Polyhydroxyalkanoates (PHAs) synthesis and degradation by microbes and applications towards a circular economy.

In the realm of gene therapy, a pivotal moment arrived with Paul Berg’s groundbreaking identification of the first recombinant DNA in 1972. This achievement set the stage for future breakthroughs. Conditions once considered undefeatable, like melanoma, pancreatic cancer, and a host of other ailments, are now being addressed at their root cause—the genetic level. Presently, the gene therapy landscape stands adorned with 22 approved in vivo and ex vivo products, including IMLYGIC, LUXTURNA, Zolgensma, Spinraza, Patisiran, and many more. In this comprehensive exploration, we delve into a rich assortment of 16 drugs, from siRNA, miRNA, and CRISPR/Cas9 to DNA aptamers and TRAIL/APO2L, as well as 46 carriers, from AAV, AdV, LNPs, and exosomes to naked mRNA, sonoporation, and magnetofection. The article also discusses the advantages and disadvantages of each product and vector type, as well as the current challenges faced in the practical use of gene therapy and its future potential.

Current State of Human Gene Therapy: Approved Products and Vectors

/pdf/capture-selex-selection-strategy-aptamer-identification-and-gcnrth73.pdf

Capture-SELEX: Selection Strategy, Aptamer Identification, and Biosensing Application

Efficient Bioproduction of 3-Hydroxypropionic Acid from Methanol by a Synthetic Yeast Cell Factory

Combinatorial metabolic engineering enables the efficient production of ursolic acid and oleanolic acid in Saccharomyces cerevisiae.

Pichia pastoris (Komagataella phaffii) is a model organism widely used for the recombinant expression of eukaryotic proteins, and it can metabolize methanol as its sole carbon and energy source. Methanol is oxidized to formaldehyde by alcohol oxidase (AOX). In the dissimilation pathway, formaldehyde is oxidized to CO2 by formaldehyde dehydrogenase (FLD), S-hydroxymethyl glutathione hydrolase (FGH) and formate dehydrogenase (FDH).The transcriptome and metabolome of P. pastoris were determined under methanol cultivation when its dissimilation pathway cut off. Firstly, Δfld and Δfgh were significantly different compared to the wild type (GS115), with a 60.98% and 23.66% reduction in biomass, respectively. The differential metabolites between GS115 and Δfld were mainly enriched in ABC transporters, amino acid biosynthesis, and protein digestion and absorption. Secondly, comparative transcriptome between knockout and wild type strains showed that oxidative phosphorylation, glycolysis and the TCA cycle were downregulated, while alcohol metabolism, proteasomes, autophagy and peroxisomes were upregulated. Interestingly, the down-regulation of the oxidative phosphorylation pathway was positively correlated with the gene order of dissimilation pathway knockdown. In addition, there were significant differences in amino acid metabolism and glutathione redox cycling that raised our concerns about formaldehyde sorption in cells.This is the first time that integrity of dissimilation pathway analysis based on transcriptomics and metabolomics was carried out in Pichia pastoris. The blockage of dissimilation pathway significantly down-regulates the level of oxidative phosphorylation and weakens the methanol assimilation pathway to the point where deficiencies in energy supply and carbon fixation result in inefficient biomass accumulation and genetic replication. In addition, transcriptional upregulation of the proteasome and autophagy may be a stress response to resolve formaldehyde-induced DNA-protein crosslinking. 

/pdf/comparative-transcriptome-and-metabolome-analyses-reveal-the-3swpwa6n.pdf

Comparative transcriptome and metabolome analyses reveal the methanol dissimilation pathway of Pichia pastoris

In the twenty first century, biotechnology offers great opportunities and solutions to climate change mitigation, energy and food security and resource efficiency. The use of metabolic engineering to modify microorganisms for producing industrially significant chemicals is developing and becoming a trend. As a famous, generally recognized as a safe (GRAS) model microorganism, Saccharomyces cerevisiae is widely used due to its excellent operational convenience and high fermentation efficiency. This review summarizes recent advancements in the field of using metabolic engineering strategies to construct engineered S. cerevisiae over the past ten years. Five different types of compounds are classified by their metabolites, and the modified metabolic pathways and strategies are summarized and discussed independently. This review may provide guidance for future metabolic engineering efforts toward such compounds and analogues. Additionally, the limitations of S. cerevisiae as a cell factory and its future trends are comprehensively discussed.

Metabolic engineering of Saccharomyces cerevisiae for the synthesis of valuable chemicals.

