Applied Microbiology and Biotechnology

Role of β-oxidation and de novo fatty acid synthesis in the production of rhamnolipids and polyhydroxyalkanoates by Pseudomonas aeruginosa Abstract Pseudomonas aeruginosa are ubiquitous γ-proteobacteria capable of producing the biosurfactant rhamnolipids (RL) and the polymer polyhydroxyalkanoate (PHA). RL are glycolipids with high biotechnological potential, whereas PHA is used for the production of biodegradable plastics. It has been proposed that the β-oxidation pathway provides intermediates for RL biosynthesis, even when using a non-fatty acid carbon source for growth, while an intermediate of de novo fatty acid biosynthesis (FASII) pathway [(R)-3-hydroxyacyl-ACP] is used for PHA biosynthesis. The aim of this work is to study the inter-relationship of the RL and PHA biosynthetic pathways in a culture medium with a non-fatty acid carbon source, focusing on the role of FASII and β-oxidation in supplying the substrates for the first step in RL and PHA synthesis, carried out by the RhlA and PhaG enzymes, respectively. The PHA synthases (PhaC1 and PhaC2) are only able to use CoA-linked 3-hydroxy acids and the PhaG enzyme catalyzes the conversion of (R)-3-hydroxyacyl-ACP to (R)-3-hydroxyacyl-CoA, the substrate of PhaC1 and PhaC2. RhlA in turn catalyzes the synthesis of the RL precursor 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAA) by the dimerization of two 3-hydroxyalkanoic acid molecules (that have been shown to be also (R)-3-hydroxyacyl-ACP). In this work, we show that RhlA can produce both RL and PHA precursors (presumably CoA-linked HAA), that the blockage of carbon flux through β-oxidation pathway does not decrease RL titer, and that the enoyl-CoA hydratase RhlY and enoyl-CoA hydratase/isomerase RhlZ produce the main fatty acids precursor of RL using as substrate also a FASII intermediate (presumably (S)-3-hydroxyacyl-CoA).

The antitumor antibiotic rebeccamycin—challenges and advanced approaches in production processes Abstract Rebeccamycin is an antibiotic and antitumor substance isolated from the filamentous bacterium Lentzea aerocolonigenes. After its discovery, investigations of rebeccamycin focused on elucidating its structure, biological activity, and biosynthetic pathway. For potential medical application, a sufficient drug supply has to be ensured, meaning that the production process of rebeccamycin plays a major role. In addition to the natural production of rebeccamycin in L. aerocolonigenes, where the complex cell morphology is an important factor for a sufficient production, rebeccamycin can also be heterologously produced or chemically synthesized. Each of these production processes has its own challenges, and first approaches to production often lead to low final product concentrations, which is why process optimizations are performed. This review provides an overview of the production of rebeccamycin and the different approaches used for rebeccamycin formation including process optimizations.

Identification of genes involved in shea butter biosynthesis from Vitellaria paradoxa fruits through transcriptomics and functional heterologous expression Abstract Shea tree (Vitellaria paradoxa) is one economically important plant species that mainly distributes in West Africa. Shea butter extracted from shea fruit kernels can be used as valuable products in the food and cosmetic industries. The most valuable composition in shea butter was one kind of triacylglycerol (TAG), 1,3-distearoyl-2-oleoyl-glycerol (SOS, C18:0–C18:1–C18:0). However, shea butter production is limited and little is known about the genetic information of shea tree. In this study, we tried to reveal genetic information of shea tree and identified shea TAG biosynthetic genes for future shea butter production in yeast cell factories. First, we measured lipid content, lipid composition, and TAG composition of seven shea fruits at different ripe stages. Then, we performed transcriptome analysis on two shea fruits containing obviously different levels of SOS and revealed a list of TAG biosynthetic genes potentially involved in TAG biosynthesis. In total, 4 glycerol-3-phosphate acyltransferase (GPAT) genes, 8 lysophospholipid acyltransferase (LPAT) genes, and 11 diacylglycerol acyltransferase (DGAT) genes in TAG biosynthetic pathway were predicted from the assembled transcriptome and 14 of them were cloned from shea fruit cDNA. Furthermore, the heterologous expression of these 14 potential GPAT, LPAT, and DGAT genes in Saccharomyces cerevisiae changed yeast fatty acid and lipid profiles, suggesting that they functioned in S. cerevisiae. Moreover, two shea DGAT genes, VpDGAT1 and VpDGAT7, were identified as functional DGATs in shea tree, showing they might be useful for shea butter (SOS) production in yeast cell factories.

