Glucose metabolism in Pseudomonas aeruginosa is cyclic when producing Polyhydroxyalkanoates and Rhamnolipids.

Pseudomonas aeruginosa is an important chassis for production of polyhydroxyalkanoates (PHA) and rhamnolipids (RHL). Advances in the understanding of the biosynthesis metabolism of these biocompounds are crucial for increasing yield. 13C-Metabolic Flux Ratio Analysis (13C-MFA) is a technique to estimate in vivo metabolic fluxes ratios. PHA and RHL are essentially non-growth associated products of biotechnological interest and both contain hydroxyalkanoates (HAs), whose labeling patterns could be accessed by GC-MS. In this study, to reveal the relative contributions of the Entner-Doudoroff (ED) pathway and the non-oxidative Pentose Phosphate (PP) pathway to PHA and RHL production, 13C-MFA was performed in Pseudomonas aeruginosa LFM634 when supplied with labeled glucose. This bacterial strain lacks both functional EMP and the oxidative PP branch. Labeling patterns in HAs were measured. Experiments with [U-13C] glucose indicated a low flux though PP pathway. An optimal design of labeling experiment showed that [6-13C] glucose would be the best substrate to enable an estimation of the ED flux with high accuracy. Results of experiments performed with this isotope indicated that about two-thirds of glyceraldehyde 3-phosphate is recycled through a cyclic ED architecture, suggesting that P. aeruginosa utilizes that cycle to regulate the NADPH/Acetyl-CoA ratio for PHA and RHL biosynthesis.

Burkholderia sacchari (synonym Paraburkholderia sacchari): An industrial and versatile bacterial chassis for sustainable biosynthesis of polyhydroxyalkanoates and other bioproducts.

This is the first review presenting and discussing Burkholderia sacchari as a bacterial chassis. B. sacchari is a distinguished polyhydroxyalkanoates producer strain, with low biological risk, reaching high biopolymer yields from sucrose (0.29 g/g), and xylose (0.38 g/g). It has great potential for integration into a biorefinery using residues from biomass, achieving 146 g/L cell dry weight containing 72% polyhydroxyalkanoates. Xylitol (about 70 g/L) and xylonic acid [about 390 g/L, productivity 7.7 g/(L.h)] are produced by the wild-type B. sacchari. Recombinants were constructed to allow the production and monomer composition control of diverse tailor-made polyhydroxyalkanoates, and some applications have been tested. 3-hydroxyvalerate and 3-hydroxyhexanoate yields from substrate reached 80% and 50%, respectively. The genome-scale reconstruction of its metabolic network, associated with the improvement of tools for genetic modification, and metabolic fluxes understanding by future research, will consolidate its potential as a bioproduction chassis.

xylA and xylB overexpression as a successful strategy for improving xylose utilization and poly-3-hydroxybutyrate production in Burkholderia sacchari.

Despite the versatility and many advantages of polyhydroxyalkanoates as petroleum-based plastic substitutes, their higher production cost compared to petroleum-based polymers has historically limited their large-scale production. One appealing approach to reducing production costs is to employ less expensive, renewable feedstocks. Xylose, for example is an abundant and inexpensive carbon source derived from hemicellulosic residues abundant in agro-industrial waste (sugarcane bagasse hemicellulosic hydrolysates). In this work, the production of poly-3-hydroxybutyrate P(3HB) from xylose was studied to develop technologies for conversion of agro-industrial waste into high-value chemicals and biopolymers. Specifically, this work elucidates the organization of the xylose assimilation operon of Burkholderia sacchari, a non-model bacterium with high capacity for P(3HB) accumulation. Overexpression of endogenous xylose isomerase and xylulokinase genes was successfully assessed, improving both specific growth rate and P(3HB) production. Compared to control strain (harboring pBBR1MCS-2), xylose utilization in the engineered strain was substantially improved with 25% increase in specific growth rate, 34% increase in P(3HB) production, and the highest P(3HB) yield from xylose reported to date for B. sacchari (YP3HB/Xil = 0.35 g/g). This study highlights that xylA and xylB overexpression is an effective strategy to improve xylose utilization and P(3HB) production in B. sacchari.

Draft Genome Sequence of Halomonas sp. HG01, a Polyhydroxyalkanoate-Accumulating Strain Isolated from Peru.

Halomonas sp. strain HG01, isolated from a salt mine in Peru, is a halophilic aerobic heterotrophic bacterium accumulating poly-3-hydroxybutyrate and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from different carbon sources. Here, we report the draft genome sequence of this isolate, which was found to be 3,665,487 bp long, with a G+C content of 68%.

