Engineering Methyltransferase and Sulfoxide Synthase for High-Yield Production of Ergothioneine

Abstract

Ergothioneine (ERG) is an unusual sulfur-containing amino acid with antioxidant activity that can be synthesized by certain bacteria and fungi. Microbial fermentation is a promising method for ERG production. In this study, the bifunctional enzyme methyltransferase−sulfoxide synthase NcEgt1 from Neurospora crassa was truncated to obtain sulfoxide synthase T NcEgt1, which showed a higher expression level in Escherichia coli BL21(DE3). Then, the genes egtD encoding methyltransferase EgtD and egtE encoding C−S lyase EgtE from Mycobacterium smegmatis were cloned with T ncEgt1 into E. coli BL21(DE3) to produce 70 mg/L ERG. To improve ERG production, T NcEgt1 and EgtD were modified, and the resulting mutants were screened with an established high-throughput method which could directly analyze the ERG content in culture broths. After several rounds of mutation and screening, the optimal mutant MD4 was obtained and produced 290 mg/L ERG. Furthermore, a fed-batch culture was conducted in a 5 L bioreactor. After optimizing the fermentation process, the ERG yield reached 5.4 g/L after 94 h of cultivation supplemented with amino acids and glycerol, which is the highest ERG yield reported to date. The results showed that ERG production was significantly improved by modifying the key enzymes, and the engineered strains constructed in this study have potential industrial application prospects.

A Practical Method for Synthesizing Iptacopan

“…Among them, only triethyloxonium tetrafluoroborate had an orate result, raising the conversion rate to 99.9% and the yield to 74.6%. After confirming the use of Meerwein's salt and a proton sponge in combination [17,18], the necessity of the proton sponge was also verified in this paper, and it was found that the reaction conversion rate was 48.5% when the proton sponge was not added (entry 6). The results were consistent with the use of other organic bases to replace the proton sponge, with the same results when the proton sponge was not added (entry 7).…”

Engineering non-conventional yeast Rhodotorula toruloides for ergothioneine production

“…HME13 can effectively catalyze EGT to produce thiol urocanic acid, which can be detected at 311 nm on a microplate reader (Fig. 6 A) [ 25 , 42 ]. To test the feasibility of this principle, 10 μL of ergothionase crude extract was added to 190 μL of 0 − 100 mg/L EGT standard at 37 ℃ for 30 min, and then the absorbance of reaction mixture was detected with a microplate reader at 311 nm.…”

“…In 2022, Chen et al reconstituted a novel EGT production system in the E. coli by co-expression of two EGT biosynthesis genes ( tregt 1 and tregt 2) from Trichoderma reesei , which could produce 4.34 g/L EGT after 143 h of cultivation in a 2 L jar fermenter [ 24 ]. In another study, Zhang et al reported that the highest EGT production in engineered E. coli strain was 5.4 g/L after 94 h in a fed-batch cultivation with 5 L jar fermenter, which was achieved by overexpressing truncated EGT1 gene from N. crassa and egtD , egtE genes form M. smegmatis with multiple rounds of mutations [ 25 ]. As for another representative model microorganism, Saccharomyces cerevisiae , it was also used to produce EGT.…”

Co‐augmentation of a transport gene mfsT1 in Mycolicibacterium neoaurum with genome engineering to enhance ergothioneine production

“…Although small microorganisms generally have shorter cultivation cycles than large fungi, they often exhibit insufficient initial synthesis efficiency of EGT [13]. An enhanced productivity can be attained more easily through genetic modifications of small microorganisms [15] for the large-scale production of high-purity EGT. To date, genetic engineering of various microorganisms has been employed to enhance heterologous synthesis of EGT with high-copy plasmids [11,[15][16][17][18][19][20][21].…”

“…An enhanced productivity can be attained more easily through genetic modifications of small microorganisms [15] for the large-scale production of high-purity EGT. To date, genetic engineering of various microorganisms has been employed to enhance heterologous synthesis of EGT with high-copy plasmids [11,[15][16][17][18][19][20][21]. However, this approach may lead to genetic instability during the large-scale cultivation stage.…”