DNA-Aptamer-Based qPCR Using Light-Up Dyes for the Detection of Nucleic Acids.

Quantitative polymerase chain reaction (qPCR) is widely used in detection of nucleic acids, but existing methods either lack sequence-specific detection or are costly because they use chemically modified DNA probes. In this work, we apply a DNA aptamer and light-up dye-based chemistry for qPCR for nucleic acid quantification. In contrast to the conventional qPCR, in our method, we observe an exponential decrease in fluorescence upon DNA amplification. The qPCR method we developed produced consistent Ct vs log10 (DNA amount) standard curves, which have a linearfit with R2 value > 0.99. This qPCR technique was validated by quantifying gene targets from Streptococcus zooepidemicus (SzhasB) and Mycobacterium tuberculosis (MtrpoB). We show that our strategy is able to successfully detect DNA at as low as 800 copies/µL. To the best of our knowledge, this is the first study demonstrating the application of light-up dyes and DNA aptamers in qPCR.

Continuous protein refolding and purification by two-stage periodic counter-current chromatography.

Matrix-assisted refolding (MAR) has been used as an alternative to conventional dilution-based refolding to improve recovery and reduce specific buffer consumption. Size exclusion chromatography (SEC) has been extensively used for MAR because of its ability to load and refold proteins at high concentrations. However, the SEC-based batch MAR processes have the disadvantages of requiring longer columns for better separation and product dilution due to a high column-to-sample volume ratio. In this work, a modified operational scheme is developed for continuous MAR of L-asparaginase inclusion bodies (IBs) using SEC-based periodic counter-current chromatography (PCC). The volumetric productivity of the modified SEC-PCC process is 6.8-fold higher than the batch SEC process. In addition, the specific buffer consumption decreased by 5-fold compared to the batch process. However, the specific activity of the refolded protein (110-130 IU/mg) was less due to the presence of impurities and additives in the refolding buffer. To address this challenge, a 2-stage process was developed for continuous refolding and purification of IBs using different matrices in sequential PCCs. The performance of the 2-stage process is compared with literature reports on single-stage IMAC-PCC and conventional pulse dilution processes for refolding L-asparaginase IBs. The 2-stage process resulted in a refolded protein with enhanced specific activity (175-190 IU/mg) and a high recovery of 84%. The specific buffer consumption (6.2 mL/mg) was lower than the pulse dilution process and comparable to the single-stage IMAC-PCC. A seamless integration of the two stages would considerably increase the throughput without compromising other parameters. High recovery, throughput, and increased operational flexibility make the 2-stage process an attractive option for protein refolding.

Metabolic engineering of Pseudomonas taiwanensis VLB120 for rhamnolipid biosynthesis from biomass-derived aromatics.

Lignin is a ubiquitously available and sustainable feedstock that is underused as its depolymerization yields a range of aromatic monomers that are challenging substrates for microbes. In this study, we investigated the growth of Pseudomonas taiwanensis VLB120 on biomass-derived aromatics, namely, 4-coumarate, ferulate, 4-hydroxybenzoate, and vanillate. The wild type strain was not able to grow on 4-coumarate and ferulate. After integration of catabolic genes for breakdown of 4-coumarate and ferulate, the metabolically engineered strain was able to grow on these aromatics. Further, the specific growth rate of the strain was enhanced up to 3-fold using adaptive laboratory evolution, resulting in increased tolerance towards 4-coumarate and ferulate. Whole-genome sequencing highlighted several different mutations mainly in two genes. The first gene was actP, coding for a cation/acetate symporter, and the other gene was paaA coding for a phenyl acetyl-CoA oxygenase. The evolved strain was further engineered for rhamnolipid production. Among the biomass-derived aromatics investigated, 4-coumarate and ferulate were promising substrates for product synthesis. With 4-coumarate as the sole carbon source, a yield of 0.27 (Cmolrhl/Cmol4-coumarate) was achieved, corresponding to 28% of the theoretical yield. Ferulate enabled a yield of about 0.22 (Cmolrhl/Cmolferulate), representing 42% of the theoretical yield. Overall, this study demonstrates the use of biomass-derived aromatics as novel carbon sources for rhamnolipid biosynthesis.

