College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China.
Acta Biochim Biophys Sin (Shanghai). 2020 Dec 29;52(12):1420-1426. doi: 10.1093/abbs/gmaa134.
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.
β-丙氨酸（3-氨基丙酸）在工业应用中具有巨大的潜力。它可以通过化学合成路线获得，但这种方法对环境有害。众所周知，天冬氨酸-α-脱羧酶（ADC）可以在细菌中将天冬氨酸转化为β-丙氨酸。然而，由于 ADC 的活性较低，通过绿色生物途径进行工业生产β-丙氨酸的情况仍不明确。因此，提高 ADC 的活性对于降低β-丙氨酸生产成本至关重要。在本研究中，我们建立了一种用于高效 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 变体的突变文库中，我们获得了一个活性提高 33%的突变体。进一步的分析表明，ADC 中的 K43R 和 P103Q 突变显著提高了全细胞生物催化产生的β-丙氨酸产量。与之前的单荧光方法相比，我们的系统不仅可以定量β-丙氨酸的量，还可以用不同的荧光测量 ADC 的表达水平，从而能够有效地筛选出相对活性提高的 ADC 变体。用于快速筛选 ADC 的双荧光高通量系统为未来通过生物转化途径工业生产β-丙氨酸提供了一个良好的策略。
