Optimized expression of the Starmerella bombicola lactone esterase in Pichia pastoris through temperature adaptation, codon-optimization and co-expression with HAC1.

The Starmerella bombicola lactone esterase (SBLE) is a novel enzyme that, in vivo, catalyzes the intramolecular esterification (lactonization) of acidic sophorolipids in an aqueous environment. In fact, this is an unusual reaction given the unfavorable conditions for dehydration. This characteristic strongly contributes to the potential of SBLE to become a 'green' tool in industrial applications. Indeed, lactonization occurs normally in organic solvents, an application for which microbial lipases are increasingly used as biocatalysts. Previously, we described the production of recombinant SBLE (rSBLE) in Pichia pastoris (syn. Komagataella phaffii). However, expression was not optimal to delve deeper into the enzyme's potential for industrial application. In the current study, we explored codon-optimization of the SBLE gene and we optimized the rSBLE expression protocol. Temperature reduction had the biggest impact followed by codon-optimization and co-expression of the HAC1 transcription factor. Combining these approaches, we achieved a 32-fold improvement of the yield during rSBLE production (from 0.75 mg/l to 24 mg/L culture) accompanied with a strong reduction of contaminants after affinity purification.

Rhamnolipids and lactonic sophorolipids: natural antimicrobial surfactants for oral hygiene.

AIMS: To assess the efficacy of rhamnolipid (mixture of monorhamnolipid and dirhamnolipid congeners), purified monorhamnolipid, dirhamnolipid and lactonic sophorolipid biosurfactants against pathogens important for oral hygiene. METHODS AND RESULTS: Acquired and produced biosurfactants were fully characterized to allow the antimicrobial activity to be assigned to the biosurfactant congeners. Antimicrobial activity was assessed using the resazurin-aided microdilution method. Mixed rhamnolipid JBR425 (MR) and lactonic sophorolipids (LSLs) demonstrated the lowest minimum inhibitory concentration (MIC) which ranged between 100 and 400 µg ml-1 against Streptococcus mutans, Streptococcus oralis, Actinomyces naeslundii, Neisseria mucosa and Streptococcus sanguinis. Combining these biosurfactants with standard antimicrobial agents namely chlorhexidine, sodium lauryl sulphate, tetracycline HCl and ciprofloxacin showed a dramatic drop in the MIC values. In addition, in vitro studies demonstrated the biosurfactants' ability to prevent and disrupt oral pathogens biofilms. The increased permeability of microorganisms treated with biosurfactant, as shown using bisbenzimide dye, in part explains the inhibition effect. CONCLUSION: The results demonstrate that rhamnolipids and LSLs have the ability to inhibit oral pathogens both in planktonic and oral biofilm states. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings indicate the potential value of biosurfactants for both oral hygiene and the pharmaceutical industries since there is a serious need to reduce the reliance on synthetic antimicrobials and antibiotics.

Exoproteome analysis of Starmerella bombicola results in the discovery of an esterase required for lactonization of sophorolipids.

