Emerging applications of the methylotrophic yeasts

Abstract The use of methylotrophic yeasts for the production of single-cell-protein (SCP), alcohol oxidase and fine chemicals has been proposed. Fermentation technology developed for the growth of these yeasts on methanol at high cell densities has been commercialized. However, it is the production of heterologous recombinant proteins by Pichia pastoris that is emerging as the most significant application of the methylotrophic yeasts.     

Emerging applications of the methylotrophic yeasts

The use of methylotrophic yeasts for the production of single-cell-protein (SCP), alcohol oxidase and fine chemicals has been proposed. Fermentation technology developed for the growth of these yeasts on methanol at high cell densities has been commercialized. However, it is the production of heterologous recombinant proteins by Pichia pastoris that is emerging as the most significant application of the methylotrophic yeasts.     

Production of metabolites by methylotrophic yeasts

Recent advances in biotechnology of methanol-utilizing yeasts are briefly summarized. The emphasis is given to production of some fine and commercial chemicals such as formaldehyde, formate, hydrogen peroxide, dihydroxyacetone, ATP, FAD as well as proteins, specifically alcohol oxidase. The advantages of mutants and recombinants derived from methylotrophic yeasts for efficient production of various useful materials are demonstrated.     

非常规酵母天然产物合成

以解脂耶氏酵母(Yarrowia lipolytica)、巴斯德毕赤酵母(Pichia pastoris)、马克斯克鲁维酵母(Kluyveromyces marxianus)、圆红冬孢酵母(Rhodosporidium toruloides)、多形汉逊酵母(Hansenula polymorpha)为代表的非常规酵母凭借较广的底物利用谱、较强的环境耐受性等优势,已成功实现多种天然产物的高效生产。随着合成生物学及基因编辑技术的发展,针对非常规酵母代谢工程改造的工具和策略也逐渐丰富。本文介绍了几类常见的非常规酵母的生理特性、工具开发及应用现状,并总结归纳了天然产物合成优化中常用的代谢工程策略;最后讨论了现阶段非常规酵母作为天然产物合成细胞工厂的优势和不足,并对后续研究和发展趋势进行了展望。     

非传统酵母代谢工程研究进展

近30年来解脂耶氏酵母、克鲁维酵母、毕赤酵母、假丝酵母、汉逊酵母等非传统酵母因其具有天然的生理代谢优势,如快速生长、多底物利用、胁迫耐受性等,在代谢工程领域得到了广泛关注,多种基因工程改造工具正逐渐被开发用于非传统酵母的特性拓展,使其成为合成重组蛋白、生物可再生化学物质的高效细胞工厂.文中总结了非传统酵母中基因编辑工具...     

Regulation of protein synthesis in methylotrophic yeasts: Repression of methanol dissimilating enzymes by nitrogen limitation

The influence of nitrogen limitation on the regulation of the methanol oxidizing enzymes alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase in the two methylotrophic yeastsHansenula polymorpha andKloeckera sp. 2201 was studied in continuous culture. When shifted from carbon-limited growth conditions (with a mixture of glucose and methanol as carbon sources) to a nitrogen-limited environment both cultures were found to go through a transition phase where neither enhanced residual concentrations of the nitrogen source nor of one of the two carbon sources could be detected in the supernatant. As soon as nitrogen became a limiting substrate an immediate reorganisation of the cell composition was initiated: protein content of the cells dropped to approximately 40% of its initial value, glycogen was synthesized and the enzyme composition of the cells was changed. The peroxisomal enzymes alcohol oxidase and catalase in both organisms and the two dehydrogenases for formaldehyde and formate in cells ofKloeckera sp. 2201 were subject to degradation (catabolite inactivation). The measured rates of inactivation indicated that in cells ofH. polymorpha this process might be limited to peroxisomes, whereas inKloeckera sp. 2201 the degradation was found to affect peroxisomal as well as cytoplasmic enzymes. In contrast to methanol dissimilating enzymes the net rate of synthesis of hexokinase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase was not affected by this process but those enzymes were synthesized with increased rates.     

