon dioxide and solar energy. Cyanobacteria are important microbial platforms for constructing photosynthetic cell factories. Toward scaled outdoor cultivations in the future, high light and high temperature tolerances of cyanobacterial chassis strains and cell factories would be determinant properties to be optimized. We proposed a convenient strategy for rapidly improving high light and high temperature tolerances of an important cyanobacterial chassis Synechococcus elongatus PCC 7942 and the derived cell factories. Through introduction and isolation of an AtpA-C252F mutation, PCC 7942 mutants with improved high light and high temperature tolerances could be obtained in only 4 days with an antibiotics-free mode. Adopting this strategy, cellular robustness and sucrose synthesizing capacities of a PCC 7942 cell factory were successfully improved.

Adopting a Theophylline-Responsive Riboswitch for Flexible Regulation and Understanding of Glycogen Metabolism in Synechococcus elongatus PCC7942.

Cyanobacteria are supposed to be promising photosynthetic microbial platforms that recycle carbon dioxide driven into biomass and bioproducts by solar energy. Glycogen synthesis serves as an essential natural carbon sink mechanism, storing a large portion of energy and organic carbon source of photosynthesis. Engineering glycogen metabolism to harness and rewire carbon flow is an important strategy to optimize efficacy of cyanobacteria platforms. ADP-glucose pyrophosphorylase (GlgC) catalyzes the rate-limiting step for glycogen synthesis. However, knockout of glgC fails to promote cell growth or photosynthetic production in cyanobacteria, on the contrary, glgC deficiency impairs cellular fitness and robustness. In this work, we adopted a theophylline-responsive riboswitch to engineer and control glgC expression in Synechococcus elongatus PCC7942 and achieved flexible regulation of intracellular GlgC abundance and glycogen storage. With this approach, glycogen synthesis and glycogen contents in PCC7942 cells could be regulated in a range from about 40 to 300% of wild type levels. In addition, the results supported a positive role of glycogen metabolism in cyanobacteria cellular robustness. When glycogen storage was reduced, cellular physiology and growth under standard conditions was not impaired, while cellular tolerance toward environmental stresses was weakened. While when glycogen synthesis was enhanced, cells of PCC7942 displayed optimized cellular robustness. Our findings emphasize the significance of glycogen metabolism for cyanobacterial physiology and the importance of flexible approaches for engineering and understanding cellular physiology and metabolism.

Effects of Reduced and Enhanced Glycogen Pools on Salt-Induced Sucrose Production in a Sucrose-Secreting Strain of Synechococcus elongatus PCC 7942.

Sucrose and glycogen syntheses in cyanobacteria share the common precursor glucose-1-phosphate. It is generally assumed that lowering glycogen synthesis could drive more carbon toward sucrose synthesis that can be induced by salt stress among cyanobacteria. By using a theophylline-dependent riboswitch system, the expression of glgC, a key gene in glycogen synthesis, was downregulated in a quantitative manner in a sucrose-secreting strain of Synechococcus elongatus PCC 7942. We observed that the stepwise suppression of glycogen synthesis limited rather than stimulated sucrose production in the salt-stressed cells, suggesting that glycogen could serve as a carbon pool for the synthesis of sucrose. Accordingly, we generated glycogen-overproducing strains, but the increased glycogen pool alone did not stimulate sucrose production, indicating that alternative steps limit the carbon flux toward the synthesis of sucrose. Consistent with previous studies that showed that sucrose-phosphate synthase (SPS) catalyzes the rate-limiting step in sucrose synthesis, the combination of glycogen overproduction and sps overexpression resulted in increased sucrose production. Our results indicate that the glycogen and sucrose pools are closely linked in Synechococcus elongatus PCC 7942, and we propose that enhancing the glycogen pool could be a promising strategy for the improvement of sucrose production by cyanobacteria in the presence of a strong sucrose synthesis sink.IMPORTANCE Many cyanobacteria naturally synthesize and accumulate sucrose when stressed by NaCl, which provides novel possibilities for obtaining sugar feedstock by engineering of cyanobacteria. It has been assumed that glycogen synthesis competes with sucrose synthesis for the carbon flux. However, our results showed that the suppression of glycogen synthesis decreased rather than stimulated sucrose production in a sucrose-secreting strain of Synechococcus elongatus PCC 7942. This result suggests that glycogen could serve as a supportive rather than a competitive carbon pool for the synthesis of sucrose, providing new insights about the relation between glycogen synthesis and sucrose synthesis in cyanobacteria. This finding is also useful to guide metabolic engineering work to optimize the production of sucrose and possibly other products by cyanobacteria.

[An Xcmâ -generated T vector and its applications in synthetic biology].

For more economical and efficient DNA clonging, pFL-XS-T, a Biobrick-T vector was constructed based on pMD18-T vector, carrying clonging regions of XbaⅠ-XcmⅠ-XcmⅠ-SpeⅠ. The results revealed that PCR products could be conveniently inserted into pFL-XS-T vevtor digested by XcmⅠby means of TA cloning. The positive frequency of recombination can meet the experimental requirements and all the plasmids obtained meet Biobrick standard. Moreover, the pFL-XS-T is compatible with other Biobrick parts, and serves as a vector for functional DNA fragments screening.

Combinatory strategy for characterizing and understanding the ethanol synthesis pathway in cyanobacteria cell factories.

