Chemical and Biochemical Engineering Approaches in Manufacturing Polyhydroxyalkanoate ( PHA ) Biopolyesters of Tailored Structure with Focus on the Diversity of Building Blocks

Polyhydroxyalkanoates (PHA) constitute prokaryotic storage materials not only harnessing microbial cells with benefits for survival under challenging environmental conditions, but also attracting attention as biological materials with properties resembling those of currently used thermoplasts and elastomers of petrochemical origin. Strongly dependent on their monomeric composition and microstructure, PHA ́s exact material properties are predestined in statu nascendi, hence, during their biosynthesis. The present review sheds light on established and emerging strategies to produce differently composed PHA homo-, co-, ter-, and quarterpolyesters from the groups of short-, medium-, and long-chain PHA. It is shown how microbial strain selection, sophisticated genetic strain engineering based on synthetic biology approaches, advanced feeding strategies, and smart process engineering can be implemented to generate PHA of tailored monomeric composition, microstructure, molar mass, and molar mass distribution. Tailoring these parameters offers the possibility to produce customer-oriented PHA for various purposes, such as packaging materials, carriers of pharmaceutically active compounds, implants, or other emerging fields of use.


Introduction
In almost every situation of our daily life, we are confronted with plastics; in the majority of cases, plastics are polymers of petrochemical origin.Over the last decades, they are surely the most emerging group of materials to produce custom-made items applied for packaging, and uses in the building industry, transportation, electronics, agriculture, healthcare, the sport sector, or leisure industry.However, we witness increasing global concern associated with petrochemical plastics.Valid estimations report that, over the last decades, a total of 8 to 9 gigatons (Gt) of plastics has been produced worldwide. 1Considering a current global plastic production approaching 400 megatons (Mt) per year, with an incredible upward trend, especially in emerging and developing countries, it is clear that the huge amounts of spent plastics, which already surpass 150 Mt per year, have to be disposed of somehow. 2,3Based on the high recalcitrance of these non-natural polymers, which prevents them from biodegradation, disposal strategies contemporarily in use are landfilling or simple release into the ecosphere (the fate of an estimated 79 % of all plastics produced to date), or thermal conversion via incineration (estimated 12 % of all plastics produced to date), all leading to unwanted consequences, such as growing piles of plastic waste or generation of toxic and/or greenhouse gas active gaseous emissions. 1A topical issue is the severe pollution of the marine environment by roughly 2 Mt of plastic waste entering oceans via rivers, which, often as "microplastic", endangers the entire food chain. 4ecycling of petrochemical plastics only functions to a certain degree due to the need of a defined degree of purity of spent plastic, sorting accuracy, and decrease in material quality with each recycling cycle. 2,5,6s a real alternative, one can resort to solutions provided by Mother Nature.Originally found as light-refractive inclusion bodies in Bacillus megate-rium cells more than 90 years ago, polyhydroxyalkanoate (PHA) biopolyesters currently are in the scientific spotlight of microbiology, systems biology, synthetic biology, material scientists, and process engineers due to their versatile material features, which make them attractive for use in various sectors of the plastic market. 7,8From the microbiological point of view, it is more and more understood that the presence of PHA in microbial cells from the domains Bacteria and Archaea serves for protection of the cells against starvation and a range of other stress factors, which have been only recently described to be correlated with PHA biosynthesis. 9In this context, UV-irradiation, 10 oxidative stress, 11,12 hypertonic shock, 13 freezing, 14 or elevated temperature 12 are described as stressors, which effects can be alleviated by the presence of PHA.Typically, PHA synthesis in microbes is favored by limitation of a nutrient essential for cell propagation, such as nitrogen or phosphate, in parallel to excessive availability of exogenous carbon source.Fig. 1 provides an electron microscope image of PHA-rich cells of the bacterium Cupriavidus necator cultivated in a continuously operated bioreactor cascade on glucose as carbon source.
