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This study reveals the genetic and biochemical changes underlying the enhanced hyaluronan (HA) biosynthesis in Streptococcus zooepidemicus. After multiple rounds of atmospheric and room temperature plasma (ARTP) mutagenesis combined with novel bovine serum albumin/cetyltrimethylammonium bromide coupled high-throughput screening assay, the HA yield of the mutant was increased by 42.9% and reached 0.813 g L -1 with a molecular weight of 0.54 × 10 6  Da within 18 h by shaking flask culture. HA production was increased to 4.56 g L -1 by batch culture in 5-L fermenter. Transcriptome sequencing exhibits that distinct mutants have similar genetic changes. Regulation in direction of metabolic flow into the HA biosynthesis, by enhancing genes responsible for the biosynthesis of HA including hasB, glmU and glmM, weaking downstream gene (nagA and nagB) of UDP-GlcNAc and significantly down-regulating transcription of wall-synthesizing genes, resulting in the accumulation of precursors (UDP-GlcA and UDP-GlcNAc) increased by 39.74% and 119.22%, respectively. These associated regulatory genes may provide control point for engineering of the efficient HA-producing cell factory., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Objectives: Microbial production of biopolymers is typically associated with high viscosity and suitable mixing plays an important role in their production. Due to the nature of Streptococcus strains in high production of lactic acid and consequently high consumption of NaOH, which is associated with increased viscosity and reduced mixing caused by hyaluronic acid production, the injected NaOH accumulates and causes cells loss, and decreases in quantity and quality of the produced hyaluronic acid., Results: In this study, the effect of increasing dilution of media culture of Streptococcus zooepidemicus fed-batch culture during pH control by NaOH on mixing time, volumetric oxygen transfer coefficient, and increasing hyaluronic acid production in a 2-L fermenter were studied. The results showed that significant increasing dilution causes reduction mixing time, remarkable improvement volumetric oxygen transfer coefficient, hyaluronic acid production enhancement from 6.6 to 8.4 g/L, and diminution the consumption of NaOH., Conclusion: Dilution of media culture of S. zooepidemicus fed-batch culture by the pH controlling agent achieved one of the highest amounts of hyaluronic acid that was reported recently. This method does not require any automatic control and can be used at a low cost to produce other soluble extracellular biopolymers., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

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 (MW HA ) 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 MW HA (1.5 MDa). Co-supplementation of acetate resulted in enhancing the acetyl-CoA and UDP-GlcNAc levels as well as the MW HA 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 MW HA of 3.4 MDa, which was sustained at this value throughout the process. This study provides the possibility of commercially producing high MW HA using recombinant L. lactis strains.

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.

Hyaluronic acid (HA) is a biopolymer with wide biomedical and cosmetic applications, wherein the molecular weight of HA (MW HA ) 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 MW HA 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 MW HA . It was observed during batch fermentations of all these recombinant L. lactis cultures that the MW HA decreases steadily during the later part of the fermentation and the final value is 19-43% lower than the peak MW HA produced. Analysis of the fermentation data showed that the decrease in MW HA correlated strongly with the decrease in specific productivity of the culture. To overcome this decrease in MW HA , 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.

Polysaccharide peptides (or protein-bound polysaccharides, PSPs) are commonly found in mushrooms and plants and possess important nutritional properties and health benefits. The pathogenic bacterium Streptococcus zooepidemicus does not inherently produce PSPs but secretes the capsular polysaccharide hyaluronan. However, in a previous investigation of the catalytic mechanism of UDP-glucose dehydrogenase (UGDH), a PSP of peptide-bound hyaluronan was found to be produced by S. zooepidemicus through the in vivo expression of a mutant of the gene encoding UGDH. In the present study, this hyaluronan-derived PSP was structurally characterized by FT-IR, NMR, and high-performance liquid chromatography-mass spectrometry (HPLC-MS), and the data confirmed that the polysaccharide backbone, hyaluronan, is covalently bound to the side-chain peptides via an amide linkage. More importantly, the bacterial production of a PSP via this genetic modification method should inspire further research on the in vitro enzymatic synthesis of PSPs or even naturally occurring polysaccharide derivatives and may provide a theoretical foundation for investigating the in vivo synthetic mechanism of PSPs., (Copyright © 2019. Published by Elsevier Ltd.)

