“…Approximately 96% of the produced EGT was retained extracellularly. Compared to other similar Rhodotorula species used for EGT production [ 43 , 44 ], our strain showed a 300% increase from the reported EGT productivity at 0.4 mg/L/h in R. mucilaginosa DL-X01 with the same volume of fermentation broth (titer of 38.5 ± 2.7 mg/L in 96 h), and could also be further iterable engineered for better EGT production performance with CACR and high-throughput screening technologies developed in this study. Fig.…”
Background
Ergothioneine (EGT) is a distinctive sulfur-containing histidine derivative, which has been recognized as a high-value antioxidant and cytoprotectant, and has a wide range of applications in food, medical, and cosmetic fields. Currently, microbial fermentation is a promising method to produce EGT as its advantages of green environmental protection, mild fermentation condition, and low production cost. However, due to the low-efficiency biosynthetic process in numerous cell factories, it is still a challenge to realize the industrial biopreparation of EGT. The non-conventional yeast Rhodotorula toruloides is considered as a potential candidate for EGT production, thanks to its safety for animals and natural ability to synthesize EGT. Nevertheless, its synthesis efficiency of EGT deserves further improvement.
Results
In this study, out of five target wild-type R. toruloides strains, R. toruloides 2.1389 (RT1389) was found to accumulate the highest EGT production, which could reach 79.0 mg/L at the shake flask level on the 7th day. To achieve iterative genome editing in strain RT1389, CRISPR-assisted Cre recombination (CACR) method was established. Based on it, an EGT-overproducing strain RT1389-2 was constructed by integrating an additional copy of EGT biosynthetic core genes RtEGT1 and RtEGT2 into the genome, the EGT titer of which was 1.5-fold increase over RT1389. As the supply of S-adenosylmethionine was identified as a key factor determining EGT production in strain RT1389, subsequently, a series of gene modifications including S-adenosylmethionine rebalancing were integrated into the strain RT1389-2, and the resulting mutants were rapidly screened according to their EGT production titers with a high-throughput screening method based on ergothionase. As a result, an engineered strain named as RT1389-3 was selected with a production titer of 267.4 mg/L EGT after 168 h in a 50 mL modified fermentation medium.
Conclusions
This study characterized the EGT production capacity of these engineered strains, and demonstrated that CACR and high-throughput screening method allowed rapid engineering of R. toruloides mutants with improved EGT production. Furthermore, this study provided an engineered RT1389-3 strain with remarkable EGT production performance, which had potential industrial application prospects.
“…Approximately 96% of the produced EGT was retained extracellularly. Compared to other similar Rhodotorula species used for EGT production [ 43 , 44 ], our strain showed a 300% increase from the reported EGT productivity at 0.4 mg/L/h in R. mucilaginosa DL-X01 with the same volume of fermentation broth (titer of 38.5 ± 2.7 mg/L in 96 h), and could also be further iterable engineered for better EGT production performance with CACR and high-throughput screening technologies developed in this study. Fig.…”
Background
Ergothioneine (EGT) is a distinctive sulfur-containing histidine derivative, which has been recognized as a high-value antioxidant and cytoprotectant, and has a wide range of applications in food, medical, and cosmetic fields. Currently, microbial fermentation is a promising method to produce EGT as its advantages of green environmental protection, mild fermentation condition, and low production cost. However, due to the low-efficiency biosynthetic process in numerous cell factories, it is still a challenge to realize the industrial biopreparation of EGT. The non-conventional yeast Rhodotorula toruloides is considered as a potential candidate for EGT production, thanks to its safety for animals and natural ability to synthesize EGT. Nevertheless, its synthesis efficiency of EGT deserves further improvement.
Results
In this study, out of five target wild-type R. toruloides strains, R. toruloides 2.1389 (RT1389) was found to accumulate the highest EGT production, which could reach 79.0 mg/L at the shake flask level on the 7th day. To achieve iterative genome editing in strain RT1389, CRISPR-assisted Cre recombination (CACR) method was established. Based on it, an EGT-overproducing strain RT1389-2 was constructed by integrating an additional copy of EGT biosynthetic core genes RtEGT1 and RtEGT2 into the genome, the EGT titer of which was 1.5-fold increase over RT1389. As the supply of S-adenosylmethionine was identified as a key factor determining EGT production in strain RT1389, subsequently, a series of gene modifications including S-adenosylmethionine rebalancing were integrated into the strain RT1389-2, and the resulting mutants were rapidly screened according to their EGT production titers with a high-throughput screening method based on ergothionase. As a result, an engineered strain named as RT1389-3 was selected with a production titer of 267.4 mg/L EGT after 168 h in a 50 mL modified fermentation medium.
Conclusions
This study characterized the EGT production capacity of these engineered strains, and demonstrated that CACR and high-throughput screening method allowed rapid engineering of R. toruloides mutants with improved EGT production. Furthermore, this study provided an engineered RT1389-3 strain with remarkable EGT production performance, which had potential industrial application prospects.
“…The strain R. mucilaginosa DL‐X01 was isolated from vinegar in our previous work 6 . Yeast peptone dextrose (YPD) broth contained 10 g L −1 yeast extract, 20 g L −1 peptone, and 20 g L −1 dextrose.…”
Section: Methodsmentioning
confidence: 99%
“…The suspension was centrifuged at 8000 × g for 5 min, and the supernatant was filtered with a 0.22 μm pore membrane. The EGT extract was analyzed by high‐performance liquid chromatography (Agilent Technologies Inc., Santa Clara, CA, USA) according to our previous conditions 6 …”
Section: Methodsmentioning
confidence: 99%
“…EGT was first discovered in Claviceps purpurea by Charles Tanret in 1909 4 . Subsequently, researchers have found accumulation of this substance in a variety of fungi and bacteria, such as Neurospora crassa , Rhodotorula mucilaginosa , and Mycobacterum smegmatis , 5‐7 all of which can synthesize EGT using l ‐histidine (His) as the starting substrate. There are different pathways of EGT biosynthesis in fungi and bacteria.…”
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