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Characterization of Pellicle Inhibition in Gluconacetobacter xylinus 53582 by a Small Molecule, Pellicin, Identified by a Chemical Genetics Screen  [PDF]
Janice L. Strap, Andrew Latos, Isaac Shim, Dario T. Bonetta
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0028015
Abstract: Pellicin ([2E]-3-phenyl-1-[2,3,4,5-tetrahydro-1,6?-benzodioxocin-8-yl]prop-2-en-1-one)was identified in a chemical genetics screen of 10,000 small molecules for its ability to completely abolish pellicle production in Gluconacetobacter xylinus. Cells grown in the presence of pellicin grew 1.5 times faster than untreated cells. Interestingly, growth in pellicin also caused G. xylinus cells to elongate. Measurement of cellulose synthesis in vitro showed that cellulose synthase activity was not directly inhibited by pellicin. Rather, when cellulose synthase activity was measured in cells that were pre-treated with the compound, the rate of cellulose synthesis increased eight-fold over that observed for untreated cells. This phenomenon was also apparent in the rapid production of cellulose when cells grown in the presence of pellicin were washed and transferred to media lacking the inhibitor. The rate at which cellulose was produced could not be accounted for by growth of the organism. Pellicin was not detected when intracellular contents were analyzed. Furthermore, it was found that pellicin exerts its effect extracellularly by interfering with the crystallization of pre-cellulosic tactoidal aggregates. This interference of the crystallization process resulted in enhanced production of cellulose II as evidenced by the ratio of acid insoluble to acid soluble product in in vitro assays and confirmed in vivo by scanning electron microscopy and powder X-ray diffraction. The relative crystallinity index, RCI, of pellicle produced by untreated G. xylinus cultures was 70% while pellicin-grown cultures had RCI of 38%. Mercerized pellicle of untreated cells had RCI of 42%, which further confirms the mechanism of action of pellicin as an inhibitor of the cellulose I crystallization process. Pellicin is a useful tool for the study of cellulose biosynthesis in G. xylinus.
Revealing Differences in Metabolic Flux Distributions between a Mutant Strain and Its Parent Strain Gluconacetobacter xylinus CGMCC 2955  [PDF]
Cheng Zhong, Fei Li, Miao Liu, Xiao-Ning Yang, Hui-Xia Zhu, Yuan-Yuan Jia, Shi-Ru Jia, Luciano Piergiovanni
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0098772
Abstract: A better understanding of metabolic fluxes is important for manipulating microbial metabolism toward desired end products, or away from undesirable by-products. A mutant strain, Gluconacetobacter xylinus AX2-16, was obtained by combined chemical mutation of the parent strain (G. xylinus CGMCC 2955) using DEC (diethyl sulfate) and LiCl. The highest bacterial cellulose production for this mutant was obtained at about 11.75 g/L, which was an increase of 62% compared with that by the parent strain. In contrast, gluconic acid (the main byproduct) concentration was only 5.71 g/L for mutant strain, which was 55.7% lower than that of parent strain. Metabolic flux analysis indicated that 40.1% of the carbon source was transformed to bacterial cellulose in mutant strain, compared with 24.2% for parent strain. Only 32.7% and 4.0% of the carbon source were converted into gluconic acid and acetic acid in mutant strain, compared with 58.5% and 9.5% of that in parent strain. In addition, a higher flux of tricarboxylic acid (TCA) cycle was obtained in mutant strain (57.0%) compared with parent strain (17.0%). It was also indicated from the flux analysis that more ATP was produced in mutant strain from pentose phosphate pathway (PPP) and TCA cycle. The enzymatic activity of succinate dehydrogenase (SDH), which is one of the key enzymes in TCA cycle, was 1.65-fold higher in mutant strain than that in parent strain at the end of culture. It was further validated by the measurement of ATPase that 3.53–6.41 fold higher enzymatic activity was obtained from mutant strain compared with parent strain.
