Synthetic biology tools for engineering Yarrowia lipolytica

Abstract

The non-conventional oleaginous yeast Yarrowia lipolytica shows great industrial promise. It naturally produces certain compounds of interest but can also artificially generate non-native metabolites, thanks to an engineering process made possible by the significant expansion of a dedicated genetic toolbox. In this review, we present recently developed synthetic biology tools that facilitate the manipulation of Y. lipolytica, including 1) DNA assembly techniques, 2) DNA parts for constructing expression cassettes, 3) genome-editing techniques, and 4) computational tools.

Keywords: CRISPR-Cas9; DNA assembly; Genome editing; Genome scale metabolic models; Golden Gate; Industrial biotechnology; Metabolic engineering; Synthetic biology; Yarrowia lipolytica.

Fig. 1

Summary of DNA assembly techniques. OE-PCR is a two-step PCR. During the first step, complementary overlapping overhangs are added to the parts to be assembled. During the second step, the parts hybridize with each other and form the new assembly via extension. In the Gateway method, the gene of interest, which has been cloned into the entry vector, is transferred into the destination vector via att site recombination. The expression vector obtained is then digested to release the expression cassette and used to transform Y. lipolytica. The BioBricks technique is used to clone parts via restriction digestion and the subsequent ligation of the resultant compatible sticky ends. YaliBricks vectors were designed to have AvrII, XbaI, SpeI, and NheI endonuclease site recognition. The ligation of the compatible overhangs produces a scar that is no longer recognized by either enzyme, which allows for subsequent assembly steps using more DNA parts. In Gibson assembly, parts are synthesized to overlap by 30+ bp. Their ends are then processed by an exonuclease that creates single-stranded 3′ overhangs, which facilitates annealing. The overhangs are fused together using a polymerase, which fills in gaps within each annealed fragment; a ligase seals gaps in the assembled DNA. Golden Gate assembly exploits type II enzymes, which cut outside their recognition sites to excise parts with arbitrarily defined four-base overhangs. Through the careful selection of compatible overhangs, such parts can be assembled altogether in a defined order. In the figure, the letters A to M represent different compatible 4-nt overhangs; the yellow, orange, and red arrows represent promoters; the green arrows represent genes; the violet circles represent markers; the blue squares represent insertion sequences; and the Ts represent terminators.

Fig. 2

Chromosome editing tools and targeted genome engineering. A. Representation of the homologous recombination (HR) approach, which requires long (~1 kb) homologous flanking fragments to be efficient in Y. lipolytica. B. TALENs and Cas9 are programmable nucleases that recognize and bind to specific DNA sequences, causing double-strand breaks (DSBs), which induce non-homologous end joining (NHEJ) or HR. NHEJ introduces random insertions and deletions into the genome. Templates with homology arms can be added to take advantage of natural HR mechanisms to either modify single nucleotides or to insert new sequences. It should be noted that Cas9 introduces blunt breaks, while Fok1, the TALEN endonuclease, introduces a staggered cut (for simplicity, this difference is not shown in the figure). C. On the left, a CRISPR interference (CRISPRi) system is illustrated. The dCas9-sgRNA complex can either target the promoter inhibiting transcription initiation or target the gene sequence to prevent transcription elongation. On the right, a CRISPR activation (CRISPRa) system is illustrated. dCas9 is fused with a transcription factor and targets the upstream region of the gene, delivering the transcription factor to the promoter; this process enhances transcription efficiency. The abbreviations are as follows: gDNA: genomic DNA; sgRNA: single-guide RNA; dCas9: catalytically inactive Cas9; RNAP: RNA polymerase; TF: transcription factor; Mxi1: repressor; and VPR: synthetic activator domain (Schwartz et al., 2018).