Computational fluid dynamics analysis of Trichosporon fermentans flocculation in refined soybean oil wastewater and flocculation rate prediction method.

Trichosporon fermentans can be used to treat refined soybean oil wastewater (RSOW) and produce microbial lipids. Bioflocculation is an effective method to recover Trichosporon fermentans which accumulates intracellular oils from wastewater. During the flocculation, the hydrodynamic distribution and parameters in the reactor are important limiting factors of yeast flocculation performance. In a 0.25 L flocculation device, it was found that the appropriate range of turbulence kinetic energy was within 0.00065-0.00073 m2/s2, the dissipation rate was within 0.119-0.317 m2/s3, and the shear force was less than 0.433 Pa by computational fluid dynamics. In this case, the flocculation rate (Fr) of Trichosporon fermentans could reach more than 90%. The empirical formula associated Fr of Trichosporon fermentans with hydrodynamic parameters was obtained by Matlab, and improved in the enlargement of flocculation device, displaying an error of less than 3.03%. A conical draft tube airlift circulating reactor for flocculation was designed based on the empirical formula, and the Fr reached 91.3%. The study shows that it is feasible to predict Fr of Trichosporon fermentans according to hydrodynamic parameters by numerical simulation, and design the industrial reactor for flocculation harvesting yeasts. It is also helpful for large-scale treatment of RSOW in a safe environment.

A two-step process for pre-hydrolysis of hemicellulose in pulp-impregnated effluent with high alkali concentration to improve xylose production.

The viscose fiber production process is accompanied by the accumulation of pulp-impregnated effluent (PIE), including hemicellulose and large amounts of alkali, and discharge of PIE will cause environment pollution. This paper aims to relieve the inhibition of high concentration of alkali on xylose production from hydrolysis of hemicellulose in PIE. Based on the fact that solid acid uses H+ at the acid sites to exchange with cations in PIE and can be recycled, a two-step method including an extra pretreatment process before pre-hydrolysis (SPP) is proposed. After the alkali was removed by the H+ dissociated from solid acid in the extra pretreatment process, the pH of PIE dropped from 14 to 4, and the content of Na+ and proteins was reduced by 99.13 % and 78.51 %, respectively. After SPP, the polymerization degree of the hemicellulose decreased by 73.4 %, and the subsequent enzymatic hydrolysis process was promoted. Finally, the xylose yield of SPP followed by enzymatic hydrolysis reached 57.15 g/L, which was 145.38 % more than that of enzymatic hydrolysis alone. The load of a downstream ion purification procedure was relieved compared to that of inorganic acid hydrolysis. The development of SPP contributes to the resource utilization of high alkali concentration wastewater.

Bacterial cellulose as an oleaginous yeast cell carrier for soybean oil refinery effluent treatment and pyrolysis oil production.

Bacterial cellulose produced from soybean oil refinery effluent is a good immobilization carrier because of the large pores in its fiber network, its high water-holding capacity, and its good biocompatibility. In this study, it was applied to immobilization of oleaginous yeasts for treating soybean oil refinery effluent. The immobilization percentage reached 50%, and the removal of chemical oxygen demand and oil content reached 92.1% and 93.1%, respectively, during dynamic immobilization using a mass percentage of bacterial cellulose of 30% and an immobilization time of 24 h, which were significantly higher than those of free oleaginous yeasts or yeasts immobilized by bacterial cellulose from rich medium. The immobilized oleaginous yeasts facilitated the recovery of the yeasts and effectively treated three batches of soybean oil refinery effluent. The immobilized oleaginous yeasts recovered after soybean oil refinery effluent treatment were pyrolyzed to produce bio-oil, which contributed to more alkanes and a higher calorific value of bio-oil in the pyrolysis products as compared to those of free oleaginous yeasts. As bacterial cellulose used as an oleaginous yeast cell carrier is produced from soybean oil refinery effluent, no waste of immobilization materials is involved and an efficient waste-into-oil bioprocess is developed.

Protein scaffold optimizes arrangement of constituent enzymes in indigoidine synthetic pathway to improve the pigment production.

