Optimization of Yarrowia lipolytica-based consolidated biocatalyst through synthetic biology approach: transcription units and signal peptides shuffling

Abstract

Nowadays considerable effort is being pursued towards development of consolidated microbial biocatalysts that will be able to utilize complex, non-pretreated substrates and produce valuable compounds. In such engineered microbes, synthesis of extracellular hydrolases may be fine-tuned by different approaches, like strength of promoter, type of secretory tag, and gene copy number. In this study, we investigated if organization of a multi-element expression cassette impacts the resultant Yarrowia lipolytica transformants' phenotype, presuming that different variants of the cassette are composed of the same regulatory elements and encode the same mature proteins. To this end, Y. lipolytica cells were transformed with expression cassettes bearing a pair of genes encoding exactly the same mature amylases, but fused to four different signal peptides (SP), and located interchangeably in either first or second position of a synthetic DNA construction. The resultant strains were tested for growth on raw and pretreated complex substrates of different plant origin for comprehensive examination of the strains' acquired characteristics. Optimized strain was tested in batch bioreactor cultivations for growth and lipids accumulation. Based on the conducted research, we concluded that the positional order of transcription units (TU) and the type of exploited SP affect final characteristics of the resultant consolidated biocatalyst strains, and thus could be considered as additional factors to be evaluated upon consolidated biocatalysts optimization. Key Points • Y. lipolytica growing on raw starch was constructed and tested on different substrates. • Impact of expression cassette design and SP on biocatalysts' phenotype was evidenced. • Consolidated biocatalyst process for lipids production from starch was conducted.

Yarrowia lipolytica strains and their biotechnological applications: how natural biodiversity and metabolic engineering could contribute to cell factories improvement

“…Despite some lack of evidence for estimating the risk of reshuffling problems when using repeated sequences, the research teams who developed toolboxes for Y. lipolytica engineering tend to privilege the use of different promoters and terminators as multiple TU elements in order to minimize the possibilities for homologous recombination. Interestingly, a recent process development study using a starch-utilizing GM Y. lipolytica for lipid production suggested that, beside TU design itself, the positional order of multiple TUs was an additional factor to be considered for optimized results [189].…”

“…These new GG-compatible secretion signals constitute valuable additions to the global Y. lipolytica molecular toolbox. They were notably used in a synthetic biology study in which a TU and signal peptides shuffling approach allowed the optimization of a starch-utilizing GM Y. lipolytica strain for lipid production [189].…”

Yarrowia lipolytica Strains and Their Biotechnological Applications: How Natural Biodiversity and Metabolic Engineering Could Contribute to Cell Factories Improvement

“…Despite some lack of evidence for estimating the risk of reshuffling problems when using repeated sequences, the research teams who developed toolboxes for Y. lipolytica engineering tend to privilege the use of different promoters and terminators as multiple TU elements in order to minimize the possibilities for homologous recombination. Interestingly, a recent process development study using a starch-utilizing GM Y. lipolytica for lipid production suggested that, beside TU design itself, the positional order of multiple TUs was an additional factor to be considered for optimized results [ 226 ].…”

“…The Cre-lox recombination system from bacteriophage P1 has been used at INRA to design a user-friendly system for gene deletion and marker rescue in Y. lipolytica : any integrated loxP-bordered marker TU can be efficiently excised through transient expression of the heterologous Cre recombinase using a replicative vector [ 215 ]. Different loxP-excisable markers have been included in some of the newly developed Y. lipolytica toolboxes, the two EasyCloneYALI toolkits [ 226 ] and the Golden Gate toolkit CRISPR tools [ 260 ]. Alternatively, marker rescue can be obtained through a blaster cassette strategy, by making use of the enhanced HR rate of a Δ Ku70 genetic background to allow the use of short repeated DNA bordering sequences for excising a marker for which a counter-selection is available.…”

Hydrolytic secretome engineering in Yarrowia lipolytica for consolidated bioprocessing on polysaccharide resources: review on starch, cellulose, xylan, and inulin

“…The amylase was proved to act on rice, amaranth, and pea starches in raw state and a broad panel of liquefied starches of various plant origin. The knowledge gained from those studies was applied in the following rationale-driven optimization of Y. lipolytica –based consolidated biocatalyst through combinatorial cloning of different signal peptides and changing positional order of the α-amylase and glucoamylase genes in double gene expression cassettes (Celińska et al 2020 ). The aforementioned insect α-amylase was cloned in tandem with Thermomyces lanuginosus glucoamylase having industrially relevant characteristics (Favaro et al 2015 ).…”