Enhanced Production of Lipstatin from Streptomyces Toxytricini By

Full Paper Enhanced production of lipstatin from Streptomyces toxytricini by optimizing fermentation conditions and medium (Received February 28, 2014; Accepted April 3, 2014) Tingheng Zhu,1 Lingfei Wang,1 Weixia Wang,2 Zhongce Hu,1 Meilan Yu,3 Kun Wang,1,＊ and Zhifeng Cui1,＊ 1 College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, 310032, PR China 2 China National Rice Research Institute, Hangzhou 310006, PR China 3 Engineering Research Center for Eco-Dyeing & Finishing of Textiles, Ministry of Education, Zhejiang Sci-Tech University, Hangzhou, 310018, PR China

prophylaxis and treatment of diseases associated with This paper is concerned with optimization of fer- obesity. Lipstatin contains a β-lactone structure that probably mentation conditions for lipstatin production with accounts for the irreversible lipase inhibition by covalent Streptomyces toxytricini zjut011 by the single factor modification of the serine residue of its catalytic triad (Luthra and orthogonal tests. Five single factors of impor- et al., 2013b). tant effects on lipstatin production were explored. The wide clinical application of orlistat has promoted the L-Leucine was identified to be the most suitable pre- commercial production of lipstatin (Zohrabian, 2100). cursor for lipstatin biosynthesis and for the first Hence, investigations into the improvement of the produc- time the divalent cations Mg2+, Co2+ and Zn2+ were tions of lipstatin are of commercial importance. In an attempt found to have significant effect on enhancing lip- to improve lipstatin production, improvement of lipstatin- statin fermentation titer. The effects of the additives producing strains and fermentation processes has been on the lipstatin production were in the order of L- carried out during the past decade. leucine ＞ Mg2+ ＞ Co2+ ＞ Zn2+ ＞ octanoic acid. S. toxytricini strains with an increased lipstatin fermenta- The optimized conditions for lipstatin production tion unit have been achieved through modification and were determined as 45.72 mmol/L of L-leucine (add- improvement by utilizing physical and chemical mutagene- ed on the 4 th day), 31.1985 mmol/L of octanoic acid sis. A high lipstatin-producing strain SIPI-HJ-80 was (added on the 6th day), 12 mmol/L of Mg2+, 1 mmol/L obtained by ultraviolet ray and microwave treatment, which of Co2+ and 0.25 mmol/L of Zn2+. Under these showed an increased productivity by 2.5 times over the conditions, a maximum lipstatin of 4.208 g/ml was original strain SIPI-UM-5 (Huang et al., 2006). More recent- achieved in verification experiments in 500 ml shake ly, a N-methyl-N′-nitro-N-nitrosoguanidine (NTG) mutated flasks. strain of S. toxytricini (derived from strain ATCC 19813) was reported to have a lipstatin production of 2.88 mg/g at Key words: divalent cations; L-leucine; lipstatin; 264 h of fermentation time in a shake flask (Luthra et al., Streptomyces toxytricini 2013a). With the strain S. toxytricini ATCC 19813, 8-fold enhance- ment (from 0.097 g/L to 0.885 g/L) in lipstatin production in Introduction a shake flask was obtained through medium optimization. It is also indicated that soya oil, soya lecithin and soya bean Lipstatin is an irreversible inhibitor of pancreatic lipases flour had significant effects on lipstatin production (Luthra produced by Streptomyces toxytricini (Hochuli et al., 1987; et al., 2012). An increased lipstatin titer of 1.980 g/L was Weibel et al., 1987). Lipstatin is of considerable importance observed with selected S. toxytricini strain NRRL 15443 as the key intermediate for the preparation of tetrahydrolip- (Kumar et al., 2012). Further, a maximum lipstatin produc- statin (THL, Orlistat), which is a drug designed for the tion of 3.290 g/L was achieved via a full factorial medium

