Engineering a marine microalga Chlorella sp. as the cell factory.

The use of marine microalgae in industrial systems is attractive for converting CO2 into value-added products using saline water and sunlight. The plant nature and demonstrated industrial potential facilitate Chlorella spp. as excellent model organisms for both basic research and commercial application. However, the transformation method has not been developed in marine Chlorella spp., thus genetic engineering is hindered in exploiting the industrial potentialities of these strains. In this study, we provided a transformation protocol for the marine Chlorella strain MEM25, which showed robust characteristics, including high production of proteins and polyunsaturated fatty acids in multiple cultivation systems over various spatial-temporal scales. We showed that transformants could be obtained in a dramatically time-saving manner (comparable to Saccharomyces cerevisiae) with four functional proteins expressed properly. The transgenes are integrated into the genome and can be successfully inherited for more than two years. The development of a marine Chlorella transformation method, in combination with the complete genome, will greatly facilitate more comprehensive mechanism studies and provide possibilities to use this species as chassis for synthetic biology to produce value-added compounds with mutual advantage in neutralization of CO2 in commercial scales.

Artificial switches induce the bespoke production of functional compounds in marine microalgae Chlorella by neutralizing CO2.

To improve the CO2 tolerance of a marine microalga Chlorella sp. of which the production capacity has been demonstrated industrially, a mutant library was created and a strain hct53 was screened. Compared to the parental strain, hct53 shows a high CO2 capture capacity, while starch biosynthesis is compromised, with increases in health beneficial metabolites and antioxidant capacity. Global gene expression and genome-wide mutation distribution revealed that transcript choreography was concomitant with more active CO2 sequestration, an increase in the lipid synthesis, and a decrease in the starch and protein synthesis. These results suggest that artificial trait improvement via mutagenesis, couple with multiomics analysis, helps discover genetic switches that induce the bespoke conversion of carbon flow from "redundant metabolites" to valuable ones for functional food.

Physiological and transcriptional responses to high temperature in Arthrospira (Spirulina) platensis C1.

Arthrospira (Spirulina) platensis is a well-known commercial cyanobacterium that is used as a food and in feed supplements. In this study, we examined the physiological changes and whole-genome expression in A. platensis C1 exposed to high temperature. We found that photosynthetic activity was significantly decreased after the temperature was shifted from 35°C to 42°C for 2 h. A reduction in biomass production and protein content, concomitant with the accumulation of carbohydrate content, was observed after prolonged exposure to high temperatures for 24 h. Moreover, the results of the expression profiling in response to high temperature at the designated time points (8 h) revealed two distinct phases of the responses. The first was the immediate response phase, in which the transcript levels of genes involved in different mechanisms, including genes for heat shock proteins; genes involved in signal transduction and carbon and nitrogen metabolism; and genes encoding inorganic ion transporters for magnesium, nitrite and nitrate, were either transiently induced or repressed by the high temperature. In the second phase, the long-term response phase, both the induction and repression of the expression of genes with important roles in translation and photosynthesis were observed. Taken together, the results of our physiological and transcriptional studies suggest that dynamic changes in the transcriptional profiles of these thermal-responsive genes might play a role in maintaining cell homeostasis under high temperatures, as reflected in the growth and biochemical composition, particularly the protein and carbohydrate content, of A. platensis C1.

Draft genome sequence of Arthrospira platensis C1 (PCC9438).

Arthrospira platensis is a cyanobacterium that is extensively cultivated outdoors on a large commercial scale for consumption as a food for humans and animals. It can be grown in monoculture under highly alkaline conditions, making it attractive for industrial production. Here we describe the complete genome sequence of A. platensis C1 strain and its annotation. The A. platensis C1 genome contains 6,089,210 bp including 6,108 protein-coding genes and 45 RNA genes, and no plasmids. The genome information has been used for further comparative analysis, particularly of metabolic pathways, photosynthetic efficiency and barriers to gene transfer.

Cultivation of Spirulina platensis using pig wastewater in a semi-continuous process.

The effluent from anaerobic digestion contains organic nitrogen and phosphorus, which are both required for growth of Spirulina platensis. Effluent (20%) from the upflow anaerobic sludge blanket (UASB) from a pig farm, supplemented with 4.5 g/l sodium bicarbonate (NaHCO(3)) and 0.2 g/l urea fertilizer (46:0:0, N:P:K), was found to be not only a suitable medium for the growth of Spirulina platensis but also a low-cost alternative. Cost calculation showed that this medium is 4.4 times cheaper than modifized Zarrouk's medium. The average productivities of a semi-continuous culture grown under outdoor conditions in a 6-l scale and a 100-l pilot scale were 19.9 g/m2/d and 12 g/m(2)/d, respectively. In addition, the biomass of organisms grown in UASB effluent contained approximately 57.9% protein, 1.12% gamma-linolenic acid, and 19.5% phycocyanin. The average rates of bicarbonate, total nitrogen, and phosphorus removal were 380 mg/l/d, 34 mg/l/d, and 4 mg/l/d, respectively.

