Barrie Fong Chong

Microbial hyaluronic acid production

AbstractsHyaluronic acid (HA) is a commercially valuable medical biopolymer increasingly produced through microbial fermentation. Viscosity limits product yield and the focus of research and development has been on improving the key quality parameters, purity and molecular weight. Traditional strain and process optimisation has yielded significant improvements, but appears to have reached a limit. Metabolic engineering is providing new opportunities and HA produced in a heterologous host is about to enter the market. In order to realise the full potential of metabolic engineering, however, greater understanding of the mechanisms underlying chain termination is required.

Amplifying the cellular reduction potential of Streptococcus zooepidemicus

The valuable pharmaceutical polymer, hyaluronic acid, is produced industrially using the gram-positive bacterium Streptococcus zooepidemicus. Synthesis of this polymer is a significant energetic burden upon the microorganism hence the native NADH oxidase gene was cloned and overexpressed to increase the energy yield of catabolism during aerobic cultivation on glucose. Elevated NADH oxidase levels led to a decline in lactic acid generation and prevented ethanol formation, leaving acetate as the main fermentation product. Biomass yield increased due to the energy gained from the formation of acetate. Evaluation of the acetate flux control coefficient over a range of NADH oxidase expression levels revealed that acetate production was sensitive to the NADH oxidase level. However, at high NADH oxidase levels, the acetate flux was mainly influenced by another factor. The concomitant excretion of pyruvate at high NADH oxidase levels suggested that the flux through the pyruvate dehydrogenase enzyme complex was limiting the conversion of pyruvate to acetate.

Aerobic cultivation of Streptococcus zooepidemicus and the role of NADH oxidase

A metabolic flux model was developed for Streptococcus zooepidemicus to compare the metabolism of glucose and maltose during aerobic batch cultivation. Lactic acid was the main product of glucose metabolism whereas acetic acid was the main product of maltose metabolism. This difference was chiefly attributed to the two-fold higher flux through NADH oxidase in maltose-grown cells that enabled the ATP generation rate to remain high despite a slower maltose consumption rate. The two-fold higher flux was matched by a two-fold increase in NADH oxidase activity, 2.53 +/- 0.1 mumol NADH min(-1) mg(-1) protein on maltose versus 1.07 +/- 0.04 Rmol NADH min(-1) mg(-1) protein on glucose, indicating that NADH oxidase activity is regulated by the energy status of the cell. Surprisingly, the energy status of the cell had little impact on hyaluronic acid (HA) yield and molecular weight

Co-ordinated synthesis of gentiobiitol and sorbitol, evidence of sorbitol glycosylation in transgenic sugarcane.

Sugarcane (a Saccharum spp. interspecific hybrid) was previously engineered to synthesize sorbitol (designated as sorbitolcane). Motivated by the atypical development of the leaves in some sorbitolcane, the polar metabolite profiles in the leaves of those plants were compared against a group of control sugarcane plants. Eighty-six polar metabolites were detected in leaf extracts by GC-MS. Principal component analysis of the metabolites indicated that three compounds were strongly associated with sorbitolcane. Two were identified as sorbitol and gentiobiose and the third was unknown. Gentiobiose and the unknown compound were positively correlated with sorbitol accumulation. The unknown compound was only abundant in sorbitolcane. This compound was structurally characterized and found to be a sorbitol-glucose conjugate. (13)C NMR analysis indicated that the glucopyranose and glucitol moieties were 1,6-linked. Ligand exchange chromatography confirmed that the compound was a beta-anomer, thus identifying the compound as 6-O-beta-d-glucopyranosyl-D-glucitol, or gentiobiitol.

Early Exposure to Ethylene Modifies Shoot Development and Increases Sucrose Accumulation Rate in Sugarcane

AbstractSugarcane varieties (Saccharum spp. hybrids) that accumulate high levels of sucrose at the start of the harvest season are of considerable commercial interest. Our understanding of the factors that contribute to early sucrose accumulation in these varieties is limited. In this study we used the plant hormone ethylene to investigate the relationship between growth and early sucrose accumulation in sugarcane. The sugarcane variety KQ228 was exposed to a low concentration of the ethylene-forming compound 2-chloroethylphosphonic acid (CEPA) for a prolonged duration commencing from shoot emergence. The changes in sucrose accumulation and plant growth were investigated. Results from two glasshouse experiments revealed that the CEPA-treated plants accumulated a significantly higher amount of sucrose in their primary culm 2 and 3½ months post-germination. The treated plants had taller primary culms with many smaller internodes, smaller leaves, and a higher photosynthetic rate. Despite producing smaller internodes, treated culms were comparable in fresh weight and volume to the controls due to the compensating effect of faster internode formation. We identified three factors that may have contributed to the early accumulation of more sucrose in the treated culm: (1) the specific leaf area of young leaves was greater indicating efficient diversion of photoassimilate to sink tissue, (2) internode formation was initiated earlier, and (3) internodes continued to form at a faster rate. Consequently, a greater proportion of the internodes in the treated sugarcane matured earlier and began filling with sucrose sooner. The higher reducing sugar level in the apical region of the culm probably contributed to faster internode development. This coincided with elevated vacuolar and cell wall acid invertase gene expression that increased sucrose turnover in the vacuole and increased apoplastic uptake of reducing sugars. These findings extend our understanding of how some sugarcane varieties can naturally accumulate a high level of sucrose early in the season.

Developing sugarcane lignocellulosic biorefineries: opportunities and challenges

It is widely acknowledged that the world must decrease its overall energy reliance on fossil fuels by embracing technologies for large-scale production of renewable energy. Part of this change is due to concerns about the impact of climate change, but it is also recognized that global oil and fossil fuel supplies are finite. The production of first-generation ethanol as a biofuel to reduce oil inputs has been successful on a large scale using sugarcane juice and/or molasses (Brazil) and corn (USA). The advent of second-generation biofuels is impending as lignocellulosic processing technology improves and costs decrease. Sugarcane and related sugar crops such as sweet sorghum will be leading candidates to drive this growth in the future, as they possess considerable advantages over other broad acre cropping systems. Sugarcane biorefineries will be realized by extracting maximum value from sugarcane waste and cultivated energycane.

Advancing Energy Cane Cell Wall Digestibility Screening by Near-Infrared Spectroscopy

Breeding energy cane for cellulosic biofuel production involves manipulating various traits. An important trait to optimize is cell wall degradability as defined by enzymatic hydrolysis. We investigated the feasibility of using near-infrared spectroscopy (NIRS) combined with multivariate calibration to predict energy cane cell wall digestibility based upon fiber samples from a range of sugarcane genotypes and related species. These samples produced digestibility values ranging between 6 and 31%. To preserve the practicality of the technique, spectra obtained from crudely prepared samples were used. Various spectral preprocessing methods were tested, with the best NIRS calibration obtained from second derivative, orthogonal signal–corrected spectra. Model performance was evaluated by cross-validation and independent validation. Large differences between the performance results from the two validation approaches indicated that the model was sensitive to the choice of test data. This may be remedied by using a larger calibration training set containing diverse sample types. The best result was obtained through independent validation which produced a R 2 value of 0.86, a root mean squared error of prediction (RMSEP) of 1.59, and a ratio of prediction to deviation (RPD) of 2.7. This study has demonstrated that it is feasible and practical to use NIRS to predict energy cane cell wall digestibility.