Chlamydomonas reinhardtii chloroplast factory construction for formate bioconversion.

The photosynthetic autotrophic production of microalgae is limited by the effective supply of carbon and light energy, and the production efficiency is lower than the theoretical value. Represented by methanol, C1 compounds have been industrially produced by artificial photosynthesis with a solar energy efficiency over 10%, but the complexity of artificial products is weak. Here, based on a construction of chloroplast factory, green microalgae Chlamydomonas reinhardtii CC137c was modified for the bioconversion of formate for biomass production. By screening the optimal combination of chloroplast transport peptides, the cabII-1 cTP1 fusion formate dehydrogenase showed significant enhancement on the conversion of formate with a better performance in the maintenance of light reaction activity. This work provided a new way to obtain bioproducts from solar energy and CO2 with potentially higher-than-nature efficiency by the artificial-natural hybrid photosynthesis.

Engineering a solar formic acid/pentose (SFAP) pathway in Escherichia coli for lactic acid production.

Microbial CO2 fixation into lactic acid (LA) is an important approach for low-carbon biomanufacturing. Engineering microbes to utilize CO2 and sugar as co-substrates can create efficient pathways through input of moderate reducing power to drive CO2 fixation into product. However, to achieve complete conservation of organic carbon, how to engineer the CO2-fixing modules compatible with native central metabolism and merge the processes for improving bioproduction of LA is a big challenge. In this study, we designed and constructed a solar formic acid/pentose (SFAP) pathway in Escherichia coli, which enabled CO2 fixation merging into sugar catabolism to produce LA. In the SFAP pathway, adequate reducing equivalents from formate oxidation drive glucose metabolism shifting from glycolysis to the pentose phosphate pathway. The Rubisco-based CO2 fixation and sequential reduction of C3 intermediates are conducted to produce LA stoichiometrically. CO2 fixation theoretically can bring a 20% increase of LA production compared with sole glucose feedstock. This SFAP pathway in the integration of photoelectrochemical cell and an engineered Escherichia coli opens an efficient way for fixing CO2 into value-added bioproducts.

Biocatalysis of CO2 and CH4: Key enzymes and challenges.

Mitigating greenhouse gas emissions is a critical challenge for promoting global sustainability. The utilization of CO2 and CH4 as substrates for the production of valuable products offers a promising avenue for establishing an eco-friendly economy. Biocatalysis, a sustainable process utilizing enzymes to facilitate biochemical reactions, plays a significant role in upcycling greenhouse gases. This review provides a comprehensive overview of the enzymes and associated reactions involved in the biocatalytic conversion of CO2 and CH4. Furthermore, the challenges facing the field are discussed, paving the way for future research directions focused on developing robust enzymes and systems for the efficient fixation of CO2 and CH4.

A kinetic-assisted growth curve prediction method for Chlamydomonas reinhardtii incorporating transfer learning.

Traditional predictions of microalgal growth states rely on empirical or easily implementable kinetic models, leading to significant biases and elevated cost. This study proposes a kinetic-assisted machine learning method for predicting the growth curve of microalgal biomass under small sample conditions. Firstly, a microalgae growth kinetic model is constructed based on the logistic model. A two-stage kinetic fitting strategy is specified to account for the light-dark ratio. The Box-Behnken method is employed for experimental design. Then, using Two-stage TrAdaboost.R2 algorithm, the kinetic model is utilized as the source domain, and the experimental design data serves as the target domain for training machine learning models. The results indicate that the proposed method outperforms a single machine learning model in terms of prediction and has the potential to rapidly estimate microalgal growth trends under different conditions and accurately predict harvested biomass, potentially reducing the need for laborious, expensive, and time-consuming laboratory trials.

Enhancing photosynthetic CO2 fixation by assembling metal-organic frameworks on Chlorella pyrenoidosa.