Indigoidine is a dark-blue natural pigment with application prospect and synthesized from glutamine (Gln) by series of indigoidine synthetases (IndCs). Indigoidine production can be improved by enhancing Gln pool via supplementing Gln directly or converting metabolism glutamate (Glu) to Gln by glutamine synthetase (GlnA). But, Gln is expensive, and excess Gln inhibits indigoidine production of the recombinant strain. Supplementing Glu instead of Gln may improve the productive and economic efficiency of indigoidine, but the local activities and positions of the indigoidine pathway enzymes GlnA, Sc-IndC, and the helper protein of Sc-IndC (IndB) should be well arranged. We identified the Streptomyces chromofuscus ATCC 49982 derived IndC (Sc-IndC) as an more efficient IndC compared to other IndCs applied for constructing indigoidine-producting strains, and designed series of protein scaffold complexes with architectures of PDZ, SH3, and GBD domains (PxSyG1) to arrange the pathway enzymes. The strain recruiting GlnA, Sc-IndC, and IndB on the PDZ, SH3, and GBD domains of scaffold P1S2G1, respectively, was the most efficient. In the strain, the GlnA supplied sufficient local Gln for Sc-IndC from Glu, and the generated Gln was immediately consumed by Sc-IndC to relieve cell growth inhibition caused by Gln. The optimum Glu concentration (6 g/L) for the strain was higher than those of the strains recruiting Sc-IndC on the GBD domain, which was away from the PDZ domain recruiting GlnA. The highest titer of indigoidine was 12 g/L, which was two folds of the control without scaffold (5.8 g/L). The titer is 5 g/L higher than the control without Glu supplemented (6.9 g/L), meaning that 97% of the supplemented Glu was transformed into indigoidine. The batch fermentation with the optimum strain in a 5-L reactor achieved an indigoidine titer of 14 g/L in 60 h. To our knowledge, this was the most efficient indigoidine productivity achieved so far. The optimization strategies by protein scaffold should be applicative to other pathways with complex substrate demands. KEY POINTS: •Protein scaffold systems were designed to arrange the indigoidine synthetic pathway. •The scaffold system improved supplement of Gln for indigoidine production from Glu. •The inhibition caused by excess Gln was relieved by proper designed scaffold. •The yield and titer of indigoidine was improved by arranging the pathway enzymes. Graphical abstract.

Metabolic engineering and optimization of the fermentation medium for vitamin B12 production in Escherichia coli.

Vitamin B12 is a crucial fine chemical that is widely used in the pharmaceutical, food and chemical industries, and its production solely dependents on microbial fermentation. We previously constructed an artificial vitamin B12 biosynthesis pathway in Escherichia coli, but the yield of the engineered strains was low. Here, we removed metabolic bottlenecks of the vitamin B12 biosynthesis pathway in engineered E. coli strains. After screening cobB genes from different sources, optimizing the expression of cobN and customizing the ribosome binding sites of cobS and cobT, the vitamin B12 yield increased to 152.29 µg/g dry cell weight (DCW). Optimization of the downstream module, which converts co(II)byrinic acid a,c-diamide into adenosylcobinamide phosphate, elevated the vitamin B12 yield to 249.04 µg/g DCW. A comparison of a variety of equivalent components indicated that glucose and corn steep liquor are optimal carbon and nitrogen sources, respectively. Finally, an orthogonal array design was applied to determine the optimal concentrations of glucose and nitrogen sources including corn steep liquor and yeast extract, through which a vitamin B12 yield of 530.29 µg/g DCW was obtained. The metabolic modifications and optimization of fermentation conditions achieved in this study offer a basis for further improving vitamin B12 production in E. coli and will hopefully accelerate its industrial application.

Computational fluid dynamics and factor analysis of a novel swirling demulsified airlift loop reactor for the treatment of refined soybean oil wastewater.

A swirling demulsified airlift loop reactor (SD-ALR) was developed for the treatment of oily wastewater with yeasts. Computational fluid dynamics simulations showed that the gas holdup and liquid velocity gradient in the SD-ALR were 2.9% and 0.37 m/s higher than those in the traditional airlift loop reactor. The optimization results of the swirling demulsifier showed that the optimal number and elevation angle of the blades were 8 and 45°, and the optimal installation position was 150 mm from the bottom of the draft tube. The results of treating refined soybean oil wastewater in the SD-ALR showed that the wastewater treatment time was decreased by 8 h, and the removals of chemical oxygen demand and oil content increased by 5.10% ±â€¯0.02% and 9.55% ±â€¯0.40%, respectively, compared with those in the traditional airlift loop reactor. A volumetric mass transfer coefficient model was established for SD-ALR and oily wastewater.

Modified substrate specificity of a methyltransferase domain by protein insertion into an adenylation domain of the bassianolide synthetase.

