Abstract:Terpenoids are a large family of natural products with diversified structures and functions that are widely used in the food, pharmaceutical, cosmetic, and agricultural fields. However, the traditional methods of terpenoids production such as plant extraction and chemical synthesis are inefficient due to the complex processes, high energy consumption, and low yields. With progress in metabolic engineering and synthetic biology, microbial cell factories provide an interesting alternative for the sustainable pro… Show more
“…As the cultivation time goes by, the concentration of extracellular β-caryophyllene gradually decreases due to the volatility of β-caryophyllene. Only 5% of β-caryophyllene was detected in the extracellular after 42 h. Although most of the sesquiterpenoids are not suitable for intracellular accumulation due to their characteristics, such as cytotoxicity, evaporability, or secretion [ 38 ], our research showed that the β-caryophyllene primarily accumulated in the intracellular. Fig.…”
Background
β-Caryophyllene, a kind of bicyclic sesquiterpene, is mainly used as a spice in the food and cosmetic industries. Furthermore, it also has significant value in the pharmaceutical industry and is now considered to be used as a new fuel. As a chemical energy heterotrophic microorganism, Escherichia coli can produce a large amount of acetyl-CoA through aerobic respiration, and acetyl-CoA is the common precursor substance in the biosynthesis of all terpenoids. Therefore, E. coli has the potential to be a cell factory to produce terpenoids.
Results
A new gene of β-caryophyllene synthase (TPS7) was found by analyzing the genome of Nicotiana tabacum L. using bioinformatics methods. The gene was overexpressed in engineered E. coli with a heterogeneous mevalonate (MVA) pathway to build a recombinant strain CAR1. Subsequent cultivation experiments in shake flask of engineered strain CAR1 verified that 16.1 mg/L β-caryophyllene was detected from the fermentation broth in the shake flask after induction for 24 h with IPTG. The toxic by-product of farnesyl acetate was detected during the process, and CAR1 showed a heavily cellular accumulation of product. We constructed an engineered strain CAR2, in which the downstream genes of the MVA pathway were integrated into the E. coli chromosome, successfully increasing β-caryophyllene production to 100.3 mg/L. The highest production of β-caryophyllene during the fed-batch fermentation was 4319 mg/L. Then we employed in situ extraction fermentation to successfully increase the production of β-caryophyllene by 20% to 5142 mg/L.
Conclusion
A new sesquiterpene synthase, TPS7, from tobacco was found to be able to produce β-caryophyllene with high efficiency. Based on this, an engineered E. coli was constructed to produce a much higher concentration of β-caryophyllene than the previous studies. During the fermentation process, we observed that β-caryophyllene tends to accumulate in intracellular space, which will eventually influence the activity of engineered E. coli. As a result, we solved this by metabolism regulation and in situ extractive fermentation.
“…As the cultivation time goes by, the concentration of extracellular β-caryophyllene gradually decreases due to the volatility of β-caryophyllene. Only 5% of β-caryophyllene was detected in the extracellular after 42 h. Although most of the sesquiterpenoids are not suitable for intracellular accumulation due to their characteristics, such as cytotoxicity, evaporability, or secretion [ 38 ], our research showed that the β-caryophyllene primarily accumulated in the intracellular. Fig.…”
Background
β-Caryophyllene, a kind of bicyclic sesquiterpene, is mainly used as a spice in the food and cosmetic industries. Furthermore, it also has significant value in the pharmaceutical industry and is now considered to be used as a new fuel. As a chemical energy heterotrophic microorganism, Escherichia coli can produce a large amount of acetyl-CoA through aerobic respiration, and acetyl-CoA is the common precursor substance in the biosynthesis of all terpenoids. Therefore, E. coli has the potential to be a cell factory to produce terpenoids.
Results
A new gene of β-caryophyllene synthase (TPS7) was found by analyzing the genome of Nicotiana tabacum L. using bioinformatics methods. The gene was overexpressed in engineered E. coli with a heterogeneous mevalonate (MVA) pathway to build a recombinant strain CAR1. Subsequent cultivation experiments in shake flask of engineered strain CAR1 verified that 16.1 mg/L β-caryophyllene was detected from the fermentation broth in the shake flask after induction for 24 h with IPTG. The toxic by-product of farnesyl acetate was detected during the process, and CAR1 showed a heavily cellular accumulation of product. We constructed an engineered strain CAR2, in which the downstream genes of the MVA pathway were integrated into the E. coli chromosome, successfully increasing β-caryophyllene production to 100.3 mg/L. The highest production of β-caryophyllene during the fed-batch fermentation was 4319 mg/L. Then we employed in situ extraction fermentation to successfully increase the production of β-caryophyllene by 20% to 5142 mg/L.
