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Construction of a Genetic System for Streptomyces albulus PD-1 and Improving Poly(ε-ʟ-lysine) Production Through Expression of Vitreoscilla Hemoglobin

  • Xu, Zhaoxian (State Key Laboratory of Materials-Oriented Chemical Engineering) ;
  • Cao, Changhong (State Key Laboratory of Materials-Oriented Chemical Engineering) ;
  • Sun, Zhuzhen (State Key Laboratory of Materials-Oriented Chemical Engineering) ;
  • Li, Sha (State Key Laboratory of Materials-Oriented Chemical Engineering) ;
  • Xu, Zheng (State Key Laboratory of Materials-Oriented Chemical Engineering) ;
  • Feng, Xiaohai (State Key Laboratory of Materials-Oriented Chemical Engineering) ;
  • Xu, Hong (State Key Laboratory of Materials-Oriented Chemical Engineering)
  • Received : 2015.07.02
  • Accepted : 2015.08.03
  • Published : 2015.11.28

Abstract

Poly(ε-ʟ-lysine) (ε-PL) is a novel bioactive polymer secreted by filamentous bacteria. Owing to lack of a genetic system for most ε-PL-producing strains, very little research on enhancing ε-PL biosynthesis by genetic manipulation has been reported. In this study, an effective genetic system was established via intergeneric conjugal transfer for Streptomyces albulus PD-1, a famous ε-PL-producing strain. Using the established genetic system, the Vitreoscilla hemoglobin (VHb) gene was integrated into the chromosome of S. albulus PD-1 to alleviate oxygen limitation and to enhance the biosynthesis of ε-PL in submerged fermentation. Ultimately, the production of ε-PL increased from 22.7 g/l to 34.2 g/l after fed-batch culture in a 5 L bioreactor. Determination of the oxygen uptake rate, transcriptional level of ε-PL synthetase gene, and ATP level unveiled that the expression of VHb in S. albulus PD-1 enhanced ε-PL biosynthesis by improving respiration and ATP supply. To the best of our knowledge, this is the first report on enhancing ε-PL production by chromosomal integration of the VHb gene in an ε-PL-producing strain, and it will open a new avenue for ε-PL production.

Keywords

Introduction

Poly(ε-ʟ-lysine) (ε-PL) is one of the four homopoly (amino acid)s discovered in nature until now. It is linked by the isopeptide bond between ε-amino and α-carboxyl groups of ʟ-lysine residues [8]. Owing to its excellent properties, including antimicrobial activity, biodegradability, water solubility, edibility, and non-toxicity to humans and the environment, ε-PL has been used in many fields, such as food, medicine, cosmetics, and electronics [2,26,27]. As a novel biopolymer, ε-PL is mostly synthesized by filamentous bacteria through submerged fermentation, and various studies have reported on the achievement of efficient production of ε-PL, including pH regulation, dissolved oxygen (DO) regulation, in situ adsorption, and cheap raw materials application [1,12,16,24,25,35]. However, because of the lack of a genetic system for most ε-PL-producing strains, very little research on improving ε-PL production by genetic manipulation has yet been conducted.

In the fermentation of Streptomyces albulus PD-1, a famous ε-PL-producing strain, it was shown that the DO concentration was a significant parameter for ε-PL production, and a higher DO level (about 30% saturation) was beneficial to ε-PL biosynthesis [35]. However, owing to the intertwined hyphae and high cell density, the culture broth became viscous during the fermentation process and oxygen transfer was limited, thus inhibiting cell growth and ε-PL biosynthesis. Although this issue can be partially settled by increasing agitation rates, the corresponding high shear stress will cause undesirable effects on the mycelium, and thus decrease product yields [7,13]. Besides this, the increasing agitation rate will also lead to additional energy cost. Thus, an effective approach is urgently needed to solve the oxygen limitation problem in ε-PL production. With the development of genetics and molecular biology, genetic manipulation provides new strategies that can complement the traditional methods to solve some traditional chemical engineering problems in bioprocesses. However, as mentioned above, owing to the lack of a genetic system for most ε-PL-producing strains, no research on solving the oxygen limitation by genetic manipulation has yet been reported in the ε-PL production process.

