The present invention mainly relates to bioengineering and development of bioproeess for high level production of novel cutinase from Thermobifida fusca in genetically engineered or modified Escherichia coli BL21 (DE3) [ACCESSION NO. MTCC 5725 AND ACCESSION NO. MTCC 5726] and use thereof. It also relates to obtain the novel recombinant genes for the production of novel cutinase.

BACKGROUND OF THE INVENTION

Cutinases (EC 3.1.1.74) are carboxylic ester hydrolases which has the capabilities to act on multi-substrates like, cutin, synthetic short and long chain fatty acids, synthetic esters (Fleet ME and MacRae ND 1983, Contribution of Mineral and Petrol 83: 75-81 ; Verger R et al., 1976, Journal of Biological Chemistry 251 : 3128-3133), water insoluble triglycerides (Lauwereys M et al., 1991 : Cloning, expression and characterization of cutinase, a fungal lipolytic enzyme, In Alberghina L, Schmid RD, Verger R, editor. Lipases-Structure, function and genetic engineering vol. 16. VCH Weinheim; p. 243-251) etc. Besides substrate specificity, its robustness for temperature, organic solvent and surfactant tolerance has made it emanate industrial enzyme in various applications such as dairy, laundry and dishwashing detergent formulations (U.S. Patent 97-05736 and 97- 09664; Egmond MR and Bemmel van CJ. 1997, Impact of structural information on understanding lipolytic , function. In: Methods in Enzymology 284, Rubin B, Dennis EA editors. New York: Academic Press; p. 119-129; International Patent application WO 94/03578), oleochemical industries, degradation of plastics, synthesis of structured triglycerides, polymers and surfactants, chiral synthesis of pharmaceuticals and agro chemicals (Carvalho CML et. al., 1999, Biotechnology and Bioengineering 66: 17-34), cotton scouring (Degani O et. al., 2002, Applied Biochemistry and Biotechnology 102: 277-289) and degradation of toxic substances [Kim YH et al., 2005, Chemosphere, 60 (10), 1349-1355].

Though there are several researchers reported on cutinase which are focused on characterization and applications of cutinase in various fields but none of the researchers reported on the cost effective production optimization or production of cutinase from the recombinant microorganism. Most of the prior art reports are on production of cutinase, which are confined to fungal cutinase in wild microorganism or in recombinant form in E. coli, or Saccharomyces cerevisiae [Backlund, E et al., 2008, Journal of Biotechnology, 135(4), 358-365; Calado CRC et al., 2003, Journal of Bioscience and Bioengineering, 96(2), 141-148; Calado CRC et al., 2003, Journal of Biotechnology, 109(1-2), 147-158; Chen S et. al., 201 1, Applied Biochemistry and Biotechnology, 165(2), 666-675; Li JH et. al., 2010, Korean Journal of Chemical Engineering, 27(4), 1233-1238; He GQ et. al., 2009, Biotechnology and Bioprocess Engineering, 14(1), 46-51]. So far, the effort to tackle cost effective industrial scale production of fungal or bacterial cutinase is not successful fully . Recently, cutinase from Thermobifida fusca have been shown its superiority over other cutinases in many aspects like thermostability, highly activity in broad pH range, surfactant and organic solvent tolerance, which could have great biotechnological promise in many industrial applications [Chen S et. al., 2010, Journal of Molecular Catalysis B: Enzymatic 63: 121-127; and Chen S et. al., 2008, Journal of Biological Chemistry 283 (38): 25854-25862]. They reported that, cutinase, Tfu_0882 and Tfu_0883 from T. fusca has great thermostability, broad pH range, and extreme organic solvent and surfactant tolerance, which are the affirmative properties of an enzyme to implement in various industrial applications like esterification, transesterification, polymer modification etc. The US patent application no. 2012/0149086 discloses the invention, which relates to the field of bioengineering and a cutinase.-producing genetically engineered microorganism and use thereof. Recombinant plasmid Tfu_0883-hlyAs/pET20b (+) was constructed and transformed into E. coli BL21 (DE3) to obtain recombinant E. coli strain Tfu_0883- hlyAs/pET20b (+)/£. coli BL21 (DE3). Specific growth rate was maintained at a certain value using fed-batch fermentation mode. After fermenting 30-34 hours, the enzyme activity in the supernatant reached 700-750 U/mL. The present invention is advantageous over the US 2012/0149086 in respect of higher yield in large scale and cost-effectiveness of the process.