Mainly the price of the latter determines the production price of PHA, which calls for the use of inexpensive carbon sources for large-scale PHA production. 8In this context, biorefinery concepts are currently in status of development to study the possibility of using carbon-rich side streams of different industrial processes as substrates for PHA production. 15,16As an example, the coupling of PHA production to the rendering and animal processing industry was successfully demonstrated by feeding PHA-producing microbes low quality biodiesel fractions stemming from transesterification of animal-based waste lipids, crude glycerol, and offal materials. 17Other biorefinery concepts include PHA production from lignocellulose-rich waste like bagasse, wood or straw, 18,19 side streams of the sugar production, 20 the use of municipal wastewater, 21 waste of the pulp and paper industry, 22 surplus whey from dairy and cheese making processes, 23 side streams of olive oil production, 24 or CO 2 -rich effluent gas from power plants. 25][28][29][30] Beside the raw material aspect, downstream processing to recover PHA from microbial biomass in an efficient and environmentally sound fashion constitutes another crucial aspect of the PHA production chain.][33] Emerging trends in optimizing PHA recovery encompass application of supercritical fluids, [34][35][36][37] non-toxic solvents, 38 the use of ionic liquids, 39 and even the selective removal of non-PHA biomass via digestion by animals, such as the meal worm Tenebrio molitor. 40Moreover, genetic engineering of microbial strains is an emerging tool to facilitate PHA revovery. 41egarding microbial strain selection, we currently witness an emerging trend towards robust organisms with a broad substrate spectrum and well understood metabolome, proteome and genome.][44][45][46] Moreover, the use of different bioreactor systems, operated continuously or discontinuously, was exhaustively reviewed recently, indicating that bioreactor setup, process regime, and feeding strategy have major impact on PHA productivity, composition, and microstructure. 47

F i g . 1 -Electron microscope image of PHA-rich C. necator cells. The PHA content is well visible as white inclusion bodies ("carbonosomes"). Magnification: 20,000. Image kindly provided by E. Ingolić, FELMI-ZFE Graz.
Different groups of PHA From the application-oriented point of view, it is pivotal to produce PHA not only in a cost-efficient way, but, to an increasing extent, to design PHA and follow-up products of properties and material quality competitive with the material performance of their petrochemical counterparts.During the last years, the number of PHA of different monomeric composition, biotechnologically produced both by microbial wild type strains and genetically engineered organisms, has increased considerably.Beside rather simple aliphatic PHA homo-and heteropolyesters (the latter encompassing co-, ter-, and quarterpolyesters) which were described decades ago, more recent reports describe the production of PHA harboring unsaturated, branched, and even aromatic building blocks, many of them displaying intriguing material features regarding the degree of crystallinity (X c ), glass transition temperature (T g ) as a measure for the mobility of polymer chains, melting temperature (T m ), elongation at break (fracture strain, ε R ), tensile strength (σ R ), and others. 7,8In order to encompass all characteristic aspects of PHA present at the same time in an investigated biological sample, the expression "PHAome" was recently coined by Chen and Hajnal.This terminus describes the diversity of monomeric PHA building blocks and their distribution, PHA homopolyesters, random and blocky structured PHA heteropolyesters, occurrence of intracellular PHA blends, and different molar masses of accumulated PHA; hence, the "PHAome" reflects the whole of different types of PHA in a microbial sample at a defined time point during biosynthesis. 48The present review provides an overview of described types of PHA, compares their material characteristics, and illustrates production strategies applied to produce them by implementing suitable strain-substrate-process regime combinations.

From substrates to PHA building blocks
The principle enzymatic machinery needed for microbial PHA biosynthesis is already well explored and understood by the scientific community. 49In principle, carbon sources, such as sugars, fatty acids, glycerol, and others are converted to oxoacyl-CoA thioesters, most frequently to acetoacetyl-CoA generated by the 3-ketothiolase catalyzed condensation of the central metabolite acetyl-CoA.Now, the oxoacyl-CoA thioesters are reduced to the corresponding (R)-hydroxyacyl-CoA thioesters by the catalytic action of NADPH-dependent oxoacyl-CoA reductases; in analogy to ethanol production from the metabolic intermediate acetaldehyde by yeast, this step regenerates reducing equivalents; therefore, PHA biosynthesis can be considered as a "pseudo-fermentation" (reviewed by Braunegg et al.). 50

PHA synthases
Finally, polyester synthases, as the key enzymes of PHA biosynthesis, come into action; they catalyze the conversion of (R)-hydroxyacyl-CoA thioesters to polyesters by releasing free CoA.In general, high intracellular pools of ATP, NAD(P)H, and acetyl-CoA and low levels of free CoA are favorable for the activity of enzymes responsible for PHA biosynthesis; such conditions occur, e.g., when the tricarboxylic acid cycle or other central metabolic pathways are blocked due to the absence of a growth-essential growth factor like, e.g., nitrogen or phosphate sources. 50,51Class I and class II PHA synthases comprise enzymes consisting of only one type of subunit (PhaC) with molecular masses between 61 kDa and 73 kDa.According to their in vivo and in vitro substrate specificity, class I PHA synthases, e.g., the enzyme found in the best known PHA producer Cupriavidus necator, preferentially utilize various (R)-hydroxyacyl-CoA thioesters with the acyl group consisting of 3 to 5 carbon atoms.In contrast, class II PHA synthases (prototype organisms Pseudomonas aeruginosa or Pseudomonas putida) preferentially accept (R)-hydroxyacyl-CoA thioesters with the acyl group consisting of 6 to 14 carbon atoms as substrates.Such longer (R)-hydroxyacyl-CoA thioesters are generated by β-oxidation of the respective fatty acids.Here, it should be mentioned that the β-oxidation pathway first generates (S)-3-hydroxyacyl-CoA, which cannot be directly incorporated into mcl-PHA due to stereospecificity of the PhaC subunit, which requires (R)-isomers as substrates.Therefore, mcl-PHA biosynthesis from lipids requires specific isomerases to convert the (S)-into the (R)-isomers.Moreover, class III PHA synthases, such as those found in the prototype organism Allochromatium vinosum, encompass enzymes comprising of two different subunits, namely, the PhaC subunit with an amino acid sequence similar by about 21-28 % to class I and II PHA synthases, and the PhaE subunit with no similarity at all to class I or II PHA synthases.Class III PHA synthases preferentially accept (R)-hydroxyacyl-CoA thioesters with 3 to 5 carbon atoms.Finally, class IV PHA synthases, which catalyze PHA biosynthesis especially in B. megaterium, are similar to class III PHA synthases, but PhaE is substituted by the lower molecular PhaR subunit (reviewed by 51,52 ).

