Key Laboratory for Enzyme and Enzyme-Like Material Engineering of Heilongjiang, College of Life Science, Northeast Forestry University, Harbin, 150040, Heilongjiang, China.
Biology Institute, Qilu University of Technology (Shandong Academy of Sciences), Jinan, 250103, Shandong, China.
Microb Cell Fact. 2023 Mar 30;22(1):60. doi: 10.1186/s12934-023-02071-9.
Orsellinic acid (2,4-dihydroxy-6-methylbenzoic acid, OA) and its structural analog o-Orsellinaldehyde, have become widely used intermediates in clinical drugs synthesis. Although the research on the biosynthesis of such compounds has made significant progress, due to the lack of suitable hosts, there is still far from the industrial production of such compounds based on synthetic biology.
With the help of genome mining, we found a polyketide synthase (PKS, HerA) in the genome of the Hericium erinaceus, which shares 60% amino acid sequence homology with ArmB from Armillaria mellea, an identified PKS capable of synthesizing OA. To characterize the function of HerA, we cloned herA and heterologously expressed it in Aspergillus oryzae, and successfully detected the production of OA. Subsequently, the introduction of an incomplete PKS (Pks5) from Ustilago maydis containing only three domains (AMP-ACP-R), which was into herA-containing A. oryzae, the resulted in the production of o-Orsellinaldehyde. Considering the economic value of OA and o-Orsellinaldehyde, we then optimized the yield of these compounds in A. oryzae. The screening showed that when maltose was used as carbon source, the yields of OA and o-Orsellinaldehyde were 57.68 mg/L and 15.71 mg/L respectively, while the yields were 340.41 mg/Kg and 84.79 mg/Kg respectively in rice medium for 10 days.
Herein, we successfully expressed the genes of basidiomycetes using A. oryzae heterologous host. As a fungus of ascomycetes, which not only correctly splices genes of basidiomycetes containing multiple introns, but also efficiently produces their metabolites. This study highlights that A. oryzae is an excellent host for the heterologous production of fungal natural products, and has the potential to become an efficient chassis for the production of basidiomycete secondary metabolites in synthetic biology.
獐牙菜苦酸（2,4-二羟基-6-甲基苯甲酸，OA）及其结构类似物奥氏小檗醛已成为临床药物合成中广泛使用的中间体。尽管此类化合物的生物合成研究已经取得了重大进展，但由于缺乏合适的宿主，基于合成生物学的此类化合物的工业生产仍远远没有实现。
借助基因组挖掘，我们在香菇基因组中发现了一种聚酮合酶（PKS，HerA），其与鉴定出的能够合成 OA 的奥氏小檗菌 ArmB 具有 60%的氨基酸序列同源性。为了表征 HerA 的功能，我们克隆了 herA 并在米曲霉中异源表达，成功检测到了 OA 的产生。随后，将仅包含三个结构域（AMP-ACP-R）的来自玉米黑粉菌的不完整 PKS（Pks5）引入含有 herA 的米曲霉中，导致了奥氏小檗醛的产生。考虑到 OA 和奥氏小檗醛的经济价值，我们随后优化了米曲霉中这些化合物的产量。筛选结果表明，当以麦芽糖为碳源时，OA 和奥氏小檗醛的产量分别为 57.68 mg/L 和 15.71 mg/L，而在大米培养基中培养 10 天，产量分别为 340.41 mg/Kg 和 84.79 mg/Kg。
本文成功地使用米曲霉异源宿主表达了担子菌的基因。作为一种子囊菌，米曲霉不仅正确拼接了含有多个内含子的担子菌基因，而且还能高效地产生它们的代谢产物。本研究表明，米曲霉是真菌天然产物异源生产的优秀宿主，有潜力成为合成生物学中担子菌次生代谢产物生产的有效底盘。
