Optimizing a Fed-Batch High-Density Fermentation Process for Medium Chain-Length Poly(3-Hydroxyalkanoates) in Escherichia coli.

Production of medium chain-length poly(3-hydroxyalkanoates) [PHA] polymers with tightly defined compositions is an important area of research to expand the application and improve the properties of these promising biobased and biodegradable materials. PHA polymers with homopolymeric or defined compositions exhibit attractive material properties such as increased flexibility and elasticity relative to poly(3-hydroxybutyrate) [PHB]; however, these polymers are difficult to biosynthesize in native PHA-producing organisms, and there is a paucity of research toward developing high-density cultivation methods while retaining compositional control. In this study, we developed and optimized a fed-batch fermentation process in a stirred tank reactor, beginning with the biosynthesis of poly(3-hydroxydecanoate) [PHD] from decanoic acid by ß-oxidation deficient recombinant Escherichia coli LSBJ using glucose as a co-substrate solely for growth. Bacteria were cultured in two stages, a biomass accumulation stage (37°C, pH 7.0) with glucose as the primary carbon source and a PHA biosynthesis stage (30°C, pH 8.0) with co-feeding of glucose and a fatty acid. Through iterative optimizations of semi-defined media composition and glucose feed rate, 6.0 g of decanoic acid was converted to PHD with an 87.5% molar yield (4.54 g L-1). Stepwise increases in the amount of decanoic acid fed during the fermentation correlated with an increase in PHD, resulting in a final decanoic acid feed of 25 g converted to PHD at a yield of 89.4% (20.1 g L-1, 0.42 g L-1 h-1), at which point foaming became uncontrollable. Hexanoic acid, octanoic acid, 10-undecenoic acid, and 10-bromodecanoic acid were all individually supplemented at 20 g each and successfully polymerized with yields ranging from 66.8 to 99.0% (9.24 to 18.2 g L-1). Using this bioreactor strategy, co-fatty acid feeds of octanoic acid/decanoic acid and octanoic acid/10-azidodecanoic acid (8:2 mol ratio each) resulted in the production of their respective copolymers at nearly the same ratio and at high yield, demonstrating that these methods can be used to control PHA copolymer composition.

Utilization of L-glutamate as a preferred or sole nutrient in Pseudomonas aeruginosa PAO1 depends on genes encoding for the enhancer-binding protein AauR, the sigma factor RpoN and the transporter complex AatJQMP.

BACKGROUND: Glutamate and aspartate are preferred nutrients for a variety of microorganisms. In the case for many Pseudomonas spp., utilization of these amino acids is believed to be dependent on a transporter complex comprised of a periplasmic-solute binding protein (AatJ), two permease domains (AatQM) and an ATP-binding component (AatP). Notably, expression of this transporter complex is hypothesized to be regulated at the transcriptional level by the enhancer-binding protein AauR and the alternative sigma factor RpoN. The purpose of the current study was to determine the biological significance of the putative aatJ-aatQMP operon and its regulatory aauR and rpoN genes in the utilization of L-glutamate, L-glutamine, L-aspartate and L-asparagine in Pseudomonas aeruginosa PAO1. RESULTS: Deletion of the aatJ-aatQMP, aauR or rpoN genes did not affect the growth of P. aeruginosa PAO1 on L-glutamate, L-glutamine, L-aspartate and L-asparagine equally. Instead, only growth on L-glutamate as the sole carbon source was abolished with the deletion of any one of these genes. Interestingly, growth of the aauR mutant on L-glutamate was readily restored via plasmid-based expression of the aatQMP genes, suggesting that it is the function of AatQMP (and not AatJ) that is limiting in the absence of the aauR gene. Subsequent analysis of beta-galactosidase reporters revealed that both aatJ and aatQ were induced in response to L-glutamate, L-glutamine, L-aspartate or L-asparagine in a manner dependent on the aauR and rpoN genes. In addition, both aatJ and aatQ were expressed at reduced levels in the absence of the inducing-amino acids and the regulatory aauR and rpoN genes. The expression of the aatJ-aatQMP genes is, therefore, multifaceted. Lastly, the expression levels of aatJ were significantly higher (> 5 fold) than that of aatQ under all tested conditions. CONCLUSIONS: The primary function of AauR in P. aeruginosa PAO1 is to activate expression of the aatJ-aatQMP genes in response to exogenous acidic amino acids and their amide derivatives. Importantly, it is the AauR-RpoN mediated induction of the aatQMP genes that is the pivotal factor enabling P. aeruginosa PAO1 to effectively utilize or consume L-glutamate as a sole or preferred nutrient.

Increased Production of the Value-Added Biopolymers Poly(R-3-Hydroxyalkanoate) and Poly(γ-Glutamic Acid) From Hydrolyzed Paper Recycling Waste Fines.

