High yield recombinant penicillin G amidase production and export into the growth medium using Bacillus megaterium
© Yang et al; licensee BioMed Central Ltd. 2006
Received: 19 September 2006
Accepted: 28 November 2006
Published: 28 November 2006
During the last years B. megaterium was continuously developed as production host for the secretion of proteins into the growth medium. Here, recombinant production and export of B. megaterium ATCC14945 penicillin G amidase (PGA) which is used in the reverse synthesis of β-lactam antibiotics were systematically improved.
For this purpose, the PGA leader peptide was replaced by the B. megaterium LipA counterpart. A production strain deficient in the extracellular protease NprM and in xylose utilization to prevent gene inducer deprivation was constructed and employed. A buffered mineral medium containing calcium ions and defined amino acid supplements for optimal PGA production was developed in microscale cultivations and scaled up to a 2 Liter bioreactor. Productivities of up to 40 mg PGA per L growth medium were reached.
The combination of genetic and medium optimization led to an overall 7-fold improvement of PGA production and export in B. megaterium. The exclusion of certain amino acids from the minimal medium led for the first time to higher volumetric PGA activities than obtained for complex medium cultivations.
The Gram positive bacterium Bacillus megaterium has several advantages over other microbial host-systems for the production and secretion of recombinant proteins . In contrast to Escherichia coli it has a high capacity for protein export . Compared to Bacillus subtilis, B. megaterium reveals an useful plasmid stability and a low intrinsic protease activity . Important prerequisites for a biotechnological application of this organism include an efficient transformation system, multiple compatible, freely replicating plasmids and the possibility to integrate a heterologous gene into the genome [3, 4]. Recently, production of heterologous exoproteins by B. megaterium was further improved by use the exoprotease NprM-deficient B. megaterium strain MS941 [5, 6] and by the coexpression of the signal peptidase gene sipM . However, some bottlenecks were still observed for the production and secretion of some of the studied heterologous proteins. The multidomain and high molecular weight dextransucrase DsrS (Mr = 180,000) from Leuconostoc mesenteroides aggregated extracellularly during high cell density cultivation . The heterologous gene of the Thermobifida fusca hydrolase (tfh) was only successfully expressed in B. megaterium after its codon bias was adapted to B. megaterium codon usage . Other unknown limiting factors contained in the employed semi-defined medium repressed protein production and secretion in high cell density cultivation .
Here, we report on the expression of the penicillin G amidase gene (pga) isolated from B. megaterium ATCC14945 in derivatives of B. megaterium DSM319. This homologous penicillin G amidase (PGA) has a relative molecular mass of 90,000 consisting of two autocatalytically processed subunits (α, β) . The function of PGA in nature is not yet fully understood. B. megaterium may produce PGA extracellularly to degrade phenylacetylated compounds in order to generate phenylacetic acid (PAA) which can be used as carbon source . In industry, PGA is used for the production of new β-lactam antibiotics. It hydrolyzes penicillin G yielding phenyl acetate and 6-aminopenicillanic acid (6-APA). The 6-APA provides the molecular core of all β-lactams to which D-amino acid derivatives can be substituted to create novel antibiotics, e.g. amoxicillin. PGA of B. megaterium is industrially used for the outlined reverse synthesis reaction due to its higher synthesis rate compared to E. coli PGA [11, 12]. The intensively studied E. coli PGA is predominantly exported into the periplasm . In contrast, using B. megaterium to secrete homologous B. megaterium PGA directly into the growth medium should facilitate its purification and consequently decrease the downstream processing and final production costs.
In this contribution we tested directed molecular strategies for the stepwise improvement of PGA production and secretion using B. megaterium.
Rationale of the experimental approach for PGA production in B. megaterium
First, in order to stabilize the desired product PGA in the growth medium the influence of calcium ions and the extracellular protease NprM on enzyme stability and activity were investigated. Subsequently, the leader peptide of the extracellular lipase LipA from B. megaterium was tested for the improvement of PGA export. Gene induction using the xylA promoter was analyzed in a xylA mutant strain to prevent inducer utilization. Finally, medium optimization and up scaling were approached systematically.