Growth of Burkholderia sacchari LFM 101 cultivated in glucose, sucrose and glycerol at different temperatures

Polyhydroxyalkanoates (PHAs) have attracted major industrial interest as alternatives to conventional plastics. They are produced by several bacteria as cytoplasmic inclusions when nutrients are in limited supply. Among the many factors influencing bacterial growth, the effect of temperature on both specific growth rates and growth yields in terms of carbon source intake is of considerable interest. This study aimed to evaluate the influence of the bacterium Burkholderia sacchari LFM 101 on growth and PHA production, using glucose, sucrose or glycerol as a carbon source, at 30 and 35 °C. The results showed that B. sacchari cultured with glucose at 35 °C presented both higher productivity and polymer yield in dried cell mass. There were no differences in growth rates (max) in sucrose and glucose. The growth conditions studied were not favorable to glycerol consumption due to limitations in the energy supply from glycerol.(AU)

Growth of Burkholderia sacchari LFM 101 cultivated in glucose, sucrose and glycerol at different temperatures

Polyhydroxyalkanoates (PHAs) have attracted major industrial interest as alternatives to conventional plastics. They are produced by several bacteria as cytoplasmic inclusions when nutrients are in limited supply. Among the many factors influencing bacterial growth, the effect of temperature on both specific growth rates and growth yields in terms of carbon source intake is of considerable interest. This study aimed to evaluate the influence of the bacterium Burkholderia sacchari LFM 101 on growth and PHA production, using glucose, sucrose or glycerol as a carbon source, at 30 and 35 °C. The results showed that B. sacchari cultured with glucose at 35 °C presented both higher productivity and polymer yield in dried cell mass. There were no differences in growth rates (max) in sucrose and glucose. The growth conditions studied were not favorable to glycerol consumption due to limitations in the energy supply from glycerol.

Quantifying NAD(P)H production in the upper Entner-Doudoroff pathway from Pseudomonas putida KT2440.

Despite the lack of biochemical information, all available in silico metabolic models of Pseudomonas putida KT2440 consider NADP as the only cofactor accepted by the glucose-6-phosphate dehydrogenases. Because the Entner-Doudoroff pathway is the main glycolytic route in this bacterium, determining how much NADH and NADPH are produced in the reaction catalyzed by these enzymes is very important for the correct interpretation of metabolic flux distributions. To determine the actual cofactor preference of the glucose-6-phosphate dehydrogenase encoded by the zwf-1 gene (PputG6PDH-1), the major isoform during growth on glucose, we purified this protein and studied its kinetic properties. Based on simple kinetic principles, we estimated the in vivo relative production of NADH and NADPH during the oxidation of glucose-6-phosphate (G6P). Contrary to the general assumption, our calculations showed that the reaction catalyzed by PputG6PDH-1 yields around 1/3 mol of NADPH and 2/3 mol of NADH per mol of oxidized G6P. Additionally, we obtained data suggesting that the reaction catalyzed by the 6-phosphogluconate dehydrogenase is active during growth on glucose, and it also produces NADH. These results indicate that the stoichiometric matrix of in silico models of P. putida KT2440 must be corrected and highlight the importance of considering the physiological concentrations of the involved metabolites to estimate the actual proportion of NADH and NADPH produced by a dehydrogenase.

Draft Genome Sequence of Pseudomonas sp. Strain LFM046, a Producer of Medium-Chain-Length Polyhydroxyalkanoate.

Pseudomonas sp. LFM046 is a medium-chain-length polyhydroxyalkanoate (PHAMCL) producer capable of using various carbon sources (carbohydrates, organic acids, and vegetable oils) and was first isolated from sugarcane cultivation soil in Brazil. The genome sequence was found to be 5.97 Mb long with a G+C content of 66%.

Draft Genome Sequence of the Polyhydroxyalkanoate-Producing Bacterium Burkholderia sacchari LMG 19450 Isolated from Brazilian Sugarcane Plantation Soil.

Burkholderia sacchari LMG 19450, isolated from the soil of a sugarcane plantation in Brazil, accumulates large amounts of polyhydroxyalkanoates from sucrose, xylose, other carbohydrates, and organic acids. We present the draft genome sequence of this industrially relevant bacterium, which is 7.2 Mb in size and has a G+C content of 64%.

Polyhydroxyalkanoate biosynthesis and simultaneous remotion of organic inhibitors from sugarcane bagasse hydrolysate by Burkholderia sp.

Burkholderia sp. F24, originally isolated from soil, was capable of growth on xylose and removed organic inhibitors present in a hemicellulosic hydrolysate and simultaneously produced poly-3-hydroxybutyrate (P3HB). Using non-detoxified hydrolysate, Burkholderia sp. F24 reached a cell dry weight (CDW) of 6.8 g L(-1), containing 48 % of P3HB and exhibited a volumetric productivity (PP3HB) of 0.10 g L(-1) h(-1). Poly-3-hydroxybutyrate-co-3-hydroxyvalerate copolymers (P3HB-co-3HV) were produced using xylose and levulinic acid (LA) as carbon sources. In shake flask cultures, the 3HV content in the copolymer increased from 9 to 43 mol% by adding LA from 1.0 to 5.0 g L(-1). In high cell density cultivation using concentrated hemicellulosic hydrolysate F24 reached 25.04 g L(-1) of CDW containing 49 % of P3HB and PP3HB of 0.28 g L(-1 )h(-1). Based on these findings, second-generation ethanol and bioplastics from sugarcane bagasse is proposed.

