italic> of the Whole nif Genes of Klebsiella oxytoca,a Nitrogen Fixer in the Rhizosphere of Rice

We cloned in E. coli the whole 17 nif genes (nifQ-J) of Klebsiella oxytoca NG13 using pBR322 as a vector, and constructed a recombinant plasmid, pNOW25 (nif⁺, Ap^r, 42.6 kb). A non nif DNA fragment was deleted from the plasmid with XhoI, and a smaller plasmid, pNOK31 (nif+, Ap^r, 31.1 kb), was reconstructed.

We constructed the restriction map of the cloned nif genes. The map was the same as that of the K. pneumoniae M5a1 nif genes as to the EcoRI, HindIII, BamHI and XhoI sites, but differed considerably in the PstI, SalI and BglII sites.

E. coli KO60 containing pNOW25 or pNOK31 can grow on a N-free medium. The acetylene reduction activities of KO60 (pNOW25) and KO60 (pNOK31) were 280 nmol and 390 nmol/48 hr per 7 ml of N-free liquid medium, whereas the activity of K. oxytoca NG13 was 3800 nmol. Thus, the expressed activity of the nif system of K. oxytoca is rather low in E. coli even if the nif genes are cloned on a multicopy plasmid.     

Cloning and Expression of the Pseudomonas Gene for Utilization of d-Leucine in Escherichia coli

Two genes of Pseudomonas putida (IFO 12996) which code for enzymes participating in amino acid metabolism, were cloned in Escherichia coli C600 using pBR322 as a vector. pST7549 is a 7.9 kb hybrid plasmid DNA which is composed of four SalI fragments (0.3, 1.4, 1.9 and 4.3 kb), and codes for β-isopropylmalate dehydrogenase (EC 1.1.1.85) in l-leucine biosynthesis. The enzyme activity in the crude extract from E. coli C600 bearing pST7549 was 80 ~ 90% lower than that of E. coli K12 or P. putida. When the foreign SalI fragments derived from P. putida were subcloned, a 1.9 kb SalI fragment was found to encode β-isopropylmalate dehydrogenase and it did not contain the promoter of P. putida DNA. Plasmid pST6961 has a 1.8 kb insert derived from the P. putida DNA in the SalI site of pBR322. E. coli cells carrying this recombinant plasmid show no leucine racemase activity and no d-leucine transaminase activity, but five-times higher d-leucine oxidation activity than the host strain, E. coli. Enzymological studies have suggested that plasmid pST6961 codes for d-amino acid dehydrogenase, a key enzyme in d-amino acid metabolism.     

Expression of klebsiella his and nif genes in serratia marcescens,Erwinia herbicola and Proteus mirabilis

Plasmid pRD1, an R plasmid of the P incompatibility group which carries his and nif genes from Klebsiella pneumoniae in addition to drug resistance markers derived from RP4, was transferred to His^- mutants of Serratia marcescens, Erwinia herbicola and Proteus mirabilis. His⁺ transconjugants were obtained at low but different frequencies according to recipient genus. Transconjugants all acquired the drug resistance, and were Nif⁺ in S. marcescens and E. herbicola, having acetylene-reducing activities of the same order of magnitude as the parent K. pneumoniae and fixing ¹⁵N₂. No evidence for nif expression in P. mirabilis transconjugants was obtained though the nif genes were present.     

Impacts of gene bioaugmentation with pJP4-harboring bacteria of 2,4-D-contaminated soil slurry on the indigenous microbial community

Gene bioaugmentation is a bioremediation strategy that enhances biodegradative potential via dissemination of degradative genes from introduced microorganisms to indigenous microorganisms. Bioremediation experiments using 2,4-dichlorophenoxyacetic acid (2,4-D)-contaminated soil slurry and strains of Pseudomonas putida or Escherichia coli harboring a self-transmissible 2,4-D degradative plasmid pJP4 were conducted in microcosms to assess possible effects of gene bioaugmentation on the overall microbial community structure and ecological functions (carbon source utilization and nitrogen transformation potentials). Although exogenous bacteria decreased rapidly, 2,4-D degradation was stimulated in bioaugmented microcosms, possibly because of the occurrence of transconjugants by the transfer of pJP4. Terminal restriction fragment length polymorphism analysis revealed that, although the bacterial community structure was disturbed immediately after introducing exogenous bacteria to the inoculated microcosms, it gradually approached that of the uninoculated microcosms. Biolog assay, nitrate reduction assay, and monitoring of the amoA gene of ammonia-oxidizing bacteria and nirK and nirS genes of denitrifying bacteria showed no irretrievable depressive effects of gene bioaugmentation on the carbon source utilization and nitrogen transformation potentials. These results may suggest that gene bioaugmentation with P. putida and E. coli strains harboring pJP4 is effective for the degradation of 2,4-D in soil without large impacts on the indigenous microbial community.     