Process optimization for the rapid conversion of calcite into hydroxyapatite microspheres for chromatographic applications.

Microsphere hydroxyapatite (HAp) is widely used in various biomedical and chromatographic applications. The work described in this manuscript focuses on a dissolution precipitation method for production of HAp microspheres. This method overcomes certain drawbacks of conventional preparation methods used for HAp preparation, which produce polydisperse particles and are time-consuming and expensive. In the present work, the calcium carbonate (calcite) particles were directly and rapidly converted into HAp microspheres using an inexpensive dissolution precipitation method. The effects of the reaction temperature, time, and mechanical stirring rates were studied, and the reaction parameters were optimized. As confirmed by the XRD studies, the higher reaction temperature and time promote complete HAp conversion, while calcite residues were observed for lower temperatures and times. SEM images show the influence of reaction parameters on the surface microstructure of the microspheres produced. It was observed that the HAp microspheres undergo disintegration at a higher stirring rate. The reaction parameters optimized in this work were ideal for preparing HAp microspheres. The resultant HAp particles were utilized as matrices for chromatographic separation of protein mixtures.

Inactivation behavior and intracellular changes in Escherichia coli during electro-oxidation process using Ti/Sb-SnO2/PbO2 anode: Elucidation of the disinfection mechanism.

This study investigates the behavior and intracellular changes in Escherichia coli (model organism) during electro-oxidation with Ti/Sb-SnO2/PbO2 anode in a chlorine free electrochemical system. Preliminary studies were conducted to understand the effect of initial E. coli concentration and applied current density on disinfection. At an applied current density 30 mA cm-2, 7 log reduction of E. coli was achieved in 75 min. The role of reactive oxygen species' (ROS) in E.coli disinfection was evaluated, which confirmed hydroxyl (•OH) radical as the predominant ROS in electro-oxidation. Observations were carried out at cell and molecular level to understand E.coli inactivation mechanism. Scanning electron microscopy images confirmed oxidative damage of the cell wall and irreversible cell death. Intracellular and extracellular protein quantification and genetic material release further confirmed cell component leakage due to cell wall rupture and degradation due to •OH radical interaction. Change in cell membrane potential suggests the colloidal nature of E. coli cells under applied current density. Plasmid deoxyribonucleic acid degradation study confirmed fragmentation and degradation of released genetic material. Overall, effective disinfection could be achieved by electro-oxidation, which ensures effective inactivation and prevents regrowth of E. coli. Disinfection of real wastewater was achieved in 12 min at an applied current density 30 mA cm-2. Real wastewater study further confirmed that effective disinfection is possible with a low cost electrode material such as Ti/Sb-SnO2/PbO2. Energy consumed during disinfection was determined to be 4.978 kWh m-3 for real wastewater disinfection at applied current density 30 mA cm-2. Cost of operation was estimated and stability of the electrode was studied to evaluate the feasibility of large scale operation. Relatively low energy and less disinfection time makes this technology suitable for field scale applications.

Continuous refolding of L-asparaginase inclusion bodies using periodic counter-current chromatography.

Chromatography-based refolding is emerging as a promising alternative to dilution-refolding of solubilized inclusion bodies (IBs). The advantages of this matrix-assisted refolding (MAR) lie in its ability to reduce aggregate formation, leading to better recovery of active protein, and enabling refolding at higher protein concentration. However, batch chromatography has the disadvantage of ineffective solvent utilization, under-utilization of resin, and low throughput. In this work, we overcome these challenges by using a 3-column Periodic Counter-current Chromatographic (PCC) system for continuous refolding of IBs, formed during the production of L-asparaginase by recombinant E. coli cultures. Initial experiments were conducted in batch processes using single-column immobilized metal-affinity chromatography. Different gradient operations were designed to improve the protein loading for the single-column, batch-MAR processes. Optimized conditions, based on the batch-MAR experiments, were used for designing the continuous-MAR processes using the PCC system. The continuous-MAR experiments were carried out over 3 cycles (∼ 30 h) in the PCC system. A detailed quantitative comparison based on recovery, throughput, buffer consumption, and resin utilization was made for the three modes of operation: pulse-dilution, single-column batch-MAR, and 3-Column PCC-based continuous-MAR processes. While recovery (73%) and throughput (11 mg/h) were the highest in PCC, specific buffer consumption (6.9 ml/mg) was the least. Also, during PCC operation, resin utilization improved by 92% in comparison to the single-column batch-MAR process. These quantitative comparisons clearly establish the advantages of the continuous-MAR process over the batch-MAR and other conventional refolding techniques.