The CO2 concentration at ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is crucial to improve photosynthetic efficiency for biomass yield. However, how to concentrate and transport atmospheric CO2 towards the Rubisco carboxylation is a big challenge. Herein, we report the self-assembly of metal-organic frameworks (MOFs) on the surface of the green alga Chlorella pyrenoidosa that can greatly enhance the photosynthetic carbon fixation. The chemical CO2 concentrating approach improves the apparent photo conversion efficiency to about 1.9 folds, which is up to 9.8% in ambient air from an intrinsic 5.1%. We find that the efficient carbon fixation lies in the conversion of the captured CO2 to the transportable HCO3- species at bio-organic interface. This work demonstrates a chemical approach of concentrating atmospheric CO2 for enhancing biomass yield of photosynthesis.

Crosslinker Nanocarriers Delivery to Chloroplasts for In Vivo Mapping of Photosynthetic Membrane Protein Complexes in Living Chlamydomonas reinhardtii Cells.

Photosynthesis, as the core of solar energy biotransformation, is driven by photosynthetic membrane protein complexes in plants and algae. Current methods for intracellular photosynthetic membrane protein complex analysis mostly require the separation of specific chloroplasts or the change of the intracellular environment, which causes the missing of real-time and on-site information. Thus, we explored a method for in vivo crosslinking and mapping of photosynthetic membrane protein complexes in the chloroplasts of living Chlamydomonas reinhardtii (C. reinhardtii) cells under cultural conditions. Poly(lactic-co-glycolic acid) (PLGA) and poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) nanoparticles were fabricated to deliver bis(succinimidyl)propargyl with a nitro compound (BSPNO) into the chloroplasts to crosslink photosynthetic membrane protein complexes. After the in vivo crosslinked protein complexes were extracted and digested, mass spectrometry was employed to detect lysine-specific crosslinked peptides for further elucidating the protein conformations and interactions. With this method, the weak interactions between extrinsic proteins in the luminal side (PsbL and PsbH) and the core subunits (CP47 and CP43) in photosynthetic protein complexes were directly captured in living cells. Additionally, the previously uncharacterized protein (Cre07.g335700) was bound to the light-harvesting proteins, which was related to the biosynthesis of light-harvesting antennae. These results indicated that in vivo analysis of photosynthetic protein complexes based on crosslinker nanocarriers was expected to not only figure out the difficulty in the study of photosynthetic protein complexes in living cells but also provide an approach to explore transient and weak interactions and the function of uncharacterized proteins.

The Carbon Source Effect on the Production of Ralstonia eutropha H16 and Proteomic Response Underlying Targeting the Bioconversion with Solar Fuels.

Chemoautotrophic bacterium Ralstonia eutropha H16 can fix CO2 to bioplastic and is potentially useful for CO2 neutralization. Targeting the solar fuel-based plastic biomanufactory, the polyhydroxybutyrate (PHB) production between heterotrophy and chemoautotrophy conditions was evaluated and the proteomic responses of the R. eutropha H16 cells to different carbon and energy sources were investigated. The results show that the chemoautotrophic mode hardly affected the cellular PHB accumulation capacity. Benefited from the high coverage proteome data, the global response of R. eutropha H16 to different carbon and energy sources was presented with a 95% KEGG pathway annotation, and the genome-wide location-related protein expression pattern was also identified. PHB depolymerase Q0K9H3 was found as a key protein responding to the low carbon input while CO2 and H2 were used, and will be a new regulation target for further high PHB production based on solar fuels.

Cultivation of fractionated cells from a bioactive-alkaloid-bearing marine sponge Axinella sp.

Sponges are among the most primitive multicellular organisms and well-known as a major source of marine natural products. Cultivation of sponge cells has long been an attractive topic due to the prominent evolutionary and cytological significance of sponges and as a potential approach to supply sponge-derived compounds. Sponge cell culture is carried out through culturing organized cell aggregates called 'primmorphs.' Most research culturing sponge cells has used unfractionated cells to develop primmorphs. In the current study, a tropical marine sponge Axinella sp., which contains the bioactive alkaloids, debromohymenialdisine (DBH), and hymenialdisine (HD), was used to obtain fractionated cells and the corresponding primmorphs. These alkaloids, DBH and HD, reportedly show pharmacological activities for treating osteoarthritis and Alzheimer's disease. Three different cell fractions were obtained, including enriched spherulous cells, large mesohyl cells, and small epithelial cells. These cell fractions were cultivated separately, forming aggregates that later developed into different kinds of primmorphs. The three kinds of primmorphs obtained were compared as regards to appearance, morphogenesis, and cellular composition. Additionally, the amount of alkaloid in the primmorphs-culture system was examined over a 30-d culturing period. During the culturing of enriched spherulous cells and developed primmorphs, the total amount of alkaloid declined notably. In addition, the speculation of alkaloid secretion and some phenomena that occurred during cell culturing are discussed.

