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The macronuclear genome of the Antarctic psychrophilic marine ciliate Euplotes focardii reveals new insights on molecular cold adaptation.

ABSTRACT: The macronuclear (MAC) genomes of ciliates belonging to the genus Euplotes species are comprised of numerous small DNA molecules, nanochromosomes, each typically encoding a single gene. These genomes are responsible for all gene expression during vegetative cell growth. Here, we report the analysis of the MAC genome from the Antarctic psychrophile Euplotes focardii. Nanochromosomes containing bacterial sequences were not found, suggesting that phenomena of horizontal gene transfer did not occur recently, even though this ciliate species has a substantial associated bacterial consortium. As in other euplotid species, E. focardii MAC genes are characterized by a high frequency of translational frameshifting. Furthermore, in order to characterize differences that may be consequent to cold adaptation and defense to oxidative stress, the main constraints of the Antarctic marine microorganisms, we compared E. focardii MAC genome with those available from mesophilic Euplotes species. We focussed mainly on the comparison of tubulin, antioxidant enzymes and heat shock protein (HSP) 70 families, molecules which possess peculiar characteristic correlated with cold adaptation in E. focardii. We found that α-tubulin genes and those encoding SODs and CATs antioxidant enzymes are more numerous than in the mesophilic Euplotes species. Furthermore, the phylogenetic trees showed that these molecules are divergent in the Antarctic species. In contrast, there are fewer hsp70 genes in E. focardii compared to mesophilic Euplotes and these genes do not respond to thermal stress but only to oxidative stress. Our results suggest that molecular adaptation to cold and oxidative stress in the Antarctic environment may not only be due to particular amino acid substitutions but also due to duplication and divergence of paralogous genes.

SUBMITTER: Mozzicafreddo M

PROVIDER: S-EPMC8455672 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The α-amylases are endo-acting enzymes that hydrolyze starch by randomly cleaving the 1,4-α-d-glucosidic linkages between the adjacent glucose units in a linear amylose chain. They have significant advantages in a wide range of applications, particularly in the food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii (EfAmy) is an alkaline enzyme, different from most of the α-amylases characterized so far. Furthermore, EfAmy has the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at a low temperature and high thermolability, which is the major drawback of cold-active enzymes in industrial applications. In this work, we applied site-directed mutagenesis combined with rational design to generate a cold-active EfAmy with improved thermostability and catalytic efficiency at low temperatures. We engineered two EfAmy mutants. In one mutant, we introduced Pro residues on the A and B domains in surface loops. In the second mutant, we changed Val residues to Thr close to the catalytic site. The aim of these substitutions was to rigidify the molecular structure of the enzyme. Furthermore, we also analyzed mutants containing these combined substitutions. Biochemical enzymatic assays of engineered versions of EfAmy revealed that the combination of mutations at the surface loops increased the thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for the changes in the biochemical properties are discussed by analyzing the three-dimensional structural model.IMPORTANCE Cold-adapted enzymes have high specific activity at low and moderate temperatures, a property that can be extremely useful in various applications as it implies a reduction in energy consumption during the catalyzed reaction. However, the concurrent high thermolability of cold-adapted enzymes often limits their applications in industrial processes. The α-amylase from the psychrophilic Antarctic ciliate Euplotes focardii (named EfAmy) is a cold-adapted enzyme with optimal catalytic activity in an alkaline environment. These unique features distinguish it from most α-amylases characterized so far. In this work, we engineered a novel EfAmy with improved thermostability, substrate binding affinity, and catalytic efficiency to various extents, without impacting its pH preference. These characteristics can be considered important properties for use in the food, detergent, and textile industries and in other industrial applications. The enzyme engineering strategy developed in this study may also provide useful knowledge for future optimization of molecules to be used in particular industrial applications.

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The macronuclear genome of the Antarctic psychrophilic marine ciliate Euplotes focardii reveals new insights on molecular cold adaptation.

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