Evolutionary engineering of Lactobacillus bulgaricus reduces enzyme usage and enhances conversion of lignocellulosics to D-lactic acid by simultaneous saccharification and fermentation.

BACKGROUND: Simultaneous saccharification and fermentation (SSF) of pre-treated lignocellulosics to biofuels and other platform chemicals has long been a promising alternative to separate hydrolysis and fermentation processes. However, the disparity between the optimum conditions (temperature, pH) for fermentation and enzyme hydrolysis leads to execution of the SSF process at sub-optimal conditions, which can affect the rate of hydrolysis and cellulose conversion. The fermentation conditions could be synchronized with hydrolysis optima by carrying out the SSF at a higher temperature, but this would require a thermo-tolerant organism. Economically viable production of platform chemicals from lignocellulosic biomass (LCB) has long been stymied because of the significantly higher cost of hydrolytic enzymes. The major objective of this work is to develop an SSF strategy for D-lactic acid (D-LA) production by a thermo-tolerant organism, in which the enzyme loading could significantly be reduced without compromising on the overall conversion. RESULTS: A thermo-tolerant strain of Lactobacillus bulgaricus was developed by adaptive laboratory evolution (ALE) which enabled the SSF to be performed at 45 °C with reduced enzyme usage. Despite the reduction of enzyme loading from 15 Filter Paper Unit/gLCB (FPU/gLCB) to 5 FPU/gLCB, we could still achieve ~ 8% higher cellulose to D-LA conversion in batch SSF, in comparison to the conversion by separate enzymatic hydrolysis and fermentation processes at 45 °C and pH 5.5. Extending the batch SSF to SSF with pulse-feeding of 5% pre-treated biomass and 5 FPU/gLCB, at 12-h intervals (36th-96th h), resulted in a titer of 108 g/L D-LA and 60% conversion of cellulose to D-LA. This is one among the highest reported D-LA titers achieved from LCB. CONCLUSIONS: We have demonstrated that the SSF strategy, in conjunction with evolutionary engineering, could drastically reduce enzyme requirement and be the way forward for economical production of platform chemicals from lignocellulosics. We have shown that fed-batch SSF processes, designed with multiple pulse-feedings of the pre-treated biomass and enzyme, can be an effective way of enhancing the product concentrations.

Biosynthesis of Hyaluronic acid polymer: Dissecting the role of sub structural elements of hyaluronan synthase.

Hyaluronic acid (HA) based biomaterials have several biomedical applications. HA biosynthesis is catalysed by hyaluronan synthase (HAS). The unavailability of 3-D structure of HAS and gaps in molecular understanding of HA biosynthesis process pose challenges in rational engineering of HAS to control HA molecular weight and titer. Using in-silico approaches integrated with mutation studies, we define a dictionary of sub-structural elements (SSE) of the Class I Streptococcal HAS (SeHAS) to guide rational engineering. Our study identifies 9 SSE in HAS and elucidates their role in substrate and polymer binding and polymer biosynthesis. Molecular modelling and docking assessment indicate a single binding site for two UDP-substrates implying conformationally-driven alternating substrate specificities for this class of enzymes. This is the first report hypothesizing the involvement of sites from SSE5 in polymer binding. Mutation at these sites influence HA production, indicating a tight coupling of polymer binding and synthase functions. Mutation studies show dispensable role of Lys-139 in substrate binding and a key role of Gln-248 and Thr-283 in HA biosynthesis. Based on the functional architecture in SeHAS, we propose a plausible three-step polymer extension model from its reducing end. Together, these results open new avenues for rational engineering of Class I HAS to study and regulate its functional properties and enhanced understanding of glycosyltransferases and processive enzymes.