PHA depolymerases
Intracellular PHA degradation starts with the catalytic action of PHA depolymerases generating the (R)-3-hydroxyalkanoate monomers and oligomers thereof, which are reversibly oxidized by (R)-3-hydroxybutyrate dehydrogenase to acetoacetate, which subsequently undergoes conversion acetoacetyl-CoA by acetoacetyl-CoA synthetase, and, finally, is hydrolyzed by the reversible enzyme 3-ketothiolase to acetyl-CoA.Both intracellular PHA depolymerases, which are needed to maintain the cyclic PHA metabolism in cells, and extracellular PHA depolymerases, which are important for PHA biodegradation, are reported (reviewed by Aneja and Charles). 53In a nutshell, intracellular depolymerases are more substrate-specific, while extracellular depolymerases are rather simple esterases, which are also found in mushrooms; they are responsible, e.g., for compostability and biodegradability of PHA.Moreover, intracellular PHA depolymerization always occurs to a certain extent in parallel to PHA polymerization, even under conditions favoring PHA formation, which causes the steady assembly and disassembly of PHA in living cells ("cyclic nature of the PHA metabolism"). 54owever, under conditions boosting PHA biosynthesis, the activity of intracellular PHA depolymerases is drastically lower than that of PHA synthases.If PHA depolymerases are completely absent, as it is the case in recombinant Escherichia coli harboring PHA synthesis genes, but not the genes encoding PHA depolymerases, the production of ultra-high molecular mass PHA of several MDa can be obtained. 55Similar effects were observed after inactivating PHA depolymerase in the genome of Azotobacter vinelandii. 56More recently, PHA depolymerases were shown to be specific for oxoester linkages of PHA; thioester bonds in analogue polythioesters (PTEs) are not cleaved by these enzymes. 57ort chain length PHA (scl-PHA)

Typical features of scl-PHA
In dependence on the type of monomeric building blocks, short chain length PHA (scl-PHA) are differentiated from medium and long chain length PHA (mcl-PHA and lcl-PHA).Low glass transition temperatures (T g typically between -40 °C and -20 °C), a highly amorphous character (degree of crystallinity X c not exceeding 40 %), wide-ranging melting intervals, low melting temperature typically far below 100 °C, and low degrees of polymerization (molecular mass in most cases below 100 kDa) differentiate mcl-PHA and lcl-PHA on the one hand from scl-PHA on the other hand.In the scl-PHA case, high T g around 0 °C and more, sharp melting points exceeding 180 °C in the case of highly crystalline PHA, and high molecular masses (up to the MDa range) are typical material features. 58Concerning polydispersity indices (P i ) for molecular mass distribution in a PHA sample, generally lower values are reported for mcl-PHA than for scl-PHA, hence, scl-PHA display a more heterogeneous distribution of PHA chains of different length in a given sample (reviewed by Zinn). 59 merly known as Alcaligenes eutrophus, Ralstonia eutropha, or Wautersia eutropha, 60 a member of the bacterial family of Burkholderiaceae, is the best investigated scl-PHA production strain with a completely decrypted genome, 61 which makes this strain intriguing for genetic modification. 62

4-hydroxybutyrate (4HB) containing scl-PHA
As an exception among scl-PHA building blocks, 4HB is the only well-studied achiral monomer found in natural PHA.4HB was first described as PHA constituent by Y. Doi´s team, who discovered this novel monomer in PHA samples produced by C. necator when being supplied with butyric acid and the 4HB-precursors 4-hydroxybutyric acid or 4-chlorobutyric acid; feeding with butyric acid alone resulted in accumulation of PHB homopolyester.Presence of 4HB in PHA was confirmed by NMR, and the strong decrease in crystallinity in parallel to increasing 4HB fraction in PHA was noticed by the authors.Based on these results, this group carried out more in-depth investigations on the properties of this novel class of PHA using X-ray diffraction and DSC studies; they came to the conclusion that the integration of 4HB building blocks into the highly crystalline PHB matrix causes a considerably stronger drop in lattice crystallinity than shown for comparable fractions of 3HV in PHBHV copolyesters. 