Rapid micro-assays for amylolytic activities determination: customization and validation of the tests

Białas

et al. 2019

Yeast

Self Cite

Fed-batch cultivation is the preferred bioprocessing strategy applied in microbial production of proteins. Feeding strategy is crucial parameters to be optimized upon development of a fed-batch process. In this study, we investigated impact of different feeding strategies on production of recombinant enzymatic protein in Yarrowia lipolytica cultures. From amongst tested strategies, comprising intermittent and continuous feedings, also in cascade with respiratory factors, intermittent feeding executed after complete exhaustion of glycerol from the medium, with moderate amplitude of osmolarity, was the most beneficial in terms of the secretory enzyme amount, its volumetric productivity and specific activity. Because adopted feeding strategies strongly modulated osmolarity of the cultures, the effect of osmotic pressure on production of the target heterologous protein was investigated in a series of batch cultivations with addition of osmoactive compounds (NaCl, sorbitol, sucrose, and glycerol) at different concentrations. Although obvious promoting effect of the osmoactive substances on the enzyme production was clear, no straightforward correlation between the medium osmolarity and the target enzyme's specific activity could be observed. These results suggest that not only the level of osmolarity but also chemical character of the osmoactive compound have both important impact on the production of secretory proteins in Y. lipolytica cultures.

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Feeding strategy impacts heterologous protein production in Yarrowia lipolytica fed‐batch cultures—Insight into the role of osmolarity

Kubiak

Białas

et al. 2019

Yeast

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“…The amount of residual starch contained in the post-culturing media was used as a measure of the recombinant strains' amylolytic activity. The protocol for starch concentration assessment (microSIT) was described previously (Borkowska et al 2019) with modifications regarding preparation of raw starchbased samples (described below). Each of the batch cultivations was analyzed in technical duplicate.…”

Section: Analysis Of Starch Hydrolysis Degreedetermination Of Residuamentioning

confidence: 99%

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

Celińska

Appl Microbiol Biotechnol

Korpys-Woźniak

et al. 2020

Self Cite

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.

“…The amount of residual starch contained in the post-culturing media was used as a measure of the recombinant strains' amylolytic activity. The protocol for starch concentration assessment (microSIT) was described previously [63] with modifications regarding preparation of raw starch-based samples (described below). Each of the batch cultivations was analyzed in technical duplicate.…”

Section: Analysis Of Substrate Consumption Degree -Determination Of Rmentioning

confidence: 99%

“…Details on sensitivity and range of the analytical methods are given in the original report, where the micro-assays were described [63]. The raw starch-containing samples were processed correspondingly, with the difference that the starch granules were initially cooked prior to the test.…”

Section: Analysis Of Substrate Consumption Degree -Determination Of Rmentioning

confidence: 99%

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

Celińska

Korpys-Woźniak

et al. 2020

Preprint

Self Cite

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.