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.
Highlights
Efficient biorefining and bioprocessing rely on exploitation of renewable substrates, like residual biomass, which in its raw, non-pretreated form is mainly composed of complex biopolymers
We aimed at optimization of starch-utilizing Y. lipolytica strains through manipulation with multi-gene construction design in terms of signal peptides (SP) and transcription units (TU) order
It has been recently pointed that starch-rich waste and by-product streams generated by bakery, confectionery, and wheat-milling plants could be employed as the sole raw materials for generic cultivation media suitable for microorganisms (Tsakona et al 2014; Tsakona et al 2019)
Summary
Efficient biorefining and bioprocessing rely on exploitation of renewable substrates, like residual biomass, which in its raw, non-pretreated form is mainly composed of complex biopolymers. It is thought that multiple transformations, as required for the former strategy, may negatively impact the overall fitness of the transformed cells, which at some level may impose significant limitations to a given bioprocess The latter strategy, relying on construction of a multi-gene expression cassette, bears its limitations. We pursued optimization of Y. lipolytica-based consolidated biocatalyst To this end, we used previously determined optimal fusions between signal peptides (SP) and polypeptides having amylolytic activities (Celińska et al 2018), and organized them in tandem expression cassettes bearing two TUs. Further, we investigated if the order of TUs within the expression cassettes and type of SP in the pre-proteins’ impact expression of heterologous genes and the resultant strains’ characteristics. Current waste treatments for these streams comprise animal feed, composting, or disposal in landfills, while could be used for “green” production of high-value added products through microbial transformations
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
