BioDesign Research

Fungi are nature's recyclers, allowing for ecological nutrient cycling and, in turn, the continuation of life on Earth. Some fungi inhabit the human microbiome where they can provide health benefits, while others are opportunistic pathogens that can cause disease. Yeasts, members of the fungal kingdom, have been domesticated by humans for the production of beer, bread, and, recently, medicine and chemicals. Still, the great untapped potential exists within the diverse fungal kingdom. However, many yeasts are intractable, preventing their use in biotechnology or in the development of novel treatments for pathogenic fungi. Therefore, as a first step for the domestication of new fungi, an efficient DNA delivery method needs to be developed. Here, we report the creation of superior conjugative plasmids and demonstrate their transfer via conjugation from bacteria to 7 diverse yeast species including the emerging pathogen Candida auris. To create our superior plasmids, derivatives of the 57 kb conjugative plasmid pTA-Mob 2.0 were built using designed gene deletions and insertions, as well as some unintentional mutations. Specifically, a cluster mutation in the promoter of the conjugative gene traJ had the most significant effect on improving conjugation to yeasts. In addition, we created Golden Gate assembly-compatible plasmid derivatives that allow for the generation of custom plasmids to enable the rapid insertion of designer genetic cassettes. Finally, we demonstrated that designer conjugative plasmids harboring engineered restriction endonucleases can be used as a novel antifungal agent, with important applications for the development of next-generation antifungal therapeutics.

Ryan R. Cochrane

Arina Shrestha

Mariana M. Severo de Almeida

Michelle Agyare-Tabbi

Stephanie L. Brumwell

Samir Hamadache

Jordyn S. Meaney

Daniel P. Nucifora

Henry Heng Say

Jehoshua Sharma

Maximillian P. M. Soltysiak

Cheryl Tong

Katherine Van Belois

Emma J. L. Walker

Marc-André Lachance

Gregory B. Gloor

David R. Edgell

Rebecca S. Shapiro

Bogumil J. Karas

Superior Conjugative Plasmids Delivered by Bacteria to Diverse Fungi

细菌传递给多种真菌的优越接合质粒

Maltose is a naturalα-(1,4)-linked disaccharide with wide applications in food industries and microbial fermentation. However, maltose has scarcely been used forin vitrobiosynthesis, possibly because its phosphorylation by maltose phosphorylase (MP) yieldsβ-glucose 1-phosphate (β-G1P) that cannot be utilized byα-phosphoglucomutase (α-PGM) commonly found inin vitrosynthetic enzymatic biosystems previously constructed by our group. Herein, we designed anin vitrosynthetic enzymatic reaction module comprised of MP,β-phosphoglucomutase (β-PGM), and polyphosphate glucokinase (PPGK) for the stoichiometric conversion of each maltose molecule to two glucose 6-phosphate (G6P) molecules. Based on this synthetic module, we further constructed twoin vitrosynthetic biosystems to produce bioelectricity and fructose 1,6-diphosphate (FDP), respectively. The 14-enzyme biobattery achieved a Faraday efficiency of 96.4% and a maximal power density of 0.6 mW/cm2, whereas the 5-enzymein vitroFDP-producing biosystem yielded 187.0 mM FDP from 50 g/L (139 mM) maltose by adopting a fed-batch substrate feeding strategy. Our study not only suggests new application scenarios for maltose but also provides novel strategies for the high-efficient production of bioelectricity and value-added biochemicals.

Guowei Li

Xinlei Wei

Ranran Wu

Wei Zhou

Yunjie Li

Zhiguang Zhu

Chun You

BioDesign Res

Stoichiometric Conversion of Maltose for Biomanufacturing by
            <i>In Vitro</i>
            Synthetic Enzymatic Biosystems

