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.

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.

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.

Tuning of acyl-ACP thioesterase activity directed for tailored fatty acid synthesis.

Medium-chain fatty acids have attracted significant attention as sources of biofuels in recent years. Acyl-ACP thioesterase, which is considered as the key enzyme to determine the carbon chain length, catalyzes the termination of de novo fatty acid synthesis. Although recombinant medium-chain acyl-ACP thioesterase (TE) affects the fatty acid profile in heterologous cells, tailoring of the fatty acid composition merely by engineering a specific TE is still intractable. In this study, the activity of a C8-C10-specific thioesterase FatB2 from Cuphea hookeriana on C10-ACP was quantified twice as high as that on C8-ACP based on a synthetic C8-C16 acyl-ACP pool in vitro. Whereas in vivo, it was demonstrated that ChFatB2 preferred to accumulate C8 fatty acids with 84.9% composition in the ChFatB2-engineered E. coli strain. To achieve C10 fatty acid production, ChFatB2 was rationally tuned based on structural investigation and enzymatic analysis. An I198E mutant was identified to redistribute the C8-ACP flow, resulting in C10 fatty acid being produced as the principal component at 57.6% of total fatty acids in vivo. It was demonstrated that the activity of TE relative to ß-ketoacyl-ACP synthases (KAS) directly determined the fatty acid composition. Our results provide a prospective strategy in tailoring fatty acid synthesis by tuning of TE activities based on TE-ACP interaction.

Structural and biochemical characterization of MCAT from photosynthetic microorganism Synechocystis sp. PCC 6803 reveal its stepwise catalytic mechanism.

Malonyl-coenzyme A: acyl-carrier protein transacylase (MCAT) catalyzes the transfer of malonyl group from malonyl-CoA to the holo-acyl carrier protein (Holo-ACP), yielding malonyl-ACP. The overall reaction has been extensively studied in heterotrophic microorganisms, while its mechanism in photosynthetic autotrophs as well as the stepwise reaction information remains unclear. Here the 2.42 Å crystal structure of MCAT from photosynthetic microorganism Synechocystis sp. PCC 6803 is presented. It demonstrates that Arg113, Ser88 and His188 constitute catalytic triad. The second step involved ACP-MCAT-malonyl intermediate is speed-limited instead of the malonyl-CoA-MCAT intermediate in the first step. Therefore His87, Arg113 and Ser88 render different contributions for the two intermediates. Additionally, S88T mutant initializes the reaction by H87 deprotonating S88T which is different from the wild type.

Enhancement of L-2-haloacid dehalogenase expression in Pseudomonas stutzeri DEH138 based on the different substrate specificity between dehalogenase-producing bacteria and their dehalogenases.

There was no direct correlation in substrate specificity between the metabolism of Pseudomonas stutzeri DEH138 and its corresponding dehalogenase. Dehalogenase substrates that could be dehalogenated might not be degraded by DEH138 or vice versa. Basing on this, different approaches to enhance L-2-haloacid dehalogenase (L-DEX) production in DEH138 via the combination of non-halogenated compounds with different inducers were applied. The optimum approach to obtain more L-DEX from DEH138 was the combination of DL-lactate and DL-2-chlorobutyrate, with 5.7-fold greater production and 11.7-fold greater productivity of the enzyme after optimization.

Protein-protein interaction network of the marine microalga Tetraselmis subcordiformis: prediction and application for starch metabolism analysis.

Under stressful conditions, the non-model marine microalga Tetraselmis subcordiformis can accumulate a substantial amount of starch, making it a potential feedstock for the production of fuel ethanol. Investigating the interactions of the enzymes and the regulatory factors involved in starch metabolism will provide potential genetic manipulation targets for optimising the starch productivity of T. subcordiformis. For this reason, the proteome of T. subcordiformis was utilised to predict the first protein-protein interaction (PPI) network for this marine alga based on orthologous interactions, mainly from the general PPI repositories. Different methods were introduced to evaluate the credibility of the predicted interactome, including the confidence value of each PPI pair and Pfam-based and subcellular location-based enrichment analysis. Functional subnetworks analysis suggested that the two enzymes involved in starch metabolism, starch phosphorylase and trehalose-phosphate synthase may be the potential ideal genetic engineering targets.

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.

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.

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.

Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of MCAT from Synechocystis sp. PCC 6803.

Malonyl-coenzymeA:acyl-carrier protein transacylase (MCAT), which catalyzes the transfer of the malonyl group from malonyl-CoA to acyl-carrier protein (ACP), is an essential enzyme in type II fatty-acid synthesis. The enzyme MCAT from Synechocystis sp. PCC 6803 (spMCAT), the first MCAT counterpart from a cyanobacterium, was cloned, purified and crystallized in order to determine its three-dimensional crystal structure. A higher-quality crystal with better diffraction was obtained by crystallization optimization. The crystal diffracted to 1.8 Šresolution and belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 43.22, b = 149.21, c = 40.59 Å. Matthews coefficient calculations indicated that the crystal contained one spMCAT molecule in the asymmetric unit with a Matthews coefficient of 2.18 Å(3) Da(-1) and a solvent content of 43.65%.

