In this study we aimed at optimization of starch-utilizing Y. lipolytica strains through manipulation with multigene construction design in terms of SPs and TUs order. Consequently to our previous studies we used amylolytic activities and starchy substrates as an easy to follow model, conferring strains with consolidated biocatalyst characteristics. Nevertheless, 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 [38, 39]. Such food waste streams emerge as a potential feedstock for the synthesis of microbial bioproducts, including lipids, in the frame of the circular economy concept. Within that concept, optimization of consolidated biocatalysts able to grow in starchy substrate-based, complex media gains significant importance.
For reliable comparison of different recombinant strains, it is necessary to analyze several sub-clones and to assure minimum variability in the parameters that are not a subject of the analysis. Synthetic DNA construction can be easily monitored, while integration site within the host genome is more difficult to direct, especially in the case of host that show preference towards NHEJ mechanisms, as Y. lipolytica . It is accepted that integration of a recombinant DNA construction into less or more transcriptionally active site in the Y. lipolytica genome, or the number of copies integrated with the host genomic DNA may impact expression level of transgenes and consequently resultant phenotype [45, 46]. In the present study we used zeta flanking elements and nonspecific integration into Po1h zeta-less strain, which is a commonly accepted, reliable strategy [36, 47]. Recent studies in both P. pastoris and Y. lipolytica demonstrated reasonable neutrality between different integration loci from amongst the commonly used integration targets [48, 49]. It has been revealed that nonspecifically integrated transformants showed highly uniform expression that was comparable to specific integration, suggesting that nonspecific integration does not necessarily influence expression . To minimize a risk of sub-clonal variation due to the cassette copy number or integration site, after transformation, five positive transformants bearing each type of construct were pre-screened for acquired amylolytic activity. Out of the pre-screened pool, three representative strains demonstrating negligible differences in starch consumption rate were subjected to further analyses. Such pre-selection strategy has been recently successfully used  and demonstrated that the following inter-clone variation was negligible.
Considering fundamental output of here conducted genetic manipulation, the obtained recombinant strains were conferred with the ability to grow on starch as the main carbon source, either in pre-treated or raw state. Secondly, the amount of substrate released from the biopolymer was sufficient to support growth of the recombinant strains – each time higher than WT strains, but varying, depending on the substrate and the recombinant strain variant (Fig. 3). It is well known that different starch species are highly variable in terms of their susceptibility to degradation, depending on their plant origin and characteristics of a given enzymatic activity [50–52]. Correspondingly, in our previous study we demonstrated that depending on the exploited starch type, amylolytic effect exerted by the recombinant SoAMY alpha-amylase differed dramatically . In the only previous study on construction of Y. lipolytica-based consolidated biocatalyst able to utilize starch, the Authors tested the obtained strains growth in either soluble starch, wheat raw starch, or industrial product containing starch (DZ starch; characteristics not provided) . The different starch species were used in different experiments (at different level of the biocatalysts testing), and the strains performance on those starch species was not systematically compared. The results presented in this study, demonstrate, how evaluation of a given strain’s biotechnological potential can differ, depending on the type of used starchy substrate. Furthermore, testing the obtained recombinant strains towards different starch species, introduced variability to the results, reflected by both - the overall level of the substrate degradation (the highest for CR, followed by CC, with the lowest values observed for CP) as well as the distribution of most / least efficient recombinants, representing genetic constructs differing in SPs and the positional order of TUs (Fig. 3.A.B.). For example, strains bearing G1TG2S constructs with SP1 sequences were particularly efficient in consumption of raw starch (Fig. 3.B.). Their predominance in raw starch digestion was clear when compared to the strains bearing the same order of TUs but different SP, and when compared with the strains bearing alternative organization of TUs and genes initiated with the same SP1. Corresponding conclusions were withdrawn for the strains bearing genes initiated with SP3 sequences. With respect to cooked starch utilization, SP3-equipped G1TG2S variant turned out to uniformly endow the resultant strains with efficient amylolytic phenotype. While in the case of raw starch digestion, the degree of the substrate consumption was rather comparable (RR vs RP), cooked starch of different plant origin showed highly variable susceptibility towards SoAMY and TlGAMY action, ultimately leading to variable level of the substrate consumption. Integration of results for gene expression (Fig. 4.) and starch consumption (Fig. 3.) revealed that genes initiated with 5’ sequences encoding SP1 and SP3 were expressed at higher level, which could contribute to the observed efficient amylolytic phenotype. However, no direct, straightforward relationship between a specific gene localization (G1 or G2), its expression level and the overall amylolytic activity could be observed. It suggests that differences in amylolytic activity observed between G1SG2T and G1TG2S strains do not derive directly from differences in the gene expression level. It is commonly accepted that the final enzymatic activity is not a first-order function of a given recombinant gene’s expression level, as evidenced earlier, also for Y. lipolytica .
