Production of Substituted Styrene Bioproducts from Lignin and Lignocellulose Using Engineered Pseudomonas putida KT2440

Ferulic acid is a renewable chemical found in lignocellulose from grasses such as wheat straw and sugarcane. Pseudomonas putida is able to liberate and metabolize ferulic acid from plant biomass. Deletion of the hydroxycinnamoyl‐CoA hydratase‐lyase gene (ech) produced a strain of P. putida unable to utilize ferulic and p‐coumaric acid, which is able to accumulate ferulic acid and p‐coumaric acid from wheat straw or sugar cane bagasse. Further engineering of this strain saw the replacement of ech with the phenolic acid decarboxylase padC, which converts p‐coumaric and ferulic acid into 4‐vinylphenol and the flavor agent 4‐vinylguaiacol, respectively. The engineered strain containing padC is able to generate 4‐vinylguaiacol and 4‐vinylphenol from media containing lignocellulose or Green Value Protobind lignin as feedstock, and does not require the addition of an exogenous inducer molecule. Biopolymerization of 4‐vinylguaiacol and 4‐vinylcatechol styrene products is also carried out, using Trametes versicolor laccase, to generate “biopolystyrene” materials on small scale.     

Growth of engineered Pseudomonas putida KT2440 on glucose,xylose, and arabinose: Hemicellulose hydrolysates and their major sugars as sustainable carbon sources

Lignocellulosic biomass is the most abundant bioresource on earth containing polymers mainly consisting of d ‐glucose, d ‐xylose, l ‐arabinose, and further sugars. In order to establish this alternative feedstock apart from applications in food, we engineered Pseudomonas putida KT2440 as microbial biocatalyst for the utilization of xylose and arabinose in addition to glucose as sole carbon sources. The d ‐xylose‐metabolizing strain P. putida KT2440_xylAB and l ‐arabinose‐metabolizing strain P. putida KT2440_araBAD were constructed by introducing respective operons from Escherichia coli. Surprisingly, we found out that both recombinant strains were able to grow on xylose as well as arabinose with high cell densities and growth rates comparable to glucose. In addition, the growth characteristics on various mixtures of glucose, xylose, and arabinose were investigated, which demonstrated the efficient co‐utilization of hexose and pentose sugars. Finally, the possibility of using lignocellulose hydrolysate as substrate for the two recombinant strains was verified. The recombinant P. putida KT2440 strains presented here as flexible microbial biocatalysts to convert lignocellulosic sugars will undoubtedly contribute to the economic feasibility of the production of valuable compounds derived from renewable feedstock.     

Potential of biotechnological conversion of lignocellulose hydrolyzates by Pseudomonas putida KT2440 as a model organism for a bio‐based economy

Lignocellulose‐derived hydrolyzates typically display a high degree of variation depending on applied biomass source material as well as process conditions. Consequently, this typically results in variable composition such as different sugar concentrations as well as degree and the presence of inhibitors formed during hydrolysis. These key obstacles commonly limit its efficient use as a carbon source for biotechnological conversion. The gram‐negative soil bacterium Pseudomonas putida KT2440 is a promising candidate for a future lignocellulose‐based biotechnology process due to its robustness and versatile metabolism. Recently, P. putida KT2440_xylAB which was able to metabolize the hemicellulose (HC) sugars, xylose and arabinose, was developed and characterized. Building on this, the intent of the study was to evaluate different lignocellulose hydrolyzates as platform substrates for P. putida KT2440 as a model organism for a bio‐based economy. Firstly, hydrolyzates of different origins were evaluated as potential carbon sources by cultivation experiments and determination of cell growth and sugar consumption. Secondly, the content of major toxic substances in cellulose and HC hydrolyzates was determined and their inhibitory effect on bacterial growth was characterized. Thirdly, fed‐batch bioreactor cultivations with hydrolyzate as the carbon source were characterized and a diauxic‐like growth behavior with regard to different sugars was revealed. In this context, a feeding strategy to overcome the diauxic‐like growth behavior preventing accumulation of sugars is proposed and presented. Results obtained in this study represent a first step and proof‐of‐concept toward establishing lignocellulose hydrolyzates as platform substrates for a bio‐based economy.     

