YALIcloneNHEJ: An Efficient Modular Cloning Toolkit for NHEJ Integration of Multigene Pathway and Terpenoid Production in Yarrowia lipolytica.

Tian-Qiong Shi,Qi Shen,Qi Guo,Cai-Ling Yang,Xiao-Man Sun,He Huang,Qian-Qian Peng,Ya-Wen Li,Xiao-Jun Ji,Zhi-Kui Nie

doi:10.3389/fbioe.2021.816980

Abstract

Non-homologous end-joining (NHEJ)-mediated random integration in Yarrowia lipolytica has been demonstrated to be an effective strategy for screening hyperproducer strains. However, there was no multigene assembly method applied for NHEJ integration, which made it challenging to construct and integrate metabolic pathways. In this study, a Golden Gate modular cloning system (YALIcloneNHEJ) was established to develop a robust DNA assembly platform in Y. lipolytica. By optimizing key factors, including the amounts of ligase and the reaction cycles, the assembly efficiency of 4, 7, and 10 fragments reached up to 90, 75, and 50%, respectively. This YALIcloneNHEJ system was subsequently applied for the overproduction of the sesquiterpene (-)-α-bisabolol by constructing a biosynthesis route and enhancing the flux in the mevalonate pathway. The resulting strain produced 4.4 g/L (-)-α-bisabolol, the highest titer reported in yeast to date. Our study expands the toolbox of metabolic engineering and is expected to enable a highly efficient production of various terpenoids.

Highlights

The monocyclic sesquiterpene (-)-α-bisabolol has important applications in the fields of medicine, food, and biofuels (Han et al, 2016; Lim et al, 2021)—for example, Melo et al (2017) found that oral formulations of (-)-α-bisabolol can reduce oral injury in rodents to a certain extent
Introducing a truncated HMG1 gene coding for HMG-CoA reductase, acetoacetyl-CoA thiolase (ERG10) encoding acetyl-CoA thiolase, and ACS1 gene coding for acetyl-CoA synthetase into S. cerevisiae genome, the titer increased to 124 mg/L (Kim et al, 2021)
The strains with plasmids encoding gene fragments were cultivated at 37°C in lysogeny broth (LB) medium supplemented with 100 μg/ml ampicillin, while the strains with the backbone plasmids with or without assembled building blocks were cultivated at 37°C in LB supplemented with 50 μg/ml kanamycin, 40 μg/ml X-Gal, and 0.05 μM IPTG

Summary

Introduction

The monocyclic sesquiterpene (-)-α-bisabolol has important applications in the fields of medicine, food, and biofuels (Han et al, 2016; Lim et al, 2021)—for example, Melo et al (2017) found that oral formulations of (-)-α-bisabolol can reduce oral injury in rodents to a certain extent. The synthesis of (-)-α-bisabolol has been previously achieved in Escherichia coli with the highest titer of 23.4 g/L (Lim et al, 2021). Due to the possible infection risk of E. coli by bacteriophage, lots of researchers preferred to use fungi as the host to synthesize (-)-α-bisabolol—for example, some researchers attempted to use Saccharomyces cerevisiae to synthesize (-)-αbisabolol heterologously. Introducing a truncated HMG1 gene coding for HMG-CoA reductase, ERG10 encoding acetyl-CoA thiolase, and ACS1 gene coding for acetyl-CoA synthetase into S. cerevisiae genome, the titer increased to 124 mg/L (Kim et al, 2021). In order to improve the level, metabolic engineering strategies, including optimizing the mevalonate pathway and the lipid synthesis pathway, were adopted. The (-)-α-bisabolol titer was improved from 8 to 364 mg/L in yeast (Ma et al, 2021). The production of (-)-α-bisabolol in fungi is still very low (Table 1)

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Conclusion