Directed Evolution Improves the Enzymatic Synthesis of L-5-Hydroxytryptophan by an Engineered Tryptophan Synthase

Abstract

L-5-Hydroxytryptophan is an important amino acid that is widely used in food and medicine. In this study, L-5-hydroxytryptophan was synthesized by a modi ed tryptophan synthase. A direct evolution strategy was applied to engineer tryptophan synthase from Escherichia coli to improve the e ciency of L-5hydroxytryptophan synthesis. Tryptophan synthase was modi ed by error-prone PCR. A high activity mutant enzyme (V231A/K382G) was obtained by a high-throughput screening method. The activity of mutant enzyme (V231A/K382G) is 3.79 times higher than that of its parent, and k cat /K m of the mutant enzyme (V231A/K382G) was 4.36 mM − 1 •s − 1 . The mutant enzyme (V231A/K382G) reaction conditions for the production of L-5-hydroxytryptophan were 100 mmol/L L-serine at pH 8.5 and 35°C for 15 h, reaching a yield of L-5-hydroxytryptophan of 86.7%. Directed evolution is an effective strategy to increase the activity of tryptophan synthase.

“Biosynthesis of psilocybin and its nonnatural derivatives by a promiscuous psilocybin synthesis pathway in Escherichia coli”

“…Significant effort toward identifying, evaluating, and improving tryptophan synthase and decarboxylase enzymes has been undertaken by a range of researchers (Phillips, 2004; Romney et al, 2017; Watkins‐Dulaney et al, 2021). Tryptophan synthases sourced from various organisms including Neurospora crassa (Hall et al, 1962; Saito & Rilling, 2006), Escherichia coli (Crowley et al, 2012; Held & Smith, 1970; Miles & Phillips, 1985; Wilcox, 1974; Xu et al, 2020, 2021), Salmonella typhimurium (Cash et al, 2004; Francis et al, 2017; Miles & Phillips, 1985; Phillips et al, 1995; Sloan & Phillips, 1992; Welch & Phillips, 1999), Salmonella enterica (Goss & Newill, 2006; Hoffarth et al, 2021; Smith et al, 2014; Winn et al, 2008), Pyrococcus furiosus (Boville, Scheele, et al, 2018; Buller et al, 2015; Herger et al, 2016; Murciano‐Calles et al, 2016), Psilocybe cubensis (Blei et al, 2018), and Thermotoga maritima (Boville, Romney, et al, 2018) have generally been characterized to have activity on a broad range of substrates. These tryptophan synthases are also highly engineerable with recent studies enabling standalone activity from the tryptophan synthases β‐subunit (TrpB) (Buller et al, 2015; Murciano‐Calles et al, 2016) and activity under mild reaction conditions (Boville, Romney, et al, 2018; Rix et al, 2020).…”

“…Escherichia coli (Crowley et al, 2012;Held & Smith, 1970;Miles & Phillips, 1985;Wilcox, 1974;Xu et al, 2020Xu et al, , 2021, Salmonella typhimurium (Cash et al, 2004;Francis et al, 2017;Miles & Phillips, 1985;Phillips et al, 1995;Sloan & Phillips, 1992;Welch & Phillips, 1999), Salmonella enterica (Goss & Newill, 2006;Hoffarth et al, 2021;Smith et al, 2014;Winn et al, 2008), Pyrococcus furiosus (Boville, Scheele, et al, 2018;Buller et al, 2015;Herger et al, 2016;Murciano-Calles et al, 2016), Psilocybe cubensis (Blei et al, 2018), and Thermotoga maritima (Boville, Romney, et al, 2018) have generally been characterized to have activity on a broad range of substrates.…”

l-Serine Biosensor-Controlled Fermentative Production of l-Tryptophan Derivatives by Corynebacterium glutamicum

“…However, the extraction method generates pollutants to the environment because of the organic solvents used, and the T5H for biosynthesis is unstable when expressed in microorganisms and requires a special cofactor 5,6,7,8-tetrahydrobiopterin that must be generated through additional enzymatic reactions [70]. Recently, directed evolution was performed on E. coli tryptophan synthase to generate a mutant with increased catalytic activity towards 5-HTP conversion from L-serine and 5-HI, reaching a yield of 86.7% [71]. In this study, 42 ± 0.91% conversion was observed for 5-HTP.…”