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Metabolic engineering Saccharomyces cerevisiae for de novo production of the sesquiterpenoid (+)-nootkatone.
Microbial Cell Factories ( IF 6.4 ) Pub Date : 2020-02-03 , DOI: 10.1186/s12934-020-1295-6
Xiangfeng Meng 1 , Hui Liu 1 , Wenqiang Xu 1 , Weixin Zhang 1 , Zheng Wang 1 , Weifeng Liu 1
Affiliation  

BACKGROUND (+)-Nootkatone is a highly valued sesquiterpenoid compound, exhibiting a typical grapefruit aroma and various desired biological activities for use as aromatics and pharmaceuticals. The high commercial demand of (+)-nootkatone is predominately met by chemical synthesis, which entails the use of environmentally harmful reagents. Efficient synthesis of (+)-nootkatone via biotechnological approaches is thus urgently needed to satisfy its industrial demand. However, there are only a limited number of studies that report the de novo synthesis of (+)-nootkatone from simple carbon sources in microbial cell factories, and with relatively low yield. RESULTS As the direct precursor of (+)-nootkatone biosynthesis, (+)-valencene was first produced in large quantities in Saccharomyces cerevisiae by overexpressing (+)-valencene synthase CnVS of Callitropsis nootkatensis in combination with various mevalonate pathway (MVA) engineering strategies, including the expression of CnVS and farnesyl diphosphate synthase (ERG20) as a fused protein, overexpression of a truncated form of the rate-limiting enzyme 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (tHMG1), and downregulating the squalene synthase enzyme (ERG9). These approaches altogether brought the production of (+)-valencene to 217.95 mg/L. Secondly, we addressed the (+)-valencene oxidation by overexpressing the Hyoscyamus muticus premnaspirodiene oxygenase (HPO) variant (V482I/A484I) and cytochrome P450 reductase (ATR1) from Arabidopsis thaliana. However, (+)-valencene was predominantly oxidized to β-nootkatol and only minor amounts of (+)-nootkatone (9.66 mg/L) were produced. We further tackled the oxidation of β-nootkatol to (+)-nootkatone by screening various dehydrogenases. Our results showed that the short-chain dehydrogenase/reductase (SDR) superfamily dehydrogenases ZSD1 of Zingiber zerumbet and ABA2 of Citrus sinensis were capable of effectively catalyzing β-nootkatol oxidation to (+)-nootkatone. The yield of (+)-nootkatone increased to 59.78 mg/L and 53.48 mg/L by additional overexpression of ZSD1 and ABA2, respectively. CONCLUSION We successfully constructed the (+)-nootaktone biosynthesis pathway in S. cerevisiae by overexpressing the (+)-valencene synthase CnVS, cytochrome P450 monooxygenase HPO, and SDR family dehydrogenases combined with the MVA pathway engineering, providing a solid basis for the whole-cell production of (+)-nootkatone. The two effective SDR family dehydrogenases tested in this study will serve as valuable enzymatic tools in further optimizing (+)-nootkatone production.

中文翻译:

代谢工程酿酒酵母,用于从头生产倍半萜(+)-新酮。

背景技术(+)-Nootkatone是一种高价值的倍半萜类化合物,具有典型的葡萄柚香气和用作芳香剂和药物的各种所需的生物活性。化学合成主要满足了(+)-Nootkatone的高商业需求,这需要使用对环境有害的试剂。因此,迫切需要通过生物技术方法高效合成(+)-Nootkatone,以满足其工业需求。然而,仅有极少数的研究报道了微生物细胞工厂从简单碳源从头合成(+)-Nootkatone的方法,并且产率相对较低。结果作为(+)-Nootkatone生物合成的直接前体,(+)-瓦伦烯是在酿酒酵母中首先通过过量表达Callitropsis nootkatensis的(+)-瓦伦烯合酶CnVS并结合各种甲羟戊酸途径(MVA)工程策略(包括CnVS和法呢基二磷酸合酶(ERG20)的表达)而大量生产的作为一种融合蛋白,限速酶3-羟基-3-甲基戊二酰辅酶A(HMG-CoA)还原酶(tHMG1)的截短形式过表达,并下调鲨烯合酶(ERG9)。这些方法总共使(+)-瓦伦烯的产量达到217.95 mg / L。其次,我们通过过量表达拟南芥的Hyoscyamus muticus早春螺二烯加氧酶(HPO)变体(V482I / A484I)和细胞色素P450还原酶(ATR1)解决了(+)-瓦伦烯氧化问题。然而,(+)-瓦伦烯主要被氧化为β-Nootkatol,仅产生少量的(+)-Nootkatone(9.66 mg / L)。通过筛选各种脱氢酶,我们进一步解决了将β-Nootkatol氧化为(+)-Nootkatone的问题。我们的结果表明,姜黄短链脱氢酶/还原酶(SDR)超家族脱氢酶ZSD1和中华柑桔的ABA2能够有效催化β-notototol氧化为(+)-nootkatone。通过额外的ZSD1和ABA2的过表达,(+)-Nootkatone的产量分别增加到59.78 mg / L和53.48 mg / L。结论我们通过与MVA途径工程相结合,过量表达了(+)-瓦伦烯合酶CnVS,细胞色素P450单加氧酶HPO和SDR家族脱氢酶,成功构建了酿酒酵母中的(+)-Nootaktone生物合成途径,为(+)-Nootkatone的全细胞生产提供了坚实的基础。在这项研究中测试的两种有效的SDR家族脱氢酶将作为进一步优化(+)-Nootkatone生产的有价值的酶工具。
更新日期:2020-02-04
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