key: cord-0011140-wryl7wu6
authors: Moradali, M. Fata; Rehm, Bernd H. A.
title: Bacterial biopolymers: from pathogenesis to advanced materials
date: 2020-01-28
journal: Nat Rev Microbiol
DOI: 10.1038/s41579-019-0313-3
sha: 7211a6eed3ad41894dcd22d468afd266893e4505
doc_id: 11140
cord_uid: wryl7wu6

Bacteria are prime cell factories that can efficiently convert carbon and nitrogen sources into a large diversity of intracellular and extracellular biopolymers, such as polysaccharides, polyamides, polyesters, polyphosphates, extracellular DNA and proteinaceous components. Bacterial polymers have important roles in pathogenicity, and their varied chemical and material properties make them suitable for medical and industrial applications. The same biopolymers when produced by pathogenic bacteria function as major virulence factors, whereas when they are produced by non-pathogenic bacteria, they become food ingredients or biomaterials. Interdisciplinary research has shed light on the molecular mechanisms of bacterial polymer synthesis, identified new targets for antibacterial drugs and informed synthetic biology approaches to design and manufacture innovative materials. This Review summarizes the role of bacterial polymers in pathogenesis, their synthesis and their material properties as well as approaches to design cell factories for production of tailor-made bio-based materials suitable for high-value applications.

Biopolymers are produced by living organisms and are synthesized by processive enzymes that link building blocks such as sugars, amino acids or hydroxy fatty acids to yield high molecular weight molecules. Bacteria can synthesize various classes of these biopolymers, such as polysaccharides (composed of sugars and/or sugar acids connected by glycosidic linkages), polyamides (composed of amino acids connected by peptide bonds), polyesters (composed of hydroxy fatty acids linked by ester bonds) and polyphosphates (polyPs; composed of inorganic phosphates linked by anhydride bonds). For decades, scientific efforts have been focusing on under standing biosynthesis pathways of bacterial polymers because of their involvement in bacterial pathogenicity and persistence. These polymeric substances can func tion as storage molecules, as protective capsular layers surrounding cells and as major matrix components of biofilms, which are involved in 60-80% of all human bacterial infections [1] [2] [3] . However, research on the physico chemical properties of biopolymers also sheds light on their utility for medical and industrial applications. Recent advances in synthetic biology and bioengineering methods allowed the production of innovative biopoly mers with uses or potential applications in medicine (for example, hyaluronate as a biomaterial), as additives in cosmetic products, as additives in food (for example, xanthan and dextran) and as biopolyesters in packag ing [4] [5] [6] . The rational design of biopolymerproducing cell factories has increasingly attracted research and commercial interest.

Although biopolymer synthesis consumes chemical energy and nutrients, it is maintained by bacteria as biopolymers enable them to persist and grow under a wide range of often unfavourable conditions, including exposure to immune responses of the host during infec tion. These polymers have diverse biological functions, such as adhesion, energy storage or protection, and their synthesis is regulated in response to environmental stimuli 7 . Their physicochemical properties are important for bacterial behaviours, such as translocation, attach ment onto biotic or abiotic surfaces, invasion, protection and persistence. For example, bacteria produce extra cellular polymeric substances, which is a general term referring to various bacterial polymeric substances that entangle themselves into a matrix that encases bacterial cells. Production of extracellular polymeric substances is essential for the formation of biofilms, which are highly structured microbial communities 2, 8 and one of the most persistent forms of life on Earth. As biofilm formation is the hallmark of many chronic infections 2,9 , a large body of research has been conducted to understand the role of bacterial biopolymers in biofilm formation and in pathogenesis. Such bacterial biopolymers and their biosynthesis and biological functions provide targets for developing novel antibacterial drugs.

On the other hand, extensive research has been focu sed on harnessing the unique material properties of bacterial polymers, such as cellulose 10 , dextran 11 , xan than 12 and polyesters 13 , in industrial production for medical and technical uses. Over the past few decades, Processive Continuous catalytic reactions by an enzyme without releasing its substrate.

An interdisciplinary research field that involves the application of engineering principles to biology aiming at (re)designing and fabricating biological components and systems.

Engineered cells that have been reprogrammed for enhanced production of desired compounds. genome sequencing and advanced molecular techniques have generated a large set of data not only providing insight into the role of bacterial polymers in pathogen esis but also for engineering bacteria as cell factories that produce tailormade biobased materials. Such renewable and biodegradable materials could replace oilbased commodity materials and would also advance development of novel highvalue biomaterials to pro vide solutions for unmet medical needs as they are often inherently biocompatible 14 . This Review highlights recent advances in our understanding of bacterial biopolymers, reflecting on their biological function and their use as biobased materials.