63Later, the impact of different 4HB fractions in poly(3HB-co-4HB) copolyesters on T m , T g , and storage modulus (E′) was investigated; it was shown that these parameters decreased with increasing 4HB fraction in PHA; yield stress and breaking stress only slightly decreased with the increase in the 4HB contents, while the elongation at break strongly increased.Moreover, improved thermal stability was observed for molded P(3HB-co-4HB) copolyester sheets with increased 4HB fractions. 64In addition, it was shown that poly(3HB-co-4HB) copolyesters are readily biodegradable and biocompatible, with the 4HB fraction considerably impacting the degradation rate. 65Direct comparison between poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (poly(3HB-co-3HV)) and poly(3HB-co-4HB) films showed that the latter is faster degraded by random chain scission in phosphate saline buffer than poly(3HB-co-3HV); adding PHB depolymerase to the degradation setups, it was demonstrated that degradation can be accelerated significantly by enzymatic action.Also in these bio-catalyzed experiments, degradation was faster for poly(3HB-co-4HB) films than shown for poly(3HB-co-3HV) or PHB.In all cases, bio-catalyzed degradation was reported to occur via surface erosion. 66These experiments opened the door for the broad biomedical application of poly(3HB-co-4HB) and the homopolyester poly(4HB).Türesin and colleagues resorted to poly(3HB-co-3HV) and poly(3HB-co-4HB) copolyesters of random distribution to manufacture biodegradable rod-shaped implants; these implants carried Sulperazone ® and Duocid ® , antibiotics applied to treat chronic osteomyelitis.The local in vivo release of these antibiotics by copolyester degradation was studied, showing that the type of antibiotic, drug loading, and additional implant surface coating significantly affect the profile of in vitro drug liberation.For Sulperazone ® , the rate and period of release from poly(3HB-co-4HB) implants were strongly dependent on the loading with the active ingredient.However, drug dissolution occurred significantly faster than PHA degradation, indicating that the release phenomenon was rather determined by the drug dissolution rate than on PHA degradation or diffusion.When implants were coated with the same type of PHA copolyester, the initial burst effect was significantly reduced, and the liberation rate considerably decreased.Constant drug release from coated implants lasted more than two weeks, while release from uncoated implants did not even take a week.In the case of using Duocid ® , the generated implants displayed rather smooth surfaces; here, drug release was considerably higher than observed for implants loaded with Sulperazone ® . 67emarkably, the homopolyester poly(4HB) has material features completely different from those observed for other scl-PHA, such as PHB or poly(3HB-co-3HV); it has an outstanding elongation at break of up to 1000 %, which makes it highly stretchable and flexible.For other "biopolymers" such as poly(glycolic acid) (PGA), poly(lactic acid) (PLA), or PHB, this value amounts to about 3-6 %, for poly(ε-caprolactone) (PCL), an elongation at break of about 60 % was reported.Oriented poly(4HB) fibers have tensile strength values of about 545 MPa, which is higher than shown for, e.g., PP sutures (410-460 MPa); this makes such biological fibers interesting as suture material.Moreover, poly(4HB) sutures have a significantly lower Young's modulus than other marketed monofilament sutures. 68The company Tepha, Inc., USA, markets a range of PHA-based biomedical products.E.g., poly(4HB)-made TephaFLEX ® sutures are US Food and Drug Administration (FDA) approved.By Tepha, Inc., poly(4HB) homopolyester is obtained by a specially engineered cultivation process based on transgenic Escherichia coli K12 as working horse.A range of additional surgical materials based on poly(4HB) or poly(3HB-co-4HB), e.g., meshes, threads, or films with advantageous mechanical features are produced by Tepha, Inc. (reviewed by 69 ).Regarding in vivo degradability, poly(4HB) absorption rate is only 8-52 weeks, which is considerably faster than reported for PHB.