BACKGROUND: Creating designer molecules using a combination of select domains from polyketide synthases and/or nonribosomal peptide synthetases (NRPS) continues to be a synthetic goal. However, an incomplete understanding of how protein-protein interactions and dynamics affect each of the domain functions stands as a major obstacle in the field. Of particular interest is understanding the basis for a class of methyltransferase domains (MT) that are found embedded within the adenylation domain (A) of fungal NRPS systems instead of in an end-to-end architecture. RESULTS: The MT domain from bassianolide synthetase (BSLS) was removed and the truncated enzyme BSLS-ΔMT was recombinantly expressed. The biosynthesis of bassianolide was abolished and N-desmethylbassianolide was produced in low yields. Co-expression of BSLS-ΔMT with standalone MT did not recover bassianolide biosynthesis. In order to address the functional implications of the protein insertion, we characterized the N-methyltransferase activity of the MT domain as both the isolated domain (MTBSLS) and as part of the full NRPS megaenzyme. Surprisingly, the MTBSLS construct demonstrated a relaxed substrate specificity and preferentially methylated an amino acid (L-Phe-SNAC) that is rarely incorporated into the final product. By testing the preference of a series of MT constructs (BSLS, MTBSLS, cMT, XLcMT, and aMT) to L-Phe-SNAC and L-Leu-SNAC, we further showed that restricting and/or fixing the termini of the MTBSLS by crosslinking or embedding the MT within an A domain narrowed the substrate specificity of the methyltransferase toward L-Leu-SNAC, the preferred substrate for the BSLS megaenzyme. CONCLUSIONS: The embedding of MT into the A2 domain of BSLS is not required for the product assembly, but is critical for the overall yields of the final products. The substrate specificity of MT is significantly affected by the protein context within which it is present. While A domains are known to be responsible for selecting and activating the biosynthetic precursors for NRPS systems, our results suggest that embedding the MT acts as a secondary gatekeeper for the assembly line. This work thus provides new insights into the embedded MT domain in NRPSs, which will facilitate further engineering of this type of biosynthetic machinery to create structural diversity in natural products.

Optimal Design of Plant Canopy Based on Light Interception: A Case Study With Loquat.

Canopy architecture determines the light distribution and light interception in the canopy. Reasonable shaping and pruning can optimize tree structure; maximize the utilization of land, space and light energy; and lay the foundation for achieving early fruiting, high yield, health and longevity. Due to the complexity of loquat canopy architecture and the multi-year period of tree growth, the variables needed for experiments in canopy type training are hardly accessible through field measurements. In this paper, we concentrated on exploring the relationship between branching angle and light interception using a three-dimensional (3D) canopy model in loquat (Eriobotrya japonica Lindl). First, detailed 3D models of loquat trees were built by integrating branch and organ models. Second, the morphological models of different loquat trees were constructed by interactive editing. Third, the 3D individual-tree modeling software LSTree integrated with the OpenGL shadow technique, a radiosity model and a modified rectangular hyperbola model was used to calculate the silhouette to total area ratio, the distribution of photosynthetically active radiation within canopies and the net photosynthetic rate, respectively. Finally, the influence of loquat tree organ organization on the light interception of the trees was analyzed with different parameters. If the single branch angle between the level 2 scaffold branch and trunk is approximately 15° and the angles among the level 2 scaffold branches range from 60 to 90°, then a better light distribution can be obtained. The results showed that the branching angle has a significant impact on light interception, which is useful for grower manipulation of trees, e.g., shoot bending (scaffold branch angle). Based on this conclusion, a reasonable tree structure was selected for intercepting light. This quantitative simulation and analytical method provides a new digital and visual method that can aid in the design of tree architecture.

Escherichia coli Strain Designed for Characterizing in Vivo Functions of Nicotinamide Adenine Dinucleotide Analogues.

An Escherichia coli strain was constructed for the efficient import of nicotinamide adenine dinucleotide (NAD) analogues into cells by limiting extracellular degradation while expressing an efficient NAD importer. In vivo functions of three NAD analogues were characterized. Nicotinamide hypoxanthine dinucleotide was identified as an inhibitor of NAD synthesis. Nicotinamide cytosine dinucleotide had excellent biocompatibility and was used for characterizing a growth-dependent degradation of in vivo nicotinamide cofactors.

Fluorimetric Detection of Candida albicans Using Cornstalk N-Carbon Quantum Dots Modified with Amphotericin B.