Primary and secondary metabolites produced in Salvia miltiorrhiza hairy roots by an endophytic fungal elicitor from Mucor fragilis.

Salvia miltiorrhiza is one of the most commonly used medicinal materials in China. In recent years, the quality of S. miltiorrhiza has attracted much attention. Biotic and abiotic elicitors are widely used in cultivation to improve the quality of medicinal plants. We isolated an endophytic fungus, Mucor fragilis, from S. miltiorrhiza. We compared the effects of endophytic fungal elicitors with those of yeast extract together with silver ion, widely used together as effective elicitors, on S. miltiorrhiza hairy roots. Seventeen primary metabolites (amino acids and fatty acids) and five secondary metabolites (diterpenoids and phenolic acids) were analyzed after elicitor treatment. The mycelium extract promoted the accumulation of salvianolic acid B, rosmarinic acid, stearic acid, and oleic acid in S. miltiorrhiza hairy roots. Additionally, qPCR revealed that elicitors affect the accumulation of primary and secondary metabolites by regulating the expression of key genes (SmAACT, SmGGPPS, and SmPAL). This is the first detection of both the primary and secondary metabolites of S. miltiorrhiza hairy roots, and the results of this work should help guide the quality control of S. miltiorrhiza. In addition, the findings confirm that Mucor fragilis functions as an effective endophytic fungal elicitor with excellent application prospect for cultivation of medicinal plants.

Quantitative analysis on photon numbers received per cell for triggering ß-carotene accumulation in Dunaliella salina.

Accumulation of ß-carotene in Dunaliella salina is highly dependent on light exposure intensity and duration, but quantitative analysis on photon numbers received per cell for triggering ß-carotene accumulation is not available so far. In this study, experiment results showed that significant ß-carotene accumulation occurred after at least 8 h illumination at 400 µmol photons·m-2·s-1. To quantify the average number of photons received per cell, correlations of light attenuation with light path, biomass concentration, and ß-carotene content were, respectively, established using both Lambert-Beer and Cornet models, and the latter provided better simulation. Using Cornet model, average number of photons received per cell (APRPC) was calculated and proposed as a parameter for ß-carotene accumulation, and constant APRPC was maintained by adjusting average irradiance based on cell concentration and carotenoids content changes during the whole induction period. It was found that once APRPC reached 0.7 µmol photons cell-1, ß-carotene accumulation was triggered, and it was saturated at 9.9 µmol photons cell-1. This study showed that APRPC can be used as an important parameter to precisely simulate and control ß-carotene production by D. salina.

A Quick Look Back at the Microalgal Biofuel Patents: Rise and Fall.

Microalgae is a promising organism as the feedstock of the next generation biofuels, as well as high value nature products, such as astaxanthin, normally under certain stress cultivation conditions. With the clear industrialization targets, there have been two waves of microalgae R&D from the last century and showed obvious energy-driven trends. The overall R&D came into a valley now, however, the promising is still there. So here, from the industrialization point of view, the patent evolution concerning the microalgae for biofuels in the second wave were reviewed and summarized. These technology information will help the scientists to join together with industry to drive the next boost.

Effects of florfenicol on growth, photosynthesis and antioxidant system of the non-target organism Isochrysis galbana.