The yeast Starmerella bombicola secretes sophorolipids, a family of biosurfactants that find applications in green household products and cosmetics. Over the past years, a gene cluster was discovered that is responsible for the entire synthesis of the open (acidic) form of these molecules from glucose, fatty acids and acetyl-CoA building blocks. However, a significant fraction of the natural product is obtained as a ring closed form (lactonic). Both genetic and proteomic approaches hitherto failed to discover an enzyme responsible for the esterification reaction required for the ring closure step. We hypothesized that this enzyme is extracellularly secreted. Therefore, we characterized the composition of the S. bombicola exoproteome at different time points of the growth and compared it with known yeast exoproteomes. We identified 44 proteins, many of them commonly found in other fungi. Curiously, we discovered an enzyme with homology to Pseudozyma antarctica lipase A. A deletion mutation of its gene resulted in complete abolishment of the sophorolipid lactonization providing evidence that this might be the missing enzyme in the sophorolipid biosynthetic pathway. BIOLOGICAL SIGNIFICANCE: Growing concern about the impact of chemical processes on the environment increases consumers' demand for bio-based products. Lately, the household care and personal care sectors show increasing interest in naturally occurring biosurfactants, which constitute environment-friendly alternatives for chemical surfactants, typically derived from mineral oils. A particular group of biosurfactants, sophorolipids, already found their way to the market, being used in a range of household detergent products and in cosmetics. This work describes how proteomic approaches have led to the completion of our knowledge on the biosynthetic pathway of sophorolipids as performed by Starmerella bombicola, a fungus used in the industrial production of these biosurfactants. Moreover, we proved that by creating a deletion mutant in the lactone esterase discovered in this study, we can shape the biosynthesis towards custom-made sophorolipids with desired functions. Herewith, we demonstrate the potential of proteomics in industrial biotechnology.

The 'LipoYeasts' project: using the oleaginous yeast Yarrowia lipolytica in combination with specific bacterial genes for the bioconversion of lipids, fats and oils into high-value products.

The oleochemical industry is currently still dominated by conventional chemistry, with biotechnology only starting to play a more prominent role, primarily with respect to the biosurfactants or lipases, e.g. as detergents, or for biofuel production. A major bottleneck for all further biotechnological applications is the problem of the initial mobilization of cheap and vastly available lipid and oil substrates, which are then to be transformed into high-value biotechnological, nutritional or pharmacological products. Under the EU-sponsored LipoYeasts project we are developing the oleaginous yeast Yarrowia lipolytica into a versatile and high-throughput microbial factory that, by use of specific enzymatic pathways from hydrocarbonoclastic bacteria, efficiently mobilizes lipids by directing its versatile lipid metabolism towards the production of industrially valuable lipid-derived compounds like wax esters (WE), isoprenoid-derived compounds (carotenoids, polyenic carotenoid ester), polyhydroxyalkanoates (PHAs) and free hydroxylated fatty acids (HFAs). Different lipid stocks (petroleum, alkane, vegetable oil, fatty acid) and combinations thereof are being assessed as substrates in combination with different mutant and recombinant strains of Y. lipolytica, in order to modulate the composition and yields of the produced added-value products.

Sophorolipid production by Candida bombicola on oils with a special fatty acid composition and their consequences on cell viability.

Sophorolipids production by the yeast Candia bombicola is most favourable when glucose is used as a carbon source in combination with a hydrophobic carbon source such as a common vegetable oil. Most vegetable oils are comprised of C16-C18 fatty acids, an ideal range for sophorolipid production. The use of oils with either shorter or longer fatty acids, such has coconut oil or meadowfoam oil, respectively, was evaluated. Such oils did not contribute to enhanced sophorolipid production when compared to cultures run on glucose as the sole carbon source. Moreover, a toxic effect of medium-chain fatty acids towards stationary C. bombicola cells was demonstrated.

Development and application of a screening assay for glycoside phosphorylases.

Glycoside phosphorylases (GPs) are interesting enzymes for the glycosylation of chemical molecules. They require only a glycosyl phosphate as sugar donor and an acceptor molecule with a free hydroxyl group. Their narrow substrate specificity, however, limits the application of GPs for general glycoside synthesis. Although an enzyme's substrate specificity can be altered and broadened by protein engineering and directed evolution, this requires a suitable screening assay. Such a screening assay has not yet been described for GPs. Here we report a screening procedure for GPs based on the measurement of released inorganic phosphate in the direction of glycoside synthesis. It appeared necessary to inhibit endogenous phosphatase activity in crude Escherichia coli cell extracts with molybdate, and inorganic phosphate was measured with a modified phosphomolybdate method. The screening system is general and can be used to screen GP enzyme libraries for novel donor and acceptor specificities. It was successfully applied to screen a residue E649 saturation mutagenesis library of Cellulomonas uda cellobiose phosphorylase (CP) for novel acceptor specificity. An E649C enzyme variant was found with novel acceptor specificity toward alkyl beta-glucosides and phenyl beta-glucoside. This is the first report of a CP enzyme variant with modified acceptor specificity.

