PHA synthase engineering toward superbiocatalysts for custom-made biopolymers

Poly-3-hydroxyalkanoates [P(3HA)s] are biologically produced polyesters that have attracted much attention as biodegradable polymers that can be produced from biorenewable resources. These polymers have many attractive properties for use as bulk commodity plastics, fishing lines, and medical uses that are dependent on the repeating unit structures. Despite the readily apparent benefits of using P(3HA)s as replacements for petrochemical-derived plastics, the use and distribution of P(3HA)s have been limited by their cost of production. This problem is currently being addressed by the engineering of enzymes involved in the production of P(3HA)s. Polyhydroxyalkanoate (PHA) synthase (PhaC) enzymes, which catalyze the polymerization of 3-hydroxyacyl-CoA monomers to P(3HA)s, were subjected to various forms of protein engineering to improve the enzyme activity or substrate specificity. This review covers the recent history of PHA synthase engineering and also summarizes studies that have utilized engineered PHA synthases.     

The Role of Hyaluronic Acid Precursor Concentrations in Molecular Weight Control in Streptococcus zooepidemicus

The biosynthetic pathway responsible for the production of hyaluronic acid (HA) has been thoroughly studied; however, many aspects remain elusive regarding the mechanisms that control molecular weight (MW). Previously, we demonstrated a positive correlation between MW and the concentration of the HA precursor sugar UDP-N acetylglucosamine (UDP-GlcNAc). To further investigate the role of UDP-GlcNAc in MW control, we increased the intracellular concentration of this monomer using both feeding strategies and genetic engineering approaches. Feeding cells glucosamine dramatically increased intracellular levels of UDP-GlcNAc, but unexpectedly, produced HA of a lower MW. This was subsequently attributed to an equally dramatic decrease in the level of the other HA precursor sugar UDP-glucuronic acid (UDP-GlcUA). Feeding cells a mixture of glucose and GlcNAc addressed this imbalance of precursor sugars, leading to an increase in both UDP-GlcNAc and UDP-GlcUA; however, no significant increase in MW was observed. Despite the increase in UDP-sugars, RNA sequencing identified no increase in the expression of the genes involved in production of HA. Returning to genetic engineering approaches to examine UDP-GlcNAc and MW, genes known to contribute to the production of UDP-GlcNAc were over-expressed, both individually and together. Using this approach, UDP-GlcNAc and MW increased. At lower levels of UDP-GlcNAc, the positive correlation between UDP-GlcNAc levels and MW was maintained, however this relationship stalled at higher concentrations of UDP-GlcNAc. Taken together, these results suggest that while optimising HA precursor levels using feeding or genetic engineering approaches can improve HA MW, there is a point at which excess availability of precursors is no longer advantageous. Once precursor concentrations are addressed, it would seem that other uncharacterised factor(s) (e.g. rate of HA synthesis) also contribute to HA MW control.     

Heparin interacting protein mediated assembly of nano-fibrous hydrogel scaffolds for guided stem cell differentiation

A new methodology is developed to conjugate hyaluronic acid (HA) hydrogel with novel nano-fibrous architectures via non-covalent assembly that specifically allows for targeted adipose-derived stem cells (ASCs) differentiation and soft tissue engineering. The assembly of non-covalently associated hydrogel network produced via the interaction of a low molecular weight heparin (LMWH) modified HA derivative and heparin interacting protein (HIP). The multifunctional star poly(ethylene glycol) (PEG) and HIP copolymer has the capability to mediate the non-covalent assembly of nano-fibrous HA hydrogel networks via affinity interactions with LMWH. The effect of the HIP mediation on in vitro gelation, rheological characteristics, degradation, equilibrium swelling, adipose-derived stem cells (ASCs) proliferation and differentiation of nano-fibrous hydrogel is examined. The results suggest the potential utility of this unique design of the bioactive nano-fibrous HA hydrogel in directing the differentiation of ASCs and adipogenesis in ECM-mimetic scaffolds in vitro. These studies demonstrate that this nano-fibrous HA hydrogel can render the formulation of a therapeutically effective platform for in vitro adipogenesis applications.     

