Strategies for Efficient Expression of Heterologous Monosaccharide Transporters in Saccharomyces cerevisiae

In previous work, we developed a Saccharomyces cerevisiae strain (DLG-K1) lacking the main monosaccharide transporters (hxt-null) and displaying high xylose reductase, xylitol dehydrogenase and xylulokinase activities. This strain proved to be a useful chassis strain to study new glucose/xylose transporters, as SsXUT1 from Scheffersomyces stipitis. Proteins with high amino acid sequence similarity (78–80%) to SsXUT1 were identified from Spathaspora passalidarum and Spathaspora arborariae genomes. The characterization of these putative transporter genes (SpXUT1 and SaXUT1, respectively) was performed in the same chassis strain. Surprisingly, the cloned genes could not restore the ability to grow in several monosaccharides tested (including glucose and xylose), but after being grown in maltose, the uptake of 14C-glucose and 14C-xylose was detected. While SsXUT1 lacks lysine residues with high ubiquitinylation potential in its N-terminal domain and displays only one in its C-terminal domain, both SpXUT1 and SaXUT1 transporters have several such residues in their C-terminal domains. A truncated version of SpXUT1 gene, deprived of the respective 3′-end, was cloned in DLG-K1 and allowed growth and fermentation in glucose or xylose. In another approach, two arrestins known to be involved in the ubiquitinylation and endocytosis of sugar transporters (ROD1 and ROG3) were knocked out, but only the rog3 mutant allowed a significant improvement of growth and fermentation in glucose when either of the XUT permeases were expressed. Therefore, for the efficient heterologous expression of monosaccharide (e.g., glucose/xylose) transporters in S. cerevisiae, we propose either the removal of lysines involved in ubiquitinylation and endocytosis or the use of chassis strains hampered in the specific mechanism of membrane protein turnover.

In Vitro and In Silico Approaches for the Evaluation of Antimicrobial Activity, Time-Kill Kinetics, and Anti-Biofilm Potential of Thymoquinone (2-Methyl-5-propan-2-ylcyclohexa-2,5-diene-1,4-dione) against Selected Human Pathogens

Thymoquinone (2-methyl-5-propan-2-ylcyclohexa-2,5-diene-1,4-dione; TQ), a principal bioactive phytoconstituent of Nigella sativa essential oil, has been reported to have high antimicrobial potential. Thus, the current study evaluated TQ’s antimicrobial potential against a range of selected human pathogens using in vitro assays, including time-kill kinetics and anti-biofilm activity. In silico molecular docking of TQ against several antimicrobial target proteins and a detailed intermolecular interaction analysis was performed, including binding energies and docking feasibility. Of the tested bacteria and fungi, S. epidermidis ATCC 12228 and Candida albicans ATCC 10231 were the most susceptible to TQ, with 50.3 ± 0.3 mm and 21.1 ± 0.1 mm zones of inhibition, respectively. Minimum inhibitory concentration (MIC) values of TQ are in the range of 12.5–50 µg/mL, while minimum biocidal concentration (MBC) values are in the range of 25–100 µg/mL against the tested organisms. Time-kill kinetics of TQ revealed that the killing time for the tested bacteria is in the range of 1–6 h with the MBC of TQ. Anti-biofilm activity results demonstrate that the minimum biofilm inhibitory concentration (MBIC) values of TQ are in the range of 25–50 µg/mL, while the minimum biofilm eradication concentration (MBEC) values are in the range of 25–100 µg/mL, for the tested bacteria. In silico molecular docking studies revealed four preferred antibacterial and antifungal target proteins for TQ: D-alanyl-D-alanine synthetase (Ddl) from Thermus thermophilus, transcriptional regulator qacR from Staphylococcus aureus, N-myristoyltransferase from Candida albicans, and NADPH-dependent D-xylose reductase from Candida tenuis. In contrast, the nitroreductase family protein from Bacillus cereus and spore coat polysaccharide biosynthesis protein from Bacillus subtilis and UDP-N-acetylglucosamine pyrophosphorylase from Aspergillus fumigatus are the least preferred antibacterial and antifungal target proteins for TQ, respectively. Molecular dynamics (MD) simulations revealed that TQ could bind to all four target proteins, with Ddl and NADPH-dependent D-xylose reductase being the most efficient. Our findings corroborate TQ’s high antimicrobial potential, suggesting it may be a promising drug candidate for multi-drug resistant (MDR) pathogens, notably Gram-positive bacteria and Candida albicans.