Characterization of cell growth and starch production in the marine green microalga Tetraselmis subcordiformis under extracellular phosphorus-deprived and sequentially phosphorus-replete conditions.

Microalgal starch is a potential feedstock for biofuel production. Nutrient stress is widely used to stimulate starch accumulation in microalgae. Cell growth and starch accumulation in the marine green microalga Tetraselmis subcordiformis were evaluated under extracellular phosphorus deprivation with initial cell densities (ICD) of 1.5, 3.0, 6.0, and 9.0×106 cells mL⁻¹. The intracellular stored phosphorus supported cell growth when extracellular phosphorus was absent. The maximum starch content of 44.1% was achieved in the lowest ICD culture, while the maximum biomass productivity of 0.71 g L⁻¹ day⁻¹, starch concentration of 1.6 g L⁻¹, and starch productivity of 0.30 g L⁻¹ day⁻¹ were all obtained in the culture with the ICD of 3.0×106 cells mL⁻¹. Appropriate ICD could be used to regulate the intracellular phosphorus concentration and maintain adequate photosynthetic activity to achieve the highest starch productivity, along with biomass and starch concentration. The recovery of phosphorus-deprived T. subcordiformis in medium containing 0.5, 1.0, or 6.0 mM KH2PO4 was also tested. Cell growth and starch accumulation ability could be recovered completely. A phosphorus pool in T. subcordiformis was shown to manipulate its metabolic activity under different environmental phosphorus availability. Though lower starch productivity and starch content were achieved under phosphorus deprivation compared with nitrogen- or sulfur-deprived conditions, the higher biomass and starch concentration make T. subcordiformis a good candidate for biomass and starch production under extracellular phosphorus deprivation.

Purification and characterization of a dehalogenase from Pseudomonas stutzeri DEH130 isolated from the marine sponge Hymeniacidon perlevis.

2-haloacid dehalogenases are enzymes that are capable of degrading 2-haloacid compounds. These enzymes are produced by bacteria, but so far they have only been purified and characterized from terrestrial bacteria. The present study describes the purification and characterization of 2-haloacid dehalogenase from the marine bacterium Pseudomonas stutzeri DEH130. P. Stutzeri DEH130 contained two kinds of 2-haloacid dehalogenase (designated as Dehalogenase I and Dehalogenase II) as detected in the crude cell extract after ammonium sulfate fractionation. Both enzymes appeared to exhibit stereo-specificity with respect to substrate. Dehalogenase I was a 109.9-kDa enzyme that preferentially utilized D-2-chloropropropionate and had optimum activity at pH 7.5. Dehalogenase II, which preferentially utilized L-2-chloropropionate, was further purified by ion-exchange chromatography and gel filtration. Purified Dehalogenase II appeared to be a dimeric enzyme with a subunit of 26.0-kDa. It had maximum activity at pH 10.0 and a temperature of 40 °C. Its activity was not inhibited by DTT and EDTA, but strongly inhibited by Cu²âº, Zn²âº, and Co²âº. The K(m) and V(max) for L-2-chloropropionate were 0.3 mM and 23.8 µmol/min/mg, respectively. Its substrate specificity was limited to short chain mono-substituted 2-halocarboxylic acids, with no activity detected toward fluoropropionate and monoiodoacetate. This is the first report on the purification and characterization of 2-haloacid dehalogenase from a marine bacterium.

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.

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.

Efficient purification with high recovery of vanadium bromoperoxidase from Corallina officinalis.

A novel, simple and highly efficient process for purifying vanadium bromoperoxidase from Corallina officinalis is reported. The key innovation is adding 0.5 mM sodium orthovanadate to the crude cell extract followed by heating at 70°C for 2 h, by which a 5.4-fold purification with a 100% activity recovery was achieved. Combining the heat treatment with ammonium sulfate precipitation and DEAE-52 column chromatography, the overall yield was 84%, 3.8 times greater than the highest yield previously reported. Finally, a specific activity of 310 U/mg, a 27-fold purification of the crude enzyme solution was produced.

Deoxyuridines from the marine sponge associated actinomycete Streptomyces microflavus.

One new nucleoside derivative, named 3-acetyl-5-methyl-2'-deoxyuridine (1), along with two known compounds 3,5-dimethyl-2'-deoxyuridine (2) and 3-methyl-2'-deoxyuridine (3), were isolated from the cultures of Streptomyces microflavus. This strain was an associated actinomycete isolated from the marine sponge Hymeniacidon perlevis collected from the coast of Dalian (China). Their structures were elucidated by detailed NMR and MS spectroscopic analysis as well as comparison with literature data.

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.

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.