Biotechnology Journal最新文献

Hydrophobic feedstocks such as waste cooking oil have recently been considered for microbial biotransformation due to their abundance, low cost, and unique advantage for lipid-derived fermentation products. Most fermentations with hydrophobic substrates are conducted at the tube or flask scale (less than 1 L total volume) or with the hydrophobic substrate comprising a small fraction of the media. Low substrate concentrations require additional feeding. Alternatively, high concentrations do not require significant dilution of the oil feedstock, which reduce volumetric requirements for larger scale fermentations. However, high-oil-density fermentations complicate efficient mixing and mass transfer challenges which are exacerbated at larger scales. To address this, computational fluid dynamics (CFD) models were explored to simulate three-phase (hydrophobic, hydrophilic, and gaseous) bench (3 L) and pilot scale (4000 L) bioreactors, highlighting challenges and potential considerations. Bioreactor fermentations of Yarrowia lipolytica strain L36DGA1 with substrate loadings of 5%, 10%, 20%, 30%, 40%, and 50% (v/v) waste cooking oil were also conducted, representing one of the highest concentrations in the reported literature. This work supports future research into and implementation of high-oil-density fermentations at the bench and pilot bioreactor scale.

When constructing cell factories, it is crucial to reallocate intracellular resources towards the synthesis of target compounds. However, imbalanced resource allocation can lead to a tradeoff between cell growth and production, reducing overall efficiency. Reliable gene expression regulation tools are needed to coordinate cell growth and production effectively. The orthogonal translation system, developed based on genetic code expansion (GCE), incorporates non-canonical amino acids (ncAAs) into proteins by assigning them to expanded codons, which enables the control of target protein expression at the translational level in an ncAA-dependent manner. However, the stringency of this regulatory tool remains inadequate. This study achieved strict translational-level control of the orthogonal translation system by addressing the abnormal leakage caused by the arabinose-inducible promoter. Further validation was conducted on the relationship between ncAA concentration and expression level, as well as the host's adaptability to the system. Subsequently, the system's applicability across multiple Escherichia coli hosts was verified by examining the roles of RF1 (peptide chain release factor 1) and endogenous TAG codons. By combining this strategy with inducible promoters, dual-level regulation of target gene expression at both transcriptional and translational levels was achieved and the dynamic range was further increased to over 20-fold. When using ncAA to control the expression of T7 RNA polymerase (T7 RNAP), the leakage expression was reduced by 82.7%, mitigating the low production efficiency caused by extensive leakage in the T7 system. As proof of concept, the strategy enhanced the production of alcohol dehydrogenase (ADH) by 9.82-fold, demonstrating its excellent capability in controlling gene expression in developing cell factories.

Saccharomyces boulardii, as a probiotic yeast, has shown great potential in regulating gut health and treating gastrointestinal diseases. Due to its unique antimicrobial and immune-regulating functions, it has become a significant subject of research in the field of probiotics. In this study, we aim to enhance the antioxidant properties of S. boulardii by producing l-ergothioneine (EGT). We first constructed a double knockout of ura3 and trp1 gene in S. boulardii to facilitate plasmid-based expressions. To further enable effective genome editing of S. boulardii, we implemented the PiggyBac system to transpose the heterologous gene expression cassettes into the chromosomes of S. boulardii. By using enhanced green fluorescent protein (EGFP) as the reporter gene, we achieved random chromosomal integration of EGFP expression cassette. By using PiggyBac transposon system, a great variety of EGT-producing strains was obtained, which is not possible for the conventional single target genome editing, and one best isolated top producer reached 17.50 mg/L EGT after 120 h cultivation. In summary, we have applied the PiggyBac transposon system to S. boulardii for the first time for genetic engineering. The engineered probiotic yeast S. boulardii has been endowed with new antioxidant properties and produces EGT. It has potential applications in developing novel therapeutics and dietary supplements for the prevention and treatment of gastrointestinal disorders.

Boldenone (BD), a protein anabolic hormone, is commonly used to treat muscle damage, osteoporosis, and off-season muscle building in athletes. Traditional BD synthesis methods rely on chemical processes, which are costly and environmentally impactful. Therefore, developing a more sustainable and economical biosynthetic pathway is crucial for BD production. This study aimed to achieve efficient production of BD. Firstly, the catalytic performance of 17β-hydroxysteroid dehydrogenase and 3-ketosteroid-Δ¹-dehydrogenase was improved through enzyme engineering, and their expression in the new strain of Mycobacterium neoaurum was enhanced using metabolic engineering. These improvements significantly increased BD production to 4.05 g/L, with a significant decrease in by-product generation. To further increase the yield, a multi-enzyme fusion expression system was constructed, and a key cell wall gene kasB was knocked out, resulting in a spatial-time yield of BD reaching 1.02 g/(L·d). Subsequent optimization of the transformation system further increased the BD production to 5.56 g/L, with a spatiotemporal yield of 1.39 g/(L·d). The green biosynthetic route of phytosterol one-step conversion to BD developed in this study lays the foundation for industrial production.

L-asparaginases (EC 3.5.1.1) are amidohydrolase enzymes that predominantly catalyze conversion of L-asparagine to L-aspartic acid and ammonia. In addition, some exhibit secondary L-glutaminase activity. Escherichia coli and Erwinia chrysanthemi L-asparaginases are widely used in the pharmaceutical industry to produce therapeutically important compounds. In the therapeutic use of enzymes, bacterial L-asparaginases can trigger immune responses, leading to a high rate of adverse effects that diminish the effectiveness of the treatment. This situation has forced scientists to search for promising L-asparaginases from new sources. Yeast L-asparaginases could be useful in reducing toxicity and enhancing efficacy but they have been poorly studied to date. Here, we characterized the yeast Lachancea thermotolerans L-asparaginase (LtASNase) purified by affinity chromatography. It has a specific activity of 313.8 U/mg and a high k_cat value (312.4 s). We demonstrated through a semi-rational design that the mutations of Lys99 show varying effects on catalytic activity, with the Lys99Ala mutant increasing specific activity 3.3-fold. Furthermore, the in vitro antileukemic activity of the non-formulated form of Lys99Ala LtASNase was evaluated against SUP-B15 and REH cell lines. The results demonstrated that LtASNase exhibits significant antileukemic potential, comparable to commercial type II bacterial enzymes. The understanding of the mutant L-asparaginases examined in this study will significantly contribute to the development of new and more effective yeast-derived asparaginases.