Rational engineering of industrial S. cerevisiae: towards xylitol production from sugarcane bagasse

BACKGROUND: Sugarcane hemicellulosic material is a compelling source of usually neglected xylose that could figure as feedstock to produce chemical building blocks of high economic value, such as xylitol. In this context, Saccharomyces cerevisiae strains typically used in the Brazilian bioethanol industry are a robust chassis for genetic engineering, given their robustness towards harsh operational conditions and outstanding fermentation performance. Nevertheless, there are no reports on the use of these strains for xylitol production using sugarcane hydrolysate. RESULTS: Potential single-guided RNA off-targets were analyzed in two preeminent industrial strains (PE-2 and SA-1), providing a database of 5'-NGG 20 nt sequences, and guidelines for the fast and cost-effective CRISPR-editing of such strains. After genomic integration of a NADPH-preferring xylose reductase (XR), FMYX (SA-1 hoΔ::xyl1) and CENPKX (CEN.PK-122 hoΔ::xyl1) were tested in varying cultivation conditions for xylitol productivity to infer the influence of the genetic background. Near-theoretical yields were achieved for all strains, however, the industrial consistently outperformed the laboratory strain. Batch fermentation of raw sugarcane bagasse hydrolysate with remaining solid particles represented a challenge for xylose metabolization and 3.65 ± 0.16 g/L xylitol titre was achieved by FMYX. Finally, quantification of NADPH - cofactor implied in XR activity - revealed that FMYX has 33% more available cofactors than CENPKX. CONCLUSIONS: Although widely used in several S. cerevisiae strains, this is the first report of CRISPR-Cas9 editing major yeast of the Brazilian bioethanol industry. Fermentative assays of xylose consumption revealed that NADPH availability is closely related to mutant strains' performance. We also pioneer the use of sugarcane bagasse as a substrate for xylitol production. Finally, we demonstrate how industrial background SA-1 is a compelling chassis for the second-generation industry, given its inhibitor tolerance and better redox environment that may favor the production of reduced sugars.

New Strategies for Efficient Expression of Heterologous Sugar Transporters in Saccharomyces cerevisiae

: In our previous work we had developed an hxt-null Saccharomyces cerevisiae strain displaying high xylose reductase, xylitol dehydrogenase and xylulokinase activities that proved to be useful as a chassis strain to study new xylose transporters, as SsXUT1 from Scheffersomyces stipitis. Spathaspora passalidarum and Spathaspora arborariae have in their genomes genes with high sequence similarity (78-80%) to SsXUT1. To characterize these putative transporter genes (SpXUT1 and SaXUT1, respectively) they were expressed in the same chassis strain as SsXUT1. Surprisingly, the cloned genes could not restore the ability to grow in several monosaccharides tested, although the strains expressing the SsXUT1 and SpXUT1 permeases, after growth on maltose, showed the presence of 14C-glucose and 14C-xylose transport activity. An important feature of these permeases is that SsXUT1 lacks lysine residues in its N-terminal domain with high-confidence ubiquitinylation potential, and has only one at the C-terminal domain, while the SpXUT1 transporter had several of such residues at its C-terminal domain. When the SpXUT1 gene was cloned in a truncated version lacking such lysine residues, the permease allowed grow on glucose or xylose, and even promoted xylose fermentation by the hxt-null strain. In another approach, we deleted two arrestins known to be involved in sugar transporter ubiquitinylation and endocytosis (ROD1 and ROG3), but only the rog3Δ strain allowed modest growth on these sugars. Taken together, these results suggest that to allow efficient sugar transporter expression in S. cerevisiae the lysines involved in transporter endocytosis should be removed from the sequence of the permease.

