Abstract

β-Alanine (3-aminopropionic acid) holds great potential in industrial application. It can be obtained through a chemical synthesis route, which is hazardous to the environment. It is well known that l-aspartate-α-decarboxylase (ADC) can convert l-aspartate to β-alanine in bacteria. However, due to the low activity of ADC, industrial production of β-alanine through the green biological route remains unclear. Thus, improving the activity of ADC is critical to reduce the cost of β-alanine production. In this study, we established a dual-fluorescence high-throughput system for efficient ADC screening. By measuring the amount of β-alanine and the expression level of ADC using two different fluorescence markers, we can rapidly quantify the relative activity of ADC variants. From a mutagenesis library containing 2000 ADC variants, we obtained a mutant with 33% increased activity. Further analysis revealed that mutations of K43R and P103Q in ADC significantly improved the yield of β-alanine produced by the whole-cell biocatalysis. Compared with the previous single-fluorescence method, our system can not only quantify the amount of β-alanine but also measure the expression level of ADC with different fluorescence, making it able to effectively screen out ADC variants with improved relative activity. The dual-fluorescence high-throughput system for rapid screening of ADC provides a good strategy for industrial production of β-alanine via the biological conversion route in the future.

中文翻译：

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开发用于高通量筛选L-天门冬氨酸-α-脱羧酶的双荧光报告系统

摘要

β-丙氨酸（3-氨基丙酸）在工业上具有巨大的潜力。可以通过对环境有害的化学合成途径获得。众所周知，l-天门冬氨酸-α-脱羧酶（ADC）可以将l-细菌中β-丙氨酸的天冬氨酸。然而，由于ADC的低活性，通过绿色生物途径的β-丙氨酸的工业生产仍不清楚。因此，提高ADC的活性对于降低β-丙氨酸的生产成本至关重要。在这项研究中，我们建立了用于高效ADC筛选的双荧光高通量系统。通过使用两种不同的荧光标记物测量β-丙氨酸的量和ADC的表达水平，我们可以快速定量ADC变体的相对活性。从包含2000个ADC变体的诱变文库中，我们获得了活性提高33％的突变体。进一步的分析表明，ADC中K43R和P103Q的突变显着提高了全细胞生物催化产生的β-丙氨酸的产量。与以前的单荧光法相比，我们的系统不仅可以量化β-丙氨酸的量，还可以测量具有不同荧光的ADC的表达水平，从而能够有效筛选出具有相对活性的ADC变体。用于快速筛选ADC的双荧光高通量系统为将来通过生物转化途径工业生产β-丙氨酸提供了一个很好的策略。