Patchoulol, a plant-derived sesquiterpene compound, is widely used in perfumes, cosmetics, and pharmaceuticals. Microbial production provides a promising alternative approach for the efficient and sustainable production of patchoulol. However, there are no systematic engineering studies on Komagataella phaffii aimed at achieving high-yield patchoulol production. Herein, by fusion overexpression of FPP synthase and patchoulol synthase (ERG20LPTS), increasing the precursor supply, adjusting the copy number of ERG20LPTS and PTS, and combined with adding auxiliary carbon source and methanol concentration optimization, we constructed a high-yield patchoulol-producing strain P6H53, which produced 149.64 mg/L patchoulol in shake-flask fermentation with methanol as the substrate. In fed-batch fermentation, strain P6H53 achieved the highest production (2.47 g/L, 21.48 mg/g DCW, and 283.25 mg/L/d) to date in a 5 L fermenter. This study will lay a good foundation for the development of K. phaffii as a promising chassis microbial cell for the synthesis of patchoulol and other sesquiterpenes with methanol as the carbon source.

Overproduction of Patchoulol in Metabolically Engineered Komagataella phaffii.

Malachite green oxalate (MG) is a kind of veterinary drug, which is freely soluble in water and hazardous to aquatic products, resulting in food toxicity and human health problems. The demand for effective and sensitive detection of MG residues is increasing in food safety. In this work, three DNA aptamers MG-36-12/16/17 targeting MG with good affinity (Kd values were 169.78, 71.94, and 102.46 μM, respectively) were obtained by Capture-SELEX. Furthermore, MG-36-12, MG-76-16-6A, and MG-36-17 were found to perform sensitively and specifically to detect MG as a sensing probe in a FRET fluorescent aptasensor, where the FAM-labeled aptamer and GO were employed as efficient energy donor–acceptor pair. The linear range of this aptasensor using aptamer MG-36-12 was from 1.71 to 514.29 ng/mL and the LOD was as low as 0.79 ng/mL. Additionally, the fluorescent assay using aptamer MG-36-17 to detect MG exhibited a linear relationship from 1.71 to 857.14 ng/mL and a LOD of 2.13 ng/mL. Meanwhile, the aptasensor showed high specificity to MG with no cross-reactivity to other veterinary drugs and had a mean recovery of 81.54% to 100.96% in actual water samples from the aquatic product market.

https://www.mdpi.com/2304-8158/11/6/801/pdf?version=1656495567

Selection and Application of ssDNA Aptamers for Fluorescence Biosensing Detection of Malachite Green

Abstract Current chemical analysis approaches for contaminants have failed to reveal their biotoxicity. Moreover, conventional bioassays are time consuming and exhibit poor repeatability. In this study, we performed the acute toxicity detection of various contaminants (chromium (Cr), cadmium (Cd), lead (Pb), arsenic (As), mercury (Hg), tin (Sn), nickel (Ni), and bisphenol A (BPA)) with four bioluminescent bacteria (Vibrio qinghaiensis Q67, V. fischeri, Photobacterium phosphoreum T3, and P. phosphoreum 502) using a rapid, flexible, and low‐cost bioassay. We found that the temperature affected the bacterial luminescence, and freeze‐dried cells exhibited sensitive toxic responses to contaminants. Indeed, the optimized protectants containing 12% (w/v) trehalose, 4% sucrose, and 2% sorbitol displayed better luminescence and toxic sensitivity. Furthermore, freeze‐dried powders of these strains were prepared and subjected to acute toxicity detection. The results showed that all contaminants exhibited acute toxicity toward Q67, but the other strains did not show obvious response to nickel and tin. The relative half‐maximal effective concentration (EC50) values of BPA, Cr, Cd, Pb, As, Hg, Ni, and Sn to Q67 were 0.674, 1.313, 11.137, 5.921, 4.674, 0.911, 5.941, and 54.077 mg/L, respectively. In addition, the EC50 values of contaminants toward different strains were suggested to be statistically significant. Freeze‐dried Q67 exhibited toxic responses to more contaminants than the other bioluminescent strains; therefore, Q67 was selected to be more suitable than the other strains for single and mixture toxicity detection tests. Compared with other strains, Q67 was more appropriate for the rapid screening of the mixture toxicity of contaminants in samples as a nonspecific screening sensor before the use of standard analysis approaches.

Preparation of freeze‐dried bioluminescent bacteria and their application in the detection of acute toxicity of bisphenol A and heavy metals

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