Evaluation of heterologous α‐amylase production in two expression platforms dedicated for Yarrowia lipolytica: commercial Po1g–pYLSC (php4d) and custom‐made A18–pYLTEF (pTEF)

Abstract

In view of the constantly increasing demand for cost-effective, low-energy and environmentally friendly industrial processes and household care products, enzyme production occupies an essential place in the field of biotechnology. Along with increasing demand for industrial and household care enzymes, the demand for heterologous expression platforms has also increased. Apart from the conventional hosts, e.g. Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris, routinely used in heterologous protein expression, the non-conventional ones have become more and more exploited in this field. Among the available yeast host systems, Yarrowia lipolytica appears to be an attractive alternative. The aim of this study was to compare efficiency of two Yarrowia-based expression platforms, commercial Po1g-pYLSC and custom-made A18-pYLTEF, in expression of an insect-derived, raw-starch-digesting α-amylase, to select the 'champion' system for further studies on this valuable enzyme. Both expression platforms were compared with respect to copy number of the integrated expression cassette/transformed genome, and the recombinant strains performance (Po1g-pYLSC-derived 4.29 strain, and A18-pYLTEF-derived B9 strain) during batch bioreactor cultures. Our results demonstrate that the average number of integration events into the recipient's genome was comparable for both expression systems under investigation, but with varying distribution of the multicopy integrants; and the number of the recombinant gene copies was highly correlated with the acquired amylolytic activity of the strains. Due to severe susceptibility of the recombinant AMY1 polypeptide to native proteases of the custom-made expression system, the final yield of the enzyme was substantially lower when compared to the commercial Po1g-pYLSC (reaching a maximum level of 142.84 AU/l).

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

“…Consistently, our previous studies have shown that such a mode of cultivation had beneficial effect on the production of heterologous alpha‐amylase by Y . lipolytica , as the titer of the extracellular protein was increased by 160 fold (max activity 22,979.2 AU L −1 AUl −1 h −1 AUl −1 h −1 ; Celińska, Borkowska, & Białas, ), when compared with previously obtained result in batch cultivation (142.84 AU L −1 AUl −1 h −1 AUl −1 h −1 ; Celińska, Borkowska, & Białas, ). Another interesting example of fed‐batch cultures optimization was presented in a series of papers by (Chang, Ryu, Park, & Kim, ; Chang, Ryu, Park, Kim, & Ogrydziak, ; Kim et al, ), which concerned production of rice alpha‐amylase in recombinant Y .…”

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

“…Surprisingly, micro-/macro-SIT assays were in this case more sensitive to glucoamylase activity which, taken together with the results presented in (Fig. 4b), clearly show that constituents of the crude culture media disallow accurate assessment of TlGAMY activity. This in turn, also explains poor performance of macro-/micro-SNT assays shown in (Fig.…”

“…One of the factors contributing to such an outcome could be formation of the insoluble precipitate encountered in the reactions with the crude culture media supernatants. In the macro-scale, the precipitate could be removed by simple centrifugation of the processed samples, as we did previously (Celińska et al 2015, 2016a, b, 2017). Nevertheless, additional step obviously disrupts rapid character of the methods, and thus disqualifies it from high-throughput screening of crude culture media.…”

“…Our recent studies were focused on expression of an insect (S itophilus oryzae ) gene encoding alpha-amylase ( SoAMY ) and a fungal ( Thermomyces lanuginosus ) glucoamylase ( TlGAMY ) in an efficient expression host—a nonconventional yeast species Yarrowia lipolytica (Celińska et al 2015, 2016b, 2017; Celińska et al 2018). We have demonstrated that the recombinant alpha-amylase exhibits raw starch-digesting properties (Celińska et al 2016a) which is one of the most desired, industrially relevant characteristics of the alpha-amylases (Sun et al 2010). Alpha-amylases are endoglucanases that cleave internal α-1,4-glycosidic bonds in starch to produce shorter saccharides.…”

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

“…The strains subjected to this analysis were confirmed to bear an estimated single copy of the heterologous expression cassette, using RTqPCR on genomic DNA (Additional File_3), as described previously [42]. Gene expression analysis was conducted in growth phase-synchronized cultures to minimize variability of the analysis [43].…”