Improving prodeoxyviolacein production via multiplex SCRaMbLE iterative cycles

doi:10.1007/s11705-018-1739-2

Front. Chem. Sci. Eng.

2018, Vol. 12

Issue (4) : 806-814 https://doi.org/10.1007/s11705-018-1739-2

RESEARCH ARTICLE

Improving prodeoxyviolacein production via multiplex SCRaMbLE iterative cycles

Juan Wang^1,², Bin Jia^1,², Zexiong Xie^1,², Yunxiang Li^1,², Yingjin Yuan^1,²(

)

¹. Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
². SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin 300072, China

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Abstract

The synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) system has been used to improve prodeoxyviolacein (PDV) production in haploid yeast containing chromosome synV. To rapidly and continuously generate genome diversification with the desired phenotype, the multiplex SCRaMbLE iterative cycle strategy has been developed for the screening of high PDV production strains. Whole-genome sequencing analysis reveals large duplications, deletions, and even the whole genome duplications. The deletion of YER151C is proved to be responsible for the increase. This study demonstrates that artificial DNA rearrangement can be used to accelerate microbial evolution and the production of biobased chemicals.

Keywords synthetic biology genome rearrangement prodeoxyviolacein SCRaMbLE Saccharomyces cerevisiae

Corresponding Author(s): Yingjin Yuan

Just Accepted Date: 25 May 2018 Issue Date: 03 January 2019

Cite this article:

Juan Wang,Bin Jia,Zexiong Xie, et al. Improving prodeoxyviolacein production via multiplex SCRaMbLE iterative cycles[J]. Front. Chem. Sci. Eng., 2018, 12(4): 806-814.

URL:

https://academic.hep.com.cn/fcse/EN/10.1007/s11705-018-1739-2
https://academic.hep.com.cn/fcse/EN/Y2018/V12/I4/806

Fig.1 Integration of heterogenous PDV pathway into synV yeast. (a) Tryptophan biosynthesis pathway and prodeoxyviolacein (PDV) pathway in yeast; (b) Yeast containing the PDV pathway formed green colonies; (c) HPLC analysis of PDV extraction at 610 nm

Fig.2 Characterization of the SCRaMbLE system for improving the PDV production. (a) Cre expression for SCRaMbLE (The reaction between loxPsym sites and Cre-EBD fusion proteins was achieved when cells were induced with extra galactose and estradiol); (b) SCRaMbLE synV generated random recombination of 177 loxPsym segments in synV; (c) The death rate curve of the SCRaMbLE system; (d) Multiplex SCRaMbLE iterative cycles (MuSIC)

Fig.3 Phenotypic verification and genome sequencing of the fitness-defective strain yWJR026. (a) Phenotypic diversity was observed after SCRaMbLE of yWJR001; (b) PDV production analysis of yWJR001 and yWJR026; (c) Growth assay of yWJR026 and its ancestor yWJR001 in YPD medium; (d) Deep sequencing analysis of yWJR026 (Long segment duplications were observed).

Fig.4 MuSIC for continuously improving the PDV production. (a) Culture color corresponding to PDV yield from yWJR001, yWJR006, yWJR040, yWJR085, yWJR00104 and yWJR00111; (b) HPLC analysis of PDV from cultures of the 6 strains; (c) Ratios of darker color colonies were calculated for each MuSIC cycle; (d) 10-fold dilution assays of the 6 strains

Fig.5 Deep sequencing analysis of SCRaMbLEd variations and verification. (a) Deep sequencing of five related strains; (b) Deletion of YER151C and duplication of PDV pathway were performed in yJBR001; (c) Verification of the deletion of YER151C and duplication of the PDV pathway

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