Nif plasmid from Lignobacter

Lignobacter strain K17 is able to degrade aromatic compounds and to fix atmospheric nitrogen. It was proved that capacity for nitrogen fixation by Lignobacter was plasmid mediated. Plasmid pUCS100 (17.5 Mdal) carrying nif genes was transferred from Lignobacter to Escherichia coli SK1592 and Salmonella typhimurium. The transposon Tn9 was translocated to pUCS100 to facilitate selection of Nif⁺ bacteria. E. coli SK1592 harboring the new plasmid (pUCS101) reduced acetylene under anaerobic conditions. Plasmids pUCS100 and pUCS101 were not stably maintained in E. coli and S. typhimurium.Abbreviations Mdal megadalton - CsCl-EtBr caesium chloride ethidium bromide - m.o.i. multiplicity of infection     

Mobilization ofEscherichia coli R1 silver-resistance plasmid pJT1 by Tn5-Mob intoEscherichia coli C600

Summary Escherichia coli Rl is an Ag⁺-resistant strain that, as we have shown recently, harbours at least two large plasmids, pJT1 (83 kb) and pJT2 (77 kb). Tn5-Mob was introduced into theE. coli Rl host replicon via conjugation on membrane filters. The transfer functions of plasmid RP4-4 were provided in this process and Tn5-Mob clones mated withE. coli C600 yielded Ag⁺-resistant transconjugants. This mobilization procedure allowed transfer and expression of pJT1 Ag⁺ resistance inE. coli C600. Prior to use of Tn5-Mob mobilization, it was not possible to transfer Ag⁺-resistant determinant(s) intoE. coli by conjugation or transformation including high-voltage electroporation.E. coli C600 containing PJTI and PJT2 displayed decreased accumulation of Ag⁺ similar toE. coli R1.E. coli C600 could not tolerate 0.1 and 0.5 mM Ag⁺, rapidly accumulated Ag⁺ and became non-viable. Tn5-Mob mobilization may be useful in the study of metal resistance in bacteria, especially in strains not studied for resistance mechanisms.     

Contribution of the Earthworm Lumbricus rubellus (Annelida,Oligochaeta) to the Establishment of Plasmids in Soil Bacterial Communities

The contribution of the carthworm Lumbricus rubellus in spreading plasmids from a nonindigenous bacterial species to the soil microbial community was studied with Escherichia coli strains as donor organisms. The selected donor strains harbored marker-gene tagged plasmids with different transfer properties and host ranges. Prototrophic benzoate degrading indigenous bacteria were analyzed as potential recipients. In filter-mating experiments, donor strains were mixed with bacterial cell consortia extracted from earthworm casts (feces) and incubated on nutrient agar at 28°C. Transfer was detected with the broad host range IncP plasmid pRP4luc; with the IncQ plasmid, pSUP104luc, but only when it was present in a mobilizing donor strain; and with the transposon delivery vector pUTlux. No transfer was detected with the nonmobilizable pUCluc and the mobilizable pSUP202luc, both of narrow host range. In microcosm studies with E. coli inoculated soil incubated at 12°C, transconjugants were only detected in casts of L. rubellus but not in bulk soil, indicating that the gut passage was a precondition for plasmid transfer. Plasmid pRP4luc was transferred at higher frequencies than detected in filter mating. Results of the filter matings were confirmed except that transfer of pUTlux could not be detected. The majority of transconjugants isolated in this study lost their acquired plasmid upon further cultivation. Stable transconjugants, however, were obtained and identified at the 16S rRNA gene level as members of the β- and γ-subgroups of Proteobacteria. Incubation of E. coli and selected transconjugants in soil microcosms with L. rubellus demonstrated that the gut passage resulted in a slight but significant reduction of ingested cells. In contrast to the donor strains, however, the population sizes of transconjugants in bulk soil and in casts did not decrease over time. This demonstrated that the transferred plasmids had established themselves in the soil microbial community.     

