Journal name: 3 Biotech

Title: Precursor-directed combinatorial biosynthesis of cephalosporin analogue by endolithic actinobacterium Streptomyces sp. AL51 by utilizing thiophene derivative

Kaushik Bhattacharjee1, Narasinga Rao Palepu2, Kollipara Mohan Rao2 and Santa Ram Joshi1, *

1Microbiology Laboratory, Department of Biotechnology & Bioinformatics, North-Eastern Hill University, Shillong 793022, India

2Centre for Advanced Studies in Chemistry, North-Eastern Hill University, Shillong 793022, India

* Corresponding author: Email:

Supplementary material

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Fig. S1Bayesian phylogenetic tree.2

Fig.S2 HR-APCI-MS spectrum of CAx1.3

Fig.S31H NMR spectrum (400 MHz, DMSO-d6) of CAx1.4

Fig.S413C NMR spectrum (400 MHz, DMSO- d6) of CAx1.5

Table S1Anti-bacterial activity of the cell-free extracts from the selected endolithic isolates after growing in designated media.6

Table S2Phenotypic characteristics of Streptomyces sp. AL51.7-8

Fig.S1Bayesian phylogenetic tree was reconstructed based on Metropolis-Hastings coupled Markov chain Monte Carlo (MCMC) method of analysis of 16S rRNA gene sequences of selected actinobacterial strain Streptomycetes sp. AL51 and its closest phylogenetic neighbours adopting the General Time Reversible (GTR) model with gamma distributed rates and invariant sites (GTR + I + G) of nucleotide substitution. Streptosporangium album DSM43023 (NR_026261) was included as outgroup. The consensus tree was constructed following a visually determined burn-in of 25 %. Only the branch with bootstrap values higher than 70 % are shown. Bootstrap values are indicated at the nodes (bar represents 10 % sequence divergence).

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Fig.S2HR-APCI-MS spectrum of CAx1.

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Fig.S31H NMR spectrum (400 MHz, DMSO-d6) of CAx1.

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Fig.S413C NMR spectrum (400 MHz, DMSO-d6) of CAx1.

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Table S1Anti-bacterial activity of the cell-free extracts from the selected endolithic isolates after growing in designated media.

Test organism / AK3 / AK5 / AL51 / AL52 / AM2
Staphylococcus aureus MTCC 740 / - / - / +++ / - / -
Staphylococcus epidermidis MTCC 3615 / - / - / + / - / -
Bacillus subtilis MTCC 736 / - / - / +++ / - / -
Pseudomonas aeruginosa MTCC 1688 / - / - / + / - / -
E. coli MTCC 739 / - / - / ++ / - / -
Klebsiella pneumonia MTCC 661 / - / - / ++ / - / -

Zone of inhibition: + , 1-5 mm; ++ , 5-10 mm; +++ , 10 mm and over, -, No zone of inhibition.

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Table S2Phenotypic and biochemical characteristics of endolithic isolate Streptomyces sp. AL51.

Characteristics / Streptomyces sp. AL51 / Streptomyces tyrosinilyticus NEAU-Jh3-20T
Growth on ISP-2 / Good growth / Good growth
Aerial mycelium / Pale-white / Pale-white
Substrate mycelium / Strong yellowish-brown / Strong yellowish-brown
Diffusible pigment / None / None
Growth on ISP-3 / Good growth / Good growth
Aerial mycelium / White / White
Substrate mycelium / Greenish-yellow / Greenish-yellow
Diffusible pigment / None / None
Growth on ISP-4 / None / None
Aerial mycelium / None / None
Substrate mycelium / None / None
Diffusible pigment / None / None
Growth on ISP-5 / Poor growth / Poor growth
Aerial mycelium / Greenish-yellow / Greenish-yellow
Substrate mycelium / Greenish-yellow / Greenish-yellow
Diffusible pigment / None / None
Growth on ISP-6 / Good growth / Good growth
Aerial mycelium / None / None
Substrate mycelium / Grey / Grey
Diffusible pigment / Brown / Brown
Growth on ISP-7 / Good growth / Good growth
Aerial mycelium / Yellowish -green / Yellowish -green
Substrate mycelium / Black / Black
Diffusible pigment / Black / Black
Gram's stain / + / +
Aerobic/Anaerobic / Aerobic / Aerobic
Motility / - / -
pH / 5.0-8.0, Optimum at 7.0 / 5.0-8.0, Optimum at 7.0
Tolerance of NaCl / Upto 4% / Upto 4%
Temperature / Optimum at 30°C / 15-37°C, Optimum at 28°C
Motility of spores / + / +
H2S production / + / +
Catalase / + / +
Urease / - / -
Peptonization of milk / + / +
Nitrate utilization / - / -
Degradation of aesculin / + / +
Degradation of gelatin / + / +
Degradation of cellulose / - / -
Degradation of starch / + / -
Degradation of T-80 / + / -
Degradation of T-40 / + / -
Degradation of T-20 / + / -
Degradation of L-tyrosine / + / +
Utilization of D-glucose / + / +
Utilization of D-fructose / + / +
Utilization of D-galactose / + / +
Utilization of lactose / - / -
Utilization of L-arabinose / - / -
Utilization of Maltose / + / +
Utilization of L-Rhamnose / + / +
Utilization of D-sorbitol / + / +
Utilization of sucrose / + / +
Utilization of D-xylose / - / -
Utilization of inositol / + / +
Utilization of D-Mannose / + / +
Utilization of D-Mannitol / + / +
Utilization of D-Raffinose / + / +
Utilization of D-ribose / - / -
Utilization of L-alanine / + / +
Utilization of L-arginine / + / +
Utilization of L-asparagine / + / +
Utilization of L-aspartic acid / + / +
Utilization of creatine / + / +
Utilization of L-glutamic acid / + / +
Utilization of glycine / + / +
Utilization of L-glutamine / + / +
Utilization of L-serine / + / +
Utilization of L-threonine / + / +
Utilization of L-histidine / + / Not known
Utilization of L-hydroxyproline / + / Not known
Utilization of L-methionine / + / Not known
Utilization of L- phenylalanine / + / Not known
Utilization of L-valine / + / Not known

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