World J Microbiol Biotechnol DOI 10.1007/s11274-014-1611-6
ORIGINAL PAPER
Enhancement of riboflavin production by deregulating gluconeogenesis in Bacillus subtilis Guanglu Wang • Ling Bai • Zhiwen Wang Ting Shi • Tao Chen • Xueming Zhao
•
Received: 17 April 2013 / Accepted: 19 January 2014 Ó Springer Science+Business Media Dordrecht 2014
Guanglu Wang and Ling Bai have contributed equally to this work.
overexpression of pckA obtained the opposite result. Significant enhancement of riboflavin titers up to 4.89 g/l was obtained in shake flask cultures when gapB and fbp were co-overexpressed, nevertheless the specific growth rate decreased slightly and the specific glucose uptake rate remained almost unchanged. An improvement by 21.9 and 27.8 % of the riboflavin production was achieved by cooverexpression of gapB and fbp in shake flask and fedbatch fermentation, respectively. These results imply that deregulation of gluconeogenesis is an effective strategy for production of metabolites directly stemming from the pentose phosphate pathway as well as other NADPHdemanding compounds with glucose as carbon source in B. subtilis.
Electronic supplementary material The online version of this article (doi:10.1007/s11274-014-1611-6) contains supplementary material, which is available to authorized users.
Keywords Riboflavin Bacillus subtilis Gluconeogenesis Pentose phosphate pathway
G. Wang L. Bai Z. Wang (&) T. Shi T. Chen X. Zhao Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, 92# Weijin Road, Nankai District, Tianjin 300072, People’s Republic of China e-mail: [email protected]
Abbreviations PPP Pentose phosphate pathway Ru5P Ribulose-5P FBPase Fructose 1,6-bisphosphatase MM Minimal medium CDW Cell dry weight TCA Tricarboxylic acid PEP Phosphoenolpyruvate
Abstract The regulation of metabolic flux through glycolytic versus the gluconeogenic pathway plays an important role in central carbon metabolism. In this study, we made an attempt to enhance riboflavin production by deregulating gluconeogenesis in Bacillus subtilis. To this end, gapB (code for NADPH-dependent glyceraldehyde-3phosphate dehydrogenase), fbp (code for fructose-1,6-bisphosphatase) and pckA (code for phosphoenolpyruvate carboxykinase) were overexpressed in parental strain B. subtilis RH33. Compared with RH33, overexpression of fbp and gapB resulted in approximately 18.0 and 14.2 % increased riboflavin production, respectively, while
G. Wang L. Bai Z. Wang T. Shi T. Chen X. Zhao Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin 300072, People’s Republic of China G. Wang L. Bai Z. Wang T. Shi T. Chen X. Zhao Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin 300072, People’s Republic of China
Introduction
G. Wang L. Bai Z. Wang T. Shi T. Chen X. Zhao Edinburgh-Tianjin Joint Research Centre for Systems Biology and Synthetic Biology, Tianjin University, Tianjin 300072, People’s Republic of China
Riboflavin (vitamin B2) is the direct precursor of FMN and FAD, one of the essential components in cellular physiology required by all bacteria, animals and plants. It has been
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used not only for pharmaceuticals but also for animal feed additives, cosmetics, and food industry. Currently, the fungi Ashbya gossypii, Eremothecium ashbyii, Candida flareri and the bacterium Bacillus subtilis have been successfully engineered as cell factories for riboflavin production (Stahmann et al. 2000; Kalingan and Liao 2002), whereas the commercial processes mainly rely on A. gossypii and B. subtilis. Bacillus subtilis has been a long tradition as safe and stable producers of purine nucleosides, inosine and guanosine (Sauer et al. 1998), which are the precursors for riboflavin biosynthesis. Biochemical, physiological, and genetic studies allowed the characterization of enzymes involved in riboflavin biosynthesis pathway in B. subtilis (Schallmey et al. 2004; Barbe et al. 2009; Sklyarova et al. 2012). It comprises seven enzymatic steps and starts from two precursors, ribulose-5P (Ru5P) deriving from pentose phosphate pathway (PPP) and GTP deriving from purine biosynthetic pathway (Dauner et al. 2001b). Commercial riboflavin production by B. subtilis has been developed by a combination of classical mutagenesis (Coquard et al. 1997; Perkins et al. 1999), rational metabolic engineering (Humbelin et al. 1999; Zamboni et al. 2003; Li et al. 2006; Duan et al. 2010; Hemberger et al. 2011; Wang et al. 2011) and fermentation improvement (Sauer et al. 1996; Wu et al. 2007). Among these strategies, riboflavin biosynthesis pathway deregulation and precursors supply are considered to be the major limiting factors and have been extensively studied, including enhancement of precursors supply of GTP by up-regulation of purine biosynthesis pathway (Shi et al. 2009b) and Ru5P by redirecting carbon flow through PPP (Tannler et al. 2008b; Duan et al. 2010; Wang et al. 2011), and increasing riboflavin biosynthesis activity by overexpression of key pathway bottleneck enzymes (Humbelin et al. 1999; Fischer et al. 2003; Lehmann et al. 2009). Although such manipulations successfully improved riboflavin production in B. subtilis, the achieved riboflavin yields still fall significantly short of the theoretical maximum yield, suggesting that further yield enhancement is possible and strain development is still desirable (Sauer et al. 1998; Li et al. 2006). In our laboratory, a series of approaches have been implemented in B. subtilis for riboflavin biosynthesis (Zhu et al. 2007; Shi et al. 2009a, b; Duan et al. 2010; Wang et al. 2011). In this study, we focus on the deregulation of gluconeogenesis for further improvement of riboflavin production. Gluconeogenesis refers to the central metabolic pathway in which Krebs cycle intermediates are converted to glucose. The gapB and pckA genes encode the two core enzymes required for gluconeogenesis but are dispensable for glycolysis in B. subtilis (Servant et al. 2005). Both of them are supposed to play important roles in
