Guhan Jayaraman

Hyaluronan (HA), a glycosaminoglycan with important medical applications, is commercially produced from pathogenic microbial sources. The metabolism of HA-producing recombinant generally regarded as safe (GRAS) systems needs to be more strategically engineered to achieve yields higher than native producers. Here, we use a genome-scale model (GEM) to account for the entire metabolic network of the cell while predicting strategies to improve HA production. We analyze the metabolic network of Lactococcus lactis adapted to produce HA and identify non-conventional strategies to enhance HA flux. We also show experimental verification of one of the predicted strategies. We thus identified an alternate route for enhancement of HA synthesis, originating from the nucleoside inosine, that can function in parallel with the traditionally known route from glucose. Adopting this strategy resulted in a 2.8-fold increase in HA yield. The strategies identified and the experimental results show that the cell is capable of involving a larger subset of metabolic pathways in HA production. Apart from being the first report to use a nucleoside to improve HA production, we demonstrate the role of experimental validation in model refinement and strategy improvisation. Overall, we point out that well-constructed GEMs could be used to derive efficient strategies to improve the biosynthesis of high-value products.

Multi-wavelength fluorescence spectroscopy was evaluated as a tool for on-line monitoring of recombinant Escherichia coli cultivations expressing human basic fibroblast growth factor (hFGF-2). The data sets for the various combinations of the excitation and emission spectra from batch cultivations were analyzed using principal component analysis. Chemometric models (the partial least squares method) were developed for correlating the fluorescence data and the experimentally measured variables such as the biomass and glucose concentrations as well as the carbon dioxide production rate. Excellent correlations were obtained for these variables for the calibration cultivations. The predictability of these models was further tested in batch and fed-batch cultivations. The batch cultivations were well predicted by the PLS models for biomass, glucose concentrations and carbon dioxide production rate (RMSEPs were respectively 5%, 7%, 9%). However, when tested for biomass concentrations in fed-batch cultivations (with final biomass three times higher than the highest calibration data) the models had good predictability at high growth rates (RMSEPs were 3% and 4%, respectively for uninduced and induced fed-batch cultivations), which was as good as for the batch cultivations used for developing the models (RMSEPs were 3% and 5%, respectively for uninduced and induced batch cultivations). The fed-batch cultivations performed at low growth rates exhibited much higher fluorescence for fluorophores such as flavin and NAD(P)H as compared to fed-batch cultivations at high growth rate. Therefore, the PLS models tended to over-predict the biomass concentrations at low growth rates. Obviously the cells changed their concentration of biogenic fluorophores depending on the growth rate. Although multi-wavelength fluorescence spectroscopy is a valuable tool for on-line monitoring of bioprocess, care must be taken to re-calibrate the PLS models at different growth rates to improve the accuracy of predictions. © 2011 Elsevier B.V.

Microbial production of hyaluronic acid (HA) is an attractive substitute for extraction of this biopolymer from animal tissues. Natural producers such as Streptococcus zooepidemicus are potential pathogens; therefore, production of HA by recombinant bacteria that are generally recognized as safe (GRAS) organisms is a viable alternative that is being extensively explored. However, plasmid-based expression systems for HA production by recombinant bacteria have the inherent disadvantage of reduced productivity because of plasmid instability. To overcome this problem, the HA synthesis genes (hasA-hasB and hasA-hasB-hasC) from has-operon of S. zooepidemicus were integrated into the chromosome of Lactococcus lactis by site-directed, double-homologous recombination developing strains VRJ2AB and VRJ3ABC. The chromosomal integration stabilized the genes and obviated the instability observed in plasmid-expressed recombinant strains. The genome-integrated strains produced higher molecular weight (3.5-4 million Dalton [MDa]) HA compared to the plasmid-expressed strains (2 MDa). High molecular weight HA was produced when the intracellular concentration of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) and uridine diphosphate-glucuronic acid (UDP-GlcUA) was almost equal and hasA to hasB ratio was low. This work suggests an optimal approach to obtain high molecular weight HA in recombinant strains.

Hyaluronic acid (HA) production was metabolically engineered in Lactococcus lactis by introducing the HA synthetic machinery from the has operon of the pathogenic bacterium Streptococcus zooepidemicus. This study shows that the insertion of uridine diphosphate (UDP)-glucose pyrophosphorylase (hasC) gene in addition to the HA synthase (hasA) and UDP-glucose dehydrogenase (hasB) genes has a significant impact on increasing HA production. The recombinant L. lactis NZ9000 strain transformed with the plasmid pSJR2 (co-expressing hasA and hasB genes only) produced a maximum of 107 mg/l HA in static flask experiments with varying initial glucose concentrations, while the corresponding experiments with the transformant SJR3 (co-expressing hasA, hasB, and hasC genes) gave a maximum yield of 234 mg/l HA. The plasmid cloned with the insertion of the full has operon comprising of five different genes (hasA, hasB, hasC, hasD, and hasE) exhibited structural instability. The HA yield was further enhanced in batch bioreactor experiments with controlled pH and aeration, and a maximum of 1.8 g/l HA was produced by the SJR3 culture. © 2009 Springer-Verlag.

Multivariate data analysis techniques are widely used in getting better insight into the processes in the fields like chemometrics, speech processing, biomedical signal processing and astronomy. In the present study, the problem of extracting the spectrum of a pure component from Near Infrared (NIR) mixture spectra containing a known diluent is tackled. Different multivariate data analysis methods such as Ordinary Least Square (OLS), Principal Component Regression (PCR) and Non Negative Matrix Factorization (NMF) are modified to solve the problem. It is shown that including partial knowledge such as the spectrum of the known diluent in the data analysis techniques, accounting for errors in the absorbance measurements, and imposing non-negativity constraints on absorbance and concentrations estimates, results in better estimation of the pure component spectrum. © IFAC.

