Human activities increase the concentration of atmospheric carbon dioxide (CO₂), which leads to global climate warming. Microbial CO₂ fixation is a promising green approach for carbon neutral. In contrast to autotrophic microorganisms, heterotrophic microorganisms are characterized by fast growth and ease of genetic modification, but the efficiency of CO₂ fixation is still limited. In the past decade, synthetic biology-based enhancement of heterotrophic CO₂ fixation has drawn wide attention, including the optimization of energy supply, modification of carboxylation pathway, and heterotrophic microorganisms-based indirect CO₂ fixation. This review focuses on the research progress in CO₂ fixation by heterotrophic microorganisms, which is expected to serve as a reference for peaking CO₂ emission and achieving carbon neutral by microbial CO₂ fixation.
Carbon Cycle ; Carbon Dioxide/metabolism* ; Heterotrophic Processes ; Humans ; Synthetic Biology

Lactate is an important industrial chemical and widely used in various industries. In recent years, with the increasing demand for polylactic acid (PLA), the demand for lactate raw materials is also increasing. The contradiction between the high cost and the market demand caused by the heterotrophic production of lactate attracts researchers to seek other favorable solutions. The production of lactate from photosynthetic carbon fixation by cyanobacteria is a potential new raw material supply strategy. Based on the photosynthetic autotrophic cell factory, it can directly produce high optical purity lactate from carbon dioxide on a single platform driven by solar energy. The raw materials are cheap and easy to obtain, the process is simple and controllable, the products are clear and easy to separate, and the double effects of energy saving and emission reduction and production of high value-added products are achieved at the same time, which has important research and application value. This paper reviews the development history of cyanobacteria carbon sequestration to produce lactate, summarizes its research progress and encounters technical difficulties from the aspects of metabolic basis, metabolic engineering strategy, metabolic kinetics analysis and technical application, and prospects the future of this technology.
Carbon Cycle ; Carbon Dioxide ; Cyanobacteria/genetics* ; Lactic Acid ; Metabolic Engineering ; Photosynthesis

OBJECTIVE: To explore the genetic basis for a child with clinically suspected 3-methylcrotonyl-coenzyme A carboxylase deficiency (MCCD). METHODS: Genomic DNA was extracted from peripheral blood samples of the proband and her parents. Whole exome sequencing was used to screen pathogenic variant in the proband. Suspected variant was verified by Sanger sequencing. Impact of the variant on the structure and function of protein product was analyzed by using bioinformatic software. RESULTS: Sanger sequencing showed that the proband has carried homozygous missense c.1342G>A (p.Gly448Ala) variant of the MCCC2 gene, for which her mother was a heterozygous carrier. The same variant was not detected in her father. The variant was predicted to be pathogenic by PolyPhen-2 and Mutation Taster software, and the site was highly conserved among various species. Based on the American College of Medical Genetics and Genomics standards and guidelines, the c.1342G>A (p.Gly448Ala) variant of MCCC2 gene was predicted to be likely pathogenic(PM2+PP2-PP5). CONCLUSION The homozygous missense variant of the MCCC2 gene c.1342G>A (p.Gly448Ala) probably underlay the molecular pathogenesis of the proband. Genetic testing has confirmed the clinical diagnosis.
Carbon-Carbon Ligases/genetics* ; Child ; Female ; Humans ; Male ; Mutation, Missense/genetics* ; Pedigree ; Urea Cycle Disorders, Inborn/genetics*

Environmental protection and energy supply are our two major concerns. Greenhouse gases released from energy consumption have serious impact on the environment. CO₂ fixation can be used to convert CO₂ into fuels or chemicals. However, natural carbon-fixing organisms usually have some disadvantages such as slow growth and low carbon fixation efficiency. Enhancing or remodeling CO₂ fixation pathways in model microorganisms can realize CO₂ recycling, which can further increase fuel or chemical production and reduce greenhouse gas emission. This review describes in detail metabolic engineering of CO₂ fixation pathways to improve chemical production and sugar synthesis, elaborates the role of relevant metabolic pathways and key enzymes in CO₂ fixation, introduces the application of electro-biochemical synthesis system, shows the great potential of CO₂ fixation, and prospects the future research direction of CO₂ fixation.
Carbon Cycle ; Carbon Dioxide ; Heterotrophic Processes ; Metabolic Engineering ; Metabolic Networks and Pathways