Cerulenin inhibits de novo sophorolipid synthesis of Candida bombicola.

Synthesis of medium-chain sophorolipids by Candida bombicola is a challenging objective. One of the difficulties is that the obtained sophorolipids always represent a mixture of medium-chain and native de novo formed or long-chain sophorolipids. The fatty acid moiety of de novo sophorolipids is derived from the de novo synthesis of fatty acids. Fatty acid synthesis can be blocked by the antifungal agent cerulenin, an inhibitor if the fatty acid synthase (FAS) complex acting on the beta-ketoacyl thioester synthetase reaction. The toxic effect of cerulenin on C. bombicola was evaluated and 20 mg/ml was added in the stationary growth phase. No de novo formed sophorolipids were observed when the cells were cultured on merely glucose. Also when the hydrophilic substrate, 1,12-dodedanediol, was added, no de novo formed sophorolipids were detected, leading to a reduced complexity of the sophorolipid mixture.

Rapid isolation of fungal genomic DNA suitable for long distance PCR.

A quick and reliable method for screening fungal transformants for specific genetic modifications is essential for many molecular applications. We have compared the applicability of a few rapid DNA extraction methods for Myrothecium and Aspergillus and tested the resulting DNA as to its suitability for PCR. For Myrothecium gramineum, the highest DNA concentration was obtained with the procedure described by N. Vanittanakom et al. (J Clin Microbiol 2002, 40: 1739-1742). For A. nidulans, concentrations higher than 100 ng/mul were reached with the glass bead, the LiCl, the boiling, the liquid N(2) and the protoplast-based method. Samples of M. gramineum resulting from the boiling and the liquid N(2) procedure were suitable for the amplification of fragments up to 2.3 kb. The direct use of mycelium from M. gramineum in the PCR tube can be employed for the reproducible amplification of fragments up to 1 kb. Amplification of fragments up to 4.3 kb requires the use of the Elongase Mix on samples extracted with the liquid N(2) procedure.

Transport kinetics of ectoine, an osmolyte produced by Brevibacterium epidermis.

Brevibacterium epidermis DSM 20659 is a halotolerant Gram-positive bacterium which can synthesize the osmolyte, ectoine, but prefers to take it up from its environment. The present study revealed that B. epidermis is equipped with at least one transport system for ectoine, with a maximal transport velocity of 15.7 +/- 4.3 nmol/g CDW.min. The transport requires energy (ATP) and is completely inhibited by the proton uncoupler, CCCP. The ectoine uptake system is constitutively expressed at a basal level of activity and its activity is immediately 10-fold increased by hyper-osmotic stress. Initial uptake rates are not influenced by the intensity of the hyper-osmotic shock but the duration of the increased activity of the uptake system could be directly related to the osmotic strength of the assay solution. Competition assays indicate that betaine, but not proline, is also transported by the ectoine uptake system.

Dynamics and optimal conditions of intracellular ectoine accumulation in Brevibacterium sp.

The optimal conditions for the intracellular synthesis of ectoine were determined in a halotolerant Brevibacterium sp. The size of the intracellular ectoine pool in the bacterial cells is shown to depend on the external salt concentrations, type of carbon source and aeration level. In erlenmeyer flasks a maximum concentration of intracellular ectoine of about 0.9 g/l was obtained. Under controlled aeration in a 1.5 l fermentor this level could be increased to 1.2 g/l. Consecutive cell transfers to media with increasingly higher salt concentrations enabled us to reach even higher levels, up to 1.6 g/l on erlenmeyer scale. The ectoine synthesis takes place immediately after the osmotic upshock. Within one generation time, the new corresponding specific intracellular ectoine concentration is reached.