Polysaccharides. Polysaccharides are polymers com posed of sugars and/or sugar acids. They are classified into homopolymers and heteropolymers and they can be charged or noncharged, nonrepeating or repeating, and branched or unbranched. Diverse bacteria produce polysaccharides and store them inside cells (for exam ple, glycogen) or secrete them either as capsular poly saccharides that are linked to the cell surface or as free exopolysaccharides that contribute to the biofilm matrix (for example, alginate and cellulose) 4 . When motile, pathogens produce virulence factors and toxic mole cules (for example, flagella and exotoxins). However, when they switch to a sessile lifestyle, they produce different types of exopolysaccharides as matrix com ponents, such as alginate, cellulose and hyaluronate. This switch to the sessile biofilm lifestyle underlies the progression of many chronic infections as embedded or encapsulated cells are protected from immune cells and antibacterial drugs 2 (Fig. 1 ). For example, alginate within the biofilm matrix of Pseudomonas aeruginosa provides a survival advantage by protecting cells from phagocytosis 1 . Alginates (TAblE 1) interact with divalent cations to form dense hydrogels with high waterholding capacity 15, 16 . Production of cellulose (TAblE 1) provides similar advantages to enterobacterial pathogens 17, 18 . Escherichia coli produces phosphoethanolamine cel lulose, which forms mortarlike structures to stabilize proteinaceous curli fibres. These fibres mediate strong connections between cells in complex biofilms and provide resistance in highshear conditions 19, 20 . Some pathogens, such as Streptococcus pyogenes and Bacillus cereus G9241, produce a capsule of hyaluronate, a linear negatively charged heteropolysaccharide (TAblE 1) that mimics the structure of hyaluronate found in human connective tissues. Thereby, these pathogens can hide their antigenic surface from opsonization and phago cytosis 21, 22 . Serogroup B Neisseria meningitidis causes invasive meningococcal disease and produces a capsu lar polysaccharide composed of homopolymers of sialic acid (Nacetylneuraminic acid) with (α2→8)sialic acid linkages. This structure resembles polysialic acid moie ties of human tissue antigens, and such molecular mim icry imparts poor immunogenic properties on these capsular polysaccharides, making the pathogen invisi ble to the host 23 . Streptococcus pneumoniae causes severe lung infections and comprises more than 100 serotypes that produce different capsular polysaccharides to evade adaptive immune responses 24 . Secreted and capsular polysaccharides are used as antigens in conjugate vac cines (TAblE 2) . As newly emerging serotypes of patho gens such as S. pneumoniae and N. meningitidis reduce the efficacy of existing vaccines, the development of serotypeindependent vaccines is becoming increasingly attractive 25 .

Glycogen, a watersoluble polymer of α1,4linked and α1,6linked glucose, is a widespread form of car bon and energy storage that promotes survival during starvation 26 . During the intracellular phase of infec tion, glycogen can have an important role in the sur vival of pathogens, such as Mycobacterium tuberculosis, Salmonella enterica subsp. enterica serovar Typhimurium and Chlamydia trachomatis 27, 28 .

Besides their role as virulence factors, bacterial poly saccharides exhibit unique material properties (Fig. 2) . Chemical synthesis of polysaccharides is very laborious, costly and limited to low molecular weights and has been achieved for only a few types of polysaccharides. Hence, microbial cell factories are required for the manufacture of these polymers. The presence of hydrophilic groups (for example, hydroxy and carboxyl groups) on poly saccharides confers high waterbinding capacity and allows intermolecular interactions and crosslinking (for example, polymer-drug, polymer-polymer and polymerhost tissue and cell interactions). Polysaccharides can form porous hydrogels that can be used for drug delivery and controlled release of anticancer drugs 29, 30 , immobili zation of enzymes 31 , tissue engineering 30 , therapeutic cell entrapment and protection of transplanted cells from the host immune system 32,33 ( Fig. 2; TAblE 1 ). Hydrogels made of bacterial cellulose form efficient matrices, hydrogel nanofibrillar network scaffolds or fibre composites for biomedical applications; for example, in wound dress ings that deliver human epidermal keratinocytes and dermal fibroblasts 24, [34] [35] [36] . Production and application of cellulose produced by Komagataeibacter xylinus have been extensively studied 37, 38 , and a process for largescale production of bacterial cellulosebased 'rayon fibres' for use as wearable textiles has been developed. A successful example of a bacterial polysaccharide used in biomedical applications is hyaluronate produced by nonpyogenic Streptococcus zooepidemicus 39 . Commercial formulations of a gellike fluid of hyaluronate were used for injection into the knee joint to mitigate arthritis pain.

Specific enzymes naturally modify bacterial poly saccharides to change their material properties and sup port their biological functions (TAblE 1) . For example, the presence of acetyl groups on polysaccharide chains nota bly alters the structural conformation and affects chainchain interactions, solubility, waterholding capacity, viscoelasticity and molecular weight 16, [40] [41] [42] . Genetic engineering of polysaccharidemodifying enzymes in bacterial cell factories or the use of such enzymes for in vitro modification of polysaccharides allows pro duction of tailormade polysaccharides. The design of materials becomes even more versatile through blending with other polymeric and nonpolymeric components (for example, citric acid (crosslinking), stearic acid (esterifying) and plasticizers). Blending allows tailor ing of properties such as viscoelasticity, gelation degree, porosity and material strength. Such materials have gained much attention as feedstock materials or bioinks for 3D bioprinting with numerous biomedical and engi neering applications, including tissue engineering, drug delivery and drug testing 43 . Cellloaded 3D scaffolds of alginate or hyaluronate have been used successfully as an artificial extracellular matrix that provides a temporary environment to support infiltration, adhesion, prolifer ation and differentiation of various cell types, includ ing mesenchymal stem cells, fibroblasts, chondrocytes, osteoblasts and embryonic stem cells 44 . Overall, bacteria are a major natural resource for the production of a vast variety of polysaccharides with many potential industrial and medical uses (TAblE 1) .