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gluconeogenesis under CcpA-independent glucose repression (Yoshida et al. 2001). Expression of gapB and pckA is repressed by CcpN in the presence of glucose and other types of glycolytic carbon sources, like ribose or gluconate. Both of these genes are constitutively expressed during gluconeogenesis, whereas their transcription is repressed under glycolytic conditions (Blencke et al. 2003). This regulation not only preserves the high energetic efficiency of the glycolysis but also gives rise to a fourth regulatory checkpoint for the central carbon metabolism (Fillinger et al. 2000). Under glycolytic conditions, unidirectional phosphorylation of fructose-6-phosphate to fructose-1,6bisphosphate is catalyzed by 6-phosphofructokinase. However, dephosphorylation of fructose-1,6-bisphosphate is catalyzed by fructose-1,6-bisphosphatase (FBPase) under gluconeogenetic conditions. B. subtilis possesses two FBPases: class II FBPase encoded by glpX and class III FBPase encoded by fbp. Contrary to the gapB and pckA essential for gluconeogenesis, both fbp and glpX are not regulated by CcpN. FBPase appears to be a constitutive enzyme and expresses constitutively at a fairly constant level undergoing glycolysis or gluconeogenesis, whose activity is mediated by the relative intracellular concentrations of AMP and PEP (Fujita et al. 1998; Jules et al. 2009). Dauner et al. observed significant back fluxes from the TCA cycle to the lower part of glycolysis via gluconeogenic PEP carboxykinase in riboflavin-producing B. subtilis in glucose-limited chemostat cultures by metabolic flux analysis with a comprehensive isotopomer model (Dauner et al. 2001a, b, 2002). Deletion of ccpN impaired cell growth on glucose and strongly altered the distribution of intracellular fluxes, rerouting the main glucose catabolism from glycolysis to the PPP (Tannler et al. 2008a). These data implied that gluconeogenic pathway could be an important target for riboflavin production promotion. A reverse engineering strategy was devised for deregulating expression of gapB and pckA to give rise to increasing relative flux through the PPP and higher riboflavin yield by knockout of their genetic repressor CcpN in B. subtilis (Tannler et al. 2008b). 13C-based flux analysis revealed increased relative flux through the PPP in a ccpN mutant, which gave an example for further improvement of riboflavin production by deregulating crucial genes expression in gluconeogenic pathway. To our knowledge, although gluconeogenesis has been studied in some detail, a systematic study to investigate riboflavin production through modification of these three critical genes pckA, gapB and fbp has not been performed yet. Herein, we present a general strategy for increasing the availability of precursors for riboflavin biosynthesis through deregulating gluconeogenesis by redirecting more carbon flux into the oxidative
World J Microbiol Biotechnol Table 1 Bacterial strains and plasmids used in this study
a
BGSC is Bacillus Genetic Stock Center, Department of Biochemistry, the Ohio State University, Columbus, OH 43210, USA
Strain or plasmid
Relevant description (s)
Reference or source
pUC18
Ampr
Laboratory stock
pUB110
Neor
BGSCa
pSG1192
Ampr, Spcr r
BGSC
r
pHY300PLK
Amp , Tm
pUC18-Spc
Ampr, Spcr; pUC18 ? spc
This study
pUC18-P43
Ampr; pUC18 ? P43 promoter
This study
pUC18-SP
Ampr, Spcr; pUC18 ? spc ? P43 promoter
This study
pUC18-SPF
Ampr, Spcr; pUC18 ? spc ? P43 promoter ? fbp
This study
pUC18-PA
Ampr; pUC18 ? P43 ? pckA
This study
pUC18-TPA
Ampr, Tmr; pUC18 ? P43 ? pckA ? tet
This study
pUC18-PB pUC18-NPB
Ampr, Neor; pUC18 ? P43 ? gapB Ampr, Neor; pUC18 ? P43 ? gapB ? neo
This study This study
pUC18-PFB
Ampr, Spcr; pUC18 ? P43 ? fbp ? gapB
This study
Laboratory stock
E. coli Top 10
Host strain for plasmid construction
Laboratory stock
B. subtilis 168
Wild-type strain, trpC2
Laboratory stock
B. subtilis RH33
Emr, Cmr; 8-AGr, roseflavinr, Dcr
Laboratory stock
B. subtilis SPF
Emr, Cmr, Spcr; RH33::pUC18-SPF
This study
B. subtilis TPA
Emr, Cmr, Tmr; RH33::pUC18-TPA
This study
B. subtilis NPB
Emr, Cmr, Neor; RH33::pUC18-NPB
This study
B. subtilis PFB
Emr, Cmr, Spcr; RH33::pUC18-PFB
This study
B. subtilis PAB
Emr, Cmr, Spcr, Neor; RH33::pUC18-TPA, pUC18-NPB
This study
B. subtilis PFBA
Emr, Cmr, Spcr, Tmr; RH33::pUC18-PFB, pUC18-TPA
This study
PPP. PckA, gapB and fbp were chosen and overexpressed individually or combinatorially in riboflavin-producing B. subtilis RH33 to investigate the effects of deregulating gluconeogenesis on cell growth, glucose consumption, and riboflavin production.
Materials and methods Strains and plasmids Bacterial strains and plasmids used in this work are listed in Table 1. E. coli Top10 was used for plasmids construction and B. subtilis 168 was used as the P43 donor. pUB110, pSG1192 and pHY300PLK were used as antibiotics resistance gene donors of neo, spc and tet, respectively. Reference strain RH33 is resistant to 8-azaguanine, decoyinine and roseoflavin, which has multiple copies of deregulated riboflavin operons in its chromosome obtained in our previous study. A brief description of the RH33 was given by Shi et al. (2009a). The integrative vectors pUC18TPA, pUC18-NPB, pUC18-SPF, pUC18-PFB were constructed and integrated into chromosome by single crossover and the positive transformants were selected by the corresponding antibiotics and identified by PCR. The
information of primers was summarized in Supplementary Table 1. Media and growth conditions All strains were stored at -80 °C and revived by growing on Luria–Bertani (LB) agar slants. Detailed descriptions of minimal medium (MM), shake cultivation medium and fed-batch medium, and batch cultivations for physiological characterization and fermentation procedure were given by Wang et al. (2011). In brief, physiological characterizations were carried out in MM. To test the riboflavin biosynthetic activity of strains, single colony was transferred into 5 ml LB medium containing corresponding antibiotics and incubated at 41 °C in a rotatory shaker at 200 rev/min for 14 h to prepare the inocula. 2 % (v/v) inocula were added aseptically to flask (500 ml) containing 50 ml shake cultivation medium. The fermentation was conducted at 41 °C in shake flasks at 250 rev/min for 72 h. Fed-batch fermentation was carried out in a 5 l bioreactor (BaoXing, China) containing 2.35 l fermentation medium. All the experiments were carried out independently in biological triplicates, and the reported results were the average of three replicate experiments.
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Measurement of biomass and riboflavin Cell growth was monitored by measuring optical density (OD) at 600 nm (OD600). OD600 was converted to dry cell mass by multiplying with a conversion factor of 0.29. Exponential growth rate was identified by log-linear regression analysis of biomass versus cultural time. Glucose concentration in culture broth was determined enzymatically by a bioanalyzer (SBA-40E, Shandong, China). For riboflavin measurements, samples were first diluted with 0.05 M NaOH and centrifuged at 16,0009g for 2 min to remove the cells, the supernatant was then diluted by acetic acid sodium-acetate buffer solution (pH 5.0) to the linear range of the spectrophotometer and the absorbance at 444 nm was recorded (Wang et al. 2011). The riboflavin concentration was counted through standard equation which had been validated, y = (A440-0.0041) 9 DF/ 0.0321 [R2 = 0.9968, with y, the riboflavin concentration of sample (mg/l); A440, the value of absorbance at 440 nm; DF, dilution fold; A440 was controlled within the range of 0.3–0.8 by dilution].
brief, 100 ng of cDNA was used in a total reaction volume of 20 ll with 0.25 mM of each primer (See Supplementary Table 1). The fold change of each transcript in each sample relative to the control was measured in triplicates, normalized to internal control gene rrnA and calculated according to the comparative CT method (Livak and Schmittgen 2001).