The increasing atmospheric carbon dioxide levels have been correlated with global warming. Carbonic anhydrases (CA) are the fastest among the known enzymes to improve carbon capture. The capture of carbon dioxide needs high temperature and alkaline condition, which is necessary for CaCO₃ precipitation in the mineralization process. In order to use CAs for biomimetic carbon sequestration, thermo-alkali-stable CAs are, therefore, essential, and polyextremophilic microbes are one of the important sources of these enzymes. The current review focuses on both those isolated by thermophilic organisms from the extreme environments and those obtained by protein engineering techniques, and the industrial application of the immobilized CAs is also briefly addressed. To reduce the greenhouse effect and delay global warming, we think further research efforts should be devoted to broadening the scope of searching for carbonic anhydrase, modifying the technology of protein engineering and developing highly efficient immobilization strategies.
Biomimetics ; Carbon Dioxide ; Carbon Sequestration ; Carbonic Anhydrases ; Protein Engineering

OBJECTIVE: To investigate the genetic characterization of 3-hydroxyisovalerylcarnitine (C5-OH) metabolic abnormality in neonates. METHODS: Fifty two newborns with increased C5-OH, C5-OH/C3 and C5-OH/C8 detected by tandem mass spectrometry during neonatal screening were enrolled in the study. Genomic DNA was extracted from the whole blood samples of 52 cases and their parents. Seventy-nine genes associated with genetic and metabolic diseases including , were targeted by liquid capture technique. Variation information of these genes was examined by high-throughput sequencing and bioinformatic analysis, and then was classified based on the American College of Medical Genetics and Genomics (ACMG) standards and guidelines. The genetic types were classified as wild-type, -maternal-mutation, -paternal-mutation and -mutation. Wilcoxon rank-sum test was performed for the increased multiples of C5-OH calculated in neonatal screening. RESULTS: Twenty one variants (14 novel) were identified in 37 cases, 6 variants (5 novel) in 4 cases. The increased multiple of C5-OH calculated in -maternal-mutation and -mutation groups were significantly higher than that in wild-type group (all <0.05), while there was no significant difference between MCCC1-paternal-mutation group and wild-type group (>0.05). CONCLUSIONS Mutations on and genes are the major genetic causes for the increased C5-OH in neonates, and maternal single heterozygous mutation can contribute to the moderately to severely increased C5-OH.
Carbon-Carbon Ligases ; genetics ; Carnitine ; analogs & derivatives ; metabolism ; Female ; Genetic Testing ; Genetic Variation ; Humans ; Infant, Newborn ; Male ; Mutation ; Neonatal Screening ; Urea Cycle Disorders, Inborn ; genetics

Carbon nanotubes (CNTs) are nanomaterials that have been employed in generating diverse materials. We previously reported that CNTs induce cell death in macrophages, possibly via asbestosis. Therefore, we generated CNT-attached polyvinylidene fluoride (PVDF), which is an established polymer in membrane technology, and then examined whether CNT-attached PVDF is immunologically safe for medical purposes compared to CNT alone. To test this, we treated RAW 264.7 murine macrophages (RAW cells) with CNT-attached PVDF and analyzed the production of nitric oxide (NO), a potent proinflammatory mediator, in these cells. RAW cells treated with CNT-attached PVDF showed reduced NO production in response to lipopolysaccharide. However, the same treatment also decreased the cell number suggesting that this treatment can alter the homeostasis of RAW cells. Although cell cycle of RAW cells was increased by PVDF treatment with or without CNTs, apoptosis was enhanced in these cells. Taken together, these results indicate that PVDF with or without CNTs modulates inflammatory responses possibly due to activation-induced cell death in macrophages.
Apoptosis ; Asbestosis ; Cell Count ; Cell Cycle ; Cell Death* ; Fluorides* ; Homeostasis ; Inflammation ; Macrophages* ; Membranes ; Nanostructures ; Nanotubes, Carbon ; Nitric Oxide ; Polymers

OBJECTIVETo explore the molecular mechanism for a boy suspected with 3-methylcrotonyl-CoA carboxylase deficiency by neonatal screening.

