细菌纤维素合成与鉴定研究综述

细菌纤维素（BC）因其独特性能被广泛应用于医药、食品等领域，目前其高产量菌株筛选、合成成本降低及合成途径改良等成为研究热点。本文依据国内外文献并结合团队研究成果对BC合成与鉴定的相关研究进行综述。首先对BC合成菌筛选及碳源利用的研究进行了分析，总结了降低BC合成成本的研究思路。其次对鉴定菌株合成产物的方法进行了归纳，总结了不同方法的特点。然后结合本团队筛选出的BC生产菌XJL-06-4 BC合成酶基因分析结果，综述了BC合成途径、合成酶存在形式以及基因水平调控作用，为BC在分子水平上通过改变合成途径提高产量提供新思路。最后，总结BC微生物发酵生产存在的问题，多角度提出解决方案。     

Pseudouridine synthase 7 impacts Candida albicans rRNA processing and morphological plasticity

RNA can be modified in over 100 distinct ways, and these modifications are critical for function. Pseudouridine synthases catalyse pseudouridylation, one of the most prevalent RNA modifications. Pseudouridine synthase 7 modifies a variety of substrates in Saccharomyces cerevisiae including tRNA, rRNA, snRNA, and mRNA, but the substrates for other budding yeast Pus7 homologues are not known. We used CRISPR-mediated genome editing to disrupt Candida albicans PUS7 and find absence leads to defects in rRNA processing and a decrease in cell surface hydrophobicity. Furthermore, C. albicans Pus7 absence causes temperature sensitivity, defects in filamentation, altered sensitivity to antifungal drugs, and decreased virulence in a wax moth model. In addition, we find C. albicans Pus7 modifies tRNA residues, but does not modify a number of other S. cerevisiae Pus7 substrates. Our data suggests C. albicans Pus7 is important for fungal vigour and may play distinct biological roles than those ascribed to S. cerevisiae Pus7.     

GPR55 Receptor Activation by the N-Acyl Dopamine Family Lipids Induces Apoptosis in Cancer Cells via the Nitric Oxide Synthase (nNOS) Over-Stimulation

GPR55 is a GPCR of the non-CB1/CB2 cannabinoid receptor family, which is activated by lysophosphatidylinositol (LPI) and stimulates the proliferation of cancer cells. Anandamide, a bioactive lipid endocannabinoid, acts as a biased agonist of GPR55 and induces cancer cell death, but is unstable and psychoactive. We hypothesized that other endocannabinoids and structurally similar compounds, which are more hydrolytically stable, could also induce cancer cell death via GPR55 activation. We chemically synthesized and tested a set of fatty acid amides and esters for cell death induction via GPR55 activation. The most active compounds appeared to be N-acyl dopamines, especially N-docosahexaenoyl dopamine (DHA-DA). Using a panel of cancer cell lines and a set of receptor and intracellular signal transduction machinery inhibitors together with cell viability, Ca²⁺, NO, ROS (reactive oxygen species) and gene expression measurement, we showed for the first time that for these compounds, the mechanism of cell death induction differed from that published for anandamide and included neuronal nitric oxide synthase (nNOS) overstimulation with concomitant oxidative stress induction. The combination of DHA-DA with LPI, which normally stimulates cancer proliferation and is increased in cancer setting, had an increased cytotoxicity for the cancer cells indicating a therapeutic potential.     

昆明假单胞菌HL22-2海藻糖合酶基因的克隆、表达及酶学性质研究

采用热不对称交错聚合酶链式反应（Tail-PCR）克隆云南磷矿来源昆明假单胞菌（Pseudomonas kunmingensis）HL22-2的海藻糖合酶（TreS）基因HL22-2TreS，将该基因与表达载体pETM3C连接后在大肠杆菌（Escherichia coli）BL21（DE3）pLysS中进行异源表达，通过Ni-NTA柱纯化重组酶HL22-2TreS，并对其酶学特性进行分析。结果表明，HL22-2TreS基因全长3 336 bp，编码1 111个氨基酸，氨基酸序列与Genbank数据库中相关的海藻糖合酶具有极高的相似性。重组酶HL22-2TreS的分子质量约126 kDa，该酶的最适反应温度和pH值分别为40 ℃和7.0，在温度20～50 ℃及pH值6.0～9.0条件下比较稳定，Cu2+、Hg2+、Ba2+及Al3+对海藻糖合酶的活力有强烈的抑制效果。HL22-2TreS基因对麦芽糖和海藻糖的米氏常数（Km）分别为20.6 mmol/L和87.5 mmol/L，对麦芽糖具有更高的亲和性，更容易将麦芽糖转化成海藻糖。     

Self-Assembly of Proteinaceous Shells around Positively Charged Gold Nanomaterials Enhances Colloidal Stability in High-Ionic-Strength Buffers

The enzyme lumazine synthase (LS) has been engineered to self-assemble into hollow-shell structures that encapsulate unnatural cargo proteins through complementary electrostatic interactions. Herein, we show that a negatively supercharged LS variant can also form organic–inorganic hybrids with gold nanomaterials. Simple mixing of LS pentamers with positively charged gold nanocrystals in aqueous buffer spontaneously affords protein-shelled gold cores. The procedure works well with differently sized and shaped gold nanocrystals, and the resulting shelled complexes exhibit dramatically enhanced colloidal stability over a wide range of pH (4.0–10.0) and at high ionic strength (up to 1 m NaCl). They are even stable over days upon dilution in buffer. Self-assembly of engineered LS shells in this way offers an easy and attractive alternative to commonly used ligand-exchange methods for stabilizing inorganic nanomaterials.     