A Virus-Like Particle That Elicits Cross-Reactive Antibodies to the Conserved Stem of Influenza Virus Hemagglutinin

The discovery of broadly neutralizing antibodies that recognize highly conserved epitopes in the membrane-proximal region of influenza virus hemagglutinin (HA) has revitalized efforts to develop a universal influenza virus vaccine. This effort will likely require novel immunogens that contain these epitopes but lack the variable and immunodominant epitopes located in the globular head of HA. As a first step toward developing such an immunogen, we investigated whether the 20-residue A-helix of the HA2 chain that forms the major component of the epitope of broadly neutralizing antibodies CR6261, F10, and others is sufficient by itself to elicit antibodies with similarly broad antiviral activity. Here, we report the multivalent display of the A-helix on icosahedral virus-like particles (VLPs) derived from the capsid of Flock House virus. Mice immunized with VLPs displaying 180 copies/particle of the A-helix produced antibodies that recognized trimeric HA and the elicited antibodies had binding characteristics similar to those of CR6261 and F10: they recognized multiple HA subtypes from group 1 but not from group 2. However, the anti-A-helix antibodies did not neutralize influenza virus. These results indicate that further engineering of the transplanted peptide is required and that display of additional regions of the epitope may be necessary to achieve protection.     

Alteration of polysaccharide size distribution of a vertebrate hyaluronan synthase by mutation

Hyaluronan (HA) is a nonsulfated glycosaminoglycan that has long been known to play structural roles in vertebrates. Recently, it has become increasingly obvious that this linear polysaccharide has many more uses than simply scaffolding or space filler. HA has been found to be involved in development, cell signaling, cell motility, and metastasis. These roles are often dictated by the length of the HA polymer, which can vary from a few to about 10,000 sugar residues in length. Three distinct isoforms of HA synthase exist in mammals. It has been shown previously by others that each isoform produces HA that differs in size distribution, but the regulatory mechanism is not yet known. Mutations have been described that alter the size distribution of the HA produced by the streptococcal HA synthases. We show that by mutating one particular amino acid residue of a vertebrate HA synthase, depending on the introduced side chain, the size of HA produced can be either reduced or increased. We postulate that several cysteine residues and a serine residue may be involved in binding directly or indirectly to the nascent HA chain. These data support the theory that the relative strength of the interaction between the catalyst and the polymer may be a major factor in HA size control.     

A recombinant <Emphasis Type="Italic">E. coli</Emphasis> bioprocess for hyaluronan synthesis

An Escherichia coli strain, JM109, was successfully engineered into an efficient hyaluronic acid (HA) producer by co-expressing the only known class-II HA synthase from a Gram-negative bacterium (Pasteurella multocida) and uridine diphosphate-glucose dehydrogenase from E. coli K5 strain. The engineered strain produced about 0.5 g/L HA in shake flask culture and about 2.0–3.8 g/L in a fed-batch fermentation process in a 1-L bioreactor. The sharp increase in viscosity associated with HA accumulation necessitated pure oxygen supplement to maintain fermentation in aerobic regime. Precursor supply during HA synthesis was probed by glucosamine supplement, which shortens biosynthesis pathway and eliminates one step requiring ATP. HA synthesis was increased with glucosamine supplement from 2.7 to 3.7 g/L (37%), which was mirrored with a concomitant 42% decrease in pure oxygen input, suggesting a close connection between energy metabolism and precursor supply. Decoupling HA synthesis from cell growth by using fosfomycin (an inhibitor for cell wall synthesis) led to a 70% increase in HA synthesis, suggesting detrimental effects on HA synthesis from cell growth via precursor competition. This study demonstrates a potentially viable process for HA based on a recombinant E. coli strain. In addition, the precursor supply limitation identified in this study suggests new engineering targets in subsequent metabolic engineering efforts.     