Florfenicol (FFC) is one of the most universally used antibiotics in aquaculture, which is substitute for chloramphenicol extensively, while the massive residues in aquatic environment were assumed to threaten the non-target organisms. Present research investigated the effects of florfenicol on growth, chlorophyll content, photosynthesis, and antioxidant ability of Isochrysis galbana. The results showed that FFC at 0.001-1 mg/L stimulated the growth of I. galbana and increased the content of chlorophyll. In addition, photosynthesis of I. galbana was inhibited and the photosynthetic parameters were uplifted with the increased exposure duration and FFC concentration. Furthermore, superoxide dismutase (SOD), catalase (CAT) activity significantly dropped at 0.01-20 mg/L FFC, while the contents of malondialdehyde (MDA), glutathione (GSH) and reactive oxygen species (ROS) increased after 72 h exposure, indicating that FFC at high concentrations caused a serious oxidative stress on algae. The simultaneous increase of ROS disrupted the equilibration between oxidants and antioxidant systems. Under the high concentration of FFC, the excessive of ROS was generated in algae which affected the membrane permeability and further decreased the cell biomass. Present study showed that acute exposure (72 h) at the environmental relevant concentration (0.01 mg/L) cannot induce the physiological dysfunction of the microalgae I. galbana, but the feeding concentration (20 mg/L) can. Additionally, this study hinted the possible negative impacts on ecosystems with the chronic exposure even at low FFC concentration or with the uncontrolled use of FFC.

Galactolipid DGDG and Betaine Lipid DGTS Direct De Novo Synthesized Linolenate into Triacylglycerol in a Stress-Induced Starchless Mutant of Chlamydomonas reinhardtii.

The increasing demand for triacylglycerol (TAG) enriching polyunsaturated fatty acids (PUFAs) has led to a surge of interest in microalgal TAG metabolism. Polar membrane lipids serve as the desaturation carrier for PUFA, and the functional group of PUFA can be incorporated into TAG. Monogalactoglycerolipid has been found to provide the de novo synthesized oleate acyl group or the nascent polyunsaturated diacylglycerol backbone for TAG biosynthesis in the model green alga, Chlamydomonas reinhardtii. However, whether other membrane lipids take part in the formation of PUFA-attached TAG has not been clearly discovered. A time course study of glycerolipidomics in the starchless mutant of C. reinhardtii, BAFJ5, which hyper-accumulates TAG, revealed that digalactosyldiacylglycerol (DGDG) and diacylglycerol-N,N,N-trimethylhomoserine (DGTS) turned into the main components of membrane lipids, accounting for 62% of the total polar lipids, under nitrogen deprivation combined with high light conditions. In addition, the membrane lipid molecules DGDG 18:3n3/16:0 and DGTS 16:0/18:3n6 were presumed to be involved in the consecutive integration of the de novo synthesized linolenates into TAG. Based on the stoichiometry calculation, DGDG and DGTS were demonstrated to provide a major contribution to the accumulation of linolenate-attached TAG. Our study gives insights into the potential PUFA-attached TAG formation pathway mediated by the turnover of de novo synthesized DGDG and DGTS in the starchless mutant of Chlamydomonas.

A Carbon Fixation Enhanced Chlamydomonas reinhardtii Strain for Achieving the Double-Win Between Growth and Biofuel Production Under Non-stressed Conditions.

The stressed cultivations are widely used in microalgae R&D for the biofuel production with the repress on growth to a certain degree, which limits the overall productivity. The balance between the growth and energy storage compounds accumulation is a target needing the combination of both strain selection or construction and culture optimization. Here, an engineered strain of Chlamydomonas reinhardtii, in which the chloroplast type glyceraldehyde-3-phosphate dehydrogenase (cGAPDH) was overexpressed and named as P3-GAPDH, was cultured on the Algal Station platform. Compared with wild type (WT), C. reinhardtii CC137c, in Tris-acetate-phosphate (TAP) medium, the highest density of WT and P3-GAPDH were 1.23 ± 0.13 and 1.74 ± 0.09 g L-1 within 96 h, and the maximum biomass productivity was 24.30 ± 1.65 and 28.54 ± 1.43 mg L-1 h-1, respectively. In terms of the energy storage compounds, both carbohydrate and fatty acids content doubled in P3-GAPDH, from 0.13 ± 0.02 to 0.26 ± 0.04 g L-1 for carbohydrate and from 0.08 ± 0.01 to 0.16 ± 0.01 g L-1 for fatty acids, among which poly unsaturated fatty acids increased by 65.8%. Together with the continuous monitor of the chlorophyll fluorescence dynamics parameters F v/F m and F v'/F m' and pH of culture, enhanced Calvin cycle by overexpressed cGAPDH promoted the carbon conversion and subsequent energy storage compounds accumulation. C. reinhardtii P3-GAPDH strain showed the potential as a good chassis with high carbon conversion ability.

