Introduction

Our team is committed to creating a standardized construction platform based on the research background of synthetic biology. As we mentioned in the design part, the main process of synthetic biology includes four modules: Design, Build, Test and Learn. However, in the era of mechanical industry, the Build module completed by manpower is far from keeping up with the developing speed of the other three parts. Therefore, our project aims to create an efficient DNA assembly strategy, and this assembly method should meet the requirements of the machine for standardized process modeling. Here, we noticed the YeastFab technology, so we optimized and applied this technology to construct the metabolic pathway of lycopene in Saccharomyces cerevisiae, to verify the feasibility of this technology and improve the construction speed to obtain high-yield strains.

Optimization of the YeastFab

According to the YeastFab technology (see details in the Design section), we built three levels of the parts and their combination, and optimized it on the basis of the YeastFab technology by replacing the original mCherry screening marker with the ccdB toxic protein gene to increase the positive rates, improve the building accuracy, and facilitate the future machine operation. The method of the YeastFab Assembly uses two type IIS restriction endonucleases and assemble DNA fragments by the Golden Gate method.

Level 1: Build standards and reusable parts: Promoter-ORF-Terminator

First, we constructed the designed standard vectors (pccdK2_P, pccdK2_O, and pccdK2_T) to carry the parts of each section. In addition, standardized design of primers and construction methods were used to achieve the cloning and characterization of a large number of promoters. When constructing the promoter library, we firstly collected a lot of promoters from the genome of Saccharomyces cerevisiae, screened and characterized them, and finally selected the promoters with the best structures as our standard parts. In terms of the ORFs, we used the genes of key enzymes involved in lycopene synthetic pathway and the genes of all key enzymes in the MVA pathway to construct a standard part library. Terminators are also from the genome of yeast, and we have constructed a series of terminators as a standard basic part library. By doing this, we built our basic parts. The building results showed that the positive rates could reach over 90%. (Check the Results section for details.)

Figure 1. Level1 builds the standard system and conditions.

Figure 2. Agarose gel images for the PCR products of the standardized parts.

Level 2: Build the transcription unit

In the construction of this level, the combination of promoter, ORF and terminator still utilized the standardized system, by subcloning all these elements into the corresponding standardized Level 2 vector. These standard parts were assembled into a transcription unit in a single reaction. We successfully constructed a large number of different combinations of TU2-P-crtE-T, TU4-P-crtI-T and TU5-P-crtB-T, and the positive rate of our clones could be close to 100% in all the construction of the Level 2. (Check the Results section for the full results.)

Figure 3. Level2 builds the standard system and conditions.

Figure 4. Agarose gel image of colony PCR to screen for a Level 2 construct.

Level 3: Builds the full pathway

After the successful assembly of the transcription units, we transferred TU2-P-CRTE-T, TU4-P-CRTI-T and TU5-P-CRTB-T transcription units carrying selection markers and homologous arms into yeast for recombinant integration into the genome and subsequent exogenous gene expression. Gene integration was verified by the PCR of genomic DNA, and lycopene production was determined by HPLC. Except for the successful integration synthesis pathway of lycopene ， the superimposed genome has also optimized of the synthesis pathway of lycopene precursor. Therefore, we achieved a breakthrough in yield, which improved from 1.5mg/g DCW to 3.6mg/g DCW. (Check the Results section for the full results.)

Figure 5. Plate of yeast colonies with different colors.	Figure 6. PCR results with extracted yeast genomic DNA as templates.

Cross species validation

In order to achieve broad applications of the YeastFab technology, we have further improved this technology. We chose Aspergillus oryzae as the biological chassis for the cross-species application test. We wanted to optimize the terpenoid metabolic pathway in A.Oryzae, so we constructed the metabolic pathway (Figure 7). The same method and system in the previous description were used to construct the Level 1 and Level 2. In the integration of Level 3, we chose a slightly different strategy from Saccharomyces cerevisiae. Because A.Oryzae does not have strong homologous recombination ability as S.Cerevisiae has, CRISPR/Cas9 technology was employed to integrate the metabolic pathways into its genome. We also achieved a very high positive rate in assembling plasmids carrying metabolic pathways in vitro. Each plasmid of the metabolic pathways was combined with plasmid expressing the Cas9 protein for co-transformation. Cas9 protein cleaved the genome to create an incision, and the metabolic pathways were subsequently integrated into the genome at the cleaved site. TLC detection showed that all the related metabolites were produced, indicating that this synthesis pathway could also be successfully expressed in A.Oryzae. The results strongly proved that we have achieved the cross-species application of the YeastFab technology. The cross-species application still meets the conditions of mechanical operation, and can be potentially used for efficient DNA assembly with a machiner.

Figure 7. Exogenous metabolic pathways introduced into A.oryzae.

Figure 8. Agarose gel image of restriction enzyme digested plasmids.

Line 1/2：Digestion of SalI: predicted bands are 316, 8494 and 6316 bps.

Line 3/4：Digestion of HindIII: predicted bands are 11541 and 3583 bps.

Figure 9. Results of TCL detection.

In each of the 7 groups, the left well is the negative control sample, and the right well is the experimental sample. Only one experimental group (the third from the left) produced an irrelevant product. The integration efficiency of the gene clusters was 94.117%

Conclusion

Using the optimized YeastFab construction process, we have successfully constructed different levels of basic standard parts, a combination part library and the lycopene synthesis pathway in S. cerevisiae in a short period of time, which demonstrating the high standardization and efficiency of our DNA assembly strategy. The standardization of the system conditions and high positive rate were also presented. Finally, we successfully applied the YeastFab technology to constructing a set of metabolic pathways in A.oryzae, which is a big leap in promoting the YeastFab technology. The success of A.Oryzae is expected to prove that the YeastFab is an efficient DNA assembly strategy that can be adapted to abundant species.

The basic modules and operation process in our machine construction are shown in the Hardware section.