Optimized prodigiosin production with Pseudomonas putida KT2440 using parallelized noninvasive online monitoring

The red pigment prodigiosin is of high pharmaceutical interest, due to its potential applications as an antitumor drug and antibiotic agent. As previously demonstrated, Pseudomonas putida KT2440 is a suitable host for prodigiosin production, as it exhibits high tolerance toward the antimicrobial properties of prodigiosin. So far, prodigiosin concentrations of up to 94 mg/L have been achieved in shake flask cultivations. For the characterization and optimization of the prodigiosin production process, the scattered light of P. putida and fluorescence of prodigiosin was measured. The excitation and emission wavelengths for prodigiosin measurement were analyzed by recording 2D fluorescence spectra. The strongest prodigiosin fluorescence was obtained at a wavelength combination of 535/560 nm. By reducing the temperature to 18 °C and using 16 g/L glucose, the prodigiosin concentration was more than doubled compared with the initial cultivation conditions. The obtained results demonstrate the capabilities of parallelized microscale cultivations combined with noninvasive online monitoring of fluorescence for rapid bioprocess development, using prodigiosin as a molecule of current biotechnological interest.     

多功能降解菌Pseudomonas putida KT2440-DOP的构建与降解特性研究

三唑磷水解酶基因为研究发现的一个新的广谱有机磷水解酶基因,通过PCR从有机磷降解菌株Ochrobactrumsp.mp-4总DNA扩增了tpd,将tpd定向克隆到pBBRMCS-5载体上,构建重组质粒pTPD,在辅助质粒pRK2013的帮助下,通过三亲接合将pTPD转移到模式菌株Pseudomonas putidaKT2440中,获得的工程菌PseudomonasputidaKT2440-DOP可以降解多种有机磷农药及芳香烃化合物;KT2440-DOP的有机磷水解酶活较出发菌株MP-4提高了一倍左右,且遗传性状稳定。     

Direct biosynthesis of adipic acid from lignin-derived aromatics using engineered Pseudomonas putida KT2440

Lignin is one largely untapped natural resource that can be exploited as a raw material for the bioproduction of value-added chemicals. Meanwhile, the current petroleum-based process for the production of adipic acid faces sustainability challenges. Here we report the successful engineering of Pseudomonas putida KT2440 strain for the direct biosynthesis of adipic acid from lignin-derived aromatics. The devised bio-adipic acid route features an artificial biosynthetic pathway that is connected to the endogenous aromatics degradation pathway of the host at the branching point, 3-ketoadipoyl-CoA, by taking advantage of the unique carbon skeleton of this key intermediate. Studies of the metabolism of 3-ketoadipoyl-CoA led to the discovery of crosstalk between two aromatics degradation pathways in KT2440. This knowledge facilitated the formulation and implementation of metabolic engineering strategies to optimize the carbon flux into the biosynthesis of adipic acid. By optimizing pathway expression and cultivation conditions, an engineered strain AA-1 produced adipic acid at 0.76 g/L and 18.4% molar yield under shake-flask conditions and 2.5 g/L and 17.4% molar yield under fermenter-controlled conditions from common aromatics that can be derived from lignin. This represents the first example of the direct adipic acid production from model compounds of lignin depolymerization.     

pH-stat fed-batch process to enhance the production of cis, cis-muconate from benzoate by Pseudomonas putida KT2440-JD1

Pseudomonas putida KT2440-JD1 is able to cometabolize benzoate to cis, cis-muconate in the presence of glucose as growth substrate. P. putida KT2440-JD1 was unable to grow in the presence of concentrations above 50 mM benzoate or 600 mM cis, cis-muconate. The inhibitory effects of both compounds were cumulative. The maximum specific uptake rate of benzoate was higher than the specific production rate of cis, cis-muconate during growth on glucose in the presence of benzoate, indicating that a benzoate derivative accumulated in the cells, which is likely to be catechol. Catechol was shown to reduce the expression level of the ben operon, which encodes the conversion of benzoate to cis, cis-muconate. To prevent overdoses of benzoate, a pH-stat fed-batch process for the production of cis, cis-muconate from benzoate was developed, in which the addition of benzoate was coupled to the acidification of the medium. The maximum specific production rate during the pH-stat fed-batch process was 0.6 g (4.3 mmol) g dry cell weight(-1) h(-1), whereas 18.5 g L(-1) cis, cis-muconate accumulated in the culture medium with a molar product yield of close to 100%. Proteome analysis revealed that the outer membrane protein H1 was upregulated during the pH-stat fed-batch process, whereas the expression of 10 other proteins was reduced. The identified proteins are involved in energy household, transport, translation of RNA, and motility.     