Polyamides. Bacteria can produce polyamides or poly(amino acid) chains (Fig. 3a) , such as secreted poly(γdglutamic acid) (γPGA) and poly(εllysine) (εPL) or the intracellular cyanophycin (a copolymer of laspartic acid and larginine), which can func tion as capsules or biofilm matrix 45, 46 , or as storage materials 47, 48 , respectively (Fig. 1) . Similarly to the role of polysaccharides in the biofilm matrix, a polyamide capsule or biofilm is poorly immunogenic and con ceals pathogens such as Bacillus anthracis from surveil lance by the immune system 45 When produced by bacterial pathogens, secreted biopolymers can function as virulence factors, whereas intracellular polymers are mainly reserve materials that increase survival during starvation. The switch from motility to sessility of bacterial pathogens is a strategic decision that is often connected with the production of exopolysaccharides. Pathogens benefit from the production of high molecular weight polysaccharides as they are an integral part of the biofilm matrix and interact with counterions and other polymers to form a hydrogel-like niche 2, 16 . Furthermore, they protect embedded bacterial cells from environmental stresses, the immune systems and antimicrobial treatment. This lifestyle transition underlies the establishment of many chronic and hard to eradicate infections. Capsular polysaccharides are attached to the cell surface and protect the pathogen from phagocytosis and antimicrobial drugs. Glycogen is an intracellular storage polysaccharide that promotes the survival of some pathogens during the intracellular phase of infection. Polyhydroxyalkanoates (PHAs) are highly reduced biopolyesters that function as storage compounds that increase bacterial fitness and potentially function as an electron sink in anaerobic zones of biofilms 54, 56 . PHA-metabolizing enzymes are produced under specific nutritional and environmental stresses to enhance bacterial survival. Polyamides function as bacterial capsules or slimes to protect cells 45 or as intracellular storage material. Bacillus anthracis, which can cause lethal infections, produces such a capsule. Polyphosphates (polyPs) are chains of condensed phosphates that function as a storage material with high energy-rich bonds. The metabolism of polyP is positively correlated with the production of virulence factors 64, 67 . Extracellular DNA (eDNA) mediates the surface adhesion of cells and stabilizes the biofilm matrix through interaction with other secreted polymers and cations 1 . Proteinaceous components such as fimbriae, pili and flagella are extracellular self-assembling nanostructures that contribute to surface attachment, the formation of the biofilm matrix and/or bacterial motility. (TAblE 1) . εPL has antibacterial properties as it disrupts membrane integrity, and its crosslinked form was used in antimicrobial coatings 52, 53 .

Polyesters. Polyhydroxyalkanoates (PHAs) such as poly((R)3hydroxybutyrate) are bacterially synthesized bioplastics. They are linear polyesters that are synthe sized and assembled into hydrophobic spherical inclu sions and they function in carbon and energy storage 7 ( Fig. 3b; TAblE 1 ). Although a wide range of Grampositive and Gramnegative bacteria produce PHAs, the roles of PHAs and their metabolizing genes in the context of persistence and pathogenesis remain largely unknown. PHAmutants of P. aeruginosa showed reduced attach ment to glass surfaces and reduced stress tolerance in biofilms, suggesting a possible contribution of PHA to persistence during infection 54 . In the plantpathogen Xanthomonas oryzae pv. oryzae, which causes major losses in rice production, the regulatory protein PhaR not only represses PHA synthesis but also affects production of extracellular polymeric substances, the bacterial life style, phenotypic changes and virulence 55 . PHAs are pro posed as a sink for electrons under anaerobic conditions; that is, in the absence of terminal electron acceptors such as oxygen, they enhance survival 56,57 (Fig. 1 ). PHA synthe sis and mobilization are regulated in response to environ mental stimuli, such as nutritional and environmental stresses, providing a survival advantage 55 .

PHAs have been considered as unique biobased plas tics that can be bioengineered, chemically modified and processed into highvalue medical materials (for example, sutures, tissue engineering scaffolds, drug carriers and particulate vaccines) or lowvalue commodity bioplastics (TAblE 1) . Production of tailormade PHAs via bioengi neering, physical blending and chemical modification resulted in improved material properties, which met specifications for industrial and medical applications 58, 59 . An exciting innovative approach engineered bacterial cell factories to assemble PHA inclusions that are densely coated with functional proteins of interest. These func tionalized PHA beads were stable after separation from the bacterial cell mass and showed promising perfor mance as vaccines, immunodiagnostics, bioseparation resins, enzyme carriers and tools for recombinant protein production 60, 61 . The functionality of these nonporous proteincoated PHA beads was further tunable by con trolled encapsulation into porous alginate microspheres, which allowed flowthrough applications. This study is an example of the tremendous materials design space pro vided by bioengineering of polymers and by blending of polymers to generate functional composite materials 62 .

Polyphosphates. PolyP is a polymer of condensed phos phates (three to several hundred inorganic phosphates) that is highly negatively charged and rich in 'highenergy' anhydride bonds. It functions as an energystorage poly mer 63,64 ( Fig. 3c; TAblE 1 ). PolyP synthesis is an evolu tionarily ancient ability of bacteria, and polyPs, besides functioning in phosphate storage, also provide chemical energy for biosynthesis pathways, function as a buffer against alkalis and as a metalchelating agent and con tribute to channel complexes for the uptake of DNA 7, 64, 65 .

PolyPs also regulate cell signalling and thereby affect bacterial lifestyle, persistence, viability, growth, stress tolerance and virulence [66] [67] [68] (Fig. 1 ). Due to their eminent energystorage feature, indus try has increasingly considered polyPs to drive energy consuming enzymecatalysed reactions (TAblE 1) . They are also considered as morphogenetically active bio materials in regenerative medicine, such as in cartilage repair and bone regeneration 69 Bacterial polysaccharides are important biomaterials due to their unique material properties, including solubility , rheological characteristics, viscoelastic properties, interaction with cations, ionic strength, crosslinking, gelation, water retention, extendibility and stability under different conditions. Hence, polysaccharides have been applied as natural viscosifiers, thickeners, stabilizers, gel and film formers, and additives or have been processed into nanostructures (for example, nanoparticles and nanotubes), microspheres, microcapsules, sponges, hydrogels, foams, elastomers and fibres 43, 44, 156 .

