| Saccharomyces cerevisiae is an important platform organism for the synthesis of a great number of natural products. However, the efficient assembly of controllable and genetically stable heterogeneous biosynthetic pathways in S. cerevisiae still remains a major challenge. Carotenoids are a diverse class of high value-added tetraterpene that are widely used as nutrient supplements, antioxidants and anticancer agents. In this study, we developed a set of toolbox and strategies for biosynthetic pathway assembly; in addition, we improved the beta-carotene and lycopene production in S. cerevisiae by combining metabolic engineering and directed evolution strategies.Firstly, we amplified the CrtE, CrtYB and CrtI genes from the cDNA of Xanthophyllomyces dendrorhous for biosynthetic pathway construction on high-copy 2μ plasmids, and a beta-carotene-producing S. cerevisiae strain was obtained. However, the genetic instability of the 2μ plasmids-based pathway assembly strategy inspired us to explore other assembly methods for genomic integration.To integrate long pathways into the yeast genome effectively, based on the Cre/loxP recombination system, a set of pMRI plasmids were constructed. The "pop-out" efficiency of the loxP-KanMX-pBR322ori-loxP structure reached 70%, which guaranteed the efficient recycling of the same selection marker for pathway integration. Furthermore, a decentralized assembly strategy was proposed for pathway assembly. By engineering the GAL regulation system of the yeast, the Gal4 promoter inhibitor Ga180 was disrupted, as a result, the expression of gene under the control of GAL promoter could be solely controlled by the glucose concentration in medium. Using the strategy mentioned above, two copies of CrtYB and CrtI and one copy of CrtE and tHMG1 under the control of GAL1/GAL10 promoter were integrated into the yeast genome, and a total carotenoid production of 11 mg/g DCW (72.57 mg/L) was achieved in shake-flask culture. In addition, the engineered yeast strain exhibited high genetic stability after 20 generations of subculture.Balanced utilization of metabolic intermediates is a key issue for production improvement of target products in microbes. In this study, using a simple colorimetric screening method, carotenoid production was employed as a reporter for promoter strength comparison, the strong promoters were then used for overexpression of target genes and the weak promoters were used for down-regulation of genes in competing pathways. Through sequential control of the downstream, upstream, and competing pathways of FPP, the crucial metabolic node in the biosynthesis of terpenoids, in a predetermined order, a carotenoid production of 20.79 mg/g DCW (1156 mg/L) was achieved, which was increased by 700-times when compared with that of the CrtYB and CrtI integrated strain. Quantitative PCR analysis of the regulated genes confirmed that the transcription patterns were controlled in a sequential manner as expected.Moreover, to construct yeast strains for lycopene production, the directed evolution strategy was employed for engineering the key enzymes in the carotenoid biosynthetic pathway. A CrtYB mutant, CrtYB11M, the lycopene cyclase domain of which was disrupted while the phytoene synthase domain was retained intact, and a CrtE mutant, CrtE03M, the carotenoid production of which was improved by 1.16 times, were obtained by directed evolution. Meanwhile, by adjusting the copy numbers of the Crt genes, the strain YXWPD-14 which showed the highest capacity of lycopene and biomass accumulation was constructed. A carotenoid production of 1.6 g/L (24.4 mg/g DCW) was achieved after 120 h of fermentation. HPLC analysis revealed that more than 90% of the carotenoid product was lycopene. |
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