Increased recombinant PGA production and secretion using B. megaterium by the addition of calcium ions
Characterization of secreted B. megaterium PGA
The pga gene was initially cloned with the 5' region encoding its mature signal peptide SP pga . SDS-PAGE analysis of the extracellular proteins of recombinant B. megaterium carrying pRBBm23 (encoding SP pga - PGA) revealed two subunits of PGA with relative molecular masses (Mr) of 27,000 (α-chain) and 57,000 (β-chain) (Fig. 1). The N-terminal amino acid analysis of both recombinant exported proteins indicated that the α-chain started at amino acid residue 25 (GEDKNEGVKVVR) while the N-terminal amino acid sequence of the β-chain SNAAIVGSEKSATGN corresponded to residues 266 to 279. Hence, the α- and β-subunit of PGA range from residue 25 to 265 and from 266 to 802 with calculated molecular masses of 27,753 Da and 61,394 Da, respectively. These calculated masses corresponded well to the experimentally observed masses of the subunits and suited perfectly the report by Panbangred et al. . The native signal peptide sequence was deduced as MKTKWLISVIILFVFIFPQNLVFA.
The signal peptide of the extracellular lipase LipA increases PGA export in B. megaterium
Stepwise improvement of PGA production and export using B. megaterium
PGA activity [U gCDW -1]
PGA [mg L-1]
MM + 0.5 × AA
MM + 1 × AA
MM + 2 × AA
MM + 1 × AA
Construction of a B. megaterium strain deficient in the utilization of the gene expression inducing xylose
Next, early and late induction of gene expression by the addition of xylose were compared. When the inducer xylose was added right at the beginning of the cultivation, the maximal specific activity was reached 7.5 h after the start of cultivation. Similar final activities were reached when xylose was added at an OD578nm of 0.4 (data not shown). An induction of gene expression at higher optical density, e.g. at OD578nm 4, led to a faster appearance of PGA activity after induction, but just half the amount of PGA was obtained compared to the early induction (data not shown). Hence, 5 g L-1 xylose was added right at the beginning of the cultivation.
Optimization of the complex growth medium
From complex to mineral medium
Upscale of PGA production using B. megaterium to a 2 Liter bioreactor
Next, the obtained improvements in the bioreactor were compared to a bioreactor cultivation performed at the beginning of the study. This comparison of the described complex and minimal medium with a pH-controlled batch cultivation of B. megaterium strain MS941 carrying pRBBm23 (encoding SP pga -PGA) using A5 semi-defined medium excluding calcium ions (Fig. 7) provided insights into the improvement process via the different described steps. In cultivations using either LB or minimal medium, PGA secretion started in the exponential phase, whereas in a cultivation using semi-defined A5 medium it started in the stationary phase. Finally, only 4.2 mg PGA per Liter growth medium were obtained using strain MS941 carrying pRBBm23 (encoding SP pga -PGA) in A5 medium. Hence, using the newly constructed strain YYBm1 deficient in xylose utilization, the signal peptide of LipA, an optimized minimal medium supplemented with calcium ions and a defined mix of amino acids the volumetric PGA productivity was improved 7-fold resulting in 29.0 mg PGA per Liter growth medium.
We systematically optimized B. megaterium for the recombinant production of PGA. Some unexpected observations were made. A potential metalloprotease was exclusively produced by B. megaterium MS941 and YYBm1 cultivated in medium containing tryptone from Oxoid and not in the presence of Bacto tryptone. The Oxoid tryptone was characterized by its higher content in arginine, aspartic acid, and tyrosine. This might have provided an external stimuli of unknown nature which induced expression of the metalloprotease gene. Determination of the corresponding mRNA levels via Northern blot analysis might help to shed some light on the observed phenomena. Similarly, PGA production was also influenced by the tryptone source as well as the amino acid composition and content of the growth medium. The observed production pattern might be the result of a complex interplay of various factors influencing growth, protein production and export as well as stress responses. Usually complex media provide better growth due to the supplement of the full set of known and unknown essential growth factors. Nevertheless, the supply of C-, N-, S- sources and other growth factors in an excess often causes stress and other regulatory responses to optimize the bacterial metabolism towards the environmental stimuli. As a consequence intracellular amino acid synthesis and protein production and export might be decreased. A system biotechnology approach with the systematic high throughput determination of transcriptome, cytoplasmic proteome, secretome and especially the metabolome for the various growth and protein production conditions will finally help us to determine the exact cellular parameters involved in the observed protein production behaviour. This information might provide a solid bases for the directed further metabolomic engineering of B. megaterium for optimal protein production and export.
