Small urban wetlands are widely distributed and susceptible to human activities, serving as important sources and sinks of carbon. Microorganisms play a crucial role in carbon cycle, while limited studies have been conducted on the microbial diversity in small urban wetlands and the functions of microbiome in carbon fixation and metabolism. To probe into the microbiome-driven carbon cycling in small urban wetlands and dissect the composition and functional groups of microbiome, we analyzed the relationships between the microbiome structure, element metabolism pathways, and habitat physicochemical properties in sediment samples across three small wetlands in Huzhou City, and compared them with natural wetlands in the Zoige wetland. High-throughput sequencing of 16S rRNA gene amplicons and metagenomics was employed to determine the species and functional groups. Sixty medium to high-quality metagenome-assembled genomes (MAGs) were constructed, including 55 bacterial and 5 archaeal taxa, and their potential in driving elemental cycles were analyzed, with a focus on carbon fixation. Several bacterial species were found to encode a nearly complete carbon fixation pathway, including the Calvin cycle, the reductive tricarboxylic acid cycle, the Wood-Ljungdahl pathway, and the reductive glycine pathway. There were several potential novel carbon-fixing bacterial members, such as those belonging to Syntrophorhabdus (Desulfobacterota) and UBA4417 (Bacteroidetes), which had high relative abundance in the wetland microbiome. Unveiling the genetic potential of these functional groups to facilitate element cycling is of great scientific importance for enhancing the carbon sequestration capacity of small urban wetlands.
Wetlands ; Microbiota/genetics* ; Carbon Cycle/genetics* ; Bacteria/classification* ; RNA, Ribosomal, 16S/genetics* ; China ; Cities ; Geologic Sediments/microbiology* ; Archaea/classification* ; Metagenomics ; Metagenome

Microalgae possess high photosynthetic efficiency, robust adaptability, and substantial biomass, serving as excellent biological resources for large-scale cultivation. Malic enzyme (ME), a ubiquitous metabolic enzyme in living organisms, catalyzes the decarboxylation of malate to produce pyruvate, CO₂, and NAD(P)H, playing a role in multiple metabolic pathways including energy metabolism, photosynthesis, respiration, and biosynthesis. In this study, we identified the Scenedesmus quadricauda malic enzyme gene (SqME) and its biological functions, aiming to provide excellent target genes for the genetic improvement of higher plants. Based on the RNA-seq data from S. quadricauda under the biofilm cultivation mode with high CO₂ and light energy transfer efficiency and small water use, a highly expressed gene (SqME) functionally annotated as ME was cloned. The physicochemical properties of the SqME-encoded protein were systematically analyzed by bioinformatics tools. The subcellular localization of SqME was determined via transient transformation in Nicotiana benthamiana leaves. The biological functions of SqME were identified via genetic transformation in Nicotiana tabacum, and the potential of SqME in the genetic improvement of higher plants was evaluated. The ORF of SqME was 1 770 bp, encoding 590 amino acid residues, and the encoded protein was located in chloroplasts. SqME was a NADP-ME, with the typical structural characteristics of ME. The ME activity in the transgenic N. tabacum plant was 1.8 folds of that in the wild-type control. Heterologous expression of SqME increased the content of chlorophyll a, chlorophyll b, and total chlorophyll by 20.9%, 26.9%, and 25.2%, respectively, compared with the control. The transgenic tobacco leaves showed an increase of 54.0% in the fluorescence parameter NPQ and a decrease of 30.1% in Fo compared with the control. Moreover, the biomass, total lipids, and soluble sugars in the transgenic tobacco leaves enhanced by 20.5%, 25.7%, and 9.5%, respectively. On the contrary, the starch and protein content in the transgenic tobacco leaves decreased by 22.4% and 12.2%, respectively. Collectively, the SqME-encoded protein exhibited a strong enzymatic activity. Heterologous expressing of SqME could significantly enhance photosynthetic protection, photosynthesis, and biomass accumulation in the host. Additionally, SqME can facilitate carbon metabolism remodeling in the host, driving more carbon flux towards lipid synthesis. Therefore, SqME can be applied in the genetic improvement of higher plants for enhancing photosynthetic carbon fixation and lipid accumulation. These findings provide scientific references for mining of functional genes from S. quadricauda and application of these genes in the genetic engineering of higher plants.
Nicotiana/genetics* ; Photosynthesis/physiology* ; Malate Dehydrogenase/biosynthesis* ; Plant Leaves/genetics* ; Scenedesmus/enzymology* ; Carbon Cycle/genetics* ; Lipid Metabolism/genetics* ; Plants, Genetically Modified/metabolism*

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*

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

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

OBJECTIVE3-Methylcrotonyl-coenzyme A carboxylase deficiency (MCCD) is an autosomal recessive inborn error of leucine catabolism. The cases suspected as MCCD detected by neonatal screening are not rare. The aim of the study was to investigate the clinical outcomes in cases suspected as MCCD by neonatal screening. The second aim was to investigate the mutation spectrum of MCC gene in Chinese population and hotspot mutation.