Besides the desired material properties, high purity and the purification process are crucial for the use of bacterial polysaccharides as high-value biomaterials 32, 43 . d-Glc, d-glucose; d-GlcA , d-glucuronic acid; GlcNAc, N-acetylglucosamine; l-GulA , l-guluronic acid; d-ManA , d-mannuronic acid.

A branch of medicine aiming at regrowing, repairing or replacing damaged or diseased cells, organs or tissues.

NAture reviews | MiCroBiology vehicles 71 . Owing to their capacity to interact with posi tively charged polymers (for example, alginate and hya luronate), inorganic cations (for example, Ca 2+ , Mg 2+ , Zn 2+ , Fe 2+ , Na + and K + ), or basic organic components (for example, amino acids, polyamines and peptides), polyPs can be processed into hydrogels or nanoparticles for bone biomineralization 69, 70 and other biomedical applications 72 . Importantly, the physical properties, such as mechani cal strength, stability and functionality, of polyPbased complex hydrogels or nanoparticles vary with the type of interacting counterions or blended polymers. This varia tion provides substantial design space to generate a range of desired material properties for 'smart biomaterials' and bioinks in regenerative medicine 71, 73 (Fig. 3c) . As no abi otic polyP minerals have been found on Earth, living organisms, in particular bacteria, are unique sources of polyP. Bacteria belonging to the genera Mycobacterium and Corynebacterium produce polyP granules with a high yield and therefore are potential production strains for the manufacture of polyPs 74 .

Bacteria also produce other types of biopolymers, including extracel lular DNA and proteinaceous components (Fig. 1) . They are not only important in bacterial pathogenesis but are also considered for development of biobased materials. 45, 47 . Due to their biodegradability, non-toxicity and modifiability , bacterial polyamides have been considered as substitutes for chemically synthesized polymers that can be processed into formulations for industrial, biomedical, pharmaceutical and cosmetic applications 50 . b | Polyhydroxyalkanoates (PHAs), such as polyhydroxybutyrate, are natural polyesters that are synthesized into hydrophobic spherical inclusions from (R)-3-hydroxybutyric acid. PHAs have been classified into short-chain-length PHAs (PHA SCL ; containing constituents with 3-5 carbon atoms) and medium-chain-length PHAs (PHA MCL ; containing constituents with 6-14 carbon atoms), which are primarily produced by pseudomonads 58 . Synthases and other PHA-binding proteins decorate the surface of PHA inclusions. PHAs are unique bio-based materials processed as bioplastics or bioengineered functionalized nanoparticles for uses in medicine and industry. PHA nanobeads can function as effective platforms for enzyme immobilization, protein purification, bioseparation, drug or vaccine delivery , tissue engineering, diagnostics and imaging 58 . c | Polyphosphates are composed of orthophosphates (inorganic phosphates, three to several hundred phosphates) linked by phosphoanhydride (P-O-P) bonds. They contribute to energy storage and can be processed into hydrogels or nanoparticles for various applications (TAblE 1) . The phosphate and counterions such as Ca 2+ and Sr 2+ are released on hydrolysis and can be used for bone biomineralization, as smart bioinks for generating 3D scaffolds and for cell bioprinting of regeneratively active patient-specific osteoarticular implants [69] [70] [71] 73 . Polyphosphate or collagen hydrogels were formulated for improving tissue integration of meshes to improve the outcome of surgical hernia repair 72 .

biomaterials with the ability to respond to stimuli or physiological conditions at the micrometre to nanometre scale.

Extracellular DNA arises when a cell lyses and releases intracellular DNA. In biofilms, lysis of a subpopulation of cells contributes extracellular DNA to the biofilm archi tecture, for example in the stalks of mushroomshaped microcolonies of P. aeruginosa 75 . Due to its high negative charge, extracellular DNA has multi faceted roles, includ ing in the adhesion and stability of the biofilm matrix through interaction with positively charged polysaccha rides (for example, Pel) and cations, it is a nutrient source during starvation and it confers antibiotic resistance 1 . Secreted polypeptides, for example composed of alter nating hydrophilic and hydrophobic amino acid resi dues, and proteins, such as fimbrillin, pilin and flagellin, can be molecular building blocks of extracellular selfassembling structures, such as functional amyloids (for example, curli fibres), fimbriae, pili and flagella. These selfassembling structures can form nanofibres or nanotubes and mediate cell adhesion to biotic and abiotic surfaces, development of the biofilm matrix or motility during pathogenesis. Several features of these structures make them attractive for applications, including the precise arrangement of protein building blocks in self assembling structures, their high surface area to volume ratio and polymorphic transformation in response to physical and chemical stimulations. These features ren der them valuable biobased materials and biotemplates for fabrication of novel nanostructures, nanodevices and multilayer lattices applicable in bioengineering and nanomedicine (for example, drug delivery) [76] [77] [78] .

The genetic programmability and ease of engineer ing of extracellular DNA, polypeptides and proteins make them fascinating programmable biomaterial platforms that are hardly achievable for other biopoly mer types, including polysaccharides and polyesters 79 . Straightforward genetic programmability has generated much recent interest in developing engineered living materials; that is, living cells are engineered to auton omously selfassemble entire materials with novel and tunable properties for a variety of purposes, such as microbial electrosynthesis, biosensors, electronic moni toring devices and bioremediation 79, 80 . We only scratch the surface of the vast scope of selfassembling structures produced by bacteria and their applications, but they have been reviewed extensively elsewhere [80] [81] [82] [83] .

