Development of new dominant selectable markers for the nonconventional yeasts Ogataea polymorpha and Candida famata

Nonconventional yeast Candida famata and Ogataea polymorpha are interesting organisms for basic and applied studies. O. polymorpha is methylotrophic thermotolerant yeast capable of xylose alcoholic fermentation whereas C. famata is capable of riboflavin overproduction. Still, the new tools for molecular research of these species are needed. The aim of this study was to develop the new dominant selective markers for C. famata and O. polymorpha usable in metabolic engineering experiments. In this work, the BSD gene from Aspergillus terreus coding for blasticidin S deaminase, O. polymorpha AUR1 gene required for sphingolipid synthesis and IMH3 gene, which encodes IMP dehydrogenase, were tested as the new dominant selective marker genes. Our results showed that AUR1 and IMH3 genes could be used as dominant selective markers for O. polymorpha with frequencies of transformation of 40 and 20 transformants per microgram of DNA, respectively. The IMH3 gene was successfully used as the marker for construction of O. polymorpha strains with increased ethanol production from xylose due to overexpression of TAL1, TKL1 and AOX1 genes. The BSD gene from A. terreus, conferring resistance to blasticidin, was found to be efficient for selection of C. famata transformants.     

Biodegradation of Ammonia Nitrogen Using a Novel Candida sp. Strain N6 Immobilization

固定化假丝酵母菌株N6降解氨氮的研究

王 凯^1,2，孙亚文¹，李斯琪¹，张瑛洁¹，程喜全¹，马 军²

（1. 哈尔滨工业大学(威海)， 海洋科学与技术学院，山东 威海 264209；

2. 哈尔滨工业大学 市政与环境工程学院，哈尔滨 150090）

创新点说明：

发现一株假丝酵母菌株，具有较高的氨氮降解能力，采用固定化技术，将其固定化后用于模拟生活污水的处理效果仍然显著，表明将该菌固定化后具有较高的应用价值。

研究目的：

将高氨氮处理效率的菌株N6固定化，以期通过固定化的形式将其应用于废水处理中。

研究方法：

从山东威海金海湾排污口分离出一株假丝酵母菌，该菌株具有较高的氨氮处理效率。采用包埋固定化技术将N6固定，考察了固定化N6的氨氮降解能力以及稳定性，通过单因素以及正交试验确定了固定化材料以及最佳固定化条件。利用SBR反应器探究固定化N6的应用情况。

研究结果：

1）当固定化材料PVA, SA, CaCl2的浓度分别为9%, 1.5%, 2%时最适合N6固定化，在此条件下，固定化N6的氨氮去除率达到了97.97%。

2）稳定性测试表明固定化N6具有很强的稳定性，固定化小球在测试条件下未出现破碎现象。

3）固定化N6的最佳固定化条件为：固定化24h，N6接种量3%，pH 8。

4） 固定化N6在SBR反应器中表现出非常好的氨氮降解能力，去除率稳定在95%-99%。

5）在固定化N6的应用过程中，最适添加量为15%。

结论：

固定化高效菌株用于废水处理具有广阔的前景。高氨氮处理效率的假丝酵母N6固定化后氨氮降解能力仍然较强。固定化N6具有潜在的应用价值，将固定化N6应用到SBR中，具有较高且稳定的氨氮去除率。

关键词：氨氮废水；固定化；假丝酵母菌属；脱氮效率

     

Effect of co-fermentation and sequential fermentation of Candida versatilis and Lactococcus lactis subsp. cremoris on unsalted pork hydrolysates components

In this study, the effect of co-fermentation and sequential fermentation with a typical halophilic soy sauce yeast Candida versatilis and a non-halophilic dairy bacterium Lactococcus lactis subsp. cremoris in pork hydrolysates was explored for the first time with regard to their viability, physicochemical changes and volatile compound production. It was observed that the growth of C. versatilis was suppressed (only 0.6 - 1.5 log CFU mL⁻¹ increase), whilst L. lactis subsp. cremoris was stimulated (total cell counts exceeded 9.0 log CFU mL⁻¹) in mixed-cultures relative to the respective monocultures. There were no significant differences between co-inoculation and sequential inoculation regarding glucose consumption, organic acid production and free amino acids utilisation. Some distinct volatile compounds such as methionol, 6-methyl-5-hepten-2-one and gamma-nonalactone were found in both co-inoculated and sequentially inoculated samples, with slightly different concentrations. These results suggest that the mixed-inoculation of C. versatilis and L. lactis have a positive impact on the volatile compounds formation in pork hydrolysates.     

