In this study, we investigated the bacterial diversity of Moutai Daqu, Zhongxing Daqu and Zhengjiu Daqu from Guizhou Province of China by high-throughput sequencing method. Sequencing results showed that 35 bacterial families were detected in the three Daqu samples. More interestingly, Sporolactobacillaceae, Streptomycetaceae, Pseudomonadaceae, Actinopolysporaceae, Caulobacteraceae, Pseudonocardiaceae, Nocardiaceae and Methlobacteriaceae were reported foe the first time in Moutaiflavour Daqu using high-throughput sequencing method. In particular, Thermoactinomycetaceae and Bacillaceae were demonstrated to be the common dominant bacteria in the three samples. These new data offer a more complete view of comprehensive of high-throughput sequencing method to understand bacteria diversity of Moutai-flavour Daqu.
By exploiting versatile P450 enzymes, whole-cell biocatalysis can be performed to synthesize valuable compounds in Escherichia coli. However, the insufficient supply of heme limits the whole-cell P450 biocatalytic activity. Here a strategy for improving intracellular heme biosynthesis to enhance the catalytic efficiencies of P450s is reported. After comparing the effects of improving heme transport and biosynthesis on P450 activities, intracellular heme biosynthesis is optimized through the integrated expression of necessary synthetic genes at proper ratios and the assembly of rate-limiting enzymes using DNA-guided scaffolds. The intracellular heme level is fine-tuned by the combined use of mutated heme-sensitive biosensors and small regulatory RNA systems. The catalytic efficiencies of three different P450s, BM3, sca-2, and CYP105D7, are enhanced through fine-tuning heme biosynthesis for the synthesis of hydroquinone, pravastatin, and 7,3′,4′-trihydroxyisoflavone as example products of chemical intermediate, drug, and natural product, respectively. This strategy of fine-tuned heme biosynthesis will be generally useful for developing whole-cell biocatalysts involving hemoproteins.
Whole‐Cell Biocatalysts
In the article 2205580, Xinrui Zhao and co‐workers report an engineered Escherichia coli strain capable of improved heme supply suitable for whole‐cell P450 biocatalysis. The intracellular heme level is fine‐tuned by the combined use of mutated heme‐sensitive biosensor and small regulatory RNA systems.
The hydroxylation is an important way to generate the functionalized derivatives of flavonoids. However, the efficient hydroxylation of flavonoids by bacterial P450 enzymes is rarely reported. Here, a bacterial P450 sca-2mut whole-cell biocatalyst with an outstanding 3′-hydroxylation activity for the efficient hydroxylation of a variety of flavonoids was first reported. The whole-cell activity of sca-2mut was enhanced using a novel combination of flavodoxin Fld and flavodoxin reductase Fpr from Escherichia coli. In addition, the double mutant of sca-2mut (R88A/S96A) exhibited an improved hydroxylation performance for flavonoids through the enzymatic engineering. Moreover, the whole-cell activity of sca-2mut (R88A/S96A) was further enhanced by the optimization of whole-cell biocatalytic conditions. Finally, eriodictyol, dihydroquercetin, luteolin, and 7,3′,4′-trihydroxyisoflavone, as examples of flavanone, flavanonol, flavone, and isoflavone, were produced by whole-cell biocatalysis using naringenin, dihydrokaempferol, apigenin, and daidzein as the substrates, with the conversion yield of 77%, 66%, 32%, and 75%, respectively. The strategy used in this study provided an effective method for the further hydroxylation of other high value-added compounds.
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