title: Development of a modularized two-step (M2S) chromosome integration technique for integration of multiple transcription units inSaccharomyces cerevisiae.
abstract: BACKGROUND
  Saccharomyces cerevisiaehas already been used for heterologous production of fuel chemicals and valuable natural products. The establishment of complicated heterologous biosynthetic pathways inS. cerevisiaebecame the research focus of Synthetic Biology and Metabolic Engineering. Thus, simple and efficient genomic integration techniques of large number of transcription units are demanded urgently.
  
  
  RESULTS
  An efficient DNA assembly and chromosomal integration method was created by combining homologous recombination (HR) inS. cerevisiaeand Golden Gate DNA assembly method, designated as modularized two-step (M2S) technique. Two major assembly steps are performed consecutively to integrate multiple transcription units simultaneously. In Step 1, Modularized scaffold containing a head-to-head promoter module and a pair of terminators was assembled with two genes. Thus, two transcription units were assembled with Golden Gate method into one scaffold in one reaction. In Step 2, the two transcription units were mixed with modules of selective markers and integration sites and transformed intoS. cerevisiaefor assembly and integration. In both steps, universal primers were designed for identification of correct clones. Establishment of a functional β-carotene biosynthetic pathway inS. cerevisiaewithin 5 days demonstrated high efficiency of this method, and a 10-transcriptional-unit pathway integration illustrated the capacity of this method.
  
  
  CONCLUSIONS
  Modular design of transcription units and integration elements simplified assembly and integration procedure, and eliminated frequent designing and synthesis of DNA fragments in previous methods. Also, by assembling most parts in Step 1 in vitro, the number of DNA cassettes for homologous integration in Step 2 was significantly reduced. Thus, high assembly efficiency, high integration capacity, and low error rate were achieved.
authors: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Ave, Tianjin Airport Economic Park, Tianjin, 300308 China.
authors: Siwei
authors: S
authors: Li
authors: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Ave, Tianjin Airport Economic Park, Tianjin, 300308 China.
authors: Wentao
authors: W
authors: Ding
authors: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Ave, Tianjin Airport Economic Park, Tianjin, 300308 China.
authors: Xueli
authors: X
authors: Zhang
authors: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Ave, Tianjin Airport Economic Park, Tianjin, 300308 China.
authors: Huifeng
authors: H
authors: Jiang
authors: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Ave, Tianjin Airport Economic Park, Tianjin, 300308 China.
authors: Changhao
authors: 0000-0002-1940-8511
authors: C
authors: Bi
keywords: Chromosome integration; DNA assembly; Module; Transcription unit