β-ionone is a commercially attractive industrial fragrance produced naturally from the cleavage of the pigment β-carotene in plants. While the production of this ionone is typically performed using chemical synthesis, environmentally friendly and consumer-oriented biotechnological production is gaining increasing attention. A convenient cell factory to address this demand is the yeast Saccharomyces cerevisiae. However, current β-ionone titers and yields are insufficient for commercial bioproduction. In this work, we optimized S. cerevisiae for the accumulation of high amounts of β-carotene and its subsequent conversion to β-ionone. For this task, we integrated systematically the heterologous carotenogenic genes (CrtE, CrtYB and CrtI) from Xanthophyllomyces dendrorhous using markerless genome editing CRISPR/Cas9 technology; and evaluated the transcriptional unit architecture (bidirectional or tandem), integration site, and impact of gene dosage, first on β-carotene accumulation, and later, on β-ionone production. A single-copy insertion of the carotenogenic genes in high expression loci of the wild-type yeast CEN.Pk2 strain yielded 4 mg/gDCW of total carotenoids, regardless of the transcriptional unit architecture employed. Subsequent fine-tuning of the carotenogenic gene expression enabled reaching 16 mg/gDCW of total carotenoids, which was further increased to 32 mg/gDCW by alleviating the known pathway bottleneck catalyzed by the hydroxymethylglutaryl-CoA reductase (HMGR1). The latter yield represents the highest total carotenoid concentration reported to date in S. cerevisiae for a constitutive expression system. For β-ionone synthesis, single and multiple copies of the carotene cleavage dioxygenase 1 (CCD1) gene from Petunia hybrida (PhCCD1) fused with a membrane destination peptide were expressed in the highest β-carotene-producing strains, reaching up to 33 mg/L of β-ionone in the culture medium after 72-h cultivation in shake flasks. Finally, interrogation of a contextualized genome-scale metabolic model of the producer strains pointed to PhCCD1 unspecific cleavage activity as a potentially limiting factor reducing β-ionone production. Overall, the results of this work constitute a step toward the industrial production of this ionone and, more broadly, they demonstrate that biotechnological production of apocarotenoids is technically feasible.

中文翻译：

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工程酿酒酵母用于过度生产 β-紫罗兰酮及其前体 β-胡萝卜素

β-紫罗兰酮是一种具有商业吸引力的工业香料，由植物中色素 β-胡萝卜素的裂解自然产生。虽然这种紫罗兰酮的生产通常使用化学合成进行，但环保和面向消费者的生物技术生产越来越受到关注。满足这种需求的一种方便的细胞工厂是酿酒酵母。然而，目前的 β-紫罗兰酮滴度和产量不足以进行商业生物生产。在这项工作中，我们优化了酿酒酵母，以积累大量 β-胡萝卜素，然​​后将其转化为 β-紫罗兰酮。对于这项任务，我们使用无标记基因组编辑 CRISPR/Cas9 技术系统地整合了来自 Xanthophyllomyces dendrorhous 的异源类胡萝卜素基因（CrtE、CrtYB 和 CrtI）；并评估了转录单位结构（双向或串联）、整合位点和基因剂量的影响，首先是对 β-胡萝卜素积累的影响，然后是对 β-紫罗兰酮生产的影响。无论采用何种转录单位结构，在野生型酵母 CEN.Pk2 菌株的高表达位点中单拷贝插入类胡萝卜素基因都产生了 4 mg/gDCW 的总类胡萝卜素。随后对类胡萝卜素基因表达的微调使总类胡萝卜素达到 16 mg/gDCW，通过缓解由羟甲基戊二酰辅酶 A 还原酶 (HMGR1) 催化的已知途径瓶颈，进一步增加到 32 mg/gDCW。后者的产量代表迄今为止在酿酒酵母中报告的组成型表达系统中最高的总类胡萝卜素浓度。对于β-紫罗兰酮合成，来自矮牵牛 (PhCCD1) 的胡萝卜素裂解双加氧酶 1 (CCD1) 基因的单拷贝和多拷贝与膜目标肽融合在产 β-胡萝卜素最高的菌株中表达，β-紫罗兰酮的产量高达 33 mg/L在摇瓶中培养 72 小时后的培养基。最后，对生产菌株的情境化基因组规模代谢模型的询问指出 PhCCD1 非特异性切割活性是减少 β-紫罗兰酮生产的潜在限制因素。总的来说，这项工作的结果是朝着这种紫罗兰酮的工业生产迈出的一步，更广泛地说，它们证明了类胡萝卜素的生物技术生产在技术上是可行的。