The primary impediment to industrial application of an enzyme is its production in large scale, which needs to fulfill the very essential requirements like, low cost of production, inexpensive media formulation and higher yield. One of the best ways to tackle the production problems is to use a recombinant microorganism, which is built for higher production at lower cost. The present inventors have solved the above problems of prior art and achieved higher level production of a recombinant novel cutinase, Cut_l or Cut_2 from T. fusca in a genetically modified E. coli BL 21 (DE3) using expression vector pET22b (+).

OBJECTS OF THE INVENTION

The primary object of the present invention is to overcome the difficulties in production of T. fusca cutinase to meet the industrial requirement by construction of recombinant cutinase for large scale production in genetically engineered microorganism Accession No. MTCC 5725 and Accession No. MTCC 5726.

The another object of the present invention is the higher level production of recombinant T. fusca cutinase in genetically modified microorganism by screening a low cost high density medium formulation, which is suitable even for large scale fermentation. The further object of the present invention is to obtain the novel recombinant genes for the production of novel cutinase.

The yet another object of the present invention is production of novel cutinase by devising the cost effective production process.

STATEMENT OF INVENTION

A novel cutinase producing genetically engineered microorganism, E. coli BL21 (DE3) characterized in that it is carrying recombinant plasmid pET22b(+)-ctti _/ [Accession No. MTCC 5725] having the cut _1 gene sequence as in FIG 1 (A) or amino acid sequence as in FIG 2 (A) or pET22b(+)-cut_2 [Accession No. MTCC 5726] having the cut J gene sequence as in FIG 1 (B) or amino acid sequence as in FIG 2 (B).

A method of construction of novel cutinase producing genetically engineered microorganism E. coli BL21 (DE3) comprising:

i. cutinase, cut_l or cut_2 gene amplified by PCR using Thermobifida fusca NRRL B- 8184 genomic DN A as template;

. pET22b(+) and purified cut_l or cut_2 PCR amplicon are subjected to enzymatic double digestion, and the purified, ligated products are transformed into E. coli DH5a competent cells to obtain expression construct pET22b(+)-c«i_i or pET22b(+)-cwi_2; in. the recombinant plasmid pET22b(+)-cw/_7 or pET22b(+)-cw _2 is transformed into E. coli BL21 (DE3) to obtain genetically engineered microorganism expressing Cut l [Accession No. MTCC 5725] or Cut_2 [Accession No. MTCC 5726].

A method of production of a novel cutinase comprises:

i. Culturing the genetically engineered microorganism, E. coli BL21 (DE3) as claimed in claim 1 in growth medium with the initial cell density of the culture is Αόοο nm 0.05 which is grown in seed culture medium and incubated with stirring; ii. After culturing for 3 to 4 hours or when A ₆ oo nm reaches 0.75 the IPTG is added for induction and the culture was grown further for 30-40 hrs for novel cutinase production in production medium.