Bacterial polymer synthesis Synthesis pathways and their regulation. Genome sequencing, functional genomics, advanced molecular tools and techniques, and new biochemical and bio physical approaches enhance our understanding of the biosynthesis of bacterial biopolymers. Vast DNA and protein databases combined with in silico approaches provide insights into biosynthesis pathways and the structure and function of key biosynthesis proteins. All these advancements have created a solid foundation for the design of cell factories for enhanced production of polymers or to produce tailormade polymers. Current synthetic biology approaches that use DNA foundries are the nextgeneration tools for the design of cell factories and allow precision engineering of production strains 84 . Biosynthesis pathways for representative bacterial poly mers are illustrated in Fig. 4 . In addition to a better understanding of biosynthesis pathways and enzymes, knowledge of the molecular mechanisms of synthesis, modification and, if required, secretion informs produc tion of novel tailormade polymers. For example, many bacterial polysaccharides are enzymatically modified at the polymer level, such as acetylated, deacetylated, epimerized or phosphoethanolaminated, and these modifications affect material properties, such as visco elasticity and gelforming capacity 40 . Genes encoding enzymes involved in polymerization and modifications of polysaccharides are usually coclustered in one main operon (Fig. 4) . Potent specific promoters often con trol these operons and the transcription of the entire biosynthesis gene cluster 4 .

Biosynthesis of polymers in bacteria is controlled by regulatory networks that process environmental signals and mediate responses through transcrip tional and posttranslational regulation (Fig. 4) . At the transcriptional level, transcription factors activate promoters that control the expression of function ally related genes. Such transcription factors include sigma factors, which are subunits of RNA poly merase, and regulatory proteins that bind to DNA regions upstream of biosynthesis genes. Some sigma factors are sequestered by antisigma factors and are released on exposure to external stimuli. For example, AlgU is a sigma factor that binds to the core RNA polymerase and thereby mediates binding to a specific promoter region upstream of the alginate biosynthesis gene cluster. AlgU is sequestered by the membranebound antisigma factor MucA in P. aeruginosa 85 and likely other pseudomonads under conditions that are not permissive to alginate production. On environmen tally induced destabilization of this complex, such as in response to cell envelope stress or on mutation of the mucA gene (for example, adaptive mutation dur ing chronic infection), AlgU is released and activates transcription of the alginate biosynthesis gene cluster. An engineered MucAinactivated strain 86 constitu tively produced high yields of alginate, which might be an avenue for enhanced production of bacterial alginate. Furthermore, small noncoding RNAs, twocomponent systems, regulatory RNAbinding proteins and second messengers (such as cdiGMP and cdiAMP) are involved in signal processing and complex regu latory networks that control polymer syn thesis in bacteria 1, 87 . Improved understanding of these regulatory complexities via systems biology will inform synthetic biology approaches for efficient production of polymeric materials.

Bacteria have highly processive enzymes for the production of biopolymers with high mole cular weights (molecular mass >100 kDa) that cannot be achieved by chemical synthesis. Many bacterial exopoly saccharides have high molecular weights, for example mole cular mass ~3.9 MDa for alginate from P. aeruginosa 40 89 , and this affects polymer properties, bacterial patho genesis and evasion of host immunity and antimicrobial

Facilities using advanced software, automation or robotics and analytical approaches for faster, easier and scalable assembly of DNA to develop advanced cell factories.

Small molecules that relay signals received by cell-surface receptors to effector proteins and regulate cellular processes.

A research field focusing on understanding relationships between networks of biological processes through computational and mathematical approaches.

NAture reviews | MiCroBiology treatment. In the context of biobased material devel opment, bacterial polysaccharides with high molecular weights have gained much attention as materials that are biocompatible and have high water retention capacity, excellent gelling properties and a long halflife under physiological conditions. Bacterial biopolymers can also be a source of biologically active oligomers (molecular mass usually <10 kDa). Such olig omers can be used as therapeutic drugs for applications such as promotion of angiogenesis, inhibition of tumour progression or induc tion of the production of proinflammatory mediators, antiinflammatory substances and antibiofilm agents [90] [91] [92] [93] [94] [95] [96] . They can also be a source of valuable monomers, such as rare sugar monomers (for example, fucose) 97 , which are in high demand as precursors for pharmaceuticals and nutraceuticals. Another example is hydroxyalkanoic acid monomers that can be obtained from the hydrolysis of PHAs, which are precursors for several antibiotics 98 . There are various mechanisms for controlling the degree of polymerization of poly saccharides. They include sub strate tethering, as described for mycobacterial galactan. In this example, the acceptor of a lipidlinked initiator 

Food additives that not only supplement the diet but also provide health and medical benefits.

www.nature.com/nrmicro oligosaccharide binds to a specific site on the glycosyl transferase and facilitates processive polymerization resulting in longer polymer chains 99 . Another mechanism is the coupling of polymerization with modifications, for example the processivity of alginate polymerization in P. aeruginosa, which is linked to in situ enzymatic mod ifications (that is, epimerization and acetylation) 40, 100, 101 . The chain length determinant protein Wzz also controls the degree of polymerization of, for example, lipopoly saccharide O antigens. In the absence of Wzz, O antigens are randomly distributed and of shorter chain length than in the presence of Wzz 102, 103 . Substrate concentration is another regulatory mechanism, as it affects the rate of polymerization, the yield of the final product and the molecular weight. For example, high concentrations of the UDPNacetylglucosamine substrate increased the molecular weight of hyalorunate 104 . Finally, the degree of polymerization also depends on the copy numbers of polymerase and synthases. For example, the presence of a number of different synthases competing for sub strates reduced polymer chain lengths of alginate 40 and polyhydroxybutyrate (PHB) 105 .