麦芽糖的化学计量转化用于体外合成酶学生物制造系统

Plants are complex systems hierarchically organized and composed of various cell types. To understand the molecular underpinnings of complex plant systems, single-cell RNA sequencing (scRNA-seq) has emerged as a powerful tool for revealing high resolution of gene expression patterns at the cellular level and investigating the cell-type heterogeneity. Furthermore, scRNA-seq analysis of plant biosystems has great potential for generating new knowledge to inform plant biosystems design and synthetic biology, which aims to modify plants genetically/epigenetically through genome editing, engineering, or re-writing based on rational design for increasing crop yield and quality, promoting the bioeconomy and enhancing environmental sustainability. In particular, data from scRNA-seq studies can be utilized to facilitate the development of high-precision Build–Design–Test–Learn capabilities for maximizing the targeted performance of engineered plant biosystems while minimizing unintended side effects. To date, scRNA-seq has been demonstrated in a limited number of plant species, including model plants (e.g.,Arabidopsis thaliana), agricultural crops (e.g.,Oryza sativa), and bioenergy crops (e.g.,Populusspp.). It is expected that future technical advancements will reduce the cost of scRNA-seq and consequently accelerate the application of this emerging technology in plants. In this review, we summarize current technical advancements in plant scRNA-seq, including sample preparation, sequencing, and data analysis, to provide guidance on how to choose the appropriate scRNA-seq methods for different types of plant samples. We then highlight various applications of scRNA-seq in both plant systems biology and plant synthetic biology research. Finally, we discuss the challenges and opportunities for the application of scRNA-seq in plants.

Md Torikul Islam

Yang Liu

Md Mahmudul Hassan

Paul E. Abraham

Jean Merlet

Alice Townsend

Daniel Jacobson

C. Robin Buell

Gerald A. Tuskan

Xiaohan Yang

Advances in the Application of Single-Cell Transcriptomics in Plant Systems and Synthetic Biology

植物系统和合成生物学中单细胞转录组学应用的进展

Probiotics are the treasure of the microbiology fields. They have been widely used in the food industry, clinical treatment, and other fields. The equivocal health-promoting effects and the unknown action mechanism were the largest obstacles for further probiotic’s developed applications. In recent years, various genome editing techniques have been developed and applied to explore the mechanisms and functional modifications of probiotics. As important genome editing tools, CRISPR-Cas systems that have opened new improvements in genome editing dedicated to probiotics. The high efficiency, flexibility, and specificity are the advantages of using CRISPR-Cas systems. Here, we summarize the classification and distribution of CRISPR-Cas systems in probiotics, as well as the editing tools developed on the basis of them. Then, we discuss the genome editing of probiotics based on CRISPR-Cas systems and the applications of the engineered probiotics through CRISPR-Cas systems. Finally, we proposed a design route for CRISPR systems that related to the genetically engineered probiotics.

Ling Liu

Shimaa Elsayed Helal

Nan Peng

CRISPR-Cas-Based Engineering of Probiotics

基于CRISPR-Cas的益生菌工程

Reducing crop loss to diseases is urgently needed to meet increasing food production challenges caused by the expanding world population and the negative impact of climate change on crop productivity. Disease-resistant crops can be created by expressing endogenous or exogenous genes of interest through transgenic technology. Nevertheless, enhanced resistance by overexpressing resistance-produced genes often results in adverse developmental affects. Upstream open reading frames (uORFs) are translational control elements located in the 5′untranslated region (UTR) of eukaryotic mRNAs and may repress the translation of downstream genes. To investigate the function of three uORFs from the 5′-UTR ofACCELERATED CELL 11(uORFsACD11), we develop a fluorescent reporter system and find uORFsACD11function in repressing downstream gene translation. Individual or simultaneous mutations of the three uORFsACD11lead to repression of downstream translation efficiency at different levels. Importantly, uORFsACD11-mediated translational inhibition is impaired upon recognition of pathogen attack of plant leaves. When coupled with thePATHOGENESIS-RELATED GENE 1(PR1) promoter, the uORFsACD11cassettes can upregulate accumulation ofArabidopsis thalianaLECTIN RECEPTOR KINASE-VI.2 (AtLecRK-VI.2) during pathogen attack and enhance plant resistance toPhytophthora capsici. These findings indicate that the uORFsACD11cassettes can be a useful toolkit that enables a high level of protein expression during pathogen attack, while for ensuring lower levels of protein expression at normal conditions.

Gan Ai

Jin Liu

Xiaowei Fu

Tianli Li

Hai Zhu

Ying Zhai

Chuyan Xia

Weiye Pan

Jialu Li

Maofeng Jing

Danyu Shen

Ai Xia

Daolong Dou

Making Use of Plant uORFs to Control Transgene Translation in Response to Pathogen Attack

利用植物uORFs调控转基因翻译以响应病原体攻击

Protein engineering aimed at increasing temperature tolerance through iterative mutagenesis and high-throughput screening is often labor-intensive. Here, we developed a deep evolution (DeepEvo) strategy to engineer protein high-temperature tolerance by generating and selecting functional sequences using deep learning models. Drawing inspiration from the concept of evolution, we constructed a high-temperature tolerance selector based on a protein language model, acting as selective pressure in the high-dimensional latent spaces of protein sequences to enrich those with high-temperature tolerance. Simultaneously, we developed a variant generator using a generative adversarial network to produce protein sequence variants containing the desired function. Afterward, the iterative process involving the generator and selector was executed to accumulate high-temperature tolerance traits. We experimentally tested this approach on the model protein glyceraldehyde 3-phosphate dehydrogenase, obtaining 8 variants with high-temperature tolerance from just 30 generated sequences, achieving a success rate of over 26%, demonstrating the high efficiency of DeepEvo in engineering protein high-temperature tolerance.

