Acetic and citric acids effect the type II secretion system and decrease the metabolic activities of salmon spoilage-related Rahnella aquatilis KM05.

Rahnella aquatilis causes seafoods to spoil by metabolizing sulfur-containing amino acids and/or proteins, producing H2S in products. The type II secretion system (T2SS) regulates the transport of proteases from the cytoplasm to the surrounding environment and promotes bacterial growth at low temperatures. To prevent premature fish spoilage, new solutions for inhibiting the T2SS of bacteria should be researched. In this study, global transcriptome sequencing was used to analyze the spoilage properties of R. aquatilis KM05. Two of the mapped genes/coding sequences (CDSs) were matched to the T2SS, namely, qspF and gspE, and four of the genes/CDSs, namely, ftsH, rseP, ptrA and pepN, were matched to metalloproteases or peptidases in R. aquatilis KM05. Subinhibitory concentrations of citric (18 µM) and acetic (41 µM) acids caused downregulation of T2SS-related genes (range from - 1.0 to -4.5) and genes involved in the proteolytic activities of bacteria (range from - 0.5 to -4.0). The proteolytic activities of R. aquatilis KM05 in vitro were reduced by an average of 40%. The in situ experiments showed the antimicrobial properties of citric and acetic acids against R. aquatilis KM05; the addition of an acidulant to salmon fillets limited microbial growth. Citric and acetic acids extend the shelf life of fish-based products and prevent food waste.

Multiple region high resolution melting-based method for accurate differentiation of food-derived yeasts at species level resolution.

Yeasts comprise a divergent group of microorganisms playing an important role in foods production. To monitor the production processes, validate authenticity and safety of foods, reliable methods of yeast identification and differentiation are needed. Nowadays, traditional PCR/sequencing-based methods are replaced by rapid techniques not requiring post-PCR processing. In the present study, we developed a three region-based qPCR-HRM protocol, for rapid differentiation of yeast species isolated from food products. The three targeted fragments (26S rDNA, 18S rDNA, ITS) were carefully analyzed to dock the primers at inter-species conservative regions, flanking polymorphic regions of ∼200 bp. Thirty eight yeast strains were used as a training material. The collection of yeast spanned Pichia, Clavispora, Candida, Yarrowia, Kluyveromyces, Saccharomyces, and Wickerhamomyces genera. MALDI-TOF mass spectrometry and conventional rDNA sequencing were used for validation. Conducted studies demonstrated that when used individually, each of the three regions analyzed by qPCR-HRM possessed its own yeast species-specific limitations, sometimes leading to inaccurate taxonomic classification. On the other hand, simultaneous analysis of the three proposed regions resulted in rapid and adequate differentiation of all the yeast strains at species-level resolution.

A new set of reference genes for comparative gene expression analyses in Yarrowia lipolytica.

Accurate quantitation of gene expression levels require sensitive, precise and reproducible measurements of specific transcripts. Normalization to a reference gene is the most common practice to minimize the impact of the uncontrolled variation. The fundamental prerequisite for an accurate reference gene is to be stably expressed amongst all the samples included in the analysis. In the present study we aimed to assess the expression level and stability of a panel of 21 genes in Yarrowia lipolytica throughout varying conditions, covering composition of the culturing medium, growth phase and strain-wild type and recombinant burdened with heterologous protein overexpression. The panel of the selected candidate genes covered those essential for growth and maintenance of metabolism and homologs of commonly used internal references in RT-qPCR. The candidate genes expression level and stability were assessed and the data were processed using dedicated computational tools (geNorm and NormFinder). The results obtained here indicated genes unaffected by the burden of overexpression (TEF1, TPI1, UBC2, SRPN2, ALG9-like, RYL1) or by the culture medium used (ACT1, TPI1, UBC2, SEC61, ODC, CLA4, FKS1, TPS1), as well as those the least (SSDH, ODC, GPD) and the most (SEC62, TPI1, IPP1) suitable for normalization of RT-qPCR data in Y. lipolytica.

