Construction of a controllable β‐carotene biosynthetic pathway by decentralized assembly strategy in Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae is an important platform organism for the synthesis of a great number of natural products. However, the assembly of controllable and genetically stable heterogeneous biosynthetic pathways in S. cerevisiae still remains a significant challenge. Here, we present a strategy for reconstructing controllable multi-gene pathways by employing the GAL regulatory system. A set of marker recyclable integrative plasmids (pMRI) was designed for decentralized assembly of pathways. As proof-of-principle, a controllable β-carotene biosynthesis pathway (∼16 kb) was reconstructed and optimized by repeatedly using GAL10-GAL1 bidirectional promoters with high efficiency (80-100%). By controling the switch time of the pathway, production of 11 mg/g DCW of total carotenoids (72.57 mg/L) and 7.41 mg/g DCW of β-carotene was achieved in shake-flask culture. In addition, the engineered yeast strain exhibited high genetic stability after 20 generations of subculture. The results demonstrated a controllable and genetically stable biosynthetic pathway capable of increasing the yield of target products. Furthermore, the strategy presented in this study could be extended to construct other pathways in S. cerevisisae.

Cadmium-induced Perturbations in Growth, Oxidative Defense System, Catalase Gene Expression and Fruit Quality in Tomato

“…Roma. Ample information is available in the literature that highlights the negative influence of Cd on plant growth (Xie et al, 2014). The decrease in tomato growth under Cd stress could be due to Cd-induced altered water relations and photosynthetic pigments (Riaz et al, 2014), oxidataive damage (Saidi et al, 2013), altered nutrients uptake and thus reduced cell expansion.…”

Highly efficient biosynthesis of astaxanthin in Saccharomyces cerevisiae by integration and tuning of algal crtZ and bkt

“…The crtZ (KP866868) and bkt (D45881.1) genes were amplified from the cDNA of H. pluvialis Flotow N-212 using primers designed based on the conserved regions of these genes among different algal species, and analyzed by the Codon Adaptation Tool (JCAT) (http://www.jcat.de/) according to the codon preference of S. cerevisiae. The OcrtZ (KP866869) and Obkt (KP866870) sequences with optimized codons were then synthesized by Sangon Biotech (Shanghai, China) and cloned into pUMRIs, which were modified from the previously reported pMRIs (Xie et al 2014a) by insertion of a URA3 cassette to facilitate 5-fluoroorotic acid counterselection and thus simplify the marker recycling process. The constructed plasmids were subsequently inserted into the yeast chromosome to build an astaxanthin synthesis pathway.…”

“…All strains in this work were constructed based on the YXWP-53 strain developed previously (Xie et al 2014a). Firstly, the URA3 marker in the GAL80 locus was replaced by the disruption cassette consisting of a LEU2 selection marker and the upstream and downstream homologous regions of the GAL80 ORF, and then, the resulting strain was selected on a SD (LEU − ) plate.…”

“…In the present study, we cloned the β-carotene hydroxylase and ketolase genes (crtZ and bkt) from H. pluvialis and introduced them into S. cerevisiae by the decentralized assembly strategy to construct an astaxanthin producer based on a β-carotene-hyperproducing parent strain (YXWP-53) recently developed in our group (Xie et al 2014a). The expression of carotenoid biosynthetic genes was under the control of GAL promoters in S. cerevisiae which were regulated by glucose using a modified GAL regulation system.…”

“…Therefore, S. cerevisiae as a generally recognized as safe (GRAS) strain is a more attractive host for the production of natural astaxanthin, especially for feed, food, and pharmaceutical applications. In our previous study, a decentralized assembly strategy was proposed for genomic integration of biosynthetic pathways, which facilitated the efficient construction of a β-carotene biosynthesis pathway in S. cerevisiae (Xie et al 2014a). For this pathway, the critical steps are the sequential reactions catalyzed by phytoene synthase, phytoene desaturase, and lycopene cyclase encoded by crtB (crtYB), crtI, and crtY (crtYB), respectively.…”