biostudies-literatureLiu KNational Key Research and Development Program of China65https://www.ebi.ac.uk/biostudies/studies/S-EPMC11089744biostudies-literatureUnknown17(1)<h4>Background</h4>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.<h4>Results</h4>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.<h4>Conclusions</h4>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.Biotechnology for biofuels and bioproductsEngineering non-conventional yeast Rhodotorula toruloides for ergothioneine production.PMC110897442021YFC2100300Liu KXiong LWei DKe JWang FXiang GLiu TLi LfalseEngineering non-conventional yeast Rhodotorula toruloides for ergothioneine production.<h4>Background</h4>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.<h4>Results</h4>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.<h4>Conclusions</h4>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.2024-01-01T00:00:00Z2024 May2026-06-01T17:28:56.633Z2026-04-08T14:15:45.31ZS-EPMC110897443874116910.1186/s13068-024-02516-2