Vitreoscilla hemoglobin (VHb) was the first bacterial hemoglobin discovered in nature [31]. Its main function is to bind oxygen, especially under oxygen-limited conditions, and deliver the oxygen to the terminal respiratory oxidase, thus enhancing bacterial respiration and oxidative phosphorylation [4,21]. Recent studies have demonstrated that heterogeneous expression of VHb can significantly enhance the production of many valuable compounds in microorganisms [11,17,28,29,39]. Thus, whether the heterogeneous expression of VHb could improve S. albulus PD-1 to produce more ε-PL will be of great significance.

In the present study, to open up the possibility of genetic manipulation for S. albulus PD-1, we directed our efforts toward the development of a genetic system for it. By using the established system, the VHb expression cassette was integrated into the chromosome of the ε-PL-producing strain for the first time. The fermentation results indicated that the ε-PL titer was significantly improved by the expression of VHb. Furthermore, to investigate the effects of VHb on S. albulus PD-1 in submerged fermentation, the oxygen uptake rate, transcriptional levels of ε-PL synthetase gene, and ATP level were also detected. This study would open a new avenue for enhancing ε-PL biosynthesis by genetic manipulation and become a good example for solving oxygen-limited problems in submerged fermentation processes.

 

Materials and Methods

Microorganisms, Plasmids, and Media

S. albulus PD-1 (Accession No. M2011043), a well-known ε-PL-producing strain, was employed as the wild-type strain in this study. Escherichia coli ET12567 (pUZ8002) was employed as the donor in intergeneric conjugal transfer. The site-specific integration vector, pIB139, which is a pSET152 derivative with a strong constitutive ermE* promoter, was used for intergeneric conjugal transfer and VHb expression.

Luria-Bertani (LB) medium (yeast extract, 5 g/l; tryptone, 10 g/l;NaCl, 10 g/l) was used for E. coli cultivation. Mannitol-soy flour (MS) agar medium (mannitol, 20 g/l; soy flour, 20 g/l; agar, 20 g/l), AS-1 (soluble starch, 5 g/l; yeast extract, 10 g/l; L-arginine, 0.5 g/1;ʟ-alanine, 0.2 g/l; Na2SO4, 10 g/l; NaCl, 2.5 g/l; agar, 20 g/l; pH 7.5), ISP-2 (yeast extract, 4 g/l; malt extract, 10 g/l; glucose, 4 g/l;agar, 20 g/l), and ISP-4 (soluble starch, 10 g/l; (NH4)2SO4, 2 g/l;MgSO4·7H2O, 2 g/l; CaCO3, 2 g/l; NaCl, 1 g/l; K2HPO4, 1 g/l;ZnSO4, 1 mg/l; FeSO4·7H2O, 1 mg/l; MnCl2, 1 mg/l; agar, 20 g/l;pH 7.2) media were used for conjugal transfer. Medium 3G (M3G) (glucose, 50 g/l; (NH4)2SO4, 10 g/l; yeast extract, 5 g/l; KH2PO4, 1.36 g/l; K2HPO4, 0.8 g/l; MgSO4·7H2O, 0.5 g/l; ZnSO4·7H2O, 0.04 g/l; FeSO4·7H2O, 0.03 g/l; pH 6.8) was used for seed culture, as well as for fed-batch fermentation of S. albulus PD-1.

Conjugal Transfer Method

Conjugal transfer was conducted as described previously with some modifications [15]. E. coli ET12567 (pUZ8002) harboring plasmid pIB139 was grown in 10 ml of LB medium to an OD600 of 0.45 in the presence of kanamycin (50 μg/ml), chloramphenicol (50 μg/ml), and apramycin (50 μg/ml). Cells were then washed twice to remove the antibiotics and were subsequently resuspended in 500 μl of fresh LB medium. While washing the E. coli cells, S. albulus PD-1 spores (about 108) were suspended in 500 μl of LB medium. Subsequently, E. coli donor cells and S. albulus PD-1 spores were mixed thoroug hly and spread on 20 ml of agarmedium (MS, ISP-2, ISP-4, or AS-1) containing 10 mM MgCl2. The plates were incubated at 30℃ for about 18 h and then overlaid with 1 ml of sterile water containing 0.05 mg of nalidixic acid and 0.1 mg of apramycin. The plates were further incubated at 30℃ for about 3 days, and the ex-conjugants were counted.