In the meanwhile, several strain/substrate combinations were reported for biosynthesis of 4HB-harboring PHA.Using wheat straw hydrolysate as main carbon source and γ-butyrolactone (GBL) as 4HB precursor, Cesário et al. reported P(3HB-co-4HB) biosynthesis by Burkholderia sacchari DSM 17165.These authors also found that, in contrast to GBL, 1,4-butanediol is no suitable 4HB precursor for these organisms, and demonstrated the impact of different glucose/GBL ratios on the PHA fraction in CDM for fed-batch cultivation setups.While cultivation on pure glucose yielded 49.2 % of PHB homopolyester in CDM, only 7.1 % poly(3HB-co-4HB) copolyester (0.046 mol mol -1 4HB in PHA) were obtained on GBL as sole carbon source. 70Later, Miranda de Sousa Dias et al. used the same organism in controlled bioreactor cultivations on saccharose from a Brazilian sugar cane mill as main carbon source, with and without co-feeding of GBL.On saccharose only, PHB homopolyester was produced at a volumetric productivity of 1.29 g L -1 h -1 , a mass fraction of 0.52 g PHB per g biomass, and a final PHB concentration of 36.5 g L -1 .Co-feeding of GBL resulted in formation of poly(3HB-co-4HB) at a volumetric productivity of 1.87 g L -1 h -1 , a mass fraction of 0.72 g PHA per g biomass, and a final PHA concentration of 53.7 g L -1 .Thermoanalysis revealed improved material properties of the copolyester in terms of reduced T m (161 °C vs. 178-187 °C obtained for the homopolyester PHB) and decreased degree of crystallinity X c (24 % vs. 71 %), indicating its enhanced suitability for polymer processing. 71In another study, Cesário et al. demonstrated the high-cell density production of poly(3HB-co-4HB) by C. necator on crude glycerol and GBL, and of poly(3HB-co-3HV-co-4HB) from crude glycerol, GBL, and the 3HV-related precursor compound propionic acid. 72In accordance with previous findings by Lee et al., 73 propionic acid did not only act as 3HV precursor, but also boosted the conversion yield of GBL to 4HB.Analogous to other types of scl-PHA, high molecular masses of about 1 MDa were obtained for the produced biopolyesters. 72Similar attempts were accomplished by Hermann-Krauss et al. using the haloarchaeon Haloferax mediterranei to produce poly(3HB-co-4HB) from CGP, a side-product of biodiesel production, plus GBL as 4HB precursor; in this case, the terpolyester poly(3HB-co-3HV-co-4HB) was produced. 74This surprising outcome is based on a metabolic particularity of this organism, namely, 3HV production from structurally unrelated carbon sources such as sugars or glycerol.Additional poly(3HB-co-3HV-co-4HB) terpolyesters were produced by Aziz and colleagues, who cultivated Cupriavidus sp.USMAA2-4, an organism isolated from a Malaysian lake, in a high-productive DO-stat fed-batch process in a 2 L bioreactor on oleic acid, pentanol (3HV precursor), and GBL.Based on the substrate mix, fractions of 3HB, 3HV, and 4HB in PHA varied between 62 and 86 % (3HB), 9 and 13 % (3HV), and 4-24 % (4HB), respectively.Similar to previous studies, presence of 3HV and 4HB considerably reduced the crystallinity of the produced PHA samples, thus enhancing the physical and thermomechanical polyester properties, which was especially evident by lower melting points and improved elongation at break.Moreover, it was revealed that the tensile strength of the terpolyesters increases with increasing M w . 75n recent time, strategies have been developed to produce poly(3HB-co-4HB) in an inexpensive and efficient way.In this context, the osmophilic halobacterium Halomonas bluephagenesis TD01, an organism naturally not producing 4HB-containing PHA, was genetically engineered by inserting the gene orfZ, which encodes Clostridium kluyveri 4HB-CoA transferase, making this strain capable of poly(3HB-co-4HB) production when cultured on glucose and the 4HB precursor GBL.Pilot scale cultivations in 1-m³ bioreactors resulted in 83 g L -1 CDM, 0.61 g poly(3HB-co-4HB) per g CDM, and 16 mol-% 4HB in the copolyester; remarkably, it was possible to carry out these cultivations under open, non-sterile conditions due to the high salinity of the cultivation medium (60 g L -1 NaCl). 76Janwen et al. accomplished further engineering of this strain to make it capable of producing poly(3HB-co-4HB) from glucose as sole carbon source; here, comparative genome analysis was applied for "pathway debugging", resulting in 25 mol-% 4HB in the copolyester. 77These activities were followed by further studies, which demonstrated the viability of using the wild type form of this strain on a larger scale (5 m³) for PHB production under continuous and non-sterile conditions, using waste gluconate as inexpensive substrate. 