(-)-α-Bisabolol Production in Engineered Escherichia coli Expressing a Novel (-)-α-Bisabolol Synthase from the Globe Artichoke Cynara cardunculus var. Scolymus.

(-)-α-Bisabolol is a functional ingredient in various health and cosmetic products and has antibacterial, anti-inflammatory, and wound healing properties. (-)-α-Bisabolol is chemically synthesized and produced by steam distillation of essential oils extracted from Brazilian Candeia (Eremanthus erythropappus). To sustainably produce pure (-)-α-bisabolol, we previously engineered Escherichia coli to produce 9.1 g/L (-)-α-bisabolol via heterologous mevalonate pathways and (-)-α-bisabolol synthase (BOS) from German chamomile, Matricaria recutita (MrBOS). BOS has only been reported in MrBOS and Brazilian Candeia (EeBOS). The limited availability of BOS has made it difficult to achieve high titer and yield and large-scale (-)-α-bisabolol production. We identified a novel BOS in globe artichoke (CcBOS) and examined its functionality in vitro and in vivo. CcBOS showed higher catalytic efficiency and (-)-α-bisabolol production rates than those from MrBOS or EeBOS. In fed-batch fermentation, CcBOS generated the highest reported (-)-α-bisabolol titer to date (23.4 g/L). These results may facilitate economically viable industrial (-)-α-bisabolol production.

Directed evolution of glycosyltransferase for enhanced efficiency of avermectin glucosylation.

Avermectin, produced by Streptomyces avermitilis, is an active compound protective against nematodes, insects, and mites. However, its potential usage is limited by its low aqueous solubility. The uridine diphosphate (UDP)-glycosyltransferase (BLC) from Bacillus licheniformis synthesizes avermectin glycosides with improved water solubility and in vitro antinematodal activity. However, enzymatic glycosylation of avermectin by BLC is limited due to the low conversion rate of this reaction. Thus, improving BLC enzyme activity is necessary for mass production of avermectin glycosides for field application. In this study, the catalytic activity of BLC toward avermectin was enhanced via directed evolution. Three mutants from the BLC mutant library (R57H, V227A, and D252V) had specific glucosylation activity for avermectin 2.0-, 1.8-, and 1.5-fold higher, respectively, than wild-type BLC. Generation of combined mutations via site-directed mutagenesis led to even further enhancement of activity. The triple mutant, R57H/V227A/D252V, had the highest activity, 2.8-fold higher than that of wild-type BLC. The catalytic efficiencies (Kcat/Km) of the best mutant (R57H/V227A/D252V) toward the substrates avermectin and UDP-glucose were improved by 2.71- and 2.29-fold, respectively, compared to those of wild-type BLC. Structural modeling analysis revealed that the free energy of the mutants was - 1.1 to - 7.1 kcal/mol lower than that of wild-type BLC, which was correlated with their improved activity. KEY POINTS: • Directed evolution improved the glucosylation activity of BLC toward avermectin. • Combinatorial site-directed mutagenesis led to further enhanced activity. • The mutants exhibited lower free energy values than wild-type BLC.

Adaptive laboratory evolution of Escherichia coli lacking cellular byproduct formation for enhanced acetate utilization through compensatory ATP consumption.

Acetate has attracted great attention as a carbon source to develop economically feasible bioprocesses for sustainable bioproducts. Acetate is a less-preferred carbon source and a well-known growth inhibitor of Escherichia coli. In this study, we carried out adaptive laboratory evolution of an E. coli strain lacking four genes (adhE, pta, ldhA, and frdA) involved in acetyl-CoA consumption, allowing the efficient utilization of acetate as its sole carbon and energy source. Four genomic mutations were found in the evolved strain through whole-genome sequencing, and two major mutations (in cspC and patZ) mainly contributed to efficient utilization of acetate and tolerance to acetate. Transcriptomic reprogramming was examined by analyzing the genome-wide transcriptome with different carbon sources. The evolved strain showed high levels of intracellular ATP by upregulation of genes involved in NADH and ATP biosynthesis, which facilitated the production of enhanced green fluorescent protein, mevalonate, and n-butanol using acetate alone. This new strain, given its high acetate tolerance and high ATP levels, has potential as a starting host for cell factories targeting the production of acetyl-CoA-derived products from acetate or of products requiring high ATP levels.

A designed whole-cell biosensor for live diagnosis of gut inflammation through nitrate sensing.

Microbes reprogrammed using advanced genetic circuits are envisaged as emerging living diagnostics for a wide range of diseases and play key roles in regulating gut microbiota to treat disease-associated symptoms in a non-invasive manner. Here, we developed a designer probiotic Escherichia coli that senses and responds to nitrate, a biomarker of gut inflammation. To this end, we first employed the NarX-NarL two-component regulatory system in E. coli to construct a nitrate-responsive genetic circuit. Next, we optimized the nitrate biosensor for the best performance using measures of sensitivity and specificity. We then introduced this genetic circuit into a probiotic E. coli Nissle 1917. We demonstrated that the designed biosensor can sense elevated nitrate levels during gut inflammation in mice with native gut microbiota. Moreover, using Boolean AND gate, we generated a genetically encoded biosensor for simultaneous sensing of the thiosulfate and nitrate biomarkers, thus increasing the tool's specificity for diagnosing gut inflammation. The nitrate-responsive genetic circuit will enable new approaches for non-invasive diagnostics of inflammation-associated diseases.

Metabolic pathway of 3,6-anhydro-D-galactose in carrageenan-degrading microorganisms.

Complete hydrolysis of κ-carrageenan produces two sugars, D-galactose and 3,6-anhydro-D-galactose (D-AnG). At present, however, we do not know how carrageenan-degrading microorganisms metabolize D-AnG. In this study, we investigated the metabolic pathway of D-AnG degradation by comparative genomic analysis of Cellulophaga lytica LIM-21, Pseudoalteromonas atlantica T6c, and Epulopiscium sp. N.t. morphotype B, which represent the classes Flavobacteria, Gammaproteobacteria, and Clostridia, respectively. In this bioinformatic analysis, we found candidate common genes that were believed to be involved in D-AnG metabolism. We then experimentally confirmed the enzymatic function of each gene product in the D-AnG cluster. In all three microorganisms, D-AnG metabolizing genes were clustered and organized in operon-like arrangements, which we named as the dan operon (3,6-d-anhydro-galactose). Combining bioinformatic analysis and experimental data, we showed that D-AnG is metabolized to pyruvate and D-glyceraldehyde-3-phosphate via four enzyme-catalyzed reactions in the following route: 3,6-anhydro-D-galactose â†’ 3,6-anhydro-D-galactonate â†’ 2-keto-3-deoxy-D-galactonate (D-KDGal) â†’ 2-keto-3-deoxy-6-phospho-D-galactonate â†’ pyruvate + D-glyceraldehyde-3-phosphate. The pathway of D-AnG degradation is composed of two parts: transformation of D-AnG to D-KDGal using two D-AnG specific enzymes and breakdown of D-KDGal to two glycolysis intermediates using two DeLey-Doudoroff pathway enzymes. To our knowledge, this is the first report on the metabolic pathway of D-AnG degradation.