Nuclear genome shuffling significantly increases production of chloroplast-based recombinant protein in Chlamydomonas reinhardtii
Nuclear genome shuffling significantly increases production of chloroplast-based recombinant protein in Chlamydomonas reinhardtii
复制标题
DOI：
10.1016/j.algal.2019.101523
复制
发表时间：
2019-08-01
影响因子：
5.1
通讯作者：
Mayfield, Stephen P.
中科院分区：
文献类型：
--
作者：
Fields, Francis J.;Ostrand, Joseph T.;Mayfield, Stephen P.
藻类生物技术为生产可持续和环境友好的产品带来了巨大的希望，但是在藻类可以用作商业相关的生产平台之前，必须开发出更强大的微藻菌株。在这里，我们展示了一种及时有效的方法，通过诱变和基因组改组的组合，显着提高藻类中所需的表型。在这项研究中，我们设计了一个菌株的衣原体reinhardtii产生重组绿色荧光蛋白的叶绿体，然后进行核基因组的各种轮的紫外线诱导的诱变和基因组改组通过有性重组，然后通过分离的菌株与增强的重组蛋白的表达，使用荧光激活细胞分选（FACS）。最大的成功来自于首先UV诱变宿主菌株以在核基因组中产生突变，然后通过FACS分离最佳GFP表达突变体，然后通过将其与选定的野生型菌株交配来改组该突变体的核基因组，然后再次通过FACS分离最佳后代。使用该策略，在< 3个月内进化出表达超过2%总可溶性蛋白（TSP）的GFP的新菌株，比以0.14%TSP表达GFP的初始重组菌株增加了15倍。在不改变GFP基因本身或降低菌株生长速率的情况下实现叶绿体GFP表达的这种显著增加，证明了这种策略在商业相关时间范围内增强特定表型并显著改善藻类物种内的产物产生的有效性。
Algae biotechnology holds great promise for the production of sustainable and environmentally friendly products, however more robust strains of microalgae must be developed before algae can be used as a commercially relevant production platform. Here we demonstrate a timely and effective approach to significantly improve a desired phenotype in algae through a combination of mutagenesis and genome shuffling. In this study, we engineered a strain of Chlamydomonas reinhardtii to produce recombinant green fluorescent protein in the chloroplast and then subjected the nuclear genome to various rounds of UV-induced mutagenesis and genome shuffling via sexual recombination, followed by isolation of strains with enhanced recombinant protein expression using fluorescence-activated cell sorting (FACS). The greatest success came from first UV-mutagenizing the host strain to create mutations in the nuclear genome, followed by FACS to isolate the best GFP-expressing mutant, then shuffling the nuclear genome of that mutant by mating it with selected wild-type strains, and then again isolating the best progeny via FACS. Using this strategy, a novel strain was evolved in < 3 months to express GFP over 2% total soluble protein (TSP), a 15-fold increase from the initial recombinant strain which expressed GFP at 0.14% TSP. This significant increase in GFP expression from the chloroplast was accomplished without altering the GFP gene itself or reducing the growth rate of the strain, demonstrating the effectiveness of this strategy to enhance a specific phenotype and significantly improve product production within an algal species in a commercially relevant timeframe.