Applied Microbiology and Biotechnology

Efficient synthesis of an antiviral drug intermediate using an enhanced short-chain dehydrogenase in an aqueous-organic solvent system Abstract (2R,3S)-N-tert-Butoxycarbonyl-3-amino-1-chloro-2-hydroxy-4-phenylbutane (1b) is key for the synthesis of the antiviral drug atazanavir. It can be obtained via the stereoselective bioreduction of (3S)-3-(N-Boc-amino)-1-chloro-4-phenyl-butanone (1a) with short-chain dehydrogenase/reductase (SDR). However, the stereoselective bioreduction of this hydrophobic and bulky substrate still remained a challenge because of the steric hindrance effect and low mass transfer rate. In this study, SDR isolated from Novosphingobium aromaticivorans (NaSDR) having low activity to 1a, which was engineered to enhance catalytic efficiency through active pocket iterative saturation mutagenesis (ISM). The obtained mutant (muSDR) (G141V/I195L) had 3.57 times higher kcat than the wild type (WT) towards 1a. Molecular docking analysis revealed considerable differences in the distance between the substrate and catalytic residues in WT and mutant SDR. Moreover, muSDR reduced 15 ketones with excellent enantioselectivity, indicating broad substrate acceptance. After optimization of expression and reaction conditions, the conversion was completed in a scale-up reaction (500 mL) using 50% toluene with 500 mM substrate without additional NADH. These results show that muSDR may be a valuable biocatalyst for future industrial applications.

A novel transcriptional regulator RXE1 modulates the essential transactivator XYR1 and cellulase gene expression in Trichoderma reesei Abstract XYR1 is the key transcription activator for cellulase gene expression in the model filamentous fungus Trichoderma reesei, which is widely applied in the industry due to its excellent capability of secreting a large quantity of cellulases. Despite the essential role of XYR1, the regulation of its expression in T. reesei cellulolytic response is poorly understood. In this study, we identified a transcription factor RXE1 exhibiting strong binding activity to the xyr1 promoter using yeast one-hybrid screen. RXE1 homologs exist in quite a few filamentous fungi but none of them have been assessed regarding their functional involvement in plant cell wall degradation. Knockdown of rxe1 in T. reesei using a copper-mediated RNAi system not only abrogated conidiation, but also remarkably compromised xyr1 and cellulase gene expression. The defective cellulase but not conidia production in the rxe1-knockdown strain was fully rescued by the constitutive expression of XYR1. Our study thus identified a novel transcriptional regulator controlling xyr1 and cellulase gene expression, which will contribute to elaborating the intricate network of cellulase gene regulation in T. reesei.

Harnessing yeast metabolism of aromatic amino acids for fermented beverage bioflavouring and bioproduction Abstract Aromatic amino acid metabolism in yeast is an important source of secondary compounds that influence the aroma and flavour of alcoholic beverages and foods. Examples are the higher alcohol 2-phenylethanol, and its acetate ester, 2-phenylethyl acetate, which impart desirable floral aromas in wine, beer and baker's products. Beyond this well-known influence on the organoleptic properties of alcoholic beverages and foods, there is a growing interest in understanding and modulating yeast aromatic amino acid metabolism. The tryptophan derivatives melatonin and serotonin have bioactive properties and exert positive effects on human health, and aromatic amino acids are also the precursors of products of industrial interest, such as nutraceuticals, fragrances, and opium-derived drugs. This mini-review presents current knowledge on the formation of compounds from aromatic amino acids by Saccharomyces cerevisiae, from genetic and environmental influences on their flavour impacts in alcoholic beverages to their potential as bioactive compounds, and the use of yeast as microbial factories for the production of commercially relevant aromatic compounds.

Xanthobacter-dominated biofilm as a novel source for high-value rhamnose Abstract Rhamnose is a high-value carbohydrate used in flavorings, aromatics, and pharmaceuticals. Current demand for rhamnose is filled through plant-based sources; however, microbially originated rhamnolipids have been proposed as an alternative source. A mixed microbial biofilm, cultured from a wastewater sludge, was found to comprise > 8 dry weight% rhamnose when provided volatile fatty acids as carbon source, and 24 dry weight% when given glucose. The latter rhamnose concentration is a fourfold higher production mass than the current plant-based origin and is competitive with yields from pure microbial cultures. The biofilm was characterized based on total carbohydrate production at varying nutrient levels, individual carbohydrate monomer production from varying organic acid substrates, and microbial community composition—based on 16s rRNA. Biofilm carbohydrate production was maximized at a C:N ratio of 28 (mol:mol). The production of rhamnose varied significantly based on carbon substrate; glucose had the greatest yield of rhamnose, followed by propionic acid, lactic acid, acetic acid, valeric acid, and butyric acid. Microbial community analysis indicated an abundance of organisms within the Xanthobacter genus, which is known to produce rhamnose as zeaxanthin rhamnoside. Rhamnose production was heavily correlated with ribose production (R2 = 0.96). Results suggest that mixed microbial biofilms could be a competitive source of monomeric rhamnose that may be produced from mixed organic waste streams of variable composition via volatile fatty acids and glucose.