Construction of Recombinant Plasmid pIB139-vgb

The VHb gene (vgb) (Accession No. AF292694, gifted by the Institute of Biochemistry and Cell Biology, SIBS, CAS, China) was amplified by PCR using the following primers: P1 (5'-GGAATTCCATATGGTGCTGGACCAGCAAAC-3', NdeI site underlined) and P2 (5'-GCTCTAGATTATTCAACCGCTTGAG-3', XbaI site underlined). Subsequently, the PCR product was digested with NdeI and XbaI, gel purified, and ligated into the corresponding sites of pIB139 to generate pIB139-vgb (Fig. 1A). The plasmid pIB139-vgb was identified by restriction digestion and DNA sequencing. Then, the identified recombinant plasmid was introduced into S. albulus PD-1 via the conjugal transfer method mentioned above.

Fig. 1.(A) Construction of recombinant plasmid pIB 139-vgb. and (B) PCR amplification for identification of the ex-conjugant. Lane M, DL 5000 marker; lane 1, the PCR fragment using the chromosome of S. albulus PD-1 as template; lane 2, the PCR fragment using the chromosome of ex-conjugant as template.

Cultivation Conditions

Submerged fermentation experiments were performed in flasks and a bioreactor. For seed cultures, a loop of 1-week-old fully grown spores was inoculated into 100 ml of M3G medium contained in a 500 ml flask and then incubated at 30℃ and 200 rpm for 24 h as stock culture. To explore the VHb effects on ε-PL biosynthesis in flask fermentation, especially under oxygen-limited conditions, 10% seeds were inoculated into 45, 90, 135, and 180 ml of M3G medium in 500 ml flasks, and the yield of ε-PL was determined after the cultivation was incubated at 30℃ and 200 rpm for 72 h. Fed-batch fermentations were conducted in a 5 L bioreactor (KoBio Tech Co., Ltd., Korea) by using a two-stage pH control strategy [12,34]. In the first stage, the pH was controlled at 6.0 for cell growth, and in the second stage, the pH was controlled at 4.0 for ε-PL biosynthesis. When the glucose concentration in the culture broth decreased to about 10 g/l, the feeding solution (glucose, 500 g/l; (NH4)2SO4, 50 g/l) was pumped into the broth to keep the glucose concentration at approximately 10 g/l.

Determination of Cell Growth, Glucose Concentration, pH, DO, and ATP Level

Cell growth was measured in terms of dry cell weight (DCW). The harvested culture sample was filtered; the mycelia were washed a nd d ried a t 65℃ until constant weight was a chieved. Glucose concentration in the culture broth was determined by using a biosensor (SBA-40C; Shandong Science Academy, China). The yield of ε-PL was determined by high-performance liquid chromatography, following the method reported previously [34]. Intracellular ATP concentration was quantified by a chemiluminescence response method as reported before [35]. The pH and DO were measured by probes of the bioreactor. The oxygen uptake rate (OUR) of bacteria was analyzed with a process mass spectrometer (SHP8400PMS-162R). All assays were performed in triplicate, and experimental errors were <5%.

Analysis for the Biological Activity of VHb

The CO-difference spectral analysis was performed to determine the biological activity of VHb according to the previously described method [39].

Analysis of the Transcriptional Level of ε-PL Synthetase Gene

As the fermentation process reached 110 h, the maximum specific ε-PL production rate was reached. At this moment, the transcriptional level of the ε-PL synthetase gene (pls) was determinated by quantitative real-time PCR (qRT-PCR) according to the method mentioned before [35]. The relative gene transcriptional level was calculated by the 2-ΔΔCt method, with hrdB (a housekeeping sigma factor gene) as the endogenous control gene [32,38]. For pls and hrdB, the following primers were used: pls-F, 5'-CGGATTCGTCCAACTCCT-3' and pls-R, 5'-GACGATGATCAGCCACCA-3'; hrdB-F, 5'-CGACTACACCAAGGGCTACA-3' and hrdB-R, 5'-TTGTTGATGACCTCGACCAT-3'.