＊ Corresponding authors: Dr. Zhifeng Cui and Dr. Kun Wang, College of Biological and Environmental Engineering, Zhejiang University of Technol- ogy, Hangzhou, 310032, PR China. Tel & Fax: +81‒571‒88352103 E-mail: [email protected]; [email protected] None of the authors of this manuscript has any financial or personal relationship with other people or organizations that could inappropriately influence their work. Enhanced production of lipstatin from Streptomyces toxytricini by optimizing fermentation conditions and medium 107 optimization by using this strain in a shake flask (Luthra et Plating/agar slant culture medium (g/L): soluble starch al., 2013b). 10 g, wheat flour 5 g, yeast extract 1 g, casein 1 g, KH2PO4 Early studies have elucidated that the carbon skeleton of 0.5 g, MgSO4 0.5 g, and agar 20 g. Seed culture medium lipstatin molecule is biosynthesized via Claisen condensa- (g/L): soya bean flour 20 g, glycerol 20 g, and yeast extract tion of two fatty acid precursors, the 8-carbon atoms (octano- 5 g. Fermentation medium (g/L): soya bean flour 32.5 g, ic acid), and 14-carbon atoms (tetra deca-5, 8- dienoic acid) corn gluten meal 8 g, sunflower oil 100 g, lecithins 25 g, (Eisenreich et al., 1997; Goese et al., 2000, 2001). As per glycerol 22.5 g, Tween 80 0.05 g, CaCO3 0.8 g, vitamin E this biosynthetic pathway and the basic building unit of 0.025 g, and vitamin C 0.125 g. Media pH was adjusted to lipstatin, oils rich in fatty acids are good resources for around 7.2 before autoclaving at 121°C for 20 min. precursors. Linoleic acid contains the same number of Slant culture was incubated at 28°C for 166‒192 h. From double bonds as lipstatin chain and it is the basic building an eggplant bottle slant, 5 fresh single colonies were unit. Further experiments with feeding putative intermedi- transferred into a 250 ml flask with 10 ml sterilized water ates in the process of fermentation indicated that linoleic and a few glass beads. The flask was then shaken at 220 rpm acid, caprylic acid and N-formyl-L-leucine or preferably and 28°C for 15 min to form a bacterial suspension. The cell L-leucine are effective precursors of lipstatin biosynthesis suspension was calculated with a blood-counting chamber. (Schuhr et al., 2002; Eisenreich et al., 2003; Demirev et al., Five percent (v/v) suspension was inoculated into 1,000 ml 2010). flask containing 100 ml of seed medium and grown at 28°C Acyl-CoA carboxylases (ACCase) are involved in with 220 rpm in a shaker incubator for 30 h. Then, 10% (v/v) important primary or secondary metabolic pathways by seed cultures were inoculated into the 500 ml flask contain- providing key extender units for the biosynthesis of fatty ing 50 ml of fermentation medium and incubated in an acids and polyketide natural products through a carboxyl- orbital shaking incubator at 28°C with 220 rpm for 7 days. ation reaction. In the genus Streptomyces two types of Experimental design for single-factor effect on lipstatin acyl-CoA carboxylase were found: acetyl-CoA carboxylase production. The effects of seed age, fermentation time, (ACCase) and propionyl-CoA carboxylase (PCCase) flask volume charge, divalent cations and precursors on (Rodriguez and Gramajo, 1999). In S. toxytricini, the acetyl- lipstatin yield were studied individually. For each experi- CoA carboxylase gene cluster is the pccB gene locus contain- mental condition, five replicates were used, and the standard ing accA3, pccB, and pccE, which has been demonstrated to deviation was calculated. Thereafter, the culture broth was be involved in the secondary metabolism. Disruption of the used for lipstatin HPLC quantification. pccB and pccE genes caused a lipstatin production reduction Effects of seed age and fermentation time on lipstatin as much as 80%, indicating their critical role in lipstatin production. Seeds cultured for 22, 26, 30 and 34 h were biosynthesis (Demirev et al., 2009, 2010). The pccB gene inoculated into fermentation medium separately. The residu- encodes a β subunit of ACCase functioning as carboxyl- al seeds were checked and observed with microscopy. The transferase in the activation of ACCase along with biotinyl- pH of the culture broth was also measured. The biomass was ation mediated by Bp1 protein, which needs divalent cations measured in terms of percentage mycelial volume. as a cofactor (Demirev et al., 2011). After inoculation of the fermentation flasks, different In this study, the effect of divalent cations and precursors fermentation time treatments were designed. Four groups of on lipstatin yield has been examined. The fermentation fermentation flasks under identical conditions were taken process has also been improved by the single factor and away for lipstatin assay at the time points of 5, 6, 7, and orthogonal tests. By using the optimized fermentation media 8 days. and conditions, the S. toxytricini strain zjut011 yielded a Effects of fermentation flask volume charge and water significant increase in lipstatin production. supplement on lipstatin production. In 500 ml fermentation flasks, two charge volumes of 30 ml and 50 ml medium Materials and Methods were compared for lipstatin production. To the 500 ml fermentation flasks charged with 30 ml and 50 ml medium, Streptomyces strain, medium and culture characteriza- water was supplemented according to the experimental tion. The actinomycetes Stretomyces toxytricini used in design described in Table 1. this study is zjut011, which is a UV-mutated strain. The Effects of precursors on lipstatin production. L -Leucine strain was stored in 20% glycerol at -80°C. The culture was and octanoic acid were tested as precursors of lipstatin. maintained at 4°C on agar slants. L-Leucine was dissolved in formic acid for a stock solution

