Synthetic biology and metabolic engineering of actinomycetes for natural product discovery

Actinomycetes are one of the most valuable sources of natural products with industrial and medicinal importance. After more than half a century of exploitation, it has become increasingly challenging to find novel natural products with useful properties as the same known compounds are often repeatedly re-discovered when using traditional approaches. Modern genome mining approaches have led to the discovery of new biosynthetic gene clusters, thus indicating that actinomycetes still harbor a huge unexploited potential to produce novel natural products. In recent years, innovative synthetic biology and metabolic engineering tools have greatly accelerated the discovery of new natural products and the engineering of actinomycetes. In the first part of this review, we outline the successful application of metabolic engineering to optimize natural product production, focusing on the use of multi-omics data, genome-scale metabolic models, rational approaches to balance precursor pools, and the engineering of regulatory genes and regulatory elements. In the second part, we summarize the recent advances of synthetic biology for actinomycetal metabolic engineering including cluster assembly, cloning and expression, CRISPR/Cas9 technologies, and chassis strain development for natural product overproduction and discovery. Finally, we describe new advances in reprogramming biosynthetic pathways through polyketide synthase and non-ribosomal peptide synthetase engineering. These new developments are expected to revitalize discovery and development of new natural products with medicinal and other industrial applications.     

pBV220载体中外源基因高效表达的自动化设计

pBV220载体是国内科学家构建的原核系统表达载体,目前仍在广泛应用.但是,实现 外源基因的高效表达需要综合考虑诸如RNA二级结构等多种因素,极其耗时费力．为此,基于我们提出的pBV220载体中外源基因高效表达数学模型,编写了外源基因高效表达的自动化设计软件,并可定性用于原核系统其它载体中外源基因表达水平分析,最终为加快实验进程提供帮助     

表达EB病毒主要膜蛋白gp350／220的非复制型重组痘苗病毒的构建

利用非复制痘苗病毒质粒载体pNEOCK11β75及pNEOCK,改造了表达EB病毒主要膜蛋白gp350/22的复制型重组痘苗病毒VMA,构建了非复制型重组痘苗病毒VMA△CK。该病毒能在鸡胚原代成纤维细胞（CEF）中正常繁殖,而在人源细胞中不能正常繁殖。在CEF中连续传代至第25代,经PCR证明,该病毒符合非复制型重组痘苗病毒的特征。经免疫荧光及免疫酶斑法证实,VMA△CK可稳定表达gp350/220,且表达水平与VAM无明显差异。VMA△CK经腹腔免疫Balb/C小鼠,4周后能诱生一定水平的抗gp350/220特异性抗体,加强免疫2周后该抗体水平明显升高。这一结果类似于VMA免疫Balb/C小鼠的结果,初免后,VMA△CK且抗痘苗抗体水平明显低于VMA免疫组;加强免疫2周后,两组小鼠的抗痘苗抗体水平趋于一致。上述结果证明,所构建的非复制痘苗病毒不影响目的抗原的表达,也不影响该抗原的免疫原性,但导致病毒毒力下降,而且用该病毒免疫小鼠后小鼠抗痘苗病毒载体的免疫反应明显下降。     

Niedergang der Insekten

The Decline of Insects – Why moths and other insects are becoming rarer Fifty years of quantitative study of abundance of night-flying insects attracted to ultraviolet light revealed a decrease of more than 80 per cent in moths since the start of the study in 1969 and of 96 per cent in the other (small) insects for the margins of the arable land subjected to conventional farming, but no changes in insect abundance in several areas in the city of Munich. There, at present more insects fly despite light pollution than on the borders of agricultural land on the countryside. The trend of abundance in the quite insect rich riverine forests is inconclusive, and probably due to fluctuations caused by the uninhibited growth of the trees, which are no longer used for fire wood. Concerning agriculture the key factor is overfertilisation which creates conditions too cold and too wet due to vegetation which became much too dense. Additionally the extensive application of pesticides reduces insect population. Second to conventional agriculture, however, is the overzealous cleaning (in fact a shaving) of marginal land in towns, villages and along roads, called the duly public maintenance. This highly destructive practice could be cancelled quite simply.     

