Metabolic engineering of Pseudomonas sp. strain VLB120 as platform biocatalyst for the production of isobutyric acid and other secondary metabolites
© Lang et al.; licensee BioMed Central Ltd. 2014
Received: 1 August 2013
Accepted: 29 December 2013
Published: 7 January 2014
Over the recent years the production of Ehrlich pathway derived chemicals was shown in a variety of hosts such as Escherichia coli, Corynebacterium glutamicum, and yeast. Exemplarily the production of isobutyric acid was demonstrated in Escherichia coli with remarkable titers and yields. However, these examples suffer from byproduct formation due to the fermentative growth mode of the respective organism. We aim at establishing a new aerobic, chassis for the synthesis of isobutyric acid and other interesting metabolites using Pseudomonas sp. strain VLB120, an obligate aerobe organism, as host strain.
The overexpression of kivd, coding for a 2-ketoacid decarboxylase from Lactococcus lactis in Ps. sp. strain VLB120 enabled for the production of isobutyric acid and isobutanol via the valine synthesis route (Ehrlich pathway). This indicates the existence of chromosomally encoded alcohol and aldehyde dehydrogenases catalyzing the reduction and oxidation of isobutyraldehyde. In addition we showed that the strain possesses a complete pathway for isobutyric acid metabolization, channeling the compound via isobutyryl-CoA into valine degradation. Three key issues were addressed to allow and optimize isobutyric acid synthesis: i) minimizing isobutyric acid degradation by host intrinsic enzymes, ii) construction of suitable expression systems and iii) streamlining of central carbon metabolism finally leading to production of up to 26.8 ± 1.5 mM isobutyric acid with a carbon yield of 0.12 ± 0.01 g gglc -1.
The combination of an increased flux towards isobutyric acid using a tailor-made expression system and the prevention of precursor and product degradation allowed efficient production of isobutyric acid in Ps. sp. strain VLB120. This will be the basis for the development of a continuous reaction process for this bulk chemicals.
KeywordsIsobutyric acid Isobutanol Pseudomonas Fermentative Valine synthesis route
The finite nature of fossil resources necessitates the development of new technologies for the production of chemicals based on natural renewable feedstocks. So far, approximately 90% of all chemicals are produced from fossil-based supplies, but the world market for bio-based chemicals is expected to increase from $US 3.6 billion in 2011 to $US 12.2 billion in 2021 [1–3]. The rapid progress in the fields of metabolic engineering and systems biology accelerates this development and allows the synthesis of non-natural and non-inherent products . Atsumi et al.  showed the production of higher alcohols via the amino acid catabolism by integrating the Ehrlich pathway into Escherichia coli. 2-keto acid intermediates were decarboxylated to the corresponding aldehydes using the 2-keto acid decarboxylase Kivd from Lactococcus lactis and were further converted by (host intrinsic) alcohol dehydrogenases to higher alcohols. Zhang et al.  demonstrated the successful oxidation of isobutyraldehyde to isobutyric acid in E. coli, by overexpressing an aldehyde dehydrogenase. Isobutyric acid is mainly used as a precursor for methacrylic acid production and has a market size of about 2.7 ∙ 106 t a-1. In addition, isobutyric acid can be utilized for the production of sucrose acetoisobutyrate, texanol or di-isobutyrate . Isobutyric acid can be chemically produced by reacting propene, carbon monoxide, and water in the presence of strong acids . This chemical route suffers from its dependency on fossil products of oil refining and natural gas processes and the involvement of compounds harmful to the environment such as sulfuric acid, hydrogen fluoride, and boron fluoride .
A major bottleneck of current bioprocesses is the susceptibility of microorganisms to toxic compounds [9, 10]. This results in low product titers and limited process stabilities having a negative impact on overall productivity. The cost competitiveness of biocatalytic processes is determined by these parameters  and it is indispensable to overcome these limitations. Pseudomonas species are known to exhibit mechanism enabling the adaptation to toxic environmental conditions . Importantly, these organisms are able to form stable surface associated microbial communities (biofilms), which have been described as potent alternative to planktonic cells regarding their application as biocatalyst, especially when solvents or otherwise toxic compounds are involved in the processes . Biofilms provide increased process stability and therefore potentially allow, in contrast to commonly used planktonic cells, a continuous process mode [14, 15].
Pseudomonads have a huge potential for bioremediation, and possess a rich pathway repertory for the degradation of a variety of non-natural and non-inherent toxic compounds such as aromatic organics [16–19]. Carbon is utilized in Pseudomonas wild types almost without the production of byproducts such as acetate , lactate, glycerol or ethanol, which improves carbon yields and simplifies downstream processing in comparison to organisms like Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae. Under anaerobic and micro aerobic conditions, E. coli needs to produce mixed-acids directly from the key-precursor pyruvate to maintain an optimal redox and carbon environment for isobutyric acid synthesis, finally limiting the final carbon yield and affecting the downstream processing. In aerobic environment E. coli is known for its overflow metabolism under glucose excess conditions resulting in acetate formation .
So far, mainly aromatic compounds are produced using recombinant Pseudomonas strains. The fermentative de-novo synthesis of e.g. phenol, p-coumarate, p-hydroxystyrene, t-cinnamate, and several polyhydroxyalkanoates [9, 23–27] from glucose have been reported as well as biotransformations using toluene or styrene for the production of 3-methylcatechol and (S)-styrene oxide [28, 29]. Moreover, Nielsen et al.  reported the fermentative production of 1-butanol using an engineered Pseudomonas putida S12. Here the term ‘fermentation’ describes synthesis of the target product by whole cells directly from the added carbon and energy source, whereas ‘biotransformation’ refers to processes, where in addition to the growth substrate a biotransfomation substrate is added .
This study aims to establish the solvent tolerant, genome-sequenced Pseudomonas sp. strain VLB120 [29, 32] as a platform biocatalyst for the fermentative production of isobutyric acid. Therefore the Ehrlich pathway is incorporated into a Pseudomonad with the aim to produce isobutyric acid via the valine synthesis pathway. In this work, strain specific limitations of Pseudomonas like low expression levels of recombinant genes and product and precursor degradation were solved by means of metabolic engineering.
Overexpression of a 2-keto acid decarboxylase encoding gene in Pseudomonas sp. strain VLB120 allows the synthesis of isobutyric acid and isobutanol
Influence of yeast extract on product titers of isobutyric acid, isobutanol and 3-methyl-1-butanol
Isobutyric acid [μM]
5.4 ± 1.3
43.1 ± 0.4
50.0 ± 1.4
M9* + 5 g L-1 yeast extract
56.4 ± 1.5
72.1 ± 2.9
236.5 ± 0.4
Product and precursor degradation in Pseudomonas sp. strain VLB120
Growth rate (μ) and biomass yield (Y) of Pseudomonas sp. strain VLB120 on different carbon sources
Y (gcdw/gcarbon source)
0.41 ± 0.01
0.64 ± 0.01
0.24 ± 0.03
0.39 ± 0.05
0.24 ± 0.01
0.52 ± 0.01
0.15 ± 0.02
0.59 ± 0.04
0.025 ± 0.001
Deletion of branched chain 2-keto acid dehydrogenase (bkd) strongly increases product titers in 2-KIV biotransformations
Random mutagenesis to prevent isobutyric acid degradation
In general, 2-KIV biotransformations showed a maximum yield of approximately 50% isobutyric acid from 2-KIV. This compound serves as a precursor for leucine, valine and panthothenic acid synthesis which negatively influences the final yield (Figure 1).