The seed culture medium having the pH 7.4 comprises 0.5% Peptone, 0.5% NaCl, 0.15% Beef extract, 0.15% Yeast extract and 0.01% ampicillin. The production medium having the pH 7 comprises 0.4% (v/v) glycerol, 0.024% MgS0 ₄ or 1% N-Z amine, 0.05% NaCl, 0.1% NH ₄ C1, 0.3% KH2PO4, 0.6% Na ₂ HPO ₄ '7H ₂ 0 and 0.01% ampicillin. The production medium having the pH 7.3 comprises 1.2% to 2% Tryptone, 2.4% Yeast extract, 0.4% (v/v) Glycerol, 0.115% to 0.23% KH ₂ P0 ₄ , 1.25% K ₂ HP0 ₄ and 0.01% ampicillin. The production medium further comprises 0.049% to 0.061% MgS0 ₄ and 0.005% _. NaCl. The IPTG is added at a final concentration of 0.1 to 1 mM and incubation temperature is maintained at 37° C with the stirring rotation speed of 250 rpm.

SUMMARY OF THE INVENTION

The present invention focuses on development of an inexpensive, high level production method of full length cutinase, Cut_l and Cut_2 from Thermobiflda fusca in a genetically engineered microorganism, E, coli BL21 (DE3) by construction of recombinant cutinase. According to the first aspect of invention recombinant cutinase was constructed by using genomic DNA of Thermobiflda fusca NRRL B-8184 as template for PCR amplification. Cut_l and Cut_2 genes from Thermobiflda fusca NRRL B-8184 was inserted in to an expression vector pET22b(+). The gene cassette was expressed in frame with pelB signal sequence for periplasmic localization under IPTG inducible T7 promoter.

Second aspect of invention was high level production of recombinant cutinase, Cut_l and Cut_2 in genetically engineered microorganism, E. coli BL21 (DE3). In order to develop a suitable process for large scale production of cutinase and to maximize the recombinant cutinase production in soluble form, in genetically engineered E. coli BL21 (DE3), expression optimization was done using different media and by varying physiochemical parameters.

The process for production of recombinant novel cutinase Cut_l and Cut_2 from Thermobifida fusca, wherein the said process comprises:

i. construction of recombinant novel cutinase expression cassette in vector pET22b(+) ii. Expression of recombinant novel cutinase, Cut_l and Cut_2 in fusion with (His)6 tag in genetically engineered microorganism, E. coli BL21 (DE3)

iii. Optimization of physiochemical parameters for economic production of recombinant novel cutinase Cut l and Cut_2

iv. High level production of recombinant novel cutinase in different media formulations. Recombinant cutinase was constructed by cloning a PCR amplicon of Cut_l and Cut_2 amplified from genomic DNA of Thermobifida fusca in to expression vector pET22b(+), inducible by IPTG. Recombinant construct was transformed into genetically engineered E. coli BL21 (DE3) and expressed. Expression parameters were optimized by varying different physiochemical parameters like growth temperature, concentration of inducer, IPTG, cell density at the time of induction, which are the decisive factors for successful high level expression of recombinant protein in genetically engineered microorganism, E. coli BL21 (DE3) in soluble form. In consideration of high level production of recombinant protein in genetically engineered microorganism, E. coli BL21 (DE3), various media compositions were evaluated presuming the fact of effect of media compositions on high level production of recombinant proteins in genetically engineered microorganism to make it technically and economically feasible process in the commercial level. A remarkable 11 fold increase in production in terms of enzyme yield (U/ml) was observed for recombinant cutinase in one of the production medium as compared to wild type production in Thermobifida fusca. A BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING

Figure 1 (A) Nucleotide sequence of full length cut_l gene of Thermobiftda fusca NR L

B-8184 as confirmed by gene sequencing.

Figure 1 (B) Nucleotide sequence of full length cut_2 gene of Thermobiflda fusca NRRL B-8184 as confirmed by gene sequencing.

Figure 2 (A) Amino acid sequence of corresponding full length cut_l gene of Thermobiflda fusca NRRL B-8184.

Figure 2 (B) Amino acid sequence of corresponding full length cut_2 gene of Thermobiflda fusca NRRL B-8184.