Results Overexpression of pckA, gapB, fbp gene in RH33
Fresh samples of cell cultures harvested at the exponential phase grown under the same condition in MM medium were used to isolated total RNA by using RNAprep pure Kit DP430 (Tiangen, Beijing, China) following the manufacturer’s instructions. RNA samples were then reversed transcribed into cDNA using Quant Reverse Transcriptase with random primers (Tiangen, Beijing, China). The qRTPCR was carried out by Light CyclerÒ 480 II (Roche, Basel, Switzerland) with Real Master Mix (SYBR Green) according to the manufacturer’s instructions as follows. In
For overexpression of pckA, gapB and fbp in RH33, a series of integrative vectors pUC18-TPA, pUC18-NPB, pUC18-SPF and pUC18-PFB were constructed (summarized in Table 1). All these vectors were integrated into chromosome by single crossover recombination. Identification by PCR and agarose gel electrophoresis (Fig. 1), integrative vectors were successfully introduced into RH33 or RH33 derivative strains. Individual integration of pUC18-TPA, pUC18-NPB, pUC18-SPF and pUC18-PFB into RH33 chromosome gave corresponding positive transformants, which were denoted as B. subtilis TPA, B. subtilis NPB, B. subtilis SPF and B. subtilis PFB, respectively. In addition, pUC18-NPB was introduced into TPA genome to obtain strain PAB for co-overexpression of pckA and gapB. Besides, pUC18-PFB was introduced into TPA genome to obtain strain PFBA for co-overexpression of pckA, gapB and fbp. In order to assess the transcription levels of gluconeogenetic genes under the control of the constitutive P43 promoter, we measured the relative expression levels of pckA, gapB and fbp in each mutant strains by qRT-PCR. As expected, the transcription levels
Fig. 1 Confirmation of plasmid integration into recipient genome by PCR with appropriate primers. Lane 1–9 show amplified DNA generated from different recombinant template. Lane M: 1 kb DNA Ladder; Lane 1, RH33 (negative control for amplification by using primer pUC18-F and pUC18-L); Lane 2, TPA (primer pair: PckAcheck-U and PckA-check-L); Lane 3, NPB (primer pair: GapB-check-
U and GapB-check-L); Lane 4, PAB (primer pair: PckA -check-U and PckA -check-L); Lane 5, PAB(primer pair: GapB -check-U and GapB -check-L); Lane 6, SPF(primer pair: SPF-check-U and SPF-check-L); Lane 7, PFB (primer pair: pUC18-F and pUC18-L); Lane 8, PFBA (primer pair: pUC18-F and pUC18-L); Lane 9, PFBA (primer pair: PckA-check-U and PckA-check-L)
Analysis of gene expression by quantitative RT-PCR
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Fig. 2 Determination of relative expression levels of pckA, gapB and fbp in different mutant strains by qRT-PCR during exponential phase in minimal medium. The average values with strain RH33 were set as 100 %. Values are the averages with standard deviations from three independent measurements
of each target gene were significantly increased about 18to 45-fold in the recombinant, suggesting their anticipated overexpression and successful molecular manipulation (Fig. 2). Growth and metabolic characteristics in different genetic modification strains For the purpose of estimating the effects of gene modification on the metabolic characterization of riboflavin producing strains, mutants were compared with respect to their growth and productivity characteristics in shake flask. Results were shown in Table 2; expression of pckA alone did not increase riboflavin productivity, instead of exhibiting a slightly lower growth and riboflavin productivity. In comparison, expression of gapB increased riboflavin productivity slightly (from 0.047 ± 0.003 to 0.050 ± 0.003 mmol/g CDW/h). While co-expression of pckA and gapB only resulted in an about 4.3 % increase in the riboflavin productivity from 0.047 ± 0.003 to 0.049 ± 0.003 mmol/g CDW/h. Surprisingly, overexpression of fbp alone in SPF significantly enhanced the riboflavin
Fig. 3 Riboflavin production of different B. subtilis strains in shake flask
productivity from 0.047 ± 0.003 to 0.052 ± 0.004 mmol/ g CDW/h. Meanwhile, the biomass productivity decreased slightly with glucose as carbon source. Moreover, cooverexpression of gapB and fbp improved riboflavin productivity approximately 12.8 %. Consistently, co-overexpression of pckA, gapB and fbp got similar results with slightly lower riboflavin productivity 0.052 ± 0.004 mmol/g CDW/h compared to that of strain PFB. It was observed that glucose uptake rate remained almost unaffected by the genetic modifications and the specific growth rate decreased with increasing riboflavin production, which verified the redistribution of carbon flux towards PPP. Influence of overexpression of pckA, gapB, fbp on riboflavin production and biomass formation in shake flask cultivation To gain further insight into how pckA, gapB and fbp overexpression influence the riboflavin production, we tested the performances of the mutants in shake flask
Table 2 Growth and metabolic characterization of B. subtilis strains during exponential growth (OD600 between 0.3 and 0.6) cultivated in minimal medium Parameter Specific glucose uptake rate (mmol/g CDW/h) Specific growth rate (/h) Specific riboflavin production rate (mmol/g CDW/h)
RH33 4.2 ± 0.4
TPA 4.2 ± 0.3
NPB 4.1 ± 0.1
PAB 4.1 ± 0.2
SPF 4.1 ± 0.3
PFB 4.1 ± 0.3
PFBA 4.1 ± 0.4
0.26 ± 0.02
0.22 ± 0.02
0.24 ± 0.03
0.24 ± 0.03
0.22 ± 0.03
0.22 ± 0.03
0.22 ± 0.02
0.047 ± 0.003
0.045 ± 0.002
0.050 ± 0.003
0.049 ± 0.003
0.052 ± 0.004
0.053 ± 0.003
0.052 ± 0.004
Results represent the mean values with standard deviations from three independent measurements
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The specific growth rate of PFB decreased from 0.26 ± 0.02 to 0.22 ± 0.01 h-1 and riboflavin productivity improved approximately 12.8 %, which is consistent with the results in shake flask cultivation in MM (Table 2). It was implied that the co-overexpression of the crucial genes in gluconeogenesis was beneficial for riboflavin precursors supply which facilitated riboflavin production. As a conclusion, deregulating gluconeogenesis by cooverexpressing gapB and fbp simultaneously could successfully improve riboflavin production in B. subtilis.
Discussion Fig. 4 Growth, residual glucose and riboflavin production profile during fed-batch fermentation in a 5 l bioreactor of B. subtilis RH33 and PFB. Biomass: RH33 (filled diamond); PFB (filled triangle); Riboflavin: RH33 (filled circle); PFB (filled square)
cultivation. Experimental results indicated that riboflavin synthesis levels varied apparently in various mutants as compared with parental strain under the same cultivation condition (Fig. 3). For SPF, fbp overexpression led to a substantial promotion in riboflavin synthesis and yielded a final concentration of 4.73 g/l, which was 18.0 % higher than that of RH33. When pckA was overexpressed in strain TPA, riboflavin synthesis even dropped slightly. In contrast, co-overexpression of pckA and gapB in PAB resulted in as much as about 12.0 % enhancement in riboflavin synthesis. And similar result was observed when gapB sole expression in NPB, whose riboflavin production increased by 14.2 %. According to the above results, we speculate that co-overexpression of fbp and gapB might have an important impact on riboflavin synthesis. As expected, approximately 21.9 % promotion in riboflavin production and 4.89 g/l accumulation concentration was observed in PFB, which proved our speculation. Overexpressed pckA in mutant strain PFB did not further improve riboflavin production. Only 4.81 g/l riboflavin accumulated in flask cultivation which is even lower than that of PFB. Riboflavin production in fed-batch cultivation With the purpose of examining the potential of large scale fermentation of PFB, we investigated the performance of the strains in fed-batch fermentation. As Fig. 4 indicated that the cell growth traits were similar between the parental strain RH33 and engineered strain PFB. In comparison to RH33, the cell density of PFB slightly decreased with glucose as carbon source in fed-batch fermentation, while the riboflavin production of PFB significantly increased. An average of 27.8 % increase in riboflavin titer was achieved by PFB, up to 13.36 g/l in 2-day fermentation.
