School of Life Sciences and Medicine, Shandong University of Technology, Zibo 255049, China.
Int J Mol Sci. 2024 Sep 9;25(17):9741. doi: 10.3390/ijms25179741.
Bilirubin (BR) is an important ingredient of a valuable Chinese medicine, Calculus bovis. Over recent decades, increasing evidence has confirmed that BR offers health benefits in cardiovascular health, stroke, diabetes, and metabolic syndrome. However, BR is mainly produced by extraction from pig bile. In this study, we assembled an efficient pathway for BR production by metabolic engineering of . First, heme oxygenase (HO1) and biliverdin reductase were co-expressed in . HPLC and LC-MS confirmed the accumulation of BR in the recombinant cells. To improve BR production, the catalytic abilities of HO1 from different species were investigated. In addition, the outermembrane-bound heme receptor (ChuA) and the enzymes involved in heme biosynthesis were overexpressed among which ChuA, 5-aminolevulinic acid dehydratase (HemB), protoporphyrin oxidase (HemG), and ferrochelatase (HemH) were found to enhance BR accumulation in . In addition, expression of ferredoxin (Fd) was shown to contribute to efficient conversion of heme to BR in . To increase supply of NADPH, isocitrate dehydrogenase (IDH), NAD kinase (nadK), NADP-specific glutamate dehydrogenase (gdhA), and glucose-6-phosphate 1-dehydrogenase (ZWF) were overexpressed and were found to enhance BR accumulation when these proteins were expressed with a low-copy plasmid pACYCduet-1. Modular optimization of the committed genes led to a titer of 17.2 mg/L in strain M1BHG. Finally, fed-batch fermentation was performed for the strains M1BHG and M1, resulting in accumulation of 75.5 mg/L and 25.8 mg/L of BR, respectively. This is the first report on biosynthesis of BR through metabolic engineering in a heterologous host.
胆红素（BR）是一种重要的中药——牛黄的有效成分。近几十年来，越来越多的证据证实 BR 对心血管健康、中风、糖尿病和代谢综合征有益。然而，BR 主要是从猪胆汁中提取得到的。在这项研究中，我们通过代谢工程组装了一个生产 BR 的有效途径。首先，在 中共同表达血红素加氧酶（HO1）和胆红素还原酶。HPLC 和 LC-MS 证实 BR 在重组 细胞中积累。为了提高 BR 的产量，研究了不同物种的 HO1 的催化能力。此外，还过表达了外膜结合血红素受体（ChuA）和血红素生物合成相关的酶，其中 ChuA、5-氨基酮戊酸脱水酶（HemB）、原卟啉氧化酶（HemG）和亚铁螯合酶（HemH）被发现可提高 中的 BR 积累。此外，铁氧还蛋白（Fd）的表达有助于 在 中有效地将血红素转化为 BR。为了增加 NADPH 的供应，过表达了异柠檬酸脱氢酶（IDH）、NAD 激酶（nadK）、NADP 特异性谷氨酸脱氢酶（gdhA）和葡萄糖-6-磷酸 1-脱氢酶（ZWF），当这些蛋白在低拷贝质粒 pACYCduet-1 上表达时，发现它们可以提高 BR 的积累。关键基因的模块化优化使菌株 M1BHG 的产量达到 17.2mg/L。最后，对菌株 M1BHG 和 M1 进行分批补料发酵，分别积累了 75.5mg/L 和 25.8mg/L 的 BR。这是首次在异源宿主中通过代谢工程生产 BR。
