Sarah A. Lee

A co-fermentation strategy to consume sugar mixtures effectively

We report a new approach for the simultaneous conversion of xylose and glucose sugar mixtures into products by fermentation. The process simultaneously uses two substrate-selective strains of Escherichia coli, one which is unable to consume glucose and one which is unable to consume xylose. The xylose-selective (glucose deficient) strain E. coli ZSC113 has mutations in the glk, ptsG and manZ genes while the glucose-selective (xylose deficient) strain E. coli ALS1008 has a mutation in the xylA gene. By combining these two strains in a single process, xylose and glucose are consumed more quickly than by a single-organism approach. Moreover, we demonstrate that the process is able to adapt to changing concentrations of these two sugars, and therefore holds promise for the conversion of variable sugar feed streams, such as lignocellulosic hydrolysates.

A substrate‐selective co‐fermentation strategy with Escherichia coli produces lactate by simultaneously consuming xylose and glucose

We describe a new approach for the simultaneous conversion of xylose and glucose sugar mixtures which potentially could be used for lignocellulosic biomass hydrolysate. In this study we used this approach to demonstrate the production of lactic acid. This process uses two substrate‐selective strains of Escherichia coli, one which is unable to consume glucose and one which is unable to consume xylose. In addition to knockouts in pflB encoding for pyruvate formate lyase, the xylose‐selective (glucose deficient) strain E. coli ALS1073 has deletions of the glk, ptsG, and manZ genes while the glucose‐selective (xylose deficient) strain E. coli ALS1074 has a xylA deletion. By combining these two strains in a single process the xylose and glucose in a mixed sugar solution are simultaneously converted to lactate. Furthermore, the biomass concentrations of each strain can readily be adjusted in order to optimize the overall product formation. This approach to the utilization of mixed sugars eliminates the problem of diauxic growth, and provides great operational flexibility. Biotechnol. Bioeng. 2009; 102: 822–827.

Glucose repression of xylitol production in Candida tropicalis mixed-sugar fermentations

Glucose repressed xylose utilization inCandida tropicalis pre-grown on xylose until glucose reached approximately 0–5 g l−1. In fermentations consisting of xylose (93 g l−1) and glucose (47 g l−1), xylitol was produced with a yield of 0.65 g g−1 and a specific rate of 0.09 g g−1 h−1, and high concentrations of ethanol were also produced (25 g l−1). If the initial glucose was decreased to 8 g l−1, the xylitol yield (0.79 g g−1) and specific rate (0.24 g g−1 h−1) increased with little ethanol formation (<5 g l−1). To minimize glucose repression, batch fermentations were performed using an aerobic, glucose growth phase followed by xylitol production. Xylitol was produced under O2 limited and anaerobic conditions, but the specific production rate was higher under O2 limited conditions (0.1–0.4 vs. 0.03 g g−1 h−1). On-line analysis of the respiratory quotient defined the time of xylose reductase induction.

Ground kenaf core as a filtration aid

Abstract The objective of this study was to examine the filtration characteristics of ground kenaf core, a waste material generated during the production of kenaf bast fibers for paper. The constant-pressure precoat filtration characteristics of ground kenaf core were compared to commercial diatomaceous earth (DE). Three challenge solutions were studied: a yeast solution, a bacterial solution, and a standard silica-particle solution. The kenaf and DE both satisfactorily permitted removal of all silica particles from solution without noticable flux degradation over the course of the filtration. The kenaf and DE also removed yeast particles. In this case, the flux loss with time was lower with the DE precoated filter than in the kenaf precoated filter. The DE precoat excluded less than 10% of the bacterial particles from solution, whereas the kenaf removed about 40% of these small particles.

Detection of methyl salicylate using bi-enzyme electrochemical sensor consisting salicylate hydroxylase and tyrosinase

Volatile organic compounds have been recognized as important marker chemicals to detect plant diseases caused by pathogens. Methyl salicylate has been identified as one of the most important volatile organic compounds released by plants during a biotic stress event such as fungal pathogen infection. Advanced detection of these marker chemicals could help in early identification of plant diseases and has huge significance for agricultural industry. This work describes the development of a novel bi-enzyme based electrochemical biosensor consisting of salicylate hydroxylase and tyrosinase enzymes immobilized on carbon nanotube modified electrodes. The amperometric detection using the bi-enzyme platform was realized through a series of cascade reactions that terminate in an electrochemical reduction reaction. Electrochemical measurements revealed that the sensitivity of the bi-enzyme sensor was 30.6±2.7µAcm(-2)µM(-1) and the limit of detection and limit of quantification were 13nM (1.80ppb) and 39nM (5.39ppb) respectively. Interference studies showed no significant interference from the other common plant volatile compounds. Synthetic analyte studies revealed that the bi-enzyme based biosensor can be used to reliably detect methyl salicylate released by unhealthy plants.