Plasmid-mediated transfer of nitrogen-fixing capability to bacteria from the rhizosphere of grasses

Summary Cells of a non-nitrogen-fixing, drug-sensitive Enterobacter cloacae strain, isolated from the rhizosphere of Festuca heterophylla, were mated to Escherichia coli cells harboring plasmid pRD1. This plasmid carries the nitrogen-fixation (nif⁺) genes as well as three markers of drug resistance. After mating, triple-resistant Enterobacter transferants could be selected. These were screened for plasmids, acetylene reduction, and stability of the transferred markers.Transferants contained plasmid pRD1. Of 48, 43 were acetylene-reducing and therefore carried the nif⁺ genes. Triple-resistance was stable upon passage in liquid minimal medium, but the number of cells with nif⁺ genes decreased. Both the triple-resistant and the nif⁺ genotypes decreased in complete medium, although by different rates, depending on the particular line. The most stable line, M14, was chosen and checked further.Samples taken after 8–14 passages in minimal medium contained cells with different genotypes, plasmid sizes smaller than the original plasmid pRD1 and no free plasmids. Progeny of the latter cells, in addition to being triple-resistant, were the best acetylene reducers. It is concluded that in these cells the plasmid pRD1 with all its relevant genes had become integrated into the recipients' chromosome.Grass seedlings were inoculated with the bacteria containing integrated plasmid pRD1. They were then planted into pots with sterile ash and watered with a nutrient salt solution of limited nitrogen content. Sampling after 8 weeks showed that the inoculated bacteria were preserved, as demonstrated by their triple-resistance. They could also still fix nitrogen.     

Poly(3-hydroxyalkanoate) polymerase synthesis and in vitro activity in recombinant Escherichia coli and Pseudomonas putida

We tested the synthesis and in vitro activity of the poly(3-hydroxyalkanoate) (PHA) polymerase 1 from Pseudomonas putida GPo1 in both P. putida GPp104 and Escherichia coli JMU193. The polymerase encoding gene phaC1 was expressed using the inducible PalkB promoter. It was found that the production of polymerase could be modulated over a wide range of protein levels by varying inducer concentrations. The optimal inducer dicyclopropylketone concentrations for PHA production were at 0.03% (v/v) for P. putida and 0.005% (v/v) for E. coli. Under these concentrations the maximal polymerase level synthesized in the E. coli host (6% of total protein) was about three- to fourfold less than that in P. putida (20%), whereas the maximal level of PHA synthesized in the E. coli host (8% of total cell dry weight) was about fourfold less than that in P. putida (30%). In P. putida, the highest specific activity of polymerase was found in the mid-exponential growth phase with a maximum of 40 U/g polymerase, whereas in E. coli, the maximal specific polymerase activity was found in the early stationary growth phase (2 U/g polymerase). Our results suggest that optimal functioning of the PHA polymerase requires factors or a molecular environment that is available in P. putida but not in E. coli.     

Plasmid Transfer Detection in Soil using the Inducible lPR System Fused to Eukaryotic Luciferase Genes

We report a model system for plasmid transfer analysis using the regulated lambda phage right promoter, λPr, fused to luc and lucOR as repoter genes. We have demonstrated that the systems cI857-λPr::luc and cI857-λPr::lucOR are temperature-inducible in Escherichia coli but not in other Gram-negative bacteria analyzed, enabling detection of luminescence when plasmids were mobilized from E. coli to those Gram-negative backgrounds. Using light for the detection, we have observed plasmid transfer from E. coli harboring RK2 and R388 derived plasmids to Pseudomonas putida KT2440 (co-introduced with donors) and to indigenous microorganisms, in vitro and in nonsterile soil microcosms. The importance of nutrients for an efficient plasmid transfer in nonsterile soil microcosms has been confirmed. When plasmid transfer experiments were carried out into nonsterile soil microcosms, significant populations of indigenous transconjugants arose. This system provides efficient marker genes and avoids the use of antibiotics for the selection of transconjugants.     