Reject fines, a waste stream of short lignocellulosic fibers produced from paper linerboard recycling, are a cellulose-rich paper mill byproduct that can be hydrolyzed enzymatically into fermentable sugars. In this study, the use of hydrolyzed reject fines as a carbon source for bacterial biosynthesis of poly(R-3-hydroxyalkanoate) (PHA) and poly(γ-glutamic acid) (PGA) was investigated. Recombinant Escherichia coli harboring PHA biosynthesis genes were cultivated with purified sugars or crude hydrolysate to produce both poly(R-3-hydroxybutyrate) (PHB) homopolymer and medium chain length-containing copolymer (PHB-co-MCL). Wild-type Bacillus licheniformis WX-02 were cultivated with crude hydrolysate to produce PGA. Both PHB and short chain-length-co-medium chain-length (SCL-co-MCL) PHA yields from crude hydrolysate were a 2-fold improvement over purified sugars, and the MCL monomer fraction was decreased slightly in copolymers produced from crude hydrolysate. PGA yield from crude hydrolysate was similarly increased 2-fold. The results suggest that sugars from hydrolyzed reject fines are a viable carbon source for PHA and PGA biosynthesis. The use of crude hydrolysate is not only possible but beneficial for biopolymer production, eliminating the need for costly separation and purification techniques. This study demonstrates the potential to divert a lignocellulosic waste stream into valuable biomaterials, mitigating the environmental impacts of solid waste disposal.

Effect of acetate as a co-feedstock on the production of poly(lactate-co-3-hydroxyalkanoate) by pflA-deficient Escherichia coli RSC10.

Developing Escherichia coli strains that are tolerant to acetate toxicity is important in light of an increased interest in the efficient utilization of lignocellulosic biomass feedstocks for the biosynthesis of value-added products. In this study, four strains known to produce polyhydroxyalkanoates (PHAs) from the typical hemicellulosic sugar xylose were tested for their tolerance to acetate. E. coli RSC10 was found to be tolerant of acetate, both in growth and fermentation studies. In the presence of acetate the strain showed a >2-fold increase in overall yields compared to using xylose alone as the feedstock. More importantly, the strain was found to be able to utilize acetate as a feedstock for biosynthesis of PHAs, with complete depletion of acetate (25 mM) at 9 h when acetate was the sole feedstock. Higher concentrations of acetate showed greater inhibition of fermentation than growth with a reduction of 90% in PHA yields at 100 mM. Additionally, the present work provides data to support the potential of acetate as a modulator for the control of composition of PHAs that incorporate lactate (LA) monomers into the copolymer from hemicellulose derived sugars.

Enhancing poly(3-hydroxyalkanoate) production in Escherichia coli by the removal of the regulatory gene arcA.

Recombinant Escherichia coli is a desirable platform for the production of many biological compounds including poly(3-hydroxyalkanoates), a class of naturally occurring biodegradable polyesters with promising biomedical and material applications. Although the controlled production of desirable polymers is possible with the utilization of fatty acid feedstocks, a central challenge to this biosynthetic route is the improvement of the relatively low polymer yield, a necessary factor of decreasing the production costs. In this study we sought to address this challenge by deleting arcA and ompR, two global regulators with the capacity to inhibit the uptake and activation of exogenous fatty acids. We found that polymer yields in a ΔarcA mutant increased significantly with respect to the parental strain. In the parental strain, PHV yields were very low but improved 64-fold in the ΔarcA mutant (1.92-124 mg L-1) The ΔarcA mutant also allowed for modest increases in some medium chain length polymer yields, while weight average molecular weights improved by approximately 1.5-fold to 12-fold depending on the fatty acid substrate utilized. These results were supported by an analysis of differential gene expression, which showed that the key genes (fadD, fadL, and fadE) encoding fatty acid degradation enzymes were all upregulated by 2-, 10-, and 31-fold in an ΔarcA mutant, respectively. Additionally, the short chain length fatty acid uptake genes atoA, atoE and atoD were upregulated by 103-, 119-, and 303-fold respectively, though these values are somewhat inflated due to low expression in the parental strain. Overall, this study demonstrates that arcA is an important target to improve PHA production from fatty acids.

Consolidated bioprocessing of poly(lactate-co-3-hydroxybutyrate) from xylan as a sole feedstock by genetically-engineered Escherichia coli.

