Biodiversity Research Center, Academia Sinica, 115, Taipei, Taiwan.
Biotechnol Biofuels. 2013 Feb 4;6(1):19. doi: 10.1186/1754-6834-6-19.
Many microorganisms possess enzymes that can efficiently degrade lignocellulosic materials, but do not have the capability to produce a large amount of ethanol. Thus, attempts have been made to transform such enzymes into fermentative microbes to serve as hosts for ethanol production. However, an efficient host for a consolidated bioprocess (CBP) remains to be found. For this purpose, a synthetic biology technique that can transform multiple genes into a genome is instrumental. Moreover, a strategy to select cellulases that interact synergistically is needed.
To engineer a yeast for CBP bio-ethanol production, a synthetic biology technique, called "promoter-based gene assembly and simultaneous overexpression" (PGASO), that can simultaneously transform and express multiple genes in a kefir yeast, Kluyveromyces marxianus KY3, was recently developed. To formulate an efficient cellulase cocktail, a filter-paper-activity assay for selecting heterologous cellulolytic enzymes was established in this study and used to select five cellulase genes, including two cellobiohydrolases, two endo-β-1,4-glucanases and one beta-glucosidase genes from different fungi. In addition, a fungal cellodextrin transporter gene was chosen to transport cellodextrin into the cytoplasm. These six genes plus a selection marker gene were one-step assembled into the KY3 genome using PGASO. Our experimental data showed that the recombinant strain KR7 could express the five heterologous cellulase genes and that KR7 could convert crystalline cellulose into ethanol.
Seven heterologous genes, including five cellulases, a cellodextrin transporter and a selection marker, were simultaneously transformed into the KY3 genome to derive a new strain, KR7, which could directly convert cellulose to ethanol. The present study demonstrates the potential of our strategy of combining a cocktail formulation protocol and a synthetic biology technique to develop a designer yeast host.
许多微生物拥有能够高效降解木质纤维素材料的酶，但它们没有大量生产乙醇的能力。因此，人们尝试将这些酶转化为发酵微生物，作为生产乙醇的宿主。然而，一种有效的综合生物加工（CBP）宿主仍有待发现。为此，需要一种能够将多个基因转化为基因组的合成生物学技术。此外，还需要一种选择协同作用的纤维素酶的策略。
为了用 CBP 生物乙醇生产工程化酵母，最近开发了一种称为“基于启动子的基因组装和同时过表达”（PGASO）的合成生物学技术，该技术可以同时转化和表达克鲁维酵母 Kluyveromyces marxianus KY3 中的多个基因。为了制定有效的纤维素酶混合物，本研究建立了一种用于筛选异源纤维素酶的滤纸活性测定法，并用于从不同真菌中选择五个纤维素酶基因，包括两个纤维二糖水解酶、两个内切-β-1,4-葡聚糖酶和一个β-葡萄糖苷酶基因。此外，选择了一个真菌纤维二糖转运蛋白基因将纤维二糖转运到细胞质中。这六个基因加上一个选择标记基因，使用 PGASO 一步组装到 KY3 基因组中。我们的实验数据表明，重组菌株 KR7 可以表达五个异源纤维素酶基因，并且 KR7 可以将结晶纤维素转化为乙醇。
包括五个纤维素酶、一个纤维二糖转运蛋白和一个选择标记基因在内的七个异源基因被同时转化到 KY3 基因组中，从而衍生出一种新的菌株 KR7，它可以直接将纤维素转化为乙醇。本研究表明，我们的组合鸡尾酒配方协议和合成生物学技术策略开发设计酵母宿主的潜力。
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