Table 1. Water supplementation in the process of lipstatin production in a fermentation shaking-ﬂask. Treatments Volume charge (ml) Method of water supplementation

I 30 Control II 30 Add the reduced water every day by weighing the flask III 30 Add 5 ml water every day from the 5th day of fermentation IV 50 Control V 50 Add the reduced water every day VI 50 Add 5 ml water every day from the 5th day of fermentation VII 50 Add the reduced water every day from the 4th day VIII 50 Add two volumes of the reduced water every day 108 ZHU et al. of 0.6 g/ml. For octanoic acid, origin fluid was used. The and octanoic acid. Statistical analysis of variance (ANOVA) precursors sterilized through a filter were added into fermen- was performed to see whether these parameters were statisti- tation medium in the ways described in Table 2. cally significant. The selected factors, their assigned levels Effects of divalent cations on lipstatin production. and the experimental design along with lipstatin production MgSO4, MnSO4･H2O, CuSO4･5H2O, ZnSO4･7H2O, data are listed in Tables 3 and 4. L-Leucine and octanoic acid FeSO4･7H2O and CoSO4･7H2O were used as divalent were added on the 4th and 6th day, respectively. cation sources and the concentration of stock solution was Lipstatin assay. Lipstatin activity in the culture broth 40 mmol/L. For each cation, five consecutive final concen- was determined by HPLC. A 0.5 ml aliquot of the fermenta- trations of 0.5, 2, 4, 6 and 8 mmol/L were tested by addition tion broth was put in a 10 ml volumetric flask with 4.5 ml into fermentation medium. acetone and sonicated it for 10 min and then soaked Orthogonal optimization of lipstatin fermentation process overnight. The extracted solution was filtered through a 5 and verification of the optimized conditions. An L16 (4 ) 0.22 µm Nylon filter and the resulting solution was injected orthogonal array design with five factors at four levels into the HPLC (Shimadzu) apparatus with a C-18 column consisting of 16 different experimental trials was used for for the estimation of lipstatin. The mobile phase was a optimization of lipstatin fermentation by S. toxytricini mixture of water and acetonitrile at the ratio of 15：85. zjut011. The five factors were Mg2+, Co2+, Zn2+, L-leucine Concentrations of lipstatin were calculated by comparison

Table 2. Addition of leucine and octanoic acid to the lipstatin fermentation process. Treatments Supply time Precursor Final concentration (mmol/L)

I ― ― Control II The 4th day Leucine 15.24 III The 4th day Leucine 30.48 IV The 6th day Leucine 15.24 V The 6th day Leucine 30.48 VI The 4th day Octanoic acid 13.866 VII The 6th day Octanoic acid 13.866

Table 3. Factors and levels of orthogonal experiment for lipstatin fermentation medium optimization.