In contrast to the complex explanations for the outlined observations the more efficient PGA production and secretion via an induction of pga gene expression at low cell densities compared to high cell densities might simply be caused by the longer gene induction and protein production time. This phenomena was observed before by our group . Once one step of protein production like protein export becomes limiting, longer protein production times increase the overall product yield. Nevertheless, produced PGA amounts (~2000 U L-1) in this study did not completely reach such of previously described B. megaterium PGA production strains (~3000 U L-1 in , ~9000 U L-1 in ) or that of the published intracellular production of the enzyme in E. coli (~30,000 U L-1 in ). Outlined enzyme activity results are not simple to compare since absolute protein amounts are not given by the mentioned PGA productions. Therefore, observed differences between the various B. megaterium production strains might be due to differences in the employed enzymatic test systems. Currently, intracellular protein production in E. coli is still more efficient compare to recombinant protein production and export with B. megaterium. Limitations in up scaling protein production processes including protein export were observed for B. megaterium [1, 6]. Again, a system biology approach should help us to identify existing bottlenecks and allow for systematic bioengineering solutions.
A systematic improvement of the recombinant production and export of B. megaterium ATCC14945 penicillin G amidase using B. megaterium was performed. The addition of 2.5 mM calcium ions increased the specific activity by 2.6-fold. Exchange of its natural signal peptide by the one of the B. megaterium extracellular lipase LipA increased secretion by 1.7-fold. A B. megaterium strain deficient in the extracellular protease NprM and in xylose utilization (ΔxylA) was developed allowing for stable extracellular proteins and long time induction of gene expression by xylose. Next, a defined minimal medium with defined amino acid additions for high yield PGA production was developed. Finally, PGA production successfully scaled up to 2 L controlled batch fermentations.
Materials and methods
Plasmids and strains
Strains, plasmids and primers used in this study.
Mutant of DSM319,lacZ-
Mutant of WH320,xylA1-spoVG-lacZ
Mutant of DSM319, ΔnprM
Mutant of MS941, ΔxylA, ΔnprM
Strain for plasmid construction
Gibco Life Technologies
Shuttle vector for cloning in E. coli (Ap r ) and gene expression under xylose control in B. megaterium (Tc r ); P xylA -MCS
pMM1520 derivative – vector for intracellular protein production
pMM1522 derivative – vector for protein secretion into the medium; P xylA -SP lipA -MCS
sp pga -pga (2,476 bp) (B. megaterium strain ATCC14945) cloned into Bsr GI/Sac I of pMM1522; P xylA -SP pga -pga
pga (2,407 bp) (B. megaterium strain ATCC14945) without coding sequence for sp pga cloned into Bgl II/Eag I of pMM1525; P xylA -SP lipA -pga with Sfo I-spacer
pRBBm48 without Sfo I-spacer; P xylA -SP lipA -pga
Shuttle vector for cloning in E. coli (Ap r ) and gene expression in B. megaterium (Ery r ); temperature sensitive B. megaterium ori
Ap r in E. coli, Cm r in B. subtilis, Cm r in E. coli, Tc r in E. coli
pHBIntE derivative with xyl A from B. megaterium DSM319 genome sequence
pYYBm4 derivative – xyl A'-cml-'xyl A
ttattagatct tggcgcc ggggaggataagaatgaagg
A xylose deficient strain was generated from B. megaterium MS941 by integration of the chloramphenicol resistance mediating cat gene into the chromosomal copy of the xylA gene via a double crossover . For the necessary construct, the xylA gene was amplified by PCR from B. megaterium MS941 genomic DNA using the primers xylA_as and xylA_s. After digestion of the PCR product, the DNA fragment was cloned into the Sac I/Sac I sites of pHBIntE . The resulting plasmid was called pYYBm4. The plasmid contained a temperature sensitive origin of replication. The cat gene was amplified by PCR from pHV33  using the primers cml_as and cml_s and cloned into the Nde I/Xba I sites of pYYBm4. The resulting plasmid was called pYYBm8. The constructed plasmid was transformed into protoplasted B. megaterium MS941. Cells were grown at a non-permissive temperature of 30°C . The double crossover was achieved by dividing the chromosomal integration process into two screenable step: First, single-crossover recombination was achieved by cultivation of the culture at 42°C and addition of 3 mg L-1 chloramphenicol. Second, excision of the carrier replicon was screened via isolation of chloramphenicol resistant bacteria deficient in xylose utilization. The new strain B. megaterium YYBm1 grew on chloramphenicol M9 agar plates and exclusively used glucose as carbon source.