Cloning, heterologous expression, and functional characterization of the nicotinate dehydrogenase gene from Pseudomonas putida KT2440

6-Hydroxynicotinate can be used for the production of drugs, pesticides and intermediate chemicals. Some Pseudomonas species were reported to be able to convert nicotinic acid to 6-hydroxynicotinate by nicotinate dehydrogenase. So far, previous reports on NaDH in Pseudomonas genus were confused and contradictory each other. Recently, Ashraf et al. reported an NaDH gene cloned from Eubacterium barkeri and suggested some deducted NaDH genes from other nine bacteria. But they did not demonstrate the activity of recombinant NaDH and did not mention NaDH gene in Pseudomonas. In this study we cloned the gene of NaDH, ndhSL, from Pseudomonas putida KT2440. NdhSL in P. putida KT2440 is composed of two subunits. The small subunit contains [2Fe2S] iron sulfur domain, while the large subunit contains domains of molybdenum cofactor and cytochrome c. Expression of recombinant ndhSL in P. entomophila L48, which lacks the ability to produce 6-hydroxynicotinate, enabled the resting cell and cell extract of engineering P. entomophila L48 to hydroxylate nicotinate. Gene knockout and recovery studies further confirmed the ndhSL function.     

Application of free-flow electrophoresis/2-dimentional gel electrophoresis for fractionation and characterization of native proteome of Pseudomonas putida KT2440

Free Flow Electrophoresis (FFE) is a liquid-based isoelectric focusing method. Unlike conventional in-gel fractionation of proteins, FFE can resolve proteins in their native forms and fractionation of subcellular compartments of the cell is also possible. To test the efficacy of the FFE method, the native cytosol proteome of a bacterium, Pseudomonas putida KT2440 was fractionated by FFE and the spectrum of protein elutes was characterized in association with 2-dimentional gel electrophoresis (2-DE). Major native proteins of P. putida KT2440 were eluted in the range of pH 4.8 approximately 6.0 in FFE, whereas the denatured proteome of P. putida KT2440 was widely distributed in the rage of pH 4 approximately 10 in the 2-DE analysis. In addition, one of the three FFE major fractions, which was eluted at pH 5.0, was further analyzed using 2-DE/MS-MS. Then, the pH range of identified proteins eluted in 2-DE/MS-MS was 4.72 approximately 5.89, indicating that observed pi values of native cytosolic proteomes in FFE were narrower than those of denatured cytosolic proteome. These results suggest that FFE fractionation and 2-DE/MS analysis may be useful tools for characterization of native proteomes of P. putida KT2440 and comparative analysis between denatured and native proteomes.     

Tolerance of plastic-encapsulated Pseudomonas putida KT2440 to chemical stress

Pseudomonas putida dried in the presence of hydroxyectoine or trehalose can withstand exposure to organic solvents and therefore can be encapsulated inside plastics such as polystyrene. Here we show that P. putida in a plastic-encapsulated dried tablet exhibits remarkable tolerance to chemical stress, comparable to that of spores of Bacillus subtilis.     

Effects of the inoculant strain Pseudomonas putida KT2442 (pNF142) and of naphthalene contamination on the soil bacterial community

The naphthalene-degrading activity of a Pseudomonas sp. strain isolated from a creosote-contaminated soil was shown to be encoded by the IncP9 plasmid pNF142 by transfer to Pseudomonas putida KT2442. The effects of the inoculant strain KT2442 (pNF142) and of naphthalene contamination on the soil bacterial community were studied in microcosms with the following treatments: (I) soil, (II) soil with naphthalene, (III) soil with naphthalene and inoculated with KT2442 (pNF142). The inoculant became the dominant bacterial population in treatment (III) as evidenced by cultivation and denaturing gradient gel electrophoresis (DGGE) analysis. The bacterial DGGE profiles revealed drastically reduced complexity due to the numerical dominance of the inoculant. However, group-specific fingerprints (beta-proteobacteria, actinobacteria) that excluded KT2442 (pNF142) showed less severe changes in the bacterial community patterns. A major effect of naphthalene on the soil bacterial community was observed in treatment (II) after 21 days. Two dominant bands appeared whose sequences showed the highest similarity to those of Burkholderia sp. RP007 and Nocardia vinaceae based on 16S rRNA gene sequencing. These bands were less intense in treatment (III). The increased abundance of RP007-like populations due to naphthalene contamination was also confirmed by PCR amplification of the phnAc gene. The nahAc and nahH genes were detected in DNA and cDNA only in treatment III. Although the inoculant strain KT2442 (pNF142) showed good survival and expression of genes involved in naphthalene degradation, this study suggests that KT2442 (pNF142) suppressed the enrichment of indigenous naphthalene degraders.     

