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Genome reprogramming for synthetic biology

Kylie Standage-Beier,Xiao Wang

《化学科学与工程前沿（英文）》 2017年第11卷第1期页码 37-45 doi: 10.1007/s11705-017-1618-2

摘要： The ability to go from a digitized DNA sequence to a predictable biological function is central to synthetic biology. Genome engineering tools facilitate rewriting and implementation of engineered DNA sequences. Recent development of new programmable tools to reengineer genomes has spurred myriad advances in synthetic biology. Tools such as clustered regularly interspace short palindromic repeats enable RNA-guided rational redesign of organisms and implementation of synthetic gene systems. New directed evolution methods generate organisms with radically restructured genomes. These restructured organisms have useful new phenotypes for biotechnology, such as bacteriophage resistance and increased genetic stability. Advanced DNA synthesis and assembly methods have also enabled the construction of fully synthetic organisms, such as J. Craig Venter Institute (JCVI)-syn 3.0. Here we summarize the recent advances in programmable genome engineering tools.

关键词： CRISPR genome engineering synthetic biology rational design

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Renewable biosynthesis of isoprene from wastewater through a synthetic biology approach: the role of

《环境科学与工程前沿（英文）》 2023年第18卷第3期 doi: 10.1007/s11783-024-1788-3

摘要：

● Engineered E. coli can use wastewater as the only feedstock to product isoprene.

关键词： Wastewater Resource recovery Genetic engineering Biosynthesis Isoprene

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Designer enzyme for green materials innovation: Lactate-polymerizing enzyme as a key catalyst

Seiichi Taguchi

《化学科学与工程前沿（英文）》 2017年第11卷第1期页码 139-142 doi: 10.1007/s11705-017-1636-0

摘要： Establishment of the regeneratable whole-cell catalyst platform for the?production of biobased polymeric materials is a?typical topic of synthetic biology. In this commentary, discovery story of a “lactate-polymerizing enzyme” (LPE)?and LPE-based?achievements for creating a new variety of polyesters with incorporated unnatural monomers are presented. Besides the importance of microbial platform itself is discussed referring to the “ballooning”- .

关键词： synthetic biology enzyme evolutionary engineering polyhydroxyalkanoate

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Improving prodeoxyviolacein production via multiplex SCRaMbLE iterative cycles

Juan Wang, Bin Jia, Zexiong Xie, Yunxiang Li, Yingjin Yuan

《化学科学与工程前沿（英文）》 2018年第12卷第4期页码 806-814 doi: 10.1007/s11705-018-1739-2

摘要：

The synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) system has been used to improve prodeoxyviolacein (PDV) production in haploid yeast containing chromosome synV. To rapidly and continuously generate genome diversification with the desired phenotype, the multiplex SCRaMbLE iterative cycle strategy has been developed for the screening of high PDV production strains. Whole-genome sequencing analysis reveals large duplications, deletions, and even the whole genome duplications. The deletion of YER151C is proved to be responsible for the increase. This study demonstrates that artificial DNA rearrangement can be used to accelerate microbial evolution and the production of biobased chemicals.

关键词： synthetic biology genome rearrangement prodeoxyviolacein SCRaMbLE Saccharomyces cerevisiae

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一种可实现合成生物传感器现场部署的增材制造方法 Article

Daniel Wolozny, John R. Lake, Paul G. Movizzo, Zhicheng Long, Warren C. Ruder

《工程（英文）》 2019年第5卷第1期页码 173-180 doi: 10.1016/j.eng.2018.12.001

摘要：

合成生物学工具可用于设计活体生物传感器，报告目标分析物的存在。虽然这些工程细胞生物传感器在实验室外具有许多潜在应用，但由于它们属于转基因生物（genetically modified organism，GMO），通常被认为具有危险性。因此，如何在实验室外使用转基因生物的同时，降低将其释放到环境中的风险就至关重要。本文描述了一种包含合成生物电路的生物传感系统。含有该系统的大肠杆菌（Escherichia coli）被置于一个特制的3D 打印的试管盖内。这些转基因生物能够检测到一种条件致病菌铜绿假单胞菌（Pseudomonas aeruginosa）的化学群体信号。在该设备中，活体生物传感器可以在不接触环境的情况下，接触感兴趣的样本。细胞可以在培养管内进行现场可视化分析，也可以送回实验室进行进一步分析。许多生物传感器缺乏现场部署所需的多功能性，由于
缺乏资源和装置，许多疾病可能无法诊断。我们的生物检测设备利用3D 打印技术，为现场部署活体生物传感器制造了一种便携式、模块化和廉价的设备。

关键词：合成生物学增材制造生物传感器

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Mobile CRISPR-Cas9 based anti-phage system in E. coli