Application of an in situ CO2-bicarbonate system under nitrogen depletion to improve photosynthetic biomass and starch production and regulate amylose accumulation in a marine green microalga Tetraselmis subcordiformis.

BACKGROUND: Microalgal starch is regarded as a promising alternative to crop-based starch for biorefinery such as the production of biofuels and bio-based chemicals. The single or separate use of inorganic carbon source, e.g., CO2 and NaHCO3, caused aberrant pH, which restricts the biomass and starch production. The present study applied an in situ CO2-NaHCO3 system to regulate photosynthetic biomass and starch production along with starch quality in a marine green microalga Tetraselmis subcordiformis under nitrogen-depletion (-N) and nitrogen-limitation (±N) conditions. RESULTS: The CO2 (2%)-NaHCO3 (1 g L-1) system stabilized the pH at 7.7 in the -N cultivation, under which the optimal biomass and starch accumulation were achieved. The biomass and starch productivity under -N were improved by 2.1-fold and 1.7-fold, respectively, with 1 g L-1 NaHCO3 addition compared with the one without NaHCO3 addition. NaHCO3 addition alleviated the high-dCO2 inhibition caused by the single CO2 aeration, and provided sufficient effective carbon source HCO3 - for the maintenance of adequate photosynthetic efficiency and increase in photoprotection to facilitate the biomass and starch production. The amylose content was also increased by 44% under this CO2-bicarbonate system compared to the single use of CO2. The highest starch productivity of 0.73 g L-1 day-1 under -N cultivation and highest starch concentration of 4.14 g L-1 under ±N cultivation were both achieved with the addition of 1 g L-1 NaHCO3. These levels were comparable to or exceeded the current achievements reported in studies. The addition of 5 g L-1 NaHCO3 under ±N cultivation led to a production of high-amylose starch (59.3% of total starch), which could be used as a source of functional food. CONCLUSIONS: The in situ CO2-NaHCO3 system significantly improved the biomass and starch production in T. subcordiformis. It could also regulate the starch quality with varied relative amylose content under different cultivation modes for diverse downstream applications that could promote the economic feasibility of microalgal starch-based biofuel production. Adoption of this system in T. subcordiformis would facilitate the CO2 mitigation couple with its starch-based biorefinery.

The heat-tolerance evaluation of an Isochrysis zhangjiangensis mutant generated by atmospheric and room temperature plasmas.

Isochrysis zhangjiangensis is widely used in the marine aquaculture as larval feed, especially for filter feeding cultures, as well as a good candidate for biofuels. However, the optimal cultivation temperature for I. zhangjiangensis is below 30 °C and this stain is seriously affected by high temperature, which causes the limited application during the summer. I. zhangjiangensis IM130005 is a strain generated by atmospheric and room temperature plasmas with relative higher growth rate and lipid production than the wide strain (WT), with the ability to tolerate several hours' high temperature during the outdoor cultivation. Here, a detailed comparison was performed by continuous monitoring growth, chlorophyll fluorescence and fatty acid profile between IM13005 and WT under a mimic temperature shock to the summer outdoor cultivation. Based on a nearly 20% increase of total fatty acid in IM13005, which was majorly contributed by saturated or monounsaturated FAs in form of neutral lipids, within 5 h under the heat shock, the fatty acids and lipids synthesis variation were postulated as the physiological reason for the high temperature tolerance.

Production and structural characterization of a new type of polysaccharide from nitrogen-limited Arthrospira platensis cultivated in outdoor industrial-scale open raceway ponds.