 

Results and Discussion

Construction of a Genetic System for S. albulus PD-1

With the development of molecular biology, intensive genetic engineering applications have been applied in biotechnology [19,20]. In such applications, the target DNA fragments should be transferred into candidate cells first. Streptomycetes are well known for their ability to produce many valuable secondary metabolites. In order to obtain the genetically modified streptomycetes, one of the key challenges is delivering the target DNA fragments into living streptomycetes cells efficiently. However, owing to the slow growth and thick cell wall of most streptomycetes, the genetic system of these organisms has obstacles compared with manipulations in E. coli, yeast, and other commonly used industrial strains [18]. With the continuous efforts of researchers, some kinds of genetic systems were developed for streptomycetes, including polyethylene glycol-mediated method, electrotransformation, and intergeneric conjugal transfer [5,10,18,22,23]. Among the three methods, intergeneric conjugal transfer has been performed in many streptomycetes because of its high transformation efficiency, and reproducibility. Thus, we attempted to construct an intergeneric conjugal transfer system for S. albulus PD-1.

It has been reported that the type of medium used has a significant effect on the efficiency of conjugation. Thus, four representative media (AS-1, ISP-2, ISP-4, and MS) were used for conjugal transfer for S. albulus PD-1 [3,22]. As shown in Table 1, the highest transformation frequency (4.0 ± 0.5 × 10-7 per recipient) was observed when MS medium was used for conjugal transfer. Thus, MS medium was selected as the most appropriate medium and was employed in the subsequent experiments. To confirm whether pIB139 was integrated into the chromosome of S. albulus PD-1, the apramycin resistance gene was amplified from the chromosome of transformants, but not from wild-type strain S. albulus PD-1 (data not shown). In particular, the integration of plasmid pIB139 had no effect on cell growth and ε-PL production of S. albulus PD-1, and it was stable after passing several generations. In previous studies, Hamano et al. [9] made a lot of effort to construct the genetic system for S. albulus IFO 14147, which is the only ε-PL-producing strain owning the genetic systems. In their studies, they spent much time constructing free-replicating plasmids for S. albulus IFO 14147. Even though the free-replicating plasmids have many uses for genetic operation, stable maintenance of a free-replicating plasmid requires selection via expensive antibiotics. Compared with free-replicating plasmids, the plasmid pIB139 (capable of integration into ϕ31 attB site in S. albulus PD-1) could be more stable in streptomycetes, which would not only reduce the cost of additional antibiotics but also prevent the contamination caused by antibiotics. Thus, pIB139 was used for VHb expression in the following work.

Table 1.Effect of medium type on the intergeneric conjugation efficiency.

Construction of S. albulus PD-2

For heterologous expression of VHb in S. albulus PD-1, the vgb was cloned and placed under the control of ermE* promoter in plasmid pIB139 to create pIB139-vgb (Fig. 1A). Subsequently, the plasmid pIB139-vgb was introduced into S. albulus PD-1 via the established genetic system. The ex-conjugant was designated as S. albulus PD-2, which was verified by PCR amplification by using the primer pair P1/P2. A PCR product of 450 bp was obtained during electrophoresis when chromosomal DNA from S. albulus PD-2 was used as the PCR template, whereas no band was observed when chromosomal DNA from S. albulus PD-1 was used (Fig. 1B). This observation confirmed that the vgb had been successfully integrated into the chromosome of S. albulus PD-2.

The biological activity of VHb was measured using the CO-difference spectra. The CO-difference spectra showed a significant VHb CO-binding absorbance peak at 420 nm from the crude cell extract of S. albulus PD-2 compared with that of the wild-type strain (Fig. 2). This typical peak of VHb protein demonstrated that the expressed VHb in S. albulus PD-2 was biologically active [17,39]. Taken together, by using the established genetic system, the biologically active VHb was successfully expressed in S. albulus PD-2.

Fig. 2.CO-difference spectral analysis of crude extracts of S. albulus PD-1 and S. abulus PD-2.