Combined T7 overexpression of valine synthesis and Ehrlich pathway genes showed increased enzyme activity for each single protein
Specific activities of AlsS, IlvC, IlvD, Kivd in cell extracts of Pseudomonas sp. strain VLB120, Pseudomonas sp. strain T7 pPAPC-Km, pPDPK and Ps. sp. strain VLB120 pCom10-kivd
Specific activity (μmol min-1mgtotal protein -1)
VLB120 wild type
0.033 ± 0.009
0.0257 ± 0.0017*
0.0075 ± 0.0004
VLB120 T7 pPAPC-Km, pPDPK
3.494 ± 0.299
0.1476 ± 0.0425*
0.1955 ± 0.0023
2.96 ± 0.24
All proteins showed higher specific activities in the desired production host as compared to the wild type strain. Under the fermentation conditions used, Kivd activity in Ps. sp. strain pCOM10-kivd was not even detectable, being in line with the eGFP expression study (Additional file 1: Figure S2), and demonstrated the advantage of T7 expression in Ps. sp. strain VLB120.
Overexpression of the 2-KIV and Ehrlich pathway in the NTG mutant C18 T7 results in stable isobutyric acid production
Deletion of pathways competing for 2-ketoisovalerate and pyruvate increased carbon yield
In the present study, different variants of recombinant Ps. sp. strain VLB120 have been constructed by means of directed and random mutagenesis, which are capable of producing significant amounts of C4-precursors from glucose in aerobic fermentations with the focus on isobutyric acid synthesis.
Isobutyraldehyde metabolism in Ps. sp. strain VLB120
Overexpressing simply one gene encoding for a branched chain 2-ketoacid decarboxylase (KivD) in Ps. sp. strain VLB120, enabled this organism to produce isobutyric acid, isobutanol, and 3-methyl-1-butanol, directly from glucose. Isobutyraldehyde was not detected in the fermentation broth, indicating direct intracellular conversion of this compound. Based on genome analysis, 32 aldehyde dehydrogenases (Additional file 1: Table S2), and about 19 alcohol dehydrogenases (Additional file 1: Table S3) have been identified in Ps. sp. strain VLB120 capable to either reduce or oxidize isobutyraldehyde. Among these enzymes are ALDHs homologs known to be highly active towards isobutyraldehyde (e.g. PVLB12825, a 42% homolog to padA of E .coli K12). In addition, the strain harbors ADHs known to be able to catalyze the reduction of isobutyraldehyde (e.g. PVLB10545, a 86% homolog to adhP of E. coli K12). However, gene deletions of these candidate genes did not significantly improve product titers (data not shown). Rodriguez et al. , aiming to overproduce isobutyraldehyde in E. coli BW25113, identified eight ADHs in the genome of E. coli BW25113 and observed that the final product titer could only be increased by combining multiple ADH deletions. While in E. coli BW25113 the degradation of isobutyric acid is not described , the present work shows that Ps. sp. strain VLB120 harbors a complete pathway for isobutyric acid utilization (Figure 1), which enabled Ps. sp. strain VLB120 to grow on isobutanol, isobutyraldehyde and isobutyric acid (Table 2). The existence of an isobutyric acid degradation pathway was already described for P. putida, Candida rugosa, Yarrowia lipolytica and Desulfococcus multivorans and used in mutant strains of the first three species for the synthesis of 3-hydroxyisobutyric acid using isobutyric acid as precursor [36, 42–45].
NTG-random mutagenesis to prevent isobutyric acid degradation
To prevent undesired isobutyric acid degradation, several mutants were created by random mutagenesis. The apparent correlation between isobutyric acid and 3-hydroxyisobutyric acid accumulation (in mutants B57, B83 and D67) indicates the mmsAB operon (PVLB_03765, PVLB_03770)  coding for methylmalonate-semialdehyde-dehydrogenase and 3–hydroxyisobutyrate dehydrogenase to be involved in the reaction of isobutyric acid to isobutyryl-CoA. Exemplarily, the respective DNA loci were sequenced in mutants B83 and C18. The DNA sequencing revealed, that mmsB is altered in variant B83. A transition (G/C → A/T) of nucleotide 413 occurred, confirming that this special transition is favored using NTG mutagenesis . On the protein level, this mutation leads to an exchange of the strictly conserved residue Gly137 with asparagine  and thereby explains the non-active 3-hydroxyisobutyirc acid dehydrogenase in mutant B83. Mutant C18 shows no alteration in the mmsAB operon. In this case, most probably a broad substrate range acetyl-CoA-synthetase or a global regulator involved in isobutyric acid degradation was affected by NTG mutagenesis [45, 49].
Fermentative production of isobutyric acid in engineered Ps. sp. strain VLB120
The efficient fermentative production of isobutyric acid requires the overexpression of the genes of the valine synthesis pathway (alsS, ilvC, ilvD), a keto acid-decarboxylase (kivd) and a highly expressed gene for an aldehyde dehydrogenase (ALDH). While ilvC, ilvD and the ALDH gene are expressed homologously, alsS and kivd are derived from other species and show an altered codon usage, which often results in lower expression levels . In order to maximize expression levels and overcome promoter related problems like catabolite repression by various carbon sources , the so far used Palk promoter was replaced by a T7 RNA polymerase based system. Using eGFP fluorescence as a fast and easily detectable read-out for expression (Additional file 1: Figure S2), the performance of this system was evaluated confirming recently published results for P. putida KT2440 T7 . In addition, enzyme activity assays of single pathway genes proved successful overexpression in Ps. sp. strain VLB120 T7 (Table 3).
Overexpression of the 2-KIV synthesis pathway genes and kivd in the T7 variants of Ps. sp. strain VLB120 resulted in drastically increased isobutyric acid titers (Figure 4/ Table 1). In addition, the variant C18 T7 showed an increased stability of the formed product, which confirms the results of the 2-KIV biotransformations (Figure 2) under fermentative conditions. During glucose utilization, gluconate accumulated in Ps. sp. strain VLB120. This phenomenon is well understood and described in Pseudomonas species, which are known to metabolize glucose exclusively via the Entner-Doudoroff pathway, using 6-phosphogluconate as key intermediate . Ps. sp. strain VLB120 possesses a glucose dehydrogenase (PVLB_05240), but is lacking a gluconate dehydrogenase, which prevents 2-ketogluconate formation. The incomplete conversion of gluconate in mutant strain C18 T7 is comparable to the behavior of Pseudomonas putida KT2440 during poly-hydroxyalkonate synthesis observed by Poblete-Castro et al. . In their work, they enhanced the production of PHAs by the deletion of glucose dehydrogenase without affecting the specific growth rate. One may speculate, that this deletion leads to an increased flux towards pyruvate and thus more precursors for isobutyric acid synthesis are available.
