Selection by UV Mutagenesis and Physiological Characterization of Mutant Strains of the Yeast Saprochaete suaveolens (Former Geotrichum fragrans) with Higher Capacity to Produce Flavor Compounds

Yeast volatile organic compounds (VOCs), i.e. low molecular weight organic acids, alcohols and esters, are considered as potential and sustainable sources of natural aromas that can replace commonly used artificial flavors in food and other industrial sectors. Although research generally focuses on the yeast Saccharomyces cerevisiae, other so-called unconventional yeasts (NCY) are beginning to attract the attention of researchers, particularly for their ability to produce alternative panels of VOCs. With this respect, a Saprochaete suaveolens strain isolated from dragon fruit in Reunion Island was shown to produce α-unsaturated esters from branched-chain amino acids (BCAAs) such as isobutyl, isoamyl or ethyl tiglate, which are rarely found in other yeasts strains. Given that β-oxidation allows the growth of S. suaveolens on BCAAs as sole carbon source, we developped a method based on UV mutagenesis to generate mutants that can no longer grow on BCAAs, while redirecting the carbon flow towards esterification of α-unsaturated esters. Among the 15,000 clones generated through UV irradiation, we identified nine clones unable to grow on BCAAs with one of them able to produce eight times more VOCs as compared to the wild-type strain. This higher production of α-unsaturated esters in this mutant strain coincided with an almost complete loss of enoyl-CoA hydratase activity of the β-oxidation pathways and with a twofold increase of acyl-CoA hydrolase with not significant changes in the enzymes of the Ehrlich pathway. Moreover, from our knowledge, it constituted the first example of VOCs enhancement in a microbial strain by UV mutagenesis.

Transglutaminase from UV Mutated Bacillus cereus NRC215: Production, Purification, and Characterization

Transglutaminase (EC 2.3.2.13, TGase) recorded the highest activity (0.101 U/ml) in bacterial isolate NRC215. 16S rRNA sequencing revealed that NRC215 was identified as Bacillus cereus NRC215 under accession number MT229271 in the NCBI database. UV irradiation was employed to improve TGase production. Five rifampin (RIF) resistant mutants were only isolated from UV-treated Bacillus cereus NRC215 for three minutes. The best mutant, BCrif5, exhibiting induced rifampin resistance, gave TGase with higher activity (0.148 U/ml). The ISSR PCR technique was employed to detect these new rearrangements resulting from UV mutagenesis between the wild-type strain and its mutants. Moreover, TGase has been purified by three-step procedures resulting in a recovery of 28 and 34.63% for wild and BCrif5 strains, respectively. The optimal purified TGase activity was exhibited at pH 7 for wild strain while the mutant BCrif5 at pH 5.0 and 40 °C for both wild and BCrif5 strains. Bacillus cereus NRC215 TGase was activated by Ba+2 (102.50 and 107.06%), while it was inhibited by Cu+2 (30% and 22.35%) for wild and BCrif5 strains, respectively. It could be concluded that Bacillus cereus NRC215 is a promising strain for TGase production, which is beneficial as a food additive.

APOBEC mutagenesis is low in most types of non-B DNA structures, unlike other types of cancer mutagenesis

While somatic mutations are known to be enriched in genome regions with non-canonical DNA secondary structure, the impact of particular mutagens still needs to be elucidated. Here, we demonstrate that in human cancers, the APOBEC mutagenesis is not enriched in direct repeats, mirror repeats, short tandem repeats, and G-quadruplexes, and even decreased below its level in B-DNA for cancer samples with very high APOBEC activity. In contrast, we observe that the APOBEC-induced mutational density is positively associated with APOBEC activity in inverted repeats (cruciform structures), where the impact of cytosine at the 3'-end of the hairpin loop is substantial. Surprisingly, the APOBEC-signature mutation density per TC motif in the single-stranded DNA of a G-quadruplex (G4) is lower than in the four-stranded part of G4 and in B-DNA. The APOBEC mutagenesis, as well as the UV-mutagenesis in melanoma samples are absent in Z-DNA regions, due to depletion of their mutational signature motifs.

