Dicarboxylic acid production by Yarrowia lipolytica strains

[摘要] English:Dicarboxylic acids are value added products, which can be prepared by diterminal oxidation of n-alkanes by bacteria or yeasts with a block in β-oxidation. Yarrowia lipolytica is one of the alkane-utilizing yeasts for which genetic tools have been developed. Y lipolytica has a complex set of five acyl Coenzyme A (CoA) oxidase isozymes (encoded by POXI through POX5 genes) with different substrate specificities. Abundant information is available on dioic acid accumulation from n-alkanes by C. maltosa and C. tropicalis strains deficient in β-oxidation. In many cases β-oxidation had been blocked at the level of the acyl CoA oxidases. In comparison very little information was available .on dioic acid accumulation by Yarrowia lipolytica. We had through our collaboration with Dr J-M Nicaud of the INRA-CNRS in France access to the above mentioned series of Y lipolytica strains with the acyl CoA oxidase encoding genes disrupted. It thus became the purpose of my studies to investigate dioic acid .accumulation by Y lipolytica strains with impaired β-oxidation. In order to study the growth of different Yarrowia lipolytica strains on or in the presence of a range of liquid and solid n-alkanes or alkane derivatives we required a rapid method using small samples to estimate biomass production. Insoluble hydrophobic substrates iriterfere with turbidimetric measurements. This problem is more severe if the hydrophobic substrate is a solid. It is not possible to efficiently separate Y lipolytica cells from a hydrophobic substrate by centrifugation in the presence of an organic solvent, because Y lipolytica cells are hydrophic and a large percentage of cells cling to the water/solvent interface. We established that pre-treatment of samples for turbidimetric analysis with 5 M NaOH, thus increasing the pH of samples to 14, abolished to a large extent the hydrophobicity of Y lipolytica cells. We also established that washing of the cells with cyclohexane and NaOH when cells were harvested by filtration, did not result in any significant loss in biomass. Based on these observations we developed a simple, cost effective sample preparation procedure for turbidimetric analysis, which gave accurate, repeatable turbidity measurements with no Interference from the hydrophobic substrates. This method involved the pre-treatment of small samples (500 µl)with 5 M NaOH plus cyclohexane, prior to harvesting the biomass by centrifugation. Sample preparation was carried out in microcentrifuge tubes and turbidimetric measurements were done with a microtitre plate reader. The newly developed procedure was used to investigate the toxicity of dodecane and hexadecane as well as their terminal and diterminal oxidation products to Y lipolytica wild type strain H222. The alkanes and dicarboxylic acids were never toxic to Y lipolytica. Dodecanol severely inhibited growth of Y lipolytica strain H222 in YP media with glucose whereas in a semi-synthetic YNB medium without additional carbon source dodecanal and dodecanoic acid were the most toxic. Hexadecanol and hexadecanoic acid did not inhibit growth of Y lipolytica in yP medium with glucose, but were toxic to Y. lipolytica in a semi-synthetic YNB medium without glucose. The results obtained in the first round of experiments indicated to us the possibility of preparing dodecanol-tolerant strains. Two dodecanol-tolerant strains were subsequently prepared. The first H222A was prepared by step-wise increasing dodecanol concentrations in YP broth supplemented with glucose to 7.5% (v/v). The second strain MTL Y35A was prepared on YP agar plates without glucose by step-wise increasing dodecanol concentrations to 8.5% (v/v). Dodecanedioic acid was not accumulated by the dodecanol-tolerant strains H222A or by the triple POX-deleted strain MTL Y35A, when grown on glucose in the presence of dodecanol. Two dodecanol concentration were tested 3 % and 10 % (v/v). Dioic acid accumulation from C 12 and C 16 alkanes and alkane degradation intermediates was investigated using Y lipolytica wild type strain W29 as well as the POX deleted strains MTLY21 (∆POX2, POX3), MTLY35 (∆POX2, POX3, POX5) and MTLY37 (∆POX2, POX3, POX4, POX5). The quadruple-deleted strain MTLY37 was the only strain that was able to accumulate dioic acids from alkanes, alkanols and monocarboxylic acids. Dodecane was the best alkane substrate for dioic acid accumulation yielding 7 mg/ml dodecanedioic acid after 144h (23% w/v conversion). Lauric acid did not yield any dioic acid (probably due to toxicity), ·but 5 mg/ml hexadecanedioic acid was accumulated from hexadecanoic acid after 48h. All the strains accumulated dodecanedioic acid from the diterminal functionalised 1,12 dodecane diol and eo-hydroxy dodecanoic acid. The quadruple-deleted strain MTL Y37 accumulated a maximum concentration of 20 mg/ml dodecanedioic acid after 48h from 1,12 dodecanediol, while the triple-deleted strain MTL Y35 accumulated 18 mg/ml dodecanedioic acid after 48h from 12-hydroxydodecanoic acid.

[发布机构] University of the Free State