Application of NAD-dependent polyol dehydrogenases for enzymatic mannitol/sorbitol production with coenzyme regeneration.

D-Mannitol and D-sorbitol were produced enzymatically from D-fructose using NAD-dependent polyol dehydrogenases. For the production of D-mannitol the Leuconostoc mesenteroides mannitol dehydrogenase could be used. Gluconobacter oxydans cell extract contained however both mannitol and sorbitol dehydrogenase. When this cell extract was used, the reduction of D-fructose resulted in a mixture of D-sorbitol and D-mannitol. To determine the optimal bioconversion conditions the polyol dehydrogenases were characterized towards pH- and temperature-optimum and -stability. As a compromise between enzyme activity and stability, the bioconversion reactions were performed at pH 6.5 and 25 degrees C. Since the polyol dehydrogenases are NADH-dependent, an efficient coenzyme regeneration was needed. Regeneration of NADH was accomplished by formate dehydrogenase-mediated oxidation of formate into CO2.

Enzymatic conversion of the clavan exopolysaccharide by Streptomyces sp. YSDL-20.

A screening programme was set up to isolate microorganisms able to hydrolyse the complex biopolymer clavan produced by Clavibacter michiganensis subsp. michiganensis LMG 5604. This valuable exopolysaccharide is very rich in L-fucose (37.5% w/w), a rare sugar, used in the medical field (Vanhooren, 1999). A microorganism capable of depolymerizing the polymer may decrease the high viscosity during clavan batch fermentations and remove the limitations of the oxygen transfer and consequently increase the clavan yield. It could also release free L-fucose or L-fucose rich oligosaccharides. An actinomycete, designated YSDL-20, isolated from a soil sample, was able to depolymerize this biopolymer. Based on its morphology and molecular characteristics, this strain could only be identified as Streptomyces sp.. On clavan, this strain displays good growth (17.5 g DCW/l after 96 h of cultivation) characterized by filamentous growth during the earlier days of cultivation followed by sporulation after 4 days. The flow behaviour of the Clavibacter broth was characterized, the fermentation culture broth behaves as a pseudoplastic fluid. The viscosity of the culture broth as well as of the purified clavan EPS, decreases when lyophilised supernatant of Streptomyces sp. YSDL-20 was added, indicating clavanase action. The viscosity decreases by 26% when the Clavibacter culture broth was incubated during 18 h with the crude Streptomyces enzyme source, whereas a 82% viscosity drop was observed, when the purified clavan EPS (10 g/l) was incubated with the lyophilised Streptomyces supernatant for 5 h.

Optimized synthesis of L-sorbose by C(5)-dehydrogenation of D-sorbitol with Gluconobacter oxydans.

The optimization of L-sorbose synthesis by regiospecific dehydrogenation of D-sorbitol using Gluconobacter oxydans is reported. The current L-sorbose production processes that are based on G. oxydans and other bacterial strains are suboptimal as to yield and rate of L-sorbose synthesis. One reason for these problems is the toxicity that is induced by the substrate D-sorbitol when used in concentrations of >10% (w/v). This phenomenon significantly limits the potentials of L-sorbose production from an industrial point of view. The goal of this study was to develop a fast production process that yields L-sorbose in stoichiometric amounts starting from D-sorbitol concentrations that exceed 10% (w/v). A gradual improvement of the inoculum build-up procedure, culture medium composition, and process parameters ultimately led to a theoretically maximal L-sorbose productivity (200 g L(-1) of L-sorbose from 200 g L(-1) of D-sorbitol in 28 h of fermentation) using a Gluconobacter oxydans mutant strain that was selected under conditions of substrate inhibition. Because the D-sorbitol/L‐sorbose bioconversion is used to mass-produce vitamin C, the procedure reported here will contribute to a more efficient and more economic synthesis of vitamin C.