Optimization of isobutyric acid production by the deletion of competing pathways
To prevent unproductive 2-KIV depletion via isobutyryl-CoA or various amino acid synthesis pathways (Figure 1), the influence of the genes bkd, ilvE, leuA, pycAB and panB was investigated. The strongest effect on isobutyric acid titers was measured for the mutants ΔpanB and Δbkd. By deleting subunit A of the branched chain α-keto acid dehydrogenase complex (bkd), growth on 2-KIV could be completely inhibited in Ps. sp. strain VLB120. Growth could be restored by overexpressing the decarboxylase gene kivd, channeling 2-KIV over isobutyraldehyde (Figure 1). The mutant strain shows drastically increased product concentrations of isobutyric acid and isobutanol during 2-KIV biotransformations, while during growth on glucose (fermentation) this effect seems to be insignificant, as the production rate of Ps. sp VLB120Δbkd C18-T7 harboring Δbkd is comparable to Ps. sp VLB120. Under fermentative conditions comprising high glucose und only low 2-KIV concentrations the activation of 2-KIV to isobutyryl-CoA seems to be only a minor carbon sink. Similar observations have been reported by Lu et al.  for Ralstonia eutropha. Deletion of ilvE helped to prevent undesired carbon loss, this confirms similar results reported for C. glutamicum and R. eutropha[55–57].
Deletion of panB had the strongest impact on carbon yield for isobutyric acid synthesis. panB is the first gene of pantothenic acid synthesis pathway, and in addition a precursor for CoA . The deletion of panB seems to increase 2-KIV availability, as also described for C. glutamicum[59, 60], and thereby results in an increased carbon yield of 0.12 ± 0.01 g g-1, which is about 25% of the theoretical maximum. panB deletion leads to mutants unable to grow on minimal medium (most probably connected to the disability to synthesize CoA).
For E. coli BW25113 10 g L-1 on a 5 mL scale using 40 g L-1 glucose has been reported  while for Ps. sp. strain VLB120 2 g L-1 have been observed using only 20 g L-1 carbon source. Experiments have been conducted in shake flasks with glucose excess where gluconate temporarily accumulated resulting in a pH drop to pH 6.8 which was even more pronounced during isobutyric acid formation. To prevent this pH drop, hampering catalyst robustness, glucose needs to be limited and the pH controlled. It is to be expected, that in such a controlled environment (bioreactor) higher final product titers and better carbon yields will be reached.
Isobutyric acid production and beyond
In consequence, the application of the demonstrated design principles, the use of the genome sequence of Ps. sp. strain VLB120 and the established metabolic engineering tools, will give access to a far broader product spectrum. Based on the engineered, highly active 2-KIV synthesis pathway the production of isobutanol, 3-methyl-1-butanol, 3-hydroxyisobutyric acid, isobutyraldehyde, valine and D-pantothenate would be feasible by slight pathway modifications. Beside the synthesis of isobutyric acid, we were already able to detect the Ehrlich pathway products isobutanol and 3-methyl-1-butanol, which are interesting bulk chemicals [33, 61]. More over 3-hydroxyisobutytric acid was accumulated in the not optimized mutant B83 with remarkable titers, being highly valuable synthons for the fine chemical industry .
The combination of an increased flux towards isobutyric acid using a tailor-made expression system and the prevention of precursor and product degradation allowed efficient production of isobutyric acid in Ps. sp. strain VLB120. This work experimentally verifies the genome derived metabolic network structure. A true platform organism was designed, which has the ability to produce an even wider product spectrum directly from glucose by only slight changes on the level of cell metabolism.
In general, studies investigating Pseudomonas for the fermentative production of chemicals are few . But clear benefits like low/no byproduct formation during glucose fermentation, high tolerance towards toxic substances , improved NAD(P)H regeneration rates under stress conditions , and a diverse gene repertoire for the processing of organic molecules underline the potential of Pseudomonads for industrial applications. The strain engineering of this novel organism sets the stage for the development of aerobic biofilm based processes for the continuous production of isobutyric acid and other secondary metabolites. Apart from increasing product titers, the long-term catalyst robustness is a most important but often neglected issue in host engineering, which will be a key focus in future studies regarding this newly introduced organism.
Chemicals, bacterial strains, and plasmids
Bacterial strains and plasmids used in this study
E. coli DH5α
supE44, DlacU169 ( f80 lacZ DM15), hsdR17 (rk-mk+), recA1, endA1, thi1, gyrA, relA
E. coli DH5α ʎ-pir
ʎ-pir lysogen of DH5a
Ps. sp. strain VLB120
Ps. sp. strain VLB120 T7
PlacUV5 T7 RNA pol, lacI q
Ps. sp. strain VLB120Δbkd
VLB120 with deletion of bkd, encoding subunit A of the branched-chain α-keto acid dehydrogenase
Ps. sp. strain VLB120Δbkd A10, A21, B57, B83, C18, D67, D76, E82
NTG-mutants of Pseudomonas sp. strain VLB120 Δbkd
Ps. sp. strain VLB120Δbkd C18 T7
PlacUV5 T7 RNA pol, lacI q
Ps. sp. strain VLB120Δbkd ΔilvE C18 T7
Ps. sp. strain VLB120Δbkd C18 ΔilvE, encoding branched-chain amino acid aminotransferase
Ps. sp. strain VLB120Δbkd ΔpycAB C18 T7
Ps. sp. strain VLB120Δbkd C18 ΔpycAB, encoding pyruvate decarboxylase
Ps. sp. strain VLB120Δbkd ΔleuA C18 T7
Ps. sp. strain VLB120Δbkd C18 ΔleuA, encoding 2-isopropylmalate synthase
Ps. sp. strain VLB120Δbkd ΔpanB C18 T7
Ps. sp. strain VLB120Δbkd C18 ΔpanB, encoding 2-ketoisovalerate hydroxymethyltransferase
Ps. sp. strain VLB120ΔbkdΔilvE ΔpanB C18 T7
Ps. sp. strain VLB120Δbkd ΔilvE C18 ΔpanB, encoding 2-ketoisovalerate hydroxymethyltransferase
Ps. sp. strain VLB120Δbkd ΔilvE ΔleuA C18 T7
Ps. sp. strain VLB120Δbkd ΔilvE C18 ΔleuA, encoding 2-isopropylmalate synthase
Ps. sp. strain VLB120Δbkd ΔilvE ΔleuA ΔpanB C18 T7
Ps. sp. strain VLB120Δbkd ΔilvE ΔleuA C18 T7ΔpanB, encoding 2-ketoisovalerate hydroxymethyltransferase
Ampr , Geneart delivery vector containing kivd
Kmr , broad host range expression vector, alk promoter, oriT, alkS regulator gene,
pCOM10 containing kivd
pCOM10 containing eGFP
Cmr, mob lacI q Km r PT7Ф10
pBR22b containing kivd
pBR22b containing alsS
pBR22b containing ilvC
pBR22b containing ilvD
Cmr, Kmr, pBR22b-alsS with BoxI blunt end inserted Kmr
pBR22b-alsS-km additionally containing ilvC
pCOM10 containing eGFP
Cmr, Kmr pBR22b-eGFP with BoxI blunt end inserted Kmr
Gmr, broad-host-range expression vector, alk promoter, oriT, alkS regulator gene
pCOM8 containing ilvD
pCOM8-T7-ilvD containing kivd
Gmr, suicide vector, P15A ori sac B RP4 pBluescriptSK MCS
Kmr, Gmr, pJQ200SK with hdp disrupted by Kmr gene, flanked by loxP recombination sites
Nick Wierckx (unpublished data)
Gm r , encodes cre recombinase
Nick Wierckx (unpublished data)
pUC18, mini-Tn7-Gm, PlacUV5 T7 RNA pol, lacI q
pUC18, mini-Tn7-Gm, PlacUV5 T7 RNA pol, lacI q
Kmr, oriR6K, lacZa with two flanking I-SceI sites
Gmr, ori RK2, xylS, Pm→ I-SceI
Cultivation of bacterial strains
Ps. sp. strain VLB120 was used as production host, while E. coli DH5α and E. coli DH5α (λ-pir) served as host strains for all plasmid based DNA manipulations. Ps. sp. strain VLB120 and E. coli strains were cultivated at 200 rpm, 30°C or 37°C (Infors AG, Bottmingen, Switzerland), respectively. For all cloning purposes, transformations and first precultures, cells were cultivated in lysogeny broth (LB, ). All other cultivations were performed in M9* minimal medium  which was supplemented with 1 mL L-1 US* trace element solution  and 2 mL L-1 1 M MgSO4 solution. For fermentations and cultivations used to prepare cell extracts, 5 g L-1 yeast extract and 0.001% (w/v) thiamine solution was added. Depending on the experiment, either glucose (5 g L-1 or 20 g L-1), isobutyric acid (10 mM), isobutyraldehyde (10 mM), isobutanol (10 mM), succinate (10 mM) or 2-ketoisovalerate (10 mM) served as carbon sources. All media were supplemented with appropriate antibiotics (kanamycin 50 μg mL-1 for E. coli and Ps. sp. strain VLB120, gentamycin 25 μg mL-1 for E. coli and Ps. sp. strain VLB120, chloramphenicol 34 μg mL-1 for E. coli).