Protein acetylation regulates xylose metabolism during adaptation of Saccharomyces cerevisiae

Background As the second most abundant polysaccharide in nature, hemicellulose can be degraded to xylose as the feedstock for bioconversion to fuels and chemicals. To enhance xylose conversion, the engineered Saccharomyces cerevisiae with xylose metabolic pathway is usually adapted with xylose as the carbon source in the laboratory. However, the mechanism under the adaptation phenomena of the engineered strain is still unclear. Results In this study, xylose-utilizing S. cerevisiae was constructed and used for the adaptation study. It was found that xylose consumption rate increased 1.24-fold in the second incubation of the yYST12 strain in synthetic complete-xylose medium compared with the first incubation. The study figured out that it was observed at the single-cell level that the stagnation time for xylose utilization was reduced after adaptation with xylose medium in the microfluidic device. Such transient memory of xylose metabolism after adaptation with xylose medium, named “xylose consumption memory”, was observed in the strains with both xylose isomerase pathway and xylose reductase and xylitol dehydrogenase pathways. In further, the proteomic acetylation of the strains before and after adaptation was investigated, and it was revealed that H4K5 was one of the most differential acetylation sites related to xylose consumption memory of engineered S. cerevisiae. We tested 8 genes encoding acetylase or deacetylase, and it was found that the knockout of the GCN5 and HPA2 encoding acetylases enhanced the xylose consumption memory. Conclusions The behavior of xylose consumption memory in engineered S. cerevisiae can be successfully induced with xylose in the adaptation. H4K5Ac and two genes of GCN5 and HPA2 are related to xylose consumption memory of engineered S. cerevisiae during adaptation. This study provides valuable insights into the xylose adaptation of engineered S. cerevisiae.

Reductive enzymatic dynamic kinetic resolution affording 115 g/L (S)-2-phenylpropanol

Background Published biocatalytic routes for accessing enantiopure 2-phenylpropanol using oxidoreductases afforded maximal product titers of only 80 mM. Enzyme deactivation was identified as the major limitation and was attributed to adduct formation of the aldehyde substrate with amino acid residues of the reductase. Results A single point mutant of Candida tenuis xylose reductase (CtXR D51A) with very high catalytic efficiency (43·103 s−1 M−1) for (S)-2-phenylpropanal was found. The enzyme showed high enantioselectivity for the (S)-enantiomer but was deactivated by 0.5 mM substrate within 2 h. A whole-cell biocatalyst expressing the engineered reductase and a yeast formate dehydrogenase for NADH-recycling provided substantial stabilization of the reductase. The relatively slow in situ racemization of 2-phenylpropanal and the still limited biocatalyst stability required a subtle adjustment of the substrate-to-catalyst ratio. A value of 3.4 gsubstrate/gcell-dry-weight was selected as a suitable compromise between product ee and the conversion ratio. A catalyst loading of 40 gcell-dry-weight was used to convert 1 M racemic 2-phenylpropanal into 843 mM (115 g/L) (S)-phenylpropanol with 93.1% ee. Conclusion The current industrial production of profenols mainly relies on hydrolases. The bioreduction route established here represents an alternative method for the production of profenols that is competitive with hydrolase-catalyzed kinetic resolutions.