Efecto de la sacarosa en la producción de celulosa por Gluconacetobacter xylinus en cultivo estático  [PDF]
Rubén Jaramillo L.,Wladimir Tobio J.,José Escamilla M.
Revista MVZ Córdoba , 2012,
Abstract: Objetivo. Determinar el efecto de sacarosa en la productividad de BC por Gluconacetobacter xylinus IFO 13693 en condición estática. Materiales y métodos. La síntesis de celulosa bacteriana (BC) por Gluconacetobacter xylinus se llevo a cabo en un cultivo estático discontinuo a temperatura ambiente, en presencia de sacarosa como la principal fuente de carbono a concentraciones iniciales de 0.8 a 7.6 % (p/v). Las concentraciones remanentes de BC, sacarosa, glucosa y fructosa se determinaron cada semana. Para la cinética de la hidrólisis de la sacarosa y formación de celulosa y el coeficiente de rendimiento del producto se utilizo el software Microcal Origin 6.0 . Resultados. En la cuarta semana los valores de BC se encontraron entre 32.5 a 39.5 g/L para las diferentes concentraciones de sacarosa. La cinética para la hidrólisis de sacarosa se ajusta al modelo de Michaelis-Menten, con una Vmax de 0.0002 mol L-1 h-1 y Km de 0.018 M. La producción de BC se ajusta al modelo propuesto por Marx-Figini y Pion, con un valor de la pendiente (kc), entre 0.0018 y 0.0024 h-1 para las diferentes concentraciones iniciales de sacarosa. Los coeficientes de rendimiento tienen valores de 0.8 a 2.4 g de BC producida/g de sacarosa consumida. Conclusiones. La hidrólisis de sacarosa, el consumo de glucosa y fructosa se refleja en la síntesis de celulosa. La hidrólisis de sacarosa y la producción de BC se ajustan a los modelos de Michaelis-Menten y al propuesto por Marx-Figini y Pion, respectivamente. Finalmente, el rendimiento depende de la concentración de sacarosa.
N-acetylglucosamine 6-Phosphate Deacetylase (nagA) Is Required for N-acetyl Glucosamine Assimilation in Gluconacetobacter xylinus  [PDF]
Vikas Yadav, Bruce Panilaitis, Hai Shi, Keiji Numuta, Kyongbum Lee, David L. Kaplan
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0018099
Abstract: Metabolic pathways for amino sugars (N-acetylglucosamine; GlcNAc and glucosamine; Gln) are essential and remain largely conserved in all three kingdoms of life, i.e., microbes, plants and animals. Upon uptake, in the cytoplasm these amino sugars undergo phosphorylation by phosphokinases and subsequently deacetylation by the enzyme N-acetylglucosamine 6-phosphate deacetylase (nagA) to yield glucosamine-6-phosphate and acetate, the first committed step for both GlcNAc assimilation and amino-sugar-nucleotides biosynthesis. Here we report the cloning of a DNA fragment encoding a partial nagA gene and its implications with regard to amino sugar metabolism in the cellulose producing bacterium Glucoacetobacter xylinus (formally known as Acetobacter xylinum). For this purpose, nagA was disrupted by inserting tetracycline resistant gene (nagA::tetr; named as ΔnagA) via homologous recombination. When compared to glucose fed conditions, the UDP-GlcNAc synthesis and bacterial growth (due to lack of GlcNAc utilization) was completely inhibited in nagA mutants. Interestingly, that inhibition occured without compromising cellulose production efficiency and its molecular composition under GlcNAc fed conditions. We conclude that nagA plays an essential role for GlcNAc assimilation by G. xylinus thus is required for the growth and survival for the bacterium in presence of GlcNAc as carbon source. Additionally, G. xylinus appears to possess the same molecular machinery for UDP-GlcNAc biosynthesis from GlcNAc precursors as other related bacterial species.
木葡糖酸醋杆菌株型对细菌纤维素产量与性能的影响
Effects of Different Gluconacetobacter xylinus Strains on Yield and Properties of Bacterial Cellulose
 [PDF]