Hyaluronic acid (HA) is a natural biopolymer and has long been attracting the attention of biotechnology industry due to its various biological functions. HA production with natural producer Streptococcus equi subsp. zooepidemicus has not been preferred because it has many drawbacks due to its pathogenicity. Therefore, in the present study, Streptococcal hyaluronan synthase gene (hasA) was introduced and expressed in Lactococcus lactis, through the auto inducible NICE system and the effect of nisin amount on the production of HA was examined. Newly constructed plasmid was transformed into L. lactis CES15, produced 6.09 g/l HA in static flask culture after three hours of induction period with initial 7.5 ng/ml nisin concentration within total six hours of incubation. The highest HA titer value ever was reported for recombinant HA-producing L. lactis by examining the effect of initial nisin concentration. We have shown that initial nisin concentration, which used to initiate the auto-inducing mechanism of NICE system and consequently hyaluronan synthase expression, has a direct and significant effect on the produced HA amount. Recently constructed recombinant L. lactis CES15 strain provide significant advantages for industrial HA production than those in literature in terms of production time, energy demand, carbon usage, and safety status.

Objective: Streptococcus equi subsp. zooepidemicus (GCS) is mainly used to produce hyaluronic acid (HA) in the industry. GCS secretes the hemolysis toxin (streptolysin S, SLS) that causes hemolysis in the host cells. Therefore, the safety of HA produced by GCS is concerned. We constructed an engineering strain, to produce commercial HA without SLS by knocking out saga., Method: The sagA of GCS was knocked out by the thermosensitive delivery vector system pJR700. The sagA mutant was identified through PCR with primers homologous to the flanking regions and SLS analysis. The yield of HA, HA molecular weight and virulence factors such as streptolysin Hylc, hyaluronate lyase, glyceraldehyde-3-phosphate dehydrogenase and cell surface proteins were determined by spectrophotometer and SDS-PAGE., Result: We constructed successfully the in-frame deletion sagA mutant strain of GCS. In the sagA mutant, HA titer increased more than 30% than that of the wild type strain and no SLS hemolytic activity was detected. Compared to the wild type strain the sagA mutant decreased the quality of surface proteins, hemolytic Hylc activity and glyceraldehyde-3-phosphate dehydrogenase activity. The activities of hyaluronidase and cell were increased in the sagA mutant., Conclusion: The sagA not only expressed hemolysis S but also regulated production of HA, the quality of surface proteins and activities of hyaluronidase, hemolysis Hylc and glyceraldehydes-3-phosphate dehydrogenase in Streptococcus equi subsp. zooepidemicus.

Hyaluronic acid, or HA, is a rigid and linear biopolymer belonging to the class of the glycosaminoglycans, and composed of repeating units of the monosaccharides glucuronic acid and N-acetylglucosamine. HA has multiple important functions in the human body, due to its properties such as bio-compatibility, lubricity and hydrophilicity, it is widely applied in the biomedical, food, health and cosmetic fields. The growing interest in this molecule has motivated the discovery of new ways of obtaining it. Traditionally, HA has been extracted from rooster comb-like animal tissues. However, due to legislation laws HA is now being produced by bacterial fermentation using Streptococcus zooepidemicus, a natural producer of HA, despite it being a pathogenic microorganism. With the expansion of new genetic engineering technologies, the use of organisms that are non-natural producers of HA has also made it possible to obtain such a polymer. Most of the published reviews have focused on HA formulation and its effects on different body tissues, whereas very few of them describe the microbial basis of HA production. Therefore, for the first time this review has compiled the molecular and genetic bases for natural HA production in microorganisms together with the main strategies employed for heterologous production of HA.

