Reconstitution of the peptidoglycan cytoplasmic precursor biosynthetic pathway in cell-free system and rapid screening of antisense oligonucleotides for Mur enzymes

Bacterial peptidoglycan is the cell wall component responsible for various biological activities. Its cytoplasmic precursor UDP-N-acetylmuramyl pentapeptide is biosynthesized by the first six enzymes of peptidoglycan synthetic pathways (Mur enzymes), which are all proved to be important targets for antibiotic screening. In our present work, the genes encoding Mur enzymes from Escherichia coli were co-expressed in the cell-free protein synthesis (CFPS) system, and the activities of Mur enzymes derived from CFPS system were validated by the synthesis of the final product UDP-N-acetylmuramyl pentapeptide. Then this in vitro reconstituted Mur biosynthetic pathway was used to screen a panel of specific antisense oligonucleotides for MurA and MurB. The selected oligonucleotides were proved to eliminate the expression of Mur enzymes, and thus inhibit the Mur biosynthetic pathway. The present work not only developed a rapid method to reconstruct and regulate a biosynthetic pathway in vitro, but also may provide insight into the development of novel antibiotics targeting on peptidoglycan biosynthetic pathway.     

太行山北段地区荆条灌丛和三裂绣线菊灌丛群落谱系结构

灌丛是太行山地区最重要的生态系统类型之一, 灌丛群落生物多样性的维持及其生态系统服务功能对京津冀地区的生态安全具有重要作用。本研究选择太行山最具代表性的两种灌木群落——荆条(Vitex negundo var. heterophylla)灌丛和三裂绣线菊(Spiraea trilobata)灌丛为研究对象, 利用净亲缘关系指数(net relatedness index, NRI)和多元回归等方法探究了两种灌丛群落构建机制的异同及主要的环境影响因子, 同时还利用谱系结构主坐标分析(principal coordinates of phylogenetic structure, PCPS)对决定群落谱系结构的关键系统发育节点进行了探究。结果表明: 两种灌丛群落内灌木植物的物种多样性没有显著差异, 但谱系结构差异显著。三裂绣线菊群落表现出显著的谱系发散趋势, 而荆条群落谱系聚集程度高于三裂绣线菊群落, 但未表现出显著的谱系结构。三裂绣线菊灌丛群落构建的驱动机制是生态位分化, 荆条灌丛中生境过滤作用有所增加, 与生态位分化共同驱动其群落构建过程。与干旱胁迫相关的生境过滤因素增加是荆条灌丛群落谱系聚集程度增加的重要原因。PCPS二维排序结果表明: 荆条灌丛群落谱系趋向聚集与其群落内缺乏蔷薇目、壳斗目等亲缘关系较远的类群有关, 而三裂绣线菊灌丛群落内物种则包含较多的演化分支。总体而言, 环境过滤不是决定太行山地区灌丛群落构建的主要驱动因素, 但水分条件仍然是影响区域群落谱系结构的重要因素。     

A 3-hydroxy-3-methylglutaryl-CoA synthase-based probe for the discovery of the acyltransferase-less type I polyketide synthases

Acyltransferase (AT)-less type I polyketide synthases (PKSs) produce complex natural products due to the presence of many unique tailoring enzymes. The 3-hydroxy-3-methylglutaryl coenzyme A synthases (HCSs) are responsible for β-alkylation of the growing polyketide intermediates in AT-less type I PKSs. In this study, we discovered a large group of HCSs, closely associated with the characterized and orphan AT-less type I PKSs through in silico genome mining, sequence and genome neighbourhood network analyses. Using HCS-based probes, the survey of 1207 in-house strains and 18 soil samples from different geographic locations revealed the vast diversity of HCS-containing AT-less type I PKSs. The presence of HCSs in many AT-less type I PKSs suggests their co-evolutionary relationship. This study provides a new probe to study the abundance and diversity of AT-less type I PKSs in the environment and microbial strain collections. Our study should inspire future efforts to discover new polyketide natural products from AT-less type I PKSs.     