The molecular weight (Mw) of hyaluronic acid (HA) determines its suitability for medical and cosmetic applications. Here, we characterize in vitro and in vivo HA synthesis of streptococcal HA synthases (HASs) with a special focus on HA Mw. To date, four streptococcal HA producers are described (Streptococcus equi subsp. equi, S. equi subsp. zooepidemicus, S. pyogenes, and S. uberis). We identified two more potential HA producers in this study: S. iniae and S. parauberis. Indeed, the HA Mw produced by the different streptococcal HASs differs in vitro. To exploit these different HA Mw synthesis capacities, Lactococcus lactis strains expressing the streptococcal HASs were constructed. HA of different Mw was also produced in vivo by these engineered strains, strongly suggesting that the protein sequences of the HASs influence HA Mw. Since the HA Mw in vivo is also influenced by metabolic factors like specific growth rate and HA precursor availability, these were also determined. In summary, the maximal Mw of HA synthesized is specific for the individual synthase, while any decrease from the maximal HA Mw is influenced by physiological and metabolic factors. The results open new avenues for Mw-tailored HA synthesis.

Hyaluronic acid has a wide range of biomedical applications and its commercial value is highly dependent on its purity and molecular weight. This study highlights the utility of aqueous two-phase separation as a primary recovery step for hyaluronic acid and for removal of major protein impurities from fermentation broths. Metabolically engineered cultures of a lactate dehydrogenase mutant strain of Lactococcus lactis (L. lactis NZ9020) were used to produce high-molecular-weight hyaluronic acid. The cell-free fermentation broth was partially purified using a polyethylene glycol/potassium phosphate system, resulting in nearly 100% recovery of hyaluronic acid in the salt-rich bottom phase in all the aqueous two-phase separation experiments. These experiments were optimized for maximum removal of protein impurities in the polyethylene glycol rich top phase. The removal of protein impurities resulted in substantial reduction of membrane fouling in the subsequent diafiltration process, carried out with a 300 kDa polyether sulfone membrane. This step resulted in considerable purification of hyaluronic acid, without any loss in recovery and molecular weight. Diafiltration was followed by an adsorption step to remove minor impurities and achieve nearly 100% purity. The final hyaluronic acid product was characterized by Fourier-transform IR and NMR spectroscopy, confirming its purity.

Fermentation-derived products are in greater demand to meet the increasing global market as well as to overcome environmental problems. In this work, Escherichia coli has been metabolically engineered with acrylate pathway genes from Clostridium propionicum for the conversion of d-lactic acid to propionic acid. The introduced synthetic pathway consisted of seven genes encoding the enzymes propionate CoA-transferase (Pct), lactoyl-CoA dehydratase (Lcd) and acryloyl-CoA reductase (Acr). The engineered strain synthesised propionic acid at a concentration of 3.7 ± 0.2 mM upon fermentation on glucose. This low production level could be attributed to the low activity of the recombinant enzymes in particular the rate-limiting enzyme, Acr. Interestingly, the recombinant pathway caused an increased lactate production in E. coli with a yield of 1.9 mol/mol of glucose consumed along with a decrease in other by-products. Down-regulation of the pfl (pyruvate formate lyase) genes and a possible inhibition of Pfl activity by the acrylate pathway intermediate, acryloyl-CoA, could have reduced carbon flow to the Pfl pathway with a concomitant increase in lactate production. This study reports a novel way of synthesising propionic acid by employing a non-native, user-friendly organism through metabolic engineering. © 2012 Springer-Verlag.

The production of recombinant proteins in Lactococcus lactis is often limited by several process and biological constraints. Recombinant gene expression in the P170 system of L. lactis is triggered at a pH below 6.5 by lactic acid accumulation. This work has used static flask, batch bioreactor and chemostat studies to independently investigate various factors affecting production of recombinant streptokinase. These factors are induction strength, complex-nutrient supplementation, specific growth rate and the cellular response to acid-stress termed as acid tolerance response (ATR). In the P170 system, induction strength is mainly a function of lactate concentration. Recombinant protein production is enhanced by increasing induction strength and complex-nutrient supplementation. However, acid-induced cellular stress-response has a deleterious effect on recombinant protein productivity. It is seen that suppression of ATR has the most predominant effect on enhancing recombinant protein productivity. The insights obtained from batch studies were used to investigate fed-batch processes, both during complete development of ATR and during suppression of ATR. During complete ATR-development, a 25-fold enhancement of volumetric productivity was achieved in fed-batch culture, due to a synergistic combination of increased glucose-feed rates and addition of complex-nutrients to the feed. A combination of these factors and ATR-suppression gave rise to nearly 60-fold increase in volumetric productivity in fed-batch culture over batch processes.

The potential advantages of hyaluronic acid (HA) production by metabolically-engineered Lactococcus lactis is constrained by the lower molecular weight and yield of HA obtained in these strains, compared to natural producers. Earlier studies have correlated lower HA yield with excessive lactate production in L. lactis cultures (Chauhan et al., 2014). In the present study, a three-fold increase was observed in the amount as well as molecular weight of HA produced by recombinant ldh-mutant L. lactis strains. The diversion from lactate production in the ldh-mutant strains resulted in excess ethanol and acetoin production and higher NAD+/NADH ratio in these cultures. The initial NAD+/NADH ratio showed a positive correlation with HA molecular weight as well as with the HA-precursor ratio (UDP-GlcUA/UDP-GlcNAc). The influence of NAD+/NADH ratio on regulation of the concerned metabolic pathways was assessed by transcriptional analysis of key genes having putative binding sites of the NADH-binding transcriptional factor, Rex.