Exploiting the diversity of streptococcal hyaluronan synthases for the production of molecular weight-tailored hyaluronan.

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.

Enhancement of acetyl-CoA by acetate co-utilization in recombinant Lactococcus lactis cultures enables the production of high molecular weight hyaluronic acid.

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.

Co-culture of Lactobacillus delbrueckii and engineered Lactococcus lactis enhances stoichiometric yield of D-lactic acid from whey permeate.

D-Lactic acid (D-LA) is an enantiomer of lactic acid, which has a niche application in synthesis of poly-lactic acid based (PLA) polymer owing to its contribution to the thermo-stability of stereo-complex PLA polymer. Utilization of renewable substrates such as whey permeate is pivotal to economically viable production of D-LA. In present work, we have demonstrated D-LA production from whey permeate by Lactobacillus delbrueckii and engineered Lactococcus lactis. We observed that lactose fermentation by a monoculture of L. delbrueckii yields D-LA and galactose as major products. The highest yield of D-LA obtained was 0.48 g g-1 when initial lactose concentration was 30 g L-1. Initial lactose concentration beyond 20 g L-1 resulted in accumulation of glucose and galactose, and hence, reduced the stoichiometric yield of D-LA. L. lactis naturally produces L-lactic acid (L-LA), so a mutant strain of L. lactis (L. lactis Δldh ΔldhB ΔldhX) was used to prevent L-LA production and engineer it for D-LA production. Heterologous over-expression of D-lactate dehydrogenase (ldhA) in the recombinant strain L. lactis TSG1 resulted in 0.67 g g-1 and 0.44 g g-1 of D-LA yield from lactose and galactose, respectively. Co-expression of galactose permease (galP) and α-phosphoglucomutase (pgmA) with ldhA in the recombinant strain L. lactis TSG3 achieved a D-LA yield of 0.92 g g-1 from galactose. A co-culture batch process of L. delbrueckii and L. lactis TSG3 achieved an enhanced stoichiometric yield of 0.90 g g-1 and ~45 g L-1D-LA from whey permeate (lactose). This is the highest reported yield of D-LA from lactose substrate, and the titres can be improved further by a suitably designed fed-batch co-culture process.

Production of controlled molecular weight hyaluronic acid by glucostat strategy using recombinant Lactococcus lactis cultures.

Hyaluronic acid (HA) is a biopolymer with wide biomedical and cosmetic applications, wherein the molecular weight of HA (MWHA) is an important quality parameter that determines its suitability for the targeted application. To produce HA with desired molecular weight, it is important to identify parameters that offer tunability and control of MWHA at a desired value during fermentation. In this work, two tunable parameters, viz. glucose concentration and combination of HA biosynthetic genes expressed, were used to produce HA of different molecular weights. Three recombinant strains of Lactococcus lactis were constructed, using a combination of the has-operon genes from Streptococcus zooepidemicus (hasA, hasB, hasE) and the α-phosphoglucomutase gene (pgmA) from L. lactis. Batch fermentations of these recombinant strains at different initial glucose concentrations enabled production of HA with different molecular weights. Co-expression of hasABE was observed to be particularly effective in improving the MWHA. It was observed during batch fermentations of all these recombinant L. lactis cultures that the MWHA decreases steadily during the later part of the fermentation and the final value is 19-43% lower than the peak MWHA produced. Analysis of the fermentation data showed that the decrease in MWHA correlated strongly with the decrease in specific productivity of the culture. To overcome this decrease in MWHA, a glucostat strategy was successfully devised which could maintain a high value of specific productivity throughout the glucostat phase and result in constant-MW HA production. Glucostat processes were designed with the three recombinant L. lactis strains at two different glucose concentrations to produce constant molecular weight HA ranging from 0.4 to 1.4 MDa. This is the first report of its kind in literature that demonstrates production of controlled MW HA over a wide range by using a combination of tunable parameters and suitable process control strategies.

