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Limited genetic tools for non-model bacteria are one of the limiting factors for genetic studies. This review compiles genetic tools used for three non-model alpha-proteobacteria, such as Zymomonas mobilis , Cereibacter ( Rhodobacter) sphaeroides , and Novosphingobium aromaticivorans , which hold significant potential to produce industrially essential bioenergy compounds due to their distinctive metabolic pathways and resilience in extreme environments. Each of these strains has a unique genetic profile that enables them to efficiently carry out key reactions relevant to producing bioenergy compounds, such as converting sugars into bioenergy compounds and breaking down lignotoxins. Genetic tools can further optimize these strains for enhanced bioenergy compound production. This review explores the metabolic advantages of these organisms. It highlights the available array of genetic toolkits that can be shared among them to unlock their full potential for sustainable biofuel production. Graphical abstract

Abstract In this study, we have isolated an endophytic fungal strain Lasiodiplodia theobromae from non-Taxus host plant Piper nigrum. The strain L. theobromae identity was confirmed by morphological characteristics and internal transcribed spacer sequence analysis. Taxol produced by L. theobromae was observed to be identical to the authentic taxol as analyzed by chromatography and spectroscopy methods. The quantity of taxol produced by the fungus was estimated to be 247 μg L−1, and fungal taxol showed potent cytotoxic activity towards cancer cell line. Evidence to support the independent production of taxol by L. theobromea, the gene encoding 10-deacetylbacccation-III-O-acetyltransferase (DBAT), as well as, for the first time, open reading frame (ORF) of WRKY1 transcription factor (TF) were cloned and sequenced. The predicted amino sequence of L. theobromae dbat gene shared high homology with the taxol-producing plant and fungal dbat gene. Not only dbat gene, ORF of WRKY1 TF too shared high homology with Taxus chinensis WRKY1 TF ORF. To the best of our knowledge, this is the first report on cloning of dbat gene and its transcription factor from endophytes of non-Taxus host plant. Cloning and sequence analysis of 10-deacetylbaccatin III-10-O-acetyl transferase gene and WRKY1 transcription factor from taxol-producing endophytic fungus Lasodiplodia theobromea.

10-deacetylbaccatin III-10-[beta]-O-acetyltransferase (DBAT) is a key rate-limiting enzyme of the Taxol biosynthetic pathway, which is uncharacterized in Taxol-producing endophytic fungi. Here, an open reading frame of DBAT was cloned from the Taxol-producing endophytic fungus Lasiodiplodia theobromae (LtDBAT). The LtDBAT enzyme was heterologously expressed and purified by the affinity and gel filtration chromatography methods. The molecular weight of the purified protein was 49 kDa and its identity was confirmed by western blot. The purified LtDBAT enzyme was capable of catalyzing 10-deacetylbaccatin III into baccatin III, as shown by liquid chromatography--mass spectroscopy. The mass spectra of baccatin III were identical to the authentic baccatin III. The LtDBAT enzyme was characterized and the kinetic parameters of catalysis were determined. In addition, localization of LtDBAT was performed by using confocal microscopy and the result showed that the enzyme was localized in lipid droplets. Together, this study provides biochemical insights into the fungal recombinant DBAT enzyme that is involved in the Taxol biosynthetic pathway. In the near future, engineering of the LtDBAT enzyme and the Taxol biosynthetic pathway in endophytic fungi could be an eco-friendly and economically feasible alternative source for production of Taxol and its precursors.

ABSTRACT 10-deacetylbaccatin III-10-β-O-acetyltransferase (DBAT) is a key rate-limiting enzyme of the Taxol biosynthetic pathway, which is uncharacterized in Taxol-producing endophytic fungi. Here, an open reading frame of DBAT was cloned from the Taxol-producing endophytic fungus Lasiodiplodia theobromae (LtDBAT). The LtDBAT enzyme was heterologously expressed and purified by the affinity and gel filtration chromatography methods. The molecular weight of the purified protein was 49 kDa and its identity was confirmed by western blot. The purified LtDBAT enzyme was capable of catalyzing 10-deacetylbaccatin III into baccatin III, as shown by liquid chromatography–mass spectroscopy. The mass spectra of baccatin III were identical to the authentic baccatin III. The LtDBAT enzyme was characterized and the kinetic parameters of catalysis were determined. In addition, localization of LtDBAT was performed by using confocal microscopy and the result showed that the enzyme was localized in lipid droplets. Together, this study provides biochemical insights into the fungal recombinant DBAT enzyme that is involved in the Taxol biosynthetic pathway. In the near future, engineering of the LtDBAT enzyme and the Taxol biosynthetic pathway in endophytic fungi could be an eco-friendly and economically feasible alternative source for production of Taxol and its precursors. Biochemical insights into the biotechnologically important fungal recombinant 10-deacetylbaccatin III-10-I2-O-acetyltransferase enzyme.

The ability to regulate genetic circuits and metabolic pathways is central to cellular control. The existing toolkit is predominantly comprised of local transcription regulators that are unsuitable for exerting control at a global genome-wide scale. Bacterial sigma factors are ideal global regulators as together they direct the RNA polymerase to thousands of transcription sites. Here, we redesigned the promoter specificity of the housekeeping sigma factor, sigma-70, toward five orthogonal promoter targets not recognized by the native sigma-70. These orthogonal sigma-70 factors were developed by screening a pooled library of computationally designed variants of the -35 DNA recognition helix, each tailored to a specific target promoter. In the redesigned sigma factors new target-specific interactions facilitate new promoter recognition. Activity of the top performing redesigned sigma-70s varied across the promoter targets and ranged from 17% to 77% of native sigma-70 on its canonical active promoter. These orthogonal sigma factors represent a new suite of regulators for global transcriptional control.