链球菌生物合成透明质酸的分子机理与基因工程菌构建进展

透明质酸（Hyaluronan, HA）是由葡萄糖醛酸和N-乙酰氨基葡萄糖为双糖单位交替连接而成的粘多糖物质。目前构建基因工程菌成为提高产量和改善品质的重要手段。本文从发酵菌种、操纵子、关键酶和工程菌构建等方面,综述了链球菌HA生物合成的分子机理,分析了当前生产中存在的问题,并提出了解决问题的方法。     

Genetic strategies for improving hyaluronic acid production in recombinant bacterial culture

Hyaluronic acid (HA) is a biopolymer of repeating units of glucuronic acid and N-acetylglucosamine. Its market was valued at USD 8.9 billion in 2019. Traditionally, HA has been obtained from rooster comb-like animal tissues and fermentative cultures of attenuated pathogenic streptococci. Various attempts have been made to engineer a safe micro-organism for HA synthesis; however, the HA titres obtained from these attempts are in general still lower than those achieved by natural, pathogenic producers. In this scenario, ways to increase HA molecule length and titres in already constructed strains are gaining attention in the last years, but no recent publication has reviewed the main genetic strategies applied to improve HA production on heterologous hosts. In light of that, we hereby compile the advances made in the engineering of micro-organisms to improve HA synthesis.     

Fabrication of porous polycaprolactone/hydroxyapatite (PCL/HA) blend scaffolds using a 3D plotting system for bone tissue engineering

For tissue engineering and regeneration, a porous scaffold with interconnected networks is needed to guide cell attachment and growth/ingrowth in three-dimensional (3D) structure. Using a rapid prototyping (RP) technique, we designed and fabricated 3D plotting system and three types of scaffolds: those from polycaprolactone (PCL), those from PCL and hydroxyapatite (HA), and those from PCL/HA and with a shifted pattern structure (PCL/HA/SP scaffold). Shifted pattern structure was fabricated to increase the cell attachment/adhesion. The PCL/HA/SP scaffold had a lower compressive modulus than PCL and PCL/HA scaffold. However, it has a better cell attachment than the scaffolds without a shifted pattern. MTT assay and alkaline phosphatase activity results for the PCL/HA/SP scaffolds were significantly enhanced compared to the results for the PCL and PCL/HA scaffolds. According to their degree of cell proliferation/differentiation, the scaffolds were in the following order: PCL/HA/SP > PCL/HA > PCL. These 3D scaffolds will be applicable for tissue engineering based on unique plotting system.     

透明质酸在间充质干细胞向软骨细胞分化中的应用

随着组织工程学的发展,利用间充质干细胞(mesenchymal stem cells,MSCs)定向分化为软骨细胞,用于治疗骨性关节炎、关节创伤等因素造成的软骨缺损的研究方兴未艾。透明质酸(hyaluronic acid,HA) 是一种酸性多糖类生物大分子,亦是软骨基质的主要成分之一。由于其优良的生物相容性、可降解等特性,HA已成为优良的天然生物材料,其作为支架材料应用于软骨缺损修复已有一段历史。近年来又发现,HA除作为载体支架材料外,还可作为调节因子应用于MSCs向软骨细胞分化。以下将对近年来利用HA应用于MSCs向软骨细胞分化的研究进行总结,旨在为以MSCs为基础的组织工程化软骨的临床应用奠定基础。     

Reductive alkylation of hyaluronic acid for the synthesis of biocompatible hydrogels by click chemistry

Hyaluronan (HA) based hydrogels have been synthesized combining chemical modification of the polysaccharide by partial oxidation, reductive amination and 'click chemistry'. HA was oxidized by 4-acetamido-TEMPO-mediated reaction, using sodium hypochlorite as primary oxidant and NaBr in buffered pH, so that the produced aldehyde moieties (hemiacetals) were trapped in situ by adding primary amines containing azide or alkyne-terminal groups. The structure of the reaction products, oxidized-HA and primary amines bonded to HA, was elucidated using 2D NMR spectroscopy. SEC-MALLS analysis of the modified substrates showed a negligible degradation of the polysaccharide using this procedure. Furthermore, azido- and alkynyl derivatives underwent cross-linking by click chemistry into hydrogels, which were characterized by NMR, FT-IR, swelling degree and mechanical properties. Possible application of the material as scaffold for tissue engineering was tested by seeding and proliferation of chondrocytes for up to 15 days.     

