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

Abstract

Background: 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, gene copy number etc. In this study, we investigated if organization of a multi-element expression cassette impacts the resultant Y. lipolytica transformants’ phenotype, presuming that different variants of the cassette are composed of the same regulatory elements and encode the same hydrolases. Results: 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 pre-treated complex substrates of different plant origin for comprehensive examination of the strains’ acquired characteristics. The best strain’s performance was tested in batch bioreactor cultivations for growth and lipids accumulation. Conclusions: 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.

‘Small volume—big problem’: culturing Yarrowia lipolytica in high-throughput micro-formats

“…1 for modeling kLa in such a wide set of different vessels is burdened with uncertainty. Nevertheless, estimations of the impact of relative culture volume change or culturing system format on the relative change in oxygen availability (expressed in kLa or oxygen transfer velocity, OTV; Table S1) show good agreement between different , at different kLa parameter settings (Mendeley data [11]). Y. lipolytica strain used in this experiment was JMY2810 (genotype: MATa, ura3::pTEF-RedStar2-LEU2-Zeta-URA3ex-pTEF-empty, leu2-270, xpr2-322; phenotype: ΔAEP, ΔAXP, suc + , ura + , leu + , intracellular RedStarII, Zeta platform).…”

“…To quantitatively describe this problem, we previously conducted a small 'proof-of-concept' experiment by culturing Y. lipolytica strain synthesizing a fluorescent reporter protein (rProt) in different volumes ( [11], Table 1). Growth and fluorescence from the intracellular rProt, as well as substrate and metabolite concentration, were analyzed (Fig.…”

“…Equation 1 was developed for standard glass Erlenmeyer flasks with hydrophilic walls, shaking frequencies of 50-500 rpm, relative filling volumes of 4-20% (the relative filling volume is defined as the filling volume divided by the nominal flask volume), shaking diameters of 1.25-10 cm, and nominal flask volume between 50 and 1000 mL [43,44,50]. The 'proof-ofconcept' experiment [11] covered vessels beyond those indicated (in volume and geometry), so the use of Eq. 1 for modeling kLa in such a wide set of different vessels is burdened with uncertainty.…”

“…studies [11,16,18,26,29,59]. For example, for a culture run in O_48-well format, a 40% decrease in volume caused an increase in kLa by 63% in a study by [29], while decreasing the volume by 60% led to an increase in kLa parameter by 93% in the 'proof-of-concept' experiment [11]. Changing the culturing format from O_96-well to O_24-well triggered an increase in the kLa parameter by 9% [16] or 2% [11].…”

“…For example, for a culture run in O_48-well format, a 40% decrease in volume caused an increase in kLa by 63% in a study by [29], while decreasing the volume by 60% led to an increase in kLa parameter by 93% in the 'proof-of-concept' experiment [11]. Changing the culturing format from O_96-well to O_24-well triggered an increase in the kLa parameter by 9% [16] or 2% [11]. Going down from a test tube culture to O_96-well decreased the kLa level by 46% [59], or 54% [11].…”