Influence of pH on the molecular weight of poly-3-hydroxybutyric acid (P3HB) produced by recombinant Escherichia coli.

The production of ultrahigh molecular weight poly-3-hydroxybutyric acid (P3HB) from carbohydrates by recombinant Escherichia coli harboring genes from Ralstonia eutropha was evaluated. In shaken-flask experiments, E. coli XL1 Blue harboring plasmid pSK::phaCAB produced P3HB corresponding to 40 and 27% of cell dry weight from glucose and xylose, respectively. Cultures in bioreactor using glucose as the sole carbon source at variable pH values (6.0, 6.5, or 7.0) allowed the production of P3HB with molecular weight varying between 2.0 and 2.5 MDa. These figures are significantly higher than the values often obtained by natural bacterial strains (0.5-1.0 MDa). Contrary to reports of other authors, no influence of pH was observed on the molecular weight of the polymer produced. Using xylose, P3HB with high molecular weight was also produced, indicating the possibility to produce these polymers from lignocellulosic materials.

The diversity of polyketide synthase genes from sugarcane-derived fungi.

The chemical ecology and biotechnological potential of metabolites from endophytic and rhizosphere fungi are receiving much attention. A collection of 17 sugarcane-derived fungi were identified and assessed by PCR for the presence of polyketide synthase (PKS) genes. The fungi were all various genera of ascomycetes, the genomes of which encoded 36 putative PKS sequences, 26 shared sequence homology with ß-ketoacyl synthase domains, while 10 sequences showed homology to known fungal C-methyltransferase domains. A neighbour-joining phylogenetic analysis of the translated sequences could group the domains into previously established chemistry-based clades that represented non-reducing, partially reducing and highly reducing fungal PKSs. We observed that, in many cases, the membership of each clade also reflected the taxonomy of the fungal isolates. The functional assignment of the domains was further confirmed by in silico secondary and tertiary protein structure predictions. This genome mining study reveals, for the first time, the genetic potential of specific taxonomic groups of sugarcane-derived fungi to produce specific types of polyketides. Future work will focus on isolating these compounds with a view to understanding their chemical ecology and likely biotechnological potential.

Cloning and overexpression of the xylose isomerase gene from Burkholderia sacchari and production of polyhydroxybutyrate from xylose.

A different organization for the xyl operon was found in different genomes of Burkholderia and Pseudomomas species. Degenerated primers were designed based on Burkholderia genomes and used to amplify the xylose isomerase gene (xylA) from Burkholderia sacchari IPT101. The gene encoded a protein of 329 amino acids, which showed the highest similarity (90%) to the homologous gene of Burkholderia dolosa. It was cloned in the broad host range plasmid pBBR1MCS-2, which partially restored growth and polyhydroxybutyrate production capability in xylose to a B. sacchari xyl- mutant. When xylA was overexpressed in the wild-type strain, it was not able to increase growth and polyhydroxybutyrate production, suggesting that XylA activity is not limiting for xylose utilization in B. sacchari.

Disruption of the 2-methylcitric acid cycle and evaluation of poly-3-hydroxybutyrate-co-3-hydroxyvalerate biosynthesis suggest alternate catabolic pathways of propionate in Burkholderia sacchari.

The objective of the present work was to evaluate the relevance of the 2-methylcitric acid cycle (2MCC) to the catabolism of propionate in Burkholderia sacchari. Two B. sacchari mutants unable to grow on propionate were obtained: one disrupted in acnM, and the other in acnM and prpC deleted. An operative 2MCC significantly reduces the bacterial ability to incorporate 3-hydroxyvalerate (3HV) into a biodegradable copolyester accumulated from carbohydrates plus propionate. The efficiency of the mutants in converting propionate to 3HV units (Y(3HV/prp)) increased from 0.09 g*g(-1) to 0.81-0.96 g*g(-1), indicating that acnM and prpC are both essential for growth on propionate. None of the mutations resulted in achievement of the maximum theoretical Y3HV/prp (1.35 g*g(-1)). When increasing concentrations of propionate were supplied, decreasing values of Y3HV/prp were observed. The results obtained corroborate the hypothesis of the presence of other propionate catabolic pathways in B. sacchari. The 2MCC would be the more operative pathway, but a second pathway, which remains to be elucidated, would assume more importance under propionate concentrations of 1 g*L(-1) or higher. The efficiency in converting propionate to 3HV units can be improved by decreasing the propionate concentrations, owing to the role of the 2MCC.