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Background    Saccharomyces cerevisiae  has already been used for heterologous production of fuel chemicals and valuable natural products. The establishment of complicated heterologous biosynthetic pathways in  S. cerevisiae  became 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) in  S. cerevisiae  and 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 into  S. cerevisiae  for assembly and integration. In both steps, universal primers were designed for identification of correct clones. Establishment of a functional β-carotene biosynthetic pathway in  S. cerevisiae  within 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.    Electronic supplementary material  The online version of this article (doi:10.1186/s13068-016-0645-4) contains supplementary material, which is available to authorized users.

biotechnology for biofuels

Development of a modularized two-step (M2S) chromosome integration technique for integration of multiple transcription units in Saccharomyces cerevisiae