Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Apartado 1095, E-41080, Sevilla, Spain.
Int J Food Microbiol. 2010 Oct 15;143(3):150-60. doi: 10.1016/j.ijfoodmicro.2010.08.009. Epub 2010 Aug 17.
HAP4 encodes a transcriptional activator of respiration-related genes and so, redirection from fermentation to respiration flux should give rise to an increase in biomass production in Saccharomyces cerevisiae transformants that overexpress HAP4. With this aim, three bakers' yeasts, that is, V1 used for lean doughs, its 2-deoxy-D-glucose resistant derivative DOG21, and V3 employed for sweet doughs, were transformed with integrative cassettes that carried HAP4 gene under the control of constitutive promoter pTEF2; in addition VTH, DTH and 3TH transformants were selected and characterized. Transformants showed increased expression of HAP4 and respiration-related genes such as QCR7 and QCR8 with regard to parental, and similar expression of SUC2 and MAL12; these genes are relevant in bakers' industry. Invertase (Suc2p) and maltase (Mal12p) activities, growth and sugar consumption rates in laboratory (YPD) or industrial media (MAB) were also comparable in bakers' strains and their transformants, but VTH, DTH and 3TH increased their final biomass production by 9.5, 5.0 and 5.0% respectively as compared to their parentals in MAB. Furthermore, V1 and its transformant VTH had comparable capacity to ferment lean doughs (volume increase rate and final volume) while V3 and its transformant 3TH fermented sweet doughs in a similar manner. Therefore transformants possessed increased biomass yield and appropriate characteristics to be employed in bakers' industry because they lacked drug resistant markers and bacterial DNA, and were genetically stable.
HAP4 编码呼吸相关基因的转录激活因子，因此，将酵母发酵通量重新导向呼吸通量应该会导致过表达 HAP4 的酿酒酵母转化体的生物量增加。为此，我们使用三种面包酵母（用于制作低脂面团的 V1、其 2-脱氧-D-葡萄糖抗性衍生物 DOG21 以及用于甜面团的 V3）进行了转化，这些酵母整合了带有组成型启动子 pTEF2 控制的 HAP4 基因的盒；此外，还选择和表征了 VTH、DTH 和 3TH 转化体。与亲本相比，转化体表现出 HAP4 和呼吸相关基因（如 QCR7 和 QCR8）的表达增加，而 SUC2 和 MAL12 的表达相似；这些基因在面包行业中很重要。在实验室（YPD）或工业培养基（MAB）中，转化体的转化体的蔗糖酶（Suc2p）和麦芽糖酶（Mal12p）活性、生长和糖消耗速率在亲本酵母和其转化体之间也相当，但与亲本相比，VTH、DTH 和 3TH 分别将最终生物量提高了 9.5%、5.0%和 5.0%。此外，V1 及其转化体 VTH 具有可比的发酵低脂面团的能力（体积增加率和最终体积），而 V3 及其转化体 3TH 以类似的方式发酵甜面团。因此，转化体具有更高的生物量产量和适当的特性，可以用于面包行业，因为它们缺乏耐药标记物和细菌 DNA，并且具有遗传稳定性。