BACKGROUND: Photosynthetic production of chemicals and fuels by recycling CO2 in cyanobacteria is a promising solution facing energy shortage and resource declination. Ethanol is an attractive and demonstrative biofuel product, and ethanol synthesis in cyanobacteria has been achieved by assembling of a pathway consisting of pyruvate decarboxylase (PDCzm) and alcohol dehydrogenase II (slr1192). For enabling more powerful ethanol photosynthetic production, an optimized and balanced catalyzing route was required. In this work, we provided a paradigm for systematically characterizing and optimizing the PDCzm-slr1192 pathway from engineered cyanobacteria strains, combining in vitro reconstitution, genetic engineering and feeding-cultivation. RESULTS: We reconstituted the PDCzm-slr1192 pathway in vitro and performed specific titration assays for enzymes, substrates, cofactors, and metal ions. In the in vitro system, K 50 of PDCzm was 0.326 µM, with a V max of 2.074 µM/s; while for slr1192, the values were 0.109 µM and 1.722 µM/s, respectively. Titration response discrepancy indicated that PDCzm rather than slr1192 was the rate-limiting factor for ethanol synthesis. In addition, a 4:6 concentration ratio of PDCzm-slr1192 would endow the reaction with a maximal specific catalytic activity. Titration assays for other components were also performed. K m values for NADPH, pyruvate, TPP, Mg(2+) and acetaldehyde were 0.136, 6.496, 0.011, 0.104, and 0.393 mM, respectively. We further constructed Synechocystis mutant strains with diverse PDCzm-slr1192 concentrations and ratios, and compared the growth and ethanol synthesis performances. The results revealed that activities of PDCzm indeed held control over the ethanol generation capacities. We performed pyruvate-feeding treatment with the newly developed Syn-YQ4 strain, and confirmed that improvement of pyruvate supply would direct more carbon flow to ethanol formation. CONCLUSIONS: We systematically characterized and optimized the PDCzm-slr1192 pathway in engineered cyanobacteria for ethanol production. Information gained from in vitro monitoring and genetic engineering revealed that for further enhancing ethanol synthesis capacities, PDCzm activities needed enhancement, and the PDCzm-slr1192 ratio should be improved and held to about 1:1.5. Considering actual metabolites concentrations of cyanobacteria cells, enhancing pyruvate supply was also a promising strategy for further updating the current ethanol photosynthetic cell factories.

Capillary ion chromatography-mass spectrometry for simultaneous determination of glucosylglycerol and sucrose in intracellular extracts of cyanobacteria.

A capillary ion chromatography-mass spectrometry (MS) method was proposed to determine glucosylglycerol (GG), sucrose, and five other carbohydrates. MS conditions and make-up flow parameters were optimized. This method is accurate and sensitive for simultaneous analysis of carbohydrates, with mean correlation coefficients of determination greater than 0.99, relative standard deviation of 0.91-2.81% for eight replicates, and average spiked recoveries of 97.3-104.9%. Limits of detection of sodium adduct were obtained with MS detection in selected ion mode for GG (0.006mg/L), sucrose (0.02mg/L), and other carbohydrates (0.03mg/L). This method was successfully applied to determine GG and sucrose in intracellular extracts of salt-stressed cyanobacteria.

Exploring the photosynthetic production capacity of sucrose by cyanobacteria.

Because cyanobacteria are photosynthetic, fast-growing microorganisms that can accumulate sucrose under salt stress, they have a potential application as a sugar source for the biomass-derived production of renewable fuels and chemicals. In the present study, the production of sucrose by the cyanobacteria Synechocystis sp. PCC6803, Synechococcus elongatus PCC7942, and Anabaena sp. PCC7120 was examined. The three species displayed different growth curves and intracellular sucrose accumulation rates in response to NaCl. Synechocystis sp. PCC6803 was used to examine the impact of modifying the metabolic pathway on the levels of sucrose production. The co-overexpression of sps (slr0045), spp (slr0953), and ugp (slr0207) lead to a 2-fold increase in intracellular sucrose accumulation, whereas knockout of ggpS (sll1566) resulted in a 1.5-fold increase in the production of this sugar. When combined, these genetic modifications resulted in a fourfold increase in intracellular sucrose accumulation. To explore methods for optimizing the transport of the intracellular sucrose to the growth medium, the acid-wash technique and the CscB (sucrose permease)-dependent export method were evaluated using Synechocystis sp. PCC6803. Whereas the acid-wash technique proved to be effective, the CscB-dependent export method was not effective. Taken together, these results suggest that using genetic engineering, photosynthetic cyanobacteria can be optimized for efficient sucrose production.

De novo biosynthesis of biodiesel by Escherichia coli in optimized fed-batch cultivation.

Biodiesel is a renewable alternative to petroleum diesel fuel that can contribute to carbon dioxide emission reduction and energy supply. Biodiesel is composed of fatty acid alkyl esters, including fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs), and is currently produced through the transesterification reaction of methanol (or ethanol) and triacylglycerols (TAGs). TAGs are mainly obtained from oilseed plants and microalgae. A sustainable supply of TAGs is a major bottleneck for current biodiesel production. Here we report the de novo biosynthesis of FAEEs from glucose, which can be derived from lignocellulosic biomass, in genetically engineered Escherichia coli by introduction of the ethanol-producing pathway from Zymomonas mobilis, genetic manipulation to increase the pool of fatty acyl-CoA, and heterologous expression of acyl-coenzyme A: diacylglycerol acyltransferase from Acinetobacter baylyi. An optimized fed-batch microbial fermentation of the modified E. coli strain yielded a titer of 922 mg L(-1) FAEEs that consisted primarily of ethyl palmitate, -oleate, -myristate and -palmitoleate.