BACKGROUND: Carbohydrates are major biomass source in fuel-targeted biorefinery. Arthrospira platensis is the largest commercialized microalgae with good environmental tolerance and high biomass production. However, the traditional target of A. platensis cultivation is the protein, which is the downstream product of carbohydrates. Aiming to provide the alternative non-food carbohydrates source, the feasible manipulation technology on the cultivation is needed, as well as new separation methodology to achieve maximum utilization of overall biomass. RESULTS: The present study aimed to demonstrate the feasibility of industrially producing carbohydrate-enriched A. platensis and characterize the structure of the polysaccharide involved. Cultivated in industrial-scale outdoor open raceway ponds under nitrogen limitation, A. platensis accumulated maximally 64.3%DW of carbohydrate. The maximum biomass and carbohydrate productivity reached 27.5 g m-2 day-1 and 26.2 g m-2 day-1, respectively. The efficient extraction and purification of the polysaccharides include a high-pressure homogenization-assisted hot water extraction followed by flocculation with a non-toxic flocculant ZTC1 + 1, with the polysaccharide purity and total recovery reaching 81% and 75%, respectively. The purified polysaccharide was mainly composed of (1→3)(1→4)- or (1→3)(1→2)-α-glucan with a molecular weight of 300-700 kDa, which differed from the commonly acknowledged glycogen. CONCLUSIONS: By the way of controlled nitrogen limitation, the high carbohydrate production of A. platensis in the industrial scale was achieved. The α-glucan from A. platensis could be a potential glucose source for industrial applications. A non-toxic separation method of carbohydrate was applied to maintain the possibility of utilization of residue in high-value field.

Expanding of Phospholipid:Diacylglycerol AcylTransferase (PDAT) from Saccharomyces cerevisiae as Multifunctional Biocatalyst with Broad Acyl Donor/Acceptor Selectivity.

Triacylglycerols are considered one of the most promising feedstocks for biofuels. Phospholipid:diacylglycerol acyltransferase (PDAT), responsible for the last step of triacylglycerol synthesis in the acyl-CoA-independent pathway, has attracted much attention by catalyzing membrane lipid transformation. However, due to lack of biochemical and enzymatic studies, PDAT has not carried forward in biocatalyst application. Here, the PDAT from Saccharomyces cerevisiae was expressed in Pichia pastoris. The purified enzymes were studied using different acyl donors and acceptors by thin layer chromatography and gas chromatography. In addition of the preferred acyl donor of PE and PC, the results identified that ScPDAT was capable of using broad acyl donors such as PA, PS, PG, MGDG, DGDG, and acyl-CoA, and ScPDAT was more likely to use unsaturated acyl donors comparing 18:0/18:1 to 18:0/18:0 phospholipids. With regard to acyl acceptors, ScPDAT preferred 1,2 to 1,3-diacylglycerol (DAG), while 12:0/12:0 DAG was identified as the optimal acyl acceptor, followed by 18:1/18:1 and 18:1/16:0 DAG. Additionally, ScPDAT reveals esterification activity that can utilize methanol as acyl acceptor to generate fatty acid methyl esters. The results fully expand the enzymatic selectivity of ScPDAT and provide fundamental knowledge for synthesis of triacylglycerol-derived biofuels.

Stereoselective catalysis controlled by a native leucine or variant isoleucine wing-gatekeeper in 2-haloacid dehalogenase.

Comprehensively understanding enzymatic stereoselectivity will assist in the creation of new enzymes for producing optically pure compounds for chemical applications. The essential features for selecting enantiomers are controlled by particular residues or regions of the enzymes. We report a stereoselective mechanism in the D-2-haloacid dehalogenase HadD AJ1, in which L288 is identified as a gatekeeper in the access channel that strictly recognizes D-enantiomers. Mutagenesis of L288 to isoleucine (I) enlarges the size of the channel and allows the enzyme to accommodate L-enantiomers. Furthermore, the wing flip of I288 induces hydrophobic interactions with the L-enantiomer and directly affects the catalytic efficiency. The results illustrate the dynamic catalytic mechanisms of Leu-Ile gatekeepers and provide knowledge for unveiling the basis of stereospecificity in biocatalysts.