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The development of rational metabolic engineering strategies to improve bioprocesses depends critically on a deep understanding of cellular metabolism. Gluconeogenesis, one of the most important pathways in central carbon metabolism, has been exclusively studied as described in introduction. Furthermore, modification of gluconeogenesis has been successfully employed to redirect flux toward the PPP in C. glutamicum (Becker et al. 2005, 2007). Since the metabolic regulation of gluconeogenesis seems similar for different organisms, deregulation of gluconeogenesis could lead to similar effects in other bacteria. Also it has been demonstrated that gluconeogenesis could be another important point of focus to improve production of riboflavin (Tannler et al. 2008a, b). Therefore, pckA, gapB and fbp were chosen and conducted to redirect carbon flow of gluconeogenesis into PPP for further investigation of metabolic characterization of riboflavin producing B. subtilis. Both fbp and glpX genes encode FBPases and are functionally equivalent to each other in B. subtilis. Besides, FBPases are proved to be constitutive enzymes and transcribed at an equal level under gluconeogenic conditions. It’s worth noting that, DglpX mutant presented approximately 30-fold activation of FBPase activity due to the activation of fbp in the presence of 1 mM PEP under in vitro condition. On the contrary, 1 mM PEP completely abolished any detectable FBPase activity in the fbp knockout mutant (Jules et al. 2009). This indicates that fbp is more suitable for manipulation to up-regulate gluconeogenesis. Consequently, fbp-encoded FBPase was chosen and overexpressed in riboflavin-producing strains. In contrast to overexpression of glyceraldehyde-3-phosphate dehydrogenase or phosphoenolpyruvate (PEP) carboxykinase, overexpression of FBPase allows the most promising improvement of riboflavin production by 18.0 % in B. subtilis grown on glucose. Becker and his partner’s research may offer clue to explain these promising results. In their research, amplified expression of FBPase via the promoter of the gene encoding elongation factor TU
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(EFTU) increased the lysine yield in the feedback-deregulated lysine-producing strain C. glutamicum lysCfbr by 40 % on glucose. 13C metabolic flux analysis on glucose indicated that the overexpression of FBPase caused a redirection of carbon flux from glycolysis toward PPP, and the relative flux into PPP was increased approximately 10 %, which led to increased NADPH supply required for lysine biosynthesis in C. glutamicum (Becker et al. 2005). However, in our research, enhancement of riboflavin production is derived from improved precursor Ru5P supply by redirecting more carbon flux into PPP (Wang et al. 2011), rather than increased NADPH supply. As a consequence, a high PPP flux led to an increased formation of NADPH, and higher catabolic NADPH formation occurred than was necessary to satisfy the anabolic demands. The riboflavin-producing B. subtilis could deal with this apparent NADPH overproduction to close its metabolic balance by two potential balancing mechanisms. First, a putative transhydrogenase-like mechanism could catalyze the oxidation of NADPH by reducing NAD?, which was considered to be the most plausible though no such transhydrogenase gene information has been annotated for B. subtilis (Dauner et al. 2002; Ruhl et al. 2010). Second, redox cycles formed by enzymes with NADPH oxidase activity could effectively achieve a balance. In the present work, NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (encoded by gapB) was activated in the presence of glucose, whose sole or combinational overexpression with other genes in gluconeogenic pathway resulted in increased riboflavin production. Derepressed synthesis of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase have been proved to lead to a metabolic jamming, resulting in high hexose phosphate concentrations that caused the increase of PPP flux (Tannler et al. 2008a), and the corresponding consumption of NADPH occurred by reducing NADP?. We therefore are more inclined to the second or the combination of these two potential balancing mechanisms in this work. Largely in agreement with results obtained from B. subtilis RB50::pRF69 (Tannler et al. 2008b), co-overexpression of pckA and gapB under promoter P43 resulted in 12 % improved riboflavin production, while overexpression of pckA or gapB separately presented different effects. Overexpression of pckA got the negative results. A possible reason is that derepression of pckA under glycolytic condition causes the growth defect observed in shake flask cultivation due to ATP dissipation via extensive futile cycling through the pyruvate carboxylase, PEP carboxykinase, and pyruvate kinase (Sauer and Eikmanns 2005; Ruhl et al. 2010). In particular, the high pckA flux limits the amount of oxaloacetate available for aspartate synthesis by the aspB-encoded aspartate aminotransferase and/or for reaction with acetyl coenzyme A to fuel the TCA cycle
(Tannler et al. 2008a). Another potential explanation for this phenomenon is that metabolites such as PEP and glycerate-3-phosphate cannot be further involved in gluconeogenic reactions without expression of gapB-encoded NADP?-dependent glyceraldehyde-3-phosphate dehydrogenase in B. subtilis TPA. Thus, carbon flux cannot be redirected towards the PPP, instead of leading to deleterious effects on cell growth and riboflavin yield. Co-expression of the gapB and fbp led to 21.9 and 27.8 % increased riboflavin production in shake flask and fed-batch cultivation, respectively. Based on our data conclusively, we recommend an extended application of gapB and fbp overexpression for the production of products directly stemming from the PPP as well as other NADPHdemanding compounds in microorganisms like B. subtilis, C. glutamicum and E. coli. Acknowledgments We thank Dr. Zhenquan Lin for the help of qRT-PCR analysis. This work was supported by National Program on Key Basic Research Project (2011CBA00804, 2012CB725203), National Natural Science Foundation of China (NSFC-21206112, NSFC-21176182), National High-tech R&D Program of China (2012AA022103, 2012AA02A702) and the Innovation Foundation of Tianjin University (1308).
References Barbe V, Cruveiller S, Kunst F, Lenoble P, Meurice G, Sekowska A, Vallenet D, Wang T, Moszer I, Medigue C, Danchin A (2009) From a consortium sequence to a unified sequence: the Bacillus subtilis 168 reference genome a decade later. Microbiology 155(Pt 6):1758–1775 Becker J, Klopprogge C, Zelder O, Heinzle E, Wittmann C (2005) Amplified expression of fructose 1,6-bisphosphatase in Corynebacterium glutamicum increases in vivo flux through the pentose phosphate pathway and lysine production on different carbon sources. Appl Environ Microbiol 71(12):8587–8596 Becker J, Klopprogge C, Herold A, Zelder O, Bolten CJ, Wittmann C (2007) Metabolic flux engineering of L-lysine production in Corynebacterium glutamicum-over expression and modification of G6P dehydrogenase. J Biotechnol 132(2):99–109 Blencke HM, Homuth G, Ludwig H, Mader U, Hecker M, Stulke J (2003) Transcriptional profiling of gene expression in response to glucose in Bacillus subtilis: regulation of the central metabolic pathways. Metab Eng 5(2):133–149 Coquard D, Huecas M, Ott M, van Dijl JM, van Loon AP, Hohmann HP (1997) Molecular cloning and characterisation of the ribC gene from Bacillus subtilis: a point mutation in ribC results in riboflavin overproduction. Mol Gen Genet 254(1):81–84 Dauner M, Bailey JE, Sauer U (2001a) Metabolic flux analysis with a comprehensive isotopomer model in Bacillus subtilis. Biotechnol Bioeng 76(2):144–156 Dauner M, Storni T, Sauer U (2001b) Bacillus subtilis metabolism and energetics in carbon-limited and excess-carbon chemostat culture. J Bacteriol 183(24):7308–7317 Dauner M, Sonderegger M, Hochuli M, Szyperski T, Wuthrich K, Hohmann HP, Sauer U, Bailey JE (2002) Intracellular carbon fluxes in riboflavin-producing Bacillus subtilis during growth on two-carbon substrate mixtures. Appl Environ Microbiol 68(4):1760–1771