For cultivations in minimal media, Ps. sp. strain VLB120 was cultivated in two subsequent precultures prior to the main culture. First, the strain was cultivated for 8 h in tubes filled with 5 mL LB overnight. 50 μL of this culture was used as inoculum for a fresh 5 mL M9* culture. After overnight growth 50 mL M9* medium in a 250 mL flask were inoculated to an initial OD450 0.2. Ps. sp. strain VLB120 cultures were either grown under aerobic conditions in open baffled shake flasks or under micro aerobic conditions in sealed baffled shake flasks to prevent the evaporation of volatile compounds.
For experiments investigating the utilization of different carbon sources by Ps. sp. strain VLB120 preculture derived cells were washed with M9* salt solution before inoculating fresh M9* medium complemented with the respective carbon source.
Random mutagenesis by N-methyl-N’-nitro-N-nitrosoguanidine (NTG)
Random mutagenesis by NTG was performed according to Adelberg et al. and Martin et al. [37, 40]. 5 mL M9*-medium (pH 7.4, 10 mM succinate as carbon source) was inoculated with 50 μL of an 8 h LB culture of Ps. sp. strain VLB120Δbkd and cultured overnight. This culture was used to inoculate the main culture (250 mL M9*, 10 mM succinate). Early exponential phase cells were harvested by centrifugation (10 min, 4.500 g, 4°C), washed and concentrated in CpI-Buffer (127 mM Na2HPO4∙2H2O, 36.2 mM Citric acid, pH 6.0) to an OD450 of 6 before 80 μg mL-1 NTG was added. Cells were incubated for 30 min, washed, resuspended in LB and stored for screening in 10% (w/v) glycerol at −80°C.
Screening for NTG-mutants unable to degrade isobutyric acid
To screen for mutants with changed properties regarding isobutyric acid degradation, 5 mL M9* medium containing 10 mM succinate were inoculated with NTG treated cells (see above). After overnight cultivation, cells were harvested by centrifugation, washed in identical medium without carbon source, and used to inoculate fresh 5 mL M9* medium with 10 mM isobutyric acid as sole carbon source to an OD450 of 0.2. After reaching an OD450 of 0.4, solid ampicillin was added up to its solubility limit (13 mg mL-1) and cultivation was continued for additional 8 hours. Afterwards, cells were harvested by centrifugation, washed twice with M9* medium, plated in appropriate dilutions to M9* plates containing 10 mM succinate as carbon source, and incubated at 30°C for 48 h. Mutants were replica plated on M9* medium containing 10 mM isobutyric acid and on LB medium. Mutants unable to grow in the presence of isobutyric acid as sole carbon source were selected for further investigations.
For the preparation of cell extracts, cells were cultivated in M9*(pH 7.4, 20 g L-1 glucose) and depending on the plasmid used induced either with 1 mM IPTG or 0.05% DCPK during exponential phase (OD450 0.5). After 4 h of induction, cells were harvested by centrifugation, washed, and resuspended to cell density of OD450 100 in ice cold buffer (50 mM Kpi pH 7.0, 0.5 mM dithiothreitol). Cells were disrupted by three passages through a precooled French press (5.5 MPa, SLM-Aminco, Rochester, NY, USA) and cellular debris was removed by centrifugation (20 min, 17,000 g, 4°C). Cell extracts were always prepared freshly and kept on ice. Total protein concentrations were determined using the Bradford protein assay .
The activity of acetolactate synthase (AlsS) was assayed according to Lang et al. , using 50 mM Kpi buffer (pH 7.0). The activity of ketol-acid reductoisomerase (IlvC) was determined according to Leyval et al. . The assay was coupled to the activity of AlsS using 50 mM pyruvate as substrate instead of 2-acetolactate. The activity of dihydroxy acid dehydratase (IlvD) was quantified according to Atsumi et al. . The substrate, 2,3-dihydroxy-isovalerate was purchased from SelectLab Chemicals GmbH (Bönen, Germany). The assay was conducted at 30°C for 30 min. 2-keto acid decarboxylase (Kivd) activity was determined by measuring the isobutyraldehyde concentration using HPLC analysis. Therefore, 500 μL 50 mM potassium phosphate buffer (pH 7.0) containing 10 mM MgCl2, 0.5 mM thiamine diphosphate, cell free extract, and 10 mM 2-KIV was incubated at 30°C for 30 min. The reaction was started by the addition of 2-KIV and stopped by the addition of 10 μL pure perchloric acid.
Cell densities were monitored by measuring the optical density at 450 nm (OD450) using a spectrophotometer (Biochrom Libra S12, Cambridge, UK). One OD450 unit corresponds to 0.186 gcdw L-1 for Ps. sp. strain VLB120.
Organic acids, sugars, alcohols and aldehydes were detected by HPLC (LaChrom Elite, Merck Hitachi, Darmstadt, Germany) equipped with a Trentec 308R-Gel.H ion exclusion column (300 × 8 mm, Trentec Analysentechnik, Gerlingen, Germany). The following conditions were applied; temperature: 40°C, isocratic flow rate: 1.0 ml min-1, solvent: 5 mM H2SO4, injection volume: 20 μL. Analytes were detected either by a UV (λ = 210 nm) or RI detector.