Epitope mapping of gp350/220 conserved domain of epstein barr virus to develop nasopharyngeal carcinoma (npc) vaccine

Nasopharyngeal carcinoma (NPC) is a malignant tumor in the nasopharyngeal epithelial cells that caused by many factors, one of which is the viral infection of EBV (Epstein Barr Virus). The standard treatments to cure NPC still have not been encouraging. The prevention through vaccination is an effective way to stop the disease. However, EBV vaccine being able to cover all variants of virus is still not available yet. Therefore, we identified the conserved region of glycoprotein 350/220 of EBV which has immunogenic and antigenic properties. The glycoprotein 350/220 is viral surface protein responsible to bind CR2 receptor, mediated EBV to enter the host cell. The conserved domain is crucial for EBV in infecting host cells. Further, by blocking CR2 binding domain of gp350/220 using antibody will inhibit EBV's spreading, and provoke an immune system to eliminate the virus in a patient. Glycoprotein 350/220 from all variants of Epstein-Barr virus was retrieved from NCBI. The conserved domain of gp350/220 was identified by aligning the protein sequences and structures. The polymorphic structure was used as a template for docking analysis to identify the resemblance of amino acid from polymorphic variants of gp350/220 that binds CR2. The epitope mapping of gp350/220 was done by Discotope BepiPred method. The result revealed that the conserved region of gp350/220 was predicted to have an epitope, QNPVYLIPETVPYIKWDNC residue, and it does not have any similarities to the human's cell surface protein. Therefore, it can be used as a reference to develop vaccine to prevent NPC.     

Chromatin remodeling and epigenetic regulation of the CrabpI gene in adipocyte differentiation

Retinoic acid (RA) acts by binding to nuclear RA receptors (RARs) to regulate a broad spectrum of downstream target genes in most cell types examined. In cytoplasm, RA binds specifically to cellular retinoic acid binding proteins I (CRABPI), and II. Although the function of CRABPI in animals remains the subject of debate, it is believed that CRABPI binding facilitates RA metabolism, thereby modulating the concentration of RA and the type of RA metabolites in cells. The basal promoter of the CrabpI gene is a housekeeping promoter that can be regulated by thyroid hormones (T3), DNA methylation, sphinganine, and ethanol acting on its upstream regulatory region. T3 regulation of CrabpI is mediated by the binding of thyroid hormone receptor (TR) to a TR response element (TRE) approximately 1 kb upstream of the basal promoter. Specifically, in the adipocyte differentiation process, T3 regulation is bimodal and closely associated with the cellular differentiation status: T3 activates CrabpI in predifferentiated cells (e.g., mesenchymal precursors or fibroblasts), but suppresses this gene once cells are committed to adipocyte differentiation. These disparate effects are functions of T3-triggered differential recruitment of coregulatory complexes in conjunction with chromatin looping/folding that alters the configuration of this genomic locus along adipocyte differentiation. Subsequent sliding, disassembly and reassembly of nucleosomes occur, resulting in specific changes in the conformation of the basal promoter chromatin at different stages of differentiation. This chapter summarizes studies illustrating the epigenetic regulation of CrabpI expression during adipocyte differentiation. Understanding the pathways regulating CrabpI in this specific context might help to illuminate the physiological role of CRABPI in vivo. This article is part of a special issue entitled: Retinoid and Lipid Metabolism.     

Kidins220/ARMS mediates the integration of the neurotrophin and VEGF pathways in the vascular and nervous systems

Signaling downstream of receptor tyrosine kinases controls cell differentiation and survival. How signals from different receptors are integrated is, however, still poorly understood. In this work, we have identified Kidins220 (Kinase D interacting substrate of 220 kDa)/ARMS (Ankyrin repeat-rich membrane spanning) as a main player in the modulation of neurotrophin and vascular endothelial growth factor (VEGF) signaling in vivo, and a primary determinant for neuronal and cardiovascular development. Kidins220(-/-) embryos die at late stages of gestation, and show extensive cell death in the central and peripheral nervous systems. Primary neurons from Kidins220(-/-) mice exhibit reduced responsiveness to brain-derived neurotrophic factor, in terms of activation of mitogen-activated protein kinase signaling, neurite outgrowth and potentiation of excitatory postsynaptic currents. In addition, mice lacking Kidins220 display striking cardiovascular abnormalities, possibly due to impaired VEGF signaling. In support of this hypothesis, we demonstrate that Kidins220 constitutively interacts with VEGFR2. These findings, together with the data presented in the accompanying paper, indicate that Kidins220 mediates the integration of several growth factor receptor pathways during development, and mediates the activation of distinct downstream cascades according to the location and timing of stimulation.     