78Recent development with this auspicious microorganism involved further genetic engineering via genome-wide random mutagenesis in order to make the strain resistant to toxic metabolites such as ethanol or short carboxylic acids during high cell density cultivation.Further genetic engineering of obtained strains was carried out for overexpression of PHA synthesis genes, resulting in 90 g L -1 CDM and 0.79 g PHA per g CDM in 7 L bioreactor setups, which is considerably higher than values obtained with the wild type strain (81 g L -1 and 0.97 g g -1 , respectively). 79Other representatives of Halomonas sp. are currently being investigated for production of PHB homopolyester under non-sterile conditions, such as Halomonas halophila, a strain which converts various inexpensive carbon sources, such as lignocellulose hydrolysates, molasses, or hydrolyzed cheese whey, for biomass growth and PHB formation; up to 0.8 g PHB per g CDM were obtained under halophilic cultivation conditions (66 g L -1 NaCl).Remarkably, the authors demonstrated that molecular mass of the produced PHB strongly depends on the salinity of the cultivation medium; higher salinity (up to 100 g L -1 ) increases M w to up to 810 kDa, which is considerably higher than about 420 kDa obtained under moderately halophilic conditions (20 g L -1 NaCl).At the same time, polydispersity of the polymer by trend increases with increasing salinity.This finding allows fine-tuning molecular mass by triggering the salt concentration. 44s shown by Lv and colleagues, clustered regularly interspaced short palindromic repeats interference (CRISPRi) can be used to trigger poly(3HBco-4HB) biosynthesis in E. coli by parallel fine-tuning gene expression and regulating the expression of multiple genes.For this purpose, a pathway was constructed in E. coli for poly(3HB-co-4HB) biosynthesis from glucose.The native gene sad, which encodes succinate semi-aldehyde dehydrogenase in E. coli, was expressed under the control of CRISPRi using five specially designed single guide RNAs (sgRNAs) to regulate the carbon flux to 4HB biosynthesis; poly(3HB-co-4HB) with 1-9 mol-% 4HB were obtained with this system.Moreover, succinate, generated by succinyl-CoA synthetase and succinate dehydrogenase, respectively, (encoded by genes sucC, sucD and sdhA, sdhB) was directed favorably towards the 4HB precursor by applying selected sgRNAs (sucC2, sucD2, sdhB2 and sdhA1) by CRISPRi.Depending on the expression levels of down-regulated gene, the resulting molar 4HB fraction in copolyesters ranged from 1.4 to 18.4 mol-%. 80

Sulfur-containing scl-PHA analogues
Apart from naturally occurring scl-PHA, which constitute polyoxoesters of alkanoic acids, biosynthesis of polythioesters (PTEs), polymers of non-naturally-occurring mercaptoalkanoic acids, such as poly (3-mercaptopropionate), also find attention in the relevant scientific literature. 81These materials are biosynthesized by the action of PHA synthases in recombinant prokaryotes (Cupriavidus sp., E. coli, and Advenella sp.) starting from organosulfur compounds, namely, mercaptoalkanoic acids, with 3-mercaptopropionate (3MP) being the best described among suchprecursors. 82As a major shortcoming, the preparation of these mercaptoalkanoic acids is exceedingly expensive, which currently hampers an economically feasible production of these compounds on the industrial scale.However, PTEs revealed lower crystallinity and, for some cases, higher thermal stability in comparison to their corresponding oxoester analogues (PHAs), which makes them stimulating for different technological applications.In addition, PTEs display astonishing persistence against biodegradation; no biodegradation of PTEs was observed when using a variety of bacteria and fungi capable of degrading PHA polyoxoesters. 83To date, no organisms or habitats have been detected were PTE becomes biodegraded. 82Remarkably, copolyesters of 3HB and 3MP, poly(3HB-co-3MP), are readily degraded by natural depolymerases due to their specificity for the oxoester bonds. 57When aspiring a cost-efficient production of persistent organosulfur polymers, it would be indispensable to develop metabolic pathways allowing for de novo biosynthesis of the sulfur-containing compounds. 82dium chain length PHA (mcl-PHA)

Typical features of mcl-PHA
Mcl-PHA, typically containing monomers with 6 to 14 carbon atoms, are considerably less crystalline than scl-PHA.Some mcl-PHA contain building blocks with chemical groups, e.g., olefinic or epoxy-groups, which allow for their chemical or enzymatic post-synthetic modification in order to finetune the polymer properties.Macroscopically, mcl-PHA often have the appearance of biological rubbers or latexes; due to their remarkably low T g values, their amorphous structure does not easily change into a crystalline structure, which prevents them from becoming brittle even at temperatures well below freezing.Pseudomonas putida (formerly known as Pseudomonas oleovorans) is the mcl-PHA production strain most frequently used by research groups all over the world. 