Team:GDSYZX/Results

• 1. Analysis of RNA- Seq data
• 2. Selection of candidate promoters
• 3. Construction of recombinant Streptomyces
• 4. Screening of efficient feather-degrading strains
  • 4.1 Determination of keratinase activities
  • 4.2 Determination of amino acid content
  • 4.3 Determination of peptide content
  • 4.4 Determination of peptide and amino acid content
  • 4.5 Analysis of feather degradation rate
• 5. Growth conditions of various SCUT-3 strains
• Future Prospects and Concern
• References

RESULTS

1.Analysis of RNA- Seq data

In order to find appropriate promoters, we obtained RNA-Seq datas from wild type SCUT-3 cultured on feather and chitin. Based on RNA-Seq data, using TPM as the gene expression intensity evaluation index, 9 candidate promoters were selected in total (Figure 1). These nine promoters are pro1380, pro2953, pro3035, pro3040, pro3071, pro05270, pro15290, pro22610 and pro24880. The lengths of these promoters are shown in Table 1.

Figure 1. Expression strength of promoters. A: promoters selected from SCUT-3 cultured on feather; B: promoters selected from SCUT-3 cultured on chitin.

Table 1 The name and length of candidate promoters.

Promter name	Promoter length (bp)
pro1380	300
pro2953	303
pro3035	403
pro3040	263
pro3071	193
pro15290	218
pro22610	152
pro24880	461

2. Selection of candidate promoters

By using the genome of SCUT-3 as template, the candidate promoters were amplified by PCR. We successfully cloned 8 kinds promoters, including pro1380, pro2953, pro3035, pro3040, pro3071, pro05270, pro15290, pro22610 and pro24880. The PCR results of each promoter are shown in Figure 2.

Figure 2. The colony PCR results of each promoter-eGFP recombinant expression vector. A: The colony PCR results ofpro1380/ pro2953/ pro3035/ pro3040/ pro3071-eGFP and recombinant expression vector. Lane 1: DNA marker. Lane 2~5：pro1380. Lane 6~9：pro2953. Lane 10~12：pro3035. Lane 13~14：pro3040. Lane 15~16：pro3071. B: The colony PCR results of pro15290-eGFP and recombinant expression vector. Lane 1: DNA marker. Lane 2~11：pro15290. C: The colony PCR results of pro24880-eGFP and recombinant expression vector. Lane 1: DNA marker. Lane 2~11：pro24880. D: The colony PCR results of pro22610-eGFP and recombinant expression vector. Lane 1: DNA marker. Lane 2~11：pro22610.

To verify the actual expression strength of the selected promoters, we chose enhanced green fluorescent protein eGFP as the reporter gene. After replacing the ermE promoter located on the Streptomyces integrative plasmid pSET152 with the promoters cloned, we obtained the recombinant pSET152 vector carrying different promoters. The colony PCR results are shown in Figure 3.

Figure 3. The colony PCR results of each promoter-eGFP recombinant expression vector. A: Lane 1: DNA marker. Lane 2~5：pro ermE. B: Lane 6: DNA marker. Lane 1~5：pro1380. Lane 7~11：pro3035. C: Lane 6: DNA marker. Lane 1~5：pro3040. Lane 7~11：pro22610. D: Lane 6: DNA marker. Lane 1~5：pro15290. Lane 7~11：pro22610. E: Lane 6: DNA marker. Lane 1~5：pro24880.

pSET152 is an integrated plasmid. After the recombinant pSET152 vector was introduced into SCUT-3, the plasmid will be integrated into the genome of SCUT-3. Therefore, we can design primers with the plasmid base sequence to identify positive conjugant. After identification, 8 promoters-eGFP expression vectors were successfully integrated into the SCUT-3 genome respectively, as shown in Figure 4.

Figure 4. The colony PCR results of SCUT-3 genome integrated with pSET152-promoter-eGFP vector. A: Lane 1: DNA marker. Lane 2：SCUT-3. Lane 3~4：SCUT-3 genome integrated with pSET152-ermE-eGFP vector. B: Lane 1: DNA marker. Lane 2~8：SCUT-3 genome integrated with pSET152-pro1380/pro3035/pro3040/pro3071/pro15290/pro22610/pro24880-eGFP vector respectively. Lane 10: SCUT-3.

We detected the fluorescence intensity of SCUT-3 integrated with different pSET152-promoter-eGFP recombinant expression vector. We found that among all the 8 screened promoters, the fluorescence intensity of pro1380 and pro24880 were significantly higher than ermE, and the the fluorescence intensity of pro3040, pro15290, pro15290 were lower than ermE, as shown in Figure 5.

Figure 5. Comparison of eGFP fluorescence intensity driven by different promoters.

