(R)-2-(4-hydroxyphenoxy)propionic acid (HPOPA) is a key intermediate for the preparation of aryloxyphenoxypropionic acid herbicides (R-isomer). In order to improve the HPOPA production from the substrate (R)-2-phenoxypropionic acid (POPA) with Beauveria bassiana CCN-A7, static cultivation and H 2 O 2 addition were attempted and found to be conducive to the task at hand. This is the first report on HPOPA production under static cultivation and reactive oxygen species (ROS) induction. On this premise, the cultivation conditions and fermentation medium compositions were optimized. As a result, the optimal carbon source, organic nitrogen source, and inorganic nitrogen source were determined to be glucose, peptone, and ammonium sulfate, respectively. The optimal inoculum size and fermentation temperature were 13.3% and 28°C, respectively. The significant factors including glucose, peptone, and H 2 O 2 , identified based on Plackett-Burman design, were further optimized through Central Composite Design (CCD). The optimal concentrations were as follows: glucose 38.81 g/l, peptone 7.28 g/l, and H 2 O 2 1.08 g/l/100 ml. Under the optimized conditions, HPOPA titer was improved from 9.60 g/l to 19.53 g/l, representing an increase of 2.03- fold. The results obtained in this work will provide novel strategies for improving the biosynthesis of hydroxy aromatics.
Check now
Introduction
Aryloxyphenoxypropionic acid-type herbicide is one of the best kinds of herbicides. It is non-toxic to crops, has a long expiration date, and degrades easily in the soil [1, 2]. Actually, two isomers (R- and S-isomer) exist in aryloxyphenoxypropionic acid-type herbicide [3], and the R-type herbicide has proved to be much more effective than the S-type one [4]. Therefore, the production of enantiomerically pure R-type herbicides is more promising. At present, the enantiomerically pure aryloxyphenoxypropionic acid-type herbicides (R-isomer) such as fenoxaprop-P-ethyl, clodinafop-propargyl, propaquizafop, and metamifop, etc. [5, 6] are mainly synthesized from (R)-2-(4-hydroxyphenoxy)propionic acid (HPOPA). As a key intermediate, HPOPA has been in increasing demand in the herbicide industry and HPOPA production has drawn more attention in recent years [7].
As one of the most common biocatalysts, entomopathogenic fungus Beauveria bassiana has multiple applications, especially in the hydroxylation of saturated and aromatic carbon atoms, Baeyer-Villiger/sulfide oxidation, keto-alcohol/alkene redox reaction, heteroatom dealkylation and epoxide, ester hydrolysis and glucosidation [8-12]. In our earlier study, a wild-type B. bassiana ZJB [7, 13] capable of hydroxylating (R)-2- phenoxypropionic acid (POPA) at C-4 site into HPOPA, was obtained from environmental samples and the derivative strain B. bassiana CCN-A7 was obtained after multi-round mutagenesis. The HPOPA titer of B. bassiana CCN-A7 was 9.6 g/l after agitated fermentation for 7 d in 30-ml medium containing 20 g/l POPA. To improve the HPOPA production of B. bassiana CCN-A7, the optimization of the nutrition components and culture conditions was performed in this work. In recent years, static cultivation methods were successfully applied in the production of various biochemicals, as for example, ganoderic acid biosynthesis by Ganoderma lucidum, cordycepin production by Cordyceps militaris, and bacterial nanocellulose production [14-16]. Herein, we attempted to investigate the effect of agitation speeds on HPOPA production by B. bassiana CCN-A7, and found that static cultivation was a more suitable fermentation mode for HPOPA production compared with the traditional agitated cultivation.
In many organisms, reactive oxygen species (ROS) are highly reactive molecules mainly including superoxide radicals (O2&#;), hydrogen peroxide (H2O2), and hydroxyl radicals (HO&#;), all of which function in regulating various physiological processes [12,17-19]. There has been increasing evidence that oxidative stress could cause the post- translational modification of enzymes and thus change enzymes activity rapidly and reversibly. As reported, in glycolysis pathway, the key enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) could be inactivated after exposure to various oxidant [20]. Therefore, it is expected that the targeted metabolites biosynthesis capacity of microbial species could be significantly enhanced when a reasonable ROS treatment is designed [21]. In this study, the influences of induced ROS on HPOPA production of B. bassiana CCN-A7 were investigated using an ROS generator (external H2O2). The external H2O2 was added into the fermentation medium of B. bassiana CCN- A7 and its concentration in the medium was optimized to improve HPOPA production. It is a novel strategy to enhance HPOPA biosynthesis via ROS induction.
In order to rapidly develop a practically feasible bioprocess, the comprehensive and systematical optimization of nutritional components and culture conditions is necessary [22]. Generally, medium optimization with the one-factor-at-a-time method is very laborious and time-consuming work, and the determination of suitable conditions is often not well guaranteed [23, 24]. The Plackett-Burman (PB) design is usually used as a preliminary optimization technique and provides unbiased estimation of all the variables by running only a few experiments [25]. Response Surface Method (RSM) is a collective statistical technique based on experiment designing, model building, evaluating the effects of factors, and finding the optimum conditions. RSM has become more popular than the conventional methods, mainly because the interaction among several variables can also be evaluated by mathematical modeling, when little information about the process is available [26-28]. In RSM, the most frequently used design of experiments (DOE) is Central Composite Design (CCD), in which slope-rotatability can be easily achieved. When it is difficult to extend the design region beyond the defined variable bounds, a face- centered CCD can be chosen [29].
huimeng Product Page
The purpose of this work was to improve HPOPA production with B. bassiana CCN-A7 through optimization of the culture conditions under static cultivation and ROS induction. The effects of significant variables (medium components including H2O2 supplement) on HPOPA production were investigated through PB design and CCD. As a result, HPOPA production was improved significantly.
Comments
All Comments (0)