59This organism has the beneficial feature of incorporating functionalized (unsaturated, halogenated, epoxy-group harboring, etc.) monomers in growing mcl-PHA chains when supplied with suitable functionalized precursor compounds.In this context, a typical rubber-like material was produced already in 1993 by de Koning and colleagues, who generated a mcl-PHA consisting of saturated and unsaturated building blocks by co-feeding Pseudomonas oleovorans with mixtures of n-octane and 1-octene.From the praxis-oriented perspective, the obtained material displayed inappropriate material features in terms of too low melting point and insufficient crystallization rate; however, the authors were able to profit from the functional (olefinic) groups, and subjected the polyester to post-synthetic modification by crosslinking the polyester chains via their pendant unsaturated groups with electron-beam irradiation.The final material revealed constant properties over a temperature range from -20 °C to 170 °C, and was still biodegradable. 84Regarding the physical properties, mcl-PHA typically show expedient elongation at break of some 100 %; for the scl-PHA PHB, this value is only about 6 %.Comparing values for tensile strength of different types of PHA, most scl-PHA representatives have values of about 30 -40 MPa, which is drastically higher than the value of about 10 MPa, which is usually measured for mcl-PHA.In this context, the scl-PHA homopolyester poly(4HB) constitutes a remarkable exception, having an elongation to break of about 1000 %; moreover, the T g of P(4HB) (about -50 °C) is in the same range as for mcl-PHA. 58Regarding molecular mass, mcl-PHA produced by class II PHA synthases have typically lower molecular weights (50-500 kDa) than scl-PHA produced by the enzymatic action of class I synthase. 85As an example, Rodrigues and colleagues recently reported the production of mcl-PHA copolyesters mainly consisting of 3-hydroxyhexanoate (3HHx) and 3-hydroxy-9-octadecenoate when cultivating the strains C. necator (IPT 026 and IPT 027) and Burkholderia cepacia (IPT 119 and IPT 400) on crude palm oil.These polymers, highly amorphous and thermostable materials, revealed molecular masses typically low for mcl-PHA (170-400 kDa). 86Fig. 3 illustrates the mcl-PHA building blocks discussed in the present review.

Mcl-PHA biosynthesis via b-oxidation of fatty acids
Typically, mcl-PHA is produced by microbes via β-oxidation of fatty acids towards mixtures of (S)-acyl-CoAs of different length, which, after enzymatic isomerization to the corresponding (R)acyl-CoAs, are typically polymerized towards struc-turally related mcl-PHA copolyesters by class II synthases. 87Therefore, mcl-PHA generally constitute heteropolyesters of different building blocks with six or more carbon atoms.As an example, the strains Pseudomonas citronellolis and Pseudomonas chlororaphis accumulated mcl-PHA of typical sticky, resin-like character, consisting of 3HHx, 3-hydroxyheptanoate (3HHp), 3-hydroxyoctanoate (3HO), 3-hydroxynonanote (3HN), and 3-hydroxydecanoate (3HD), when cultivated on fatty acids methyl esters in fed-batch bioreactor setups. 88,89In 2007, Liu and Chen demonstrated that β-oxidation weakened mutants of P. putida KT2442, which were generated by knocking out 3-ketoacyl-CoA thiolase and 3-hydroxyacyl-CoA dehydrogenase, when cultivated on tetradecanoic acid as sole carbon source, produce mcl-PHA with high shares (up to 49 %) of 3-hydroxytetradecanoate (3HTD), and low shares (3 %) of 3HHx if compared to the wild type strain.Remarkably, at the same time, the authors found oligomers of 3HTD in the cells, which evidences the restricted potential of the strain of polymerizing 3HAs of such long chain length.Moreover, increased shares of 3HTD resulted in higher crystallinity and tensile strength than reported for typical mcl-PHA with building blocks not longer than 3-hydroxydodecanoate (3HDD). 90

Mcl-PHA biosynthesis via fatty acid de novo biosynthesis
In addition, mcl-PHA production starting from fatty acid de novo synthesis from unrelated carbon sources, such as sugars or glycerol, was described. 91

F i g . 3 -Monomers of mcl-PHA in PHA polyesters discussed in the present review