Conclusion
A new sesquiterpene synthase, TPS7, from tobacco was found to be able to produce β-caryophyllene with high efficiency. Based on this, an engineered E. coli was constructed to produce a much higher concentration of β-caryophyllene than the previous studies. During the fermentation process, we observed that β-caryophyllene tends to accumulate in intracellular space, which will eventually influence the activity of engineered E. coli. As a result, we solved this by metabolism regulation and in situ extractive fermentation.
“…As an oleaginous yeast, Y. lipolytica possesses an active tricarboxylic acid cycle and a pentose phosphate pathway, which supports high fluxes of acetyl‐CoA, NADPH, and the ATP supply by metabolizing a variety of carbon sources (Muhammad et al, 2020; Zhang et al, 2022). The characteristic gives it great potential for the production of terpenoids and lipids.…”
The sesquiterpene α-humulene is an important plant natural product, which has been used in the pharmaceutical industry due to its anti-inflammatory and anticancer activities. Although phytoextraction and chemical synthesis have previously been applied in α-humulene production, the low efficiency and high costs limit the development. In this study, Yarrowia lipolytica was engineered as the robust cell factory for sustainable α-humulene production. First, a chassis with high α-humulene output in the cytoplasm was constructed by integrating α-humulene synthases with high catalytic activity, optimizing the flux of mevalonate and acetyl-CoA pathways.Subsequently, the strategy of dual cytoplasmic-peroxisomal engineering was adopted in Y. lipolytica; the best strain GQ3006 generated by introducing 31 copies of 12 different genes could produce 2280.3± 38.2 mg/l (98.7 ± 4.2 mg/g dry cell weight) α-humulene, a 100-fold improvement relative to the baseline strain. To further improve the titer, a novel strategy for downregulation of squalene biosynthesis based on Cu 2+ -repressible promoters was firstly established, which significantly improved the α-humulene titer by 54.2% to 3516.6 ± 34.3 mg/l. Finally, the engineered strain could produce 21.7 g/l α-humulene in a 5-L bioreactor, 6.8-fold higher than the highest α-humulene titer reported before this study.Overall, system metabolic engineering strategies used in this study provide a valuable reference for the highly sustainable production of terpenoids in Y. lipolytica.
“…Recently, several groups developed various host cells, advanced strategies, and tools for the microbial production of terpenoids ( Carsanba et al, 2021 ; Zhang et al, 2021 ; Rinaldi et al, 2022 ). However, as shown in Table 3 , only a few studies explored the biorefining of various forms of organic waste to produce terpenoid flavor and fragrance compounds ( Table 3 ; Yao et al, 2020 ; Zhuang et al, 2020 ; Styles et al, 2021 ; Szotkowski et al, 2021 ; Wei et al, 2021 ).…”
Section: Discussionmentioning
confidence: 99%
“…Y. lipolytica is a GRAS yeast that, due to its endogenous mevalonate pathway (MVA), high capacity to generate acetyl coenzyme A, efficient lipid metabolism, and wide substrate scope, is rapidly emerging as a promising tool for the production of terpenoids ( Ma et al, 2019 ; Li et al, 2021 ; Zhang et al, 2021 ). Metabolic engineering strategies and synthetic biology tools raise the possibility of redirecting microbial carbon fluxes in Y. lipolytica to create efficient cellular pathways for the production of natural compounds ( Schwartz et al, 2016 ; Liu et al, 2022 ).…”
This study demonstrates the feasibility of establishing a natural compound supply chain in a biorefinery. The process starts with the biological or chemical hydrolysis of food and agricultural waste into simple and fermentative sugars, followed by their fermentation into more complex molecules. The yeast strain, Yarrowia lipolytica, was modified by introducing high membrane affinity variants of the carotenoid cleavage dioxygenase enzyme, PhCCD1, to increase the production of the aroma compound, β-ionone. The initial hydrolysis process converted food waste or sugarcane bagasse into nutrient-rich hydrolysates containing 78.4 g/L glucose and 8.3 g/L fructose, or 34.7 g/L glucose and 20.1 g/L xylose, respectively. During the next step, engineered Y. lipolytica strains were used to produce β-ionone from these feedstocks. The yeast strain YLBI3120, carrying a modified PhCCD1 gene was able to produce 4 g/L of β-ionone with a productivity of 13.9 mg/L/h from food waste hydrolysate. This is the highest yield reported for the fermentation of this compound to date. The integrated process described in this study could be scaled up to achieve economical large-scale conversion of inedible food and agricultural waste into valuable aroma compounds for a wide range of potential applications.
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