METHODSPCR and Sanger sequencing were used to identify potential mutations of MCCC1 and MCCC2 genes. SIFT and Polyphen-2 software was used to predict the effect of variant on the protein function and conservation of the variant across various species. Human Splicing Finder and Swiss-PdbViewer4.1.0 were applied to analyze the possible mechanism of the variant.

RESULTSFor the proband, a compound heterozygous mutation was discovered in the MCCC1 gene, namely c.539G>T (p.G180V) and c.704_711del (p.A235Vfs*4), which were inherited from his father and mother, respectively. The two mutations have disrupted the protein conformation, which in turn may impact the function of MCC protein.

CONCLUSIONThe compound heterozygous mutations of the MCCC1 gene may contribute to the 3-methylcrotonyl-CoA carboxylase deficiency manifested by the patient.

Amino Acid Sequence ; Base Sequence ; Carbon-Carbon Ligases ; chemistry ; deficiency ; genetics ; DNA Mutational Analysis ; Heterozygote ; Humans ; Infant, Newborn ; Male ; Models, Molecular ; Mutation ; Neonatal Screening ; methods ; Protein Conformation ; Sequence Homology, Amino Acid ; Urea Cycle Disorders, Inborn ; diagnosis ; genetics

Carboxysomes are extremely efficient microcompartments committed to CO2 fixation due to tailored CO2-concentrating mechanism (CCM). In cyanobacteria and some chemoautotrophs, carboxysomes as organelle-like microbodies encapsulate ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase (CA). Together with active inorganic carbon uptake transporters, carboxysomes accumulate HCO3(-) in the cytoplasm, leading to high efficiency of carbon fixation. Based on the elucidation of structures and functionalities, heterologous production of carboxysomes has been achieved so far. In fact, the genes encoding either vacant carboxysome shell or only interior components have been characterized. This review summarizes the discovery along with types, showcases molecular structures and roles of carboxysomes in CCM, and presents their broad applications in metabolic engineering.
Biological Transport ; Carbon ; metabolism ; Carbon Cycle ; Carbon Dioxide ; metabolism ; Cyanobacteria ; metabolism ; Metabolic Engineering

OBJECTIVETo study the effects of hypoxia of different duration on movement and proliferation of human epidermal cell line HaCaT.

METHODS(1) HaCaT cells in logarithmic phase were cultured in RPMI 1640 medium containing 10% FBS (the same culture method below). Cells were divided into control group (routine culture) and hypoxia for 1, 3, 6 h groups according to the random number table (the same grouping method below), with 6 wells in each group. Cells in the 3 hypoxia groups were cultured in incubator containing 5% CO2, 2% O2, and 93% N2 (the same hypoxic condition below) for corresponding duration. Range of movement of cells in 3 hours was observed under live cell imaging workstation, and their curvilinear and rectilinear movement speeds were calculated at post observation hour (POH) 1, 2, 3. (2) HaCaT cells in logarithmic phase were divided into control group (routine culture) and hypoxia for 1, 3, 6, 9, 12, 24 h groups, with 20 wells in each group. Cells in the 6 hypoxia groups were cultured under hypoxic condition for corresponding duration. Proliferation of cells was examined with cell counting kit and microplate reader (denoted as absorbance value). (3) HaCaT cells in logarithmic phase were divided into control group (routine culture) and hypoxia for 1, 3, 6, 24 h groups, with 5 wells in each group. Cells in the 4 hypoxia groups were cultured under hypoxic condition for corresponding duration. Protein expression of proliferating cell nuclear antigen (PCNA) was determined with Western blotting. Data were processed with one-way analysis of variance and Dunnett- t test.