Co-occurrence of functional modules derived from nicotine-degrading gene clusters confers additive effects in Pseudomonas sp. JY-Q Abstract Pseudomonas sp. JY-Q was isolated from nicotine-rich environment and could degrade and tolerate high-content nicotine. Its specific genetic architecture comprised duplicated homologous nicotine-degrading clusters for different functional modules on the whole pathway. Its adaptive and genomic properties caused our concern whether the duplicated homologous gene clusters confer additive effects on nicotine degradation and result in strain JY-Q strong capability. After deletion of representative genes from duplicated homologous gene clusters of upstream module Nic1, midstream module Spm, and downstream module Nic2, the nicotine degradation efficiency of the wild type and mutant strains were examined. As the first genes of clusters Nic1-1 and Nic1-2, nicA2 and nox are both involved in nicotine degradation, but nox exhibited more contribution to nicotine metabolism due to the higher transcriptional amount of nox than that of nicA2. Likewise, the sub-clusters spm1 and spm2 showed additive effect on nicotine metabolism. As two hpo-like genes of clusters Nic2-1 and Nic2-2, hpo1, and hpo2 also showed additive effect on the nicotine degrading, but hpo1 provided more contribution than hpo2. The third hpo-like gene in cluster NA (nicotinic acid degrading), nicX is not necessary for 2,5-dihydroxypyridine transformation when hpo1 and hpo2 exist. A variety of transposases and integrases observed around Nic1 and Nic2 cluster genes suggests that the duplicated genes could evolve from horizontal gene transfer (HGT)-related dissemination. This study provide an insight into a novel adaptability mechanism of strains in extreme environment such as high nicotine concentration, and potential novel targets to enhance strain synthesis/degradation ability for future applications.

Engineered biosynthesis of cyclic lipopeptide locillomycins in surrogate host Bacillus velezensis FZB42 and derivative strains enhance antibacterial activity Abstract Locillomycins are cyclic lipononapeptides assembled by a nonlinear hexamodular NRPS and have strong antibacterial activity. In this study, we genetically engineered Bacillus velezensis FZB42 as a surrogate host for the heterologous expression of the loc gene cluster for locillomycins. The fosmid N13 containing whole loc gene cluster was screened from the B. velezensis 916 genomic library. Subsequently, a spectinomycin resistance cassette, and the cassette fused with an IPTG inducible promoter Pspac, was introduced in the fosmid N13 using λ Red recombination system, respectively. The resulting fosmids, designated N13+Spec and N13+PSSpec, were used for the transformation of B. velezensis FZB42 to obtain derivative strains FZBNPLOC and FZBPSLOC. RT-PCR and qRT-PCR results revealed the efficient heterologous expression of the loc gene cluster in both derivative strains. Particularly, there was positive correlation between the derivative FZBPSLOC strain and the enhanced production of locillomycins upon addition of the inducer IPTG with the highest production of locillomycins at 15-fold more than that of B. velezensis 916. This overproduction of locillomycins was also related to the enhancement of antibacterial activity against methicillin-resistant Staphylococcus aureus, and exhibited moderate changes in its hemolytic activity. Together our findings demonstrate that the nonlinear hexamodular NRPS, encoded by the loc gene cluster from B. velezensis 916, is sufficient for the biosynthesis of cyclic lipononapeptide locillomycins in the surrogate host B. velezensis FZB42. Moreover, the FZBPSLOC strain will also be useful for further development of novel locillomycins derivatives with improved antibacterial activity.

A novel multiplex xMAP assay for generic detection of avian, fish, and ruminant DNA in feed and feedstuffs Abstract The identification of animal species in feed and feedstuffs is important for detecting contamination and fraudulent replacement of animal components that might cause health and economic problems. A novel multiplex assay, based on xMAP technology and the generic detection of closely related species, was developed for the simultaneous differential detection of avian, fish, and ruminant DNA in products. Universal primers and probes specific to avian, fish, or ruminant species were designed to target a conserved mitochondrial DNA sequence in the 12S ribosomal RNA gene (rRNA). The assay specificity was validated using samples of 27 target and 10 nontarget animal species. The limits of detection of the purified DNA were determined to be 0.2 pg/μL–0.1 ng/μL by testing the meat samples of six species and four feedstuffs. The detection sensitivity of the experimental mixtures was demonstrated to be 0.01% (weight percentage). The assay's suitability for practical application was evaluated by testing feed samples; unlabeled animal ingredients were detected in 32% of the 56 samples. The assay differentially detected the three targeted categories of animal species in less than 2 h, reflecting improvements in speed and efficiency. Based on these results, this novel multiplex xMAP assay provides a reliable and highly efficient technology for the routine detection of animal species in feed and other products for which this information is needed.