Due to the advanced fluorescence property of N-carbon quantum dots (N-CQDs), a new method to detect pathogenic fungi by newly synthesized cornstalk N-CQDs modified with water-soluble amphotericin B (N-CQDs@AmpB) was developed. Specifically, N-CQDs with blue fluorescence were initially synthesized according to a previous report and modified with amphotericin B on their surfaces. Subsequently, the as-prepared N-CQDs@AmpB was used to detect Candida albicans, exhibiting a linear range of 2.60 × 105 to 1.99 × 108 cfu/mL and a detection limit of 1124 cfu/mL. Compared with other common methods, the method largely shortened the detection time and enabled the process to be performed with minimal interference from complex samples such as beef sausage. The high cost of water-soluble amphotericin B may hamper the large-scale application of the new detection method using N-CQDs@AmpB. Thus, alcohol-soluble amphotericin B was used in subsequent experiments, confirming its potential to broaden avenues for the detection of fungi.

Trichosporon fermentans biomass flocculation from soybean oil refinery wastewater using bioflocculant produced from Paecilomyces sp. M2-1.

The soybean oil refinery (SOR) wastewater contains a high concentration of chemical oxygen demand (COD) and lipid, so the direct emissions of SOR wastewater will result in environmental pollution and waste of resources. Oleaginous yeast Trichosporon fermentans can consume organic materials in SOR wastewater to synthesize microbial oil, which achieves the purpose of SOR wastewater resource utilization. The effective harvesting technology of oleaginous yeasts can improve the utilization efficiency. In this study, Paecilomyces sp. M2-1 with high flocculating activity was isolated. The flocculants produced by M2-1 (MBF2-1) include 75% (w/w) polysaccharides, rely on cations, and display the flocculation percentage of above 77% in the range of pH 2-11. Especially under alkaline conditions, the flocculation percentage can be kept above 97%. The results of scanning electron microscope observation and zeta potential measurements suggested that the bridging, net trapping, and sweeping were the main flocculation mechanism of MBF2-1. MBF2-1 could flocculate T. fermentans that was used to reduce the organic matter in SOR wastewater and to produce microbial oil. Under the optimum conditions, the flocculation percentage of MBF2-1 against T. fermentans from SOR wastewater can reach 95%. Fatty acid content percent in microbial oil from T. fermentans was not almost affected by flocculation of MBF2-1. Moreover, MBF2-1 can further remove 55% and 53% of COD and oil content in the fermented SOR wastewater, respectively. The properties and high flocculating percentage displayed by MBF2-1 indicated its potential application prospect in oleaginous yeast harvest and food industry wastewater treatment.

Refined soybean oil wastewater treatment and its utilization for lipid production by the oleaginous yeast Trichosporon fermentans.

BACKGROUND: The release of refined soybean oil wastewater (RSOW) with a high chemical oxygen demand (COD) and oil content burdens the environment. The conversion of RSOW into lipids by oleaginous yeasts may be a good way to turn this waste into usable products. RESULTS: The oleaginous yeast Trichosporon fermentans was used for treating the RSOW without sterilization, dilution, or nutrient supplementation. It was found that the COD and oil content of the RSOW were removed effectively; microbial oil was abundantly produced in 48 h; and the phospholipids in the RSOW tended to contribute to a higher biomass and microbial lipid content. With Plackett-Burman design and response surface design experiments, the optimal wastewater treatment conditions were determined: temperature 28.3 °C, amount of inoculum 5.9% (v/v), and initial pH 6.1. The optimized conditions were used in a 5-L bioreactor to treat the RSOW. The maximum COD degradation of 94.7% was obtained within 40 h, and the removal of the oil content was 89.9%. The biomass was 7.9 g/L, the lipid concentration was 3.4 g/L, and the lipid content was 43% (w/w). The microbial oil obtained, with a main component of unsaturated fatty acids, was similar to vegetable oils and was suggested as a potential raw material for biodiesel production. CONCLUSION: Trichosporon fermentans can be effectively used for RSOW treatment, and lipid production and can complete pretreatment and biochemical treatment simultaneously, allowing the utilization of RSOW, which both solves an environmental problem and positively impacts the use of resources. These results provide valuable information for developing and designing more efficient waste-into-lipid bioprocesses.

Rhodium(iii)-catalyzed three-component cascade synthesis of 6H-benzo[c]chromenes through C-H activation.

A three-component cascade reaction is described that provides concise access to 6H-benzo[c]chromenes via Rh(iii)-catalyzed annulation of aryl ketone O-acyloximes, quinones and acetone. Acetone acts as a co-solvent and as a reactant. This reaction shows high efficiency, atom- and step-economy, good substrate scope, excellent functional group compatibility and gives the products in good yields.

