Gene Editing of Candida glycerinogenes by Designed Toxin-Antitoxin Cassette.

Candida glycerinogenes is an industrial yeast with excellent multistress resistance. However, due to the diploid genome and the lack of meiosis and screening markers, its molecular genetic operation is limited. Here, a gene editing system using the toxin-antitoxin pair relBE from the type II toxin-antitoxin system in Escherichia coli as a screening marker was constructed. The RelBE complex can specifically and effectively regulate cell growth and arrest through a conditionally controlled toxin RelE switch, thereby achieving the selection of positive recombinants. The constructed editing system achieved precise gene deletion, replacement, insertion, and gene episomal expression in C. glycerinogenes. Compared with the traditional amino acid deficiency complementation editing system, this editing system produced higher biomass and the gene deletion efficiency was increased by 3.5 times. Using this system, the production of 2-phenylethanol by C. glycerinogenes was increased by 11.5-13.5% through metabolic engineering and tolerance engineering strategies. These results suggest that the stable gene editing system based on toxin-antitoxin pairs can be used for gene editing of C. glycerinogenes to modify metabolic pathways and promote industrial applications. Therefore, the constructed gene editing system is expected to provide a promising strategy for polyploid industrial microorganisms lacking gene manipulation methods.

Transcription factor Hap5 induces gsh2 expression to enhance 2-phenylethanol tolerance and production in an industrial yeast Candida glycerinogenes.

2-Phenylethanol (2-PE) is an important flavor compound but also impairs cell growth severely, which in turn blocks its bioproduction. However, the molecular mechanism of 2-PE tolerance is unclear. In this study, a superb 2-PE stress-tolerant and producing yeast, Candida glycerinogenes, was selected to uncover the underlying mechanism of 2-PE tolerance. We discovered that Hap5 is an essential regulator to 2-PE resistance, and its induction by 2-PE stress occurs at the post-transcriptional level, rather than at the transcriptional level. Under 2-PE stress, Hap5 is activated and imported into the nucleus rapidly. Then, the nuclear Hap5 binds to the glutathione synthetase (gsh2) promoter via CCAAT box, to induce the expression of gsh2 gene. The increased gsh2 expression contributes to enhanced cellular glutathione content, and consequently alleviates ROS accumulation, lipid peroxidation, and cell membrane damage caused by 2-PE toxicity. Specifically, increasing the expression of gsh2 is effective in improving not just 2-PE tolerance (33.7% higher biomass under 29 mM 2-PE), but also 2-PE production (16.2% higher). This study extends our knowledge of 2-PE tolerance mechanism and also provides a promising strategy to improve 2-PE production.

[Cloning of the gene encoding a key enzyme involved in production of glycerol in Candida glycerinogenes].

Candida glycerinogenes WL2002-5, an excellent glycerol producer, has been used for industrial scale fermentation of glycerol by an aerobic process. However, our knowledge about glycerol biosynthesis at the molecular level and genetic background of this yeast species lags far behind those of model yeasts such as Saccharomyces cerevisiae et al. In this report, inverse primers, in conjunction with degenerated primers, were used to amplify the NAD+-dependent glycerol 3-phosphate dehydrogenase (GPD) encoding gene from C. glycerinogenes. The completed nucleotide sequence of the coding, as well as flanking genomic regions was determined (GenBank accession No. EU186536). DNA sequence analysis revealed the present of the open reading frame (ORF) of 1,167 bp, encoding a polypeptide with 388-amino-acid with a molecular mass of 42,695 Da. The CgGPD did not exhibit significant sequence similarity with others described in other eukaryotic systems by comparative analysis. However, it consisted of two typical functional domains which belong to almost all eukaryotic GPDs: a co-enzyme binding domain in the N-terminal, and a catalytic domain. Moreover, some relevant features involved in initiation, regulation and stress response element of gene transcription were observed in the nucleotide sequence of the 5'-non-coding regions. Heterologous expression of CgGPD gene in S. cerevisiae improved its glycerol production significantly. In conclusion, the functional CgGPD has been cloned and identified successfully from C. glycerinogenes genome.