《化学科学与工程前沿（英文）》 2022年第16卷第8期页码 1281-1289 doi: 10.1007/s11705-022-2141-7

摘要： Escherichia coli is one of the most important microbial cell factories, but infection by bacteriophages in the environment may have a huge impact on its application in industrial production. Here, we developed a mobile CRISPR-Cas9 based anti-phage system for bacteriophages defense in E. coli. Two conjugative plasmids pGM1 (phosphoglucomutase 1) and pGM2 carrying one and two guide RNAs, respectively, were designed to defend against a filamentous phage. The results showed that the pGM1 and pGM2 could decrease the phage infection rate to 1.6% and 0.2% respectively in infected cells. For preventing phage infection in E. coli, the pGM2 decreased the phage infection rate to 0.1%, while pGM1 failed to block phage infection. Sequence verification revealed that point mutations in protospacer or protospacer adjacent motif sequences of the phage genome caused loss of the defense function. These results support the potential application of MCBAS in E. coli cell factories to defend against phage infections.

关键词： phage infections anti-phage CRISPR-Cas9 conjugative transfer synthetic biology

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Dendritic cell vaccines in cancer immunotherapy: from biology to translational medicine

Hongmei Xu, Xuetao Cao

《医学前沿（英文）》 2011年第5卷第4期页码 323-332 doi: 10.1007/s11684-011-0172-4

摘要：

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Synthetically engineered microbes reveal interesting principles of cooperation

Michael D. Dressler,Corey J. Clark,Chelsea A. Thachettu,Yasmine Zakaria,Omar Tonsi Eldakar,Robert P. Smith

《化学科学与工程前沿（英文）》 2017年第11卷第1期页码 3-14 doi: 10.1007/s11705-016-1605-z

摘要： Cooperation is ubiquitous in biological systems. However, if natural selection favors traits that confer an advantage to one individual over another, then helping others would be paradoxical. Nevertheless, cooperation persists and is critical in maintaining homeostasis in systems ranging from populations of bacteria to groupings of mammals. Developing an understanding of the dynamics and mechanisms by which cooperation operates is critical in understanding ecological and evolutionary relationships. Over the past decade, synthetic biology has emerged as a powerful tool to study social dynamics. By engineering rationally controlled and modulatable behavior into microbes, we have increased our overall understanding of how cooperation enhances, or conversely constrains, populations. Furthermore, it has increased our understanding of how cooperation is maintained within populations, which may provide a useful framework to influence populations by altering cooperation. As many bacterial pathogens require cooperation to infect the host and survive, the principles developed using synthetic biology offer promise of developing novel tools and strategies to treat infections, which may reduce the use of antimicrobial agents. Overall, the use of engineered cooperative microbes has allowed the field to verify existing, and develop novel, theories that may govern cooperative behaviors at all levels of biology.

关键词： synthetic biology engineered bacteria cooperation cheater quorum sensing

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微生物药物产业现状与发展趋势

于晴,黄婷婷,邓子新

《中国工程科学》 2021年第23卷第5期页码 69-78 doi: 10.15302/J-SSCAE-2021.05.009

摘要：

微生物来源的天然产物药物具有结构多样、活性优良等优点，临床应用潜力巨大。本研究系统分析了我国微生物药物产业发展现状，梳理了微生物种质资源利用、优良菌种筛选和发酵工艺优化、菌株工程化改造、新型微生物药物创制等技术趋势，助力我国破解产业瓶颈、加速产业升级。我国现代微生物药物产业已经具备了坚实的资源和技术基础，但与国际领先水平有一定的差距。本研究针对产业面临的机遇与挑战，从建立统筹创新的微生物医药大科学装置、加强微生物药物基础研究和技术自主研发、构建战略性人才引育系统、形成体系化产业激励政策等四个方面提出了发展建议。

关键词：微生物药物发展趋势生物活性微生物代谢合成生物学

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Engineering of

Ruizhao Wang,Xiaoli Gu,Mingdong Yao,Caihui Pan,Hong Liu,Wenhai Xiao,Ying Wang,Yingjin Yuan

