Cost-Effective Production of L-DOPA by Tyrosinase-Immobilized Polyhydroxyalkanoate Nanogranules in Engineered Halomonas bluephagenesis TD01

Abstract

3,4-dihydroxyphenyl-L-alanine (L-DOPA) is a preferred drug for Parkinson’s disease, with an increasing demand worldwide that mainly relies on costly and environmentally problematic chemical synthesis. Yet, biological L-DOPA production is unfeasible at the industrial scale due to its low L-DOPA yield and high production cost. In this study, low-cost Halomonas bluephagenesis TD01 was engineered to produce tyrosinase TyrVs-immobilized polyhydroxyalkanoate (PHA) nanogranules in vivo, with the improved PHA content and increased immobilization efficiency of TyrVs accounting for 6.85% on the surface of PHA. A higher L-DOPA-forming monophenolase activity of 518.87 U/g PHA granules and an L-DOPA concentration of 974.36 mg/L in 3 h catalysis were achieved, compared to those of E. coli. Together with the result of L-DOPA production directly by cell lysates containing PHA-TyrVs nanogranules, our study demonstrated the robust and cost-effective production of L-DOPA by H. bluephagenesis, further contributing to its low-cost industrial production based on next-generation industrial biotechnology (NGIB).

Development and Application of Transcription Terminators for Polyhydroxylkanoates Production in Halophilic Halomonas bluephagenesis TD01

“…Wild type H. bluephagenesis TD01 (Collection No. CGMCC4353) was used for terminator validation (Tan et al, 2011 ), and its phaC gene knockout mutant H. bluephagenesis TDΔC (Zhao et al, 2021 ) served as another host for PHA production. A series of recombinant H. bluephagenesis TD strain harboring validation plasmids of different promoters (pMCS-P lib -sfGFP) constructed previously (Zhao et al, 2021 ) was used to test the activities of different promoters.…”

“…CGMCC4353) was used for terminator validation (Tan et al, 2011 ), and its phaC gene knockout mutant H. bluephagenesis TDΔC (Zhao et al, 2021 ) served as another host for PHA production. A series of recombinant H. bluephagenesis TD strain harboring validation plasmids of different promoters (pMCS-P lib -sfGFP) constructed previously (Zhao et al, 2021 ) was used to test the activities of different promoters. E. coli and H. bluephagenesis TD cells were cultured in LB and 60 LB medium (5 g/L yeast extract, 10 g/L tryptone, 60 g/L NaCl), respectively, at 37°C and 200 rpm on a rotary shaker.…”

“…For the construction of PhaC expression vector, the porin promoter, the phaC gene amplified from H. bluephagensis genome, and the selected terminators were assembled into the pSEVA341 vector via the Golden Gate method. The expression vectors were further transformed into H. bluephagensis TDΔC, a mutant of H. bluephagensis with phaC gene knockout in the genome by CRISPR/Cas 9 in a previous study (Zhao et al, 2021 ), to generate recombinant PHA producers TDΔC(A01–G05).…”

“…Different promoters with various activities (Porin, H19060, H18740, and ML03285) previously reported (Zhao et al, 2021 ), were employed to test the effects of promoter activity on the Terminator efficiency. The validation plasmids contained these different promoters which were located upstream of the sfGFP gene as well as a fixed T7 terminator flanked by sfGFP and mRFP genes.…”

Banana peel tyrosinase immobilized in agar-agar organic matrix “beads” for L-DOPA production in improvised reactors: A Box-Behnken response methodology-based approach

“…(Agarwal et al, 2019). The cresolase-mediated ohydroxylation of tyrosine results in the production of L-DOPA (Xu et al, 2012;Zhao et al, 2021). Of the numerous several biological methods useful in the production of L-DOPA in literature, the use of immobilized enzymes offers a better approach due to its inherent importance such as reusability, easy separation and stability against denaturation (Min et al, 2014).…”