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

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.

Production of Raw Starch-Digesting Amylolytic Preparation in Yarrowia lipolytica and Its Application in Biotechnological Synthesis of Lactic Acid and Ethanol.

Sustainable economy drives increasing demand for raw biomass-decomposing enzymes. Microbial expression platforms exploited as cellular factories of such biocatalysts meet requirements of large-volume production. Previously, we developed Yarrowia lipolytica recombinant strains able to grow on raw starch of different plant origin. In the present study, we used the most efficient amylolytic strain as a microbial cell factory of raw-starch-digesting (RSD) amylolytic preparation composed of two enzymes. The RSD-preparation was produced in fed-batch bioreactor cultures. Concentrated and partly purified preparation was then tested in simultaneous saccharification and fermentation (SSF) processes with thermotolerant Kluyveromyces marxianus for ethanol production and Lactobacillus plantarum for production of lactic acid. These processes were conducted as a proof-of-concept that application of the novel RSD-preparation supports sufficient starch hydrolysis enabling microbial growth and production of targeted molecules, as the selected strains were confirmed to lack amylolytic activity. Doses of the preparation and thermal conditions were individually adjusted for the two processes. Additionally, ethanol production was tested under different aeration strategies; and lactic acid production process was tested in thermally pre-treated substrate, as well. Conducted studies demonstrated that the novel RSD-preparation provides satisfactory starch hydrolyzing activity for ethanol and lactic acid production from starch by non-amylolytic microorganisms.

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

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.

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

High-throughput function-based screening techniques remain the major bottleneck in the novel biocatalysts development pipeline. In the present study, we customized protocols for amylolytic activity determination (Somogyi-Nelson and starch-iodine tests) to micro-volume thermalcycler-based assays (linearity range 60-600 µM of reducing sugar, R2 = 0.9855; 0-2 mg/mL of starch, R2 = 0.9921, respectively). Exploitation of a thermalcycler enabled rapid and accurate temperature control, further reduction of reagents and samples volumes, and limited evaporation of the reaction mixtures, meeting several crucial requirements of an adequate enzymatic assay. In the optimized micro-volume Somogyi-Nelson protocol, we were able to reduce the time required for high-temperature heating sixfold (down to 5 min) and further increase sensitivity of the assay (tenfold), when compared to the previous MTP-based protocol. The optimized microassays have complementary scope of specificities: micro-starch-iodine test for endoglucanases, micro-Somogyi-Nelson test for exoglucanases. Due to rapid, micro-volume and high-throughput character, the methods can complement toolbox assisting development of novel biocatalysts and analysis of saccharides-containing samples.

Genetic engineering of Ehrlich pathway modulates production of higher alcohols in engineered Yarrowia lipolytica.

Microbial cells can produce a vast spectrum of chemical compounds, including those most desired by the global chemical market, for example, higher alcohols, which are promising alternative fuels and chemical feedstock. In the current research, we investigated the effects of the Ehrlich pathway genetic engineering on higher alcohols production in Yarrowialipolytica, which directly follows our previous findings concerning elucidation of putative molecular identities involved in this pathway. To this end, we constructed two alternative expression cassettes composed of previously identified genes, putatively involved in the Ehrlich pathway in Y. lipolytica, and cloned them under the control of constitutive pTEF promoter, and by this released them from extensive native regulation. The effects of the pathway engineering were investigated upon provision of different Ehrlich pathway-inducing amino acids (L-Phe, L-Leu, L-Ile and L-Val). In general, amplification of the Ehrlich pathway in many cases led to increased formation of a respective higher alcohol from its precursor. We observed interesting effects of aminotransferase BAT2 deletion on synthesis of 2-phenylethanol and its acetate ester, significant relationship between L-Val and L-Phe catabolic pathways and extensive 'cross-induction' of the derivative compounds synthesis by non-direct precursors.

Robust signal peptides for protein secretion in Yarrowia lipolytica: identification and characterization of novel secretory tags.