Immunocytochemical evidence for the acidic nature of peroxisomes in methylotrophic yeasts

The possible acidic nature of the peroxisomal matrix present in intact yeast cells was studied immunocytochemically, using the weak base DAMP as a probe. Spheroplasts of methanol-grown Candida boidinii and Hansenula polymorpha were regenerated and incubated with DAMP. After immunogold labelling, using antibodies against DAMP, a specific accumulation of gold particles was observed on the peroxisomal profiles. This labelling was absent in controls, performed in the presence of ionophores or chloroquine. These results support earlier observations, that in intact cells a pH-gradient exists across the peroxisomal membrane. Experiments, carried out on osmotically swollen spheroplasts indicated that maintenance of this pH-gradient is strongly related to the cell's integrity.     

甲醇酵母代谢工程研究进展

甲醇酵母由于独特优点被认为是绿色生物制造的潜在宿主.特别是其天然甲醇利用性能有望建立甲醇生物转化路线,拓展生物炼制底物,具有重要经济价值和环保意义.文中综述了代谢工程改造甲醇酵母合成蛋白质和化学品的最新研究进展,并比较了其与模式生物酿酒酵母作为细胞工厂的优缺点.随后,分析了甲醇酵母代谢工程改造面临的挑战,并展望了潜在解...     

Application of anhydrobiosis and dehydration of yeasts for non-conventional biotechnological goals

Dehydration of yeast cells causes them to enter a state of anhydrobiosis in which their metabolism is temporarily and reversibly suspended. This unique state among organisms is currently used in the production of active dry yeasts, mainly used in baking and winemaking. In recent decades non-conventional applications of yeast dehydration have been proposed for various modern biotechnologies. This mini-review briefly summarises current information on the application of dry yeasts in traditional and innovative fields. It has been shown that dry yeast preparations can be used for the efficient protection, purification and bioremediation of the environment from heavy metals. The high sorption activity of dehydrated yeasts can be used as an interesting tool in winemaking due to their effects on quality and taste. Dry yeasts are also used in agricultural animal feed. Another interesting application of yeast dehydration is as an additional stage in new methods for the stable immobilisation of microorganisms, especially in cases when biotechnologically important strains have no affinity with the carrier. Such immobilisation methods also provide a new approach for the successful conservation of yeast strains that are very sensitive to dehydration. In addition, the application of dehydration procedures opens up new possibilities for the use of yeast as a model system. Separate sections of this review also discuss possible uses of dry yeasts in biocontrol, bioprotection and biotransformations, in analytical methods as well as in some other areas.     

Engineering the elongation factor Tu for efficient selenoprotein synthesis

Selenocysteine (Sec) is naturally co-translationally incorporated into proteins by recoding the UGA opal codon with a specialized elongation factor (SelB in bacteria) and an RNA structural signal (SECIS element). We have recently developed a SECIS-free selenoprotein synthesis system that site-specifically—using the UAG amber codon—inserts Sec depending on the elongation factor Tu (EF-Tu). Here, we describe the engineering of EF-Tu for improved selenoprotein synthesis. A Sec-specific selection system was established by expression of human protein O⁶-alkylguanine-DNA alkyltransferase (hAGT), in which the active site cysteine codon has been replaced by the UAG amber codon. The formed hAGT selenoprotein repairs the DNA damage caused by the methylating agent N-methyl-N′-nitro-N-nitrosoguanidine, and thereby enables Escherichia coli to grow in the presence of this mutagen. An EF-Tu library was created in which codons specifying the amino acid binding pocket were randomized. Selection was carried out for enhanced Sec incorporation into hAGT; the resulting EF-Tu variants contained highly conserved amino acid changes within members of the library. The improved UTu-system with EF-Sel1 raises the efficiency of UAG-specific Sec incorporation to >90%, and also doubles the yield of selenoprotein production.     