Diels-Alder Click cross-linked hyaluronic acid hydrogels for tissue engineering

Hyaluronic acid (HA) is a naturally occurring polymer that holds considerable promise for tissue engineering applications. Current cross-linking chemistries often require a coupling agent, catalyst, or photoinitiator, which may be cytotoxic, or involve a multistep synthesis of functionalized-HA, increasing the complexity of the system. With the goal of designing a simpler one-step, aqueous-based cross-linking system, we synthesized HA hydrogels via Diels-Alder "click" chemistry. Furan-modified HA derivatives were synthesized and cross-linked via dimaleimide poly(ethylene glycol). By controlling the furan to maleimide molar ratio, both the mechanical and degradation properties of the resulting Diels-Alder cross-linked hydrogels can be tuned. Rheological and degradation studies demonstrate that the Diels-Alder click reaction is a suitable cross-linking method for HA. These HA cross-linked hydrogels were shown to be cytocompatible and may represent a promising material for soft tissue engineering.     

Novel hyaluronic acid–methotrexate conjugates for osteoarthritis treatment

Hyaluronic acid (HA) provides synovial fluid viscoelasticity and has a lubricating effect. Injections of HA preparations into the knee joint are widely used as osteoarthritis therapy. The current HA products reduce pain but do not fully control inflammation. Oral methotrexate (MTX) has anti-inflammatory efficacy but is associated with severe adverse events. Based on the rationale that a conjugation of HA and MTX would combine the efficacy of the two clinically evaluated agents and avoid the risks of MTX alone, we designed HA–MTX conjugates in which the MTX connects with the HA through peptides susceptible to cleavage by lysosomal enzymes. Intra-articular injection of our HA–MTX conjugate (conjugate 4) produced a significant reduction of the knee swelling in antigen-induced arthritis rat, whereas free MTX, HA or a mixture of HA and MTX showed no or marginal effects on the model. The efficacy of conjugate 4 was almost the same as that of MTX oral treatment. Conjugate 4 has potential as a compound for the treatment of osteoarthritis.     

Are hyaluronan receptors involved in three-dimensional cell migration?

Hyaluronan (HA), an unbranched polysaccharide consisting of repeated glucuronic acid/N-acetylglucosamine disaccharide units, is ubiquitously present in the extracellular matrix of many tissues (for a more comprehensive review see: Fraser et al., 1997). Increased amounts of hyaluronan are produced by solid tumors and tumor-associated fibroblasts, and tumor-induced HA is correlated with poor prognosis. HA is well known to stimulate the migration of a large variety of cell types. Stimulation of cell migration by HA has been explained by different mechanisms. HA was shown to specifically bind to cell surface receptors, and inhibition of HA-receptor function was demonstrated to decrease cell migration and tumor growth. On the other hand, HA as a large hydrophilic molecule is also known to modulate the extracellular packing of collagen and fibrin, leading to increased fiber size and porosity of extracellular substrates. Hence a modified matrix architecture might similarly account for increased locomotion of cells. In this review, we attempted to summarize the available data on HA-induced cell migration, with particular emphasis on the role of HA receptors in three-dimensional cell migration. Although the HA receptor CD44 has been shown to mediate migration of cells over two-dimensional hyaluronan-coated surfaces in vitro, there is only little evidence that HA-binding to CD44 or other HA receptors has major impact on the locomotion of cells through three-dimensional matrices in vivo. We showed recently that the promigratory effect of HA in fibrin gels is largely due to HA-mediated modulation of fibrin polymerization. By increasing the porosity of fibrin gels, HA strongly accelerates cell migration. The porosity of matrices therefore appears as an important and probably underestimated determinant of cell migration and tumor spread.     

Higher titer hyaluronic acid production in recombinant Lactococcus lactis

Abstract

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.     

