Flavonoids are a group of valuable compounds that have a variety of health benefits. (2S)-Naringenin is an important flavonoid precursor that can synthesize almost all flavonoids.
However, current metabolic engineering strategies do not fully utilize the intracellular carbon source for the synthesis of (2S)-naringenin. Insufficient precursors and loss of carbon metabolic flux severely limit the microbial heterologous synthesis of (2S)-naringenin.
Recently, Jingwen Zhou's team at Jiangnan University adopted a systems engineering strategy to produce (2S)-naringenin at a high level. Eventually,After fermentation in a 5L bioreactor, the (2S)-naringenin titer reached 34206 mg l, the highest titer reported to date. This study not only provides the possibility for the efficient bioproduction of flavonoids, but also provides an idea for the biosynthesis of natural products through the centralized utilization of intracellular carbon sources.
Flavonoids are a group of plant secondary metabolites containing more than 15,000 compounds found in almost all fruits, vegetables, and medicinal plants. Due to their various physiological activities, such as anti-cancer, anti-inflammatory, antioxidant, anti-malarial, and anti-leishmania, flavonoids have been widely used in chemical, pharmaceutical, food, cosmetic, agrochemical and other industries.
2S)-Naringenin production method mainly relies on extraction from orange peel, but there are limitations such as low production efficiency, long culture cycle, and high extraction cost, which is neither economical nor environmentally friendlyIn addition, (2S)-naringenin and its derivatives still face challenges in terms of production.
Therefore, the synthesis of microorganisms (2S)-naringenin represented by Saccharomyces cerevisiae has become a hot topic in the industry.
in natural plants(2S)-Naringenin biosynthesis requires two precursors p-coumaric acidwithMalonyl-CoADeficiencies in coumaric acid and malonyl-CoA affect the production of (2S)-naringentin. Among them, p-coumaric acid is synthesized from phenylalanine or tyrosine through the phenylalanine ammonia lyase (PAL) pathway and tyrosine ammonilyase (TAL) pathway, respectively.
The team systematically studied the effects of tyrosine and shikimic acid pathway-related genes on (2S)-naringenin biosynthesis. The yeast strain E32 used is derived from the team's previous work.
Figure |2s)-Naringenin biosynthesis of Saccharomyces cerevisiae systems engineering (**above**).
First, variants such as shikimic kinase derived from Escherichia coli were introduced into E32 to give strains HB01 and HB02. However, the (2s)-naringenin titer was not increased;In addition, the overexpression of the inner courtyard TYR1 gene in Saccharomyces cerevisiae did not significantly increase the (2S)-naringenin titer.
Next, the team constructed ARO8 overexpression and ARO9 knockout strains, HB04 and HB05, respectively. Among them, aro9 is involved in the catabolism of aromatic amino acids. The titer of (2S)-naringenin in strain HB04 is 1604mg L with a titer of 196 in strain HB05 (2S)-Naringenin3mg/l。
HB06 is constructed by replacing the promoter of the branching pathway PHA2 gene with glucose-regulated promoter PHXT1. Compared to strain E32, the (2S)-naringenin titer was increased by 196% to 1793mg/l。
To determine the effect of p-coumaric acid on (2S)-naringenin biosynthesis, the researchers constructed strain HB12, which was cultured in YPD medium supplemented with 500 mg L of p-coumarin for 120 hours, and the (2S)-naringenin titer increased to 3876mg/l 。
The team also explored the effect of multiple conditions on the titer of (2S)-naringenin, and finally co-expressed and knocked out the genes TYR1, PHA2, ARO1, ARO2, ARO3, ECAROL and ARO8 to generate strain HB32, resulting in a (2S)-naringenin titer of 3475mg/l。This suggests that the synergistic effect of the PAL and TAL pathways, as well as the upstream aromatic amino acid pathway, enhances the ability of the engineered strain to synthesize (2S)-naringenin.
To generate 1 molecule (2S)-naringenin, 3 molecules of malonyl-CoA are required in addition to 1 molecule of p-coumaric acid. Therefore, the team also explored limitations targeting the malonyl-CoA biosynthesis pathway.
First, TAT1 was replaced with the acetyl-CoA synthase mutation gene SEACSL641P from Salmonella enterica, and knocking out this gene increased p-coumaric acid production. The (2S)-naringenin titer in the resulting strain HB36 is 4528mg/l。
In combination with ALD6 overexpression, the resulting strain HB37 produced (2S)-naringenin 79% 。
Since acetyl-CoA is a central molecule in cellular metabolism and an important precursor of this metabolism, the team expects that the enhancement of acetyl-CoA** will facilitate the conversion to malonyl-CoA. The end result was that increasing malonyl-CoA** slightly increased the production of (2S)-naringenin.
The authors also described the carbon flux transport capacity between subcellular organelles and cytoplasm. The peroxisome citrate synthase gene CIT2 and the mitochondrial pyruvate carrier protein genes MPC1 and MPC3 were overexpressed alone in strain HB40, and the (2S)-naringenin yield was not increased. However, when the genes (strain HB50) were overexpressed together, the yield increased by 40 compared to strain HB94%。
Subsequently, the mitochondrial carrier protein gene was overexpressed to give HB53, and the (2S)-naringenin titer reached 7296mg/l。
The heterologous citrate synthase gene RTCIT1 from S. koraispora was introduced into HB53 to enhance mitochondrial citric acid synthesis, resulting in a (2S)-naringenin titer of 771 in strain HB545mg/l。
Further attempts to transfer oxaloacetate from the cytoplasm** into the mitochondria did not observe a significant change in (2S)-naringenin titers. The accumulation of acidic intermediates may affect pH homeostasis, leading to cell growth defects during fermentation. Therefore, the team added a 1% CA CO3 neutralizer to the YPD medium of strain HB54 to increase the (2S)-naringenin titer to 9862mg/l；Finally, when the strain HB54 was grown in a 5L bioreactor fed-batch fermentation, the titer increased to 34206mg/l。
Table |Comparison of heterologous production (2S)-naringenin by different strains (**above**).
Overall, in this study, the team utilized Saccharomyces cerevisiae to achieve high levels of (2S)-naringenin production by using strategies such as endogenous pathway enhancement, multi-pathway synergistic engineering, and enzyme engineering to address the precursor** problem.
The resulting engineered strain alleviates the problem of flavonoid precursor deficiency and reduces the accumulation of by-products. These engineered strains can be used as the basis for the biosynthesis of high-value flavonoids and provide a promising platform for flavonoid production.
It is worth noting that as early as 2022, Zhou's team screened from flavonoid-rich plants and obtained four chalcone synthase (CHS) with higher activity than the commonly used chalcone synthase gene (PHCHS). In these CHSs, SJCHS1 increased the (2S)-naringenin titer by 4838% 。The team combined the optimization of the natural precursor pathway of Saccharomyces cerevisiae with a more active CHS to further increase the (2S)-naringenin titer to 203 in shake flasks49mg/l。
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