Highly thermostable GH51 α-arabinofuranosidase from Hungateiclostridium clariflavum DSM 19732 Abstract Arabinofuranosidase plays an essential role in the process of hydrolysis of arabinoxylan (AX). Thermostable, versatile, and efficient arabinofuranosidase is thus of great interest for the biorefinery industry. A GH51 arabinofuranosidase, Abf51, from Hungateiclostridium clariflavum DSM 19732 was heterogeneously expressed in Escherichia coli. Abf51 was found to have an optimal pH and temperature of 6.5 and 60 °C, respectively, with very high thermostability. At the optimal working temperature (60 °C), Abf51 retained over 90% activity after a 2-day incubation and over 60% activity after a 6-day incubation. Abf51 could effectively remove the arabinofuranosyls from three kinds of AX oligosaccharides [23-α-l-arabinofuranosyl-xylotriose (A2XX), 32-α-l-arabinofuranosyl-xylobiose (A3X), and 2333-di-α-l-arabinofuranosyl-xylotriose (A2 + 3XX)], which characterized as either single substitution or double substitution by arabinofuranosyls on terminal xylopyranosyl units. The maximal catalytic efficiency (Kcat/Km) was observed using p-nitrophenyl-α-l-arabinofuranoside (pNPAF) as a substrate (205.0 s−1 mM−1), followed by using A3X (22.8 s−1 mM−1), A2XX (6.9 s−1 mM−1), and A2 + 3XX (0.5 s−1 mM−1) as substrates. Moreover, the presence of Abf51 significantly stimulated the saccharification level of AX (18.5 g L−1) up to six times along with a β-xylanase as well as a β-xylosidase. Interestingly, in our survey of top thermostable arabinofuranosidases, most members were found from GH51, probably due to their owning of (β/α)8-barrel architectures. Our results suggested the great importance of GH51s as candidates for thermostable, versatile, and efficient arabinofuranosidases toward industry application.

Haloferax volcanii as immobilised whole cell biocatalyst: new applications for halophilic systems Abstract Enzyme-mediated synthesis of pharmaceutical compounds is a 'green' alternative to traditional synthetic chemistry, and microbial engineering opens up the possibility of using whole cells as mini-factories. Whole-cell biocatalysis reduces cost by eliminating expensive enzyme purification and cofactor addition steps, as well as resulting in increased enzyme stability. Haloferax volcanii is a model halophilic archaeon encoding highly salt and organic solvent tolerant enzymes such as alcohol dehydrogenase (HvADH2), which catalyses the reduction of aldehydes and ketone in the presence of NADPH/NADH cofactor. A H. volcanii strain for constitutive HvADH2 expression was generated using a strong synthetic promoter (p.syn). The strain was immobilised in calcium alginate beads and repeatedly used as a whole-cell biocatalyst. The reduction of acetophenone, used as test substrate, was very successful and high yields were detected from immobilised whole cells over repeated biotransformation cycles. The immobilised H. volcanii retained stability and high product yields after 1 month of storage at room temperature. This newly developed system offers halophilic enzyme expression in its native environment, high product yield, stability and reusability without the addition of any expensive NADPH/NADH cofactor. This is the first report of whole cell–mediated biocatalysis by the halophilic archaeon H. volcanii.

