细菌信号传导调控元件的合成生物学研究进展

合成生物学的一个重要目标是设计、改造微生物(主要指细菌),使其能够自主执行复杂任务,如合成重要生物基产品(药物、生物燃料等)、疾病治疗以及环境修复等,造福人类社会.要完成这些任务,细菌必须依赖其信号传导系统,根据环境变化作出正确及时的应答.在长期进化过程中,细菌产生了众多不同的信号传导系统,给我们提供了大量宝贵的信号传导调控元件.通过对这些调控元件的合成生物学设计、改造,我们可以给细菌装备全新的信号传导系统,从而使其能够在工业生物技术及生物医学等应用中执行设定任务.     

人工小RNA调控元件在合成生物学中的应用北大核心CSCD

合成生物学通过改造天然系统或创造生物元件、模块和系统赋予生命体新的功能,为农业、能源、制造业及医学进步带来了巨大推动力。对元件、模块或系统的精准、定量及高效调控将对合成生命系统的控制至关重要。细菌小RNA是一类长度在50–300 bp且通常不具备翻译能力的功能小分子,在环境胁迫响应、代谢变化适应和细菌毒力控制过程中发挥着不可替代的调控作用。近年来,基于天然小RNA设计构建的人工小RNA调控元件的工作日益丰富,实现了对目的基因甚至通路的有效抑制或激活。人工小RNA分子小、灵活性高,可程序化且易于设计,几乎不会对宿主细胞造成代谢负担,因此在合成生物学中具备广泛应用前景。为促进对人工小RNA的机理理解及应用拓展,本文围绕若干人工小RNA调控元件进行了系统介绍及比较;此外,总结了其在合成生物学中的代表性应用;最后,对其未来优化方向进行了讨论。     

钙调磷酸酶信号调控真菌生长代谢、毒力及抗逆性能

钙调磷酸酶是一种丝氨酸/苏氨酸（Ser/Thr）蛋白磷酸酶,在真菌中普遍保守,上游信号途径由Ca²⁺通道（Cch1）、转运蛋白（Mid1）、钙离子感应蛋白（CaM）、钙调蛋白依赖性磷酸酶等组成。钙调磷酸酶受钙离子和钙调蛋白调节,在调控真菌Ca²⁺稳态的钙信号级联途径中发挥着中心作用,通过钙信号级联途径参与生物学过程,调控真菌生长、发育和毒力形成来响应外界环境因素的变化,使真菌能够适应不同环境,维持正常的生命活动。本文综述了真菌钙调磷酸酶信号的组成和上下游信号转导途径、调控细胞生长代谢、毒力形成以及抗逆性能调控的研究进展;结合对真菌代谢产物合成的调控作用,对钙调磷酸酶信号作为重要合成生物学元件及调控开关进行了展望。     

噬菌体重组酶系统在合成生物学中的应用*

合成生物学技术采用工程化设计理念,对生物体进行有目标的设计、改造乃至重新合成,对重塑非自然功能的“人造生命”具有重要意义。噬菌体重组系统具有高效、精确和广谱适用性等特点,在基因工程、代谢工程以及生物治疗等合成生物学领域得到了广泛的应用。从基因电路、体内遗传改造和体外重组等方面全面阐述了噬菌体重组系统在合成生物学研究的现状及热点,对当前该系统的局限性进行了探讨,并就未来的研究和发展趋势进行了展望。     

代谢物感知失调的分子致病机理

代谢是最基础的生命活动.生物代谢网络由代谢酶和代谢物共同构成.相对于对代谢酶性质的深刻认识,对代谢物生理重要性的认识停留在物质代谢与能量代谢的水平.近年来,本研究组及国际上其他实验室的工作发现,代谢物具有信号传导功能.细胞内的代谢物水平通过不同的机制被感知,其信号通过不同的化学基础在蛋白质间相互传递并调控包括代谢、表观遗传和信号通路等重要生理过程.代谢物的失调也因此通过这些生理改变而导致人类疾病.本文对本研究组近年来在该领域的相关进展进行综述并讨论其理论及转化价值.     