Unlabelled: During Streptococcus zooepidemicus fermentation, most carbon sources are used to synthesize lactic acid, which can inhibit strain growth and hyaluronic acid production. Here, we expressed bacterial haemoglobin (Vhb) in Strep. zooepidemicus. Due to highly efficient oxygen use, only 15·26 g l(-1) lactic acid was produced, which is 0·73 times the quantity produced by the control strain. Compared with the control strain (1·61 g l(-1) ), hyaluronic acid (HA) production in this strain did not substantially increase, only to 2·16 g l(-1) . Next, we used a series of N-methyl-N'-nitro-N-nitroso-guanidine (NTG) treatments and selection programmes. Finally, we generated a hyaluronidase-negative and rifampin-resistant mutant strain that produces high levels of HA. The optimum carbon concentration for maximum hyaluronic acid production is only 30 g l(-1) of sucrose, which is lower than the control strain (60 g l(-1) ). The oxygen transfer rate coefficient KL a increased significantly to 372 ± 53 h(-1) from 18 ± 4 h(-1) of the control. The optimum carbon source for this strain is 21 g l(-1) of sucrose, 9 g l(-1) of maltose and 5 g l(-1) of glutamic acid. Hyaluronic acid accumulated at 6·7 g l(-1) in the culture broth. However, the molecular weight of HA decreased from 1835 KDa (Control) to 429 kDa. The prepared low-molecular weight HA could function as potential antiangiogenic substances, antiviral and antitumour agents to possibly be used as functional food ingredients., Significance and Impact of the Study: Hyaluronic acid (HA) has been used for a wide range of applications in health, cosmetic and clinical fields. During fermentation of Streptococcus to produce HA, 80-85% of the carbon source is used to produce lactic acid and acetic acid, and only approx. 5 and 10% of the carbon source is used to produce HA and biomass respectively. Here, we expressed bacteria haemoglobin (Vhb) in Streptococcus zooepidemicus, which can dramatically inhibit lactic acid production. After NTG treatments and selection programmes, we identified a mutant strain with highly efficient hyaluronic acid production (6·7 g l(-1) ) under economic fermentation conditions., (© 2016 The Society for Applied Microbiology.)

The biosynthetic pathway for hyaluronic acid (HA) has been proposed; however, a thorough genetic and functional analysis is required to further elucidate the roles of genes involved in HA production. Previously, we developed a markerless gene-deletion system for Streptococcus zooepidemicus and confirmed that hasA is essential for HA synthesis. Here, we constructed a comprehensive set of deletion mutants and investigated the roles of ten additional predicted genes in the HA synthetic pathway. Phenotypic assays revealed that all ten genes play a role in cell growth and/or HA synthesis. As expected, the deletion of hasA or hasB abolished HA production with little effect on growth, while the deletion of genes that are also required for peptidoglycan biosynthesis (hasE, glmM, and glmS) significantly reduced cell growth and HA production. Either of the glmU homologues (hasD and gcaD) was sufficient for optimal growth and the mucoid phenotype, while no double mutant could be isolated. Of the two UDP-glucose pyrophosphorylase (UGPase) paralogues, the operon-encoded hasC1 was responsible for 65 % of the activity, while hasC2 was responsible for the remaining 35 %. The deletion of hasC1 had no effect on cell growth and caused only a moderate decrease in the UDP-glucose level and HA production. The deletion of both hasC1 and hasC2 resulted in a severe growth defect and negligible UDP-glucose accumulation, HA production, and pyrophosphorylase activity. Of the two phosphoglucomutase paralogues, pgm1 and pgm2, the former is responsible for around 10 % of activity, while the latter is responsible for 90 %. The deletion of pgm1 showed no apparent effect on HA synthesis and growth, while the deletion of pgm2 resulted in the abolishment of HA synthesis and a significantly slower growth. These results should guide the metabolic engineering of S. zooepidemicus to improve HA productivity and quality.