Factors (mmol/L) Level Mg2+ Co2+ Zn2+ Leucine Octanoic acid 1 6 0.125 0.125 15.24 6.933 2 8 0.25 0.25 30.48 13.866 3 10 0.5 0.5 45.72 20.799 4 12 1 1 60.96 31.1985

Table 4. Results and analysis of orthogonal experiment for lipstatin fermentation medium optimization. A B C D E Test number Lipstatin titer (µg/ml) Mg Co Zn Leucine Octanoic acid 1 1 1 1 1 1 3,283.77 2 1 2 2 2 2 2,254.63 3 1 3 3 3 3 2,916.054 4 1 4 4 4 4 2,991.87 5 2 1 2 3 4 4,164.23 6 2 2 1 4 3 2,152.136 7 2 3 4 1 2 3,023.902 8 2 4 3 2 1 2,854.782 9 3 1 3 4 2 2,779.644 10 3 2 4 3 1 3,641.594 11 3 3 1 2 4 2,582.622 12 3 4 2 1 3 4,068.596 13 4 1 4 2 3 2,904.86 14 4 2 3 1 4 3,536.19 15 4 3 2 4 1 3,380.65 16 4 4 1 3 2 3,810.844 k1 2,861.581 3,283.126 2,957.343 3,478.115 3,290.199 k2 3,048.762 2,896.137 3,467.026 2,649.224 2,967.255 k3 3,268.114 2,975.807 3,021.668 3,633.181 3,010.412 k4 3,408.136 3,431.523 3,140.557 2,826.075 3,318.728 Range 546.555 535.386 509.683 983.957 351.473 Enhanced production of lipstatin from Streptomyces toxytricini by optimizing fermentation conditions and medium 109 of peak areas with those standard lipstatin. Data analysis. The statistical analysis of data was carried out using the Origin 8.0 Software. A* means statistically signiﬁcant (p ＜ 0.01) when comparing each treatment to the control.

Results and Discussion

Effects of seed age and fermentation time on lipstatin production Twenty-two-, 26-, 30- and 34-h-old seeds were compared for lipstatin production. The microscopic observation of seeds showed that the seed broth turned very viscous and the Fig. 2. The effect of volume charge of flask in lipstatin fermentation. color deepened with fermentation time prolonged. These Star marker significant difference at the level of p＜0.05. seeds of different culture periods gave a near lipstatin titer and no significant difference was observed among them. The 30-h seed culture gave the highest lipstatin titer; thus the seed of 30 h was chosen to inoculate the fermentation medium for lipstatin production. At this time point the pH of the seed broth was 7.66. Effects of four fermentation periods (5, 6, 7 and 8 days) on lipstatin production were then investigated. The results showed that the lipstatin titer increased significantly from the 5th day, reaching its highest level on the 7th day, and then decreased by the 8th day. In these periods, however, as the culture time increased only a slight increase in biomass was observed and the broth pH decreased from 7.16 to 6.96. These results are shown in Fig. 1.

Effects of fermentation flask volume charge and water supplementation on lipstatin production The effect of different volumes of the fermentation Fig. 3. The effect of water supplement in lipstatin fermentation. medium in shake flasks on the production of lipstatin by S. Star marker significant difference compared to control at the level of toxytricini zjut011was investigated. The lipstatin production p＜0.01. was markedly influenced by the volume of the fermentation medium contained in the shaken flasks. In 500 ml fermentation flasks, the group charged with 50 ml medium obtained a dissolved oxygen level in the fermentation broth is one of statistically significant increase in lipstatin titer compared to the key parameters influencing the fermentation titer (Luthra the group charged with 30 ml (Fig. 2).The water supplement and Dubey, 2012). In theory, the larger the charged volume in the process of lipstatin fermentation combined with these in a shaking-flask is, the lower the dissolved oxygen level is. two charge volumes was investigated further. The result The viscosity, however, was increased with the prolonged showed that the lipstatin production got the highest titer of culture time and then the dissolved oxygen level decreased, 1,899.55 µg/ml when the fermentation shaking-flask was especially when the charge volume was smaller. Therefore, charged with 50 ml medium and supplemented with reduced the stimulation of lipstatin production could be obtained by water every day (treatment V, Fig. 3). As S. toxytricini maintaining an aerobic condition by loading a suitable production of lipstatin is a strictly aerobic process, the volume of medium combined with supplementation of water