Figure 3 (A) Effect of medium on growth of genetically modified microorganism. Growth pattern of E. coli BL21 (DE3) pET22b (+)-cut_l in Production medium 1(·), Production medium 2(o), Production medium3( Y), Production medium4(A), Production medium5(e). Figure 3 (B) Effect of medium on growth of genetically modified microorganism. Growth pattern of E. coli BL21(DE3)pET22b(+)-ct _2 in Production medium 1 (·), Production medium2(o), Production medium3(T), Production medium 4(Δ), Production medium5(-_). Figure 4 (A) Effect of medium on production of cutinase. Production pattern of E. coli BL21 (DE3) pET22b(+)-cni_i in Production medium 1 (·), Production medium 2 (o), Production medium 3 (T), Production medium 4 (Δ), Production medium 5 (■).

Figure 4 (B) Effect of medium on production of cutinase. Production pattern of E. coli BL21 (DE3) pET22b(+)-CMi_2 in Production medium 1 (·), Production medium 2 (o), Production medium 3 ( Y), Production medium 4 (Δ), Production medium 5 (■).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is projected towards providing an easy way of producing novel cutinase from T. fusca in a genetically engineered microorganism, E. coli BL21 (DE3). The invention particularly relates to a method or a process for high level production of cutinase in soluble form in genetically engineered E. coli BL21 (DE3), which is implementable for scale up at fermenter level for cost effective production. The various steps in the method of the present invention are described more fully hereinafter as examples.

Materials and methods

Chemicals and enzymes:

High fidelity PhusionTaq, dNTPs, ligase and restriction enzymes were obtained from New England Biolabs Inc., USA. Plasmid isolation kit was purchased from HiMedia Pvt. Ltd., India. Genomic DNA isolation kit was procured from Sigma Aldrich, USA: All chemicals used in analytical techniques were from Sigma, USA and the chemicals for preparation of medium were from Merck, USA.

Bacterial strain vector and plasmid:

pET22b(+) expression vector and E. coli BL21 (DE3) expression host were procured from Novagen Inc., USA. DH5a was procured from Microbial Type Culture Collection (MTCC), Chandigarh, India. T. fusca NRRL B-8184 strain used as a source of cutinase gene was obtained from ARS culture collection (NRRL), USA.

Activity assay:

Cutinase activity against p-nitropheiiyl butyrate (pNPB) was determined by measuring the amount of p-nitrophenol released by hydrolysis of pNPB. The production of p-nitrophenol was monitored at 405nm. The standard assay was measured by addition of 2-5 μΐ crude enzyme from cell lysate in 1 ml reaction volume containing 1 mM pNPB as a substrate in 50 mM Potassium phosphate buffer (pH 8) containing 4% THF, 10 mM Sodium deoxycholate at 50°C. One unit of enzyme activity is defined as release of 1 μιηοΐ of p- nitrophenol per min. Example 1

Cloning of cut l and cut_2 in an expression vector pET22b (+)

Genomic DNA of Thermobifida fusca NRRL B-8184 was used as a template for amplification of genes. cut_l gene was amplified by PCR using cutlF and cutlR primers as described below and cut_2 gene was amplified by PCR using cut2F and cut2R primers as described below. Both genes are expressed as fusion protein to C-terminal (His)6 tag. cutlF: 5 '-GGA ATTCGGATCC A ATGCCCCCGCATGCGGCGCG-3 '

cutlR: 5 '-GAAGCTTCTCGAGG AAGGGGC AGGTGGAGCG-3 '

cut2F: 5 '-GGAATTCGGATCC AATGGCTGTGATGACCCCCCG-3 '

cut2R: 5 '-GA AGCTTCTCGAGGAACGGGC AGGTGGAGC-3 '

Genomic DNA was prepared using genomic DNA isolation kit following manufacturer's instructions. High fidelity taq polymerase was used for amplification of genes. Following PCR condition was used for the amplification of genes in 50 μΐ reaction volume; Initial denaturation at 98°C for 3 min, and then 20 cycles of denaturation at 98°C for 10 Sec, annealing and elongation at 72°C for 45 sec and final extension at 72°C for 10 min. Upon 1% agarose gel electrophoresis the amplicon showed the size of ~ 1 kb for both cut_l and cut_2 genes.