Reasons for Performing Study: Streptococcus zooepidemicus causes opportunist respiratory and other infections in the horse. Capsule expression is highly variable and known to affect resistance to phagocytosis. Most clinical isolates producing small, dry colonies at 37°C produce mucoid colonies at temperatures below 35°C., Objectives: The aim was to understand the molecular basis of increased capsule expression by equine isolates of S. zooepidemicus at temperatures lower than 35°C., Study Design: Cross-sectional observational study., Methods: Capsule production by groups of equine S. zooepidemicus strains was determined at 23, 30, 35 and 37°C. Hyaluronidase (HylC) at 23 and 37°C was measured by quantitative enzyme-linked immunosorbent assay. Expressions of hasA and hylC at 23 and 37°C were measured by quantitative reverse transcriptase-PCR. The covRS genes in representative S. zooepidemicus were sequenced and checked for mutations., Results: Colonies of randomly selected S. zooepidemicus strains became mucoid or showed marked increase in colony mucoidy following the change in temperature to 23°C. Expression of hasA at 23°C was 45- to 700-fold greater than at 37°C. Transcription of hylC at 23°C was 2.5- to 200-fold greater than at 37°C, yet enzyme concentrations in cultures were significantly higher at 37°C (P<0.05), suggesting that production of HylC is regulated post transcriptionally. The covRS gene in S. zooepidemicus was not mutated as seen in isolates of Streptococcus pyogenes with increased capsule production at 25°C., Conclusions: Sensitivity of capsule expression to temperature above 35°C but not HylC by the general population of equine S. zooepidemicus indicates that capsule is not required for extended colonisation nor for opportunist invasion. Instead, capsule production at lower than body temperature may reflect adaptation to life on skin and mucosal surfaces, where hyaluronic acid contributes to adhesion and resistance to desiccation. Pathogenicity of S. zooepidemicus following opportunist invasion is possibly dependent on factors other than capsule that may be co-regulated with HylC. The Summary is available in Chinese - see Supporting information., (© 2014 EVJ Ltd.)

Hyaluronic acid (HA), a natural high molecular weight polysaccharide, is produced by Streptococcus zooepidemicus. However, Streptococcus has several drawbacks including its potential to produce exotoxins, so there is demand for an alternative HA source. Here, a recombinant HA biosynthesis operon, as well as the HA biosynthesis operon of S. zooepidemicus were introduced into L. lactis using the nisin-controlled expression system, respectively. HA was successfully synthesized by recombinant L. lactis. Furthermore, overexpression of the endogenous enzymes directing the synthesis of precursor sugars was effective at increasing HA production, and increasing the supply of UDP-activated monosaccharide donors aided synthesis of monodisperse HA polysaccharides. Besides GRAS host strain (L. lactis) and NICE system, the selecting marker (lacF gene) of the recombinant strain is also food grade. Therefore, HA produced by recombinant L. lactis overcomes the problems associated with Streptococcus and provides a source of food-grading HA appropriate for widespread biotechnological applications.