In pathogenic bacteria, biopolymers are often major virulence factors and contribute to the persis tence of infections in different ways, including antigenic mimicry (for example, polysialic acid and hyaluronate) 23 , hiding of the antigenic cell surface to evade opsonization and phagocytosis (for example, hyaluronate, alginate and γPGA) 1,106,107 and as a barrier against antimicrobial drugs and toxic molecules 108 . Therefore, biopolymer synthe sis and function might be new targets for anti microbial drugs to overcome persistent infection, antibiotic resist ance and antibiotic persistence [109] [110] [111] . Although this is an emerging field, there is evidence for the success of such approaches. For example, inhibition of the inter action between Alg44 and cdiGMP, which is required for alginate polymerization, by a class of thiolbenzo triazoloquinazolinone compounds reduced alginate secretion by P. aeruginosa by up to 30% (rEF. 112 ). OligoG CF5/20 (an alginate oligomer) decreased the thick ness of mucus in cystic fibrosis lungs and destabilized the biofilm matrix and the extracellular polymeric net work 113, 114 . Furthermore, biopolymerdegrading enzymes (such as alginate lyases 115, 116 and amylases 117 ) degraded the biofilm matrix. Finally, natural products (such as quercetin and curcumin) reduced the production of alginate and other polysaccharides by reducing the expression of the quorum sensingregulated genes and concomitantly virulence factors such as pyocyanin, pro tease and elastase in P. aeruginosa, Klebsiella pneumoniae and Yersinia enterocolitica [118] [119] [120] (TAblE 2) .

Over the past decade, knowledge of the biosynthesis of bacterial polymers together with systems biology and synthetic biology has revolutionized the rational engineering of cell factories, which has increased production yields and/or led to production of innovative biobased materials (Fig. 5) . As mentioned earlier, bio synthesis of bacterial polymers requires the engagement of complex cellular processes from gene expression to provision of enzymes and proteins, central metabolism, and regulatory and signalling systems leading to intra cellular assembly or secretion across the cell envelope.

Hence, design of cell factories for production of novel biopolymers requires integration of the complexity of cellular and metabolic process and extensive experimen tation to combine the relevant genetic information. DNA foundries use advanced software, robotic and analyti cal approaches to allow automated 'design-build-test' engineering cycles for the highthroughput development of desired cell factories through synthetic assembly of genetic elements. DNA foundries have higher experi mental consistency and lower costs than manual oper ations. Furthermore, modular genetic elements, such as promoters, terminators, ribosomebinding sites, orthog onal polymerases, untranslated regions, signal peptides, putative stabilization modules, genetic effectors and protein folding enhancers, provide a dynamic plat form to tune gene expression and protein production. Striking advancements include the introduction of inducible and/or controllable genetic switches, such as T7 polymerasebased expression systems 121 , pro grammable T7based synthetic transcription fac tors 122 , the riboTite system 123 , vector engineering 124 and CriSPr-Cas 125 tools, to allowed finetuned expression of endogenous or heterologous genes. Furthermore, CRISPR-Cas9 has been successfully used to simultane ously manipulate several genes. CriSPr interference has been successfully used to redirect metabolic flux towards Intermediates of central metabolism are diverted towards the provision of precursors for polymer synthesis. Four general mechanisms for the production of polysaccharides in bacteria are shown. Synthesis of some secreted non-repeating polysaccharides, such as alginate and cellulose, is mediated by multiprotein complexes, usually consisting of a polymerase, a copolymerase, carbohydrate-modifying enzymes and secretion subunits. The genes encoding such functionally related protein subunits are co-clustered in large operons, such as the alg and bcs operons. Some polysaccharides, such as xanthan, are produced through the Wzy-dependent polysaccharide synthesis mechanism. In this pathway the repeating sugar units and their linked lipid carriers are assembled by several glycosyltransferases at the cytoplasmic membrane, followed by flipping across the cytoplasmic membrane, the final polymerization step in the periplasm and secretion. However, the synthesis of some polysaccharides, such as hyaluronate, dextrans and levans, is less complex and is mediated by a single enzyme. Dextrans and levans are synthesized outside the cell by sugar transferases that convert disaccharides into polysaccharides and use the energy that is released by hydrolysis of the glycosidic bond of the disaccharides. Modification of secreted polysaccharides (for example, acetylation, deacetylation, epimerization and phosphoethanolamine (pEtN) addition) can occur during translocation of nascent polymers across the cell envelope. Polyhydroxyalkanoates (for example, polyhydroxybutyrate (PHB)) are synthesized by a polyhydroxyalkanoate synthase that coverts hydroxyacyl-CoA derivatives of central metabolism into intracellular polyesters. Enzymatic processes independent of ribosomal protein biosynthesis synthesize polyamides. Dashed lines indicate multiple enzymatic steps, a circled plus sign indicates positive correlation and a circled minus sign indicates negative correlation. ABC, ATP-binding cassette; CPS, capsular polysaccharide; FA , fatty acid de novo biosynthesis; Fru-6-P, fructose 6-phosphate; Glc, glucose; GlcA , glucuronic acid; GlcNAc, N-acetylglucosamine; Glc-1-P, glucose 1-phosphate; Glc-6-P, glucose 6-phosphate; KDPG, 2-keto-3-deoxy-6-phosphogluconate pathway ; LPS, lipopolysaccharide; ManA , mannuronic acid; polyP, polyphosphate; RBP, RNA-binding protein; SM, second messenger ; sRNA small non-coding RNA ; TCA , tricarboxylic acid; TCS, two-component system; TF, transcription factor.

