Division of Biotechnology, Department of Chemistry, Center for Chemistry & Chemical Engineering, Lund University, Box 124, 221 00, Lund, Sweden.
Appl Microbiol Biotechnol. 2024 Aug 17;108(1):442. doi: 10.1007/s00253-024-13256-6.
The antioxidant molecule protocatechuic acid (PCA) can also serve as a precursor for polymer building blocks. PCA can be produced in Escherichia coli overexpressing 3-dehydroshikimate dehydratase (DSD), an enzyme that catalyses the transformation of 3-dehydroshikimate to PCA. Nevertheless, optimizing the expression rate of recombinant enzymes is a key factor in metabolic engineering when producing biobased chemicals. In this study, a degenerate synthetic promoter approach was investigated to improve further the production of PCA. By limited screening of a randomized promoter library made using pSEVA221 plasmid in E. coli, three novel synthetic constitutive promoters were selected that increased the PCA yield from glucose by 10-21% compared to the inducible T7-promoter. RT-qPCR analysis showed that the DSD gene, regulated by the synthetic promoters, had high expression during the exponential phase, albeit the gene expression level dropped 250-fold during stationary phase. Besides the increased product yield, the synthetic promoters avoided the need for a costly inducer for gene expression. Screening of the entire promoter library is likely to provide more positive hits. The study also shows that E. coli transformed with the DSD gene on either pSEVA221 or pCDFDuet plasmids exhibit background PCA levels (~ 0.04 g/L) in the absence of a transcriptional regulatory element. KEY POINTS: • Degenerate synthetic promoters are remarkable tools to produce protocatechuic acid. • The constitutive synthetic promoters did not affect the growth rate of the bacterial host. • The use of constitutive synthetic promoters avoids the need for the costly inducer.
抗氧化分子原儿茶酸（PCA）也可以作为聚合物构建块的前体。在过表达 3-脱氢莽草酸脱水酶（DSD）的大肠杆菌中可以生产 PCA，该酶催化 3-脱氢莽草酸转化为 PCA。然而，在生产生物基化学品时，优化重组酶的表达率是代谢工程的关键因素。在这项研究中，研究了一种简并合成启动子方法，以进一步提高 PCA 的产量。通过在大肠杆菌中使用 pSEVA221 质粒对随机启动子文库进行有限筛选，选择了三个新的合成组成型启动子，与诱导型 T7-启动子相比，葡萄糖生产 PCA 的产量提高了 10-21%。RT-qPCR 分析表明，受合成启动子调控的 DSD 基因在指数生长期具有高表达水平，尽管在稳定期基因表达水平下降了 250 倍。除了提高产物产量外，合成启动子还避免了对基因表达所需的昂贵诱导剂的需求。对整个启动子文库进行筛选可能会提供更多的阳性结果。该研究还表明，在没有转录调节元件的情况下，在 pSEVA221 或 pCDFDuet 质粒上转化了 DSD 基因的大肠杆菌会产生背景 PCA 水平（约 0.04 g/L）。关键点：• 简并合成启动子是生产原儿茶酸的重要工具。• 组成型合成启动子不影响细菌宿主的生长速度。• 使用组成型合成启动子避免了昂贵诱导剂的需求。