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World J Microbiol Biotechnol Duan YX, Chen T, Chen X, Zhao XM (2010) Overexpression of glucose-6-phosphate dehydrogenase enhances riboflavin production in Bacillus subtilis. Appl Microbiol Biotechnol 85(6):1907– 1914 Fillinger S, Boschi-Muller S, Azza S, Dervyn E, Branlant G, Aymerich S (2000) Two glyceraldehyde-3-phosphate dehydrogenases with opposite physiological roles in a nonphotosynthetic bacterium. J Biol Chem 275(19):14031–14037 Fischer M, Haase I, Kis K, Meining W, Ladenstein R, Cushman M, Schramek N, Huber R, Bacher A (2003) Enzyme catalysis via control of activation entropy: site-directed mutagenesis of 6,7dimethyl-8-ribityllumazine synthase. J Mol Biol 326(3):783–793 Fujita Y, Yoshida K, Miwa Y, Yanai N, Nagakawa E, Kasahara Y (1998) Identification and expression of the Bacillus subtilis fructose-1, 6-bisphosphatase gene (fbp). J Bacteriol 180(16): 4309–4313 Hemberger S, Pedrolli DB, Stolz J, Vogl C, Lehmann M, Mack M (2011) RibM from Streptomyces davawensis is a riboflavin/ roseoflavin transporter and may be useful for the optimization of riboflavin production strains. BMC Biotechnol 11:119 Humbelin M, Griesser V, Keller T, Schurter W, Haiker M, Hohmann HP, Ritz H, Richter G, Bacher A, van Loon APGM (1999) GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone 4-phosphate synthase are rate-limiting enzymes in riboflavin synthesis of an industrial Bacillus subtilis strain used for riboflavin production. J Ind Microbiol Biotechnol 22(1):1–7 Jules M, Le Chat L, Aymerich S, Le Coq D (2009) The Bacillus subtilis ywjI (glpX) gene encodes a class II fructose-1,6bisphosphatase, functionally equivalent to the class III Fbp enzyme. J Bacteriol 191(9):3168–3171 Kalingan AE, Liao CM (2002) Influence of type and concentration of flavinogenic factors on production of riboflavin by Eremothecium ashbyii NRRL 1363. Bioresour Technol 82(3):219–224 Lehmann M, Degen S, Hohmann HP, Wyss M, Bacher A, Schramek N (2009) Biosynthesis of riboflavin: screening for an improved GTP cyclohydrolase II mutant. FEBS J 276(15):4119–4129 Li XJ, Chen T, Chen X, Zhao XM (2006) Redirection electron flow to high coupling efficiency of terminal oxidase to enhance riboflavin biosynthesis. Appl Microbiol Biotechnol 73(2):374–383 Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4):402–408 Perkins JB, Sloma A, Hermann T, Theriault K, Zachgo E, Erdenberger T, Hannett N, Chatterjee NP, Williams V, Rufo GA, Hatch R, Pero J (1999) Genetic engineering of Bacillus subtilis for the commercial production of riboflavin. J Ind Microbiol Biotechnol 22(1):8–18 Ruhl M, Zamboni N, Sauer U (2010) Dynamic flux responses in riboflavin overproducing Bacillus subtilis to increasing glucose limitation in fed-batch culture. Biotechnol Bioeng 105(4):795– 804 Sauer U, Eikmanns BJ (2005) The PEP–pyruvate–oxaloacetate node as the switch point for carbon flux distribution in bacteria. FEMS Microbiol Rev 29(4):765–794
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Sauer U, Hatzimanikatis V, Hohmann HP, Manneberg M, van Loon AP, Bailey JE (1996) Physiology and metabolic fluxes of wildtype and riboflavin-producing Bacillus subtilis. Appl Environ Microbiol 62(10):3687–3696 Sauer U, Cameron DC, Bailey JE (1998) Metabolic capacity of Bacillus subtilis for the production of purine nucleosides, riboflavin, and folic acid. Biotechnol Bioeng 59(2):227–238 Schallmey M, Singh A, Ward OP (2004) Developments in the use of Bacillus species for industrial production. Can J Microbiol 50(1):1–17 Servant P, Le Coq D, Aymerich S (2005) CcpN (YqzB), a novel regulator for CcpA-independent catabolite repression of Bacillus subtilis gluconeogenic genes. Mol Microbiol 55(5):1435–1451 Shi S, Chen T, Zhang Z, Chen X, Zhao X (2009a) Transcriptome analysis guided metabolic engineering of Bacillus subtilis for riboflavin production. Metab Eng 11(4–5):243–252 Shi SB, Shen Z, Chen X, Chen T, Zhao XM (2009b) Increased production of riboflavin by metabolic engineering of the purine pathway in Bacillus subtilis. Biochem Eng J 46(1):28–33 Sklyarova SA, Kreneva RA, Perumov DA, Mironov AS (2012) The characterization of internal promoters in the Bacillus subtilis riboflavin biosynthesis operon. Russ J Genet 48(10):967–974 Stahmann KP, Revuelta JL, Seulberger H (2000) Three biotechnical processes using Ashbya gossypii, Candida famata, or Bacillus subtilis compete with chemical riboflavin production. Appl Microbiol Biotechnol 53(5):509–516 Tannler S, Fischer E, Le Coq D, Doan T, Jamet E, Sauer U, Aymerich S (2008a) CcpN controls central carbon fluxes in Bacillus subtilis. J Bacteriol 190(18):6178–6187 Tannler S, Zamboni N, Kiraly C, Aymerich S, Sauer U (2008b) Screening of Bacillus subtilis transposon mutants with altered riboflavin production. Metab Eng 10(5):216–226 Wang Z, Chen T, Ma X, Shen Z, Zhao X (2011) Enhancement of riboflavin production with Bacillus subtilis by expression and site-directed mutagenesis of zwf and gnd gene from Corynebacterium glutamicum. Bioresour Technol 102(4):3934–3940 Wu QL, Chen T, Gan Y, Chen X, Zhao XM (2007) Optimization of riboflavin production by recombinant Bacillus subtilis RH44 using statistical designs. Appl Microbiol Biotechnol 76(4):783– 794 Yoshida K, Kobayashi K, Miwa Y, Kang CM, Matsunaga M, Yamaguchi H, Tojo S, Yamamoto M, Nishi R, Ogasawara N, Nakayama T, Fujita Y (2001) Combined transcriptome and proteome analysis as a powerful approach to study genes under glucose repression in Bacillus subtilis. Nucleic Acids Res 29(3):683–692 Zamboni N, Mouncey N, Hohmann H-P, Sauer U (2003) Reducing maintenance metabolism by metabolic engineering of respiration improves riboflavin production by Bacillus subtilis. Metab Eng 5(1):49–55 Zhu Y, Chen X, Chen T, Zhao X (2007) Enhancement of riboflavin production by overexpression of acetolactate synthase in a pta mutant of Bacillus subtilis. FEMS Microbiol Lett 266(2):224– 230
ORIGINAL PAPER
Enhancement of riboflavin production by deregulating gluconeogenesis in Bacillus subtilis Guanglu Wang • Ling Bai • Zhiwen Wang Ting Shi • Tao Chen • Xueming Zhao
•
Received: 17 April 2013 / Accepted: 19 January 2014 Ó Springer Science+Business Media Dordrecht 2014
Guanglu Wang and Ling Bai have contributed equally to this work.
overexpression of pckA obtained the opposite result. Significant enhancement of riboflavin titers up to 4.89 g/l was obtained in shake flask cultures when gapB and fbp were co-overexpressed, nevertheless the specific growth rate decreased slightly and the specific glucose uptake rate remained almost unchanged. An improvement by 21.9 and 27.8 % of the riboflavin production was achieved by cooverexpression of gapB and fbp in shake flask and fedbatch fermentation, respectively. These results imply that deregulation of gluconeogenesis is an effective strategy for production of metabolites directly stemming from the pentose phosphate pathway as well as other NADPHdemanding compounds with glucose as carbon source in B. subtilis.
Electronic supplementary material The online version of this article (doi:10.1007/s11274-014-1611-6) contains supplementary material, which is available to authorized users.