Role of Candida albicans-Secreted Aspartyl Proteinases (Saps) in Severe Early Childhood Caries

Candida albicans is strongly associated with severe early childhood caries (S-ECC). However, the roles of secreted aspartyl proteinases (Saps), an important virulence factor of C. albicans, in the progress of S-ECC are not clear. In our study, the Saps activities were evaluated by the yeast nitrogen base–bovine serum albumi (YNB–BSA) agar plate method and by the MTT method with bovine serum albumin (BSA) as the substrate. Genotypes of C. albicans and gene expression of Sap1–5 were evaluated. The relationships of Saps activities and genotypes with S-ECC were analyzed. The results showed that enzyme activities of Saps in the S-ECC group were significantly higher than those in the caries free (CF) group (p < 0.05). Genotypes A, B and C were detected in the S-ECC group, and genotypes A and C were detected in the CF group. In the genotype A group, Saps activity in the S-ECC group was significantly different from that in the CF group (p < 0.05). The gene expression level of Sap1 in the S-ECC group was significantly higher than that in the CF group (p = 0.001), while Sap4 expression was significantly lower than that in the CF group (p = 0.029). It can be concluded that Sap1–5 are the predominant proteinase genes expressed in C. albicans from dental biofilm and Sap1 may play an important role in the development of S-ECC.     

Potent Synergy between Spirocyclic Pyrrolidinoindolinones and Fluconazole against Candida albicans

A spiroindolinone, (1S,3R,3aR,6aS)‐1‐benzyl‐6′‐chloro‐5‐(4‐fluorophenyl)‐7′‐methylspiro[1,2,3a,6a‐tetrahydropyrrolo[3,4‐c]pyrrole‐3,3′‐1H‐indole]‐2′,4,6‐trione, was previously reported to enhance the antifungal effect of fluconazole against Candida albicans. A diastereomer of this compound was synthesized, along with various analogues. Many of the compounds were shown to enhance the antifungal effect of fluconazole against C. albicans, some with exquisite potency. One spirocyclic piperazine derivative, which we have named synazo‐1, was found to enhance the effect of fluconazole with an EC₅₀ value of 300 pM against a susceptible strain of C. albicans and going as low as 2 nM against some resistant strains. Synazo‐1 exhibits true synergy with fluconazole, with an FIC index below 0.5 in the strains tested. Synazo‐1 exhibited low toxicity in mammalian cells relative to the concentrations required for antifungal synergy.     

A two‐step inoculation of Candida etchellsii to enhance soy sauce flavour and quality

Effects of novel two‐step inoculation to enhance soy sauce special flavour in Candida etchellsii were investigated at the cell growth phase. The first‐stage consists of a 5% culture inoculum of log phase cells at 30‐day. Subsequently, a 20% culture inoculum of stationary phase cells was added at 60‐day. The resulting amino nitrogen and soluble salt‐free solid yield reached 9.15 ± 0.12 and 269.60 ± 3.15 mg L^?1 in 30 °C incubator experiments, increased by 23.1% and 17.6%, respectively, as compared to the control without culture inoculation. Maximal free amino acid yield of 58.21 ± 1.77 g L^?1 was achieved, and 39 types of volatile flavour compounds content was 17.81 ± 0.45 g L^?1, which were 1.76% and 178.7% higher than the control. A novel two‐step inoculation using the C. etchellsii yeast was developed and optimised. It was proven to be a feasible reproducible process for industrial application for the improvement of the flavour and quality of soy sauce production.