Green Microalgae Strain Improvement for the Production of Sterols and Squalene

Sterols and squalene are essential biomolecules required for the homeostasis of eukaryotic membrane permeability and fluidity. Both compounds have beneficial effects on human health. As the current sources of sterols and squalene are plant and shark oils, microalgae are suggested as more sustainable sources. Nonetheless, the high costs of production and processing still hinder the commercialization of algal cultivation. Strain improvement for higher product yield and tolerance to harsh environments is an attractive way to reduce costs. Being an intermediate in sterol synthesis, squalene is converted to squalene epoxide by squalene epoxidase. This step is inhibited by terbinafine, a commonly used antifungal drug. In yeasts, some terbinafine-resistant strains overproduced sterols, but similar microalgae strains have not been reported. Mutants that exhibit greater tolerance to terbinafine might accumulate increased sterols and squalene content, along with the ability to tolerate the drug and other stresses, which are beneficial for outdoor cultivation. To explore this possibility, terbinafine-resistant mutants were isolated in the model green microalga Chlamydomonas reinhardtii using UV mutagenesis. Three mutants were identified and all of them exhibited approximately 50 percent overproduction of sterols. Under terbinafine treatment, one of the mutants also accumulated around 50 percent higher levels of squalene. The higher accumulation of pigments and triacylglycerol were also observed. Along with resistance to terbinafine, this mutant also exhibited higher resistance to oxidative stress. Altogether, resistance to terbinafine can be used to screen for strains with increased levels of sterols or squalene in green microalgae without growth compromise.

Promising Anti-MRSA Activity of Brevibacillus sp. Isolated from Soil and Strain Improvement by UV Mutagenesis

Antibiotic-resistant infection is a major health problem, and a limited number of drugs are currently approved as antibiotics. Soil bacteria are promising sources in the search for novel antibiotics. The aim of the present study is to isolate and assess soil bacteria with anti-MRSA activity and improve their capabilities by UV mutagenesis. Soil samples from the upper south of Thailand were screened for antibacterial activity using the cross-streak method. Agar well diffusion was used to examine the activity of isolates against a spectrum of human pathogens. The most active isolate was identified by 16S rRNA sequencing, and the production kinetics and stability were investigated. The most promising isolate was mutated by UV radiation, and the resulting activity and strain stability were studied. The results show that isolates from the cross-streak method could inhibit Staphylococcus aureus TISTR 517 (94 isolates) and Escherichia coli TISTR 887 (67 isolates). Nine isolates remained active against S. aureus TISTR 517 and MRSA, and eight isolates inhibited the growth of E. coli TISTR 887 as assessed using agar well diffusion. The most active strain was Brevibacillus sp. SPR-20, which had the highest activity at 24 h of incubation. The active substances in culture supernatants exhibited more than 90% activity when subjected to treatments involving various heat, enzymes, surfactants, and pH conditions. The mutant M201 showed significantly higher activity (109.88–120.22%) and strain stability compared to the wild-type strain. In conclusion, we demonstrate that soil Brevibacillus sp. is a potential resource that can be subjected to UV mutagenesis as a useful approach for improving the production of anti-MRSA in the era of antibiotic resistance.

UV Mutagenesis as a Strategy to Enhance Growth and Lipid Productivity of Chlorella sp. 042

Microalgae appeared to be an alternative feedstock for renewable biodiesel production due to their capability to accumulate considerable amounts of lipids. In this study, mutagenesis using UVC light with different periods was applied to Chlorella sp. 042 to produce a microalgae strain with high lipid productivity of 45, 60, and 75 min. The Nile red fluorescence method was conducted to select a Chlorella sp. mutant with high neutral lipid and generated one mutant from every UV mutation period, M45-06, M60-02, and M75-21. All of the mutants have higher growth rates than the wild type. Chlorella sp. 042 M60-02 achieved the highest lipid productivity, with 34 mg L-1 day-1. Furthermore, as other major biochemical components, carbohydrate and protein contents were determined. Our results showed that all the mutants enhance their carbohydrate and protein contents compared to the wild type. However, mutations for more than 60 min do not intensely change the protein content of mutant microalgae. Gas chromatography-mass spectrophotometry analysis revealed that M60-02 mutant has similar FAME profiles with the wild type, which contain palmitic acid (C16:0), stearic acid (C 18:0), oleic acid (C18:1), and linoleic acid (C18:2). These results demonstrate that the UV mutation of Chlorella sp. 042 for 60 min is suitable as a source of biodiesel production.

Participation of the HIM1 gene of yeast Saccharomyces cerevisiae in the error-free branch of post-replicative repair and role Polη in him1-dependent mutagenesis

In eukaryotes, DNA damage tolerance (DDT) is determined by two repair pathways, homologous repair recombination (HRR) and a pathway controlled by the RAD6-epistatic group of genes. Monoubiquitylation of PCNA mediates an error-prone pathway, whereas polyubiquitylation stimulates an error-free pathway. The error-free pathway involves components of recombination repair; however, the factors that act in this pathway remain largely unknown. Here, we report that the HIM1 gene participates in error-free DDT. Notably, inactivation RAD30 gene encoding Polη completely suppresses him1-dependent UV mutagenesis. Furthermore, data obtained show a significant role of Polη in him1-dependent mutagenesis, especially at non-bipyrimidine sites (NBP sites). We demonstrate that him1 mutation significantly reduces the efficiency of the induction expression of RNR genes after UV irradiation. Besides, this paper presents evidence that significant increase in the dNTP levels suppress him1-dependent mutagenesis. Our findings show that Polη responsible for him1-dependent mutagenesis.