A multilayered modular genetic control circuit using standard inducible promoters and orthogonal riboswitches for tunable control of T7 rNA polymerase activity and recombinant expression of genes of interest.

A genetic system for gene editing based on naturally occurring genome editing in bacteria and archaea that confers adaptive immunity against invading phages.

A method that uses a catalytically inactive Cas9 protein and a customizable single guide rNA that binds to DNA and blocks transcription of a gene of interest.

NAture reviews | MiCroBiology PHA biosynthesis 6 . Other examples are the rational reprogramming of Komagataeibacter rhaeticus iGEM for the production of cellulosebased materials 126 and the engineering of broadhostrange vector systems to use cyanobacteria for the production of renewable bioprod ucts 127 . Recombinant production of hyaluronate, PHAs, γPGA and cyanophycin has been successfully achieved as these required reconstructions of relatively simple pathways. E. coli, Ralstonia eutropha, Pseudomonas putida and Alcaligenes latus are industrial workhorses for commercial production of PHA, and Bacillus subtilis has been used for commercial production of hyaluro nate 128 . Novel inducible systems such as lightsensing or temperaturesensing systems can act as logic gates, timers, switches and oscillators to precisely control the expression or production of desired products in response to specific inputs or inducers 129, 130 . These inducers are alternatives to chemical induction, which suffers from loss of directionality and poor control over the induc tion period. Lightsensing systems from cyanobacteria have been adapted for the photoinducible expression of specific genes in E. coli 131 and P. aeruginosa 132 .

As illustrated in Fig. 4 , the biosynthesis of biopoly mers is linked to central metabolism, which means that the engineering of highly productive cell factories requires integration of carbon, nitrogen and energy fluxes 133 . Initially, several cell factories need to be gen erated to then efficiently convert precursor substrates into polymers. For example, a genetically engineered mutant of P. aeruginosa produced ~125 g of alginate from 1 g of dry cells 40 , which suggested a predominant flux of precursor substrates into the polymer. Indeed, understanding the major points that control the flux in the biosynthesis of biopolymers and the energetic state and relevant metabolites in the cells is a key step for increasing productivity. Thus, the metabolic engi neering of synthesis pathways aims to enhance sub strate and energy flux towards biosynthesis of the desired polymer. Biosynthesis of active precursors is an energyconsuming process and is commonly based on diversion of metabolites from central metabolism and primary cellular functions (Fig. 4) . Therefore, deter mining the redox state of the cells is important when one is amending metabolic pathways and redirecting www.nature.com/nrmicro metabolites towards the desired biosynthesis pathway. The redox state is determined by factors such as elec tron carriers (for example, NADH and NAD + ), oxygen availability, the carbon and nitrogen uptake rates and the kinetics of enzymes involved in metabolic flux. For example, the balance between the concentration of hyal uronate precursors and ATP levels, which are linked to the recycling of electron carriers, was crucial for optimal production of hyaluronate 39 . In B. licheniformis, increas ing the ATP content of cells increased the production of γPGA. This was achieved by improving the respiratory electron transport chain (through the Vitreoscilla sp. haemoglobin), ATP synthesis and nitrate metabolism 134 .

In another example, weakening or abolishment of com peting pathways (for example, βoxidation of fatty acids) and boosting of NADH (or NADPH) levels increase carbon flux towards PHA biosynthesis 125 . In E. coli, a combination of multiple gene deletions and additions coupled lactate utilization and conversion with the formation of GDPfucose and, in combination with blocking of the competing colonic acid biosynthesis pathway, this strategy led to high yields of fucosylated Nacetyllactosamine oligosaccharides 135 . Such con trol elements should enhance carbon and energy flux towards the synthesis of the biopolymer, but not towards cell biomass and/or metabolic byproducts 40, 136 . Systems biology combined with metabolic engi neering using computational methods linked with highthroughput measurements of cellular processes (including metabolic pathways and gene regulatory and signalling networks) and omics data (that is, transcrip tomics, proteomics, metabolomics and fluxomics data) has greatly advanced the development and improvement of cell factories and their products 5,137 (Fig. 5) . The num ber of in silico tools and computational frameworks to support synthetic biology approaches is growing and these include the iGEM Registry of Standard Biological Parts (a collection of genetic parts), COBRA and Cameo (for gene target identification, gene knockout and gene overexpression) 138, 139 and macromolecular expression models (for computing the optimal proteome composi tion of a growing cell) 140 . In particular for biopolymers with a complex biosynthesis pathway such as polysaccha rides, computational modelling of the interplay between central metabolism and biosynthesis pathways can strongly improve bioengineering strategies. In silico genomescale metabolic flux analysis identified meta bolic engineering targets in E. coli to enhance the yields of polylactic acid and poly(3hydroxybutyrate colactate), contributing to 11% and 56% of cellular dry weight, respectively 141 . Accordingly, metabolic engineer ing of E. coli achieved the production of nonnatural tailormade polymers such as poly(lactatecoglycolate) with a broad range of material properties 142 .

Rational engineering increased the range of pro duction hosts; for example, developing the halo philic bacterium Halomonas smyrnensis AAD6 T as a biotechnological production platform that does not require costly sterilization steps (highsalt media pre vent growth of other living organisms) for the pro duction of levan, Pel exopolysaccharide, PHAs and osmoprotectants 143 .

Challenges in bacterial production of biobased mate rials. The application of bacterial biopolymers as biobased materials is expanding (TAblE 1) . Despite inherent properties such as biocompatibility and bio degradability, some bacterial biobased materials have shortcomings; for example, they do not meet specifica tions (such as consistency and purity) that are required for medical applications. In addition, bacterial fer mentation is inherently expensive and associated high production costs often prohibit commercial use.

