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.

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.

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.

Lactococcus lactis is a promising host for pathway-engineered production of high molecular weight hyaluronic acid (HA). This work investigates the effect of hemin-supplemented respiratory metabolism in an engineered L. lactis ldh mutant strain (Δldh L. lactis) as a strategy to enhance HA titer in the batch fermentation process. L. lactis lacks a functional electron transport chain (ETC) and ultimately relies on substrate-level phosphorylation for energy production. We conducted detailed investigations under different dissolved oxygen (DO) conditions and found a steady increase in HA titer with increased DO levels. We found a strong correlation between cofactor availability (NAD+ and acetyl-CoA) and the intracellular concentration of HA precursors, UDP-GlcUA and UDP-GlcNAc. We correlated the intracellular precursor levels with the HA titer obtained for the four conditions. We found that these were higher in hemin-supplemented cultures, with the best HA titer of 4.6 g/l. Finally, we observed that the molecular weight of HA (MWHA) correlates strongly with the ratio of HA precursors concentrations. We also found an inverse correlation between MWHA and HA titer across these experiments. These results can be used in optimizing the trade-off between molecular weight and HA production in bioprocesses involving recombinant L. lactis cultures.

The molecular weight of hyaluronic acid (HA) is a critical property which determines its usage in various biomedical applications. This study investigates the correlation between the availability of a critical cofactor, acetyl-CoA, the concentration of a limiting precursor, UDP-N-acetylglucosamine (UDP-GlcNAc), and the molecular weight of HA (MWHA) produced by recombinant Lactococcus lactis MKG6 cultures. This strain expressed three heterologous HA-pathway genes obtained from the has operon of Streptococcus zooepidemicus in an ldh-mutant host strain, L. lactis NZ9020. A flux balance analysis, performed using the L. lactis genome-scale metabolic network, showed a positive correlation of acetyl-CoA flux with the UDP-GlcNAc flux and the experimental data on HA productivity. To increase the intracellular levels of acetyl-CoA, acetate was supplemented as a pulse feed in anaerobic batch cultures. However, acetate is effectively utilized only in the presence of glucose and exhaustion of glucose resulted in decreasing the final MWHA (1.5 MDa). Co-supplementation of acetate resulted in enhancing the acetyl-CoA and UDP-GlcNAc levels as well as the MWHA to 2.5 MDa. This logic was extended to fed-batch cultures, designed with a pH-based feedback control of glucose feeding and pulse acetate supplementation. When the glucose feed concentration was optimally adjusted to prevent glucose exhaustion or accumulation, the acetate utilization was found to be high, resulting in significantly enhanced levels of acetyl-CoA and UDP-GlcNAc as well as a MWHA of 3.4 MDa, which was sustained at this value throughout the process. This study provides the possibility of commercially producing high MWHA using recombinant L. lactis strains.

HA molecular weight variation in Streptococcus zooepidemicus and two recombinant Lactococcus lactis strains were investigated by chemostat experiments and metabolic flux analysis (MFA). The study showed that intracellular flux ratio of UDP-GlcUA to UDP-GlcNAc correlated directly with HA molecular weight, for all the three strains. The ratio of intracellular concentration of these HA precursors also exhibited a similar trend. Phosphoglucoisomerase activity and glucose flux towards lactic acid formation were found to be the major bottlenecks for HA production in all the three strains. The study suggests that environmental conditions and genetic manipulations that balance the intracellular flux and HA precursors concentrations will result in increased molecular weight. © 2014 Elsevier Ltd.

The development of reliable kinetic models of bioprocesses from data is a challenging task. In this work, a systematic approach to develop a kinetic model of bioprocess involving single biomass from concentration data is proposed without imposing any kinetic model a priori. The proposed incremental model identification approach decomposes the model-building task into a set of sub-tasks such as determining the yield coefficients and maintenance coefficient, specific growth rate structure identification, and parameter estimation. It is shown that the proposed approach allows identifying the mechanism of product formation. The proposed approach is applied to the microbial production of Hyaluronic acid (HA), an important biopolymer, using a recombinant Lactococcus lactis MKG6. An unstructured kinetic model is developed for the HA production from data. It is shown that HA production is a growth-associated process. Further, the specific growth rate of HA production is identified from a set of rate candidates. It is revealed that the specific growth rate in the HA production follows the non-competitive HA inhibition model. The parameters obtained by the incremental identification are further refined to obtain statistically optimal estimates using the simultaneous model identification. Validation of the identified kinetic model of HA production on new experimental data shows that the proposed approach leads to a reliable kinetic model with the optimal parameter estimates.

Hyaluronic acid (HA) is a high-value polysaccharide with many biomedical applications. Microbial production of HA is now replacing the traditional extraction method from rooster combs. Production of medical-grade HA with defined characteristics requires controlled process conditions because there are many fermentation process parameters that affect the microbial synthesis of HA. This necessitates the development of online tools for monitoring multiple analytes during microbial fermentation. Here, we describe the application of in situ transflectance spectroscopy for online quantification of seven major fermentation analytes, viz. biomass, glucose, lactate, formate, ethanol, acetate and HA in metabolically engineered Lactococcus lactis fermentations. The near-infrared spectral information acquired from synthetic mixtures and untransformed L. lactis fermentations were used to develop chemometric models. Based on principal component analysis and partial least squares regression methods, analyte-specific models were developed for quantification. These models were then independently validated for fermentation analytes from four different recombinant L. lactis strains. The chemometric model developed for HA based on recombinant L. lactis fermentation data and pure HA standards could accurately predict HA concentrations under homolactic conditions. The online estimation of HA was found to be poor under heterolactic conditions due to the overlapping absorbance of acetate produced in these cultures. Alternatively, an independent model based on yield correlation was successfully developed for indirect real-time quantification of HA.