Comparison of ε-PL Production for Different Loading Volumes in Flask Culture

Heterologous expression of VHb has been demonstrated in many strains to improve cell growth and secondary metabolite productivity, especially under oxygen-limited conditions [33,39]. To reveal the effect of VHb expression on ε-PL production, S. albulus PD-1 and S. albulus PD-2 were cultured in 500 ml flasks first, with increasing loading volumes from 50 ml to 200 ml. As shown in Table 2, the loading volume had a strong effect on cell growth and ε-PL production. In all these cases, the concentration of ε-PL and DCW decreased with the increasing loading volume. According to a report, the increasing liquid volume decreases the volumetric oxygen mass transfer rate and leads to oxygen limitation [30]. This phenomenon could be the main reason for the decrease in ε-PL production and cell growth. However, the expression of VHb in S. albulus PD-2 was functional for reducing the unsatisfactory effect caused by the high liquid volume. In addition, when the liquid volume was increased, the effect of VHb was more significant. With 50 ml of broth in a 500 ml flask, the expression of VHb could only contribute to 0.16-fold increase of ε-PL titer. However, with 200 ml of broth in a 500 ml flask, the expression of VHb could lead to as much as 1.28-fold increase of ε-PL titer. Thus, our results supported that VHb expression in S. albulus PD-2 can solve the problems caused by limited oxygen and eventually increase ε-PL production.

Table 2.Summary of ε-PL yield in different volumes of culture broth.

Performance of Fed-Batch Operation Using Recombinant Strain

To verify the feasibility of VHb expression in actual production, S. albulus PD-1 and S. albulus PD-2 were further incubated in a 5 L bioreactor. As illustrated in Fig. 3C, for both strains, the DO concentrations in the culture broth decreased continuously at the early stage of the fermentation period. Finally, the DO concentration maintained at about 23.5% after 55 h, compared with 18.9% after 52 h in S. albulus PD-2 fermentation process. In another words, the DO level in the culture broth of S. albulus PD-2 was lower compared with that in S. albulus PD-1. A similar phenomenon has been reported for S. diastatochromogenes [17]. In previous studies, researchers found that VHb can increase the intracellular effective DO concentrations by allowing a more effective intracellular delivery of oxygen [6,28]. The lower DO in S. albulus PD-2 culture broth may be because more oxygen was transported to cells and metabolized rapidly. Therefore, the oxygen uptake rate (OUR) values of the wild-type strain and of S. albulus PD-2 were determined. As depicted in Fig. 3D, the OUR values of S. albulus PD-2 were higher than those of the wild-type strain regardless of the limited DO concentration during the 55th-168th h or not (0-55 h). The high OUR supported the increased respiration of the recombinant strain with VHb expression. The enhancement of respiration will provide more energy for both cell growth and ε-PL biosynthesis. These results explained the growth advantage (Fig. 3B) and high ε-PL production (Fig. 3A) exhibited by S. albulus PD-2.

Fig. 3.Time curve analysis of ε-PL production (A), cell growth (B), DO concentration (C), OUR (D), specific ε-PL production rate (E), and specific cell growth rate (F) of S. albulus PD-1 and S. albulus PD-2 in a 5 L bioreactor culture experiment.

Ultimately, S. albulus PD-2 obtained a final biomass concentration of 33.4 g/l and ε-PL of 34.2 g/l, which corresponded to 27.5% and 50.7% increase, respectively, compared with the wild strain. Moreover, the specific ε-PL production rate of S. albulus PD-2 was higher than that of the wild-type strain (Fig. 3E), indicating that the improvement of ε-PL production was not only due to the higher biomass volume of S. albulus PD-2 (Figs. 3B and 3F) but that the expression of VHb also strengthened the ε-PL synthesis ability of the signal cell. Recently, oxygen-vectors were added in culture broth to alleviate the oxygen-limited problem in the fermentation process of S. albulus PD-1 [35]. As a result, 30.8 g/l of ε-PL could be produced with 0.5% n-dodecane in the culture broth. Compared with oxygen-vector applications, the expression of VHb not only resulted in a higher ε-PL production but also saved the cost brought by oxygen-vectors addition. Thus, the expression of VHb provides a new strategy that can complement the existing methods for alleviating low DO concentrations in the ε-PL production process.

Changes of ATP Level in S. albulus PD-2

Until now, several hypotheses have been raised to elucidate the function of VHb. The dominant view at present is that VHb could bind oxygen and deliver the oxygen to the terminal respiratory oxidase and/or oxygenases, thus enhancing bacterial respiration and oxidative phosphorylation [4,14,21]. In short, VHb can take part in one or more steps of the respiratory chain and thus promote respiration of bacteria. The increase in OUR of S. albulus PD-2 agreed with those perspectives, and the enhancement of respiratory action may probably lead to a higher ATP, which is an essential cofactor in ε-PL biosynthesis. To verify this speculation, the ATP level was also determined during the fermentation process.

