In vitro and In vivo recombination of heterologous modules for improving biosynthesis of astaxanthin in yeast

Background : Astaxanthin is a kind of tetraterpene and has strong antioxygenic property. Concerning the safety and economy issue the biosynthesis of astaxanthin has greater potential than chemical synthesis and extraction from natural producers. However, the production of astaxanthin in microorganisms is still limited by the poor efficiency of the heterologous pathway. Results: To address the bottleneck of astaxanthin yield in microbes, we developed the in vitro and in vivo recombination methods to optimize the combination of heterologous modules of β-carotene ketolase ( crtW ) and hydroxylase ( crtZ ) from different species in engineered yeast strains. Finally, the astaxanthin yield of in vitro recombination and in vivo recombination were enhanced 2.11- to 8.51-fold and 3.05- to 9.71-fold compared to the parent strains, respectively. The highest astaxanthin producing yeast yQDD022 was obtained by the in vivo recombination with 6.05 mg/g DCW of the astaxanthin yield. Moreover, it is demonstrated that the astaxanthin producing yeast of the in vivo recombination has higher efficiency and stability than that of the in vitro recombination. Conclusions: Recombination of heterologous modules by in vitro and in vivo provides a simple and efficient way to improve the astaxanthin yield in yeast. Both the in vitro and in vivo recombination methods enable high-throughput screening of heterologous pathways by combining crtW and crtZ from different species. And the heterologous pathway constructed by the in vivo recombination is more stable than that of the in vitro recombination. This study not only found the underlying optimal combination of crtZ and crtW , but also provided a reference to greatly enhance desired compounds accumulation by evolving heterologous pathways.

Sex in a test tube: testing the benefits of in vitro recombination

The origin and evolution of sex, and the associated role of recombination, present a major problem in biology. Sex typically involves recombination of closely related DNA or RNA sequences, which is fundamentally a random process that creates but also breaks up beneficial allele combinations. Directed evolution experiments, which combine in vitro mutation and recombination protocols with in vitro or in vivo selection, have proved to be an effective approach for improving functionality of nucleic acids and enzymes. As this approach allows extreme control over evolutionary conditions and parameters, it also facilitates the detection of small or position-specific recombination benefits and benefits associated with recombination between highly divergent genotypes. Yet, in vitro approaches have been largely exploratory and motivated by obtaining improved end products rather than testing hypotheses of recombination benefits. Here, we review the various experimental systems and approaches used by in vitro studies of recombination, discuss what they say about the evolutionary role of recombination, and sketch their potential for addressing extant questions about the evolutionary role of sex and recombination, in particular on complex fitness landscapes. We also review recent insights into the role of ‘extracellular recombination’ during the origin of life. This article is part of the themed issue ‘Weird sex: the underappreciated diversity of sexual reproduction’.