Manipulation of two regulatory genes for efficient production of chromomycins in Streptomyces reseiscleroticus.

BACKGROUND: Regulatory genes play critical roles in natural product biosynthetic pathways. Chromomycins are promising anticancer natural products from actinomycetes. This study is aimed to create an efficient strain for production of these molecules by manipulating the regulatory genes. RESULTS: A putative but silent chromomycin biosynthetic gene cluster was discovered in Streptomyces reseiscleroticus. Heterologous expression of the ketosynthase, chain length factor, and acyl carrier protein in Streptomyces lividans confirmed that they are responsible for the assembly of a decaketide. Two regulatory genes are present in this gene cluster, including SARP-type activator SrcmRI and PadR-like repressor SrcmRII. Either overexpression of SrcmRI or disruption of SrcmRII turned on the biosynthetic pathway of chromomycins. The production titers of chromomycin A3/A2 in R5 agar in these two strains reached 8.9 ± 1.2/13.2 ± 1.6 and 49.3 ± 4.3/53.3 ± 3.6 mg/L, respectively. An engineered strain was then constructed with both SrcmRII disruption and SrcmRI overexpression, which produced chromomycins A3 and A2 in R5 agar at 69.4 ± 7.6 and 81.7 ± 7.2 mg/L, respectively. Optimization of the culture conditions further increased the titers of chromomycins A3 and A2 respectively to 145.1 ± 15.3 and 158.3 ± 15.4 mg/L in liquid fermentation. CONCLUSIONS: This work revealed the synergistic effect of manipulation of pathway repressor and activator genes in the engineering of a natural product biosynthetic pathway. The resulting engineered strain showed the highest production titers of chromomycins by a strain of Streptomyces, providing an efficient way to produce these pharmaceutically valuable molecules.

Decoding and reprogramming fungal iterative nonribosomal peptide synthetases.

Nonribosomal peptide synthetases (NRPSs) assemble a large group of structurally and functionally diverse natural products. While the iterative catalytic mechanism of bacterial NRPSs is known, it remains unclear how fungal NRPSs create products of desired length. Here we show that fungal iterative NRPSs adopt an alternate incorporation strategy. Beauvericin and bassianolide synthetases have the same C1-A1-T1-C2-A2-MT-T2a-T2b-C3 domain organization. During catalysis, C3 and C2 take turns to incorporate the two biosynthetic precursors into the growing depsipeptide chain that swings between T1 and T2a/T2b with C3 cyclizing the chain when it reaches the full length. We reconstruct the total biosynthesis of beauvericin in vitro by reacting C2 and C3 with two SNAC-linked precursors and present a domain swapping approach to reprogramming these enzymes for peptides with altered lengths. These findings highlight the difference between bacterial and fungal NRPS mechanisms and provide a framework for the enzymatic synthesis of non-natural nonribosomal peptides.

Characterization of Three Tailoring Enzymes in Dutomycin Biosynthesis and Generation of a Potent Antibacterial Analogue.

The anthracycline natural product dutomycin and its precursor POK-MD1 were isolated from Streptomyces minoensis NRRL B-5482. The dutomycin biosynthetic gene cluster was identified by genome sequencing and disruption of the ketosynthase gene. Two polyketide synthase (PKS) systems are present in the gene cluster, including a type II PKS and a rare highly reducing iterative type I PKS. The type I PKS DutG repeatedly uses its active sites to create a nine-carbon triketide chain that is subsequently transferred to the α-l-axenose moiety of POK-MD1 at 4″-OH to yield dutomycin. Using a heterologous recombination approach, we disrupted a putative methyltransferase gene (dutMT1) and two glycosyltransferase genes (dutGT1 and dutGT2). Analysis of the metabolites of these mutants revealed the functions of these genes and yielded three dutomycin analogues SW140, SW91, and SW75. The major product SW91 in Streptomyces minoensis NRRL B-5482-ΔDutMT1 was identified as 12-desmethyl-dutomycin, suggesting that DutMT1 is the dedicated 12-methyltransferase. This was confirmed by the in vitro enzymatic assay. DutGT1 and DutGT2 were found to be responsible for the introduction of ß-d-amicetose and α-l-axenose, respectively. Dutomycin and SW91 showed strong antibacterial activity against Staphylococcus aureus and methicillin-resistant S. aureus, whereas POK-MD1 and SW75 had no obvious inhibition, which revealed the essential role of the C-4″ triketide chain in antibacterial activity. The minimal inhibitory concentration of SW91 against the two strains was 0.125 µg mL(-1), lower than that of dutomycin (0.25 µg mL(-1)), indicating that the antibacterial activity of dutomycin can be improved through biosynthetic structural modification.

