Tag Archives: SynBio

Biosynthesis of the antibiotic nonribosomal peptide penicillin in baker’s yeast

Ali R. Awan, Benjamin A. Blount, David J. Bell, William M. Shaw, Jack C.H. Ho, Robert M. McKiernan & Tom Ellis

Fungi are a valuable source of enzymatic diversity and therapeutic natural products including antibiotics. Here we engineer the baker’s yeast Saccharomyces cerevisiae to produce and secrete the antibiotic penicillin, a beta-lactam nonribosomal peptide, by taking genes from a filamentous fungus and directing their efficient expression and subcellular localization. Using synthetic biology tools combined with long-read DNA sequencing, we optimize productivity by 50-fold to produce bioactive yields that allow spent S. cerevisiae growth media to have antibacterial action against Streptococcus bacteria. This work demonstrates that S. cerevisiae can be engineered to perform the complex biosynthesis of multicellular fungi, opening up the possibility of using yeast to accelerate rational engineering of nonribosomal peptide antibiotics.

Read the full article in Nature Communications

Whole-cell biocatalysts by design

Baixue Lin and Yong Tao

Whole-cell biocatalysts provide unique advantages and have been widely used for the efficient biosynthesis of value-added fine and bulk chemicals, as well as pharmaceutically active ingredients. What is more, advances in synthetic biology and metabolic engineering, together with the rapid development of molecular genetic tools, have brought about a renaissance of whole-cell biocatalysis. These rapid advancements mean that whole-cell biocatalysts can increasingly be rationally designed. Genes of heterologous enzymes or synthetic pathways are increasingly being introduced into microbial hosts, and depending on the complexity of the synthetic pathway or the target products, they can enable the production of value-added chemicals from cheap feedstock. Metabolic engineering and synthetic biology efforts aimed at optimizing the existing microbial cell factories concentrate on improving heterologous pathway flux, precursor supply, and cofactor balance, as well as other aspects of cellular metabolism, to enhance the efficiency of biocatalysts. In the present review, we take a critical look at recent developments in whole-cell biocatalysis, with an emphasis on strategies applied to designing and optimizing the organisms that are increasingly modified for efficient production of chemicals.

Read the full article in Microbial Cell Factories

Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli

Francesca Zerbini, Ilaria Zanella, Davide Fraccascia, Enrico König, Carmela Irene, Luca F. Frattini, Michele Tomasi, Laura Fantappiè, Luisa Ganfini, Elena Caproni, Matteo Parri, Alberto Grandi and Guido Grandi

The exploitation of the CRISPR/Cas9 machinery coupled to lambda (λ) recombinase-mediated homologous recombination (recombineering) is becoming the method of choice for genome editing in E. coli. First proposed by Jiang and co-workers, the strategy has been subsequently fine-tuned by several authors who demonstrated, by using few selected loci, that the efficiency of mutagenesis (number of mutant colonies over total number of colonies analyzed) can be extremely high (up to 100%). However, from published data it is difficult to appreciate the robustness of the technology, defined as the number of successfully mutated loci over the total number of targeted loci. This information is particularly relevant in high-throughput genome editing, where repetition of experiments to rescue missing mutants would be impractical. This work describes a “brute force” validation activity, which culminated in the definition of a robust, simple and rapid protocol for single or multiple gene deletions.

Read the full article Microbial Cell Factories

Microbial response to environmental stresses: from fundamental mechanisms to practical applications

Ningzi Guan, Jianghua Li, Hyun-dong Shin, Guocheng Du, Jian Chen, Long Liu

Environmental stresses are usually active during the process of microbial fermentation and have significant influence on microbial physiology. Microorganisms have developed a series of strategies to resist environmental stresses. For instance, they maintain the integrity and fluidity of cell membranes by modulating their structure and composition, and the permeability and activities of transporters are adjusted to control nutrient transport and ion exchange. Certain transcription factors are activated to enhance gene expression, and specific signal transduction pathways are induced to adapt to environmental changes. Besides, microbial cells also have well-established repair mechanisms that protect their macromolecules against damages inflicted by environmental stresses. Oxidative, hyperosmotic, thermal, acid, and organic solvent stresses are significant in microbial fermentation. In this review, we summarize the modus operandi by which these stresses act on cellular components, as well as the corresponding resistance mechanisms developed by microorganisms. Then, we discuss the applications of these stress resistance mechanisms on the production of industrially important chemicals. Finally, we prospect the application of systems biology and synthetic biology in the identification of resistant mechanisms and improvement of metabolic robustness of microorganisms in environmental stresses.