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., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aims: Corynebacterium glutamicum was tested as an alternative host for heterologous production of hyaluronic acid (HA)., Methods and Results: A set of expression vectors containing hasA, encoding HA synthase from Streptococcus equi subsp. zooepidemicus, alone or in combination with genes encoding enzymes for HA precursor production (hasB, hasC, glmU from Pseudomonas putida KT2440) or bacterial haemoglobin (vgb from Vitreoscilla sp.) was constructed. Recombinant Coryne. glutamicum strains were cultivated in two different minimal media, CGXII and MEK700. HA was isolated from the culture broth by ethanol precipitation or ultrafiltration. Analyses of the isolated HA revealed that overall production was higher in CGXII medium (1241 mg l(-1)) than in MEK700 medium (363 mg l(-1)), but molecular weight of the product was higher in MEK700 (>1·4 MDa) than in CGXII (<270 kDa). Coexpression of hasB, hasC or glmU had no effect on HA yield and did not improve molecular weight of the product. Coexpression of vgb lowered HA yield about 1·5-fold and did not affect molecular weight of the product. Microscopy of negative-stained cultures revealed that Coryne. glutamicum produces no distinct HA capsule., Conclusions: Regulation of cell growth and gene expression level of hasA are reasonable starting points for controlling the molecular weight of HA produced by Coryne. glutamicum., Significance and Impact of the Study: Corynebacterium glutamicum has a great potential as an alternative production host for HA. The fact that Coryne. glutamicum produces no distinct HA capsule facilitates HA isolation and improves overall yield., (© 2014 The Society for Applied Microbiology.)

This study addresses the influence of upstream region sequence on the strength of has operon promoter in highly encapsulated S. equi subsp. zooepidemicus (SEZ). For this purpose, seven different strains were constructed. Each strain carries a point mutation in one of the following positions upstream of the has promoter: -43, -44, -49, and -50 bp. To facilitate measuring of the recombinant promoter relative strength, ß-glucuronidase gene was used as a reporter gene. Three mutations located in positions -49 and -50: AT, GT, and AG, positively impacted has promoter strength when compared to the wild type sequence GG. Conversely, two other mutations: TG and TT, exhibited a slight inhibitory effect. Further, three different strains carrying chromosomal mutations in the has promoter region were constructed. In two cases, the has operon is under the control of a stronger promoter and in the third strain the has operon is controlled by a weaker promoter. The laboratory fermenter scale cultivations confirmed the increase of hyaluronan yields for SEZPhasAG and SEZPhas2G, resulting 116 and 105 %, respectively. As expected, the yield of the hyaluronic acid of SEZPhas2B strain fell to 41 %.

Aim: Selection of high-mucoid morphotype of Streptococcus equi subsp. zooepidemicus (Streptococcus zooepidemicus) and study of its morphological, physiological, biochemical and technological characteristics for providing increased secretion of hyaluronic acid (HA)., Materials and Methods: Submerged cultivation was performed in 100 ml glass flasks without baffles or in 1.5 or 10 1 laboratory bioreactors. LB and MRS media were used for cultivation. Mutagenesis was carried out by UV exposure with consequent selection of mucoid phenotype. HA was determined by carbazole method or after exhaustive acid hydrolysis by reaction of N-acetylglucosamine with Morgan-Elson reagent. Total hyaluronidase activity was evaluated by viscosimeter. Determination of cell and capsule size, ability to ferment carbohydrates and other microbiological, physiological and biochemical tests were performed by standard techniques., Results: Instability of capsule phenotype of S. zooepidemicus B-8014 strain was revealed that is explained most probably by formation under certain conditions of bacterial hyaluronidase. This is confirmed by a reduction of HA concentration in cultural medium at pre- and stationary growth phases. Mucoid strain S. zooepidemicus KB-04 was obtained by mutagenesis with subsequent selection that is characterized by increased capsules. The strain was studied for HA formation. Optimization of growth medium composition, physical-chemical conditions and modes of cultivation allowed to significantly increase HA yield., Conclusion: The studies of morphologic, physiologic, biochemical and technological characteristics of the high-mucoid S. zooepidemicus KB-04 strain obtained by mutagenesis with consequent selection were performed, conditions of its cultivation and composition of growth mediu by carbon source and content of bivalent metal ions were optimized.