光遗传学技术在调控细菌生命活动中的应用

光遗传学技术利用光作为输入信号,能够精准地调控细胞的生理功能,同时具有高度的时间和空间特异性,使得构建高度动态的调控系统成为可能.近年来,随着新型光敏蛋白的发现和光照系统的创新,基于光遗传学技术的光控系统的效率得到了显著提高.通过合成生物学方法构造各种生物回路,光控系统在细菌中的应用也日益广泛.将光控系统作为输入模块,与其他生物功能模块相结合,能够实现对基因表达、蛋白质活性以及细菌生理功能的调控.本文主要介绍光遗传学技术的基本原理及其在合成生物学和调控细菌生命活动方面的应用.     

蓝藻光驱固碳合成糖类物质的技术研究进展*

糖类物质在食品、医药、日化、发酵领域有着广泛应用,对人类健康和社会发展有着重要意义。发展新型糖类物质合成技术有利于解决传统植物生物质“采集-炼制”产糖模式所面临的高成本、长周期、时空限制等风险和问题。蓝藻是一类重要的光自养微生物,也是极具潜力的新型微生物光合平台,发展蓝藻光驱固碳产糖技术有望实现二氧化碳向特定糖类产物的一站式定向转化,实现糖类物质合成的模式变革。糖类物质本身在蓝藻天然光合代谢网络中发挥重要作用,特别是卡尔文循环、糖原代谢、相容性物质代谢等几个重要生理模块的运转都是以不同糖类物质的转化来驱动的;而合成生物技术的发展又为光合产糖网络重塑和扩展注入了新的驱动力,在产品类型、合成模式及生产效率上显著提升了蓝藻光驱固碳产糖技术的发展和应用潜力。针对蓝藻光驱固碳产糖技术的发展应用,从模式、策略、产物等不同维度总结了相关进展和风险挑战,并对其未来前景和方向进行了展望。     

病原细菌效应蛋白靶向宿主细胞核研究进展*

细胞核是细胞遗传与代谢的控制中心,调控细胞对外界的响应、代谢、生长和分化等细胞活动。在细菌感染宿主细胞过程中,个别细菌来源的效应蛋白能够靶向进入宿主细胞核,影响细胞核内基因的转录、RNA剪切、DNA修复以及染色质重组等生命活动,将这些能够进入细胞核的细菌效应蛋白称之为核调节蛋白。对病原菌分泌的核调节蛋白进入宿主细胞核的方式,以及不同病原菌的核调节蛋白调控宿主细胞的生命过程进行归纳总结,从而为深入探究病原细菌感染宿主细胞的致病机理提供理论基础。     

光照调控红曲霉生长发育及聚酮类代谢产物合成的研究进展

光在自然界普遍存在并多层次全方位影响着生物的生长和代谢。红曲霉作为最早被人类驯化的微生物之一，被广泛应用于食品及医药领域。红曲霉通过蓝色、红色和绿色光感受器感知不同波长的光，而这些光通过复杂的信号通路影响菌体的生长和代谢。该文分别介绍蓝光、红光、绿光等对红曲霉生长发育以及红曲色素、桔霉素等聚酮类代谢产物合成的影响及相应光感受器的研究进展，提出了进一步探讨光调控红曲霉生长发育及聚酮类代谢产物合成的研究思路，为揭示光调控红曲霉生长发育及聚酮类代谢产物合成的机制提供参考。     

金针菇单孢菌株菌丝生长速度QTL定位分析

本研究以已经完成基因组测序的单核菌株“6-3”与“6-21”为出发菌株,配对后获得有锁状联合的异核菌株并进行出菇,收集担孢子,单孢分离获得90个菌株构成作图群体,对作图群体的每个菌株进行二代测序并测定菌丝在PDA培养基的生长速度。分析“6-3”与“6-21”两单核菌株的SNP,获得68 914个高质量SNP标记用于遗传连锁群分析,构建了14个遗传连锁群,总长度744.32cM,平均长度为53.17cM,标记间平均遗传距离为1.88cM。QTL分析获得一个控制菌丝生长速度的基因座qMGRP1-LG7,该基因座包含134个基因,富集了与物质代谢有关的通路和基因。     