Accelerating pathway evolution by increasing the gene dosage of chromosomal segments

Significance Gene duplication and divergence are cornerstones of evolution. Genetic redundancy resulting from repeated DNA provides flexibility for transient changes in copy number that may confer selective benefit under new or changing environmental conditions. This work describes a method for creating tandem arrays of specific DNA segments in a bacterium, Acinetobacter baylyi, to accelerate experimental evolution. The induced chromosomal gene amplification mimics a natural process that would otherwise occur more slowly and stochastically. The success of this approach for the evolution of novel protein function was demonstrated with studies of an enzyme that has bioenergy applications in lignin valorization. A protein variant, which emerged from Evolution by Amplification and Synthetic biology, is beneficial in a different bacterium, indicating the broad utility of the method. Experimental evolution is a critical tool in many disciplines, including metabolic engineering and synthetic biology. However, current methods rely on the chance occurrence of a key step that can dramatically accelerate evolution in natural systems, namely increased gene dosage. Our studies sought to induce the targeted amplification of chromosomal segments to facilitate rapid evolution. Since increased gene dosage confers novel phenotypes and genetic redundancy, we developed a method, Evolution by Amplification and Synthetic Biology (EASy), to create tandem arrays of chromosomal regions. In Acinetobacter baylyi, EASy was demonstrated on an important bioenergy problem, the catabolism of lignin-derived aromatic compounds. The initial focus on guaiacol (2-methoxyphenol), a common lignin degradation product, led to the discovery of Amycolatopsis genes (gcoAB) encoding a cytochrome P450 enzyme that converts guaiacol to catechol. However, chromosomal integration of gcoAB in Pseudomonas putida or A. baylyi did not enable guaiacol to be used as the sole carbon source despite catechol being a growth substrate. In ∼1,000 generations, EASy yielded alleles that in single chromosomal copy confer growth on guaiacol. Different variants emerged, including fusions between GcoA and CatA (catechol 1,2-dioxygenase). This study illustrates the power of harnessing chromosomal gene amplification to accelerate the evolution of desirable traits.

Glucose consumption in carbohydrate mixtures by phosphotransferase-system mutants of Escherichia coli

Escherichia coli lacking the glucose phosphotransferase system (PTS), mannose PTS and glucokinase are supposedly unable to grow on glucose as the sole carbon source (Curtis SJ, Epstein W. J Bacteriol 1975;122:1189-1199). We report that W ptsG manZ glk (ALS1406) grows slowly on glucose in media containing glucose with a second carbon source: ALS1406 metabolizes glucose after that other carbon source, including arabinose, fructose, glycerol, succinate or xylose, is exhausted. Galactose is an exception to this rule, as ALS1406 simultaneously consumes both galactose and glucose. The ability of ALS1406 to metabolize glucose in a xylose-glucose mixture was unchanged by an additional knockout in any single gene involved in carbohydrate transport and utilization, including agp (periplasmic glucose-1-phosphatase), galP (galactose permease), xylA (xylose isomerase), alsK (allose kinase), crr (glucose PTS enzyme IIA), galK (galactose kinase), mak (mannokinase), malE (maltose transporter), malX (maltose PTS enzyme IIBC), mglB (methyl-galactose transporter subunit), nagE (N-acetyl glucosamine PTS enzyme IICBA), nanK (N-acetyl mannosamine kinase) or pgm (phosphoglucose mutase). Glucose metabolism was only blocked by the deletion of two metabolic genes, pgi (phosphoglucose isomerase) and zwf (glucose-6-phosphate 1-dehydrogenase), which prevents the entry of glucose-6-phosphate into the pentose phosphate and Embden-Meyerhof-Parnas pathways. Carbon-limited steady-state studies demonstrated that xylose must be sub-saturating for glucose to be metabolized, while nitrogen-limited studies showed that xylose is partly converted to glucose when xylose is in excess. Under transient conditions, ALS1406 converts almost 25 % (mass) xylose into glucose as a result of reversible transketolase and transaldolase and the re-entry of carbon into the pentose phosphate pathway via glucose-6-phosphate 1-dehydrogenase.