Streptococcus equisimilis hyaluronan (HA) synthase (SeHAS) contains four cysteines (C226, C262, C281 and C367) that are conserved in the mammalian HAS family. Previous studies of single Cys-to-Ser and all possible Cys-to-Ala mutants of SeHAS found that: the Cys-null mutant is active, Cys modification inhibits HAS activity and the conserved cysteines are clustered at the membrane-enzyme interface in substrate-binding sites (Kumari K, Weigel PH. 2005. Identification of a membrane-localized cysteine cluster near the substrate binding sites of the Streptococcus equisimilis hyaluronan synthase. Glycobiology. 15:529-539). We re-examined these Cys mutants using a single technique (size exclusion chromatography-multi-angle laser light scattering) that allows simultaneous assays on the same sample for both HA synthesis activity and HA product size. Among 18 mutants compared with wild type, 4 showed no change in either function and 3 showed changes in both (decreased activity and HA size). Only one of the two functions was altered in 11 other mutants, which showed either decreased polymerizing activity or product size. No mutants made larger HA, 8 made smaller HA and 10 showed no change in HA size. Nine mutants showed no change in activity and nine were less active. The mutants fell into four of nine possible groups in terms of changes in HA size or synthesis rate (i.e. none, increased or decreased). Specific Cys residues were associated with each mutant group and the pattern of effects on both functions. Thus, the four conserved Cys residues, individually and in specific combinations, influence the rate of sugar assembly by HAS and HA product size, but their participation in one function is independent of the other.

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.

Hyaluronic acid (HA) is a natural and linear polymer composed of repeating disaccharide units of β-1, 3-N-acetyl glucosamine and β-1, 4-glucuronic acid with a molecular weight up to 6 million Daltons. With excellent viscoelasticity, high moisture retention capacity, and high biocompatibility, HA finds a wide-range of applications in medicine, cosmetics, and nutraceuticals.Traditionally HA was extracted from rooster combs, and now it is mainly produced via streptococcal fermentation. Recently the production of HA via recombinant systems has received increasing interest due to the avoidance of potential toxins. This work summarizes the research history and current commercial market of HA, and then deeply analyzes the current state of microbial production of HA by Streptococcus zooepidemicus and recombinant systems, and finally discusses the challenges facing microbial HA production and proposes several research outlines to meet the challenges.

UDP-Glucose Pyrophosphorylase (EC 2.7.7.9, UGPase) plays an important role in Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) cell envelope Hyaluronic acid (HA) biosynthesis and it is also recognized as a virulence determinant in several bacterial species. HA is valuable biopolymer used in the pharmaceutical and cosmetic industry. In addition, encapsulation by HA is considered an important virulence factor in other streptococci. Research UGPase will contribute to the vaccine development of S. zooepidemicus and the production of HA. In this study, The UGPase gene fragment (789 bp) obtained from previous research was amplified using PCR, and located by Genome walking technology (Genebank No.GQ423507). The UGPase was expressed, purified and identified using UGPase antibody. The enzyme kinetic parameters were determined, the temperature and pH of the highest activity for the cloned UGPase were 37°C, pH 7.5. The Km and Kcat value against UTP and G-1-P was 8.5 μM, 69.05 s(-1) and 36.41 μM, 48.81 s(-1), respectively. The homology-modeling was operated. Overexpression of the UGPase in S. zooepidemicus, its virulence was slightly affected, and HA yield reduced. Real-time PCR was carried out to determine the UGPase expression levels of both SEZp and SEZugp in different grow period, the level is high in logarithmic phase and low in Decline phase.

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.

Hyaluronic acid is a biopolymer with valuable applications in the pharmaceutical and cosmetic industries. Streptococcus equi subspecies zooepidemicus cells transformed with a nisin-inducible, empty plasmid control displayed higher molecular weight. This increase in molecular weight is independent of the nisin promoter or antibiotic resistance. Using 2D DIGE followed by mass spectrometry, we identified up-regulation of the last step in UDP-N-acetyl-glucosamine biosynthesis (GlmU) and down-regulation of the first step in peptidoglycan biosynthesis (MurA) as possible mechanism for the plasmid effect. Over-expression of GlmU to further increase activity had no effect on UDP-N-acetyl-glucosamine levels or molecular weight, while over-expression of MurA reduced UDP-N-acetyl-glucosamine levels and molecular weight. Global transcriptional analysis revealed that differential regulation of GlmU and MurA activity was not reflected in transcription levels. This results, suggest that regulation is at a translational or post-translational level. Differential expression of two clp proteases may explain this effect as well as the small but significant changes in transcription levels of nearly 300 genes.