Function, essentiality, and expression of cytochrome P450 enzymes and their cognate redox partners in Mycobacterium tuberculosis: are they drug targets? Abstract This review covers the current knowledge of the cytochrome P450 enzymes (CYPs) of the human pathogen Mycobacterium tuberculosis (Mtb) and their endogenous redox partners, focusing on their biological function, expression, regulation, involvement in antibiotic resistance, and suitability for exploitation as antitubercular targets. The Mtb genome encodes twenty  CYPs and nine associated redox partners required for CYP catalytic activity. Transposon insertion mutagenesis studies have established the (conditional) essentiality of several of these enzymes for in vitro growth and host infection. Biochemical characterization of a handful of Mtb CYPs has revealed that they have specific physiological functions in bacterial virulence and persistence in the host. Analysis of the transcriptional response of Mtb CYPs and redox partners to external insults and to first-line antibiotics used to treat tuberculosis showed a diverse expression landscape, suggesting for some enzymes a potential role in drug resistance. Combining the knowledge about the physiological roles and expression profiles indicates that, at least five Mtb CYPs, CYP121A1, CYP125A1, CYP139A1, CYP142A1, and CYP143A1, as well as two ferredoxins, FdxA and FdxC, can be considered promising novel therapeutic targets.

Cobalamin is produced by Acetobacter pasteurianus DSM 3509 Abstract Only a few cobalamin-producing bacterial species are known which are suitable for food fermentations. The strain of Acetobacter pasteurianus DSM 3509 was found to have the capability to synthesize cobalamin. A survival test and a preliminary genetic study of the gene of uroporphyrinogen-III synthase indicated the ability to synthesize cobalamin. By a modified microbiological assay based on Lactobacillus delbrueckii ssp. lactis DSM 20355, 4.57 ng/mL of cyanocorrinoids and 0.75 ng/mL of noncorrinoid growth factors were detected. The product extracted and isolated by immunoaffinity chromatography in its cyanide form had the similar UV spectrum as standard cyanocobalamin and Coα-[α-(7-adenyl)]-(Coβ-cyano) cobamide also known as pseudovitamin B12 produced by Lactobacillus reuteri DSM 20016. The chromatographically separated product of A. pasteurianus was subjected to mass spectrometrical analysis. There, its fragmentation pattern turned out to be equivalent to that of cyanocobalamin also produced by Propionibacterium freudenreichii ssp. freudenreichii DSM 20271 and clearly differs from pseudovitamin B12. Due to the presence of this species in several food applications, there might be cobalamin residues in food fermented with these bacteria.

Directed aryl sulfotransferase evolution toward improved sulfation stoichiometry on the example of catechols Abstract Sulfation is an important way for detoxifying xenobiotics and endobiotics including catechols. Enzymatic sulfation occurs usually with high chemo- and/or regioselectivity under mild reaction conditions. In this study, a two-step p-NPS-4-AAP screening system for laboratory evolution of aryl sulfotransferase B (ASTB) was developed in 96-well microtiter plates to improve the sulfate transfer efficiency toward catechols. Increased transfer efficiency and improved sulfation stoichiometry are achieved through the two-step screening procedure in a one-pot reaction. In the first step, the p-NPS assay is used (detection of the colorimetric by-product, p-nitrophenol) to determine the apparent ASTB activity. The sulfated product, 3-chlorocatechol-1-monosulfate, is quantified by the 4-aminoantipyrine (4-AAP) assay in the second step. Comparison of product formation to p-NPS consumption ensures successful directed evolution campaigns of ASTB. Optimization yielded a coefficient of variation below 15% for the two-step screening system (p-NPS-4-AAP). In total, 1760 clones from an ASTB-SeSaM library were screened toward the improved sulfation activity of 3-chlorocatechol. The turnover number (kcat = 41 ± 2 s−1) and catalytic efficiency (kcat/KM = 0.41 μM−1 s−1) of the final variant ASTB-M5 were improved 2.4- and 2.3-fold compared with ASTB-WT. HPLC analysis confirmed the improved sulfate stoichiometry of ASTB-M5 with a conversion of 58% (ASTB-WT 29%; two–fold improvement). Mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) confirmed the chemo- and regioselectivity, which yielded exclusively 3-chlorocatechol-1-monosulfate. For all five additionally investigated catechols, the variant ASTB-M5 achieved an improved kcat value of up to 4.5-fold and sulfate transfer efficiency was also increased (up to 2.3-fold).