Consolidated bioprocessing of lignocellulose is an attractive strategy for the sustainable production of petroleum-based alternatives. One of the underutilized sources of carbon in lignocellulose is the hemicellulosic fraction which largely consists of the polysaccharide xylan. In this study, Escherichia coli JW0885 (pyruvate formate lyase activator protein mutant, pflA(-)) was engineered to express recombinant xylanases and polyhydroxyalkanoate (PHA)-producing enzymes for the biosynthesis of poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)] from xylan as a consolidated bioprocess. The results show that E. coli JW0885 was capable of producing P(LA-co-3HB) when xylan was the only feedstock and different feeding and growth parameters were examined in order to improve upon initial yields. The highest yields of P(LA-co-3HB) copolymer obtained in this study occurred when xylan was added during mid-exponential growth after cells had been grown at high shaking-speeds (290 rpm). The results showed an inverse relationship between total PHA production and LA-monomer incorporation into the copolymer. Proton nuclear magnetic resonance ((1)H NMR), gel permeation chromatography (GPC), and differential scanning calorimetry (DSC) analyses corroborate that the polymers produced maintain physical properties characteristic of LA-incorporating PHB-based copolymers. The present study achieves the first ever engineering of a consolidated bioprocessing bacterial system for the production of a bioplastic from a hemicelluosic feedstock.

The metabolism of (R)-3-hydroxybutyrate is regulated by the enhancer-binding protein PA2005 and the alternative sigma factor RpoN in Pseudomonas aeruginosa PAO1.

A variety of soil-dwelling bacteria produce polyhydroxybutyrate (PHB), which serves as a source of energy and carbon under nutrient deprivation. Bacteria belonging to the genus Pseudomonas do not generally produce PHB but are capable of using the PHB degradation product (R)-3-hydroxybutyrate [(R)-3-HB] as a growth substrate. Essential to this utilization is the NAD+-dependent dehydrogenase BdhA that converts (R)-3-HB into acetoacetate, a molecule that readily enters central metabolism. Apart from the numerous studies that had focused on the biochemical characterization of BdhA, there was nothing known about the assimilation of (R)-3-HB in Pseudomonas, including the genetic regulation of bdhA expression. This study aimed to define the regulatory factors that govern or dictate the expression of the bdhA gene and (R)-3-HB assimilation in Pseudomonas aeruginosa PAO1. Importantly, expression of the bdhA gene was found to be specifically induced by (R)-3-HB in a manner dependent on the alternative sigma factor RpoN and the enhancer-binding protein PA2005.This mode of regulation was essential for the utilization of (R)-3-HB as a sole source of energy and carbon. However, non-induced levels of bdhA expression were sufficient for P. aeruginosa PAO1 to grow on ( ± )-1,3-butanediol, which is catabolized through an (R)-3-HB intermediate. Because this is, we believe, the first report of an enhancer-binding protein that responds to (R)-3-HB, PA2005 was named HbcR for (R)-3-hydroxybutyrate catabolism regulator.

Soil microbial community toxic response to atrazine and its residues under atrazine and lead contamination.

Intensive use of atrazine and extensive dispersal of lead (Pb) have occurred in farmland with chemical agriculture development. However, the toxicological effect of their presence on soil microorganism remains unknown. The objective of this study was to investigate the impacts of atrazine or Pb on the soil microbiota, soil net nitrogen mineralization, and atrazine residues over a 28-day microcosm incubation. The Shannon-Wiener diversity index, typical microbe species, and a Neighbor-joining tree of typical species from sequencing denaturing gradient gel electrophoresis (DGGE) bands were determined across periodical sampling times. The results showed that the existence of atrazine or Pb (especially high concentration) in soils reduced microbial diversity (the lowest H value is 2.23) compared to the control (H = 2.59) after a 28-day incubation. The species richness reduced little (from 17~19 species to 16~17 species) over the research time. But soil microbial community was significantly affected by the incubation time after the exposure to atrazine or Pb. The combination of atrazine and Pb had a significant inhibition effect on soil net nitrogen nitrification. Atrazine and Pb significantly stimulated soil cumulative net nitrogen mineralization and nitrification. Pb (300 and 600 mg kg(-1)) accelerated the level of atrazine dissipation. The exposure might stimulate the significant growth of the autochthonous soil degraders which may use atrazine as C source and accelerate the dissipation of atrazine in soils.

Deletion of the pflA gene in Escherichia coli LS5218 and its effects on the production of polyhydroxyalkanoates using beechwood xylan as a feedstock.

Engineering of microorganisms to directly utilize plant biomass as a feedstock for the biosynthesis of value-added products such as bioplastics is the aim of consolidated bioprocessing. In previous research we successfully engineered E. coli LS5218 to produce polyhydroxyalkanoates (PHAs) from xylan. In this study we report further genetic modifications to Escherichia coli LS5218 in order to increase the lactic acid (LA) fraction in poly(lactic acid-co-3-hydroxyalkanoate) P(LA-co-HA) copolymers. Deletion of the pflA gene resulted in increased content of LA repeating units in the copolymers by over 3-fold compared with the wild type; however, this increase was offset by reduced yields in cell mass. Additionally, when acetate was used as a feedstock LA monomer incorporation reached 18.5 (mol%), which suggests that acetate can be used as a feedstock for the production of P(LA-co-HA) copolymers by E. coli.