《化学科学与工程前沿（英文）》 2017年第11卷第1期页码 89-99 doi: 10.1007/s11705-017-1628-0

摘要： The conversion of -carotene to astaxanthin is a complex pathway network, in which two steps of hydroxylation and two steps of ketolation are catalyzed by -carotene hydroxylase (CrtZ) and -carotene ketolase (CrtW) respectively. Here, astaxanthin biosynthesis pathway was constructed in by introducing heterologous CrtZ and CrtW into an existing high -carotene producing strain. Both genes and were codon optimized and expressed under the control of constitutive promoters. Through combinatorial expression of CrtZ and CrtW from diverse species, nine strains in dark red were visually chosen from thirty combinations. In all the selected strains, strain SyBE_Sc118060 with CrtW from DC263 and CrtZ from sp. strain PC-1 achieved the highest astaxanthin yield of 3.1 mg/g DCW. Protein phylogenetic analysis shows that the shorter evolutionary distance of CrtW is, the higher astaxanthin titer is. Further, when the promoter of in strain SyBE_Sc118060 was replaced from FBA1p to TEF1p, the astaxanthin yield was increased by 30.4% (from 3.4 to 4.5 mg/g DCW). In the meanwhile, 33.5-fold increase on transcription level and 39.1-fold enhancement on the transcriptional ratio of to were observed at early exponential phase in medium with 4% (w/v) glucose. Otherwise, although the ratio of to were increased at mid-, late-exponential phases in medium with 2% (w/v) glucose, the transcription level of both and were actually decreased during the whole time course, consequently leading to no significant improvement on astaxanthin production. Finally, through high cell density fed-batch fermentation using a carbon source restriction strategy, the production of astaxanthin in a 5-L bioreactor reached to 81.0 mg/L, which was the highest astaxanthin titer reported in yeast. This study provides a reference to greatly enhance desired compounds accumulation by employing the key enzyme(s) in microbes.

关键词： synthetic biology astaxanthin β-carotene hydroxylase β-carotene ketolase Saccharomyces cerevisiae

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一种使用隔离子RiboJ测量绝对定量蛋白表达量的方法 Article

余泓彬, 王铮, 许瀚月, 郭玖思, 马清格, 穆翔栩, 罗云孜

《工程（英文）》 2018年第4卷第6期页码 881-887 doi: 10.1016/j.eng.2018.09.012

摘要：

特定启动子的绝对定量蛋白表达量测定在分子生物学及合成生物学领域具有重要的意义。启动子强度一般由该启动子表达荧光蛋白的荧光强度表示。然而，对于特定启动子的绝对定量蛋白表达量测定方法目前仍不尽如人意。不同蛋白编码序列对启动子表达水平的影响及现有测定绝对定量蛋白表达量方法的复杂性，均为绝对定量蛋白表达量测定带来了挑战。我们在这里介绍了一种利用隔离子RiboJ和标准荧光曲线快速测定绝对定量蛋白表达量的方法，并通过模型对该方法进行了验证。使用这种方法，我们成功测定了Anderson启动子家族中9种组成型启动子的强度。本方法为代谢通路设计提供了更准确的数据，并为不同启动子强度的标准化提供了一种直接的方案。

关键词： RiboJ 启动子测定合成生物学

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工业5.0——仿生学和合成生物学的关联及内涵 Artical

Peter Sachsenmeier

《工程（英文）》 2016年第2卷第2期页码 225-229 doi: 10.1016/J.ENG.2016.02.015

摘要：

仿生学(模仿生物特殊本领的学科) 以及合成生物学，将和过去50年的硅芯片一样与工程开发、工业发展产生紧密联系。化学工业已经将白色生物技术应用于新工艺、新材料和资源的可持续利用中。合成生物学也已经应用到第二代生物燃料的发展中，并利用特制的微生物或生物制催化剂获取太阳能。而仿生学在制药、处理工程以及DNA存储领域的市场潜力是巨大的。“登月计划”(ProjectMoonshot) 已经明确把医疗和新材料作为关注点，一场始于美国的创造大量新分子的竞争已经开始。本文首先概述了当前的一些项目，接下来探讨了代码工程(code engineering) 及其影响，包括脱氧核糖核酸(DNA)人工合成、信号分子以及生物回路。除此之外，还探讨了涉及人造食物链和食物的仿生学前沿——合成代谢，以及原材料生物工程。这些研究将给生物学带来新思考。生物工程将和今天的数字化技术一样驱动创新。本文讨论了生物工程，特别是碳基生物燃料的应用和细胞饰变的技术与风险。大数据、分析学和海量存储将是未来的发展方向。虽然合成生物学在未来50年将和当今的数字化一样普遍且具有革新能力，但是目前它的应用和影响力还处在初级阶段。本文采用了将生物工程发展分为五个阶段(DNA分析、生物回路、最小基因组、原始细胞、异源生物学)的普遍分类方法，阐述了其对安全与保障、工业发展以及生物工程和生物技术作为跨学科领域发展的影响，同时讨论了伦理问题及公众对仿生学和合成生物学结果的公众讨论的重要性。

关键词：仿生学合成生物学生物工程生物传感器生物燃料生物武器虚拟进化原始细胞异种细胞经济意义工业5.0 德国中国

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Construction, characterization and application of a genome-wide promoter library in