Upon expression of a given protein in an expression host, its secretion into the culture medium or cell-surface display is frequently advantageous in both research and industrial contexts. Hence, engineering strategies targeting folding, trafficking, and secretion of the proteins gain considerable interest. Yarrowia lipolytica has emerged as an efficient protein expression platform, repeatedly proved to be a competitive secretor of proteins. Although the key role of signal peptides (SPs) in secretory overexpression of proteins and their direct effect on the final protein titers are widely known, the number of reports on manipulation with SPs in Y. lipolytica is rather scattered. In this study, we assessed the potential of ten different SPs for secretion of two heterologous proteins in Y. lipolytica. Genomic and transcriptomic data mining allowed us to select five novel, previously undescribed SPs for recombinant protein secretion in Y. lipolytica. Their secretory potential was assessed in comparison with known, widely exploited SPs. We took advantage of Golden Gate approach, for construction of expression cassettes, and micro-volume enzymatic assays, for functional screening of large libraries of recombinant strains. Based on the adopted strategy, we identified novel secretory tags, characterized their secretory capacity, indicated the most potent SPs, and suggested a consensus sequence of a potentially robust synthetic SP to expand the molecular toolbox for engineering Y. lipolytica.

Pichia cactophila and Kluyveromyces lactis are Highly Efficient Microbial Cell Factories of Natural Amino Acid-Derived Aroma Compounds.

The pivotal role of non-conventional yeast (NCY) species in formation of valuable aroma compounds in various food commodities is widely acknowledged. This fact inspires endeavors aiming at exploitation of food-derived NCYs as biocatalysts in natural aromas production. In this study, we isolated, characterized and evaluated aroma-producing capacity of two NCY representatives-Pichia cactophila 7.20 and Klyuveromyces lactis 6.10 strains. The strains were isolated from food-related habitats-goat-milk regional cheese and Swiss-type ripening cheese, respectively. Aroma profiles generated by the two strains cultured in a general rich medium were analyzed through solvent extraction and GC-MS analysis of the compounds retained in the culture media. Finally, the strains were tested in bioconversion cultures with branched chain- or aromatic amino acids as the sole nitrogen source, to assess capability of the strains towards formation of amino acid-derived aromas. The results showed extraordinary capacity of both strains for production of 2-phenylethanol (at more than 3 g/L) and isoamyl alcohol (approx. 1.5 g/L). A distinctive trait of 2-phenylethyl acetate synthesis at high concentrations (0.64 g/L) was revealed for P. cactophila 7.20 strain. Highly valued disulfide dimethyl as well as methionol acetate were identified amongst the aroma compounds synthesized by the strains.

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

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). Copyright © 2015 John Wiley & Sons, Ltd.

Evaluation of a recombinant insect-derived amylase performance in simultaneous saccharification and fermentation process with industrial yeasts.

Starch is the dominant feedstock consumed for the bioethanol production, accounting for 60 % of its global production. Considering the significant contribution of bioethanol to the global fuel market, any improvement in its major operating technologies is economically very attractive. It was estimated that up to 40 % of the final ethanol unit price is derived from the energy input required for the substrate pre-treatment. Application of raw starch hydrolyzing enzymes (RSHE), combined with operation of the process according to a simultaneous saccharification and fermentation (SSF) strategy, constitutes the most promising solutions to the current technologies limitations. In this study, we expressed the novel RSHE derived from an insect in Saccharomyces cerevisiae strain dedicated for the protein overexpression. Afterwards, the enzyme performance was assessed in SSF process conducted by industrial ethanologenic or thermotolerant yeast species. Comparison of the insect-derived RSHE preparation with commercially available amylolytic RSH preparation was conducted. Our results demonstrate that the recombinant alpha-amylase from rice weevil can be efficiently expressed and secreted with its native signal peptide in S. cerevisiae INVSc-pYES2-Amy1 expression system (accounting for nearly 72 % of the strain's secretome). Application of the recombinant enzyme-based preparation in SSF process secured sufficient amylolytic activity for the yeast cell propagation and ethanol formation from raw starch. (Oligo)saccharide profiles generated by the compared preparations differed with respect to homogeneity of the sugar mixtures. Concomitantly, as demonstrated by a kinetic model developed in this study, the kinetic parameters describing activity of the compared preparations were different.