Histidine kinases play important roles in the perception and signal transduction of hydrogen peroxide in the cyanobacterium, Synechocystis sp. PCC 6803.

Oxidative stress caused by reactive oxygen species and, in particular, to hydrogen peroxide (H(2)O(2)) has a major impact on all biological systems, including plants and microorganisms. We investigated the H(2)O(2)-inducible expression of genes in the cyanobacterium Synechocystis sp. PCC 6803 using genome-wide DNA microarrays. Our systematic screening of a library of mutant lines with defects in histidine kinases (Hiks) by RNA slot-blot hybridization and DNA-microarray analysis suggested that four Hiks, namely, Hik33, Hik34, Hik16 and Hik41, are involved in the perception and transduction of H(2)O(2) signals that regulate the gene expression of 26 of the 77 H(2)O(2)-inducible genes with induction factors higher than 4.0. Among the four Hiks, Hik33 was the main contributor and was responsible for 22 of the 26 H(2)O(2)-inducible genes under the control of the Hiks. By contrast to Hik33, PerR encoding putative peroxide-sensing protein is involved in the regulation of only nine H(2)O(2)-inducible genes.

Identical Hik-Rre systems are involved in perception and transduction of salt signals and hyperosmotic signals but regulate the expression of individual genes to different extents in synechocystis.

In previous studies, we characterized five histidine kinases (Hiks) and the cognate response regulators (Rres) that control the expression of approximately 70% of the hyperosmotic stress-inducible genes in the cyanobacterium Synechocystis sp. PCC 6803. In the present study, we screened a gene knock-out library of Rres by RNA slot-blot hybridization and with a genome-wide DNA microarray and identified three Hik-Rre systems, namely, Hik33-Rre31, Hik10-Rre3, and Hik16-Hik41-Rre17, as well as another system that included Rre1, that were involved in perception of salt stress and transduction of the signal. We found that these Hik-Rre systems were identical to those that were involved in perception and transduction of the hyperosmotic stress signal. We compared the induction factors of the salt stress- and hyperosmotic stress-inducible genes that are located downstream of each system and found that these genes responded to the two kinds of stress to different respective extents. In addition, the Hik33-Rre31 system regulated the expression of genes that were specifically induced by hyperosmotic stress, whereas the system that included Rre1 regulated the expression of one or two genes that were specifically induced either by salt stress or by hyperosmotic stress. Our observations suggest that the perception of salt and hyperosmotic stress by the Hik-Rre systems is complex and that salt stress and hyperosmotic stress are perceived as distinct signals by the Hik-Rre systems.

Five histidine kinases perceive osmotic stress and regulate distinct sets of genes in Synechocystis.

Microorganisms respond to hyperosmotic stress via changes in the levels of expression of large numbers of genes. Such responses are essential for acclimation to a new osmotic environment. To identify factors involved in the perception and transduction of signals caused by hyperosmotic stress, we examined the response of Synechocystis sp. PCC 6803, which has proven to be a particularly useful microorganism in similar analyses. We screened knockout libraries of histidine kinases (Hiks) and response regulators (Rres) in Synechocystis by DNA microarray and slot-blot hybridization analyses, and we identified several two-component systems, which we designated Hik-Rre systems, namely, Hik33-Rre31, Hik34-Rre1, and Hik10-Rre3, as well as Hik16-Hik41-Rre17, as the transducers of hyperosmotic stress. We also identified Hik2-Rre1 as a putative additional two-component system. Each individual two-component system regulated the transcription of a specific group of genes that were responsive to hyperosmotic stress.

Differential responses of three acyl-lipid desaturases to immediate temperature reduction occurring in two lipid membranes of Spirulina platensis strain C1.

In cyanobacteria, the elevation of unsaturated fatty acid levels in membrane lipids has been shown to play a major role in the response to temperature change. The cyanobacterium Spirulina platensis strain C1 contains three desaturases--delta9, delta12 and delta6 desaturases--which are encoded by desC, desA and desD, respectively. In light of the above, a study was conducted of the regulation of desaturase gene expression in response to temperature change in S. platensis strain C1. The two lipid membranes, thylakoid and plasma, were separated, while the expressions of the desaturase genes to the downward shift of growth temperature were studied in the translation level by Western blot analysis. The results revealed that the expression of delta9 desaturase is independent of temperature. In the case of delta12 desaturase, two forms of the enzyme were found, at 45 and 40 kDa. In terms of correlation with the results in the transcription level, it is more likely that the 45 kDa-delta12 desaturase and the 40 kDa-delta12 desaturase are translated from 1.7 kb and 1.5 kb mRNA, respectively. Taken together, the results indicate that the expression of the 40 kDa-delta12 desaturase is temperature independent, whereas, the 45 kDa protein form demonstrates a response to the immediate temperature reduction. Furthermore, the activity of delta6 desaturase in the two lipid membranes is possibly regulated by temperature reduction. However, alteration in the level of gamma-linolenic acid, the product synthesized by delta6 desaturase, was observed in the plasma membrane prior to the thylakoid membrane.