Altered Metabolic Strategies: Elaborate Mechanisms Adopted by Oenococcus oeni in Response to Acid Stress.

Oenococcus oeni plays a key role in inducing malolactic fermentation in wine. Acid stress is often encountered under wine conditions. However, the lack of systematic studies of acid resistance mechanisms limits the downstream fermentation applications. In this study, the acid responses of O. oeni were investigated by combining transcriptome, metabolome, and genome-scale metabolic modeling approaches. Metabolite profiling highlighted the decreased abundance of nucleotides under acid stress. The gene-metabolite bipartite network showed negative correlations between nucleotides and genes involved in ribosome assembly, translation, and post-translational processes, suggesting that stringent response could be activated under acid stress. Genome-scale metabolic modeling revealed marked flux rerouting, including reallocation of pyruvate, attenuation of glycolysis, utilization of carbon sources other than glucose, and enhancement of nucleotide salvage and the arginine deiminase pathway. This study provided novel insights into the acid responses of O. oeni, which will be useful for designing strategies to address acid stress in wine malolactic fermentation.

CRISPR-CBEI: a Designing and Analyzing Tool Kit for Cytosine Base Editor-Mediated Gene Inactivation.

Base editing is a promising technique, allowing precise single-base mutagenesis in genomes without double-strand DNA breaks or donor templates. Cytosine base editors (CBEs) convert cytosine to thymidine. In particular, CBEs can transform four codons, CAA, CAG, CGA, and TGG, into stop codons, providing a new means to rapidly inactivate a gene of interest and enabling loss-of-function study in recombination-deficient species and the construction of gene-inactivation libraries. However, designing single guide RNAs (sgRNAs) for gene inactivation is more complicated and more restricted in applicability than using the lustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (CRISPR/Cas9) system only, especially for researchers who do not specialize in the bioinformatics skills needed to design and evaluate sgRNAs. Here, we present a new user-friendly designing tool kit, namely, CRISPR-CBEI (cytosine base editor-mediated gene inactivation), including a Web tool and a command-line tool. The Web tool is dedicated to the design of sgRNAs for CBE-mediated gene inactivation and integrates various functions, including open reading frame (ORF) identification, CBE customization, sgRNA designing, summarizing, and front-end off-target searching against user-defined unlimited-file-size local genome files without the necessity of uploading to the server. The command-line version serves the same purpose but for a larger number of coding DNA sequences (CDSs), for instance, for designing a CBE-inactivation library in a target species which provides comprehensive evaluations of CBEs and target genomes. We envision that this tool would contribute to CBE-inactivation design.IMPORTANCE Life science has been in pursuit of precise and efficient genome editing in living cells since the very beginning of the first restriction cloning attempt. The introduction of RNA-guided CRISPR-associated (Cas) nucleases contributed to this ultimate goal through their ability to deliver a double-strand break (DSB) to a precise target location in various species, obsoleting the preceding editing tools, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). The derivative technology, base editing, combines the catalytically inactivated Cas nuclease and nucleotide deaminase and mediates the genetic modifications at single-nucleotide precision without introducing a DSB. Moreover, the cytosine base editors (CBEs) are able to transform multiple codons into stop codons, rapidly inactivating a gene of interest and enabling loss-of-function study in some recombination-deficient species. Here, we present the CRISPR-CBEI tool kit to assist the design of sgRNAs for CBE-mediated gene inactivation.