3. Construction of recombinant Streptomyces

In our previous work, we found that co-overexpression of CDO1 and SEP39 (both used constitutive promoter ermE, which was named SCUT-Ocdo-Osep39, increased SCUT-3’s feather degrading efficiency but SCUT-Ocdo-Osep39 showed lower bacterial cell copy number than wild-type SCUT-3. To obtain stronger industrial feather-degrading strains, we decided to genetically modifying these SCUT-Ocdo-Osep39 strains by replacing promoter ermE with the promoters we have chosen. Fortunately, we successfully constructed SCUT-Ocdo-p1380-Osep39, SCUT-p22610-Ocdo-Osep39 and SCUT-Ocdo-p24880-Osep39 strains by using promoter pro1380, pro22610, pro24880 respectively. The colony PCR results are shown in Figure 6.

Figure 6. .The colony PCR results of recombinant Streptomyces genome promoter. Lane 1: DNA marker. Lane 2~3：The colony PCR results of pro1380. Lane 4~5：The colony PCR results of pro22610. Lane 6~7：The colony PCR results of pro24880.

4. Screening of efficient feather-degrading strains

Feather degradation by FDB (Feather degrading bacteria) is completed by the integration of two processes, disulfide bond reduction and protease hydrolysis. CDO is a non-heme mononuclear iron enzyme that oxidizes cysteine to cysteine sulfinic acid, which is then dissimilated by aminotransferase into pyruvate and sulfite. In SCUT-3’s feather degradation, overexpression of CDO1 increased SCUT-3’s single-cell feather degradation efficiency. SEP39 is a major protease involved in SCUT-3’s keratin hydrolysis, and previous work showed that its overexpression could increase SCUT-Osep39’s keratinase activity. We previously showed that co-overexpression of CDO1 and SEP39 by using constitutive promoter ermE increased SCUT-3’s feather degrading efficiency, which implied that CDO and SEP39 play an important role in the degradation of feather. However, we also found that SCUT-Ocdo-Osep39 showed lower growth rate than wild-type SCUT-3. To increase the degradation efficiency of SCUT-3 and not affect the growth of SCUT-3, we constructed recombinant Streptomyces by using promoters we select to replace ermE.

To verify our hypothesis, the feather degradation efficiency of strain SCUT-Ocdo-p1380-sep39, SCUT-p22610-Ocdo-Osep39 and SCUT-Ocdo-p24880-Osep39 was compared with wild-type SCUT-3 and SCUT-Ocdo-Osep39. The total peptide and amino acid yields of were highest in SCUT-Ocdo-p24880-Osep39, and tests indicated that there was significant difference between SCUT-Ocdo-p24880-Osep39 and SCUT-3, SCUT-Ocdo-Osep39 on 48h.

4.1 Determination of keratinase activities

Fig. 7 showes the keratinase activities of SCUT-3 and this 4 recombinant groups in in 10% CFM fermentation at different times. From the picture, we can see SCUT-Ocdo-p24880-Osep39 demonstrated highest keratinase activity at 48 h. This affirmed that p24880 can be further used to replace ermE for feather degradation.

Figure 7 Keratinase activities detected in SCUT-3 and recombinant SCUT-3 in 10% CFM fermentation at different time.

4.2 Determination of amino acid content

According to Fig. 8, at the first 24h, the SCUT-Ocdo-p24880-Osep39 has already exhibited high amino acid content. At 48 h, the SCUT-Ocdo-p24880-Osep39 still showed highest amino acid content among all five groups, which is over 35000 μg/mL, and significantly higher than SCUT-3 and SCUT-Ocdo-Osep39.

Figure 8 Amino acid content from SCUT-3 and recombinant SCUT-3 in 10% CFM fermentation at 24 h and 48 h, *P < 0.05. P-values between groups were obtained by unpaired two-tailed Student’s t-test. All data were presented as mean ± SD.

4.3 Determination of peptide content

Figure 9 shows the content of peptide for this 5 groups in 10% CFM fermentation at 24 h and 48 h. SCUT-Ocdo-p24880-Osep39 exhibited highest content both at 24 h and 48 h, which is approximately 22 mg/mL at 48 h. The peptide content of SCUT-Ocdo-p24880-Osep39 is significantly higher than SCUT-3 and SCUT-Ocdo-Osep39.

Figure 9 Peptide content from SCUT-3 and recombinant SCUT-3 in 10% CFM fermentation at 24 h and 48 h, *P < 0.05. P-values between groups were obtained by unpaired two-tailed Student’s t-test. All data were presented as mean ± SD.

4.4 Determination of peptide and amino acid content

Figure 10 shows the feather protein conversion for this 5 groups in 10% CFM fermentation at 24 h and 48 h. SCUT-Ocdo-p24880-Osep39 exhibited highest content both at 24 h and 48 h, which is approximately 57000 μg/mL at 48 h. The peptide and amino acid content of SCUT-Ocdo-p24880-Osep39 is significantly higher than SCUT-3 and SCUT-Ocdo-Osep39.

Figure 10 Peptide and amino acid content from SCUT-3 and recombinant SCUT-3 in 10% CFM fermentation at 24 h and 48 h, *P < 0.05. P-values between groups were obtained by unpaired two-tailed Student’s t-test. All data were presented as mean ± SD.