Different behaviors of cyclic dipeptide prenyltransferases toward the tripeptide derivative ardeemin fumiquinazoline and its enantiomer Abstract In nature, cyclic dipeptide prenyltransferases catalyze regioselective Friedel-Crafts alkylations of tryptophan-containing cyclic dipeptides. This enzyme class, belonging to the dimethylallyl tryptophan synthase superfamily, is known to be flexible toward aromatic prenyl acceptors, while mostly retaining its typical regioselectivity. Ardeemin fumiquinazoline (FQ) (1), a tryptophan-containing cyclic tripeptide derivative, is assembled in Aspergillus fischeri by the non-ribosomal peptide synthetase ArdA and modified by the prenyltransferase ArdB, leading to the pharmaceutically active hexacyclic ardeemin. Therefore, 1 and its enantiomer ent-ardeemin FQ (2) constitute potential substrates for aromatic prenyltransferases. In this study, we investigated the acceptance of both enantiomers by two cyclic dipeptide C2-prenyltransferases BrePT and FtmPT1 and three C3-prenyltransferases CdpNPT, CdpC3PT, and AnaPT. LC-MS analysis of the incubation mixtures and NMR analysis of the isolated products revealed that the stereochemistry at C11 and C14 in 1 and 2 has a strong influence on their acceptance by these enzymes and the regioselectivity of the prenylation reactions. 1 was very well accepted by BrePT, FtmPT1, and CdpNPT, with C2- or C3-prenylated derivatives as predominant products, which fills the prenylation gaps by tryptophan prenyltransferases reported in a previous study. 2 was a poor substrate for all the enzymes and converted with low regioselectivity and mainly prenylated at C6 and C7 of the indole moiety.

Shape of gastrointestinal immunity with non-genetically modified Lactococcus lactis particles requires commensal bacteria and myeloid cells-derived TGF-β1 Abstract Heat-killed probiotics or microbial autologous components show multiple activities on modulating host immune responses towards tolerance or vice versus aggressiveness. Gram-positive enhancer matrix particles (GEMs), the non-genetically modified particles which composed of the cell wall derived from Lactococcus lactis (L. lactis), were used as a typical microbial molecule to investigate the mechanism of opposite immune responses generated in disparate scenarios. The results of stool 16S rRNA Illumina sequencing suggested that the overwhelming number of mice pre-administered with GEMs showed the expansion of Bacteroidetes but contraction of Verrucomicrobia. Co-administration GEMs and antibiotics could preserve the microbial diversity, even though the abundance of gut microbes was largely depleted by antibiotics. Additionally, dendritic cells (DCs) from mice receiving GEMs rather than DCs that in vitro treated with GEMs induced the expansion of regulatory T cells (Tregs), witnessing the critical role of gut flora alteration. Importantly, this alteration provided protection to alleviate dextran sulfate sodium (DSS)-induced intestinal inflammation. On the other hand, in the context of Helicobacter felis (H. felis) infection, the mice pre-administrated with GEMs exhibited a comparably potent gastric immunity with the elevated expression of IFN-γ, IL-17, and multiple anti-microbial factors, leading to the reduced burden of H. felis. However, tolerance for both DSS-induced intestinal inflammation and immunity against H. felis was depleted in a mice model lacking of transforming growth factor-β1 (TGF-β1) in myeloid cells. These findings suggest that GEMs can modulate host immune responses bidirectionally according to context, and may serve as a supplement for antibiotic treatment.