Fig. 1. Effects of fermentation time on lipstatin production. 110 ZHU et al. to reduce the viscosity. tal investigations are needed to estimate the effect of L-leucine under different fermentation conditions. Effects of precursors on lipstatin production Supplying the fermentation broth with lipstatin precursors Effects of divalent cations on lipstatin production and the methods of addition were investigated. The results The effects of six divalent cations of Mg2+, Mn2+, Cu2+, showed that the addition of L-leucine and octanoic acid Zn2+, Fe2+ and Co2+ on the lipstatin synthesis with a series of effectively activated the biosynthesis of lipstatin. When the concentrations of 0.5, 2, 4 and 8 mmol/L were investigated. fermentation broth was supplemented with 30.48 mmol/L of Among them, Mg2+, Co2+ and Zn2+ showed positive effects, L-leucine on the 4th day (treatment III) or 13.866 mmol/L while Cu2+, Mn2+ and Fe2+ showed negative effects (Fig. 5). octanoic acid on the 6th day (treatment VII), the final With the increase of concentration (from 0.5 to 8 mmol/L), lipstatin production was more than 100% higher than that in the promotion effect of Mg2+ on lipstatin was enhanced. In the control. The highest lipstatin yield reached was contrast, the promotion effect of Co2+ decreased. When the 2,637.55 mmol/L after 7 days of fermentation when supple- concentration of Mg2+, Co2+, and Zn2+ was 8, 0.5 and mented with octanoic acid on the 6th day, which was an 0.5 mmol/L respectively, the lipstatin titer was 3,506.44, improvement of 132.6% compared to that of the control 3,275.775 and 2,803.59 µg/ml, nearly three times that of the (Table 2, Fig. 4). As for the biosynthetic pathway, the carbon control group. Mn2+, Fe2+ and Cu2+ showed significant skeleton of the lipstatin molecule is biosynthesized via inhibitory effect on lipstatin production at the tested concen- Claisen condensation of two fatty acid precursors, 8-carbon trations, especially Cu2+, which showed an inhibitory effect atoms, and 14-carbon atoms fatty acids (Demirev et al., of more than 95% when compared to the control at a concen- 2010). Lipstatin contains a β-lactone ring; one of the side tration of 2 mmol/L. The inhibitory effect of Mn2+ increased chains contains two isolated double bonds and a hydroxyl with increasing concentration, its largest inhibitory effect group esterified to N-formyl-L-leucine. S. toxytricini also reaching 50% when compared to the control. Fe2+ showed a produces lipase, which liberates fatty acids in different relatively stable inhibitory effect regardless of the change in quantities. Our results reported herein suggest that the its concentration. Enzymes involved in lipstatin biosynthe- suitable fatty acids can be used for lipstatin synthesis as sis, including acyl-CoA carboxylases (ACCase), are often precursors. It is found in this study that the addition of short regulated by metal ions, which play important roles in the chain L-leucine was effective in the middle phase of modulation of enzyme activity as cofactors and thus fermentation when the synthesis of lipstatin started gradual- stimulate the lipstatin biosynthesis pathway. Our results ly, whereas the addition of long-chain octanoic acid was indicate that the stimulating effect of Mg2+ and Co2+ on more effective in the late phase of fermentation when the lipstatin production at suitable concentrations is very signifi- synthesis of lipstatin is most active. Our results provide cant, suggesting the significance of their practical applica- further evidence in support of precursors for biosynthesis of tion in the lipstatin production industry. To our knowledge, lipstatin as described by Schuhr et al. (2002). As described in no previous study has investigated the effects of metal ions a patent (US 4598089, Hadvary et al., 1988), feeding of on lipstatin fermentative production. Further investigation L-leucine or N-formyl-L-leucine during the fermentation and experimentation into divalent cations is strongly process is identified as being particularly useful in order to recommended. increase the final yield of lipstatin with S. toxytricini NRRL15443. In the study reported by Luthra and Dubey Orthogonal optimization of the fermentation process and (2012), however, they found that there is no significant verification of the optimized conditions impact of L-leucine, linoleic acid, or palmitic acid as media On the basis of single factor experiments, by means of an ingredients or as a feeding solution on lipstatin production orthogonal trial, the effects of divalent cation and precursor with strain S. toxytricini (ATCC 19813). Further experimen- factors, including Co2+, Mg2+, Zn2+, L-leucine and octanoic

