Raita Svetlana, Kuzmika Iveta, Mika Taras, Geiba Zane, Spalvins Kriss
Riga Technical University, Institute of Energy Systems and Environment, Azenes Street 12/1, Riga, Latvia.
J Ind Microbiol Biotechnol. 2024 Dec 31;52. doi: 10.1093/jimb/kuaf011.
According to the Food and Agricultural Organisation 2024 statement, developing single-cell protein technology is important to reduce the burden on conventional feed protein production sectors. In this regard, improved commercial strains rich in amino acids, especially Lys and Met, may provide a sustainable alternative source of protein in aquaculture diets. The developed and laboratory-validated methodology for creating protein-synthesizing mutants will strengthen the competitiveness of SCP production technology. The present work provides unique results on improving the protein-producing properties of wild-type Phaffia rhodozyma DSM 5626 by mutagenesis and screening on herbicide-containing medium as a selective agent for amino acid biosynthesis inhibition. Inhibitory concentrations of pure herbicide actives were determined for S-(2-aminoethyl)-L-cysteine (AEC) hydrochloride and glufosinate-ammonium (GA) for complete inhibition and strong inhibition of the DSM 5626 strain. GA at a concentration of 50 mM and 100 mM and AEC at 0.5 mM and 2.5 mM were chosen for mutant selection after chemical mutagenesis. The use of herbicides resulted in the selection of mutants with significantly improved synthesis of Met and Lys. Specifically, mutants GA6/4 and GA7/5 exhibited 37% and 26% higher Met levels, respectively, while GA6/3 had a 14% increase in Lys compared to the wild-type strain. The AEC3/9 mutant demonstrated a 35% increase in Met, 24% in Lys, 8% in Ile, and 6% in Phe, underscoring the efficacy of this screening approach in enhancing essential amino acid content. The protein quality parameters essential amino acid index and amino acid score of these mutants became higher compared with commercial strains of SCP yeast such as C. utilis, S. cerevisiae, K. marxianus, etc.
ONE-SENTENCE SUMMARY: Mutagenesis combined with selective screening using herbicides is an effective approach to enhancing amino acid biosynthesis in yeast.
根据联合国粮食及农业组织2024年的声明，开发单细胞蛋白技术对于减轻传统饲料蛋白生产部门的负担很重要。在这方面，改良富含氨基酸（尤其是赖氨酸和蛋氨酸）的商业菌株可能为水产养殖饲料提供可持续的蛋白质替代来源。已开发并经实验室验证的创建蛋白质合成突变体的方法将增强单细胞蛋白生产技术的竞争力。目前的工作通过在含除草剂的培养基上进行诱变和筛选，作为抑制氨基酸生物合成的选择剂，在改善野生型红法夫酵母DSM 5626的蛋白质生产特性方面提供了独特的结果。测定了纯除草剂活性成分对S-(2-氨基乙基)-L-半胱氨酸（AEC）盐酸盐和草铵膦（GA）的抑制浓度，以完全抑制和强烈抑制DSM 5626菌株。化学诱变后，选择浓度为50 mM和100 mM的GA以及0.5 mM和2.5 mM的AEC进行突变体筛选。使用除草剂导致选择出蛋氨酸和赖氨酸合成显著改善的突变体。具体而言，突变体GA6/4和GA7/5的蛋氨酸水平分别比野生型菌株高37%和26%，而GA6/3的赖氨酸比野生型菌株增加了14%。AEC3/9突变体的蛋氨酸增加了35%，赖氨酸增加了24%，异亮氨酸增加了8%，苯丙氨酸增加了6%，突出了这种筛选方法在提高必需氨基酸含量方面的有效性。与产朊假丝酵母、酿酒酵母、马克斯克鲁维酵母等单细胞蛋白酵母商业菌株相比，这些突变体的蛋白质质量参数必需氨基酸指数和氨基酸评分更高。
诱变结合使用除草剂进行选择性筛选是增强酵母中氨基酸生物合成的有效方法。