DJ‐1 inhibits autophagy activity of prostate cancer cells by repressing JNK–Bcl2–Beclin1 signaling

The regulation of DJ‐1 on AR signaling plays an important role in the pathogenesis of prostate cancer (PCa). DJ‐1 could alter autophagy and regulate Beclin1‐involved autophagy response through JNK‐dependent pathway. JNK is known to mediate autophagy through Bcl2–Beclin1 complex. Therefore, this study aimed to investigate the significance of autophagy in DJ‐1‐modulated PCa cells. The current studies showed that DJ‐1 overexpression in LNCaP decreased LC3 transformation and autophagosome formation. However, DJ‐1 knockdown exerted the opposite effect. Moreover, DJ‐1 silencing inhibited survival and promoted death in LNCaP, which was recovered by autophagy inhibition with 3‐MA. In addition, DJ‐1 overexpression inhibited the phosphorylation of JNK and Bcl2, and the dissociation of Beclin1 and Bcl2; while the effect of silencing DJ‐1 was completely opposite. More important, JNK activated by anisimycin inhibited the proliferation and promoted death of DJ‐1‐overexpressed LNCaP while increasing LC3 transformation and LC3‐puncta formation, but these results were reversed by the decrease of Beclin1 (by spautin‐1). In contrast, when DJ‐1 was silenced, the death of LNCaP, LC3 transformation, and LC3‐puncta formation were inhibited by JNK inhibitor SP600125, which promoted cell proliferation. However, Bcl2 inhibition (by ABT737) reversed all the effects of SP600125. Our results suggested that DJ‐1 in PCa cells could promote the growth of PCa through autophagy inhibition, and JNK–Bcl2–Beclin1 signaling played an important role in it. The study provided new insights into the role of DJ‐1 in the development of PCa.     

Streptomycin-induced ribosome engineering complemented with fermentation optimization for enhanced production of 10-membered enediynes tiancimycin-A and tiancimycin-D

Tiancimycins (TNMs) are a group of 10-membered anthraquinone-fused enediynes, newly discovered from Streptomyces sp. CB03234. Among them, TNM-A and TNM-D have exhibited excellent antitumor performances and could be exploited as very promising warheads for the development of anticancer antibody-drug conjugates (ADCs). However, their low titers, especially TNM-D, have severely limited following progress. Therefore, the streptomycin-induced ribosome engineering was adopted in this work for strain improvement of CB03234, and a TNMs high producer S. sp. CB03234-S with the K43N mutation at 30S ribosomal protein S12 was successfully screened out. Subsequent media optimization revealed the essential effects of iodide and copper ion on the production of TNMs, while the substitution of nitrogen source could evidently promote the accumulation of TNM-D, and the ratio of produced TNM-A and TNM-D was responsive to the change of carbon and nitrogen ratio in the medium. Further amelioration of the pH control in scaled up 25 L fermentation increased the average titers of TNM-A and TNM-D up to 13.7 ± 0.3 and 19.2 ± 0.4 mg/L, respectively. The achieved over 45-fold titer improvement of TNM-A, and 109-fold total titer improvement of TNM-A and TNM-D enabled the efficient purification of over 200 mg of each target molecule from 25 L fermentation. Our efforts have demonstrated a practical strategy for titer improvement of anthraquinone-fused enediynes and set up a solid base for the pilot scale production and preclinical studies of TNMs to expedite the future development of anticancer ADC drugs.     

古田山中亚热带常绿阔叶林群落物种多样性的空间变异特征

古田山常绿阔叶林的群落组成、结构及其维持机制已有许多研究, 但该地区亚热带常绿阔叶林生物多样性空间变异特征还缺乏认识。本文以古田山24 ha大样地(划分为24个1 ha小样地)为基础, 具体分析了α多样性和β多样性在1 ha尺度上的空间变异特征。结果表明: (1)群落第一、二优势物种在各小样地之间变化不大, 但第三优势种变化较大; (2) α多样性变化中, 样地间木本植物个体数量变异最大, 物种丰富度其次, Pielou均匀度指数变异性最小; (3)物种丰富度与植株个体数量、Pielou均匀度指数没有显著的相关性, 与Shannon-Wiener指数呈显著正相关; Shannon-Wiener指数与Pielou均匀度指数呈显著正相关; (4)相邻样地间物种替代速率空间变异较大, 与物种丰富度的空间变化格局有明显差异。这些结果说明尺度对认识群落结构、探讨群落维持机制有重要作用; 由于森林群落是多尺度生态过程作用的结果, 大尺度样地可能有利于更好地揭示森林群落维持机制。     