Ting Yuan,Yakun Guo,Junkai Dong,Tianyi Li,Tong Zhou,Kaiwen Sun,Mei Zhang,Qingyu Wu,Zhen Xie,Yizhi Cai,Limin Cao,Junbiao Dai

《化学科学与工程前沿（英文）》 2017年第11卷第1期页码 107-116 doi: 10.1007/s11705-017-1621-7

摘要： Promoters are critical elements to control gene expression but could behave differently under various growth conditions. Here we report the construction of a genome-wide promoter library, in which each native promoter in was cloned upstream of a yellow fluorescent protein (YFP) reporter gene. Nine libraries were arbitrarily defined and assembled in bacteria. The resulting pools of promoters could be prepared and transformed into a yeast strain either as centromeric plasmids or integrated into a genomic locus upon enzymatic treatment. Using fluorescence activated cell sorting, we classified the yeast strains based on YFP fluorescence intensity and arbitrarily divided the entire library into 12 bins, representing weak to strong promoters. Several strong promoters were identified from the most active bins and their activities were assayed under different growth conditions. Finally, these promoters were applied to drive the expression of genes in xylose utilization to improve fermentation efficiency. Together, this library could provide a quick solution to identify and utilize desired promoters under user-defined growth conditions.

关键词： synthetic biology yeast promoter activity metabolic engineering xylose utilization

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Rational Design of and Mechanism Insight into an Efficient Antifreeze Peptide for Cryopreservation

Haishan Qi,Yihang Gao,Lin Zhang,Zhongxin Cui,Xiaojie Sui,Jianfan Ma,Jing Yang,Zhiquan Shu,Lei Zhang,

《工程（英文）》 doi: 10.1016/j.eng.2023.01.015

摘要： The development of effective antifreeze peptides to control ice growth has attracted a significant amount of attention yet still remains a great challenge. Here, we propose a novel design method based on an in-depth investigation of repetitive motifs in various ice-binding proteins (IBPs) with an evolution analysis. In this way, several peptides with notable antifreeze activity were developed. In particular, a designed antifreeze peptide named AVD exhibits ideal ice recrystallization inhibition (IRI), solubility, and biocompatibility, making it suitable for use as a cryoprotective agent (CPA). A mutation analysis and molecular dynamics (MD) simulations indicated that the Thr6 and Asn8 residues of the AVD peptide are fundamental to its ice-binding capacity, while the Ser18 residue can synergistically enhance their interaction with ice, revealing the antifreeze mechanism of AVD. Furthermore, to demonstrate the cryoprotection potential of AVD, the peptide was successfully employed for the cryopreservation of various cells, which demonstrated significant post-freezing cell recovery. This work opens up a new avenue for designing antifreeze materials and provides peptide-based functional modules for synthetic biology.

关键词： Antifreeze peptides Evolution analysis Ice recrystallization inhibition Molecular dynamics simulation Cryopreservation Synthetic biology

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利用异源酶组合构建酿酒酵母中咖啡酸的生物合成 Article

刘岚清, 刘宏, 张伟, 姚明东, 李炳志, 刘夺, 元英进

《工程（英文）》 2019年第5卷第2期页码 287-295 doi: 10.1016/j.eng.2018.11.029

摘要：

微生物中构建植物源天然产物的生物合成面临着许多挑战，尤其是当需要表达与激活植物源细胞色素P450酶的时候。通过从几种细菌中筛选HpaB和HpaC两种酶，本文在酿酒酵母中构建了具有活性的4-羟基苯乙酸3-羟化酶（4 HPA3H），它可以发挥植物源细胞色素P450酶的相似作用，用于生产咖啡酸。在与一个共同的酪氨酸氨裂解酶（TAL）协同作用下，不同的异源HpaB酶和HpaC酶组合在将底物L-酪氨酸转化为目标产物咖啡酸上展现出不同能力。以铜绿假单胞菌的HpaB酶和肠沙门氏菌的HpaC酶的异源酶组合可生产最高咖啡酸产量，摇瓶培养下可达到（289.4 ± 4.6）mg·L⁻¹。酵母底盘细胞与异源酶的相容性得到了有效的改善，咖啡酸的产量比初始值提高了40倍。铜绿假单胞菌HpaB酶中黄素腺嘌呤二核苷酸（FAD）结合域周围的6个关键氨基酸残基与其他细菌来源的HpaB酶有明显区别，可能在影响酶活性方面起关键作用。综上，我们建立了一种有效的方法来构建高效的酵母系统用于合成非天然羟基苯丙烷类化合物。

关键词：酿酒酵母咖啡酸异源酶细胞色素P450酶合成生物学