甲醇酵母表达系统研究进展

甲醇酵母是一种新的基因表达系统,有着许多突出的优点。多种重组蛋白已在甲醇酵母获得成功表达.该文综述几种甲醇酵母表达系统的特点和现状。     

Biocatalysis of nitro substituted styrene oxides by non-conventional yeasts

Yeast strains (410) from more than 45 different genera were screened for the enantioselective hydrolysis of nitro substituted styrene oxides. These strains included 262 yeasts with known epoxides hydrolase activity for various other epoxides. Epoxide hydrolase activity for p-nitrostyrene oxide (pNSO) (177 strains) and m-nitrostyrene oxide (mNSO) (148 strains) was widespread in the yeasts, while activity for o-nitrostyrene oxide (oNSO) was less ubiquitous (22 strains). The strains that displayed enantioselectivity in the hydrolysis of one or more of the nitro substituted styrene oxides (35 strains) were also screened against styrene oxide (SO). Rhodosporidium toruloides UOFS Y-0471 displayed the highest enantioselectivity for pNSO (ee 55%, yield 35%) while Rhodotorula glutinis UOFS Y-0653 displayed the highest enantioselectivity for mNSO (ee >98%, yield 29%), oNSO (ee 39%, yield 19%) and SO (ee >98%, yield 19%). (R)-Styrene oxide was preferentially hydrolysed to the corresponding (R)-diol with retention of configuration at the stereogenic centre. In the case of the nitro substituted styrene oxides the absolute configurations of the remaining epoxides and the formed diols were not established.     

Biological treatment of olive mill wastewater by non-conventional yeasts

The ability of lipolytic yeasts to grow on olive mill wastewater (OMW)-based medium and to produce high-value compounds while degrading this waste, was tested. OMW collected from three-phase olive mills from the North region of Portugal were characterized and used. OMW with COD ranging from 100 g L⁻¹ to 200 g L⁻¹ were supplemented with yeast extract and ammonium chloride. Studies of OMW consumption were carried out in batch cultures of Candida rugosa, Candida cylindracea and Yarrowia lipolytica. All strains were able to grow in the OMW-based media, without dilution, to consume reducing sugars and to reduce COD. C. cylindracea was the best strain concerning the lipase production and the reduction of phenolic compounds and COD. For all strains, the phenols degradation was quite difficult, mostly when more easily degradable carbon source is still present in the medium. Among the phenolic compounds tested catechol is the most inhibitory to the cells.     

Engineering yeasts for xylose metabolism

Technologies for the production of alternative fuels are receiving increased attention owing to concerns over the rising cost of petrol and global warming. One such technology under development is the use of yeasts for the commercial fermentation of xylose to ethanol. Several approaches have been employed to engineer xylose metabolism. These involve modeling, flux analysis, and expression analysis followed by the targeted deletion or altered expression of key genes. Expression analysis is increasingly being used to target rate-limiting steps. Quantitative metabolic models have also proved extremely useful: they can be calculated from stoichiometric balances or inferred from the labeling of intermediate metabolites. The recent determination of the genome sequence for P. stipitis is important, as its genome characteristics and regulatory patterns could serve as guides for further development in this natural xylose-fermenting yeast or in engineered Saccharomyces cerevisiae. Lastly, strain selection through mutagenesis, adaptive evolution or from nature can also be employed to further improve activity.     

Effect of formaldehyde on the level of cytochrome c in methylotrophic yeasts

The effect of formaldehyde on the cytochrome system of the methylotrophic yeasts Candida tropicalis, Candida boidinii, Candida methylica, Hansenula capsulata, Hansenula polymorpha, Pichia pastoris and nonmethylotrophic yeasts Saccharomyces cerevisiae was studied. In the investigated concentration range of 0.58 – 2.5 mol · 1^?1, formaldehyde decreases the level of the reduced cytochrome c in the above-mentioned microorganisms. The kinetics of the change in the reduced cytochrome c content has two phases: a phase of slow decrease and a phase of complete disappearance of the reduced cytochrome c in the cells. The duration of the first phase shortens with increasing concentrations of formaldehyde.     

Implementing CRISPR-Cas technologies in conventional and non-conventional yeasts: Current state and future prospects

Within five years, the CRISPR-Cas system has emerged as the dominating tool for genome engineering, while also changing the speed and efficiency of metabolic engineering in conventional (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and non-conventional (Yarrowia lipolytica, Pichia pastoris syn. Komagataella phaffii, Kluyveromyces lactis, Candida albicans and C. glabrata) yeasts.Especially in S. cerevisiae, an extensive toolbox of advanced CRISPR-related applications has been established, including crisprTFs and gene drives. The comparison of innovative CRISPR-Cas expression strategies in yeasts presented here may also serve as guideline to implement and refine CRISPR-Cas systems for highly efficient genome editing in other eukaryotic organisms.