Bug mapping and fitness testing of chemically synthesized chromosome X

Author:

Wu Yi12ORCID,Li Bing-Zhi12ORCID,Zhao Meng12ORCID,Mitchell Leslie A.3ORCID,Xie Ze-Xiong12ORCID,Lin Qiu-Hui12,Wang Xia12,Xiao Wen-Hai12,Wang Ying12,Zhou Xiao12,Liu Hong12,Li Xia12ORCID,Ding Ming-Zhu12,Liu Duo12,Zhang Lu12,Liu Bao-Li12,Wu Xiao-Le12,Li Fei-Fei12,Dong Xiu-Tao12,Jia Bin12,Zhang Wen-Zheng12,Jiang Guo-Zhen12,Liu Yue12ORCID,Bai Xue12ORCID,Song Tian-Qing12ORCID,Chen Yan12ORCID,Zhou Si-Jie12,Zhu Rui-Ying12ORCID,Gao Feng12ORCID,Kuang Zheng3ORCID,Wang Xuya3,Shen Michael3,Yang Kun4ORCID,Stracquadanio Giovanni45ORCID,Richardson Sarah M.4ORCID,Lin Yicong6,Wang Lihui6ORCID,Walker Roy7ORCID,Luo Yisha7ORCID,Ma Ping-Sheng12,Yang Huanming89ORCID,Cai Yizhi7ORCID,Dai Junbiao6ORCID,Bader Joel S.4ORCID,Boeke Jef D.3ORCID,Yuan Ying-Jin12ORCID

Affiliation:

1. Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, PR China.

2. SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, 300072, PR China.

3. Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, New York University (NYU) Langone Medical Center, New York City, NY 10016, USA.

4. High Throughput Biology Center and Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA.

5. School of Computer Science and Electronic Engineering, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK.

6. Key laboratory of Industrial Biocatalysis (Ministry of Education), Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University, Beijing, 100084, PR China.

7. School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3BF, Scotland, UK.

8. BGI-Shenzhen, Shenzhen, 518083, PR China.

9. James D. Watson Institute of Genome Sciences, Hangzhou 310058, PR China.

Abstract

INTRODUCTION Design and construction of an extensively modified yeast genome is a direct means to interrogate the integrity, comprehensiveness, and accuracy of the knowledge amassed by the yeast community to date. The international synthetic yeast genome project (Sc2.0) aims to build an entirely designer, synthetic Saccharomyces cerevisiae genome. The synthetic genome is designed to increase genome stability and genetic flexibility while maintaining cell fitness near that of the wild type. A major challenge for a genome synthesis lies in identifying and eliminating fitness-reducing sequence variants referred to as “bugs.” RATIONALE Debugging is imperative for successfully building a fit strain encoding a synthetic genome. However, it is time-consuming and laborious to replace wild-type genes and measure strain fitness systematically. The Sc2.0 PCRTag system, which specifies recoded sequences within open reading frames (ORFs), is designed to distinguish synthetic from wild-type DNA in a simple polymerase chain reaction (PCR) assay. This system provides an opportunity to efficiently map bugs to the related genes by using a pooling strategy and subsequently correct them. Further, as we identify bugs in designer sequences, we will identify gaps in our knowledge and gain a deeper understanding of genome biology, allowing refinement of future design strategies. RESULTS We chemically synthesized yeast chromosome X, synX, designed to be 707,459 base pairs. A high-throughput mapping strategy called pooled PCRTag mapping (PoPM) was developed to identify unexpected bugs during chromosome assembly. With this method, the genotypes of pools of colonies with normal or defective fitness are assessed by PCRTag analysis. The PoPM method exploits the patchwork structure of synthetic and wild-type sequences observed in the majority of putative synthetic DNA integrants or meiotic progeny derived from synthetic/wild-type strain backcross. PCRTag analysis with both synthetic and wild-type specific primers, carried out with genomic DNA extracted from the two pools of clones (normal fitness versus a specific growth defect), can be used to identify regions of synthetic DNA missing from the normal fitness pool and, analogously, sections of wild-type DNA absent from the specific growth-defect pool. In this way, the defect can be efficiently mapped to a very small overlapping region, and subsequent systematic analysis of designed changes in that region can be used to identify the bug. Several bugs were identified and corrected, including a growth defect mapping to a specific synonymously recoded PCRTag sequence in the essential FIP1 ORF and the effect of introducing a loxPsym site that unexpectedly altered the the promoter function of a nearby gene, ATP2. In addition, meiotic crossover was employed to repair the massive duplications and rearrangements in the synthetic chromosome. The debugged synX strain exhibited high fitness under a variety of conditions tested and in competitive growth with the wild-type strain. CONCLUSION Synthetic yeast chromosome X was chemically synthesized from scratch, a rigorous, incremental step toward complete synthesis of the whole yeast genome. Thousands of designer modifications in synX revealed extensive flexibility of the yeast genome. We developed an efficient mapping method, PoPM, to identify bugs during genome synthesis, generalizable to any watermarked synthetic chromosome, and several details of yeast biology were uncovered by debugging. Considering the numerous gene-associated PCRTags available in the synthetic chromosomes, PoPM may represent a powerful tool to map interesting phenotypes of mutated synthetic strains or even mutated wild-type strains to the relevant genes. It may also be useful to study yeast genetic interactions when an unexpected phenotype is generated by alterations in two or more genes, substantially expanding understanding of yeast genomic and cellular functions. The PoPM method is also likely to be useful for mapping phenotype(s) resulting from the genome SCRaMbLE system. Characterization of synX and debugging by pooled PCRTag mapping. ( Top ) Design overview of synthetic chromosome X. ( Bottom ) Flow diagram of pooled PCRTag mapping (PoPM).

Funder

National Science Foundation

U.S. Department of Energy

Biotechnology and Biological Sciences Research Council

National Natural Science Foundation of China

Ministry of Science and Technology of the People’s Republic of China

Publisher

American Association for the Advancement of Science (AAAS)

Subject

Multidisciplinary

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