Description

Saccharomyces cerevisiae

The biosynthetic chassis has become increasingly diverse with the progress of research; however, when it comes to food safety, Saccharomyces cerevisiae (S. cerevisiae) has an edge. For example, between Escherichia coli (E. coli) and trispora, E. coli can release endotoxins, while the production process of trispora requires the addition of chemicals that inhibit cyclase activity; thus, the biosynthesis applications of these two chassis are somewhat controversial^[1].

S. cerevisiae was used to make wine as early as 7,000 BC in China, and the use of yeast by humans can even be traced back to 10,000 years ago according to plentiful historical literature^[2]. S. cerevisiae has been living in the symbiosis with humans for thousands of years. After passage and domestication, S. cerevisiae is generally recognized as safe (GRAS), and used as a eukaryotic model organism for whole-genome sequencing. Because of its relatively complete genomic information and strong homologous recombination ability, S. cerevisiae has great potential to be used as a biosynthetic chassis. Meanwhile, the excellent resistance to contamination (such as bacteriophage contamination), decent stress resistance, and abundant organelles make S. cerevisiae a preferred organism for industrial utilization.

However, since S. cerevisiae is a monocistronic organism, the expression of each gene requires the assistance of a promoter and a terminator. With the metabolic pathway involving five enzymes as an example, if we take the promoter, ORF and terminator as average lengths (1,000, 2,000 and 500 bps, respectively), the overall length of DNA embracing such a metabolic pathway will be more than 17,000 bps. Actually, this is just a relatively short metabolic pathway in nature, and thus it is very difficult, or even impossible, to assemble naturally complex metabolic pathways using conventional DNA assembly methods. Therefore, constructing complex metabolic pathways and signal transduction pathways in S. cerevisiae is extremely challenging. This problem is genuine not only for S. cerevisiae, but also for other eukaryotes in general.

Retarded Building module

Synthetic biology follows a logical paradigm of Design-Build-Test-Learn (DBTL). However, with the development of bioinformatics, high-throughput screening technology and artificial intelligence, the circling speed of this cycle is gradually accelerated, but this acceleration is mostly due to the reduced time windows of the Design, Test and Learn modules. In terms of the Design module, the processing throughput has increased by 10,000 times in less than five years, such as from weeks/protein to seconds/protein. The TransTermHP for terminator sequence search and analysis achieved less than 1 min/genome. The Learn module contains a large number of open-source databases, including different levels of "gene-RNA-Protein-metabolism-phenotype" analysis. However, regarding the Build module, commonly used assembly methods can only construct single-digit DNA fragments, although occasionally the assembly of over 100 DNA pieces can be achieved^[3], such as the CasHRA (Cas9 - facilitated Homologous Recombination Assembly). Overall, the Build module is still far behind the Design, Test and Learn modules.

Automation Age

Automation is becoming increasingly important in practice, because it is the most reliable way to obtain data with high quality, large volume and low bias. With the advent of automation, we are expected to solve the problem of construction inefficiency. The speed of construction and the accuracy of operation can be significantly improved by replacing humans with machines. However, due to the lack of rational design thinking, the machine is unable to cope with complex and dynamic reaction conditions, or deal with unexpected situations. Therefore, not all DNA assembly technologies can be combined with automation.

Our Plan

Automation is becoming increasingly important in practice, because it is the most reliable way to obtain data with high quality, large volume and low bias. With the advent of automation, we are expected to solve the problem of construction inefficiency. The speed of construction and the accuracy of operation can be significantly improved by replacing humans with machines. However, due to the lack of rational design thinking, the machine is unable to cope with complex and dynamic reaction conditions, or deal with unexpected situations. Therefore, not all DNA assembly technologies can be combined with automation.

Based on the analysis described above, only when the DNA assembly process is standardized, efficient and accurate, and the reaction conditions are simple enough, can the advantages of machinery be really implemented, and the efficiency of the Building module be improved. To achieve our goal, we employed the YeastFab technology from Tsinghua University, which was based on the Golden Gate DNA assembly method to enable the assembly of multiple DNA segments.

We did the DNA assembly using a universal system under standard reaction conditions, and improved the YeastFab technique. In the construction, the ccdB toxic protein was used as a screening marker to replace the original mCherry protein, and the toxicity of ccdB prevented the DNA unwinding process to improve the positive rate in a more direct way^[4]. Second, we hope to build a hardware system matching the YeastFab to further increase the efficiency. Meanwhile, such a hardware set should be fully functional and low cost, so that biological laboratories and synthetic biology enthusiasts around the world can afford it. Finally, we hope to realize the cross-species application of the YeastFab technology, so that the efficient DNA assembly strategy can be widely applied to a variety of chassis organisms, not just for yeast, and improve the efficiency of DNA construction in the entire synthetic biology.

After achieving these goals, we will package the above technologies into a standard operating manual and make it available to all iGEM teams around the world, to help worldwide biosynthesis laboratories achieve efficient DNA assembly.