Capability of iturin from Bacillus subtilis to inhibit Candida albicans in vitro and in vivo Abstract Candida albicans is a fungal pathogen that is difficult to cure clinically. The current clinic C. albicans-inhibiting drugs are very harmful to humans. This study revealed the potential of iturin fractions from Bacillus subtilis to inhibit C. albicans in free status (MIC = 32 μg/mL) and natural biofilm in vitro. The inhibition mechanism was identified as an apoptosis pathway via the decrease of mitochondrial membrane potential, the increase of the reactive oxygen species (ROS) accumulation, and the induction of nuclear condensation. For in vivo experiments, the C. albicans infection model was constructed via intraperitoneal injection of 1 × 108C. albicans cells into mice. One day after the infection, iturin was used to treat infected mice at different concentrations alone and in combination with amphotericin B (AmB) by intraperitoneal injection. The treatment with AmB alone could cause the death of infected mice, whereas treatment with 15 mg/kg iturin per day alone led to the survival of all infected mice throughout the study. After continuously treated for 6 days, all mice were sacrificed and analyzed. As results, the combination of 15 mg/kg iturin and AmB at a ratio of 2:1 had the most efficient effect to remove the fungal burden in the kidney and cure the infected mice by reversing the symptoms caused by C. albicansinfection, such as the loss of body weight, change of immunology cells in blood and cytokines in serum, and damage of organ structure and functions. Overall, iturin had potential in the development of efficient and safe drugs to cure C. albicans infection.

L-Erythrulose production with a multideletion strain of Gluconobacter oxydans Abstract Many ketoses or organic acids can be produced by membrane-associated oxidation with Gluconobacter oxydans. In this study, the oxidation of meso-erythritol to L-erythrulose was investigated with the strain G. oxydans621HΔupp BP.8, a multideletion strain lacking the genes for eight membrane-bound dehydrogenases. First batch biotransformations with growing cells showed re-consumption of L-erythrulose by G. oxydans 621HΔuppBP.8 in contrast to resting cells. The batch biotransformation with 2.8 g L−1 resting cells of G. oxydans 621HΔupp BP.8 in a DO-controlled stirred-tank bioreactor resulted in 242 g L−1 L-erythrulose with a product yield of 99% (w/w) and a space-time yield of 10 g L−1 h−1. Reaction engineering studies showed substrate excess inhibition as well as product inhibition of G. oxydans 621HΔupp BP.8 in batch biotransformations. In order to overcome substrate inhibition, a continuous membrane bioreactor with full cell retention was applied for meso-erythritol oxidation with resting cells of G. oxydans 621HΔupp BP.8. At a mean hydraulic residence time of 2 h, a space-time yield of 27 g L−1 h−1 L-erythrulose was achieved without changing the product yield of 99% (w/w) resulting in a cell-specific product yield of up to 4.4 gP gX−1 in the steady state. The product concentration (54 g L−1 L-erythrulose) was reduced in the continuous biotransformation process compared with the batch process to avoid product inhibition.

Novel metagenome-derived ornithine lipids identified by functional screening for biosurfactants Abstract Biosurfactants are amphiphilic molecules that interact with the surfaces of liquids leading to many useful applications. Most biosurfactants have been identified from cultured microbial sources, leaving a largely untapped resource of uncultured bacteria with potentially novel biosurfactant structures. To access the uncultured bacteria, a metagenomic library was constructed in Escherichia coli from environmental DNA within an E. coli, Pseudomonas putida and Streptomyces lividans shuttle vector. Phenotypic screening of the library in E. coli and P. putida by the paraffin spray assay identified a P. putida clone with biosurfactant activity. Sequence analysis and transposon mutagenesis confirmed that an ornithine acyl-ACP N-acyltransferase was responsible for the activity. Although the fosmid was not active in E. coli, overexpression of the olsB gene could be achieved under the control of the inducible T7 promoter, resulting in lyso-ornithine lipid production and biosurfactant activity in the culture supernatants. Screening for activity in more than one host increases the range of sequences that can be identified through metagenomic, since olsB would not have been identified if only E. coli had been used as a host. The potential of lyso-ornithine lipids as a biosurfactant has not been fully explored. Here, we present several biosurfactant parameters of lyso-ornithine lipid to assess its suitability for industrial application.