Isolation of transfer-negative nif-plasmids (pCE1) and their integration into the chromosome of Escherichia coli with the help of phage Mu

Summary Strain JC5466 of Escherichia coli K12 harbouring the nitrogen fixation plasmid pCE1 was lysogenized with bacteriophage Mu cts, followed by partial induction and infection with bacteriophage PRD1. This made it possible to obtain transfer-defective derivatives of pCE1, carrying Mu prophage. These derivatives could be mobilized by using the helper plasmid pME400 and it was possible to segregate the helper plasmid from the donor plasmid in the transconjugants.By incubating the strains 302 and 328 at 42°C, for induction of Mu prophage, derivatives with different plasmid contents could be obtained such as strains without plasmids, some with smaller or larger plasmids and others possessing plasmids without any visible alteration in size. Integration of the nitrogen-fixation (nif) genes into the chromosomes of the strains without plasmids and those containing a smaller plasmid, was confirmed by Southern hybridization using radioactive nifKDH DNA. Conjugation assays have shown that the plasmid is integrated into the chromosome as a unit but that it can also be excised.     

Nucleotide sequence analysis and comparison of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida.

Summary The complete nucleotide sequences of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida were determined; the DNA sequences of the lexA genes from these bacteria were 86%, 76%, 61% and 59% similar, respectively, to the Escherichia coli K12 gene. The predicted amino acid sequences of the S. typhimurium, E. carotovora and P. putida LexA proteins are 202 residues long whereas that of P. aeruginosa is 204. Two putative LexA repressor binding sites were localized upstream of each of the heterologous genes, the distance between them being 5 by in S. typhimurium and E. carotovora, as in the lexA gene of E. coli, and 3 by in P. putida and P. aeruginosa. The first lexA site present in the lexA operator of all five bacteria is very well conserved. However, the second lexA box is considerably more variable. The Ala-84 — Gly-85 bond, at which the LexA repressor of E. coli is cleaved during the induction of the SOS response, is also found in the LexA proteins of S. typhimurium and E. carotovora. Likewise, the amino acids Ser-119 and Lys-156 are present in all of these three LexA repressors. These residues also exist in the LexA proteins of P. putida and P. aeruginosa, but they are displaced by 4 and 6 residues, respectively. Furthermore, the structure and sequence of the DNA-binding domain of the LexA repressor of E. coli are highly conserved in the S. typhimurium, E. carotovora, P. aeruginosa and P. putida LexA proteins.     

Identification and characterization of the exbB, exbD and tonB genes of Pseudomonas putida WCS358: their involvement in ferric-pseudobactin transport

Catechol-cephalosporins are siderophore-like antibiotics which are taken up by cells of Pseudomonas putida WCS358 via the ferric-siderophore transport pathway. Mutants of strain WCS358 were isolated that are resistant to high concentrations of these antibiotics. These mutants failed to grow under iron-limiting conditions, and could not utilize different ferric-siderophores. The mutants fall in three complementation groups. The nucleotide sequence determination identified three contiguous open reading frames, which were homologous to the exbB, exbD and tonB genes of Escherichia coli respectively. The deduced amino acid sequence of P. putida ExbB showed 58.6% homology with its E. coli homologue, but, unlike the E. coli protein, it has a N-terminal extension of 91 amino acids. The ExbD proteins are 64.8% homologous, whereas the TonB proteins only show 27.7% homology. The P. putida exbB gene could complement an E. coli exbB mutation, but the TonB proteins were not interchangeable between the species. It is concluded that P. putida WCS358 contains an energy-coupling system between the membranes for active transport across the outer membrane, which is comprised of a TonB-like energy-transducing protein and two accessory proteins. This system is similar to, but not completely compatible with, the E. coli system.     

Intergeneric mobilization of Rhizobium nif genes to Agrobacterium and Klebsiella.

Summary The P-1 incompatibility group plasmid RP1 transfers itself from Escherichia coli J53 to the clover endosymbiont bacterium Rhizobium trifolii strain T1 at low frequency in agar surface matings. R. trifolii T1 R-plasmid recipients display a phenotype identical to the wild-type parent strain in all respects except RP1 antibiotic resistances, allowing straightforward donor counterselection and differentiation of excojugants in further intergeneric plasmid transfer experiments. Hence RP1 can readily transfer itself intergenerically from R. trifolii T1 to the related plant pathogenic organism Agrobacterium tumefaciens and to a strain of the free-living diazotroph Klebsiella pneumoniae.Using R. trifolii T1 (RP1) as donor and as recipient LBA 4006, an avirulent strain of A. tumefaciens lacking the tumour-inducing (Ti) plasmid, selection was made for intergeneric transfer of the R-plasmid and its potential as vector of nitrogen-fixation genes evaluated by subsequent indirect screening. Exconjugant Agrobacteria were obtained which carried RP1 resistance markers and, given specific physiological conditions, would reduce acetylene under air. This is the first report of expression of nif genes in a hybrid strain of A. tumefaciens and is of interest since the Ti plasmid of this organism has been suggested as a natural vector for the introduction of these genes into plants. Plasmid RP1 also cotransferred Rhizobium nif genes to KP52, a strain of K. pneumoniae M5al, with deletion by phage eduction of the chromosomal genes for histidine biosynthesis, one of the nif regulatory genes (nif A), the gene for molybdenum cofactor (nif B) and for an electron transport protein of the nitrogen-fixation pathway (nif F). KP52 exconjugants carried RP1 drug resistances and reduced acetylene under anaerobic conditions.     

