Rational design of short-chain dehydrogenase DHDR for efficient synthesis of (S)-equol.

(S)-equol, the most influential metabolite of daidzein in vivo, has aroused great attention due to the excellent biological activities. Although existing studies have accomplished the construction of its heterologous synthetic pathway in the context of anaerobicity and inefficiency of natural strains, the low productivity of (S)-equol limits its industrial application. Here, rational design strategies based on decreasing the pocket steric hindrance and fine-tuning the pocket microenvironment to systematically redesign the binding pocket of enzyme were developed and processed to the rate-limiting enzyme dihydrodaidzein reductase in (S)-equol synthesis. After iterative combinatorial mutagenesis, an effective mutant S118G/T169A capable of significantly increasing (S)-equol yield was obtained. Computational analyses illustrated that the main reason of the increased activity relied on the decreased critical distance and more stable interacting conformation. Then, the reaction optimization was performed, and the recombinant Escherichia coli whole-cell biocatalyst harboring S118G/T169A enabled the efficient conversion of 2 mM daidzein to (S)-equol, achieving conversion rate of 84.5 %, which was 2.9 times higher than that of the parental strain expressing wide type dihydrodaidzein reductase. This study provides an effective idea and a feasible method for enzyme modification and whole-cell catalytic synthesis of (S)-equol, and will greatly accelerate the process of industrial production.

CRISPR/Cas9-based toolkit for rapid marker recycling and combinatorial libraries in Komagataella phaffii.

Komagataella phaffii, a nonconventional yeast, is increasingly attractive to researchers owing to its posttranslational modification ability, strict methanol regulatory mechanism, and lack of Crabtree effect. Although CRISPR-based gene editing systems have been established in K. phaffii, there are still some inadequacies compared to the model organism Saccharomyces cerevisiae. In this study, a redesigned gRNA plasmid carrying red and green fluorescent proteins facilitated plasmid construction and marker recycling, respectively, making marker recycling more convenient and reliable. Subsequently, based on the knockdown of Ku70 and DNA ligase IV, we experimented with integrating multiple DNA fragments at a single locus. A 26.5-kb-long DNA fragment divided into 11 expression cassettes for lycopene synthesis could be successfully integrated into a single locus at one time with a success rate of 57%. A 27-kb-long DNA fragment could also be precisely knocked out with a 50% positive rate in K. phaffii by introducing two DSBs simultaneously. Finally, to explore the feasibility of rapidly balancing the expression intensity of multiple genes in a metabolic pathway, a yeast combinatorial library was successfully constructed in K. phaffii using lycopene as an indicator, and an optimal combination of the metabolic pathway was identified by screening, with a yield titer of up to 182.73 mg/L in shake flask fermentation. KEY POINTS: • Rapid marker recycling based on the visualization of a green fluorescent protein • One-step multifragment integration and large fragment knockout in the genome • A random assembly of multiple DNA elements to create yeast libraries in K. phaffii.