The PCR amplicon was purified by PCR purification kit following manufacturer's instructions. Both cut_l and cut_2 PCR product was digested with BamHI and Xhol and ligated separately into expression vector pET22b (+) double digested with the same enzymes. The ligation was performed using T4 DNA ligase at 25°C for 4 hours. The a

ligated product was transformed in to DH5a competent cells and the transformants were grown at 37°C for 8-10 hours on Luria Bertani-agar plate supplemented with 100 μg/ml ampicillin. Single colonies of the putative DH5a clones were inoculated in to 5 ml of Luria Bertani medium containing 100 g/ml ampicillin and grown for 8-10 hours at 37°C with 250 rpm in a shaking incubator. The resulting culture was centrifuged and the cell pellet was used for plasmid isolation.

Isolated putative clones were analyzed by restriction digestion with BamHI and Xhol and further confirmed by gene sequencing. Sequencing revealed the gene sequence of cut_l to be of 957 base pair (FIG 1A) and cut . to be of 903 base pair (FIG IB). BLAST (Basic Local Alignment Search Tool) result of the cut_l gene showed 100 % homology to the available triacylglycerol lipase; tfu_0882 of Thermobifida fusca YX (Acc. no.YP_288943.1). The cut_2 gene also showed 100 % homology to the available triacylglycerol lipase; ifu_0883 of Thermobifida fusca YX (Acc. No.YP_288944.1). The nucleotide sequence of cut_l and cut_2 has been deposited in NCBI with Acc. No. JN129499.1 and JN129500.1, respectively. FIG. 2 A and 2 B shows the amino acid sequence of full length Cut_l and Cut_2, respectively.

Sequence confirmed pET22b (+)-cut_l or pET22b (+)-cut_2 plasmid was transformed into expression host E. coli BL21 (DE3). Transformants were grown at 37°C for 8-10 hours on Luria Bertani-agar plate supplemented with 100 μg/ml ampicillin. Single colonies of pET22b (+)-cut_l or pET22b (+)-cut_2 were selected for further experiments as said in subsequent examples.

Example 2

Expression and expression parameter optimization for production of recombinant cutinase

Bacterial strain used: (A) E. coli BL21 (DE3) harboring pET22b(+)-cw _7 [Accession No. MTCC 5725]

(B) E. coli BL21 (DE3) harboring pET22b (+)-cut_2 [Accession No. MTCC 5726]

Expression condition optimization medium: Per liter: 10 g Tryptone, 5 g Yeast extract, 10 g NaCl, 100 mg ampicillin, pH 7 i. The standard culture method used for production condition optimization

A single colony of E. coli BL21 (DE3) harboring pET22b (+)-cut_l or pET22b (+)-cut_2 was inoculated in to 5 ml of optimization medium, incubated at 37°C and 250 rpm in a shaking incubator for 12 hrs. 50 ml of optimization medium was inoculated with the above seed culture (around 1%), adjusted for the initial cell density Aeoo nm of 0.05 and incubated at 37°C and 250 rpm in a shaking incubator. IPTG was added to the culture medium (A ₆ oonm ~ 0.75) and allowed to grow further for 30 hrs at 37°C with 250 rpm shaking in a shaking incubator.

ii. Expression parameter optimization

Experiments were performed to determine the optimal level of IPTG for higher level of novel recombinant cutinase expression by the addition of various concentration of IPTG (0.1 mM, 0.4 mM, 0.6 mM, 0.8 mM, 1 raM) at cell density A ₆ oo nm ~ 0.75 at 37°C. Temperature optimization was done by inducing the cells with optimal IPTG of 0.1 mM at cell density Αδοο nm ~ 0.75. The culture was further grown at different temperature viz., 20°C, 25°C, 30°C and 37°C. Induction opportunity was studied at optimal IPTG and temperature (0.1 mM and 37°C, respectively) by adding IPTG to the culture at different Aeoo nm viz., 0.20, 0.5, 0.75, 1, 1.5, 2.5 and 3.