The basic chemical structure has a major role in deter mining the biophysical properties of a biopolymer and its applications. For example, some PHAs have high crys tallinity that causes stiffness, brittleness, poor thermo mechanical properties (high melting temperature and low glass transition temperature), high hydrophobicity and stickiness and therefore restricts their applica tion 58, 144 . For some polysaccharides, poor mechanical stability, a lack of elastomeric properties and reduced sol ubility due to neutral charge or a high molecular weight restrict their utility. However, these biopolymers are naturally diverse in structure and can be enzymatically or chemically modified, which provides a wide range of physicochemical properties suitable for various applica tions. Improvements of biopolymers have been success fully achieved by genetic manipulation of cell factories, improving fermentation conditions and enzymatic modifications 142, [145] [146] [147] as well as blending with other biopolymers and/or chemical modifications such as crosslinking, chlorination, epoxidation, hydroxylation, carboxylation, etherification and esterification 144, 148, 149 . These approaches have extensively improved biopolymer properties such as stability, solubility, crystallinity, glass transition temperature, elasticity and permeability and have expanded the utility for biomedical applications, such as drug delivery and regenerative medicine 43, 58 .

For medical applications, the cell factory and the biopolymer must be certified as 'generally recognized as safe' (GRAS), a designation determined by the FDA that applies to substances accepted as safe. Despite advan tages (for example, biocompatibility) over synthetic materials, biomedical and biotechnological applications of bacterial biopolymers are constrained by the GRAS status of the production strain. For example, despite the extensive study of bacterial alginate biosynthesis, these alginates cannot be regarded as GRAS so far. This is also true for many products derived from Gramnegative bacteria, for which host cellderived impurities such as endotoxins might reduce product quality 150, 151 .

The GRAS standard of biopolymers requires the establishment of standard assays to demonstrate that polymers derived from bacteria meet purity criteria and are safe to be used as a medical device. Furthermore, the safety profiles should include that longterm use will not induce undesirable immune responses and potentially autoimmune diseases. Currently, the FDA and contract research organizations lack such standard assays for quality control. Development of these standard safety assays and their validation in relevant animal models will be important. Compositional analysis of bacterial biopolymers using nextgeneration, highend analy tical instruments such as advanced chromatographs

A reversible physical transition (for example, from a hard, glassy state to a soft, rubbery state) that an amorphous material undergoes in a particular temperature range.

NAture reviews | MiCroBiology and mass spectrometers will help further improve their quality control.

Also, it is clear that development of safe bacterial cell factories (for example, novel endotoxinfree and non pathogenic strains) through synthetic biology and bio engineering as well as efficient purification methods can lead to a plethora of new polymers and highvalue biomaterials. Successful examples include generation of endotoxinfree E. coli ClearColi 150 and the commercial use of highly attenuated P. aeruginosa PGN5 for produc tion of alginate 152 and nonpyogenic S. zooepidemicus for production of hyaluronate, which was purified through extensive filtration and diverse adsorbents to eliminate impurities 153 (TAblE 1) .

Successful industrialscale production of biopoly mers depends on various factors, including the cost of precursor substrates, yield over substrate rate, volumet ric productivity and the cost of downstream processing (purification). Whereas bioengineering aims at improv ing the upstream process (use of lowcost substrates and increased productivity), bioprocess optimization of the upstream and downstream processes is required for scalable and costeffective manufacture 154, 155 . Production of extracellular biopolymers is challenging because of associated high viscosity of the culture liquids, which reduces the diffusion of dissolved oxygen and ATP for mation. Therefore, strategies that could enhance toler ance of anaerobic conditions or boost energygenerating systems may enhance productivity.

Extracellular polymers that are produced by bacterial pathogens are major virulence factors. Thus, inhibition of their biosynthesis pathways represents a strategy for the treatment of bacterial infections. Owing to rising rates of antimicrobial resistance, the development of novel strategies to fight bacterial infections is in high demand. Insights into the synthesis, secretion and regu lation of biopolymers will disclose new and specific tar gets suitable for drug discovery; for example, for targets that weaken bacterial defences against the host immune defences or antimicrobial treatment (Fig. 5) .

Polymers that are produced by nonpathogenic bac teria are considered safe materials for a range of applica tions. Despite great advances in the design of cell factories for enhanced biopolymer production as well as produc tion of tailored biopolymers, challenges remain. Because of a plethora of interacting components and multiple feedback loops in complex biological systems, rational engineering of novel GRAScertified cell factories and biopolymers remains challenging. It is important to reduce this complexity through systems biology to better inform genomescale metabolic models, metabolic net work modelling and computational simulations of large data sets that feed into synthetic biology approaches. This work will provide the foundation for efficient bioengineering strategies and accurate predictions for cell factory and bioprocess development.

In this Review, we have highlighted the advances in understanding the roles of bacterial biopolymers in pathogenesis and their current and potential applications as biobased materials. We hope that this Review will guide both drug discovery programmes and the development of new biobased materials by outlining strategies to over come pitfalls and challenges associated with biopolymers as virulence factors and as innovative biobased materials.

Published online 28 January 2020

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Both authors researched data for the article, discussed the content, wrote the article and reviewed and edited the manuscript before submission.

B.H.A.R. is a co-founder and shareholder of PolyBatics Ltd, which commercializes veterinary tuberculosis diagnostic products related to protein-coated polyester spheres assembled in engineered E. coli. M.F.M. declares no competing interests.

Nature Reviews Microbiology thanks Guo-Qiang Chen, Hongwei Yu and the other, anonymous, reviewers for their contribution to the peer review of this work.

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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