RESULTS(1) Compared with that of control group, the movement area of cells was obviously expanded in hypoxia for 1, 3, 6 h groups. The longer the hypoxic treatment, the greater the increase was. At POH 1, 2, 3, the curvilinear movement speeds of cells in hypoxia for 1, 3, 6 h groups were respectively (43 ± 18), (44 ± 17), (43 ± 16) µm/h; (44 ± 16), (44 ± 14), (45 ± 14) µm/h; (55 ± 19), (54 ± 17), (56 ± 18) µm/h. They were significantly higher than those of control group [(33 ± 13), (33 ± 12), (33 ± 10) µm/h, with t values from 2.840 to 9.330, P < 0.05 or P < 0.01]. The curvilinear movement speed of cells was significantly higher in hypoxia for 6 h group than in hypoxia for 1 or 3 h group (with t values from 3.474 to 4.545, P < 0.05 or P < 0.01). There was no significant difference in the curvilinear movement speed among the observation time points within each group (with F values from 0.012 to 0.195, P values above 0.05). At POH 1, the rectilinear movement speed of cells in hypoxia for 1 h group was (22 ± 11) µm/h, which was obviously higher than that of control group [(15 ± 10) µm/h, t = 2.697, P < 0.01]. At POH 1, 2, 3, rectilinear movement speeds of cells in hypoxia for 3 and 6 h groups were respectively (19 ± 14), (12 ± 8), (10 ± 6) µm/h; (32 ± 19), (21 ± 13), (17 ± 12) µm/h. They were significantly higher than those of control group [(9 ± 7) and (6 ± 5) µm/h at POH 2 and 3, with t values from 1.990 to 8.231, P < 0.05 or P < 0.01]. The rectilinear movement speed of cells in hypoxia for 6 h group was obviously higher than that of hypoxia for 1 or 3 h group (with t values from 3.394 to 6.008, P < 0.05 or P < 0.01). The rectilinear movement speed of cells in each group decreased at POH 2 or 3 in comparison with POH 1 (with t values from -8.208 to -4.232, P values below 0.01). The rectilinear movement speed of cells in control group at POH 3 was significantly different from that at POH 2 (t = -1.967, P < 0.05). (2) The proliferation levels of cells in control group and hypoxia for 1, 3, 6, 9, 12, 24 h groups were respectively 1.11 ± 0.08, 1.36 ± 0.10, 1.39 ± 0.05, 1.38 ± 0.05, 1.10 ± 0.14, 1.06 ± 0.09, 0.99 ± 0.06 (F = 39.19, P < 0.01). Compared with that of control group, the rate of proliferation of cells was obviously increased in hypoxia for 1, 3, 6 h groups (with t values respectively 6.639, 7.403, 7.195, P values below 0.01), but obviously decreased in hypoxia for 24 h group (t = -3.136, P < 0.05). The proliferation of cells decreased in hypoxia for 9, 12, 24 h groups in comparison with hypoxia for 1, 3, 6 h groups (with t values from -10.538 to -6.775, P values below 0.01). (3) The protein expressions of PCNA of cells in control group and hypoxia for 1, 3, 6, 24 h groups were respectively 0.93 ± 0.12, 0.97 ± 0.14, 1.62 ± 0.18, 0.95 ± 0.09, 0.66 ± 0.21 (F = 20.11, P < 0.01). Compared with that of control group, the expression of PCNA was obviously increased in hypoxia for 1, 3, 6 h groups (with t values respectively 2.339, 5.783, 2.235, P < 0.05 or P < 0.01), but obviously decreased in hypoxia for 24 h group (t = -1.998, P < 0.05). The protein expression of PCNA was higher in hypoxia for 3 h group than in hypoxia for 1 or 6 h group (with t values respectively 4.312 and 3.947, P values below 0.01), and it was increased in the 3 groups in comparison with that of hypoxia for 24 h group (with t values respectively 2.011, 6.193, 3.287, P < 0.05 or P < 0.01).

CONCLUSIONSShort-time hypoxia (1, 3, 6 h) treatment can promote the movement and proliferation of HaCaT cells. Hypoxia for 6 h is the best condition to promote their movement, while hypoxia for 3 or 6 h is better for their proliferation.

Carbon Dioxide ; pharmacology ; Cell Cycle ; drug effects ; Cell Line ; Cell Movement ; physiology ; Cell Proliferation ; drug effects ; physiology ; Cells, Cultured ; Epithelial Cells ; cytology ; drug effects ; Humans ; Hypoxia ; physiopathology ; Nitric Oxide ; pharmacology ; Oxygen ; pharmacology ; Phosphorylation ; Proliferating Cell Nuclear Antigen ; Signal Transduction