College of Life and Science, Nanjing Normal University, Nanjing, 210023, JS, China.
School of Environment and Safety Engineering, Jiangsu University, 1 Xuefu Road, Zhenjiang, 212013, JS, China.
Appl Microbiol Biotechnol. 2018 Sep;102(18):7951-7962. doi: 10.1007/s00253-018-9185-0. Epub 2018 Jul 3.
Glutamine:fructose-6-phosphate aminotransferase (GFAT) catalyzes the formation of glucosamine-6-phosphate, and its gene is one of the genes essential for microbes. Using the GFAT-encoding gene can prevent the use of a drug-resistant gene as a selection marker in a bacterial system. Another unique property of the GFAT selection marker is that no particular compound is prohibited or required for creating a selective stress for a yeast. Filamentous fungi are major producers of industrial enzymes. However, there has been no report on the construction and application of the GFAT gene as a selection marker in filamentous fungi. To develop a new selection marker, the GFAT-encoding gene gfaA was deleted from the genome of the filamentous fungus Aspergillus nidulans, and the gfat gene of the straw mushroom Volvariella volvacea was used as the selection marker to mediate the transformation and overexpression of a thermostable bacterial laccase in A. nidulans. The GFAT-deficient strain A. nidulans ∆gfaA was not able to grow in the culture medium containing 0.5% yeast extract unless about 20 mM glucosamine was used to supplement to the medium. The gfat gene was amplified and inserted into the integration vector pAL5 and autonomous replication vector Prg3-AMA1-NotI for A. nidulans to generate the gfat vectors pALG and pAMAG, respectively. Using these gfat vectors, the laccase gene lcs from a hyperthermophilic bacterium was overexpressed intra- and extracellularly in A. nidulans ∆gfaA. Therefore, recombinant filamentous fungi can be constructed with gfat vectors, which can be maintained stably in host cells with the naturally occurred selective stress of a medium, forage, pulp, animal gut, wastewater, or soil.
果糖-6-磷酸氨基转移酶（GFAT）催化葡萄糖胺-6-磷酸的形成，其基因是微生物必需的基因之一。使用 GFAT 编码基因可以防止在细菌系统中使用耐药基因作为选择标记。GFAT 选择标记的另一个独特性质是，对于酵母，没有特定的化合物被禁止或需要用于创建选择性压力。丝状真菌是工业酶的主要生产者。然而，尚未有关于 GFAT 基因作为丝状真菌选择标记的构建和应用的报道。为了开发新的选择标记，从丝状真菌构巢曲霉的基因组中删除了编码 GFAT 的基因 gfaA，并使用草菇 Volvariella volvacea 的 gfat 基因作为选择标记，介导耐热细菌漆酶在 A. nidulans 中的转化和过表达。不能在含有 0.5%酵母提取物的培养基中生长的 GFAT 缺陷型 A. nidulans ∆gfaA 菌株，除非向培养基中补充约 20 mM 葡萄糖胺。扩增 gfat 基因并将其插入整合载体 pAL5 和自主复制载体 Prg3-AMA1-NotI 中，用于在 A. nidulans 中分别生成 gfat 载体 pALG 和 pAMAG。使用这些 gfat 载体，在 A. nidulans ∆gfaA 中细胞内和细胞外过表达了来自嗜热细菌的漆酶基因 lcs。因此，可以使用 gfat 载体构建重组丝状真菌，这些载体可以在宿主细胞中稳定维持，其选择性压力来源于培养基、饲料、纸浆、动物肠道、废水或土壤中的天然选择性压力。