In the only previous literature report describing starch-utilizing Y. lipolytica , the obtained strain was tested for lipids production. In that study, to maximize production of FA, the C/N ratio of the culturing medium was set at 60 and 90, resulting in enhanced FA accumulation from 4.4 to 7.2 FA %DCW (0.49 vs 0.8 total FA g/L). Even more pronounced effect was observed when the two “amylolytic” genes were expressed in “a lipid overproducer” strain, heavily modified in FA and TAG turnover net [19, 55–57]. In such background, lipids accumulation increased to 21.1 and 27% (FA %DCW; 2.44 ± 0.15 and 3.34 ± 0.13 g/L, for C/N ratio 60 and 90, respectively). In the present study, the microbial lipid accumulation in bioreactor culture (Fig. 6.) was at a level comparable to previous reports for a strain not engineered with respect to TAG turnover cultivated on carbohydrates (0.64 ± 0.08 g/L of lipids, 7.69–4.85% DCW, depending on the culturing time), even though the C/N ratio of the present medium equaled to 8.23 (not optimal for FA accumulation, but promoting growth and enzymes synthesis). Nevertheless, to generate highly efficient lipid producer from starchy wastes, the optimized cassette could be transformed in to lipid overproducer strain background, as previously . Strains optimized in this trait, upon cultivation in a medium of high C/N ratio can accumulate as much as 30–70% of total lipids [6, 20, 56]. On the other hand, we observed that here obtained, optimized strain (F215) exhibited much faster substrate utilization in time than the starch-utilizing Y. lipolytica strain constructed previously . In that previous study, after 168 h of culture the strains utilized from 49.4 ± 2.4% to 60.3 ± 6.4% of starch (29.64 to 54.27 g/L in 168 h, depending of the strain and culture medium); based provided data it was calculated that the total substrate consumption in time ranged between 0.1769 to 0.323 g/(L*h). In the present study, depending on the culturing time, substrate utilization in time reached 0.64 and 0.4 g/(L*h) at the 48 h and the end of culturing (after 48 h further consumption was negligible), with substrate utilization rate ΔS/Δt ranging 2.45–0.14 g/(L*h) (Additional File_5).
Finally, here observed FA profile, was corresponding to previous reports on FA profile in Y. lipolytica cultivated on standard substrates, like glycerol and glucose , or more complex media like sugar beet molasses , with dominance of unsaturated C18:2 and C18:1, followed by saturated form of C16:0. In previous study on cultivation of engineered Y. lipolytica on starch , FA profile was mainly represented by C18:1, but the percentage contribution of C18:2 was much lower than in the present study. On the other hand, in that previous report, longer chain FA (C24:0) were produced at detectable level, which was not the case in the current study. This difference could result from differences in technical / analytical approach. Interestingly, the observed FA profile, especially in terms of percentage content of C18:1 and C16:0, so the dominant FA detected, was also similar to FA profile observed in the other oleaginous yeast Rhodosporidium toruloides grown on cassava starch hydrolysate . The other compounds, typical for Y. lipolytica metabolism were detected at surprisingly low levels. Erythritol, mannitol and citric acid were produced at the levels below 1 g/L. Importantly, the same observation was done in , where small molecular metabolites were detected at close to zero level.