Keywords Riboflavin Bacillus subtilis Gluconeogenesis Pentose phosphate pathway
G. Wang L. Bai Z. Wang (&) T. Shi T. Chen X. Zhao Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, 92# Weijin Road, Nankai District, Tianjin 300072, People’s Republic of China e-mail: [email protected]
Abbreviations PPP Pentose phosphate pathway Ru5P Ribulose-5P FBPase Fructose 1,6-bisphosphatase MM Minimal medium CDW Cell dry weight TCA Tricarboxylic acid PEP Phosphoenolpyruvate
Abstract The regulation of metabolic flux through glycolytic versus the gluconeogenic pathway plays an important role in central carbon metabolism. In this study, we made an attempt to enhance riboflavin production by deregulating gluconeogenesis in Bacillus subtilis. To this end, gapB (code for NADPH-dependent glyceraldehyde-3phosphate dehydrogenase), fbp (code for fructose-1,6-bisphosphatase) and pckA (code for phosphoenolpyruvate carboxykinase) were overexpressed in parental strain B. subtilis RH33. Compared with RH33, overexpression of fbp and gapB resulted in approximately 18.0 and 14.2 % increased riboflavin production, respectively, while
G. Wang L. Bai Z. Wang T. Shi T. Chen X. Zhao Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin 300072, People’s Republic of China G. Wang L. Bai Z. Wang T. Shi T. Chen X. Zhao Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin 300072, People’s Republic of China
Introduction
G. Wang L. Bai Z. Wang T. Shi T. Chen X. Zhao Edinburgh-Tianjin Joint Research Centre for Systems Biology and Synthetic Biology, Tianjin University, Tianjin 300072, People’s Republic of China
Riboflavin (vitamin B2) is the direct precursor of FMN and FAD, one of the essential components in cellular physiology required by all bacteria, animals and plants. It has been
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used not only for pharmaceuticals but also for animal feed additives, cosmetics, and food industry. Currently, the fungi Ashbya gossypii, Eremothecium ashbyii, Candida flareri and the bacterium Bacillus subtilis have been successfully engineered as cell factories for riboflavin production (Stahmann et al. 2000; Kalingan and Liao 2002), whereas the commercial processes mainly rely on A. gossypii and B. subtilis. Bacillus subtilis has been a long tradition as safe and stable producers of purine nucleosides, inosine and guanosine (Sauer et al. 1998), which are the precursors for riboflavin biosynthesis. Biochemical, physiological, and genetic studies allowed the characterization of enzymes involved in riboflavin biosynthesis pathway in B. subtilis (Schallmey et al. 2004; Barbe et al. 2009; Sklyarova et al. 2012). It comprises seven enzymatic steps and starts from two precursors, ribulose-5P (Ru5P) deriving from pentose phosphate pathway (PPP) and GTP deriving from purine biosynthetic pathway (Dauner et al. 2001b). Commercial riboflavin production by B. subtilis has been developed by a combination of classical mutagenesis (Coquard et al. 1997; Perkins et al. 1999), rational metabolic engineering (Humbelin et al. 1999; Zamboni et al. 2003; Li et al. 2006; Duan et al. 2010; Hemberger et al. 2011; Wang et al. 2011) and fermentation improvement (Sauer et al. 1996; Wu et al. 2007). Among these strategies, riboflavin biosynthesis pathway deregulation and precursors supply are considered to be the major limiting factors and have been extensively studied, including enhancement of precursors supply of GTP by up-regulation of purine biosynthesis pathway (Shi et al. 2009b) and Ru5P by redirecting carbon flow through PPP (Tannler et al. 2008b; Duan et al. 2010; Wang et al. 2011), and increasing riboflavin biosynthesis activity by overexpression of key pathway bottleneck enzymes (Humbelin et al. 1999; Fischer et al. 2003; Lehmann et al. 2009). Although such manipulations successfully improved riboflavin production in B. subtilis, the achieved riboflavin yields still fall significantly short of the theoretical maximum yield, suggesting that further yield enhancement is possible and strain development is still desirable (Sauer et al. 1998; Li et al. 2006). In our laboratory, a series of approaches have been implemented in B. subtilis for riboflavin biosynthesis (Zhu et al. 2007; Shi et al. 2009a, b; Duan et al. 2010; Wang et al. 2011). In this study, we focus on the deregulation of gluconeogenesis for further improvement of riboflavin production. Gluconeogenesis refers to the central metabolic pathway in which Krebs cycle intermediates are converted to glucose. The gapB and pckA genes encode the two core enzymes required for gluconeogenesis but are dispensable for glycolysis in B. subtilis (Servant et al. 2005). Both of them are supposed to play important roles in
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gluconeogenesis under CcpA-independent glucose repression (Yoshida et al. 2001). Expression of gapB and pckA is repressed by CcpN in the presence of glucose and other types of glycolytic carbon sources, like ribose or gluconate. Both of these genes are constitutively expressed during gluconeogenesis, whereas their transcription is repressed under glycolytic conditions (Blencke et al. 2003). This regulation not only preserves the high energetic efficiency of the glycolysis but also gives rise to a fourth regulatory checkpoint for the central carbon metabolism (Fillinger et al. 2000). Under glycolytic conditions, unidirectional phosphorylation of fructose-6-phosphate to fructose-1,6bisphosphate is catalyzed by 6-phosphofructokinase. However, dephosphorylation of fructose-1,6-bisphosphate is catalyzed by fructose-1,6-bisphosphatase (FBPase) under gluconeogenetic conditions. B. subtilis possesses two FBPases: class II FBPase encoded by glpX and class III FBPase encoded by fbp. Contrary to the gapB and pckA essential for gluconeogenesis, both fbp and glpX are not regulated by CcpN. FBPase appears to be a constitutive enzyme and expresses constitutively at a fairly constant level undergoing glycolysis or gluconeogenesis, whose activity is mediated by the relative intracellular concentrations of AMP and PEP (Fujita et al. 1998; Jules et al. 2009). Dauner et al. observed significant back fluxes from the TCA cycle to the lower part of glycolysis via gluconeogenic PEP carboxykinase in riboflavin-producing B. subtilis in glucose-limited chemostat cultures by metabolic flux analysis with a comprehensive isotopomer model (Dauner et al. 2001a, b, 2002). Deletion of ccpN impaired cell growth on glucose and strongly altered the distribution of intracellular fluxes, rerouting the main glucose catabolism from glycolysis to the PPP (Tannler et al. 2008a). These data implied that gluconeogenic pathway could be an important target for riboflavin production promotion. A reverse engineering strategy was devised for deregulating expression of gapB and pckA to give rise to increasing relative flux through the PPP and higher riboflavin yield by knockout of their genetic repressor CcpN in B. subtilis (Tannler et al. 2008b). 13C-based flux analysis revealed increased relative flux through the PPP in a ccpN mutant, which gave an example for further improvement of riboflavin production by deregulating crucial genes expression in gluconeogenic pathway. To our knowledge, although gluconeogenesis has been studied in some detail, a systematic study to investigate riboflavin production through modification of these three critical genes pckA, gapB and fbp has not been performed yet. Herein, we present a general strategy for increasing the availability of precursors for riboflavin biosynthesis through deregulating gluconeogenesis by redirecting more carbon flux into the oxidative
World J Microbiol Biotechnol Table 1 Bacterial strains and plasmids used in this study
a
BGSC is Bacillus Genetic Stock Center, Department of Biochemistry, the Ohio State University, Columbus, OH 43210, USA
Strain or plasmid
Relevant description (s)
Reference or source
pUC18
Ampr
Laboratory stock
pUB110
Neor
BGSCa
pSG1192
Ampr, Spcr r
BGSC
r
pHY300PLK
Amp , Tm
pUC18-Spc
Ampr, Spcr; pUC18 ? spc
This study
pUC18-P43
Ampr; pUC18 ? P43 promoter
This study
pUC18-SP
Ampr, Spcr; pUC18 ? spc ? P43 promoter
This study
pUC18-SPF
Ampr, Spcr; pUC18 ? spc ? P43 promoter ? fbp
This study
pUC18-PA
Ampr; pUC18 ? P43 ? pckA
This study
pUC18-TPA
Ampr, Tmr; pUC18 ? P43 ? pckA ? tet
This study
pUC18-PB pUC18-NPB
Ampr, Neor; pUC18 ? P43 ? gapB Ampr, Neor; pUC18 ? P43 ? gapB ? neo
This study This study
pUC18-PFB
Ampr, Spcr; pUC18 ? P43 ? fbp ? gapB
This study
Laboratory stock
E. coli Top 10
Host strain for plasmid construction
Laboratory stock
B. subtilis 168
Wild-type strain, trpC2
Laboratory stock
B. subtilis RH33
Emr, Cmr; 8-AGr, roseflavinr, Dcr
Laboratory stock
B. subtilis SPF
Emr, Cmr, Spcr; RH33::pUC18-SPF
This study
B. subtilis TPA
Emr, Cmr, Tmr; RH33::pUC18-TPA
This study
B. subtilis NPB
Emr, Cmr, Neor; RH33::pUC18-NPB
This study
B. subtilis PFB
Emr, Cmr, Spcr; RH33::pUC18-PFB
This study
B. subtilis PAB
Emr, Cmr, Spcr, Neor; RH33::pUC18-TPA, pUC18-NPB
This study
B. subtilis PFBA
Emr, Cmr, Spcr, Tmr; RH33::pUC18-PFB, pUC18-TPA
This study
PPP. PckA, gapB and fbp were chosen and overexpressed individually or combinatorially in riboflavin-producing B. subtilis RH33 to investigate the effects of deregulating gluconeogenesis on cell growth, glucose consumption, and riboflavin production.