Lower product concentrations (< 500 μM analyte) were quantified on a Finningan Focus GC (Thermo Electron Corporation, Dreireich, Germany) with a Rt-βDex-sm column (30 m × 0.25 mm × 0.25 μm, Restek GmbH, Bad Homburg, Germany): GC oven temperature was 40°C for 5 min before it was increased to 80°C at a rate of 20°C min-1, and to 105°C at a rate of 7°C min-1. The maximum temperature of 200°C was reached at a rate of 60°C min-1 and kept for 3 min. Samples were extracted with 1:1 (v/v) ice cold diethyl ether containing 0.2 mM dodecane as internal standard.
eGFP fluorescence was measured using a microtiter plate reader (Infinite M200, Tecan, Mannedorf, Switzerland) at 488 nm and 511 nm for excitation and emission, respectively. Cells were harvested by centrifugation, washed and resuspended in 50 mM Tris buffer pH 8.0. Biomass concentrations were adjusted to OD450 0.4 using 50 mM Tris-Cl pH 8.0.
Restriction enzymes, T4 ligase, thermosensitive alkaline phosphatase, T4 polynucleotide kinase, and high-fidelity phusion DNA polymerase were purchased from Fermentas GmbH (St. Leo-Rot, Germany). All enzymes were used according to supplier’s recommendations. Plasmid DNA was isolated with a miniprep Kit (Peqlab GmbH, Erlangen, Germany). Gel extraction and DNA purification were performed with a NucleoSpin Gel and PCR clean-up Kit (Macherey-Nagel GmbH, Düren, Germany). All DNA manipulations were performed using standard procedures . Restriction sites, which were added to the respective DNA fragments using PCR amplification, are listed in the primer section (Additional file 1: Table S2).
Transformations were performed by electroporation (2500 V, Equibio EasyjecT Prima, Ashford, UK) using sucrose-treated competent cells  and glycerol-treated competent cells  for Ps. sp. strain VLB120 and E. coli DH5α, respectively.
pCOM-based plasmid construction
The kivd gene was artificially synthesized (Geneart AG, Regensburg, Germany) in pMA-RQ-kivd based on available sequence data of Lactococcus lactis subsp. lactis strain IFPL730 (GenBank: AJ746364). kivd was cut from the original delivered plasmid at artificially inserted restriction sites NdeI and AscI and ligated into pCOM10 creating pCOM10-kivd. eGFP was amplified using primers KL35/36 digested and ligated into pCOM10 to create pCOM10-eGFP.
Single gene pBR22b constructs
pCOM10-kivd was cut with NdeI and SalI and the fragment was ligated into pBR22b creating pBR22b-kivd.
The alsS gene was amplified in two fragments from genomic Bacillus subtilis DNA using primers KL1/2 and KL3/4, respectively, which were designed based on available sequence data (Genbank: AP012496 region: 3537181). The fragment was ligated into pBR22b creating pBR22b-alsS. To ensure an appropriate antibiotic selection pressure in Ps. sp. strain VLB120, a kanamycin cassette was amplified from pJQhdp::Km by primers KL11/12 and the phosphorylated fragment was ligated into dephosphorylated pBR22b-alsS cut with BoxI, creating pBR22b-alsS-Km.
ilvC (PVLB_03705) and ilvD (PVLB_01425) were amplified from genomic Ps. sp. strain VLB120 DNA using primers KL 5/6 and KL 7/8, respectively. The fragments were inserted in pBR22b creating pBR22b-ilvC and pBR22b-ilvD.
eGFP was amplified using primer KL35/36 digested and ligated into pBR22b to create pBR22b-eGFP.
Construction of pPAPC-Km and pPDPK
ilvC including the T7 promoter and the ribosomal binding site (RBS) was amplified from pBR22b-ilvC with primers KL13/14. The fragments were inserted into the dephosphorylated vector pBR22b-alsS-Km creating pPAPC-Km.
ilvD including the T7 promoter, the RBS, and the T7 terminator was amplified from pBR22b-ilvD using primers KL29/30 and the phosphorylated, digested fragment was ligated into the dephosphorylated Bst1107I cut pCOM8 creating pCOM8-ilvD. Orientation of the fragment was determined using XhoI restriction and the vector with the same orientation of ilvD and Gmr cassette was used for further work.
kivd including the T7 promoter, and the RBS was amplified from pBR22b-kivd using primers KL27/28 and the phosphorylated, cut fragment was ligated into the dephosphorylated, restricted pCOM8-ilvD creating pPDPK.
Gene deletion (Δbkd)
Deletion of the gene coding for subunit A of the branched chain α-keto acid dehydrogenase (bkd) was achieved as described earlier using pJQ200SK . For the construction of pJQ200bkd::Km, genomic Ps. sp. strain VLB120 DNA was used as template to amplify the up- and downstream fragment of bkd using primers K15/16 and K17/18. Kmr-loxP was cut from pJQhdp::Km with XbaI and purified by gel electrophoresis. All three parts were ligated in one pot and Ps. sp. strain VLB120 was transformed with the verified vector. Kanamycin resistance was used to prove genomic integration of pJQ200bkd::Km. To distinguish between single crossover and double crossover events, mutants were screened by replica plating for kanamycin resistance and gentamycin sensitivity. Positive clones were confirmed using colony PCR with primers KL15/18. Kmr-cassette was removed by using cre recombinase. Positive mutants were transformed with pTnCre and a single colony was picked to inoculate a 5 mL LB culture including 0.1 mM sodium salicylate to induce cre recombinase expression. After 4 h, the culture was appropriately diluted and resulting kanamycin sensitive colonies were cured from pTNCre by subsequent inoculation and screening for gentamycin sensitive colonies.
Gene deletions (ΔilvE, ΔleuA, ΔpanB, ΔpycAB)
To obtain all other deletion mutants, the recently published gene editing method for Pseudomonas species using the pEMG/pSW-2 system was used . This system allows faster identification of positive clones compared to the pJQ200SK method. Up- and downstream regions of the desired genes were amplified by PCR using the following primers (KL19/20 and KL21/22 for ilvE, KL23/24 and KL25/26 for pycAB, KL37/38 and KL39/40 for leuA, KL41/42 and KL43/44 for panB) and purified via gel electrophoresis. The resulting up- and downstream fragments were fused by PCR using primers (KL19/22 for ilvE, KL23/26 for pycAB, KL37/40 for leuA, KL41/44 for panB) and after gel electrophoresis, cut and ligated into the identically treated pEMG vectors. Ps. sp. strain VLB120 was transformed with the respective plasmids and single crossover mutants were identified using cPCR. Positive clones were transformed with pSW-2. I-SceI nuclease activity was already present in Ps. sp. strain VLB120 pSW-2 without additional induction of xylS with 3-methylbenzoate, thus colonies were replica plated to screen for kanamycin sensitive mutants. Gene deletion mutants were identified by cPCR using primers KL31/32 for ilvE, KL33/34 for pycAB, KL45/46 for leuA, and KL47/48 for panB. Positive clones were cured from the pSW-2 plasmid by repeated inoculation and screening for gentamycin sensitive mutants.