张少瑞, 陈琳, 钟春燕, 洪枫
Bioprocess (BP) , 2016, DOI: 10.12677/BP.2016.61002
Abstract: 木葡糖酸醋杆菌是重要的细菌纤维素生产菌。该课题以40 g/L葡萄糖为碳源,研究了静态培养时5株菌种对细菌纤维素产量与性能的影响。采用扫描电镜、X射线衍射和万能材料测试机分别对细菌纤维素进行了形貌观察、结晶度表征以及力学性能测试。研究结果表明:细菌纤维素的产量排序依次为DHU-ZCY-1 (3.9 g/L) > DHU-ATCC-1 (2.6 g/L) > DHU-ZGD-1 (2.3 g/L) > DHU-YQ-1 (1.7 g/L) > ATCC23770 (0.6 g/L);株型对细菌纤维素的含水率和结晶度基本无影响;不同菌株产的细菌纤维素湿膜的断裂拉伸强度大小依次如下:DHU-ZGD-1 (350 KPa) > DHU-ZCY-1 (150 KPa) > DHU-YQ-1 (68 KPa) > DHU-ATCC-1 (56 KPa) > ATCC23770 (35 KPa);菌株对细菌纤维素的微观结构影响甚微,纤维直径大都分布在65~91 nm之间。
Gluconacetobacter xylinus is an important bacterial cellulose (BC) producing bacterium. Effects of five strains of G. xylinus on cellulose production and properties were investigated by using D-glu- cose as carbon source. The initial concentration of glucose was 40 g/L. X-ray diffraction analysis, scanning electron microscopy and universal material testing machine were used to characterize the bacterial cellulose. The volume yield of bacterial cellulose by the five strains of G. xylinus was as follows: DHU-ZCY-1 (3.9 g/L) > DHU-ATCC-1 (2.62 g/L) > DHU-ZGD-1 (2.34 g/L) > DHU-YQ-1 (1.72 g/L) > ATCC23770 (0.61 g/L). The highest volume yield was around 7 times higher than the lowest one. Tensile strength of bacterial cellulose of the five strains was as follows: DHU-ZGD-1 > DHU-ZCY-1 > DHU-YQ-1 > DHU-ATCC-1 > ATCC23770. The results showed that unremarkable morphological difference of bacterial cellulose was found among the five strains. And there was no obvious difference in water content and crystallinity of BC obtained by the five strains. The fiber diameters were in the range between 65 nm and 91 nm.
Identification and biocellulose production of Gluconacetobacter strains isolated from tropical fruits in Thailand
Daungjai Ochaikul
Maejo International Journal of Science and Technology , 2013,
Abstract: Two hundred and four strains of biocellulose (BC)-producing Gluconacetobacter strains were isolated from 48 rotten tropical fruits collected in Thailand. Twenty-nine representative isolates were selected from each of the 16 isolation sources and identified by morphological, physiological and biochemical characteristics and 16S rRNA gene sequence analysis. The selected 29 isolates were divided into seven subgroups within the Gluconacetobacter xylinus group of the genus Gluconacetobacter and identified as Gluconacetobacter oboediens (subgroup I, five isolates), Gluconacetobacter rhaeticus (subgroup II, one isolate), Gluconacetobacter hansenii (subgroup III, seven isolates), Gluconacetobacter swingsii (subgroup IV, two isolates) and Gluconacetobacter sucrofermentans (subgroup V, two isolates). The remaining isolates were grouped into subgroups VIa (three isolates) and VIb (nine isolates). All the isolates were cultured in Hestrin-Schramm (HS) medium statically at 30 C for 7 days to determine cellulose production capability. Of the 29 isolates, isolate PAP1 (subgroup VIb, unidentified) gave the highest yield (1.15 g/L) of BC. However, the BC yield increased threefold (3.5 g/L) when D-glucose in HS medium was replaced by D-mannitol.
Screening and Identification of a Strain Producing Dihydroxyacetone During Oxidation of Glycerol
一株以甘油为底物产二羟丙酮(DHA)微生物菌种的筛选及鉴定

