Metabolic engineering of Escherichia coli for high-level production of benzyl acetate from glucose.

BACKGROUND: Benzyl acetate is an aromatic ester with a jasmine scent. It was discovered in plants and has broad applications in food, cosmetic, and pharmaceutical industries. Its current production predominantly relies on chemical synthesis. In this study, Escherichia coli was engineered to produce benzyl acetate. RESULTS: Two biosynthetic routes based on the CoA-dependent ß-oxidation pathway were constructed in E. coli for benzyl acetate production. In route I, benzoic acid pathway was extended to produce benzyl alcohol by combining carboxylic acid reductase and endogenous dehydrogenases and/or aldo-keto reductases in E. coli. Benzyl alcohol was then condensed with acetyl-CoA by the alcohol acetyltransferase ATF1 from yeast to form benzyl acetate. In route II, a plant CoA-dependent ß-oxidation pathway via benzoyl-CoA was assessed for benzyl alcohol and benzyl acetate production in E. coli. The overexpression of the phosphotransacetylase from Clostridium kluyveri (CkPta) further improved benzyl acetate production in E. coli. Two-phase extractive fermentation in situ was adopted and optimized for benzyl acetate production in a shake flask. The most optimal strain produced 3.0 ± 0.2 g/L benzyl acetate in 48 h by shake-flask fermentation. CONCLUSIONS: We were able to establish the whole pathway for benzyl acetate based on the CoA-dependent ß-oxidation in single strain for the first time. The highest titer for benzyl acetate produced from glucose by E. coli is reported. Moreover, cinnamyl acetate production as an unwanted by-product was very low. Results provided novel information regarding the engineering benzyl acetate production in microorganisms.

NADH-dependent formate dehydrogenase mutants for efficient carbon dioxide fixation.

Bioconversion of CO2 to high-valuable products is a globally pursued sustainable technology for carbon neutrality. However, low CO2 activation with formate dehydrogenase (FDH) remains a major challenge for further upcycling due to the poor CO2 affinity, reduction activity and stability of currently used FDHs. Here, we present two recombined mutants, ΔFDHPa48 and ΔFDHPa4814, which exhibit high CO2 reduction activity and antioxidative activity. Compared to FDHPa, the reduction activity of ΔFDHPa48 was increased up to 743 % and the yield in the reduction of CO2 to methanol was increased by 3.16-fold. Molecular dynamics identified that increasing the width of the substrate pocket of ΔFDHPa48 could improve the enzyme reduction activity. Meanwhile, the enhanced rigidity of C-terminal residues effectively protected the active center. These results fundamentally advanced our understanding of the CO2 activation process and efficient FDH for enzymatic CO2 activation and conversion.

Comprehensive insights into the mechanism of keratin degradation and exploitation of keratinase to enhance the bioaccessibility of soybean protein.

Keratin is a recalcitrant protein and can be decomposed in nature. However, the mechanism of keratin degradation is still not well understood. In this study, Bacillus sp. 8A6 can completely degrade the feather in 20 h, which is an efficient keratin degrader reported so far. Comprehensive transcriptome analysis continuously tracks the metabolism of Bacillus sp. 8A6 throughout its growth in feather medium. It reveals for the first time how the strain can acquire nutrients and energy in an oligotrophic feather medium for proliferation in the early stage. Then, the degradation of the outer lipid layer of feather can expose the internal keratin structure for disulfide bonds reduction by sulfite from the newly identified sulfite metabolic pathway, disulfide reductases and iron uptake. The resulting weakened keratin has been further proposedly de-assembled by the S9 protease and hydrolyzed by synergistic effects of the endo, exo and oligo-proteases from S1, S8, M3, M14, M20, M24, M42, M84 and T3 families. Finally, bioaccessible peptides and amino acids are generated and transported for strain growth. The keratinase has been applied for soybean hydrolysis, which generates 2234 peptides and 559.93 mg/L17 amino acids. Therefore, the keratinases, inducing from the poultry waste, have great potential to be further applied for producing bioaccessible peptides and amino acids for feed industry.

Green chemical and biological synthesis of cadaverine: recent development and challenges.

Cadaverine has great potential to be used as an important monomer for the development of a series of high value-added products with market prospects. The most promising strategies for cadaverine synthesis involve using green chemical and bioconversion technologies. Herein, the review focuses on the progress and strategies towards the green chemical synthesis and biosynthesis of cadaverine. Specifically, we address the specific biosynthetic pathways of cadaverine from different substrates as well as extensively discussing the origination, structure and catalytic mechanism of the key lysine decarboxylases. The advanced strategies for process intensification, the separation and purification of cadaverine have been summarized. Furthermore, the challenging issues of the environmental, economic, and applicable impact for cadaverine production are also highlighted. This review concludes with the promising outlooks of state-of-the-art applications of cadaverine along with some insights toward their challenges and potential improvements.