Construction of Pseudomonas sp. strain VLB120 T7
To establish Ps. sp. strain VLB120 as host for aerobic fermentations, the gene encoding for a T7 RNA polymerase under the control of a UV5 promoter and its regulator gene lac Iq were integrated in the genome using a Tn7-transposon. Kmr-loxP, was isolated from pJQhdp::Km with XbaI, purified by gel electrophoresis and ligated into XbaI cut, dephosporylated mini-Tn7-T7-Gm creating mini-Tn7-T7-KmLoxP. Ps. sp. strain VLB120 was transformed by electroporation with mini-Tn7-T7-KmLoxP and pTNS1 and kanamycin resistant mutants were checked by cPCR and primers KL9/10 for correct genomic integration of genes coding for lacI q and T7 RNA polymerase. The Kmr-cassette was removed as described above. To compare the effectiveness of the two different expression systems, eGFP fluorescence was used as a fast and easily detectable read-out for expression (Additional file 1: Figure S2). Depending on the carbon source, the fluorescence signal is about 3–6 fold higher in pBR22b-eGFP carrying strains, as compared to strains carrying pCOM10-eGFP. In both systems, the highest eGFP levels could be obtained on citrate as carbon source whereas in the presence of glucose only half of the fluorescence signal could be measured. The biggest difference was observed in complex medium; an approximately 6-fold higher eGFP concentration could be detected in Ps. sp. strain VLB120 pBR22b-eGFP. With regard to the fermentative production of isobutyric acid, the utilization of complex medium compounds might increase the maximum achievable product concentrations (Table 1). In addition, the eGFP expression for pBR22b as well as for pCOM10 was found to be tightly controlled in Ps. sp. strain VLB120 (T7). Without induction only a negligible amount of eGFP expression was detected.
This study was financially supported by the Graduate Cluster Industrial Biotechnology (CLIB2021) at TU Dortmund University. We are grateful to Nick Wierckx of BIRD Engineering BV, Delft and Victor de Lorenzo of 552 CNB, CSIC, Madrid for providing required plasmids.
- Erickson B, Nelson JE, Winters B: Perspective on opportunities in industrial biotechnology in renewable chemicals. Biotechnol J. 2012, 7: 176-185. 10.1002/biot.201100069.View ArticleGoogle Scholar
- Lichtenthaler FW, Peters S: Carbohydrates as green raw materials for the chemical industry. C R Chim. 2004, 7: 65-90. 10.1016/j.crci.2004.02.002.View ArticleGoogle Scholar
- SBI Reports: The World Market for Bio-Based Chemicals. 2012, Rockville, 2Google Scholar
- Lee JW, Na D, Park JM, Lee J, Choi S, Lee SY: Systems metabolic engineering of microorganisms for natural and non-natural chemicals. Nat Chem Biol. 2012, 8: 536-546. 10.1038/nchembio.970.View ArticleGoogle Scholar
- Atsumi S, Wu TY, Eckl EM, Hawkins SD, Buelter T, Liao JC: Engineering the isobutanol biosynthetic pathway in Escherichia coli by comparison of three aldehyde reductase/alcohol dehydrogenase genes. Appl Microbiol Biotechnol. 2010, 85: 651-657. 10.1007/s00253-009-2085-6.View ArticleGoogle Scholar
- Bauer W: Methacrylic acid and derivatives. Methacrylic Acid and Derivatives, vol. 23. 2012, Weinheim: Wiley-VCH, 1-12.Google Scholar
- Zhang KC, Woodruff AP, Xiong MY, Zhou J, Dhande YK: A synthetic metabolic pathway for production of the platform chemical isobutyric acid. ChemSusChem. 2011, 4: 1068-1070. 10.1002/cssc.201100045.View ArticleGoogle Scholar
- Koch H: Production of carboxylic acids. US patent. 1958, 2: 831-877.Google Scholar
- Wierckx NJ, Ballerstedt H, de Bont JA, Wery J: Engineering of solvent-tolerant Pseudomonas putida S12 for bioproduction of phenol from glucose. Appl Environ Microbiol. 2005, 71: 8221-8227. 10.1128/AEM.71.12.8221-8227.2005.View ArticleGoogle Scholar
- Fischer CR, Klein-Marcuschamer D, Stephanopoulos G: Selection and optimization of microbial hosts for biofuels production. Metab Eng. 2008, 10: 295-304. 10.1016/j.ymben.2008.06.009.View ArticleGoogle Scholar
- Stephanopoulos G: Challenges in engineering microbes for biofuels production. Science. 2007, 315: 801-804. 10.1126/science.1139612.View ArticleGoogle Scholar
- Heipieper HJ, Neumann G, Cornelissen S, Meinhardt F: Solvent-tolerant bacteria for biotransformations in two-phase fermentation systems. Appl Microbiol Biotechnol. 2007, 74: 961-973. 10.1007/s00253-006-0833-4.View ArticleGoogle Scholar
- Halan B, Buehler K, Schmid A: Biofilms as living catalysts in continuous chemical syntheses. Trends Biotechnol. 2012, 30: 453-465. 10.1016/j.tibtech.2012.05.003.View ArticleGoogle Scholar
- Halan B, Schmid A, Buehler K: Maximizing the productivity of catalytic biofilms on solid supports in membrane aerated reactors. Biotechnol Bioeng. 2010, 106: 516-527. 10.1002/bit.22732.View ArticleGoogle Scholar
- Gross R, Buehler K, Schmid A: Engineered catalytic biofilms for continuous large scale production of n-octanol and (S)-styrene oxide. Biotechnol Bioeng. 2013, 110: 424-436. 10.1002/bit.24629.View ArticleGoogle Scholar
- Wackett LP: Pseudomonas putida - a versatile biocatalyst. Nat Biotechnol. 2003, 21: 136-138. 10.1038/nbt0203-136.View ArticleGoogle Scholar
- Spanggord RJ, Spain JC, Nishino SF, Mortelmans KE: Biodegradation of 2,4-dinitrotoluene by a Pseudomonas sp. Appl Environ Microbiol. 1991, 57: 3200-3205.Google Scholar
- Haigler BE, Spain JC: Biodegradation of 4-nitrotoluene by Pseudomonas sp. strain 4NT. Appl Environ Microbiol. 1993, 59: 2239-2243.Google Scholar
- Poblete-Castro I, Becker J, Dohnt K, dos Santos VM, Wittmann C: Industrial biotechnology of Pseudomonas putida and related species. Appl Microbiol Biotechnol. 2012, 93: 2279-2290. 10.1007/s00253-012-3928-0.View ArticleGoogle Scholar
- Escapa IF, Garcia JL, Buhler B, Blank LM, Prieto MA: The polyhydroxyalkanoate metabolism controls carbon and energy spillage in Pseudomonas putida. Environ Microbiol. 2012, 14: 1049-1063. 10.1111/j.1462-2920.2011.02684.x.View ArticleGoogle Scholar
- Blank LM, Ionidis G, Ebert BE, Buhler B, Schmid A: Metabolic response of Pseudomonas putida during redox biocatalysis in the presence of a second octanol phase. FEBS J. 2008, 275: 5173-5190. 10.1111/j.1742-4658.2008.06648.x.View ArticleGoogle Scholar