大肠埃希菌热不稳定肠毒素B亚单位在婴儿双歧杆菌中的表达

目的将大肠埃希菌热不稳定肠毒素B亚单位（LTB）克隆到表达载体pBV220,使LTB基因在大肠埃希菌和双歧杆菌中稳定表达。方法将LTB基因克隆到表达载体pBV220中,再分别转化大肠埃希菌DH5a和婴儿双歧杆菌,使之表达,表达产物用SDS—PAGE鉴定。并通过家兔肠袢实验验证表达蛋白的安全性。结果LTB基因在大肠埃希菌和双歧杆菌中均可稳定表达,毒性实验证明LTB保留轻微的毒性。结论稳定表达LTB的双歧杆菌为今后的口服疫苗佐剂奠定了基础。     

Plasma appearance and tissue accumulation of non-esterified, free astaxanthin in C57BL/6 mice after oral dosing of a disodium disuccinate diester of astaxanthin (Heptax)

Oral bioavailability of natural and synthetic carotenoids is generally poor in rodents, and this has limited the ability to test these antioxidant compounds in well-defined rodent models of human disease. Various strategies have been employed, with variable success, to increase the percentage of the total oral dose absorbed by the rodent GI tract. In the current study, a novel carotenoid derivative (the disodium disuccinate diester of astaxanthin; Heptax) was administered by oral gavage in a lipophilic emulsion to C57BL/6 mice. Plasma appearance and tissue accumulation of non-esterified, free astaxanthin was studied by HPLC over 72 h after single- and multiple-dose regimens. One-time dosing of Heptax in emulsion at 500 mg/kg resulted in significant appearance of free astaxanthin in plasma (Cmax=0.2 mg/l; 381 nM) and accumulation in solid organs (e.g. liver Cmax=0.9 mg/l; 1735 nM), levels not previously reported after single carotenoid doses in rodents. At each point in the concentration/time curve (AUC), free astaxanthin levels in liver were greater than the corresponding concentration in plasma, suggesting concentrative uptake by the liver. As the ED50 as an antioxidant for non-esterified, free astaxanthin in model systems is approximately 200 nM, the current results suggest that hepatoprotection against oxidative insults may be achieved after a single dose of Heptax in these animals. In humans, where the bioavailability of oral carotenoids ranges from 40 to 60% of the total dose when given in lipophilic vehicle, much smaller oral doses may be utilized for therapeutic benefit in a particular clinical application.     

Epstein─Barr病毒膜抗原在中国仓鼠卵巢（CHO）细胞中的表达

将切去３’端穿膜序列的ＥＢ病毒膜抗原（ＭＡ）基因,插入ｐＳＶ２－ｄｈｆｒ质粒的ＳＶ４０早期启动子下游,构建了真核表达载体ｐＳＶ２－ｄｈｆｒＧＰＴＲ,使两个ＳＶ４０早期启动子分别调控ＭＡ和二氢叶酸还原酶（ｄｈｆｒ）基因。将该重组质粒转化ＣＨＯ－ｄｈｆｒ细胞,在选择培养基中筛选阳性克隆,用氨甲喋呤加压扩增,建立了表达ＥＢＶ－ＭＡ的克隆细胞系。ｗｅｓｔｅｒｎｂｌｏｔ分析证明,所表达的蛋白的分子量大约为３４０ｋｄ和２２０ｋｄ。经过细Ｓｅｐｈａｒｏｓｅ２Ｂ琼脂糖凝胶层析初步纯化的抗原与福氏佐剂混合免疫小鼠,２周后小鼠血清中出现明显的ｇｐ３４０／２２０特异性抗体,表明切去嵌膜区结构的ＥＢＶ－ＭＡ基因在ＣＨＯ细胞中的表达产物具有同天然膜抗原相似的分子量大小、糖基化程度、免疫特异性和免疫原性,可望成为ＥＢ病毒人用基因工程亚单位疫苗。