Fig. 4 shows the ATP level of S. albulus PD-2 and the wild-type strain. Amongst the variation trends, the ATP level in S. albulus PD-2 was higher than that of the wild-type strain at all times. The fact that high ATP level is a benefit for ε-PL synthetase (Pls) has been demonstrated in some ε-PL-producing strains [35,36]. As reported, ATP regulates ε-PL in two ways: first, ATP is directly involved in the assembly of ε-PL through activing lysine to lysyl-O-AMP [37]; second, a high ATP level is essential for the expression of pls [36]. The increase of transcriptional level of pls in S. albulus PD-2 also validated the improvement of ATP level indirectly (Fig. 5). Thus, we hold the opinion that with the expression of VHb in S. albulus PD-2, the respiratory action was enhanced and more ATP was generated. The high ATP level in S. albulus PD-2 stimulated ε-PL biosynthesis by improving the transcriptional level of pls and activing more lysine to participate in ε-PL assembly.

Fig. 4.Comparison of ATP level changing patterns between S. albulus PD-1 and S. albulus PD-2 in a 5 L bioreactor culture experiment.

Fig. 5.qRT-PCR analysis of the change of pls transcriptional level with VHb expression.

In conclusion, in the present study, we constructed a genetic system for S. albulus PD-1. By using the established genetic system, vgb was integrated into the chromosome of S. albulus PD-1 for the first time. The expression of VHb in S. albulus PD-1 relieved the unsatisfactory effect caused by limited oxygen in the culture broth, and significantly enhanced ε-PL production compared with the wild-type strain. By measuring the oxygen uptake rate, the transcriptional level of pls, and ATP level, it can be concluded that the expression of VHb in S. albulus PD-1 enhanced ε-PL biosynthesis through improving respiration and the ATP supply. We believe that this study will open a new avenue for ε-PL production by genetic engineering.