- Vemuri GN, Altman E, Sangurdekar DP, Khodursky AB, Eiteman MA: Overflow metabolism in Escherichia coli during steady-state growth: Transcriptional regulation and effect of the redox ratio. Appl Environ Microbiol. 2006, 72: 3653-3661. 10.1128/AEM.72.5.3653-3661.2006.View ArticleGoogle Scholar
- Nijkamp K, Westerhof RG, Ballerstedt H, de Bont JA, Wery J: Optimization of the solvent-tolerant Pseudomonas putida S12 as host for the production of p-coumarate from glucose. Appl Microbiol Biotechnol. 2007, 74: 617-624. 10.1007/s00253-006-0703-0.View ArticleGoogle Scholar
- Verhoef S, Wierckx N, Westerhof RG, de Winde JH, Ruijssenaars HJ: Bioproduction of p-hydroxystyrene from glucose by the solvent-tolerant bacterium Pseudomonas putida S12 in a two-phase water-decanol fermentation. Appl Environ Microbiol. 2009, 75: 931-936. 10.1128/AEM.02186-08.View ArticleGoogle Scholar
- Wierckx NJ, Ballerstedt H, de Bont JA, de Winde JH, Ruijssenaars HJ, Wery J: Transcriptome analysis of a phenol-producing Pseudomonasputida S12 construct: genetic and physiological basis for improved production. J Bacteriol. 2008, 190: 2822-2830. 10.1128/JB.01379-07.View ArticleGoogle Scholar
- Witholt B, Kessler B: Perspectives of medium chain length poly(hydroxyalkanoates), a versatile set of bacterial bioplastics. Curr Opin Biotechnol. 1999, 10: 279-285. 10.1016/S0958-1669(99)80049-4.View ArticleGoogle Scholar
- Wang HH, Zhou XR, Liu Q, Chen GQ: Biosynthesis of polyhydroxyalkanoate homopolymers by Pseudomonasputida. Appl Microbiol Biotechnol. 2011, 89: 1497-1507. 10.1007/s00253-010-2964-x.View ArticleGoogle Scholar
- Husken LE, Beeftink R, de Bont JAM, Wery J: High-rate 3-methylcatechol production in Pseudomonas putida strains by means of a novel expression system. Appl Microbiol Biotechnol. 2001, 55: 571-577. 10.1007/s002530000566.View ArticleGoogle Scholar
- Panke S, Witholt B, Schmid A, Wubbolts MG: Towards a biocatalyst for (S)-styrene oxide production: characterization of the styrene degradation pathway of Pseudomonas sp. strain VLB120. Appl Environ Microbiol. 1998, 64: 3546-3546.Google Scholar
- Nielsen DR, Leonard E, Yoon SH, Tseng HC, Yuan C, Prather KL: Engineering alternative butanol production platforms in heterologous bacteria. Metab Eng. 2009, 11: 262-273. 10.1016/j.ymben.2009.05.003.View ArticleGoogle Scholar
- Schrewe M, Julsing MK, Buhler B, Schmid A: Whole-cell biocatalysis for selective and productive C-O functional group introduction and modification. Chem Soc Rev. 2013, 42: 6346-6377. 10.1039/c3cs60011d.View ArticleGoogle Scholar
- Kuhn D, Buhler B, Schmid A: Production host selection for asymmetric styrene epoxidation: Escherichia coli vs. solvent-tolerant Pseudomonas. J Ind Microbiol Biotechnol. 2012, 39: 1125-1133. 10.1007/s10295-012-1126-9.View ArticleGoogle Scholar
- Connor MR, Liao JC: Engineering of an Escherichia coli strain for the production of 3-methyl-1-butanol. Appl Environ Microbiol. 2008, 74: 5769-5775. 10.1128/AEM.00468-08.View ArticleGoogle Scholar
- Marshall VD, Sokatch JR: Regulation of caline catabolism in Pseudomonas putida. J Bacteriol. 1972, 110: 1073-1081.Google Scholar
- Mooney BP, Miernyk JA, Randall DD: The complex fate of α-ketoacids. Annu Rev Plant Biol. 2002, 53: 357-375. 10.1146/annurev.arplant.53.100301.135251.View ArticleGoogle Scholar
- Goodhue CT, Schaeffe JR: Preparation of L(+) β-hydroxyisobutyric acid by bacterial oxidation of isobutyric acid. Biotechnol Bioeng. 1971, 13: 203-214. 10.1002/bit.260130204.View ArticleGoogle Scholar
- Adelberg EA, Mandel M, Chen GCC: Optimal conditions for mutagenesis by N-methyl-N’-nitro-N-nitrosoguanidine in Escherichia coli K12. Biochem Biophys Res Commun. 1965, 18: 788-795. 10.1016/0006-291X(65)90855-7.View ArticleGoogle Scholar
- Nijkamp K, Luijk N, Bont JAM, Wery J: The solvent-tolerant Pseudomonas putida S12 as host for the production of cinnamic acid from glucose. Appl Microbiol Biotechnol. 2005, 69: 170-177. 10.1007/s00253-005-1973-7.View ArticleGoogle Scholar
- Wolff JA, Macgregor CH, Eisenberg RC, Phibbs PV: Isolation and characterization of catabolite repression control mutants of Pseudomonas aeruginosa PAO. J Bacteriol. 1991, 173: 4700-4706.Google Scholar
- Martin RR, Marshall VD, Sokatch JR, Unger L: Common enzymes of branched-chain amino-acid catabolism in Pseudomonas putida. J Bacteriol. 1973, 115: 198-204.Google Scholar
- Rodriguez GM, Atsumi S: Isobutyraldehyde production from Escherichia coli by removing aldehyde reductase activity. Microb Cell Fact. 2012, 11: 90-10.1186/1475-2859-11-90.View ArticleGoogle Scholar
- Kim CH, Hong WK, Lee IY, Choi ES, Rhee SK: Enhanced production of D-β-hydroxyisobutyric acid through strain improvement. J Biotechnol. 1999, 69: 75-79. 10.1016/S0168-1656(99)00003-6.View ArticleGoogle Scholar
- Kim HS, Ju JY, Suh JH, Shin CS: Optimized fed-batch fermentation of L-β-hydroxy isobutyric acid by Yarrowia lipolytica. Bioprocess Eng. 1999, 20: 189-193.Google Scholar
- Aberhart DJ: A stereochemical study on the metabolism of isobutyrate in Pseudomonas putida. Bioorg Chem. 1977, 6: 191-201. 10.1016/0045-2068(77)90020-7.View ArticleGoogle Scholar
- Stieb M, Schink B: Anaerobic degradation of isobutyrate by methanogenic enrichment cultures and by a Desulfococcus multivorans strain. Arch Microbiol. 1989, 151: 126-132. 10.1007/BF00414426.View ArticleGoogle Scholar
- Steele MI, Lorenz D, Hatter K, Park A, Sokatch JR: Characterization of the mmsAB operon of Pseudomonas aeruginosa PAO encoding methylmalonate-semialdehyde dehydrogenase and 3-hydroxyisobutyrate dehydrogenase. J Biol Chem. 1992, 267: 13585-13592.Google Scholar
- Harper M, Lee CJ: Genome-wide analysis of mutagenesis bias and context sensitivity of N-methyl-N ’-nitro-N-nitrosoguanidine (NTG). Mutat Res-Fundam Mol Mech Mutag. 2012, 731: 64-67. 10.1016/j.mrfmmm.2011.10.011.View ArticleGoogle Scholar
- Lokanath NK, Ohshima N, Takio K, Shiromizu I, Kuroishi C, Okazaki N, Kuramitsu S, Yokoyama S, Miyano M, Kunishima N: Crystal structure of novel NADP-dependent 3-hydroxyisobutyrate dehydrogenase from Thermus thermophilus HB8. J Mol Biol. 2005, 352: 905-917. 10.1016/j.jmb.2005.07.068.View ArticleGoogle Scholar