Fig. 4. The effects of precursors supply on lipstatin fermentation. Fig. 5. The effects of divalent cations on lipstatin production. Star marker signiﬁcant difference compared to control at the level of Star marker signiﬁcant difference compared to control at the level of p＜0.01. p＜0.01. Enhanced production of lipstatin from Streptomyces toxytricini by optimizing fermentation conditions and medium 111

Table 5. ANOVA of orthogonal experiment results for lipstatin fermentation medium optimization. Factor DEVSQ Degrees of freedom F value p value Signiﬁcance Mg2+ 571,814.418 3 0.487 ＜0.1 Co2+ 679,262.779 3 0.579 ＜0.1 Zn2+ 818,762.313 3 0.697 ＜0.1 Leucine 3,454,141.781 3 2.942 ＞0.1 * Octanoic acid 346,474.167 3 0.295 ＜0.1

*Significant difference. acid, on the lipstatin production medium with S. toxytricini Demirev, A. V., Khanal, A., Hanh, N. P., Nam, K. T., and Nam, D. H. (2011) Biochemical characterization of propionyl-coenzyme A zjut011 under the condition of a shaking flask was tested and carboxylase complex of Streptomyces toxytricini. J. Microbiol., optimized. The significance of each factor on lipstatin 49, 407‒ 412. production can be seen from the corresponding arranged Demirev, A. V., Lee, J. S., Sedai, B. R., Ivanov, I. G., and Nam, D. H. values listed in Table 4. The order in which the individual (2009) Identification and characterization of acetyl-CoA carbox- factors selected in the present study affected the lipstatin ylase gene cluster in Streptomyces toxytricini. J. Microbiol., 47, 2+ 2+ 473‒ 478. production can be ranked as L-leucine ＞ Mg ＞ Co ＞ Eisenreich, W., Kupfer, E., Stohler, P., Weber, W., and Bacher, A. 2+ Zn ＞ octanoic acid. The ANOVA table demonstrates that (2003) Biosynthetic origin of a branched chain analogue of the the L-leucine is the most significant factor for lipstatin lipase inhibitor, lipstatin. J. Med. Chem., 46, 4209‒ 4212. production, as is evident from the probability value (p ＜0.1). Eisenreich, W., Kupfer, E., Weber, W., and Bacher, A. (1997) Tracer The best conditions could be selected according to k values. studies with crude U-13C-lipid mixtures. Biosynthesis of the lipase inhibitor lipstatin. J. Biol. Chem., 272, 867‒ 874. From the analytical results of the orthogonal design with Goese, M., Eisenreich, W., Kupfer, E., Stohler, P., and Weber, W. et al. zjut011, the optimized conditions for lipstatin production (2001) Biosynthesis of lipstatin. Incorporation of multiply deute- were determined as D3A4B4C2E4 corresponding to rium-labeled (5Z, 8Z)-tetradeca-5, 8-dienoic acid and octanoic 45.72 mmol/L of L-leucine (added on the 4th day), acid. J. Org. Chem., 66, 4673‒ 4678. Goese, M., Eisenreich, W., Kupfer, E., Weber, W., and Bacher, A. 31.1985 mmol/L of octanoic acid (added on the 6th day), (2000) Biosynthetic origin of hydrogen atoms in the lipase inhib- 2+ 2+ 12 mmol/L of Mg , 1 mmol/L of Co and 0.25 mmol/L of itor lipstatin. J. Biol. Chem., 275, 21192‒ 21196. Zn2+ (Tables 4 and 5). Hadvary, P., Hochuli, E. E., Kupfer, H., Lengsfeld, H., and Weibel, This optimized fermentation conditions were tested E. K. (1988) ‘Leucine Derivatives’, U.S. Pat. No.4598089. further. Under these conditions, a maximum lipstatin titer of Hochuli, E., Kupfer, E., Maurer, R., Meister, W., and Mercadal, Y. et al. (1987) Lipstatin, an