Microb Cell Fact:中科院天津工业生物所发表产糖醇酵母基因组重组

糖醇是一类重要的功能性化合物，具有低热值、低胰岛素代谢响应、防龋齿等特性。2004年，美国能源部(DOE)将其中的D-阿拉伯糖醇和木糖醇列为12类高附加值生物基化学品之一，已广泛应用于食品、医药及化工领域。目前，生产糖醇的方法主要是化学加氢法，但是该方法存在设备投资大，底物要求高等问题，因此，绿色高效的生物合成功能糖醇方法备受关注。而要实现功能糖醇的生物合成，核心在于获得高产糖醇微生物，并根据生产工艺进行有效改造。

天津工业生物所进化与代谢工程研究组在前期工作中筛选获得了一株高效产糖醇异常毕赤酵母Pichia anomala TIB-x229。P. anomala TIB-x229可以将葡萄糖转化为D-阿拉伯糖醇和核糖醇，其最高糖醇转化率可达0.57g/g。P. anomala TIB-x229也可以将木糖转化为D-阿拉伯糖醇，核糖醇和木糖醇，其最高转化率可达0.77g/g。同时，以不同比例葡萄糖和木糖混合糖为底物时，P. anomala TIB-x229也表现出良好的糖醇转化能力，并没有表现出明显的葡萄糖抑制效应，这有利于混合糖的同步有效利用。当以木糖母液废液为底物进行生物转化时，P. anomala TIB-x229也表现出良好的抗逆性和糖醇转化性能，为低成本原料利用奠定了重要基础。

为了进一步提高酵母P. anomala TIB-x229糖醇生产性能，研究人员将高碘酸盐糖醇显色筛选和基于荧光标记的流式融合子筛选进行有效的整合，构建了高效产糖醇酵母基因组重组改造筛选方法。首先，通过传统的紫外和等离子体诱变方法构建突变体亲本库，并利用显色法高通量筛选糖醇产量提高菌株作为出发菌;然后，基于高效荧光染料标记流式筛选技术，对含双荧光融合子进行分选并评价糖醇生产性能。通过两轮基因组重组筛选，实现了产糖醇酵母性能的有效提升，重组菌株GS2-3的糖醇产量较原始菌P. anomala TIB-x229提高了32.3%，为进一步实现糖醇生物制造奠定了基础。本研究构建的高效的基因组重组技术实现了菌种产糖醇性能快速高效提升，同时也为其它缺少遗传标记的非常规酵母基因组工程改造提供了解决方法。

该研究获得国家自然科学基金和863计划的支持，相关研究成果发表在Microbial Cell Factories，中科院天津工业生物所博士研究生张国强为论文的第一作者。

原文链接：

genome shuffling of the nonconventional yeast Pichia anomala for improved sugar alcohol production

原文摘要：

Background

Sugar alcohols have been widely applied in the fields of food and medicine owing to their unique properties. Compared to chemical production, microbial production of sugar alcohols has become attractive because of its environmentally friendly and sustainable characteristics. Our previous study identified the nonconventional yeast Pichia anomala TIB-x229 as a potential producer of sugar alcohols from glucose. To further improve strain performance, we combined genome shuffling with optimized high throughput screening methods for the directed improvement of nonconventional yeast and complex phenotypes.

Results

To accelerate strain improvement, a practical genome shuffling procedure was developed and successfully applied in the nonconventional yeast P. anomala to increase sugar alcohol production. Through two rounds of genome shuffling, an improved P. anomala isolate GS2-3 could produce 47.1 g/L total sugar alcohols from 100 g/L glucose, which was 32.3% higher than the original strain. In this process, a simple and accurate colorimetric assay was optimized and used for high throughput screening of sugar alcohol-producing strains. Moreover, a fluorescence-activated cell sorting method was developed to efficiently screen protoplast fusions for genome shuffling of nonconventional yeast.

Conclusion

An efficient genome shuffling procedure was developed and applied to enhance the sugar alcohol production of the nonconventional yeast P. anomala. Our results provide a general platform for strain improvement of polyol-producing microorganisms or nonconventional microorganisms in the future.

doi:10.1186/s12934-015-0303-8

作者：Qinhong Wang