Huanyu Chu

Zhenyang Tian

Lingling Hu

Hejian Zhang

Hong Chang

Jie Bai

Dingyu Liu

Lina Lu

Jian Cheng

Huifeng Jiang

High-Temperature Tolerance Protein Engineering through Deep Evolution

高温耐受性蛋白质工程通过深度进化

Bacterial infections are a major threat to the human healthcare system worldwide, as antibiotics are becoming less effective due to the emergence of multidrug-resistant strains. Therefore, there is a need to explore nontraditional antimicrobial alternatives to support rapid interventions and combat the spread of pathogenic bacteria. New nonantibiotic approaches are being developed, many of them at the interface of physics, nanotechnology, and microbiology. While physical factors (e.g., pressure, temperature, and ultraviolet light) are typically used in the sterilization process, nanoparticles and phages (bacterial viruses) are also applied to combat pathogenic bacteria. Particularly, phage-based therapies are rising due to the unparalleled specificity and high bactericidal activity of phages. Despite the success of phages mostly as compassionate use in clinical cases, some drawbacks need to be addressed, mainly related to their stability, bioavailability, and systemic administration. Combining phages with nanoparticles can improve their performance in vivo. Thus, the combination of nanotechnology and phages might provide tools for the rapid and accurate detection of bacteria in biological samples (diagnosis and typing), and the development of antimicrobials that combine the selectivity of phages with the efficacy of targeted therapy, such as photothermal ablation or photodynamic therapies. In this review, we aim to provide an overview of how phage-based nanotechnology represents a step forward in the fight against multidrug-resistant bacteria.

Marco Pardo-Freire

Pilar Domingo-Calap

Phages and Nanotechnology: New Insights against Multidrug-Resistant Bacteria

噬菌体与纳米技术：对抗多重耐药细菌的新见解

Globally, agriculture depends on industrial nitrogen fertilizer to improve crop growth. Fertilizer production consumes fossil fuels and contributes to environmental nitrogen pollution. A potential solution would be to harness nitrogenases—enzymes capable of converting atmospheric nitrogen N2to NH3in ambient conditions. It is therefore a major goal of synthetic biology to engineer functional nitrogenases into crop plants, or bacteria that form symbiotic relationships with crops, to support growth and reduce dependence on industrially produced fertilizer. This review paper highlights recent work toward understanding the functional requirements for nitrogenase expression and manipulating nitrogenase gene expression in heterologous hosts to improve activity and oxygen tolerance and potentially to engineer synthetic symbiotic relationships with plants.

Emily M. Bennett

James W. Murray

Mark Isalan

Engineering Nitrogenases for Synthetic Nitrogen Fixation: From Pathway Engineering to Directed Evolution

合成固氮酶工程：从途径工程到定向进化

ISSN	2097-2237, 2693-1257
h-index	10

出版信息	出版商: American Association for the Advancement of Science，出版周期: ，期刊类型: journal，开源期刊: 否
基本数据	创刊年份: 2019，原创研究文献占比: ，自引率:， Gold OA占比:

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Superior Conjugative Plasmids Delivered by Bacteria to Diverse Fungi

Stoichiometric Conversion of Maltose for Biomanufacturing by In Vitro Synthetic Enzymatic Biosystems

Advances in the Application of Single-Cell Transcriptomics in Plant Systems and Synthetic Biology

CRISPR-Cas-Based Engineering of Probiotics

Making Use of Plant uORFs to Control Transgene Translation in Response to Pathogen Attack

Advances in the Application of Single-Cell Transcriptomics in Plant Systems and Synthetic Biology

High-Temperature Tolerance Protein Engineering through Deep Evolution

CRISPR-Cas-Based Engineering of Probiotics

Phages and Nanotechnology: New Insights against Multidrug-Resistant Bacteria

Engineering Nitrogenases for Synthetic Nitrogen Fixation: From Pathway Engineering to Directed Evolution