上转换纳米颗粒介导的无线光遗传学技术的研究进展

光遗传学技术是结合基因工程和光学技术对生物体特定细胞进行精确调控的新兴生物技术,该技术可以特异性地兴奋或抑制靶神经元,成为解析介导特定行为神经环路的强有力的工具.传统技术依赖光纤,对脑组织有损伤且限制了动物的自由活动.新一代上转换纳米颗粒介导的无线光遗传学技术,借助近红外光组织穿透相对深的特性,能够对啮齿类动物脑组织深层核团进行无线调控,克服了传统技术中埋置光纤存在的缺陷.本文总结了上转换纳米颗粒介导的无线光遗传学技术的发展历程及现状,比较分析了这类无线光遗传学技术的优缺点,最后对该技术面临的挑战及未来前景进行了分析和展望.     

LOV to BLUF: flavoprotein contributions to the optogenetic toolkit

Optogenetics is an emerging field that combines optical and genetic approaches to non-invasively interfere with cellular events with exquisite spatiotemporal control. Although it arose originally from neuroscience, optogenetics is widely applicable to the study of many different biological systems and the range of applications arising from this technology continues to increase. Moreover, the repertoire of light-sensitive proteins used for devising new optogenetic tools is rapidly expanding. Light, Oxygen, or Voltage sensing (LOV) and Blue-Light-Utilizing flavin adenine dinucleotide (FAD) (BLUF) domains represent new contributors to the optogenetic toolkit. These small (100-140-amino acids) flavoprotein modules are derived from plant and bacterial photoreceptors that respond to UV-A/blue light. In recent years, considerable progress has been made in uncovering the photoactivation mechanisms of both LOV and BLUF domains. This knowledge has been applied in the design of synthetic photoswitches and fluorescent reporters with applications in cell biology and biotechnology. In this review, we summarize the photochemical properties of LOV and BLUF photosensors and highlight some of the recent advances in how these flavoproteins are being employed to artificially regulate and image a variety of biological processes.     

Engineering Strategy and Vector Library for the Rapid Generation of Modular Light-Controlled Protein–Protein Interactions

Optogenetics enables the spatio-temporally precise control of cell and animal behavior. Many optogenetic tools are driven by light-controlled protein–protein interactions (PPIs) that are repurposed from natural light-sensitive domains (LSDs). Applying light-controlled PPIs to new target proteins is challenging because it is difficult to predict which of the many available LSDs, if any, will yield robust light regulation. As a consequence, fusion protein libraries need to be prepared and tested, but methods and platforms to facilitate this process are currently not available. Here, we developed a genetic engineering strategy and vector library for the rapid generation of light-controlled PPIs. The strategy permits fusing a target protein to multiple LSDs efficiently and in two orientations. The public and expandable library contains 29 vectors with blue, green or red light-responsive LSDs, many of which have been previously applied ex vivo and in vivo. We demonstrate the versatility of the approach and the necessity for sampling LSDs by generating light-activated caspase-9 (casp9) enzymes. Collectively, this work provides a new resource for optical regulation of a broad range of target proteins in cell and developmental biology.     

Optogenetic delivery of trophic signals in a genetic model of Parkinson’s disease

Optogenetics has been harnessed to shed new mechanistic light on current and future therapeutic strategies. This has been to date achieved by the regulation of ion flow and electrical signals in neuronal cells and neural circuits that are known to be affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and are implicated in degenerative disorders, has never been demonstrated in an animal model of disease. Here, we reengineered the human and Drosophila melanogaster REarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1^B9 flies that exhibit loss of PTEN-induced putative kinase 1 (PINK1), a kinase associated with familial Parkinson’s disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRET via the PI3K/NF-кB pathway. Our results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to inspire novel strategies towards a spatio-temporal regulation of tissue repair.     

