Hernández-Alcántara Annel M, Chiva Rosana, Mohedano María Luz, Russo Pasquale, Ruiz-Masó José Ángel, Del Solar Gloria, Spano Giuseppe, Tamame Mercedes, López Paloma
Departamento de Biotecnología Microbiana y de Plantas, Centro de Investigaciones Biológicas Margarita Salas, CSIC, Madrid, Spain.
Instituto de Biología Funcional y Genómica, CSIC-Universidad de Salamanca, Salamanca, Spain.
Front Nutr. 2022 Oct 4;9:978831. doi: 10.3389/fnut.2022.978831. eCollection 2022.
This work describes a method for deriving riboflavin overproducing strains of by exposing three strains (BAL3C-5, BAL3C-7, and BAL3C-22) isolated from dough to increasing concentrations of roseoflavin. By this procedure, we selected one mutant overproducing strain from each parental strain (BAL3C-5 B2, BAL3C-7 B2, and BAL3C-22 B2, respectively). Quantification of dextran and riboflavin produced by the parental and mutant strains in a defined medium lacking riboflavin and polysaccharides confirmed that riboflavin was only overproduced by the mutant strains, whereas dextran production was similar in both mutant and parental strains. The molecular basis of the riboflavin overproduction by the mutants was determined by nucleotide sequencing of their operons, which encode the enzymes of the riboflavin biosynthetic pathway. We detected a unique mutation in each of the overproducing strains. These mutations, which map in the sensor domain (aptamer) of a regulatory element (the so-called FMN riboswitch) present in the 5' untranslated region of the operon mRNA, appear to be responsible for the riboflavin-overproducing phenotype of the BAL3C-5 B2, BAL3C-7 B2, and BAL3C-22 B2 mutant strains. Furthermore, the molecular basis of dextran production by the six strains has been characterized by () the sequencing of their genes encoding dextransucrases, which synthesize dextran using sucrose as substrate, and () the detection of active Dsr proteins by zymograms. Finally, the parental and mutant strains were analyzed for production of riboflavin and dextran during experimental bread making. The results indicate that the mutant strains were able to produce experimental wheat breads biofortified with both riboflavin and dextran and, therefore, may be useful for the manufacture of functional commercial breads.
本研究描述了一种通过将从面团中分离出的三株菌株（BAL3C - 5、BAL3C - 7和BAL3C - 22）暴露于浓度递增的玫瑰黄素中来获得核黄素高产菌株的方法。通过该程序，我们从每个亲本菌株中分别筛选出一株突变高产菌株（分别为BAL3C - 5 B2、BAL3C - 7 B2和BAL3C - 22 B2）。在缺乏核黄素和多糖的限定培养基中，对亲本菌株和突变菌株产生的葡聚糖和核黄素进行定量分析，结果证实只有突变菌株过量产生核黄素，而突变菌株和亲本菌株的葡聚糖产量相似。通过对编码核黄素生物合成途径中酶的操纵子进行核苷酸测序，确定了突变体过量产生核黄素的分子基础。我们在每个高产菌株中检测到一个独特的突变。这些突变位于操纵子mRNA 5'非翻译区存在的调节元件（所谓的FMN核糖开关）的传感器结构域（适体）中，似乎是BAL3C - 5 B2、BAL3C - 7 B2和BAL3C - 22 B2突变菌株核黄素高产表型的原因。此外，通过以下方式对六株菌株产生葡聚糖的分子基础进行了表征：（i）对编码葡聚糖蔗糖酶的基因进行测序，葡聚糖蔗糖酶以蔗糖为底物合成葡聚糖；（ii）通过酶谱法检测活性Dsr蛋白。最后，在实验性面包制作过程中，对亲本菌株和突变菌株的核黄素和葡聚糖产量进行了分析。结果表明，突变菌株能够生产同时富含核黄素和葡聚糖的实验性小麦面包，因此可能有助于功能性商业面包的制造。
