Selective and Sustainable Production of Sub-terminal Hydroxy Fatty Acids by a Self-Sufficient CYP102 Enzyme from Bacillus Amyloliquefaciens.

Enzymatic hydroxylation of fatty acids by Cytochrome P450s (CYPs) offers an eco-friendly route to hydroxy fatty acids (HFAs), high-value oleochemicals with various applications in materials industry and with potential as bioactive compounds. However, instability and poor regioselectivity of CYPs are their main drawbacks. A newly discovered self-sufficient CYP102 enzyme, BAMF0695 from Bacillus amyloliquefaciens DSM 7, exhibits preference for hydroxylation of sub-terminal positions (ω-1, ω-2, and ω-3) of fatty acids. Our studies show that BAMF0695 has a broad temperature optimum (over 70 % of maximal enzymatic activity retained between 20 to 50 °C) and is highly thermostable (T50 >50 °C), affording excellent adaptive compatibility for bioprocesses. We further demonstrate that BAMF0695 can utilize renewable microalgae lipid as a substrate feedstock for HFA production. Moreover, through extensive site-directed and site-saturation mutagenesis, we isolated variants with high regioselectivity, a rare property for CYPs that usually generate complex regioisomer mixtures. BAMF0695 mutants were able to generate a single HFA regiosiomer (ω-1 or ω-2) with selectivities from 75 % up to 91 %, using C12 to C18 fatty acids. Overall, our results demonstrate the potential of a recent CYP and its variants for sustainable and green production of high-value HFAs.

Universal protocol and standard-spiking strategy for profiling of host cell proteins in therapeutic growth hormone.

Liquid chromatography coupled to mass spectrometry (LC-MS) is widely used for host cell proteins (HCP) identification in antibody drug development because of its sensitivity, selectivity, and adaptability. However, LC-MS based identification of HCP in biotherapeutics produced from the prokaryotic Escherichia coli-derived growth hormone (GH) has rarely been reported. Herein, we developed a universal and powerful workflow by combining optimized sample preparation with one-dimension ultra-high performance LC-MS based shotgun proteomics to support HCP profiling in GH samples from downstream pools and the final product, which would be beneficial to direct the purification process development and compare the difference of impurity of different products for guiding the development of the biosimilar. A standard-spiking strategy was also developed to increase the depth of HCP identification. Spiking with standards enables additional identification of HCP species, which is promising for trace-level HCP analysis. Our universal and standard-spiking protocols would open an avenue for profiling HCP in biotherapeutics derived from prokaryotic host cells.

Heterologous expression and characterization of a novel subtilisin-like protease from a thermophilic Thermus thermophilus HB8.

Subtilisins are a family of serine proteases used widely throughout the detergent, leather and food industries, with the identification and development of new enzymes holding much potential value. Thermus thermophilus HB8 was examined for serine proteases and found TTHA0724 gene. Sequence analysis of this putative serine protease placed it within the subtilisin family. To obtain active T. thermophilus HB8 subtilisins, three genes encoding prepro-subtilisin, pro-subtilisin and mature-subtilisin were cloned and expressed in Escherichia coli Transetta (DE3). Although direct expression of the mature-subtilisin gene was found to produce inactive inclusion bodies, expression of the pro-subtilisin gene resulted in active mature-subtilisin, indicating that the pro-sequence of translated pro-subtilisin underwent autoproteolysis. The resulting mature-subtilisin exhibited maximal activity between 65 and 85 °C at pH 7.5. The mature-subtilisin showed good stability, maintaining 50% activity after 48 h at 75 °C and >78% activity across the pH range 5.0-9.5. Furthermore, the mature-subtilisin demonstrated broad substrate specificity, with no requirement for the presence of metal ions which are essential for other subtilisin enzymes. Despite this Cu2+ was able to increase enzyme activity, while Ca2+ partially inhibited the activity. These properties suggest that T. thermophilus HB8 mature-subtilisin has potential value in its application in many industries.