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).

Halomonas bluephagenesis TD01 is one of the ideal chassis for low-cost industrial production based on “Next Generation Industrial Biotechnology,” yet the limited genetically regulatory parts such as transcriptional terminators, which are crucial for tuned regulations on gene expression, have hampered the engineering and applications of the strain. In this study, a series of intrinsic Rho-independent terminators were developed by either genome mining or rational design, and seven of them proved to exhibit higher efficiencies than the canonical strong T7 terminator, among which three terminators displayed high efficiencies over 90%. A preliminary modeling on the sequence-efficiency relationship of the terminators suggested that the poly U sequence regularity, the length and GC content of the stem, and the number and the size of hairpin loops remarkably affected the termination efficiency (TE). The rational and de novo designs of novel synthetic terminators based on the sequence-efficiency relationship and the “main contributor” engineering strategy proved to be effective, and fine-tuned polyhydroxylkanoates production was also achieved by the regulation of these native or synthetic terminators with different efficiencies. Furthermore, a perfectly positive correlation between the promoter activity and the TE was revealed in our study. The study enriches our knowledge of transcriptional termination via its sequence–strength relationship and enables the precise regulation of gene expression and PHA synthesis by intrinsic terminators, contributing to the extensive applications of H. bluephagenesis TD01 in the low-cost production of various chemicals.

Rapidly growing industrial biotechnology and bio-manufacturing require simple and cost-effective bioseparation tools. A novel strategy of bioseparation based on the streptavidin-decorated polyhydroxyalkanoate (PHA) nano-granules was developed in this study. By fusing to the N-terminus of PHA-associated phasin protein, the streptavidin was one-step immobilized on the surface of PHA nano-granules simultaneously with the accumulation of PHA in recombinant Escherichia coli. About 1.95 g/L of PHA nano-granules (54.51 wt% of cell dry weight) were produced after 48 h bacterial cultivation. The following qualitative and quantitative characterizations demonstrated that the streptavidin accounted for approximately 6.78% of the total weight of the purified PHA nano-granules and confirmed a considerable biotin affinity of 0.1 ng biotin/μg surface protein. As a proof of concept, the nano-granules were further functionalized with biotinylated oligo(dT) for mRNA isolation and about 1.26 μg of mRNA (occupied 2.59%) was purified from 48.45 μg of total RNA, achieving good integrity and high purity with few DNA and rRNA contaminations. Moreover, the nano-granules retained more than 80% of their initial mRNA recovery efficiency after ten cycles of repeated use. The PHA-SAP nano-granules were also functionalized with biotinylated magnetic beads, allowing magnetic recovery of the PHA nano-granules from cell lysates that still needs optimization. Our study provides a novel and expandable platform of PHA nano-granules that can be further functionalized with various biological groups for bioseparation applications. The functional PHA nano-granules have a great potential to serve as bioseparation resin for large-scale purification processes after suitable optimizations for “bench-to-factory” translation, contributing to scalable and sustainable bioprocessing.