Beneficial actions of hyaluronan (HA) on arthritic joints: effects of molecular weight of HA on elasticity of cartilage matrix

Hyaluronan (HA) has viscoelastic, anti-inflammatory and protective actions in joint tissues, and is being widely used for treatment of OA and RA patients. However, the mechanisms underlying the pharmacological action of HA on OA and RA have not been fully understood. In this article, we review the molecular weight-dependent, anti-inflammatory actions of HA preparations - produced in Japan - in joint tissues, and show that the molecular weight of HA, but not its concentration, is crucial for maintenance of cartilage elasticity.     

Insight into hyaluronic acid molecular weight control

Hyaluronic acid (HA) is a ubiquitous polysaccharide found in humans, animals, bacteria, algae and molluscs. Simple yet sophisticated, HA demonstrates unique and valuable rheological properties. In solution, HA behaves as a stiffened random coil and the resultant behaviour, even at low concentrations, is far from Newtonian or ‘ideal’. These rheological properties are heavily influenced by molecular weight (MW), so it is not surprising that many of the biological functions of HA are dependent on molecular size. The current billion dollar market for HA continues to grow rapidly, both in gross production and the number of applications for its use. Increasing demand, in conjunction with a reticence to use animal-derived HA, has revitalised the market for HA produced by bacterial fermentation. Although the genes and pathways involved in bacterial production of HA are well characterised, the mechanisms that underlie HA MW control are less well understood. By performing a thorough analysis of the proposed mechanisms of MW control in bacterial fermentation, this mini-review tries to elucidate the challenges and future directions for bacterial HA biosynthesis.     

透明质酸的生物合成及其代谢工程的研究进展

透明质酸(hyaluronic acid,HA)是广泛存在于生物体内的功能性糖胺高分子聚合物,在日化、医疗和食品领域应用前景广阔.随着基因工程与代谢工程等合成生物学技术的发展,人们对HA的生物合成过程和机理解析越发深入的同时,也伴随一些新的挑战来临.该综述从分子生物学角度总结了HA的关键合成酶基因及合成途径,对不同来源...     

The distribution of renal hyaluronan and the expression of hyaluronan synthases during water deprivation in the Spinifex hopping mouse, Notomys alexis.

Hyaluronan (HA) is a glycosaminoglycan that is synthesized by a family of enzymes called hyaluronan synthases (HASs), of which there are three isoforms (HAS1, 2 and 3) in mammals. The HASs have different tissue expression patterns and function, indicating that synthesis of HA and formation of the HA matrix may be regulated by various factors. The HA matrix has an important role in renal water handling and the production of a concentrated urine. We investigated the distribution of HA and the expression of HAS1, HAS2 and HAS3 mRNAs in the kidney of the Spinifex hopping mouse, Notomys alexis, a native Australian desert rodent that is reported to produce the most concentrated urine of any mammal. After periods of three, seven and fourteen days of water deprivation, the distribution of renal HA changed considerably, and there was a general down-regulation of HAS mRNA expression. It is proposed that the regulation of HA synthesis by the different HAS isoforms during water deprivation in N. alexis, could be influenced by the molecular mass of the HA chains produced by each isoform, followed by the rate at which the individual HAS produces HA.     

The distribution of renal hyaluronan and the expression of hyaluronan synthases during water deprivation in the Spinifex hopping mouse, Notomys alexis

Hyaluronan (HA) is a glycosaminoglycan that is synthesized by a family of enzymes called hyaluronan synthases (HASs), of which there are three isoforms (HAS1, 2 and 3) in mammals. The HASs have different tissue expression patterns and function, indicating that synthesis of HA and formation of the HA matrix may be regulated by various factors. The HA matrix has an important role in renal water handling and the production of a concentrated urine. We investigated the distribution of HA and the expression of HAS1, HAS2 and HAS3 mRNAs in the kidney of the Spinifex hopping mouse, Notomys alexis, a native Australian desert rodent that is reported to produce the most concentrated urine of any mammal. After periods of three, seven and fourteen days of water deprivation, the distribution of renal HA changed considerably, and there was a general down-regulation of HAS mRNA expression. It is proposed that the regulation of HA synthesis by the different HAS isoforms during water deprivation in N. alexis, could be influenced by the molecular mass of the HA chains produced by each isoform, followed by the rate at which the individual HAS produces HA.