Synthetic metagenomics: transforming digital information into biological functions
Advantage
· Simultaneous detection of clones and quantitative fluorescent labeling - using phenotypic screening clones
· From loading and coating to picking up fully automated workflows
· From sample coating to picking, copying and rearranging records and tracking sample data
· Accurately pick more than 30,000 screened clones per day, with >98% efficiency
Researchers at the US Department of Energy's Joint Genome Research Institute ( DoE JGI ) have developed synthetic metagenomics technologies based on high-throughput screening techniques. This technology can transform the vast amount of digital information obtained by the latest generation of sequencing into tangible, observable organisms.
Introduction
Next-generation sequencing technologies have discovered millions of new genes, including all known pathways and functional activities. These novel genes include a large number of novel bioenzyme genes with potential applications in energy and environmental technologies, including basic research and applied biology techniques. One of the biggest challenges is how to convert large amounts of digital information (sequences, bioinformatics, chemical informatics) into observable, functionally active organisms (enzymatic activity, cell death, protein expression, etc.).
DoE JGI has developed synthetic metagenomics technology to overcome this challenge. Genes and pathways are synthesized in a template-independent manner, transforming digital information into biochemical molecules quickly and cost-effectively.
A major bottleneck for the commercial production of cellulosic biofuels is the lack of stable and highly active enzymes for the conversion of biomass to glucose. Ionic liquids can be used to pretreat dissolved cellulose to increase the content of fermentable sugars such as glucose. In order to find suitable industrially relevant enzymes, synthetic metagenomic methods are used. The Department of Energy's Joint Bioenergy Research Institute (JBEI) has designed enzymes for special industrial applications that have the following conditions: (i) functional enzyme activity at 70 degrees; (ii) stable at pH 4.5 (iii) Tolerant to ionic solutions. At the beginning of this project, no enzyme was able to have sufficient activity under these conditions.
Figure 1. Synthetic metagenomics process: a metagenomics research process used by DoE JGI
Standard Flow Chart for Metagenomics Research: Using the QPix460 system during the cloning and picking phase can significantly reduce the time of the entire process, increase efficiency, and eliminate labor-intensive and error-prone manual processes. The fully automated workflow can process tens of thousands of clones in a single week, and from coating to picking, with complete data record tracking, and significant cost savings. 
The first research goal was the known family of GH1s enzymes that catalyze the conversion of cellobiose to glucose . The phylogenetic approach allows us to select 200 genes from the thousands of existing sequences to achieve maximum functional diversity. Of the 200 genes, 180 were synthesized and then cloned into an expression vector using the QPix460 system ( Figure 2 ).
Figure 2. Metagenomics phylogenetic tree analysis, select representative gene families
The high-throughput automated process developed by DoE JGI includes parallel transformation, coating, and selection of multiple clones from each gene variant for sequencing. The QPix460 system makes this possible, providing high-throughput vaccination, coating and cloning on a single platform. Once the sequence is verified correctly, the optimal clone of each gene is transformed into a protein expression strain.
The protein expression of the GH1 protein producing strain was then analyzed for solubility. The protein expressed by 65% ​​of the clones was soluble and then further analyzed under the conditions described above. For example, as early quality control, the activity of a series of enzymes was analyzed under different temperature conditions, and the results showed that some of the candidate enzymes were thermally stable (Figure 3).
Figure 3. Identification of GH1 enzyme family activity and discovery of thermostable enzymes
In order to evaluate the properties of these candidate enzymes under different conditions, about 15,000 reactions are required, and screening for these reactions requires an ultra-high throughput technique. For this purpose, a mass spectrometry-based high-throughput technique , nanostructured start-up mass spectrometry ( NIMS ), was used. The enzyme - substrate reactant is spotted onto a solid support and ionized by laser. The activity of the enzyme was determined by measuring the ratio of substrate to product ( Figure 4 ), and the enzymes with the top 20 activity were selected for in-depth studies, of which 5 were active under the industrial-related conditions described above. This suggests that synthetic metagenomics can be used to quickly find enzymes with specific activities, starting with proteins predicted by public sequence databases.
in conclusion
These efforts demonstrate that the technology developed and used by DoE JGI can successfully convert digital information into biologically relevant, new-functioning enzymes. 180 proteins from the GH1 enzyme family, through the high-throughput, automated screening of the QPix460 system, finally succeeded in finding a suitable enzyme protein with new properties. This system has become an integral part of efficient, automated development and management of the metagenomics library.
Reference material
1. A nanostructure-initiator mass spectrometry-based enzyme activity assay. Northen TR, Lee JC, Hoang L, Raymond J, Hwang DR , Yannone SM, Wong CH, Siuzdak G. Proc Natl Acad Sci USA
Domestic and foreign users who currently use QPix for related applications:
1. US Department of Energy's Joint Genome Institute (DoE JGI) QPix460
2 , US recyclable product manufacturer Amyris purchased 3 QPix (developing algae to produce biofuels and converting sugar cane into ethanol)
3. Wuhan Institute of Biotechnology Synthetic Biology Laboratory QPix460
The US Department of Energy's Joint Genome Research Institute (DOE JGI) is based in Walnut Creek, Calif., and was established in 1997 to bring the Department of Energy at LBNL, Lawrence Livermore National Laboratory (LLNL) and Los Alamos National Laboratory (LANL). The Genomics Center is the first to integrate expertise and resources in DNA sequencing, information science and technology development. In 1999, in order to accelerate the completion of the Department of Energy's commitment to the Human Genome Project, the University of California, which is responsible for managing DOE JGI, will be leased out at the Walnut Creek Light Industrial Park laboratory and office in California to consolidate activities. The significant economies of scale that have been achieved have led the Joint Genetics Institute to be the first sequence analysis of target chromosomes 5, 16 and 19 published in the journal Nature. Following this achievement, DOE JGI continued to advance basic science by sequencing several typical organisms of more than 20 microbial species and contributing this information to public databases without compensation. In 2004, DOE JGI identified itself as a national user facility, and today there are more than 2,000 users worldwide. The vast majority of DOE JGI sequencing is conducted under the auspices of the Biome Sequencing Project (CSP), which measures the biosphere to characterize organisms associated with DOE bioenergy, global carbon cycle, and biogeochemical science missions. DOE JGI's largest customer is the US Department of Energy's Bioenergy Research Centers (BRCs), which were established in 2007 to accelerate the development of basic research in the next generation of cellulosic biofuels. The Institute continues to receive substantial funding from the Department of Biological and Environmental Research of the US Department of Energy Science Bureau.
Currently, DOE JGI has grown to 80,000 square feet and employs 250 employees led by internationally recognized geneticists, MD and Ph.D. Eddy Rubin. DOE JGI's partner labs include: LBNL, Lawrence Livermore National Laboratory, Los Alamos National Laboratory, Oak Ridge National Laboratory and Pacific Northwest National Laboratory (PNNL), and the Hasen Alpha Biotechnology Institute (formerly Related to the Stanford Human Genome Center).
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