Identification of a type III polyketide synthase involved in the biosynthesis of spirolaxine.

Spirolaxine is a natural product isolated from Sporotrichum laxum ATCC 15155, which has shown a variety of biological activities including promising anti-Helicobacter pylori property. To understand how this compound is biosynthesized, the genome of S. laxum was sequenced. Analysis of the genome sequence revealed two putative type III polyketide synthase (PKS) genes in this strain, Sl-pks1 and Sl-pks2, which are located adjacent to each other (~2.0 kb apart) in a tail-to-tail arrangement. Disruption of these two genes revealed that Sl-PKS2 is the dedicated PKS involved in the biosynthesis of spirolaxine. The intron-free Sl-pks2 gene was amplified from the cDNA of S. laxum and ligated into the expression vector pET28a for expression in Escherichia coli BL21-CodonPlus (DE3)-RIL. The major products of Sl-PKS2 in E. coli were characterized as alkylresorcinols that contain a C13-C17 saturated or unsaturated hydrocarbon side chain based on the spectral data. This enzyme was purified and reacted with malonyl-CoA and a series of fatty acyl-SNACs (C6-C10). Corresponding alkylresorcinols were formed from the decarboxylation of the synthesized tetraketide resorcylic acids, together with fatty acyl-primed triketide and tetraketide pyrones as byproducts. This work provides important information about the PKS involved in the biosynthesis of spirolaxine, which will facilitate further understanding and engineering of the biosynthetic pathway of this medicinally important molecule.

A specific cytochrome P450 hydroxylase in herboxidiene biosynthesis.

The anti-cholesterol natural product herboxidiene is synthesized by a noniterative modular polyketide synthase (HerB, HerC and HerD) and three tailoring enzymes (HerE, HerF and HerG) in Streptomyces chromofuscus A7847. In this work, the putative monooxygenase HerG was expressed in Escherichia coli and the purified enzyme was subjected to biochemical studies. It was identified as a cytochrome P450 enzyme responsible for the stereospecific hydroxylation at C-18. This enzyme is highly substrate-specific as it efficiently hydroxylates 18-deoxy-25-demethyl-herboxidiene, but showed no activity towards 18-deoxy-herboxidiene. The kcat/Km value for the HerG-catalyzed hydroxylation of 18-deoxy-25-demethyl-herboxidiene was determined to be 1669.70±47.36 M(-1) s(-1). In vitro co-reaction of HerG with the methyltransferase HerF and analysis of the product formation in S. chromofuscus A7847 revealed that the biosynthetic intermediate 18-deoxy-25-demethyl-herboxidiene is successively hydroxylated at C-18 by HerG and methylated at 17-OH to yield the final product herboxidiene. The minor metabolite 18-deoxy-hereboxidiene is a byproduct of the biosynthetic pathway.

Characterization of a methyltransferase involved in herboxidiene biosynthesis.

The herboxidiene biosynthetic gene cluster contains a regulatory gene and six biosynthetic genes that encode three polyketide synthases (HerB, HerC and HerD) and three tailoring enzymes (HerE, HerF and HerG). Through single crossover recombination, an integrative plasmid was inserted into the genome of Streptomyces chromofuscus ATCC 49982 between herE and herF, resulting in low-level expression of herF and the downstream herG. The mutant strain produced two new compounds, 18-deoxy-25-demethyl-herboxidiene and 25-demethyl-herboxidiene. HerF was expressed in Escherichia coli and biochemically characterized as the dedicated methyltransferase in herboxidiene biosynthesis. It prefers 25-demethyl-herboxidiene to 18-deoxy-25-demethyl-herboxidiene, suggesting that C-25 methylation is the last tailoring step.

Functional dissection and module swapping of fungal cyclooligomer depsipeptide synthetases.

BbBSLS and BbBEAS were dissected and reconstituted in Saccharomyces cerevisiae. The intermodular linker is essential for the reconstitution of the separate modules. Module 1 can be swapped between BbBEAS and BbBSLS, while modules 2 and 3 control the product profiles. BbBSLS is a flexible enzyme that also synthesizes beauvericins.