The molecular weight of hyaluronan is important for its rheological and biological function. The molecular mechanisms underlying chain termination and hence molecular weight control remain poorly understood, not only for hyaluronan synthases but also for other beta-polysaccharide synthases, e.g. cellulose, chitin, and 1,3-betaglucan synthases. In this work, we manipulated metabolite concentrations in the hyaluronan pathway by overexpressing the five genes of the hyaluronan synthesis operon in Streptococcus equi subsp. zooepidemicus. Overexpression of genes involved in UDP-glucuronic acid biosynthesis decreased molecular weight, whereas overexpression of genes involved in UDP-N-acetylglucosamine biosynthesis increased molecular weight. The highest molecular mass observed was at 3.4 +/- 0.1 MDa twice that observed in the wild-type strain, 1.8 +/- 0.1 MDa. The data indicate that (a) high molecular weight is achieved when an appropriate balance of UDP-N-acetylglucosamine and UDP-glucuronic acid is achieved, (b) UDP-N-acetylglucosamine exerts the dominant effect on molecular weight, and (c) the wild-type strain has suboptimal levels of UDP-N-acetylglucosamine. Consistent herewith molecular weight correlated strongly (rho = 0.84, p = 3 x 10(-5)) with the concentration of UDP-N-acetylglucosamine. Data presented in this paper represent the first model for hyaluronan molecular weight control based on the concentration of activated sugar precursors. These results can be used to engineer strains producing high molecular weight hyaluronan and may provide insight into similar polymerization mechanisms in other polysaccharides.

Aims: To determine the effects of the ratios of hyaluronan synthase expression level to precursor sugar UDP-GlcA biosynthesis ability on the molecular weight (MW) of hyaluronic acid (HA) in recombinant Lactococcus lactis., Methods and Results: The genes szHasA (hyaluronan synthase gene) and szHasB (UDP-glucose-6-dehydrogenase gene) of Streptococcus zooepidemicus were introduced into L. lactis under the control of nisA promoter and lacA promoter respectively, resulting in a dual-plasmid controlled expression system. The effects of the ratios of hyaluronan synthase expression level to the precursor sugar UDP-GlcA biosynthesis ability under different induction concentration collocations with nisin and lactose on the MW of HA in recombinant L. lactis were determined. The results showed that the final weight-average molecular weight () of HA correlated with the relative ratios of HasA (hyaluronan synthase) expression level to the concentration of UDP-GlcA., Conclusions: Regulating the relative ratios of HasA expression level to the precursor sugar biosynthesis ability was an efficient method to control the size of HA., Significance and Impact of the Study: This study put forward a guide to establish an efficacious way to control the size of HA in fermentation.

Dissolved oxygen (DO) has a significant effect on the molecular weight of hyaluronic acid (HA) during the fermentation of Streptococcus zooepidemicus. Therefore, to further investigate the effect of DO on the yield and molecular weight of HA, this study compared the metabolic flux distribution of S. zooepidemicus under aerobic conditions at various DO levels. The metabolic flux analysis demonstrated that the HA synthesis pathway, considered a dependent network, was little affected by the DO level. In contrast, the fluxes of lactate and acetate were greatly influenced, and more ATP was generated concomitant with acetate at a high DO level. Furthermore, the has gene expression and HA synthase activity were both repressed under anaerobic conditions, yet not obviously affected under aerobic conditions at various DO levels. Therefore, it was concluded that the HA molecular weight would seem to depend on the concomitant effect of the generation of ATP and reactive oxygen species. It is expected that this work will contribute to a better understanding of the effect of the DO level on the mechanism of the elongation of HA chains.