METHODForty-two cases (male 33, female 9) , who had higher blood 3-hydroxy-isovalerylcarnitine (C5-OH) levels(cut-off <0.6 µmol/L) detected by neonatal screening using MS/MS, were recruited to this study during Sept.2011 to Mar.2013. The C5-OH concentrations were [0.84 (0.61-20.15) µmol/L] in 42 cases at the screening recall. Five cases were firstly diagnosed as maternal MCCD, 6 cases as benign MCCD and 31 cases were suspected as MCCD. To follow up the height, weight, mental development, blood C5-OH concentrations and urinary 3-methylcrotonyl-glycine (3-MCG) and 3-hydroxy isovalerate (3-HIVA) in order to investigate the clinical outcome. The MCCC1 and MCCC2 gene mutation were analyzed for some cases. The novel gene variants were evaluated, and the influence of novel missense variants on the protein structure and function were predicted by PolyPhen-2, SIFT, UniProt and PDB software.

RESULT(1) Forty-two cases had no symptoms, their physical and mental development were normal in the last visit at the median ages of 29 months, the oldest age of follow up was nearly 9 years. (2) Gene mutation analysis was performed for 29 cases with informed consent signed by parents.Fourteen different mutations were identified in 19 cases. The mutations in MCCC1 gene accounted for 86%, the most common mutation was c.ins1680A, (accounted for 40%). Nine kinds of novel variant were detected including 211AG>CC/p.Q74P, c.295G>A/p.G99S, c.764A>C/p.H255P, c.964G>A/p. E322K, c.1331G>A/p.R444H, c.1124delT, c.39_58del20, c.1518delG, c.639+2T>A.Other 3 kinds of mutation in MCCC1 gene and 2 kinds of mutation in MCCC2 gene have been reported previously; the amino acid of mutant positions of five kinds of novel missense variant are almost highly conserved. These missense variants were predicted to cause change of human MCC protein side chain structure by changing hydrogen bonding, size of amino acid residue and electric charge, and predicted to damage the protein function possibly according to PolyPhen-2 and PDB analysis. So these novel variants may be disease-causing mutations. No mutation were detected in 10 cases. (3) Blood concentrations of C5-OH when screening, recall and end of follow-up in maternal MCCD was 3.50 (1.63-11.43), 1.84 (1.00-9.30), 0.27 (0.26-5.81) µmol/L. There was a significant downward trend.In contrast, benign MCCD group was 8.20 (3.60-9.60), 9.67 (3.88-20.15), 23.0 (5.87-49.10) µmol/L.It showed a rising trend. Children's urinary 3-MCG of benign MCCD group was found abnormally elevated in 4 cases (100%) when they were recalled.

CONCLUSIONA certain number of cases with MCCD or suspected as MCCD in this study had no symptoms and normal physical and mental development after follow-up to oldest age of nearly 9 years. The mutation in MCCC1 gene is common, nine novel mutations were found, c.ins1680A may be a hotspot mutation in Chinese population. The urinary GC/MS analysis and blood MS/MS analysis for mother should be routinely performed for all cases with high blood C5-OH level detected by neonatal screening.

Amino Acid Sequence ; Asian Continental Ancestry Group ; genetics ; Carbon-Carbon Ligases ; blood ; deficiency ; genetics ; Carnitine ; analogs & derivatives ; blood ; Child ; Child, Preschool ; DNA Mutational Analysis ; Female ; Follow-Up Studies ; Humans ; Infant ; Infant, Newborn ; Male ; Mutation ; Neonatal Screening ; Tandem Mass Spectrometry ; Urea Cycle Disorders, Inborn ; blood ; diagnosis ; enzymology ; genetics

OBJECTIVETo report on 5 patients with maternal 3-methylcrotonyl coenzyme A carboxylase deficiency (MCCD) and to confirm the clinical diagnosis through mutation analysis.

METHODSFive neonates with higher blood 3-hydroxy isovalerylcarnitine (C5-OH) concentration detected upon newborn screening with tandem mass spectrometry and their mothers were recruited. Urinary organic acids were analyzed with gas chromatography mass spectrometry. Gene mutation and protein function analysis were performed by PCR direct sequencing and PolyPhen-2 software.

RESULTSHigher blood C5-OH concentrations (5.11-21.77 μmol/L) and abnormal 3-hydroxy isovalerate and 3-methylcrotonyl glycine in urine were detected in the five asymptomatic mothers, who were diagnosed as benign MCCD. Higher C5-OH concentration was also detected in their neonates by tandem mass spectrometry, which had gradually decreased to normal levels in three neonates. Four new variations, i.e., c.ins1680A(25%), c.203C > T (p.A68V), c.572T > C (p.L191P) and c.639+5G > T were detected in the MCCC1 gene, in addition with 2 mutations [c.1406G > T (p.R469L, novel variation) and c.592C > T (p.Q198X)]. The novel variations were predicted to have affected protein structure and function.

CONCLUSIONFor neonates with higher C5-OH concentration detected upon neonatal screening, their mothers should be also tested to rule out MCCD. Mutations in MCCC1 gene are quite common.

Adult ; Amino Acid Sequence ; Base Sequence ; Carbon-Carbon Ligases ; blood ; deficiency ; genetics ; Carnitine ; analogs & derivatives ; blood ; DNA Mutational Analysis ; Female ; Genomic Imprinting ; Humans ; Infant, Newborn ; Male ; Molecular Sequence Data ; Mutation ; Neonatal Screening ; Sex Factors ; Tandem Mass Spectrometry ; Urea Cycle Disorders, Inborn ; blood ; diagnosis ; enzymology ; genetics