Phosphoproteomic Analysis of Subcutaneous and Omental Adipose Tissue Reveals Increased Lipid Turnover in Dairy Cows Supplemented with Conjugated Linoleic Acid

Phosphoproteomics is a cutting-edge technique that can be utilized to explore adipose tissue (AT) metabolism by quantifying the repertoire of phospho-peptides (PP) in AT. Dairy cows were supplemented with conjugated linoleic acid (CLA, n = 5) or a control diet (CON, n = 5) from 63 d prepartum to 63 d postpartum; cows were slaughtered at 63 d postpartum and AT was collected. We performed a quantitative phosphoproteomics analysis of subcutaneous (SC) and omental (OM) AT using nanoUPLC-MS/MS and examined the effects of CLA supplementation on the change in the phosphoproteome. A total of 5919 PP were detected in AT, and the abundance of 854 (14.4%) were differential between CON and CLA AT (p ≤ 0.05 and fold change ± 1.5). The abundance of 470 PP (7.9%) differed between OM and SC AT, and the interaction treatment vs. AT depot was significant for 205 PP (3.5% of total PP). The integrated phosphoproteome demonstrated the up- and downregulation of PP from proteins related to lipolysis and lipogenesis, and phosphorylation events in multiple pathways, including the regulation of lipolysis in adipocytes, mTOR signaling, insulin signaling, AMPK signaling, and glycolysis. The differential regulation of phosphosite on a serine residue (S777) of fatty acid synthase (FASN) in AT of CLA-supplemented cows was related to lipogenesis and with more phosphorylation sites compared to acetyl-coenzyme A synthetase (ACSS2). Increased protein phosphorylation was seen in acetyl-CoA carboxylase 1 (ACACA;8 PP), FASN (9 PP), hormone sensitive lipase (LIPE;6 PP), perilipin (PLIN;3 PP), and diacylglycerol lipase alpha (DAGLA;1 PP) in CLA vs. CON AT. The relative gene expression in the SC and OM AT revealed an increase in LIPE and FASN in CLA compared to CON AT. In addition, the expression of DAGLA, which is a lipid metabolism enzyme related to the endocannabinoid system, was 1.6-fold higher in CLA vs. CON AT, and the expression of the cannabinoid receptor CNR1 was reduced in CLA vs. CON AT. Immunoblots of SC and OM AT showed an increased abundance of FASN and a lower abundance of CB1 in CLA vs. CON. This study presents a complete map of the SC and the OM AT phosphoproteome in dairy cows following CLA supplementation and discloses many unknown phosphorylation sites, suggestive of increased lipid turnover in AT, for further functional investigation.     

The Fifth WS/DGAT Enzyme of the Bacterium Marinobacter aquaeolei VT8

Wax esters (WE) belong to the class of neutral lipids. They are formed by an esterification of a fatty alcohol and an activated fatty acid. Dependent on the chain length and desaturation degree of the fatty acid and the fatty alcohol moiety, WE can have diverse physicochemical properties. WE derived from monounsaturated long-chain acyl moieties are of industrial interest due to their very good lubrication properties. Whereas WE were obtained in the past from spermaceti organs of the sperm whale, industrial WE are nowadays mostly produced chemically from fossil fuels. In order to produce WE more sustainably, attempts to produce industrial WE in transgenic plants are steadily increasing. To achieve this, different combinations of WE producing enzymes are expressed in developing Arabidopsis thaliana or Camelina sativa seeds. Here we report the identification and characterization of a fifth wax synthase from the organism Marinobacter aquaeolei VT8, MaWSD5. It belongs to the class of bifunctional wax synthase/acyl-CoA:diacylglycerol O-acyltransferases (WSD). The protein was purified to homogeneity. In vivo and in vitro substrate analyses revealed that MaWSD5 is able to synthesize WE but no triacylglycerols. The protein produces WE from saturated and monounsaturated mid- and long-chain substrates. Arabidopsis thaliana seeds expressing a fatty acid reductase from Marinobacter aquaeolei VT8 and MaWSD5 produce WE. Main WE synthesized are 20:1/18:1 and 20:1/20:1. This makes MaWSD5 a suitable candidate for industrial WE production in planta.     

甘草黄酮合酶Ⅱ催化甘草素特异性合成7，4′-二羟基黄酮

利用同源序列比对和分子进化树分析从胀果甘草转录组数据库中挖掘并成功克隆到2个黄酮合酶基因：Gur.gene26505和Gur.gene26116。经表征Gur.gene26505为黄酮合酶Ⅱ,具有催化甘草素特异性合成7,4′-二羟基黄酮的特性,而Gur.gene26116为黄烷酮2位羟化酶,可催化甘草素生成包括7,4′-二羟基黄酮在内的三种产物。进一步通过蛋白质结构预测、分子对接和分子动力学模拟探究了黄酮合酶Ⅱ(Gur.gene26505)催化合成7,4′-二羟基黄酮特异性的原因。由于Gur.gene26505活性口袋附近特有的刚性结构β片层使大位阻苯丙氨酸残基翻转至羟化中心下方,消除了羟化产物2-羟基甘草素进入脱水中心的阻力进而发生C₂-C₃位的脱水反应特异性生成7,4′-二羟基黄酮。最后通过基因过表达、反应条件优化和强化菌体生长建立了7,4′-二羟基黄酮特异性合成的最佳细胞催化工艺,使甘草素转化率达到了76.67%。