Constructed expression plasmids pRBBm23 and pRBBm49 were transformed in B. megaterium strains MS941, YYBm1, WH320, and WH323 by protoplast transformation . All used strains are derivatives of the wild type strain DSM319. MS941 has a defined deletion in the gene of the major extracellular protease NprM . WH323 is derived from WH320 (a chemically obtained β-galactosidase deficient mutant of DSM319) by inserting the E. coli lacZ gene in the xyl A gene. Hence, YYBm1 and WH323 do not consume xylose as carbon source.
Growth medium composition
As complex medium a high salt Luria-Bertani broth (LB) medium containing of 5 g L-1 NaCl, 5 g L-1 yeast extract, and 10 g L-1 tryptone from Bacto (Heidelberg, Germany) or Oxoid (Wesel, Germany), respectively, was used.
The semi-defined A5 medium contained 30 g L-1 glucose, 5 g L-1 (NH4)2SO4, 2.2 g L-1 KH2PO4, 300 mg L-1 MgSO4 × 7H2O, 500 mg L-1 yeast extract, and 2 mL L-1 trace element solution. The trace element solution contained 40 g MnCl2 × 4H2O, 53 g CaCl2 × 2H2O, 2.5 g FeSO4 × 7H2O, 2 g (NH4)6Mo7O24 × 4H2O, and 2 g CoCl2 × 6H2O per Liter.
The minimal medium contained 50 mM MOPSO (pH 7.0), 5 mM Tricin (pH 7.0), 520 μM MgCl2 × 6H2O, 276 μM K2SO4, 50 μM FeSO4 × 7H2O, 2.5 mM CaCl2, 100 μM MnCl2 × 4H2O, 50 mM NaCl, 10 mM KCl, 37.4 mM NH4Cl, 1.32 mM K2HPO4, 0.4 % (w/v) glucose, 1 mL L-1 trace element solution, and 1 mL L-1 vitamin solution with 5 g L-1 xylose for induction of recombinant gene expression. The trace element solution contained 3.708 mg (NH4)6Mo7O24 × 4H2O, 24.73 mg H3BO3, 7.137 mg CoCl2, 2.497 mg CuSO4, 15.832 mg MnCl2, and 2.875 mg ZnSO4 per Liter. The vitamin solution consisted of 6 mg biotin, 20 mg niacin amid, 20 mg p-amino benzoate, 10 mg Ca-panthotenate, 100 mg pyridoxal/HCl, 20 mg folacid, 50 mg riboflavin, 50 mg DL-6,8-thioctic acid, and 10 mg thiamine dichloride per Liter. For medium optimization, minimal medium was supplemented with different concentrations of amino acid solution. Stock solution (10 x) was prepared separately according to the maximal solubility of amino acids in water as: 10 mg alanine, 10 mg arginine, 1 mg aspartic acid, 1 mg cysteine, 40 mg glycine, 4 mg isoleucine, 2 mg leucine, 10 mg lysine, 5 mg methionine, 5 mg proline, 2.5 mg phenylalanine, 5 mg serine, 5 mg threonine, 1.6 mg glutamic acid, 1 mg tryptophane, 55 μg tyrosine, 8 mg valine, 4 mg histidine, 3 mg asparagines, and 3 mg glutamine per Liter. The various optimized amino acid solutions (1 x) were added to the growth medium as indicated.
