Journal
JOURNAL OF BIOTECHNOLOGY
Volume 325, Issue -, Pages 119-127Publisher
ELSEVIER
DOI: 10.1016/j.jbiotec.2020.11.009
Keywords
Flavonoids; rDNA; multi-copy; copy number; feedback inhibition
Categories
Funding
- National Key Research and Development Program of China [2019YFA0904800]
- National Outstanding Youth Foundation [21822806]
- Fundamental Research Funds for the Central Universities [JUSRP52018A]
- National First-class Discipline Program of Light Industry Technology and Engineering [LITE2018-08]
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Flavonoids have notable biological activities and are widely used in various industries. This study successfully integrated multiple flavonoid pathway genes in a single-step integration method in yeast, significantly improving the production of naringenin. Optimizing the supply of donor DNA and utilizing a high titer p-coumaric acid strain as a chassis were key factors in enhancing flavonoid production.
Flavonoids have notable biological activities and have been widely used in the medicinal and chemical industries. However, single-copy integration of heterologous pathway genes limits the production of flavonoids. In this work, we designed and constructed single-step integration of multiple flavonoid (2S)-naringenin biosynthetic pathway genes in S. cerevisiae. The efficiency of the naringenin metabolic pathway gene integration into the rDNA site reached 93.7%. Subsequently, we used a high titer p-coumaric acid strain as a chassis, which eliminated feedback inhibition of tyrosine and downregulated the competitive pathway. The results indicated that increasing the supply of p-coumaric acid was effective for naringenin production. We additionally optimized the amount of donor DNA. The optimum strain produced 149.8 mg/L of (2S)-naringenin. The multi-copy integration of flavonoid pathway genes effectively improved (2S)-naringenin production in S. cerevisiae. We further analyzed the copy numbers and expression levels of essential genes (4CL and CHS) in the (2S)-naringenin metabolic pathway by qPCR. Higher copy numbers of the (2S)-naringenin metabolic pathway genes were associated with greater 4CL and CHS transcription, and the efficiency of naringenin production was higher. Therefore, multi-copy integration of genes in the (2S)-naringenin metabolic pathway was imperative in rewiring p-coumaric acid flux to enhance flavonoid production.
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