Read the full article in Applied Microbiology and Biotechnology

Development of a fast and easy method for Escherichia coli genome editing with CRISPR/Cas9

Dongdong Zhao, Shenli Yuan, Bin Xiong, Hongnian Sun, Lijun Ye, Jing Li, Xueli Zhang and Changhao Bi

Microbial genome editing is a powerful tool to modify chromosome in way of deletion, insertion or replacement, which is one of the most important techniques in metabolic engineering research. The emergence of CRISPR/Cas9 technique inspires various genomic editing methods. Read more

Easy regulation of metabolic flux in Escherichia coli using an endogenous type I-E CRISPR-Cas system

Yizhao Chang, Tianyuan Su, Qingsheng Qi and Quanfeng Liang

Clustered regularly interspaced short palindromic repeats interference (CRISPRi) is a recently developed powerful tool for gene regulation. In Escherichia coli, the type I CRISPR system expressed endogenously shall be easy for internal regulation without causing metabolic burden in compared with the widely used type II system, which expressed dCas9 as an additional plasmid.

Read the full article in Microbial Cell Factories

Synthetic Genomics team engineers Vmax™, an advantaged next-generation host organism for a wide range of biotechnology applications

Optimized system has potential to replace the workhorse E. coli by increasing speed and efficiency of protein production and cloning

Researchers from Synthetic Genomics, Inc. (SGI) announced today the development and extensive engineering of Vibrio natriegens into a next-generation biotechnology host organism Vmax™. Looking to accelerate the pace of discovery and the path to sustainable solutions, the team set out to develop a novel bacterial host that will drastically reduce the amount of time scientists spend on each experiment and workflow and to enhance productivity of the resulting new host.

After screening for the fastest-growing strain and optimizing methods for introducing DNA into those cells at high efficiencies, the team developed genome engineering tools to improve the performance of Vmax™ for common biotech applications, namely, recombinant protein expression and molecular cloning. These breakthroughs build on expertise gleaned during the creation of the first synthetic cell and first minimal cell and again position SGI at the forefront of synthetic biology.

The paper describing this work is the first peer-reviewed publication of its kind and was published online today in Nature Methods by Matthew T. Weinstock, Eric D. Hesek, Christopher M. Wilson, and Daniel G. Gibson.

“This work provides a game-changing alternative to E. coli, the organism that has been a laboratory staple for decades, and again highlights the rapid and innovative synthetic biology expertise we’ve developed at SGI. We are in the process of designing and synthesizing new Vmax™ cells that operate at even higher efficiencies and productivity as we move toward a next-generation host for protein production,” said Daniel Gibson, Vice President, DNA Technologies, SGI.

Commenting on the origin of the research, Todd Peterson, Chief Technology Officer at SGI stated, “Despite the known drawbacks and shortcomings, scientists have been necessitated to use E. coli as a laboratory host primarily because there have been no suitable alternatives. We deployed our synthetic biology expertise to develop a new host strain that will drastically improve upon the traditional methods and tools.”

Typical cloning projects using E. coli competent cells span several days starting from the time a cloning process is initiated to the time plasmid DNA is prepared. Cloning strategies employing Vmax™ developed by the SGI team shorten that time to as little as one day.

The advancements described by the team set the stage for commercialization of these next-generation cells for cloning and protein expression by SGI in the coming months. Vmax™ is compatible with most kits, reagents, growth medium, vectors, and procedures already entrenched in laboratories. Making these cells commercially available will accelerate the pace of global biotechnological research, making a far-reaching and lasting impact toward genetic exploration and discovery worldwide.

About Synthetic Genomics Inc.
Synthetic Genomics Inc. (SGI), located in La Jolla, CA, is a leader in the fields of synthetic biology and synthetic genomics, advancing genomics to better life. SGI applies its intellectual property in this rapidly evolving field to design and build biological systems solving global sustainability challenges. SGI serves three end markets: research, bioproduction, and applied products. The company’s research offerings, commercialized through its subsidiary SGI-DNA, are revolutionizing science and medicine with next-generation genomic solutions, including the world’s first DNA printer. SGI applies its integrated synthetic biology capabilities to reinvent bio-based production by improving existing production systems and developing novel, optimized production hosts. SGI develops its applied products, typically in partnership with leading global organizations, across a variety of industries including sustainable bio-fuels, sustainable crops, nutritional supplements, vaccines, and transplantable organs.

SGI-DNA, a wholly owned subsidiary of Synthetic Genomics, Inc (SGI), is responsible for all commercial aspects of SGI’s synthetic DNA business and focuses on strategic business relationships with both academic and commercial researchers. Building on the scientific advancements and breakthroughs from leading scientists such as J. Craig Venter, Ham Smith, Clyde Hutchison, Dan Gibson and their teams, SGI-DNA utilizes unique and proprietary DNA technologies to produce complex synthetic genes and reagents. SGI-DNA also offers the BioXp™ 3200 System, the world’s first DNA printer, in addition to a comprehensive suite of genomic services, including whole genome sequencing, library design, bioinformatics services, and reagent kits to enable synthetic biology.