References

  1. Bankar SB, Singhal RS. 2011. Improved poly-ε-lysine biosynthesis using Streptomyces noursei NRRL 5126 by controlling dissolved oxygen during fermentation. J. Microbiol. Biotechnol. 21: 652-658.
  2. Bankar SB, Singhal RS. 2013. Panorama of poly-ε-lysine. RSC Adv. 3: 8586-8603. https://doi.org/10.1039/c3ra22596h
  3. Cheng YQ, Tang GL, Shen B. 2002. Identification and localization of the gene cluster encoding biosynthesis of the antitumor macrolactam leinamycin in Streptomyces atroolivaceus S-140. J. Bacteriol. 184: 7013-7024. https://doi.org/10.1128/JB.184.24.7013-7024.2002
  4. Chi P, Webster D, Stark B. 2009. Vitreoscilla hemoglobin aids respiration under hypoxic conditions in its native host. Microbiol. Res. 164: 267-275. https://doi.org/10.1016/j.micres.2006.11.018
  5. Flett F, Mersinias V, Smith CP. 1997. High efficiency interg eneric conjug al transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting streptomycetes. FEMS Microbiol. Lett. 155: 223-229. https://doi.org/10.1111/j.1574-6968.1997.tb13882.x
  6. Frey A, Kallio P. 2003. Bacterial hemoglobins and flavohemoglobins: versatile proteins and their impact on microbiology and biotechnology. FEMS Microbiol. Rev. 27: 525-545. https://doi.org/10.1016/S0168-6445(03)00056-1
  7. Gao W, Chung CH, Li J, Lee JW. 2015. Enhanced production of cellobiase by marine bacterium Cellulophaga lytica LBH-14 from rice bran under optimized conditions involved in dissolved oxygen. Biotechnol. Bioprocess Eng. 20: 131-138. https://doi.org/10.1007/s12257-014-0486-6
  8. Hamano Y, Arai T, Ashiuchi M, Kino K. 2013. NRPSs and amide ligases producing homopoly(amino acid)s and homooligo(amino acid)s. Nat. Prod. Rep. 30: 1087-1097. https://doi.org/10.1039/c3np70025a
  9. Hamano Y, Nicchu I, Hoshino Y, Kawai T, Nakamori S, Takagi H. 2005. Development of gene delivery systems for the ε-poly-ʟ-lysine producer, Streptomyces albulus. J. Biosci. Bioeng. 99: 636-641. https://doi.org/10.1263/jbb.99.636
  10. Hwang YS, Lee JY, Kim ES, Choi CY. 2001. Optimization of transformation procedures in avermectin high-producing Streptomyces avermitilis. Biotechnol. Lett. 23: 457-462. https://doi.org/10.1023/A:1010377219368
  11. Jeong JW, Park KM, Chung M, Won JI. 2015. Influence of Vitreoscilla hemoglobin gene expression on 2,3-butanediol production in Klebsiella oxytoca. Biotechnol. Bioprocess Eng. 20: 10-17. https://doi.org/10.1007/s12257-014-0642-z
  12. Kahar P, Iwata T, Hiraki J, Park EY, Okabe M. 2001. Enhancement of ε-polylysine production by Streptomyces albulus strain 410 using pH control. J. Biosci. Bioeng. 91: 190-194. https://doi.org/10.1016/S1389-1723(01)80064-5
  13. Kahar P, Kobayashi K, Iwata T, Hiraki J, Kojima M, Okabe M. 2002. Production of ε-polylysine in an airlift bioreactor (ABR). J. Biosci. Bioeng. 93: 274-280. https://doi.org/10.1016/S1389-1723(02)80028-7
  14. Khosla C, Curtis J, DeModena J, Rinas U, Bailey J. 1990. Expression of intracellular hemoglobin improves protein synthesis in oxygen-limited Escherichia coli. Nat. Biotechnol. 8: 849-853. https://doi.org/10.1038/nbt0990-849
  15. Kieser T. 2000. Practical Streptomyces Genetics. John Innes Foundation.
  16. Liu SR, Zhang JM. 2015. Efficient production of ε-poly-ʟ-lysine by Streptomyces ahygroscopicus using one-stage pH control fed-batch fermentation coupled with nutrient feeding. J. Microbiol. Biotechnol. 25: 358-365. https://doi.org/10.4014/jmb.1405.05069
  17. Ma Z, Liu J, Bechthold A, Tao L, Shentu X, Bian Y, Yu X. 2014. Development of intergeneric conjugal gene transfer system in Streptomyces diastatochromogenes 1628 and its application for improvement of toyocamycin production. Curr. Microbiol. 68: 180-185. https://doi.org/10.1007/s00284-013-0461-z
  18. Mazodier P, Petter R, Thompson C. 1989. Intergeneric conjugation between Escherichia coli and Streptomyces species. J. Bacteriol. 171: 3583-3585. https://doi.org/10.1128/jb.171.6.3583-3585.1989
  19. Nozzi NE, Desai SH, Case AE, Atsumi S. 2014. Metabolic engineering for higher alcohol production. Metab. Eng. 25: 174-182. https://doi.org/10.1016/j.ymben.2014.07.007
  20. Ortiz-Marquez JCF, Nascimento MD, Zehr JP, Curatti L. 2013. Genetic engineering of multispecies microbial cell factories as an alternative for bioenergy production. Trends Biotechnol. 31: 521-529. https://doi.org/10.1016/j.tibtech.2013.05.006
