A Bacillus subtilis mutant strain overexpressing surfactin biosynthetic genes was previously constructed. In order to further increase the production of this biosurfactant, our hypothesis is that the surfactin precursors, especially leucine, must be overproduced. We present a three step approach for leucine overproduction directed by methods from computational biology. Firstly, we develop a new algorithm for gene knockout prediction based on abstract interpretation, which applies to a recent modeling language for reaction networks with partial kinetic information. Secondly, we model the leucine metabolic pathway as a reaction network in this language, and apply the knockout prediction algorithm with the target of leucine overproduction. Out of the 21 reactions corresponding to potential gene knockouts, the prediction algorithm selects 12 reactions. Six knockouts were introduced in B. subtilis 168 derivatives strains to verify their effects on surfactin production. For all generated mutants, the specific surfactin production is increased from 1.6-to 20.9-fold during the exponential growth phase, depending on the medium composition. These results show the effectiveness of the knockout prediction approach based on formal models for metabolic reaction networks with partial kinetic information, and confirms our hypothesis that precursors supply is one of the main parameters to optimize surfactin overproduction. Keywords: Abstract interpretation · Bacillus subtilis · Knockout prediction · Modeling language · SurfactinCorresponding author: Dr. François Coutte, Research Institute for Food and Biotechnology -Charles Viollette, Polytech-Lille, Université de Lille, Sciences et Technologies, 59655 Villeneuve d'Ascq, France. E-mail: francois.coutte@polytech-lille.fr Abbreviations: Acyl-CoA, acyl coenzyme A; Akb, L-2-amino-acetoacetate; BCAA, branched chain amino acid; Glu, glutamate; Gtp, guanosine triphosphate; Ile, isoleucine; Ket a , 2-keto-3-methylvalerate;Ket b , 2-keto-isovalerate; Ket c , 2-keto-isocaproate; Leu, leucine; NRPS, nonribosomal peptide synthetase; OxoGlu, oxoglutarate; P Ilv-Leu , ilv-leu operon promoter; Pyr, pyruvate; Thr, threonine; Val, valine; XML, eXtensible Markup Language; XSLT, eXtensible Stylesheet Language Transformations Biotechnology JournalSupporting information available online * These authors contributed equally to this work. Biotechnol. J. 2015Biotechnol. J. , 10, 1216Biotechnol. J. -1234 surfactin is composed of a ring of seven amino acid residues connected to a β-hydroxylated fatty acid chain of different length and isomery [1,2]. The peptide moiety contains four leucines (Fig. 1). Genetic engineering of B. subtilis has already been made in order to increase the lipopeptide production. In previous work [3], the overproduction of surfactin was obtained by replacing the native promoter of the surfactin operon (srfA) by a constitutive one and disrupted the plipastatin operon (ppsA) to save the precursor availability. The same approach was recently developed for the mycosubtilin producti...

Surfactin, a lipopeptide produced by Bacillus subtilis, is one of the most powerful biosurfactants known. This molecule consists of a cyclic heptapeptide linked to a β-hydroxy fatty acid chain. The isomery and the length of the fatty acid (FA) chain are responsible for the surfactin's activities. In this study, the gene codY, which encode for the global transcriptional regulator and the gene lpdV, located in the bkd operon (lpdV, bkdAA, bkdAB and bkdB genes), which is responsible for the last step of the branched chain amino acid (BCAA) degradation in acyl-CoA were deleted. The influence of these deletions on the quantitative and qualitative surfactin production was analysed. The surfactin production was quantified by RP-HPLC and the surfactin isoforms were characterized using LC-MS-MS and GC-MS analysis. The results obtained in the mutants showed an enhancement of surfactin specific production by a factor of 5.8 for the codY mutant and 1.4 for lpdV mutant. Moreover qualitative analysis of the lpdV mutant reveals that it mainly produced surfactin C isoform (2 fold more than the wild type) with linear FA chain. Complete analysis of the extracellular metabolites using H quantitative NMR reveals a reduced production of acetoin in this mutant. This work demonstrates for the first time an original approach to overproduce specifically surfactin with C FA chain.

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A B S T R A C TAn indigenous bacterial isolate Planococcus sp. TRC1 was found to tolerate Cr(VI) solution up to 500 mg/L concentration when grown in mineral salt media. Gene sequencing of the isolated strain using 16S rDNA technique and phylogenetic analysis confirmed that the species was 96% close to Planococcus maritimus (KP8). Studies on cell dynamics in batch bioreactor showed the maximum specific cell growth rate (μ max ) to be 0.276 h −1 . Removal of Cr(VI) was observed to be dependent on initial chromium concentration and the maximum removal of Cr(VI) (75 ± 3%) was obtained at 25 mg/L Cr(VI) solution for an incubation period of 72 h. Above this value, the removal of Cr(VI) was declined. Scanning electron microscope and Fourier transform infrared spectroscopic studies indicated that the Cr(VI) removal took place by the adsorption process on the cell outer membrane. It is observed that the adsorption process follows the Freundlich adsorption isotherm (R 2 = 0.987). Planococcus sp. TRC1 reduced 80 ± 5% chemical oxygen demand (COD) of tannery effluent (COD 7,270 ± 45 mg O 2 /L) within 48 h of batch treatment. The elimination of cytotoxicity and genotoxicity imparted by the raw tannery effluent and Cr(VI) solution after bacterial treatment is an important observation which reflects the eco-friendly behavior of the Planococcus sp. TRC1 mediated tannery effluent and aqueous Cr(VI) solution treatment. The results revealed the applicability of Planococcus sp. TRC1 in both tannery waste and Cr(VI) reduction without affecting our environment.

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