三个玉米合成群体选系的配合力及杂种优势分析

采用NCⅡ遗传交配设计,通过在黑龙江省哈尔滨和泰来的两点试验,以玉米自交系Mo17、B73、444和丹340为测验种,对从群体品综1号、中综3号和陕综5号选育的18份自交系进行配合力及杂种优势分析,以探讨群体选系在我国东北早熟玉米区的利用途径。结果表明,供试自交系间一般配合力存在较大差异;陕综5号群体选系HR14、HR17和HR15、中综3号选系HR9和HR8、品综1号选系HR4的一般配合力较高。在供试的72个组合中HR15×丹340、HR17×丹340、HR9×Mo17、HR14×丹340、HR7×B73、HR8×B73、HR6×444、HR5×丹340产量的特殊配合力及对照优势较高,表现出较高的利用潜力。依据特殊配合力及对照优势分析,中综3号选系与旅大红骨群、陕综5号选系与兰卡斯特群、品综1号选系与瑞德群遗传关系较近。结合育种实践,在我国北方早熟春玉米区陕综5号×旅大红骨、中综3号×瑞德或兰卡斯特、品综1号×旅大红骨或唐四平头可能组成较大利用潜力的杂种优势模式。     

In vitro study on the effect of cytokines and auxins addition to growth medium on the micropropagation and rooting of Paulownia species (Paulownia hybrid and Paulownia tomentosa)

This study represents an efficient preliminary protocol for in vitro mass production of two Paulownia species (Paulownia hybrid and Paulownia tomentosa) seedlings by using seed explant. Different concentrations of benzyladenine (BA) or Kinetin (Kin) (0.0, 2.0, 4.0, 6.0, 8.0 and 10.0 mg/L) were tested during multiplication stage. The number of shoots/explants was significantly increased with increasing either BA or Kin concentration; however, the shoot length significantly decreased. Data show that media fortified by BA (10 mg/L) combined with indole butyric acid (IBA) at 1.0 or 1.5 mg/L recorded the highest number of shoots/explant (9.13 and 9.25, respectively). After six weeks during the multiplication stage, data cleared that media fortified by benzyladenine (10 mg/L) combined with IBA at 0.5 mg/L recorded the highest shoot length (3.23 cm). The inclusion of indole butyric acid (IBA) or naphthalene acetic acid (NAA) at 1.0–1.5 mg/L to the medium significantly increased the number of roots/plantlets and the highest root length. The results indicated that IBA supplementation was more effective than NAA for in vitro rooting of both Paulownia species. The best treatment for multiplication was 10 mg/L and 8.0–10 mg/L BA for P. hybrid and P. tomentosa, respectively. Peat moss and sand (1:1, v/v) or peat moss and sand (1:2, v/v) were investigated as soil mixture during the adaptation stage. The results referred that Paulownia species plantlets were successfully survived (100 %) in soil mixture contained peat moss: sand (1:2, v/v). This mixture recorded the highest values of plantlet height and number of leaves/plantlets.     

Electrogenetics: Bridging synthetic biology and electronics to remotely control the behavior of mammalian designer cells

Electrogenetics, the combination of electronics and genetics, is an emerging field of mammalian synthetic biology in which electrostimulation is used to remotely program user-designed genetic elements within designer cells to generate desired outputs. Here, we describe recent advances in electro-induced therapeutic gene expression and therapeutic protein secretion in engineered mammalian cells. We also review available tools and strategies to engineer electro-sensitive therapeutic designer cells that are able to sense electrical pulses and produce appropriate clinically relevant outputs in response. We highlight current limitations facing mammalian electrogenetics and suggest potential future directions for research.     

Engineering the human gut commensal Bacteroides thetaiotaomicron with synthetic biology

The role of the microbiome in health and disease is attracting the attention of researchers seeking to engineer microorganisms for diagnostic and therapeutic applications. Recent progress in synthetic biology may enable the dissection of host–microbiota interactions. Sophisticated genetic circuits that can sense, compute, memorize, and respond to signals have been developed for the stable commensal bacterium Bacteroides thetaiotaomicron, dominant in the human gut. In this review, we highlight recent advances in expanding the genetic toolkit for B. thetaiotaomicron and foresee several applications of this species for microbiome engineering. We provide our perspective on the challenges and future opportunities for the engineering of human gut-associated bacteria as living therapeutic agents.     

总体国家安全观下合成生物学风险和应对策略研究*

合成生物学是近年来兴起的一门兼材料学、医学和信息学等学科特性的交叉学科,在促进医学进步和科技转化应用的同时,也在总体国家安全内涵中被赋予了特殊的重要地位。如何在新时期应对错综复杂的安全形势和严峻挑战,是世界各国和国际社会所面临的科技治理和生物安全的重要命题。重点介绍合成生物技术相关生物武器威胁、生物恐怖威胁、生物安全国际公约条例、生物安全伦理治理框架,总结近年来合成生物技术领域生物安全风险相关问题,提出合成生物学安全风险应对策略和国家总体安全观下科技发展建议。     

CelloSelect – A synthetic cellobiose metabolic pathway for selection of stable transgenic CHO cell lines