A genetically engineered strain of Pseudomonas putida as a useful tool for identifying new therapeutic herbicides

A genetically engineered strain of Pseudomonas putida U designed for the identification of new therapeutic herbicides has been obtained. In this bacterium, deletion of the homogentisate gene cluster (hmgRABC) confers upon this mutant huge biotechnological possibilities since it can be used: (i) as a target for testing new specific herbicides (p-hydroxy-phenylpyruvate dioxygenase inhibitors); (ii) to identify new therapeutic drugs-effective in the treatment of alkaptonuria and other related tyrosinemia - and (iii) as a source of homogentisic acid in a plant-bacterium association.     

Putting cells under pressure: a simple and efficient way to enhance the productivity of medium-chain-length polyhydroxyalkanoate in processes with Pseudomonas putida KT2440

The success of bioprocess implementation relies on the ability to achieve high volumetric productivities and requires working with high‐cell‐density cultivations. Elevated atmospheric pressure might constitute a promising tool for enhancing the oxygen transfer rate (OTR), the major growth‐limiting factor for such cultivations. However, elevated pressure and its effects on the cellular environment also represent a potential source of stress for bacteria and may have negative effects on product formation. In order to determine whether elevated pressure can be applied for enhancing productivity in the case of medium‐chain‐length polyhydroxyalkanoate (mcl‐PHA) production by Pseudomonas putida KT2440, the impact of a pressure of 7 bar on the cell physiology was assessed. It was established that cell growth was not inhibited by this pressure if dissolved oxygen tension (DOT) and dissolved carbon dioxide tension (DCT) were kept below ～30 and ～90 mg L^?1, respectively. Remarkably, a little increase of mcl‐PHA volumetric productivity was observed under elevated pressure. Furthermore, the effect of DCT, which can reach substantial levels during high‐cell‐density processes run under elevated pressure, was investigated on cell physiology. A negative effect on product formation could be dismissed since no significant reduction of mcl‐PHA content occurred up to a DCT of ～540 mg L^?1. However, specific growth rate exhibited a significant decrease, indicating that successful high‐cell‐density processes under elevated pressure would be restricted to chemostats with low dilution rates and fed‐batches with a small growth rate imposed during the final part. This study revealed that elevated pressure is an adequate and efficient way to enhance OTR and mcl‐PHA productivity. We estimate that the oxygen provided to the culture broth under elevated pressure would be sufficient to triple mcl‐PHA productivity in our chemostat system from 3.4 (at 1 bar) to 11 g L^?1 h^?1 (at 3.2 bar). Biotechnol. Bioeng. 2012; 109:451–461. © 2011 Wiley Periodicals, Inc.     

恶臭假单胞菌NA-1菌株烟酸羟基化酶活性的诱导和转化条件的研究

恶臭假单胞菌NA-1菌株的培养和产酶特性与已报道的产酶菌株粘质沙雷氏菌(Serratiamarcescens)IFO12648和荧光假单胞菌(Psudomonasfluorescens)TN5有所不同,主要反映在最适碳源及浓度、最适诱导剂浓度和最适培养温度等方面。最适的转化条件是温度为30℃,pH为7.0,烟酸的浓度为3%。采用初步优化后的条件和流加底物的方式进行4L上罐生产,恶臭假单胞菌NA-1菌株的6-羟基烟酸产率可达到108.39gL。     

基于蛋白质组学的假单胞菌耐溶剂机制研究

【目的】有机溶剂对微生物有强烈的毒害作用致使绝大多数微生物不能在较高的有机溶剂浓度下生长。为了探究微生物的耐溶剂性机制,由野生型假单胞菌Pseudomonas putida JUCS驯化获得一株能够在60%(V/V)的环己烷中生长的菌株P.putidaJUCT1。【方法】采用蛋白质二维电泳对P.putida JUCT1在不同溶剂条件下的蛋白组分表达量的差异进行分析比对。【结果】从总共22个表达量差异均超过50%的蛋白质中,选取了3个高丰度蛋白质,通过MALDI-TOF/TOF鉴定为:3-羟基异丁酸水解酶、蛋白质延伸因子EF-Ts、异分支酸水解酶超家族(编码基因分别为mmsB、tsf、PSEEN0851)。将这3个基因在大肠杆菌中重组表达,3个蛋白均能不同程度地提高E.coli JM109的耐溶剂性,其中3-羟基异丁酸脱氢酶(编码基因mmsB)对菌株的溶剂耐受性影响最为显著。【结论】证明了运用蛋白组学的方法研究微生物的耐溶剂性的可行性,并为构建适用于工业化应用的溶剂耐受性整体细胞生物催化剂提供理论依据。