4.5 Analysis of feather degradation rate

Figure 11 shows the degradation rate for different groups. SCUT-Ocdo-p24880-Osep39 showed highest degradation rate; SCUT-Ocdo-p24880-Osep39 had the highest feather-degrading ability. The degradation rate of SCUT-Ocdo-p24880-Osep39 was significantly higher than SCUT-3, but has no significant difference with SCUT-Ocdo-p1380-Osep39.

Figure 11 Comparison of the degradation rate between SCUT-3 and recombinant SCUT-3; *P < 0.05. P-values between groups were obtained by unpaired two-tailed Student’s t-test. All data were presented as mean ± SD.

5. Growth conditions of various SCUT-3 strains

Earlier studies found that co-overexpression of cdo1 and sep39 under constitutive promoter ermE resulted in SCUT-Ocdo-Osep39 yielding fewer cells than wild type SCUT and SCUT-Osep39 but more than SCUT-Ocdo. In our experiments, the bacterial growth condition tests showed that the overexpression of sep39 under promoter p24880 did not affect wild type SCUT’s growth (no significant difference compared to the wild-type) which demonstrated that the negative effect of cdo1 overexpression disappeared.

Figure 12 Bacterial growth condition of wild type SCUT-3 (A), SCUT-Ocdo-Osep39 (B), SCUT-Ocdo-p1380-Osep39 (C), SCUT-p22610-Ocdo-Osep39 (D) and SCUT-Ocdo-p24880-Osep39 (E).

Future Prospects and Concern

Because of the resulting pollution, amino acid destruction, low solubility and difficult to be digested by animals, traditional physical/chemical treatment methods for feather waste are gradually being abandoned[1]. The most promising alternative approaches involve biodegradation using bacteria. Through studies of using microbial means to convert accumulated millions tonnes of feathers into keratin, we take one step further to accomplish turning feather, instead of a burdensome waste, into rich, widely usable resource of protein and valuable raw materials for many fields.

(1) Biomedical applications of keratin. Keratin based biomaterials have been widely produced and used in various biomedical applications, such as bone tissue engineering[2, 3], wound healing[4, 5], nerve regeneration[6], skin replacement[7] and controlled drug delivery[8-10]. Our research is a simple, green, cost effective and yet efficient methodologies for the better extraction of keratin from feather. It is expected that keratin we extracted will turn into a mainstream biomaterial for clinical trials.

(2) Feather is a natural source of amino acids, peptides, and minerals that can be used in animal feed supplements and as a nitrogen fertilizer for plants[11]. Biodegradation of feather by microbial is a safe, sanitary and cost-effective technology that can play an important role in in processing feather waste.

Our study would shed light on the feather utilization mechanisms of FDB and provide a highly efficient, cost-effective way for degradation of feathers and extraction of available keratin, amino acids. Further exploration of the physiological functions of CDO and Sep39 in this Streptomyces. SCUT-3 will likely provide additional insights into biodegradation using bacteria.

Reference

[1] Li Z W, Liang S, Ke Y, et al. The feather degradation mechanisms of a new Streptomyces sp. isolate SCUT-3 [J]. Commun Biol, 2021, 3(1): 191.

[2] Li J S, Li Y, Liu X, et al. Strategy to introduce an hydroxyapatite-keratin nanocomposite into a fibrous membrane for bone tissue engineering [J]. J Mater Chem B, 2013, 1(4): 432-7.

[3] Tachibana A, Nishikawa Y, Nishino M, et al. Modified keratin sponge: Binding of bone morphogenetic protein-2 and osteoblast differentiation [J]. Journal of Bioscience and Bioengineering, 2006, 102(5): 425-9.

[4] Ham T R, Lee R T, Han S, et al. Tunable keratin hydrogels for controlled erosion and growth factor delivery [J]. Biomacromolecules, 2016, 17(1): 225-36.

[5] Nakata R, Tachibana A, Tanabe T. Preparation of keratin hydrogel/hydroxyapatite composite and its evaluation as a controlled drug release carrier [J]. Mater Sci Eng C Mater Biol Appl, 2014, 41(59-64.

[6] Lin Y C, Ramadan M, Van Dyke M, et al. Keratin gel filler for peripheral nerve repair in a rodent sciatic nerve injury model [J]. Plast Reconstr Surg, 2012, 129(1): 67-78.

[7] Xu S, Sang L, Zhang Y, et al. Biological evaluation of human hair keratin scaffolds for skin wound repair and regeneration [J]. Mater Sci Eng C Mater Biol Appl, 2013, 33(2): 648-55.

[8] Yin X C, Li F Y, He Y F, et al. Study on effective extraction of chicken feather keratins and their films for controlling drug release [J]. Biomater Sci, 2013, 1(5): 528-36.

[9] Guo J, Pan S, Yin X, et al. pH-sensitive keratin-based polymer hydrogel and its controllable drug-release behavior [J]. Journal of Applied Polymer Science, 2014.

[10] Shavandi A, SilVA T H, Bekhit A A, et al. Keratin: dissolution, extraction and biomedical application [J]. Biomater Sci, 2017, 5(9): 1699-735.

[11] Kornillowicz-kowalska T, Bohacz J. Biodegradation of keratin waste: Theory and practical aspects [J]. Waste Manag, 2011, 31(8): 1689-701.

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