XU Mei-Juan,RAO Zhi-Ming,SHEN Wei,FANG Hui-Ying,ZHUGE Jian,
徐美娟
,饶志明,沈 微,方慧英,诸葛健

微生物学通报 , 2008,
Abstract: More than 20 strains capable of producing dihydroxyacetone from glycerol were isolated from 4 different natural environment samples by using two detection methods. The strain 6-8 which could grow on medium containing glycerol as sole carbon source had a higher converting capability. Under a better culture, the highest DHA production of the strain 6-8 reached 6.4 g/L. In addition to general morphological and biochemical characteristics, the strain 6-8 was identified by 16S rDNA sequence and systematic analysis. The results showed that 16S rDNA sequence of the strain 6-8 had similarity of 99.7% with Acinetobacter sp. suggesting that the strain 6-8 is one of subspecies of Acinetobacter sp.
Screening and Identification of a Strain Producing Dihydroxyacetone During Oxidation of Glycerol
一株以甘油为底物产二羟丙酮(DHA)微生物菌种的筛选及鉴定

XU Mei-Juan,RAO Zhi-Ming,SHEN Wei,FANG Hui-Ying,ZHUGE Jian,
徐美娟
,饶志明,沈 微,方慧英,诸葛健

微生物学报 , 2008,
Abstract: More than 20 strains capable of producing dihydroxyacetone from glycerol were isolated from 4 different natural environment samples by using two detection methods. The strain 6-8 which could grow on medium containing glycerol as sole carbon source had a higher converting capability. Under a better culture, the highest DHA production of the strain 6-8 reached 6.4 g/L. In addition to general morphological and biochemical characteristics, the strain 6-8 was identified by 16S rDNA sequence and systematic analysis. The results showed that 16S rDNA sequence of the strain 6-8 had similarity of 99.7% with Acinetobacter sp. suggesting that the strain 6-8 is one of subspecies of Acinetobacter sp.
Progress in metabolic engineering of microbial production of 1,3-dihydroxyacetone
微生物法生产1,3-二羟基丙酮代谢工程研究进展

Lihui Sun,Zhongce Hu,Yuguo Zheng,Yinchu Shen,
孙丽慧
,胡忠策,郑裕国,沈寅初

生物工程学报 , 2010,
Abstract: 1,3-Dihydroxyacetone is widely used in cosmetics, medicines and food products. We reviewed the recent progress in metabolic pathways, key enzymes, as well as metabolic engineering for microbial production of 1,3-dihydroxyacetone. We addressed the research trend to increase yield of 1,3-dihydroxyacetone by improving the activity of glycerol dehydrogenase with genetic engineering, and regulating of fermentation process based on metabolic characteristic of the strain.
Effects of Osmotic Stress on Intracellular Glycerol Content and Enzyme Activity in Dunaliella salina
渗透胁迫对杜氏盐藻胞内甘油含量及相关酶活性影响

Li Zhou,Xianghong Meng,Chengsheng Liu,Lejun Yu,Xiguang Chen,
周丽
,孟祥红,刘成圣,于乐军,陈西广

植物学报 , 2006,
Abstract: Dunaliella salina is known as probably the most halotolerant eukaryotic organism living in various salt habitats, and glycerol metabolism plays an important role in its osmoregulation. We investigated the growth, intracellular glycerol content and activities of enzymes in glycerol metabolism under NaCl stress. Growth of D. salina was inhibited in solutions containing 4.0, 3.0, 0.8, and 0.6 mol.L-1 NaCl. The intracellular glycerol content accumulated and activities of glycerol 3-phosphate phosphatase and dihydroxyacetone reductase catalyzing dihydroxyacetone to glycerol were enhanced in response to hyperosmotic stress. However, glycerol content decreased and the activities of glycerol 3-phosphate phosphatase lost and dihydroxyacetone reductase catalyzing glycerol to dihydroxyacetone increased when adapting to hypo-osmotic stress. The paper discusses the association of glycerol metabolism with the osmoregulation mechanism of D. salina.
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