Rationally engineered biotransformation of p‐nitrophenol

An operon encoding enzymes responsible for degradation of the EPA priority contaminant para‐nitrophenol (PNP) from Pseudomonas sp. ENV2030 contains more genes than would appear to be necessary to mineralize PNP. To determine some necessary genes for PNP degradation, the genes encoding the proposed enzymes in the degradation pathway (pnpADEC) were assembled into a broad‐host‐range, BioBricks‐compatible vector under the control of a constitutive promoter. These were introduced into Escherichia coli DH10b and two Pseudomonas putida strains, one with a knockout of the aromatic transport TtgB and the parent with the native transporter. The engineered strains were assayed for PNP removal. E. coli DH10b harboring several versions of the refactored pathway was able to remove PNP from the medium up to a concentration of 0.2 mM; above which PNP was toxic to E. coli. A strain of P. putida harboring the PNP pathway genes was capable of removing PNP from the medium up to 0.5 mM. When P. putida harboring the native PNP degradation cluster was exposed to PNP, pnpADEC were induced, and the resulting production of β‐ketoadipate from PNP induced expression of its chromosomal degradation pathway (pcaIJF). In contrast, pnpADEC were expressed constitutively from the refactored constructs because none of the regulatory genes found in the native PNP degradation cluster were included. Although P. putida harboring the refactored construct was incapable of growing exclusively on PNP as a carbon source, evidence that the engineered pathway was functional was demonstrated by the induced expression of chromosomal pcaIJF. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Expression ofKlebsiella pneumoniae nif genes inProteus mirabilis

Self-transmissible plasmids carryinghis andnif genes fromKlebsiella pneumoniae have been introduced into threehis mutants ofProteus mirabilis: strains 5006-1, WR19 and WR20. Expression ofhis by the transconjugants was unequivocal, if slightly temperature-sensitive, but none was Nif⁺ when tested for acetylene reduction in anaerobic glucose medium using inocula from rich or glucose-minimal aerobic agar cultures. Succinate or pyruvate in place of glucose, low glucose, lower temperature or elevated Na₂MoO₄ did not allownif expression and no nitrogenase MoFe-protein peptide was detected immunologically after exposure to conditions in which diazotrophic enterobacteria, normal or genetically constructed, derepressnif.One strain,P. mirabilis WR19, carrying thehis nif Km^r plasmid pMF250 was examined in detail. Thenif activator genenifA was introduced on the plasmid pCK1. Such derivatives remained Nif^- when tested, after aerobic growth on rich agar media, with normal or low glucose, with succinate or with elevated Mo. However, pre-conditioning by aerobic growth on glucose-minimal agar led to subsequent anaerobic expression ofnif in glucose medium from pMF250 in WR19 carrying pCK1. NH ₄ ⁺ or proline could serve as N-source in the glucose-minimal agar. Maximum activity was about 5% of that ofK. pneumoniae in our assay conditions. Material cross-reacting with anti-serum to the nitrogenase MoFe protein was formed. Nitrogenase activity was not switched off by NH ₄ ⁺ .P. mirabilis WR19 (pCK1) showed NH ₄ ⁺ -constitutive temperature-sensitive kanamycin resistance (anif-related phenotype of this plasmid) in aerobic glucose minimal medium. Expression ofnif inP. mirabilis WR19 (pCK1, pMF250) was NH ₄ ⁺ -repressible despite the constitutivenifA character of pCK1 and introduction of thentrA ⁺ plasmid pMM17 did not alter this phenotype. However, pCK1 did not give rise to NH ₄ ⁺ -constitutive diazotrophy in the wild-typeK. pneumoniae M5al. A construct of WR19 carrying pMF250 and constitutiventrC plasmid (pMD45) remained Nif^- even after pre-growth on glucose-minimal media.We conclude (a) thatP. mirabilis forms a gene product functionally equivalent to that ofntrA inK. pneumoniae, (b) that it forms no functional equivalent of thentrC product in our growth conditions. The need for pre-conditioning on aerobic glucose media remains perplexing.Non-common abbreviation NFDM Nitrogen-free-Davis-Mingioli medium     