Materials and methods Strains and plasmids Bacterial strains and plasmids used in this work are listed in Table 1. E. coli Top10 was used for plasmids construction and B. subtilis 168 was used as the P43 donor. pUB110, pSG1192 and pHY300PLK were used as antibiotics resistance gene donors of neo, spc and tet, respectively. Reference strain RH33 is resistant to 8-azaguanine, decoyinine and roseoflavin, which has multiple copies of deregulated riboflavin operons in its chromosome obtained in our previous study. A brief description of the RH33 was given by Shi et al. (2009a). The integrative vectors pUC18TPA, pUC18-NPB, pUC18-SPF, pUC18-PFB were constructed and integrated into chromosome by single crossover and the positive transformants were selected by the corresponding antibiotics and identified by PCR. The
information of primers was summarized in Supplementary Table 1. Media and growth conditions All strains were stored at -80 °C and revived by growing on Luria–Bertani (LB) agar slants. Detailed descriptions of minimal medium (MM), shake cultivation medium and fed-batch medium, and batch cultivations for physiological characterization and fermentation procedure were given by Wang et al. (2011). In brief, physiological characterizations were carried out in MM. To test the riboflavin biosynthetic activity of strains, single colony was transferred into 5 ml LB medium containing corresponding antibiotics and incubated at 41 °C in a rotatory shaker at 200 rev/min for 14 h to prepare the inocula. 2 % (v/v) inocula were added aseptically to flask (500 ml) containing 50 ml shake cultivation medium. The fermentation was conducted at 41 °C in shake flasks at 250 rev/min for 72 h. Fed-batch fermentation was carried out in a 5 l bioreactor (BaoXing, China) containing 2.35 l fermentation medium. All the experiments were carried out independently in biological triplicates, and the reported results were the average of three replicate experiments.
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Measurement of biomass and riboflavin Cell growth was monitored by measuring optical density (OD) at 600 nm (OD600). OD600 was converted to dry cell mass by multiplying with a conversion factor of 0.29. Exponential growth rate was identified by log-linear regression analysis of biomass versus cultural time. Glucose concentration in culture broth was determined enzymatically by a bioanalyzer (SBA-40E, Shandong, China). For riboflavin measurements, samples were first diluted with 0.05 M NaOH and centrifuged at 16,0009g for 2 min to remove the cells, the supernatant was then diluted by acetic acid sodium-acetate buffer solution (pH 5.0) to the linear range of the spectrophotometer and the absorbance at 444 nm was recorded (Wang et al. 2011). The riboflavin concentration was counted through standard equation which had been validated, y = (A440-0.0041) 9 DF/ 0.0321 [R2 = 0.9968, with y, the riboflavin concentration of sample (mg/l); A440, the value of absorbance at 440 nm; DF, dilution fold; A440 was controlled within the range of 0.3–0.8 by dilution].
brief, 100 ng of cDNA was used in a total reaction volume of 20 ll with 0.25 mM of each primer (See Supplementary Table 1). The fold change of each transcript in each sample relative to the control was measured in triplicates, normalized to internal control gene rrnA and calculated according to the comparative CT method (Livak and Schmittgen 2001).
Results Overexpression of pckA, gapB, fbp gene in RH33
Fresh samples of cell cultures harvested at the exponential phase grown under the same condition in MM medium were used to isolated total RNA by using RNAprep pure Kit DP430 (Tiangen, Beijing, China) following the manufacturer’s instructions. RNA samples were then reversed transcribed into cDNA using Quant Reverse Transcriptase with random primers (Tiangen, Beijing, China). The qRTPCR was carried out by Light CyclerÒ 480 II (Roche, Basel, Switzerland) with Real Master Mix (SYBR Green) according to the manufacturer’s instructions as follows. In
For overexpression of pckA, gapB and fbp in RH33, a series of integrative vectors pUC18-TPA, pUC18-NPB, pUC18-SPF and pUC18-PFB were constructed (summarized in Table 1). All these vectors were integrated into chromosome by single crossover recombination. Identification by PCR and agarose gel electrophoresis (Fig. 1), integrative vectors were successfully introduced into RH33 or RH33 derivative strains. Individual integration of pUC18-TPA, pUC18-NPB, pUC18-SPF and pUC18-PFB into RH33 chromosome gave corresponding positive transformants, which were denoted as B. subtilis TPA, B. subtilis NPB, B. subtilis SPF and B. subtilis PFB, respectively. In addition, pUC18-NPB was introduced into TPA genome to obtain strain PAB for co-overexpression of pckA and gapB. Besides, pUC18-PFB was introduced into TPA genome to obtain strain PFBA for co-overexpression of pckA, gapB and fbp. In order to assess the transcription levels of gluconeogenetic genes under the control of the constitutive P43 promoter, we measured the relative expression levels of pckA, gapB and fbp in each mutant strains by qRT-PCR. As expected, the transcription levels
Fig. 1 Confirmation of plasmid integration into recipient genome by PCR with appropriate primers. Lane 1–9 show amplified DNA generated from different recombinant template. Lane M: 1 kb DNA Ladder; Lane 1, RH33 (negative control for amplification by using primer pUC18-F and pUC18-L); Lane 2, TPA (primer pair: PckAcheck-U and PckA-check-L); Lane 3, NPB (primer pair: GapB-check-
U and GapB-check-L); Lane 4, PAB (primer pair: PckA -check-U and PckA -check-L); Lane 5, PAB(primer pair: GapB -check-U and GapB -check-L); Lane 6, SPF(primer pair: SPF-check-U and SPF-check-L); Lane 7, PFB (primer pair: pUC18-F and pUC18-L); Lane 8, PFBA (primer pair: pUC18-F and pUC18-L); Lane 9, PFBA (primer pair: PckA-check-U and PckA-check-L)
Analysis of gene expression by quantitative RT-PCR
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Fig. 2 Determination of relative expression levels of pckA, gapB and fbp in different mutant strains by qRT-PCR during exponential phase in minimal medium. The average values with strain RH33 were set as 100 %. Values are the averages with standard deviations from three independent measurements
of each target gene were significantly increased about 18to 45-fold in the recombinant, suggesting their anticipated overexpression and successful molecular manipulation (Fig. 2). Growth and metabolic characteristics in different genetic modification strains For the purpose of estimating the effects of gene modification on the metabolic characterization of riboflavin producing strains, mutants were compared with respect to their growth and productivity characteristics in shake flask. Results were shown in Table 2; expression of pckA alone did not increase riboflavin productivity, instead of exhibiting a slightly lower growth and riboflavin productivity. In comparison, expression of gapB increased riboflavin productivity slightly (from 0.047 ± 0.003 to 0.050 ± 0.003 mmol/g CDW/h). While co-expression of pckA and gapB only resulted in an about 4.3 % increase in the riboflavin productivity from 0.047 ± 0.003 to 0.049 ± 0.003 mmol/g CDW/h. Surprisingly, overexpression of fbp alone in SPF significantly enhanced the riboflavin
Fig. 3 Riboflavin production of different B. subtilis strains in shake flask
productivity from 0.047 ± 0.003 to 0.052 ± 0.004 mmol/ g CDW/h. Meanwhile, the biomass productivity decreased slightly with glucose as carbon source. Moreover, cooverexpression of gapB and fbp improved riboflavin productivity approximately 12.8 %. Consistently, co-overexpression of pckA, gapB and fbp got similar results with slightly lower riboflavin productivity 0.052 ± 0.004 mmol/g CDW/h compared to that of strain PFB. It was observed that glucose uptake rate remained almost unaffected by the genetic modifications and the specific growth rate decreased with increasing riboflavin production, which verified the redistribution of carbon flux towards PPP. Influence of overexpression of pckA, gapB, fbp on riboflavin production and biomass formation in shake flask cultivation To gain further insight into how pckA, gapB and fbp overexpression influence the riboflavin production, we tested the performances of the mutants in shake flask
Table 2 Growth and metabolic characterization of B. subtilis strains during exponential growth (OD600 between 0.3 and 0.6) cultivated in minimal medium Parameter Specific glucose uptake rate (mmol/g CDW/h) Specific growth rate (/h) Specific riboflavin production rate (mmol/g CDW/h)
RH33 4.2 ± 0.4
TPA 4.2 ± 0.3
NPB 4.1 ± 0.1
PAB 4.1 ± 0.2
SPF 4.1 ± 0.3
PFB 4.1 ± 0.3
PFBA 4.1 ± 0.4
0.26 ± 0.02
0.22 ± 0.02
0.24 ± 0.03
0.24 ± 0.03
0.22 ± 0.03
0.22 ± 0.03
0.22 ± 0.02
0.047 ± 0.003
0.045 ± 0.002
0.050 ± 0.003
0.049 ± 0.003
0.052 ± 0.004
0.053 ± 0.003
0.052 ± 0.004
Results represent the mean values with standard deviations from three independent measurements
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The specific growth rate of PFB decreased from 0.26 ± 0.02 to 0.22 ± 0.01 h-1 and riboflavin productivity improved approximately 12.8 %, which is consistent with the results in shake flask cultivation in MM (Table 2). It was implied that the co-overexpression of the crucial genes in gluconeogenesis was beneficial for riboflavin precursors supply which facilitated riboflavin production. As a conclusion, deregulating gluconeogenesis by cooverexpressing gapB and fbp simultaneously could successfully improve riboflavin production in B. subtilis.