Real-time monitoring of hyaluronic acid fermentation by in situ transflectance spectroscopy.

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.

Improving the accuracy of hyaluronic acid molecular weight estimation by conventional size exclusion chromatography.

There is an unreasonably high variation in the literature reports on molecular weight of hyaluronic acid (HA) estimated using conventional size exclusion chromatography (SEC). This variation is most likely due to errors in estimation. Working with commercially available HA molecular weight standards, this work examines the extent of error in molecular weight estimation due to two factors: use of non-HA based calibration and concentration of sample injected into the SEC column. We develop a multivariate regression correlation to correct for concentration effect. Our analysis showed that, SEC calibration based on non-HA standards like polyethylene oxide and pullulan led to approximately 2 and 10 times overestimation, respectively, when compared to HA-based calibration. Further, we found that injected sample concentration has an effect on molecular weight estimation. Even at 1g/l injected sample concentration, HA molecular weight standards of 0.7 and 1.64MDa showed appreciable underestimation of 11-24%. The multivariate correlation developed was found to reduce error in estimations at 1g/l to <4%. The correlation was also successfully applied to accurately estimate the molecular weight of HA produced by a recombinant Lactococcus lactis fermentation.

Design of aqueous two-phase systems for purification of hyaluronic acid produced by metabolically engineered Lactococcus lactis.

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.

Hyaluronan production and molecular weight is enhanced in pathway-engineered strains of lactate dehydrogenase-deficient Lactococcus lactis.

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.

Chromosomal integration of hyaluronic acid synthesis (has) genes enhances the molecular weight of hyaluronan produced in Lactococcus lactis.

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.

Ratio of intracellular precursors concentration and their flux influences hyaluronic acid molecular weight in Streptococcus zooepidemicus and recombinant Lactococcus lactis.

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.

Engineering Escherichia coli with acrylate pathway genes for propionic acid synthesis and its impact on mixed-acid fermentation.

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.

Transcription analysis of hyaluronan biosynthesis genes in Streptococcus zooepidemicus and metabolically engineered Lactococcus lactis.

The has operon genes in the hyaluronan (HA) producer, Streptococcus zooepidemicus, encode for some of the critical enzymes in the HA biosynthetic pathway. Heterologous expression of different combinations of multiple has genes has resulted in increasing HA production to varying degrees in different recombinant strains. In this work, a recombinant Lactococcus lactis strain (SJR6) was constructed, with insertion of three has operon genes (hasABD) from S. zooepidemicus. The SJR6 strain was found to be a better HA producer than two previously constructed recombinant L. lactis strains (SJR2 and SJR3), containing hasAB and hasABC genes, respectively, but exhibited lower HA production than the native HA producer S. zooepidemicus. To understand the differences in HA yield between the various strains, transcriptions of the HA biosynthesis genes (has genes and their homologues) were compared at different phases of exponential growth of the L. lactis and S. zooepidemicus cultures. The mRNA levels of all the heterologous has genes were expectedly far higher than their corresponding homologues in the L. lactis strains. The relative mRNA level of the hasB-homologue, viz. ugd (encoding UDP-glucose dehydrogenase), was found to be much lower than that of other homologues, corroborating earlier reports which indicate tight transcriptional regulation of the ugd gene in L. lactis. Interestingly, all the has gene homologues were found to be up-regulated in all the recombinant L. lactis strains, when compared with the corresponding genes in the untransformed strain, L. lactis NZ9000. A transcription analysis of S. zooepidemicus cultures revealed that the has operon was down-regulated in the mid-exponential growth phase in comparison to the early- and late-exponential growth phases. The transcription analyses in this study have provided insights for the design of recombinant strains with higher HA productivity.