光遗传学工具的开发及其应用研究

细胞所处微环境的动态变化对细胞分化、细胞信号通路、个体生长以及疾病等有很大影响。光遗传学技术利用基因编码蛋白质表达并结合光控的手段为动态调控细胞信号通路、细胞定位和基因表达等方面提供了一种全新、无损、可逆、非侵入、时空特异性的研究手段。文中总结了光遗传学元件的类型以及涉及的细胞信号通路,并探讨了光控细胞信号通路的应用与未来发展前景。     

A High Speed Detection Platform Based on Surface-Enhanced Raman Scattering for Monitoring Antibiotic-Induced Chemical Changes in Bacteria Cell Wall

Rapid and accurate diagnosis for pathogens and their antibiotic susceptibility is critical for controlling bacterial infections. Conventional methods for determining bacterium''s sensitivity to antibiotic depend mostly on measuring the change of microbial proliferation in response to the drug. Such “biological assay” inevitably takes time, ranging from days for fast-growing bacteria to weeks for slow-growers. Here, a novel tool has been developed to detect the “chemical features” of bacterial cell wall that enables rapid identification of drug resistant bacteria within hours. The surface-enhanced Raman scattering (SERS) technique based on our newly developed SERS-active substrate was applied to assess the fine structures of the bacterial cell wall. The SERS profiles recorded by such a platform are sensitive and stable, that could readily reflect different bacterial cell walls found in Gram-positive, Gram-negative, or mycobacteria groups. Moreover, characteristic changes in SERS profile were noticed in the drug-sensitive bacteria at the early period (i.e., ∼1 hr) of antibiotic exposure, which could be used to differentiate them from the drug-resistant ones. The SERS-based diagnosis could be applied to a single bacterium. The high-speed SERS detection represents a novel approach for microbial diagnostics. The single-bacterium detection capability of SERS makes possible analyses directly on clinical specimen instead of pure cultured bacteria.     

Engineering rhodopsins’ activation spectra using a FRET-based approach

In the past decade, optogenetics has become a nearly ubiquitous tool in neuroscience because it enables researchers to manipulate neural activity with high temporal resolution and genetic specificity. Rational engineering of optogenetic tools has produced channelrhodopsins with a wide range of kinetics and photocurrent magnitude. Genome mining for previously unidentified species of rhodopsin has uncovered optogenetic tools with diverse spectral sensitivities. However, rational engineering of a rhodopsin has thus far been unable to re-engineer spectral sensitivity while preserving full photocurrent. Here, we developed and characterized ChroME-mTFP, a rhodopsin-fluorescent protein fusion that drives photocurrent through Förster resonance energy transfer (FRET). This FRET-opsin mechanism artificially broadened the activation spectrum of the blue-green-light-activated rhodopsin ChroME by approximately 50 nm, driving higher photocurrent at blue-shifted excitation wavelengths without sacrificing kinetics. The excitation spectra’s increase at short wavelengths enabled us to optogenetically excite neurons at lower excitation powers with shorter wavelengths of light. Increasing this rhodopsin’s sensitivity to shorter, bluer wavelengths pushes it toward dual-channel, crosstalk-free optogenetic stimulation and imaging with green-light-activated sensors. However, this iteration of FRET-opsin suffers from some imaging-light-induced photocurrent crosstalk from green or yellow light due to maintained, low-efficiency excitation at longer wavelengths.     

A photoconversion model for full spectral programming and multiplexing of optogenetic systems

Optogenetics combines externally applied light signals and genetically engineered photoreceptors to control cellular processes with unmatched precision. Here, we develop a mathematical model of wavelength‐ and intensity‐dependent photoconversion, signaling, and output gene expression for our two previously engineered light‐sensing Escherichia coli two‐component systems. To parameterize the model, we develop a simple set of spectral and dynamical calibration experiments using our recent open‐source “Light Plate Apparatus” device. In principle, the parameterized model should predict the gene expression response to any time‐varying signal from any mixture of light sources with known spectra. We validate this capability experimentally using a suite of challenging light sources and signals very different from those used during the parameterization process. Furthermore, we use the model to compensate for significant spectral cross‐reactivity inherent to the two sensors in order to develop a new method for programming two simultaneous and independent gene expression signals within the same cell. Our optogenetic multiplexing method will enable powerful new interrogations of how metabolic, signaling, and decision‐making pathways integrate multiple input signals.