Lee Danna, Lloyd Natoiya D R, Pretorius Isak S, Borneman Anthony R
The Australian Wine Research Institute, PO Box 197, Adelaide, SA, 5064, Australia.
Macquarie University, Sydney, NSW, 2109, Australia.
Microb Cell Fact. 2016 Mar 4;15:49. doi: 10.1186/s12934-016-0446-2.
Raspberry ketone is the primary aroma compound found in raspberries and naturally derived raspberry ketone is a valuable flavoring agent. The economic incentives for the production of raspberry ketone, combined with the very poor yields from plant tissue, therefore make this compound an excellent target for heterologous production in synthetically engineered microbial strains.
A de novo pathway for the production of raspberry ketone was assembled using four heterologous genes, encoding phenylalanine/tyrosine ammonia lyase, cinnamate-4-hydroxlase, coumarate-CoA ligase and benzalacetone synthase, in an industrial strain of Saccharomyces cerevisiae. Synthetic protein fusions were also explored as a means of increasing yields of the final product.
The highest raspberry ketone concentration achieved in minimal media exceeded 7.5 mg/L when strains were fed with 3 mM p-coumaric acid; or 2.8 mg/L for complete de novo synthesis, both of which utilized a coumarate-CoA ligase, benzalacetone synthase synthetic fusion protein that increased yields over fivefold compared to the native enzymes. In addition, this strain was shown to be able to produce significant amounts of raspberry ketone in wine, with a raspberry ketone titer of 3.5 mg/L achieved after aerobic fermentation of Chardonnay juice or 0.68 mg/L under anaerobic winemaking conditions.
We have shown that it is possible to produce sensorially-relevant quantities of raspberry ketone in an industrial heterologous host. This paves the way for further pathway optimization to provide an economical alternative to raspberry ketone derived from plant sources.
覆盆子酮是覆盆子中发现的主要香气化合物，天然来源的覆盆子酮是一种有价值的调味剂。生产覆盆子酮的经济诱因，再加上植物组织产量极低，因此使这种化合物成为在合成工程微生物菌株中进行异源生产的理想目标。
利用四个异源基因，即编码苯丙氨酸/酪氨酸氨裂解酶、肉桂酸-4-羟化酶、香豆酸-CoA连接酶和苯甲酰丙酮合酶，在酿酒酵母工业菌株中组装了一条生产覆盆子酮的从头合成途径。还探索了合成蛋白融合作为提高最终产物产量的一种方法。
当用3 mM对香豆酸喂养菌株时，在基本培养基中达到的最高覆盆子酮浓度超过7.5 mg/L；或从头完全合成时为2.8 mg/L，这两种情况均使用了香豆酸-CoA连接酶、苯甲酰丙酮合酶合成融合蛋白，与天然酶相比，产量提高了五倍以上。此外，该菌株被证明能够在葡萄酒中产生大量覆盆子酮，在霞多丽葡萄汁有氧发酵后，覆盆子酮滴度达到3.5 mg/L，在厌氧酿酒条件下为0.68 mg/L。
我们已经表明，在工业异源宿主中生产感官上相关量的覆盆子酮是可能的。这为进一步优化途径铺平了道路，以提供一种经济的替代植物来源的覆盆子酮的方法。