Expression of a plasmid-encoded gene for cadmium resistance of Pseudomonas putida GAM-1 in Escherichia coli

A Cd²⁺-resistant Escherichia coli C600 transformant harboring pGU100, which was derived from Cd²⁺-resistant Pseudomonas putida GAM-1, was able to grow in concentrations of CdCl₂ as high as 3.5 mM, whereas E. coli C600 could not grow in the presence of 1.5 mM CdCl₂. E. coli C600 (pGU100) possesses a Cd²⁺ efflux system. This efflux system was inhibited by 100 μM dicyclohexylcarbodiimide, indicating that the system seems to be energy-dependent. Further studies revealed that the Cd²⁺ efflux system of E. coli C600 (pGU100) can operate under proliferous conditions, but not under nonproliferous conditions.     

Narrow- and Broad-Host-Range Symbiotic Plasmids of Rhizobium spp. Strains That Nodulate Phaseolus vulgaris

Agrobacterium transconjugants containing symbiotic plasmids from different Rhizobium spp. strains that nodulate Phaseolus vulgaris were obtained. All transconjugants conserved the parental nodulation host range. Symbiotic (Sym) plasmids of Rhizobium strains isolated originally from P. vulgaris nodules, which had a broad nodulation host range, and single-copy nitrogenase genes conferred a Fix⁺ phenotype to the Agrobacterium transconjugants. A Fix⁻ phenotype was obtained with Sym plasmids of strains isolated from P. vulgaris nodules that had a narrow host range and reiterated nif genes, as well as with Sym plasmids of strains isolated from other legumes that presented single nif genes and a broad nodulation host range. This indicates that different types of Sym plasmids can confer the ability to establish an effective symbiosis with P. vulgaris.     

携带Paenibacillus polymyxa WLY78固氮基因簇(nif)的重组大肠杆菌Escherichia coli78-7转录组分析

[目的]来自Paenibacillus polymyxa WLY78的固氮基因簇（nifBHDKEfNXhesAnifV）可以转化入Escherichia coli中表达并使重组大肠杆菌合成有固氮活性的固氮酶。本文拟通过对重组大肠杆菌E.coli 78-7的转录组分析以提高其固氮能力。[方法]对固氮条件（无氧无NH₄⁺）和非固氮条件（空气和100 mmol/L NH₄⁺）培养的重组大肠杆菌E.coli 78-7进行转录组分析。[结果]nif基因在两种培养条件下显著表达,说明在重组大肠杆菌中可规避原菌中氧气和NH₄⁺对nif基因的负调控。对于固氮过程必需的非nif基因,如参与钼、硫、铁元素转运的mod、cys和feoAB,这些基因在两种培养条件下表达水平有差异。而参与铁硫簇合成的suf和isc基因簇在两条件下表达水平差异巨大。此外,参与氮代谢的基因在固氮条件下显著上调。[结论]重组大肠杆菌中与固氮相关的非nif基因在该菌的固氮过程中具有较大影响,本文对在异源宿主中调高固氮酶活性研究具有重要意义。     

Engineering alternative butanol production platforms in heterologous bacteria

Alternative microbial hosts have been engineered as biocatalysts for butanol biosynthesis. The butanol synthetic pathway of Clostridium acetobutylicum was first re-constructed in Escherichia coli to establish a baseline for comparison to other hosts. Whereas polycistronic expression of the pathway genes resulted in the production of 34 mg/L butanol, individual expression of pathway genes elevated titers to 200 mg/L. Improved titers were achieved by co-expression of Saccharomyces cerevisiae formate dehydrogenase while overexpression of E. coli glyceraldehyde 3-phosphate dehydrogenase to elevate glycolytic flux improved titers to 580 mg/L. Pseudomonas putida and Bacillus subtilis were also explored as alternative production hosts. Polycistronic expression of butanol biosynthetic genes yielded butanol titers of 120 and 24 mg/L from P. putida and B. subtilis, respectively. Production in the obligate aerobe P. putida was dependent upon expression of bcd-etfAB. These results demonstrate the potential of engineering butanol biosynthesis in a variety of heterologous microorganisms, including those cultivated aerobically.