inhibitor of pancreatic lipase, produced by 4.208 g/L was achieved. The fermentation conditions were Streptomyces toxytricini. II. Chemistry and structure elucidation. the same as those previously described for the fermentation J. Antibiot., 40, 1086‒ 1091. flask. Luthra et al. (2013) recently reported a maximum Huang, J., Chen, Z. J., Feng, J., Zheng, Y. G., and Zhao, W. J. (2006) lipstatin production of 3.290 g/L with a mutated S. toxytrici- Induced mutagenesis of lipstatin producing strain by ultraviolet ni strain by using full factorial design experiment optimiza- ray and microwave. Chin. J. Pharmaceut., 37, 12‒ 14. Kumar, M. S., Verma, V., Soni, S., and Rao, A. S. P. (2012) Identifica- tion in a shake flask. tion and process development for enhanced production of lipstatin Taken together, despite the many challenges associated from Streptomyces toxytricini (Nrrl 15443) and further with optimization for the lipstatin fermentation process, downstream processing. Adv. Biol. Technol., 11, 6‒ 11. these results are helpful in providing some new insights into Luthra, U. and Dubey, R. C. (2012) The role of linoleic acid, palmitic acid and leucine in lipstatin biosynthesis by Streptomyces toxytr- the regulation of lipstatin biosynthesis and the approach can icini. Int. J. Appl. Biol. Pharmaceut. Technol., 3, 356‒ 365. be applied in its fermentation processes for enhancing Luthra, U., Kumar, H., and Dubey, R. C. (2013a) Mutagenesis of the production. Further research is needed to obtain more lipstatin producer Streptomyces toxytricini ATCC 19813. J. information about lipstatin production with these parameters Biotechnol. Lett., 4, 68‒ 71. under the conditions scaled up from a shake flask to a Luthra, U., Kumar, H., Kulshreshtha, N., Tripathi, A., and Trivedi, A. et al. (2013b) Medium optimization for the production of lipstatin bioreactor. by Streptomyces toxytricini using full factorial design of experiment. Nat. Sci., 11, 73‒ 76. Acknowledgments Rodriguez, E. and Gramajo, H. (1999) Genetic and biochemical characterization of the α and β components of propionyl-CoA This work was supported by National Natural Science Foundation carboxylase complex of Streptomyces coelicolor A3(2). Microbi- of China (Grant No. 31272002), Open Research Fund of Zhejiang ology, 145, 3109‒ 3119. First-foremost Key Subject (2013) and Zhejiang Provincial Top Schuhr, C. A., Eisenreich, W., Goese, M., Stohler, P., and Weber, W. et Academic Discipline of Applied Chemistry and Eco-Dyeing & Finish- al. (2002) Biosynthetic precursors of the lipase inhibitor lipstatin. ing (Grant No. YR2011009). J. Org. Chem., 67, 2257‒ 2262. Weibel, E. K., Hadvary, P., Hochuli, E., Kupfer, E., and Lengsfeld, H. (1987) Lipstatin, an inhibitor of pancreatic lipase, produced by References Streptomyces toxytricini. I. Producing organism, fermentation, isolation and biological activity. J. Antibiot., 40, 1081‒ 1085. Demirev, A. V., Khanal, A., Sedai, B. R., Lim, S. K., and Na, M. K. et Zohrabian, A. (2010) Clinical and economic considerations of antiobe- al. (2010) The role of acyl-coenzyme A carboxylase complex in sity treatment: A review of Orlistat. Clinicoecon Outcomes Res., lipstatin biosynthesis of Streptomyces toxytricini. Appl. Microbi- 2, 63‒ 74. ol. Biotechnol., 87, 1129‒ 1139.