The optimal levels of IPTG concentration, temperature and cell density was found to be 0.1 mM, 37°C and A ₆ oo nm 0.75, respectively. The maximum expression of Cut_l and Cut_2 was observed to be 101 U/ml and 1 12 U/ml, respectively, after 6 hr post induction under above said condition.

Example 3

Production of novel recombinant Cut_l or Cut_2 enzymes at shake flask level.

Bacterial strains used:

(A) E. coli BL21 (DE3) harboring pET22b (+)-cutJ [Accession No. MTCC 5725]. (B) E. coli BL21 (DE3) harboring pET22b {+)-cut_2 [Accession No. MTCC 5726]. Seed culture medium: Per liter; 5 g Peptone, 5 g Sodium chloride, 1.5 g Beef extract, 1.5 g Yeast extract, 100 mg ampicillin, pH-7.4

Production media used:

Following five production medium with different medium composition was used to analyze the higher growth and production of recombinant Cut l and Cut_2.

Production medium 1: Per liter: 4 ml glycerol, 0.246 g MgS0 ₄ , 0.5 g NaCl, lg NH ₄ C1, 3g KH2PO4, 6 g Na ₂ HP0 ₄ '7H ₂ 0, 100 mg ampicillin, pH-7

Production medium 2: Per liter: 4 ml glycerol, 10 g N-Z amine, 0.5 g NaCl, 1 g NH4CI, 3 g KH ₂ P0 ₄ , 6 g Na ₂ HP0 ₄ '7H ₂ 0, 100 mg ampicillin, pH-7

Production medium 3: Per liter: 12 g Tryptone, 24 g Yeast extract, 4 ml Glycerol, 2.31 g KH ₂ P0 ₄ , 12.54 g K2HPO4, 100 mg ampicillin, pH-7.3

Production medium 4: Per liter: 12 g Tryptone, 24 g Yeast extract, 4 ml Glycerol, 2.31 g KH ₂ P0 ₄ , 12.54 g K ₂ HP0 ₄ , 0.49 g MgS0 ₄ , 0.05 g NaCl, 100 mg ampicillin, pH-7.3

Production medium 5: Per liter: 20 g Tryptone, 24 g Yeast extract, 4 ml Glycerol, 1.15 g KH ₂ P0 ₄ , 12.54 g K ₂ HP0 ₄ , 0.61 g MgS0 ₄ , 0.058 g NaCl, 100 mg ampicillin, pH-7.3 The standard culture method used for preparation of seed culture and production medium was as mentioned in example 2. The culture medium was incubated for 30 hrs at 37°C and 250 rpm in a shaking incubator.

The cell density in terms of Dry Cell Weight (DCW) for different medium respectively for Cut_l and Cut_2 was found to be 1.72g/L and 1.59 g/L in production medium 1, 0.73 g/L and 0.88 g/L in production medium 2, 3.19 g/L and 3.0 g/L in production medium 3, 4.8 g L and 4.38 g/L in production medium 4 and 5.16 g/L and 4.74 g/L in production medium 5 respectively. The growth profile of Cut l and Cut_2 in different medium is depicted in FIG 3A and 3B for Cut_l and Cut_2, respectively. The enzyme production yield after 6-9 hours of induction, in terms of U/ml respectively for Cut l and Cut_2 was found to be 203.1 U/ml and 188.0 U/ml in production medium 1, 157.6 U/ml and 165 U/ml in production medium 2, 230.0 U/ml and 236 U/ml in production medium 3, 260.0 U/ml and 263.2 U/ml in production medium 4 and 318.4 U/ml and 316.3 U/ml in production medium 5 respectively. The production profile of Cut l and Cut_2 in different medium at different time intervals is illustrated in FIG 4A and 4B for Cut l and Cut_2, respectively.