Streptococcus equi causes equine 'strangles'. Hyaluronate lyases, which degrade connective tissue hyaluronan and chondroitins, are thought to facilitate streptococcal invasion of the host. However, prophage-encoded hyaluronate lyases are hyaluronan-specific and are thought to be primarily involved in the degradation of the hyaluronan capsule of streptococci during bacteriophage infection. To understand the role of prophage-encoded hyaluronate lyases further, we have biochemically characterized such a hyaluronate lyase, SEQ2045 from S. equi, and have shown that it is produced during equine infection. Prophage-encoded hyaluronan-specific hyaluronate lyases may therefore play a more direct role in disease pathogenesis than previously thought.

Hyaluronic acid (HA) is a ubiquitous linear polysaccharide in vertebrates and also is the capsule material of some pathogenic bacteria including group A and C streptococci. In bacteria, the HA synthase occurs in an operon (has) coding for enzymes involved in the production of HA precursors. We report two new members of the has operon family from Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) and Streptococcus equi subsp. equi (S. equi). The has operon of S. zooepidemicus contains, in order, hasA, hasB, hasC, glum, and pgi, whereas these genes are separated on two operons in S. equi (hasA, hasB, hasC and hasC, glmU, pgi). The transcription start site and a sigma(70) promoter were experimentally identified 50 bp upstream of hasA in S. zooepidemicus. We performed a phylogenetic analysis of each of the has operon genes to determine the evolutionary origin(s) of the streptococcal has operon. In contrast to other capsular and exopolysaccharide operons, has operons have undergone no detectable interspecies lateral gene transfers in their construction, instead relying on intragenome gene duplication for their assembly. Specifically, hasC and glmU appear to have been duplicated into the S. zooepidemicus has operon from remotely located but near-identical paralogues most likely to improve HA productivity by gene dosage in this streptococcus. The intragene rearrangements appear to be ongoing events and the two has operons of the S. equi subspecies represent two alternatives of the same gene arrangement. A scenario for the evolution of streptococcal has operons is proposed.

Hyaluronic acid (HA) has been industrially produced using the gram-positive bacterium Streptococcus zooepidemicus. Large amount of lactic acid formation was one of the important factors that restricted cell growth and HA productivity and lowered the substrate to HA conversion efficiency in a fermentor. In this study, polyhydroxybutyrate (PHB) synthesis genes (phbCAB) of Ralstonia eutropha were cloned from the plasmid pBHR68 and were inserted into the plasmid pEU308, an expression vector for gram-positive bacteria. The plasmid was transformed into S. zooepidemicus by electroporation. beta-Ketothiolase (PhbA), acetoacetyl-CoA reductase (PhbB), and polyhydroxyalkanoate (PHA) synthase (PhbC) activity assays were carried out to demonstrate the expression of these genes. The PhbA and PhbB activities were 3.13 and 1.23 U mg(-1), respectively. No PhbC activities were detected. In shake flask studies, there was no obvious difference between the wild-type and recombinant S. zooepidemicus harboring phbCAB genes in terms of lactic acid and HA formation. However, in fermentor studies, the recombinant produced only 40 g L(-1) lactic acid and 7.5 g L(-1) HA, whereas the wild type produced 65 g L(-1) lactic acid and 5.5 g L(-1) HA. These results suggested that expression of phbCAB genes in S. zooepidemicus could help regulate HA production metabolism. Because the lactic acid formation in S. zooepidemicus was sensitive to cellular oxidation/reduction potential, it is proposed that the PHB synthesis pathway could act as a regulator to adjust the cellular oxidation/reduction potential. This is the first study demonstrating that PHA synthesis related to energy and carbon metabolism could be employed as a pathway to regulate other cellular metabolism and possibly to regulate the production of other metabolic products.

Hyaluronic 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.

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.