- Mai XH, Adams MWW: Purification and characterization of two reversible and ADP-dependent acetyl coenzyme a synthetases from the hyperthermophilic archaeon Pyrococcus furiosus. J Bacteriol. 1996, 178: 5897-5903.Google Scholar
- Terpe K: Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol. 2006, 72: 211-222. 10.1007/s00253-006-0465-8.View ArticleGoogle Scholar
- Staijen IE, Marcionelli R, Witholt B: The P alkBFGHJKL promoter is under carbon catabolite repression control in Pseudomonas oleovorans but not in Escherichia coli alk(+) recombinants. J Bacteriol. 1999, 181: 1610-1616.Google Scholar
- Troeschel SC, Thies S, Link O, Real CI, Knops K, Wilhelm S, Rosenau F, Jaeger KE: Novel broad host range shuttle vectors for expression in Escherichia coli, Bacillus subtilis and Pseudomonas putida. J Biotechnol. 2012, 161: 71-79. 10.1016/j.jbiotec.2012.02.020.View ArticleGoogle Scholar
- del Castillo T, Ramos JL, Rodriguez-Herva JJ, Fuhrer T, Sauer U, Duque E: Convergent peripheral pathways catalyze initial glucose catabolism in Pseudomonas putida: genomic and flux analysis. J Bacteriol. 2007, 189: 5142-5152. 10.1128/JB.00203-07.View ArticleGoogle Scholar
- Poblete-Castro I, Binger D, Rodrigues A, Becker J, dos Santos VAPM, Wittmann C: In-silico-driven metabolic engineering of Pseudomonas putida for enhanced production of poly-hydroxyalkanoates. Metab Eng. 2013, 15: 113-123.View ArticleGoogle Scholar
- Lu J, Brigham CJ, Gai CS, Sinskey AJ: Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha. Appl Microbiol Biotechnol. 2012, 96: 283-297. 10.1007/s00253-012-4320-9.View ArticleGoogle Scholar
- Krause FS, Blombach B, Eikmanns BJ: Metabolic engineering of Corynebacterium glutamicum for 2-ketoisovalerate production. Appl Environ Microbiol. 2010, 76: 8053-8061. 10.1128/AEM.01710-10.View ArticleGoogle Scholar
- Blombach B, Riester T, Wieschalka S, Ziert C, Youn JW, Wendisch VF, Eikmanns BJ: Corynebacterium glutamicum tailored for efficient isobutanol production. Appl Environ Microbiol. 2011, 77: 3300-3310. 10.1128/AEM.02972-10.View ArticleGoogle Scholar
- Levintow L, Novelli GD: The synthesis of coenzyme A from pantetheine - preparation and properties of pantetheine kinase. J Biol Chem. 1954, 207: 761-765.Google Scholar
- Radmacher E, Vaitsikova A, Burger U, Krumbach K, Sahm H, Eggeling L: Linking central metabolism with increased pathway flux: L-valine accumulation by Corynebacterium glutamicum. Appl Environ Microbiol. 2002, 68: 2246-2250. 10.1128/AEM.68.5.2246-2250.2002.View ArticleGoogle Scholar
- Sahm H, Eggeling L: D-pantothenate synthesis in Corynebacterium glutamicum and use of panBC and genes encoding L-valine synthesis for D-pantothenate overproduction. Appl Environ Microbiol. 1999, 65: 1973-1979.Google Scholar
- Atsumi S, Hanai T, Liao JC: Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature. 2008, 451: 86-90. 10.1038/nature06450.View ArticleGoogle Scholar
- Shimazaki M, Hasegawa J, Kan K, Nomura K, Nose Y, Kondo H, Ohashi T, Watanabe K: Synthesis of captopril starting from an optically-active beta-hydroxy acid. Chem Pharm Bull (Tokyo). 1982, 30: 3139-3146. 10.1248/cpb.30.3139.View ArticleGoogle Scholar
- Hanahan D: Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983, 166: 557-580. 10.1016/S0022-2836(83)80284-8.View ArticleGoogle Scholar
- Martinez-Garcia E, de Lorenzo V: Engineering multiple genomic deletions in gram-negative bacteria: analysis of the multi-resistant antibiotic profile of Pseudomonas putida KT2440. Environ Microbiol. 2011, 13: 2702-2716. 10.1111/j.1462-2920.2011.02538.x.View ArticleGoogle Scholar
- Chowdhury EK, Akaishi Y, Nagata S, Misono H: Cloning and overexpression of the 3-hydroxyisobutyrate dehydrogenase gene from Pseudomonas putida E23. Biosci Biotechnol Biochem. 2003, 67: 438-441. 10.1271/bbb.67.438.View ArticleGoogle Scholar
- Smits TH, Seeger MA, Witholt B, van Beilen JB: New alkane-responsive expression vectors for Escherichia coli and Pseudomonas. Plasmid. 2001, 46: 16-24. 10.1006/plas.2001.1522.View ArticleGoogle Scholar
- Rosenau F, Jaeger KE: Overexpression and secretion of biocatalysts in Pseudomonas. Enzyme Functionality. 2003, CRC PressGoogle Scholar
- Quandt J, Hynes MF: Versatile suicide vectors which allow direct selection for gene replacement in Gram-negative bacteria. Gene. 1993, 127: 15-21. 10.1016/0378-1119(93)90611-6.View ArticleGoogle Scholar
- Choi K-H, Gaynor JB, White KG, Lopez C, Bosio CM, Karkhoff-Schweizer RR, Schweizer HP: A Tn7-based broad-range bacterial cloning and expression system. Nat Methods. 2005, 2: 443-448. 10.1038/nmeth765.View ArticleGoogle Scholar
- Bertani G: Studies on lysogenesis.1. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol. 1951, 62: 293-300.Google Scholar
- Sambrook J, Russel DW: Molecular cloning - a laboratory manual. 2001, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 3Google Scholar
- Panke S, Meyer A, Huber CM, Witholt B, Wubbolts MG: An alkane-responsive expression system for the production of fine chemicals. Appl Environ Microbiol. 1999, 65: 2324-2332.Google Scholar
- Lang Z-F, Shen J-J, Cai S, Zhang J, He J, Li S-P: Expression, characterization, and site-directed mutation of a multiple herbicide-resistant acetohydroxyacid synthase (rAHAS) from Pseudomonas sp. Lm10. Curr Microbiol. 2011, 63: 145-150. 10.1007/s00284-011-9953-x.View ArticleGoogle Scholar
- Leyval D, Uy D, Delaunay S, Goergen JL, Engasser JM: Characterisation of the enzyme activities involved in the valine biosynthetic pathway in a valine-producing strain of Corynebacterium glutamicum. J Biotechnol. 2003, 104: 241-252. 10.1016/S0168-1656(03)00162-7.View ArticleGoogle Scholar
- Atsumi S, Higashide W, Liao JC: Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde. Nat Biotechnol. 2009, 27: 1177-1180. 10.1038/nbt.1586.View ArticleGoogle Scholar
- Choi K-H, Kumar A, Schweizer HP: A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation. J Microbiol Methods. 2006, 64: 391-397. 10.1016/j.mimet.2005.06.001.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.