BACKGROUND Astaxanthin is a kind of tetraterpene and has strong antioxygenic property. The biosynthesis of astaxanthin in engineered microbial chassis has greater potential than its chemical synthesis and extraction from natural producers in an environmental-friendly way. However, the cost-offsetting production of astaxanthin in engineered microbes is still constrained by the poor efficiency of astaxanthin synthesis pathway as a heterologous pathway. RESULTS To address the bottleneck of limited production of astaxanthin in microbes, we developed in vitro and in vivo recombination methods respectively in engineered yeast chassis to optimize the combination of heterologous β-carotene ketolase (crtW) and hydroxylase (crtZ) modules that were selected from different species. As a result, the in vitro and in vivo recombination methods enhanced the astaxanthin yield respectively to 2.11-8.51 folds and 3.0-9.71 folds compared to the initial astaxanthin pathway, according to the different combination of particular genes. The highest astaxanthin producing strain yQDD022 was constructed by in vivo method and produced 6.05 mg g-1 DCW of astaxanthin. Moreover, it was proved that the in vivo recombination method showed higher DNA-assembling efficiency than the in vitro method and contributed to higher stability to the engineered yeast strains. CONCLUSIONS The in vitro and in vivo recombination methods of heterologous modules provide simple and efficient ways to improve the astaxanthin yield in yeast. Both the two methods enable high-throughput screening of heterologous pathways through recombination of certain crtW and crtZ derived from different species. This study not only exploited the underlying optimal combination of crtZ and crtW for astaxanthin synthesis, but also provided a general approach to evolve a heterologous pathway for the enhanced accumulation of desired biochemical products.

中文翻译：

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异源模块的体外和体内重组，以改善酵母中虾青素的生物合成。

背景技术虾青素是一种四萜烯，具有很强的抗氧特性。在工程微生物底盘中虾青素的生物合成比其化学合成和以天然方式从自然生产者中提取具有更大的潜力。然而，虾青素在工程微生物中的成本抵消生产仍然受到虾青素合成途径作为异源途径效率低下的制约。结果为解决微生物中虾青素生产受限的瓶颈，我们分别在工程酵母底盘中开发了体外和体内重组方法，以优化选自以下来源的异源β-胡萝卜素酮醇酶（crtW）和羟化酶（crtZ）模块的组合不同的物种。结果是，根据特定基因的不同组合，体外和体内重组方法使虾青素的产量分别比初始虾青素途径提高了2.11-8.51倍和3.0-9.71倍。通过体内方法构建最高虾青素产生菌株yQDD022，并产生6.05mg g-1 DCW的虾青素。此外，已经证明，体内重组方法比体外方法显示出更高的DNA组装效率，并且对工程酵母菌株具有更高的稳定性。结论异源模块的体外和体内重组方法为提高酵母中虾青素的产量提供了简单有效的方法。两种方法都可以通过重组不同物种的某些crtW和crtZ进行高通量筛选异源途径。这项研究不仅为虾青素的合成利用了crtZ和crtW的潜在最佳组合，而且还提供了一种通用途径来发展异源途径，以增强所需生化产物的积累。