Elucidation of new xylose metabolizing pathway in Pseudomonas gessardii VXlt-16 and its correlation with xylitol production from sugarcane bagasse hydrolysate

Scientific interventions have identified lignocellulosic biomass as potential raw material for various industrial processes. However toxic byproducts released during the process result in deterioration of environment to a greater extent. Microbes can utilize these wastes for production of products of commercial value like bio-fuels, protein, organic acids and xylitol. However, high production cost and astringent operating conditions have been the major bottlenecks for its commercial production. In microbes, xylose is metabolized by xylose isomerase (XI) and xylose reductase-xylitol dehydrogenase (XR-XDH) pathways, with later having ability to transform pure xylose as well as xylose rich lignocelluloses. Efforts to find hyper producer isolates for xylitol production resulted in identification of one such isolate Pseudomonas gessardii VXlt-16 (MG770460) by 16s rDNA sequencing. Statistical optimization resulted in 7.28 folds’ increase in xylitol yield with 64.76% xylose bioconversion. Conversion of xylose to xylitol even at large scale suggests the possible application of bacterial isolate for the production of this useful product at industrial scale.

Effect of Evolutionary Adaptation on Metabolic Enzyme Activities of Thermotolerant Strain Kluyveromyces Marxianus NIRE-K1 and NIRE-K3 for Bioethanol Production

Evolutionary adaptation provides stability to the strains in the challenging environment. As extension of earlier study, the evolved strains Kluyveromyces marxianus NIRE-K1.1 and K. marxianus NIRE-K3.1 were subjected for secondary adaptation on minimal salt (MS) medium with the aim to enhance xylose utilization for ethanol production together with salt tolerance. Both the strains were adapted till saturated improvement in xylose uptake i.e., 54 generations on MS medium containing xylose. Xylose utilization increased from 14.21 to 45.80% and 10.55 to 45.31%, in evolved strains KmNIRE-K1.2 and KmNIRE-K3.2, respectively. Specific xylose reductase activity has also increased 2.04 and 3.36-folds in KmNIRE-K1.2 and KmNIRE-K3.2, respectively. Xylitol dehydrogenase activity was also increased by 2.82 and 1.35-folds in KmNIRE-K1.2 and KmNIRE-K3.2, respectively. Decrease in redox imbalance was observed in evolved strains, and hence there was a reduction in xylitol production during growth and fermentation. Xylose uptake rate increased by 2.53 and 1.5-folds in KmNIRE-K1.2 and KmNIRE-K3.2, respectively with 2.20 and 6.46-folds higher ethanol concentration, and 2.25 and 5.86-folds higher volumetric productivity, respectively. This study has demonstrated the role of evolutionary adaptation for developing robust yeast strains. KmNIRE-K1.2 and KmNIRE-K3.2 have shown enhanced ethanol production, enzyme activities and less by-product formation like xylitol during xylose metabolism.

Optimization of Xylose Reductase production from Citrobacter sp.

Xylitol is a poly-hydroxy straight-chain five-carbon alcohol that can replace sugar in daily uses without any side effects. Lowered risk of dental carries and bone demineralization further support its involvement in a healthy lifestyle. In addition, its role in the synthesis of various commercial products like glycol, ethanol, and resins etc. increases its market value and makes it one of the most valuable bio-products. Microbial fermentation is a cost-effective and eco-friendly method for xylitol production from agricultural residues as available xylose is reduced to xylitol by Xylose reductase (XR) using an equivalent amount of NADPH as a mediator for electron transfer. Previous literature emphasized the use of fungi and yeast for xylitol production rather than bacteria. In contrast to available reports, the potential of the bacterial isolate was evaluated for xylitol production. The effect of process parameters was observed on xylitol yield in terms of XR activity. Out of sixty-eight bacterial isolates obtained, XYLBV-05 was selected for XR production after screening and was identified as Citrobacter sp. based on morphological, microscopic, and biochemical characteristics. Parametric analysis increased the XR production by 4.12 folds (36.61 U/ml). Preliminary results also proved its efficiency in conversion of biomass hydrolysate to xylitol at lab scale but further efforts are needed for xylitol production using agro-industrial lignocellulosic biomass at a large scale which will not only aid in the generation of revenue as a result of value-added products but will also help in environment conservation.