Development of a dedicated Golden Gate Assembly Platform (RtGGA) for Rhodotorula toruloides

Abstract

Rhodotorula toruloides is a potential chassis for microbial cell factories as this yeast can metabolise different substrates into a diverse range of natural products, but the lack of efficient synthetic biology tools hinders its applicability. In this study, the modular, versatile and efficient Golden Gate DNA assembly system (RtGGA) was adapted to the first basidiomycete, an oleaginous yeast R. toruloides. R. toruloides CCT 0783 was sequenced, and used for the GGA design. The DNA fragments were assembled with predesigned 4-nt overhangs and a library of standardized parts was created containing promoters, genes, terminators, insertional regions, and resistance genes. The library was combined to create cassettes for the characterization of promoters strength and to overexpress the carotenoid production pathway. A variety of reagents, plasmids, and strategies were used and the RtGGA proved to be robust. The RtGGA was used to build three versions of the carotenoid overexpression cassette by using different promoter combinations. The cassettes were transformed into R. toruloides and the three new strains were characterized. Total carotenoid concentration increased by 41%. The dedicated GGA platform fills a gap in the advanced genome engineering toolkit for R. toruloides, enabling the efficient design of complex metabolic pathways.

Keywords: Golden gate assembly; Metabolic engineering; Non-conventional yeast; Oleaginous yeast; Rhodotorula toruloides; Synthetic biology.

Fig. 1

The RtGGA with A) Level 1 (Lv1) for the library of standardized parts-containing regions upstream (insUP) and downstream (insD) an insertional site, selection marker (M), promoters, genes, and terminators-and level 2 (Lv 2) by combining promoter (P), gene (G), terminator (T) into transcriptional units (TU). B) Level 3 (Lv3) RtGGA multigene expression cassette and standardized overhangs from the Y. lipolytica GGA platform (Celińska et al., 2017). The overhangs in italic are the changes made to make it compatible with R. toruloidesC) Upper box shows the reaction method proposed in the Y. lipolytica GGA platform (Celińska et al., 2017) for level 2 assembly using linearized PCR-amplified parts. The lower box shows the two-step assembly protocol adapted from Larroude et al., 2019 for the assembly of Lv2. D) Scheme of the cassettes assembled for the characterization of the promoters strength and the overexpression of the carotenoid pathway. Legend: ori – the origin of replication; black arrow– bacterial resistance gene; RFP: red fluorescence protein. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Relative expression in terms of the fold of the NAT gene under different promoters. pADH2 (light blue) was used as a reference and the asterisk indicates a difference with statistical significance (Student's t-test; p-value ≤ 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Carotenoid biosynthesis in R. toruloides according to different genome-scale models (GEM) (Dinh et al., 2019; Kim et al., 2021; Tiukova et al., 2019). Geranylgeranyl diphosphate synthase (CRTE) catalyzes the formation of precursor of carotenoids. Phytoene dehydrogenase (CRTI) and phytoene synthase/lycopene cyclase (CRTYB) are both involved in the production of, respectively, acyclic and cyclic carotenoids.

Fig. 4

A) final carotenoid titer and B) composition (HPLC method). C) Relative expression of the genes crtE, crtI, and crtYB in the strains SBY92-94 in comparison to parental strain during the exponential phase. Asterisk shows the significative statistical difference (Student's t-test; p-value ≤ 0.05) when compared to the parental strain (R. toruloides CCT 7815).