Overview

The current DNA assembly methods are inefficient and cannot meet the requirements of synthetic biology nowadays. However, the age of mechanization provides us with a chance to improve the efficiency of DNA assembly. The use of machinery can carry out a large number of single repeated operations, which means that the DNA assembly strategy can be applied to the machine with extremely high standardization and efficiency. Therefore, we have noticed the YeastFab technology.

The YeastFab technology is a strategy that adapts to the assembly and construction of a large number of DNA fragments, with high efficiency, unified system and simple operation. Our team optimizes and expands the technical content of the YeastFab. We have further improved the YeastFab’s positive rate, explored diverse ways of expressing exogenous genes, and, more importantly, achieved the first step in its cross-species application.

Golden Gate

Current cloning technologies based on site-specific recombination are efficient, simple to use, and flexible, but have the drawback of leaving recombination site sequences in the final constructs, adding extra 8 to 13 amino acids in the expressed proteins. We used a simple and rapid subcloning strategy 'Golden Gate' to transfer any DNA fragment of interest from an entry clone into an expression vector, which eliminated this shortcoming. The strategy is based on the use of type IIs restriction enzymes, which cut outside elements of their recognition sequences. With proper design of the cleavage sites, two fragments cut by type IIs restriction enzymes can be ligated into a product lacking the original restriction site. The 'Golden Gate' cloning was devised to allow one tube and one step reaction with nearly hundred percent correct recombinant plasmids after just a 5 min of restriction-ligation. This method is therefore as efficient as currently used recombination-based cloning technologies but yields recombinant plasmids that do not contain unwanted sequences in the final constructs, thus providing precision for this fundamental process of genetic manipulation.^[1]

YeastFab

When using traditional cloning methods to construct exogenous metabolic pathways, even a simple metabolic pathway needs to go through complicated and tedious steps. Moreover, if metabolic pathways with different copy numbers, multiple promoters or RBS are further constructed, it is necessary to optimize these pathways in a combination way, which is a more arduous and time-consuming task. Therefore, the application of traditional cloning methods has great limitations. Balance of gene expression includes regulation of promoter strength, and modification of endogenous and external regulatory networks. In order to achieve standardized design and rapid construction, assembly, and expression of exogenous metabolic pathways to facilitate balance of gene expression, our team used the YeastFab technology to overcome the limitations of conventional methods.

We chose both ZFNs and CRISPR/Cas9 systems to make parallel and comparative designing and studies, to find an optimal targeted nicking system.

YeastFab: Building block principle of Level 1

Each pccdK2-P/O/T vector contains recognition sites of the two type II restriction enzymes, BsaI and BsmBI. BsaI is used for the assembly of standard parts into the vector, and BsmBI is utilized to release the parts for the construction of transcription units. Both enzymes share the same overhangs as shown below. To clone each part into the Level 1 vector, a pair of primers with defined prefix sequences will be used.

YeastFab: Building block principle of Level 2

Once all desired parts are ready, it is time to assemble the transcription unit. In this part, the promoter, ORF and terminator of interest will be released from their vectors and assembled together into an accepting vector in a one-pot reaction. BsmBI is utilized to release the parts for the construction of transcription units (Figure 2).

We constructed 11 vectors totally as transcription units (Figure 3). Among these vectors, only 4 bases with prominent cleavage sites were different, and the vector selection of the transcription units was determined by how many transcription units the subsequent metabolic pathways needed to be assembled. This enables one-pot reaction assembly of up to six transcription units.

YeastFab: Building block principle of Level 3

After obtaining standardized transcription units, the transcription units are assembled to form a metabolic pathway--Level3. Each transcription unit was cut off from the corresponding vector under the action of BsaI restriction enzyme. Then, under the guidance of the designed sticky end, the target path is assembled in a fixed order (Strategy is shown in Figure 4.)

Metabolic pathway for exogenous lycopene synthesis

Selection of pathway genes

Lycopene is synthesized by the mevalonate (MVA) pathway in S. cerevisiae. The MVA pathway is the only pathway for terpenoid production in S. cerevisiae. In this pathway, acetyl-CoA is used as the starting substrate to produce isoprene pyrophosphate (IPP) and its isoform DMAPP through A series of reactions. IPP and DMAPP are converted to geraniyl pyrophosphate (GPP), and subsequently catalyzed to farniki pyrophosphate (FPP) under the catalysis of farniki pyrophosphate synthase. Further conversion of FPP to lycopene is catalyzed by additional three enzymes.

Assembly of metabolic pathways

Based on the principle of the YeastFab technology (see Design for details), we have designed the pccdK2 and TU series of vectors, and the pccdK2 series of vectors are used to carry standard basic components (promoter, ORF, terminator, homologous arm, selection marker and so on). The TU series of vectors are used to carry transcriptional units (Promoter-ORF-Termiator, i.e., POT). The selection markers on the pccdK2 and TU vectors are different from those of the YeastFab technology. We used the ccdB toxin gene to replace the mCherry gene. The ccdB toxic protein has strong toxicity to disrupt the DNA unwinding process, and can kill the yeast that fails in assembly. This is more direct and efficient than using the mCherry protein that makes the colonies pink.

Optimization: Fermentation

We tried to imrpove the yield from the perspective of fermentation, and thus modeled the fermentation process to pursue better conditions. We reviewed the literature and found that optimization through the Response Surface Methods (RSM) by response surface analysis was a commonly used method. We use software for rational experimental design and result analysis. The experimental design diagram is as follows.

After obtaining the experimental results, we used two methods to predict the optimal fermentation condition. Both the response surface analysis method and the neural network model were used to process the data separately, and the prediction of the optimal fermentation conditions were similar. We can obtain higher yield by using this method for iteration, which proves that our model is reliable.

Cross-species application

In order to realize the cross-species application of the YeastFab technology, we first tried it in Aspergillus oryzae.

We intended to construct a metabolic pathway in Aspergillus oryzae, which is a gene cluster composed of four genes, HMGS-TS, P450-71, P450-74 and P450-75.

Based on the YeastFab technology, we assembled the basic standard elements of the Aspergillus oryzae promoter, terminator and corresponding ORF(Level 1 construction). After that, we came to the transcription unit assembly(Level 2 construction). Then, we modified the YeastFab technology. Because A. oryzae does not have as strong homologous recombination ability as S. cerevisiae, it is unable to assemble linear DNA fragments carrying homologous arms for recombination under the guidance of homologous arms. Here, we introduced the CRISPR/Cas9 technology to assist homologous recombination. We constructed a plasmid carrying the Cas9 gene, and our target gene cluster was also constructed into the plasmid to perform double plasmid transformation of A. oryzae. Under the guidance of a predesigned gRNA, Cas9 protein made an incision at the target site of the genome, and the homologous arms at both ends of the exogenous metabolic pathway were homologous to the fragments at both ends of the gap (with the same sequence). Under the action of the repair mechanism of A. Oryzae, the exogenous metabolic pathway could be integrated into the genome of A. Oryzae.

Two plasmids were transferred into Aspergillus oryzae, one carrying the exogenous metabolic pathway and homologous arm, and the other carrying the Cas9 gene. The Cas9 protein is guided by a gRNA to create an incision a specific site in the genome, and the repair mechanism of A. oryzae reorganizes metabolic pathways into the genome at this cleavage site.