Fungi participate in driving home-field advantage of litter decomposition in a subtropical forest

Plant and Soil - Home-field advantage (HFA) hypothesis predicts that plant litter decomposes faster beneath the plant species from which it was derived than beneath other plant species. However, it...     

Efficient production of l-lactic acid from hydrolysate of Jerusalem artichoke with immobilized cells of Lactococcus lactis in fibrous bed bioreactors

Hydrolysate of Jerusalem artichoke was applied for the production of l-lactic acid by immobilized Lactococcus lactis cells in a fibrous bed bioreactor system. Preliminary experiments had indicated that the high quality hydrolysate, which was derived from the 40min acid treatment at 95°C and pH 1.8, was sufficient to support the cell growth and synthesis of l-lactic acid. With the addition of 5g/l yeast extract, the fermentative performance of free cell system was evidently improved. After the basal settlement of hydrolysate based fermentation, the batch mode and the fed-batch mode fermentation were carried out in the free cell system and the fibrous bed bioreactor system, respectively. In all cases the immobilized cells presented the superior ability to produce l-lactic acid. The comparison of batch mode and fed-batch mode also indicated that the growth-limiting feeding strategy could reduce the lag phase of fermentation process and enhance the production of l-lactic acid. The achieved maximum concentration of l-lactic acid was 142g/l in the fed-batch mode. Subsequent repeated-batch fermentation of the fibrous bed bioreactor system had further exhibited the persistence and stability of this system for the high production of l-lactic acid in a long term. Our work suggested the great potential of the fibrous bed bioreactor system and hydrolysate of J. artichoke in the economical production of l-lactic acid at industrial scale.     

古田山常绿阔叶林凋落量时间动态及冰雪灾害的影响

2006年10月至2009年12月期间,我们通过对古田山24 ha常绿阔叶林动态样地169个种子雨收集器的凋落物进行烘干、分类、称量和数据分析,研究了凋落量的组成特征和时间动态,以及受2008年2月特大冰雪灾害的影响.2007年和2009年凋落量分别为532.05 g/m2和375.17 g/m2,年际变化显著,这与2008年的冰雪灾害有关.2007年古田山常绿阔叶林各组分在凋落量中所占比例依次为:叶(78.99%)>枝(14.69%)>皮(3-33%)>其他(2.99%);叶凋落量中各组分所占比例依次为:常绿阔叶树种叶(78.70%)>落叶阔叶树种叶(12.37%)>针叶树种叶(8.92%),其中甜槠(Castanopsis eyrei)、木荷(Schima superba)、马尾松(Pinus massoniana)和短柄袍(Quercus serrata vat.brevipetiolata)4个优势种年凋落量合计占叶凋落总量的71.36%,它们的动态直接影响着凋落总量的变化格局.凋落量高峰发生在春季(4月)和秋冬季(10月末至12月初),其中总凋落量、叶凋落量动态呈双峰型,枝条凋落量和树皮凋落量动态为不规则峰型,其他凋落量动态为单峰型.冰雪灾害后总凋落量、叶凋落量、枝条凋落量显著减少(P<0.05),其中甜槠、杨梅叶蚊母树(Distylium myricoides)叶凋落最显著减少(P<0.05)、木荷叶凋落量减少达到边缘显著水平(P<0.1),这也反映了冰亏灾害期间森林群落的受损情况.其他凋落量(主要成分为虫粪)在4B份增加极其显著(P<0.01),说明灾后植物的枝叶出现了补偿性生长.