Optimization of 4-aminobutyric acid feeding strategy and clustered regularly interspaced short palindromic repeats activation for enhanced value-added chemicals in halophilic Chlorella sorokiniana.

Chlorella sorokiniana (CS) is a prominent microalga with vast potential as a biocarrier for carbon mitigation toward a green process. However, challenges remain in achieving high biomass levels and production rates. Therefore, a systematic feeding strategy using 4-aminobutyric acid (GABA) and CRISPR technology was applied to improve microalgal productivity. At first, GABA increased protein content by 1.4-fold, while intermittent supplementation during cultivation resulted in a 1.58-fold and 2.13-fold increase in biomass and pigment content, respectively. Under halophilic conditions, the optimal approach involved repeated feeding of 5 mM GABA at the initial and mid-log phases of growth, resulting in biomass, protein, and pigment levels of 6.74 g/L, 3.24 g/L, and 49.87 mg/L. CRISPRa mediated glutamate synthase and using monosodium glutamate (MSG) as a cheap precursor for GABA has effectively enhanced the biomass, protein, and lutein content, thus offers a cost-effective approach to commercialize high-valued chemical using algae towards a low-carbon paradigm.

High value ferulic acid biosynthesis using modular design and spent coffee ground in engineered Escherichia coli chassis.

Sophisticated genetic engineering enables microbial hosts to derive high-value aromatics in a green manner. Ferulic acid (FA) is one of the noteworthy aromatics due to its potent pharmacokinetic properties. However, the current approaches to FA biosynthesis still decamp from time- and cost-effectiveness. Herein, FA pathway was artificially reconstructed in Escherichia coli using modular designs. Comprehensive screening of E. coli lineages was reckoned for efficient synthesis of p-coumaric acid (pCA) as a precursor and FA eventually. The modular design was further advanced by harboring tyrosine transporter, adapting the heterologous codon, utilizing pCA symporter, and enriching FADH2 cofactor pools via in vivo regeneration. Taken together with simultaneous optimization of culture condition, a remarkable FA yield of 972.6 mg/L with 89.4 % conversion was achieved in 48 h, circumventing the time-consuming issue. Moreover, this study successfully exported inexpensive precursor from spent coffee ground for the first time, paving the economical way of FA biosynthesis.

Investigation of enzymatic quality and quantity using pyridoxal 5'-phosphate (PLP) regeneration system as a decoy in Escherichia coli.

Pyridoxal 5'-phosphate (PLP), an essential cofactor for multiple enzymes, was used as a protein decoy to prompt enzyme expression and activity for the first time. The best chassis, denoted as WJK, was developed using a pyridoxal kinase (PdxK) and integrated at the HK022 phage attack site of Escherichia coli W3110. When compared with the original strain, the amount and activity of lysine decarboxylase (CadA) in WJK were significantly increased by 100 % and 120 %, respectively. When supplementary nineteen amino acids as second carbon source, cell growth and protein trade-off were observed. The transcriptional levels of genes from glycolysis to TCA cycle, adhE, argH and gdhA were dominating and redirected more flux into α-ketoglutarate, thus facilitated cell growth. Stepwise improvement was conducted with pyridoxal and nitrogen-rich medium; hence, CadA activity was increased to 60 g-cadaverine/g-dry cell weight/h. By reutilizing the whole-cell biocatalysts in two repeated reactions with the supplementation of fresh cells, a total cadaverine of 576 g/L was obtained even without additional PLP. Notably, PLP decoy augment the enzymatic activities of 5-aminolevulinic acid synthase and glutamate/lysine/arginine decarboxylases by over 100 %. Finally, a conserved PLP-binding pocket, Ser-His-Lys, was identified as a vital PLP sponge site that simultaneously improved protein quality and quantity.

Synergistic effect of Trichoderma reesei cellulases on agricultural tea waste for adsorption of heavy metal Cr(VI).

This is the first attempt to study the synergistic effect between Trichoderma reesei cellulases and the abundant agricultural tea waste in absorption of heavy metal Cr(VI) as well as its kinetic model development. The properties of tea waste were first analyzed by near infrared spectroscopy (NIR), particle size distribution (PSD), X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) examination with EDX for comparison between its original (UN-TW) and cellulase-hydrolyzed (TRCEL-TW) conditions. Then, an advanced kinetic model in the form of -d[Cr(VI)]/dt = A[H+](n)e(-Ea/RT) [Cr(VI)](m)(0), which can successfully predict the time-dependent Cr(VI) concentration of various pHs, initial Cr(VI) concentrations and temperatures was developed. The demonstrated synergistic effects of T. reesei cellulases on tea waste suggested that cellulosic material provides more accessibility area for absorption of heavy metal. This study also provides an alternative approach to remove toxic Cr(VI) from aqueous solutions and extend the utilization of agricultural tea waste.

Copper ion-stimulated McoA-laccase production and enzyme characterization in Proteus hauseri ZMd44.

The novel bioelectricity-generating bacterium of Proteus hauseri ZMd44 has been first identified to produce McoA-laccase (EC 1.10.3.2) induced by copper sulphate. The optimal concentration of copper is 3 mM as supplementation at the beginning of culture or early exponential growth phase, during which laccase is predominantly synthesized. Moreover, the whole cellular and intracellular activities of laccase increase in the degrees of inducible copper concentrations. A possible mechanism for this phenomenon is that copper ions enhance the laccase genetic transcription level during the laccase synthesis thus granting this strain in copper tolerance. McoA-laccase belongs to typical type 1 (T1) Cu site laccase by electron paramagnetic resonance (EPR) analysis of intracellular enzyme. From our results, the optimal temperature and pH are 60°C and pH 2.2, respectively. The kinetic profiles show that this enzyme is stable under 50°C and in the slightly acidic environment, making it a potentially useful enzyme in dye decolorization, paper-pulp bleaching and bioremediation industries.

Partially purified Carica papaya lipase: a versatile biocatalyst for the hydrolytic resolution of (R,S)-2-arylpropionic thioesters in water-saturated organic solvents.

With the hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl thioesters in water-saturated isooctane as a model system, improvements of the specific lipase activity and thermal stability were found when a crude Carica papaya lipase (CPL) was partially purified and employed as the biocatalyst. The partially purified Carica papaya lipase (PCPL) was furthermore explored as an effective enantioselective biocatalyst for the hydrolytic resolution of (R,S)-profen thioesters in water-saturated organic solvents. The kinetic analysis in water-saturated isooctane indicated that both acyl donor and acyl acceptor have profound influences on the lipase activity, E-value, and enantioselectivity. Inversion of the enantioselectivity from (S)- to (R)-thioester was found for (R,S)-fenoprofen and (R,S)-ketoprofen thioesters that contained a bulky substituent at the meta-position of 2-phenyl moiety of the acyl part. Kinetic constants for the acylation step were furthermore estimated for elucidating the kinetic data and postulating an active site model. The thermodynamic analysis indicated that the enantiomer discrimination was driven by the difference of activation enthalpy (DeltaDeltaH) and that of activation entropy (DeltaDeltaS), yet the latter was dominated for most of the reacting systems. The postulated active site model was supported from the variation of DeltaDeltaH and DeltaDeltaS with the acyl moiety, in which a good linear enthalpy-entropy compensation relationship was also illustrated. A comparison of the performances between Candida rugosa lipase (CRL) and PCPL indicated that PCPL was superior to CRL in terms of the better thermal stability, similar or better lipase activity for the fast-reacting substrate, time-course-stability, and lower enzyme cost.