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

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.

Metabolic Engineering of Yarrowia lipolytica for Terpenoid Production: Tools and Strategies

“…It is sometimes easier to construct gene expression libraries relying on NHEJ. In some cases, NHEJ even plays an important role in gene integration. ,, However, the stochasticity of integration sites and copy numbers in NHEJ causes difficulty for precise optimization of synthetic pathways . Genome editing tools that rely on HR are difficult to function in Y. lipolytica .…”

“…After overexpressing the whole MVA pathway, the titer of αhumulene was increased by 30 times, 32 the titer of β-ionone was increased by 43.7 times, 66 and the titer of (−)-α-bisabolol was increased by as much as 97 times. 36 However, integrative overexpression of the entire MVA pathway is a challenge due to the low HR efficiency of Y. lipolytica. Therefore, most researchers chose to manipulate only the key genes.…”

Advances in Metabolic Engineering Paving the Way for the Efficient Biosynthesis of Terpenes in Yeasts

“…Isopentenyl diphosphate isomerase (IDI) is a key node for balancing the amount of IPP and DMAPP. Accordingly, its overexpression can also improve the precursor supply. , Combinatorial expression of these key genes can be achieved using the Golden Gate modular cloning system based on nonhomologous end-joining . Another strategy to enhance the flux through the MVA pathway is to overexpress the endogenous transcription activator UPC2, which is unique in S. cerevisiae .…”

“…13,95 Combinatorial expression of these key genes can be achieved using the Golden Gate modular cloning system based on nonhomologous endjoining. 96 Another strategy to enhance the flux through the MVA pathway is to overexpress the endogenous transcription activator UPC2, which is unique in S. cerevisiae. Binding to UPC2 binding sites in natural promoters of MVA pathway genes enhances enzyme expression at the transcriptional level.…”

Engineering Saccharomyces cerevisiae for enhanced (–)-α-bisabolol production

“…Shi et al overexpressed the entire MVA pathway genes in Y. lipolytica to increase the metabolic flux to FPP and optimize the copy number of MrBBS and tHMG1 , resulting in the production of 4.4 g/L in a 5 L bioreactor. This was the highest production in yeast reported to date [ 14 ]. A recent study showed that overexpression of tHMG1 , ERG10 , and ACS1 lead to a 13-fold increase in (–)-α-bisabolol biosynthesis in S. cerevisiae , but the final production was only 124 mg/L [ 11 ].…”