Cloning, expression, and purification of insect (Sitophilus oryzae) alpha-amylase, able to digest granular starch, in Yarrowia lipolytica host.

Raw-starch-digesting enzymes (RSDE) are of major importance for industrial applications, as their usage greatly simplifies the starch processing pipeline. To date, only microbial RSDE have gained considerable attention, since only microbial production of enzymes meets industrial demands. In this study, α-amylase from rice weevil (Sitophilus oryzae), the major rice pest, was cloned and expressed in Yarrowia lipolytica Po1g strain. The enzyme was secreted into the culture medium, and the peak activity (81 AU/L) was reached after only 29 h of culturing in 5-L bioreactors. Through simple purification procedure of ammonium sulfate precipitation and affinity chromatography, it was possible to purify the enzyme to apparent homogeneity (25-fold purification factor, at 5 % yield). The optimal conditions for the α-amylase activity were pH 5.0 and a temperature of 40 °C. The α-amylase studied here did not show any obligate requirement for Ca(2+) ions. The recombinant α-amylase appeared to efficiently digest granular starch from pea, amaranth, waxy corn, and waxy rice.

SpaCBA sequence instability and its relationship to the adhesion efficiency of Lactobacillus casei group isolates to Caco-2 cells.

The ability to adhere to enterocytes is one of the key features of probiotics. This process involves a number of factors, among which the important role of pili was demonstrated. Some Lactobacillus species are confirmed to have heterotrimeric spaCBA type pili. The aim of this study was to identify spaCBA pili in strains of selected Lactobacillus spp. and assess the impact of their presence and sequence polymorphism on the adhesion of these strains to enterocytes. Total 20 bacterial strains of L. rhamnosus, L. casei and L. paracasei were tested. The presence of pilus specific proteins coding genes spaA, spaB and spaC was verified by PCR in order to identify the presence of sequence polymorphism in the genes possibly affecting the structure of the spaCBA pilus. To correlate spaCBA polymorphism to adhesion capability the adhesion assay was carried out using Caco-2 cell line. The effectiveness of the adhesion was measured using a scintillation counter. The Lactobacillus strains analyzed showed the adhesion to Caco-2 enterocytes capability from 0.6% to 19.6%. The presence of spaCBA pili is a factor increasing the adhesion efficiency of Lactobacillus spp. to Caco-2 enterocytes. Lack of these structures on the surface of bacterial cells results in the reduction in adhesion efficiency, indicating its important role in the adhesion process. But not in all cases the correlation between the presence of protein spaCBA structures and adhesion efficiency was observed, what may indicate the important role of other factors in adhesion of analyzed strains to Caco-2 cells.

Evaluation of quantitative PCR measurement of bacterial colonization of epithelial cells.

Microbial colonization is an important step in establishing pathogenic or probiotic relations to host cells and in biofilm formation on industrial or medical devices. The aim of this work was to verify the applicability of quantitative PCR (Real-Time PCR) to measure bacterial colonization of epithelial cells. Salmonella enterica and Caco-2 intestinal epithelial cell line was used as a model. To verify sensitivity of the assay a competition of the pathogen cells to probiotic microorganism was tested. The qPCR method was compared to plate count and radiolabel approach, which are well established techniques in this area of research. The three methods returned similar results. The best quantification accuracy had radiolabel method, followed by qPCR. The plate count results showed coefficient of variation two-times higher than this of qPCR. The quantitative PCR proved to be a reliable method for enumeration of microbes in colonization assay. It has several advantages that make it very useful in case of analyzing mixed populations, where several different species or even strains can be monitored at the same time.