  21. Park K, Kim K, Howard A, Stark B, Webster D. 2002. Vitreoscilla hemoglobin binds to subunit I of cytochrome boubiquinol oxidases. J. Biol. Chem. 277: 33334-33337. https://doi.org/10.1074/jbc.M203820200
  22. Phornphisutthimas S, Sudtachat N, Bunyoo C, Chotewutmontri P, Panijpan B, Thamchaipenet A. 2010. Development of an intergeneric conjugal transfer system for rimocidin-producing streptomyces rimosus. Lett. Appl. Microbiol. 50: 530-536. https://doi.org/10.1111/j.1472-765X.2010.02835.x
  23. Pigac J, Schrempf H. 1995. A simple and rapid method of transformation of Streptomyces rimosus R6 and other Streptomycetes by electroporation. Appl. Environ. Microbiol. 61: 352-356.
  24. Ren XD, Chen XS, Zeng X, Wang L, Tang L, Mao ZG. 2015. Acidic pH shock induced overproduction of ε-poly-ʟ-lysine in fed-batch fermentation by Streptomyces sp. M-Z18 from agro-industrial by-products. Bioproc. Biosyst. Eng. 38: 1113-1125. https://doi.org/10.1007/s00449-015-1354-2
  25. Rong LS, Zhang JM, Yang XJ. 2012. Enhanced ε-poly-ʟ-lysine production from Streptomyces ahygroscopicus by a combination of cell immobilization and in situ adsorption. J. Microbiol. Biotechnol. 22: 1218-1223. https://doi.org/10.4014/jmb.1111.11039
  26. Shih IL, Shen MH, Van YT. 2006. Microbial synthesis of poly (ε-lysine) and its various applications. Bioresour. Technol. 97: 1148-1159. https://doi.org/10.1016/j.biortech.2004.08.012
  27. Shukla S, Singh A, Pandey A, Mishra A. 2012. Review on production and medical applications of ε-polylysine. Biochem. Eng. J. 65: 70-81. https://doi.org/10.1016/j.bej.2012.04.001
  28. Stark B, Pagilla K, Dikshit K. 2015. Recent applications of Vitreoscilla hemoglobin technology in bioproduct synthesis and bioremediation. Appl. Microbiol. Biotechnol. 94: 1-10.
  29. Suen YL, Tang H, Huang J, Chen F. 2014. Enhanced production of fatty acids and astaxanthin in Aurantiochytrium sp. by the expression of Vitreoscilla hemoglobin. J. Agric. Food Chem. 62: 12392-12398 https://doi.org/10.1021/jf5048578
  30. Tang B, Qiu B, Huang S, Yang K, Bin L, Fu F, Yang H. 2015. Distribution and mass transfer of dissolved oxygen in a multi-habitat membrane bioreactor. Bioresour. Technol. 182: 323-328. https://doi.org/10.1016/j.biortech.2015.02.028
  31. Wakabayashi S, Matsubara H, Webster D. 1986. Primary sequence of a dimeric bacterial haemoglobin from Vitreoscilla. Nature 322: 481-483. https://doi.org/10.1038/322481a0
  32. Wang G, Hosaka T, Ochi K. 2008. Dramatic activation of antibiotic production in Streptomyces coelicolor by cumulative drug resistance mutations. Appl. Environ. Microbiol. 74: 2834-2840. https://doi.org/10.1128/AEM.02800-07
  33. Wei X, Chen G. 2008. Applications of the VHb gene vgb for improved microbial fermentation processes. Methods Enzymol. 436: 273-287. https://doi.org/10.1016/S0076-6879(08)36015-7
  34. Xia J, Xu H, Feng X, Xu Z, Chi B. 2013. Poly (ʟ-diaminopropionic acid), a novel non-proteinic amino acid oligomer co-produced with poly(ε-ʟ-lysine) by Streptomyces albulus PD-1. Appl. Microbiol. Biotechnol. 97: 7597-7605. https://doi.org/10.1007/s00253-013-4936-4
  35. Xu Z, Bo F, Xia J, Sun Z, Li S, Feng X, Xu H. 2015. Effects of oxygen-vectors on the synthesis of epsilon-poly-lysine and the metabolic characterization of Streptomyces albulus PD-1. Biochem. Eng. J. 94: 58-64. https://doi.org/10.1016/j.bej.2014.11.009
  36. Yamanaka K, Kito N, Imokawa Y, Maruyama C, Utagawa T, Hamano Y. 2010. Mechanism of epsilon-poly-ʟ-lysine production and accumulation revealed by identification and analysis of an epsilon-poly-ʟ-lysine-degrading enzyme. Appl. Environ. Microbiol. 76: 5669-5675. https://doi.org/10.1128/AEM.00853-10
  37. Yamanaka K, Maruyama C, Takagi H, Hamano Y. 2008. ε-Poly-ʟ-lysine dispersity is controlled by a highly unusual nonribosomal peptide synthetase. Nat. Chem. Biol. 4: 766-772. https://doi.org/10.1038/nchembio.125
  38. Zhou T, Kim B, Zhong J. 2014. Enhanced production of validamycin A in Streptomyces hygroscopicus 5008 by engineering validamycin biosynthetic gene cluster. Appl. Microbiol. Biotechnol. 98: 7911-7922. https://doi.org/10.1007/s00253-014-5943-9
  39. Zhu H, Sun S, Zhang S. 2011. Enhanced production of total flavones and exopolysaccharides via Vitreoscilla hemoglobin biosynthesis in Phellinus igniarius. Bioresour. Technol. 102: 1747-1751. https://doi.org/10.1016/j.biortech.2010.08.085

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