For solid media 15 g agar per Liter was added. For selection of Bmegaterium deficient in xylose utilization, M9 media were used consisting of 500 mg NaCl, 1 g NH4Cl, 3 g KH2PO4, 7.5 g Na2HPO4 2H2O, 4 g glucose, 120 mg MgSO4, and 10 mg CaCl2 per Liter tap water .
B. megaterium precultures were cultivated in 50 mL of the indicated medium at 37°C and 120 rpm for 16 h. For microtiter plate cultivation, 200 μL culture medium with an adjusted initial OD578nm of 0.1 to 0.2 was transferred to a 96-well microtiter plate except the outer wells which were filled with water because of the evaporation. The plate was cultivated in the Fluoroskan Ascent fluorescence reader (Thermo electron corporation, Dreieich, Germany) at 37 C and 1,020 rpm with an orbital shaking diameter of 1 mm as described previously .
For shaking flask cultivation, B. megaterium strains were grown in 500 mL baffled Erlenmeyer flasks with 100 mL medium at 37°C and 250 rpm. Expression of the pga gene was induced by addition of 5 g L-1 xylose to the growth medium.
For bioreactor cultivation, a Biostat B2 (B. Braun, Melsungen, Germany) with 2 L working volume connected to an exhaust gas analysis unit (S710, Sick Maihak, Germany) was used. The bioreactor was inoculated with 1 % (v/v) cells and cultivated at 37°C with controlled pH at 7 as previously described [1, 8].
In microtiter plate cultivation OD580nm was measured in the Multiskan Ascent photometer (Thermo electron corporation, Dreieich, Germany). The relationship between OD580nm measured from microtiter plate and OD578nm measured from 1 cm cuvette was determined as OD578nm = 3.719 × OD580nm. For shaking flask and bioreactor cultivation samples for biomass, metabolites, and PGA activity were taken at regular intervals. The OD578nm was measured in triplicates with an Ultrospec 3100 pro spectrophotometer (Amersham Pharmacia, UK). The relationship between CDW and OD578nm was determined as CDW [g L-1] = 0.395 × OD578nm for YYBm1 and as CDW [g L-1] = 0.334 × OD578nm for MS941. The concentration of glucose and metabolites was determined by HPLC (Shimadzu, Japan) using an Aminex HPX-87H column (Biorad, USA) and 10 mM H2SO4 as the mobile phase. A flow rate of 600 μL min-1 at 60°C was used in order to separate xylose from pyruvate.
SDS-PAGE was performed using a Mini Protean 3 apparatus (Bio-Rad, USA). Proteins were stained by Coomassie Brilliant Blue G250. For N-terminal sequencing, the separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane using a Trans-Blot Semi-Dry Transfer Cell (Bio-Rad, Munich, Germany) as described by the manufacturer and the N-terminal amino acid sequence was determined by Edmann degradation.
Directly after sampling, PGA activity was measured spectrophotometrically (Ultrospec 3100 pro, Amersham Biosciences, Sweden) via release of the 6-nitro-3-phenylacetamido-benzoic acid (NIPAB) as described previously . Freshly prepared NIPAB solution was prepared by dissolving 60 mg 6-nitro-3-phenylacetamido-benzoic acid in 100 mL 50 mM Na-Phosphate buffer. After addition of the enzyme sample, the absorption was immediately measured at 405 nm and 37°C for 60 s after a 20 s delay against a standard without addition of enzyme. One unit was defined as the amount of enzyme that caused the release of 1 μmol 6-nitrophenol per minute under the test conditions. The extinction coefficient of 6-nitrophenol is 8.98 cm2 μmol-1.
This work was financially supported by the Sonderforschungsbereich 578 der Deutschen Forschungsgemeinschaft (DFG). The authors thank Jibin Sun for the discussion during the work and Dr. Anton Ross for his kindly help by developing the NIPAB assay for PGA activity test. Part of the results presented here have been communicated at the 4th Recombinant Protein Production Meeting (Barcelona, 2006).
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