Current protocols for generating stable transgenic cell lines mostly rely on antibiotic selection or the use of specialized cell lines lacking an essential part of their metabolic machinery, but these approaches require working with either toxic chemicals or knockout cell lines, which can reduce productivity. Since most mammalian cells cannot utilize cellobiose, a disaccharide consisting of two β-1,4-linked glucose molecules, we designed an antibiotic-free selection system, CelloSelect, which consists of a selection cassette encoding Neurospora crassa cellodextrin transporter CDT1 and β-glucosidase GH1-1. When cultivated in glucose-free culture medium containing cellobiose, CelloSelect-transfected cells proliferate by metabolizing cellobiose as a primary energy source, and are protected from glucose starvation. We show that the combination of CelloSelect with a PiggyBac transposase-based integration strategy provides a platform for the swift and efficient generation of stable transgenic cell lines. Growth rate analysis of metabolically engineered cells in cellobiose medium confirmed the expansion of cells stably expressing high levels of a cargo fluorescent marker protein. We further validated this strategy by applying the CelloSelect system for stable integration of sequences encoding two biopharmaceutical proteins, erythropoietin and the monoclonal antibody rituximab, and confirmed that the proteins are efficiently produced in either cellobiose- or glucose-containing medium in suspension-adapted CHO cells cultured in chemically defined media. We believe coupling heterologous metabolic pathways additively to the endogenous metabolism of mammalian cells has the potential to complement or to replace current cell-line selection systems.     

Multiplex genome editing of mammalian cells for producing recombinant heparin

Heparin is an essential anticoagulant used for treating and preventing thrombosis. However, the complexity of heparin has hindered the development of a recombinant source, making its supply dependent on a vulnerable animal population. In nature, heparin is produced exclusively in mast cells, which are not suitable for commercial production, but mastocytoma cells are readily grown in culture and make heparan sulfate, a closely related glycosaminoglycan that lacks anticoagulant activity. Using gene expression profiling of mast cells as a guide, a multiplex genome engineering strategy was devised to produce heparan sulfate with high anticoagulant potency and to eliminate contaminating chondroitin sulfate from mastocytoma cells. The heparan sulfate purified from engineered cells grown in chemically defined medium has anticoagulant potency that exceeds porcine-derived heparin and confers anticoagulant activity to the blood of healthy mice. This work demonstrates the feasibility of producing recombinant heparin from mammalian cell culture as an alternative to animal sources.     

Combinatorial pathway engineering of Bacillus subtilis for production of structurally defined and homogeneous chitooligosaccharides

Chitooligosaccharides (COSs) have a widespread range of biological functions and an incredible potential for various pharmaceutical and agricultural applications. Although several physical, chemical, and biological techniques have been reported for COSs production, it is still a challenge to obtain structurally defined COSs with defined polymerization (DP) and acetylation patterns, which hampers the specific characterization and application of COSs. Herein, we achieved the de novo production of structurally defined COSs using combinatorial pathway engineering in Bacillus subtilis. Specifically, the COSs synthase NodC from Azorhizobium caulinodans was overexpressed in B. subtilis, leading to 30 ± 0.86 mg/L of chitin oligosaccharides (CTOSs), the homo-oligomers of N-acetylglucosamine (GlcNAc) with a well-defined DP lower than 6. Then introduction of a GlcNAc synthesis module to promote the supply of the sugar acceptor GlcNAc, reduced CTOSs production, which suggested that the activity of COSs synthase NodC and the supply of sugar donor UDP-GlcNAc may be the limiting steps for CTOSs synthesis. Therefore, 6 exogenous COSs synthase candidates were examined, and the nodCM from Mesorhizobium loti yielded the highest CTOSs titer of 560 ± 16 mg/L. Finally, both the de novo pathway and the salvage pathway of UDP-GlcNAc were engineered to further promote the biosynthesis of CTOSs. The titer of CTOSs in 3-L fed-batch bioreactor reached 4.82 ± 0.11 g/L (85.6% CTOS5, 7.5% CTOS4, 5.3% CTOS3 and 1.6% CTOS2), which was the highest ever reported. This is the first report proving the feasibility of the de novo production of structurally defined CTOSs by synthetic biology, and provides a good starting point for further engineering to achieve the commercial production.     

Engineering precursor supply for the high-level production of ergothioneine in Saccharomyces cerevisiae

Ergothioneine (ERG) is an unusual sulfur-containing amino acid. It is a potent antioxidant, which shows great potential for ameliorating neurodegenerative and cardiovascular diseases. L-ergothioneine is rare in nature, with mushrooms being the primary dietary source. The chemical synthesis process is complex and expensive. Alternatively, ERG can be produced by fermentation of recombinant microorganisms engineered for ERG overproduction. Here, we describe the engineering of S. cerevisiae for high-level ergothioneine production on minimal medium with glucose as the only carbon source. To this end, metabolic engineering targets in different layers of the amino acid metabolism were selected based on literature and tested. Out of 28 targets, nine were found to improve ERG production significantly by 10%–51%. These targets were then sequentially implemented to generate an ergothioneine-overproducing yeast strain capable of producing 106.2 ± 2.6 mg/L ERG in small-scale cultivations. Transporter engineering identified that the native Aqr1 transporter was capable of increasing the ERG production in a yeast strain with two copies of the ERG biosynthesis pathway, but not in the strain that was further engineered for improved precursor supply. Medium optimization indicated that additional supplementation of pantothenate improved the strain's productivity further and that no supplementation of amino acid precursors was necessary. Finally, the engineered strain produced 2.39 ± 0.08 g/L ERG in 160 h (productivity of 14.95 ± 0.49 mg/L/h) in a controlled fed-batch fermentation without supplementation of amino acids. This study paves the way for the low-cost fermentation-based production of ergothioneine.