López Javiera, Bustos Diego, Camilo Conrado, Arenas Natalia, Saa Pedro A, Agosin Eduardo
Centro de Aromas y Sabores, DICTUC S.A., Santiago, Chile.
Department of Chemical and Bioprocess Engineering, School of Engineering, Pontificia Universidad Católica de Chile, Santiago, Chile.
Front Bioeng Biotechnol. 2020 Sep 30;8:578793. doi: 10.3389/fbioe.2020.578793. eCollection 2020.
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 . However, current β-ionone titers and yields are insufficient for commercial bioproduction. In this work, we optimized 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 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 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 for a constitutive expression system. For β-ionone synthesis, single and multiple copies of the carotene cleavage dioxygenase 1 (CCD1) gene from (CCD1) 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 CCD1 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.
β-紫罗兰酮是一种具有商业吸引力的工业香料，它由植物中色素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采用化学合成方法，但环境友好型和以消费者为导向的生物技术生产正受到越来越多的关注。满足这一需求的便捷细胞工厂是酵母。然而，目前β-紫罗兰酮的滴度和产量不足以进行商业生物生产。在这项工作中，我们针对大量β-胡萝卜素的积累及其随后向β-紫罗兰酮的转化进行了优化。为此任务，我们使用无标记基因组编辑CRISPR/Cas9技术系统地整合了来自[具体来源未明确]的异源类胡萝卜素生成基因（CrtE、CrtYB和CrtI）；并评估了转录单元结构（双向或串联）、整合位点以及基因剂量的影响，首先是对β-胡萝卜素积累的影响，随后是对β-紫罗兰酮生产的影响。在野生型酵母CEN.Pk2菌株的高表达区域单拷贝插入类胡萝卜素生成基因，无论采用何种转录单元结构，总类胡萝卜素产量均为4 mg/gDCW。随后对类胡萝卜素生成基因表达进行微调，使总类胡萝卜素产量达到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，进一步提高到32 mg/gDCW。后一产量代表了迄今为止[具体范围未明确]中组成型表达系统报道的最高总类胡萝卜素浓度。对于β-紫罗兰酮合成，在产生β-胡萝卜素最多的菌株中表达了来自[具体来源未明确]的与膜靶向肽融合的胡萝卜素裂解双加氧酶1（CCD1）基因的单拷贝和多拷贝，在摇瓶中培养72小时后，培养基中β-紫罗兰酮含量高达33 mg/L。最后，对生产菌株的上下文基因组规模代谢模型进行分析表明，CCD1的非特异性裂解活性是降低β-紫罗兰酮产量的潜在限制因素。总体而言，这项工作的结果朝着这种紫罗兰酮的工业化生产迈出了一步，更广泛地说，它们表明类胡萝卜素的生物技术生产在技术上是可行的。