Discussion Fig. 4 Growth, residual glucose and riboflavin production profile during fed-batch fermentation in a 5 l bioreactor of B. subtilis RH33 and PFB. Biomass: RH33 (filled diamond); PFB (filled triangle); Riboflavin: RH33 (filled circle); PFB (filled square)
cultivation. Experimental results indicated that riboflavin synthesis levels varied apparently in various mutants as compared with parental strain under the same cultivation condition (Fig. 3). For SPF, fbp overexpression led to a substantial promotion in riboflavin synthesis and yielded a final concentration of 4.73 g/l, which was 18.0 % higher than that of RH33. When pckA was overexpressed in strain TPA, riboflavin synthesis even dropped slightly. In contrast, co-overexpression of pckA and gapB in PAB resulted in as much as about 12.0 % enhancement in riboflavin synthesis. And similar result was observed when gapB sole expression in NPB, whose riboflavin production increased by 14.2 %. According to the above results, we speculate that co-overexpression of fbp and gapB might have an important impact on riboflavin synthesis. As expected, approximately 21.9 % promotion in riboflavin production and 4.89 g/l accumulation concentration was observed in PFB, which proved our speculation. Overexpressed pckA in mutant strain PFB did not further improve riboflavin production. Only 4.81 g/l riboflavin accumulated in flask cultivation which is even lower than that of PFB. Riboflavin production in fed-batch cultivation With the purpose of examining the potential of large scale fermentation of PFB, we investigated the performance of the strains in fed-batch fermentation. As Fig. 4 indicated that the cell growth traits were similar between the parental strain RH33 and engineered strain PFB. In comparison to RH33, the cell density of PFB slightly decreased with glucose as carbon source in fed-batch fermentation, while the riboflavin production of PFB significantly increased. An average of 27.8 % increase in riboflavin titer was achieved by PFB, up to 13.36 g/l in 2-day fermentation.
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The development of rational metabolic engineering strategies to improve bioprocesses depends critically on a deep understanding of cellular metabolism. Gluconeogenesis, one of the most important pathways in central carbon metabolism, has been exclusively studied as described in introduction. Furthermore, modification of gluconeogenesis has been successfully employed to redirect flux toward the PPP in C. glutamicum (Becker et al. 2005, 2007). Since the metabolic regulation of gluconeogenesis seems similar for different organisms, deregulation of gluconeogenesis could lead to similar effects in other bacteria. Also it has been demonstrated that gluconeogenesis could be another important point of focus to improve production of riboflavin (Tannler et al. 2008a, b). Therefore, pckA, gapB and fbp were chosen and conducted to redirect carbon flow of gluconeogenesis into PPP for further investigation of metabolic characterization of riboflavin producing B. subtilis. Both fbp and glpX genes encode FBPases and are functionally equivalent to each other in B. subtilis. Besides, FBPases are proved to be constitutive enzymes and transcribed at an equal level under gluconeogenic conditions. It’s worth noting that, DglpX mutant presented approximately 30-fold activation of FBPase activity due to the activation of fbp in the presence of 1 mM PEP under in vitro condition. On the contrary, 1 mM PEP completely abolished any detectable FBPase activity in the fbp knockout mutant (Jules et al. 2009). This indicates that fbp is more suitable for manipulation to up-regulate gluconeogenesis. Consequently, fbp-encoded FBPase was chosen and overexpressed in riboflavin-producing strains. In contrast to overexpression of glyceraldehyde-3-phosphate dehydrogenase or phosphoenolpyruvate (PEP) carboxykinase, overexpression of FBPase allows the most promising improvement of riboflavin production by 18.0 % in B. subtilis grown on glucose. Becker and his partner’s research may offer clue to explain these promising results. In their research, amplified expression of FBPase via the promoter of the gene encoding elongation factor TU
World J Microbiol Biotechnol
(EFTU) increased the lysine yield in the feedback-deregulated lysine-producing strain C. glutamicum lysCfbr by 40 % on glucose. 13C metabolic flux analysis on glucose indicated that the overexpression of FBPase caused a redirection of carbon flux from glycolysis toward PPP, and the relative flux into PPP was increased approximately 10 %, which led to increased NADPH supply required for lysine biosynthesis in C. glutamicum (Becker et al. 2005). However, in our research, enhancement of riboflavin production is derived from improved precursor Ru5P supply by redirecting more carbon flux into PPP (Wang et al. 2011), rather than increased NADPH supply. As a consequence, a high PPP flux led to an increased formation of NADPH, and higher catabolic NADPH formation occurred than was necessary to satisfy the anabolic demands. The riboflavin-producing B. subtilis could deal with this apparent NADPH overproduction to close its metabolic balance by two potential balancing mechanisms. First, a putative transhydrogenase-like mechanism could catalyze the oxidation of NADPH by reducing NAD?, which was considered to be the most plausible though no such transhydrogenase gene information has been annotated for B. subtilis (Dauner et al. 2002; Ruhl et al. 2010). Second, redox cycles formed by enzymes with NADPH oxidase activity could effectively achieve a balance. In the present work, NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (encoded by gapB) was activated in the presence of glucose, whose sole or combinational overexpression with other genes in gluconeogenic pathway resulted in increased riboflavin production. Derepressed synthesis of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase have been proved to lead to a metabolic jamming, resulting in high hexose phosphate concentrations that caused the increase of PPP flux (Tannler et al. 2008a), and the corresponding consumption of NADPH occurred by reducing NADP?. We therefore are more inclined to the second or the combination of these two potential balancing mechanisms in this work. Largely in agreement with results obtained from B. subtilis RB50::pRF69 (Tannler et al. 2008b), co-overexpression of pckA and gapB under promoter P43 resulted in 12 % improved riboflavin production, while overexpression of pckA or gapB separately presented different effects. Overexpression of pckA got the negative results. A possible reason is that derepression of pckA under glycolytic condition causes the growth defect observed in shake flask cultivation due to ATP dissipation via extensive futile cycling through the pyruvate carboxylase, PEP carboxykinase, and pyruvate kinase (Sauer and Eikmanns 2005; Ruhl et al. 2010). In particular, the high pckA flux limits the amount of oxaloacetate available for aspartate synthesis by the aspB-encoded aspartate aminotransferase and/or for reaction with acetyl coenzyme A to fuel the TCA cycle
(Tannler et al. 2008a). Another potential explanation for this phenomenon is that metabolites such as PEP and glycerate-3-phosphate cannot be further involved in gluconeogenic reactions without expression of gapB-encoded NADP?-dependent glyceraldehyde-3-phosphate dehydrogenase in B. subtilis TPA. Thus, carbon flux cannot be redirected towards the PPP, instead of leading to deleterious effects on cell growth and riboflavin yield. Co-expression of the gapB and fbp led to 21.9 and 27.8 % increased riboflavin production in shake flask and fed-batch cultivation, respectively. Based on our data conclusively, we recommend an extended application of gapB and fbp overexpression for the production of products directly stemming from the PPP as well as other NADPHdemanding compounds in microorganisms like B. subtilis, C. glutamicum and E. coli. Acknowledgments We thank Dr. Zhenquan Lin for the help of qRT-PCR analysis. This work was supported by National Program on Key Basic Research Project (2011CBA00804, 2012CB725203), National Natural Science Foundation of China (NSFC-21206112, NSFC-21176182), National High-tech R&D Program of China (2012AA022103, 2012AA02A702) and the Innovation Foundation of Tianjin University (1308).
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