In contrast to β-oxidation, fatty acid de novo synthesis directly generated the (R)-isomers of acyl-CoAs.In this context, Sathiyanarayanan and colleagues only recently reported the production of poly(3HTD-co-3HDD-co-3HD-co-3HO) by the psychrophilic bacterium Pseudomonas sp.PAMC 28620, isolated from Arctic glacier fore-field soil.Using glycerol as sole carbon source, high PHA fractions of more than 50 wt.% in CDM were achieved.Such exotic mcl-PHA copolyesters were also obtained by exchanging glycerol with carbohydrates, which revealed low crystallinity and high thermal stability; moreover, as a typical feature also for other mcl-PHA, these materials had a rather low molecular mass of only about 30 kDa. 92 Similar findings were reported by Muangwong et al., who investigated four strains isolated in Thailand from soils contaminated with used cooking oil (UCO).These strains, classified as Acinetobacter sp., Pseudomonas sp., Enterobacter sp., and Bacillus sp., accumulated mcl-PHA copolyesters consisting of 3HO and the unsaturated building block 3-hydroxy-(5Z)-dodecenoate from crude glycerol stemming from UCO-based biodiesel production.Similar to the results reported by Sathiyanarayanan et al., molecular mass amounted to about 30-40 kDa.Surprisingly, homopolyester poly(3-hydroxy-(5Z)dodecenoate) was accumulated by a Bacillus sp. when the cultivation was prolonged to 96 h. 93Genetic engineering was used by Mendonça et al., who equipped a PHA-negative mutant of B. sacchari with Aeromonas sp.PHA biosynthesis genes.This genetic construct was able to accumulate copolyesters of scl-3HA (3HB) and mcl-3HA from sucrose as sole carbon source. 94

Dual nutrient limited cultivation to produce mcl-PHA of tailored composition
Mcl-PHA biosynthesis is often based on the use of growth-inhibiting substrates like fatty acids, which drastically restricts volumetric productivity of the process; to overcome the inhibiting effect, one can resort to chemostat cultivations under a dual nutrient limited growth (DNL) regime, where both carbon and nitrogen sources are supplied continuously at concentrations immediately metabolized by the cells.The DNL concept originates from the biological law that concentration and composition of biomass and growth rate are not determined by a single limiting factor, but by the synergistic action of at least two limiting nutrients (reviewed by Zinn et al. 95 ).Substrate toxicity depends on the substrate´s concentration, hence, biomass growth is typically not influenced when the substrate is instantaneously converted and does not surpass a given threshold concentration.In the context of mcl-PHA production, DNL cultivation was tested using continuous cultivation of P. putida GPo1 on mixed carbon sources; here, mcl-PHA of unprecedented monomeric composition were produced.Mixtures of 5-phenylvalerate, octanoate, and 10-undecenoate yielded diverse poly(3-hydroxy-5-phenylvalerateco-3-hydroxyalkanoate-co-3-hydroxy-ω-alkenoate) copolyesters composed of aromatic, saturated, and unsaturated monomers.Depending on the substrate mix, the aromatic (3-hydroxy-5-phenylvalerate, 3H5PhV) fraction ranged from 0 to 0.52 mol mol -1 ; substrate feeding was accomplished at a rate according to substrate conversion by the microbes, which kept the actual substrate concentrations at almost zero.Surprisingly, increasing aromatic fraction correlated with a linear increase in the T g from -37.6 °C to -6 °C. 96Further DNL cultivation was carried out using mixtures of lactic acid and the aromatic compounds p-methylphenylvaleric acid or phenylvaleric acid, respectively, as co-substrates.When using p-methylphenylvaleric acid as co-substrate, the mcl-PHA homopolyester poly(3-hydroxy-5-p-methylphenylvalerate) was produced, a material displaying properties different to other, often completely amorphous, mcl-PHA copolyesters: it revealed significant crystallinity and a sharp T m at 99 °C, which is in contrast to other described mcl-PHA samples.Using phenylvaleric acid as co-substrate, the homopolyester poly(3H5PhV) was produced, which displayed typical mcl-PHA material properties.It should be emphasized that chemostat cultivation processes, especially using DNL substrate supply, might be the only efficient strategy to thrive PHA producing strains on toxic substrates like the described aromatic compounds. 97ng chain length PHA (lcl-PHA) Lcl-PHA are still scarcely described; these polyesters contain building blocks with more than 14 carbon atoms.The first report on a PHA containing 3-hydroxypentadecanoate (3HPD) was delivered by Barbuzzi and colleagues, who found 2 % 3HPD in PHA accumulated by Pseudomonas aeruginosa ATCC 27853 when cultivated on pentadecanoic acid as sole carbon source. 98A recent report describes lcl-PHA production by C. necator IPT027 from crude palm oil as inexpensive carbon source, which contains a high fraction of unsaturated fatty acids.This polymer consisted predominately of 3HD and diverse saturated and unsaturated building blocks with 18 carbon atoms, namely 3-hydroxyoctadecanoate, 3-hydroxy-9-octadecenoate, and 3-hydroxy-9,12-octadecadienaote.The material was of highly amorphous character and revealed low polydispersity and high thermal stability.In the same study, it was demonstrated that, switching from C. necator IPT027 to Burkholderia cepacia IPT400 while using the same substrate and cultiva-tion conditions, the polymer structure arrangement changed considerably, as evidenced by a decrease in crystallinity from X c = 48.47 % to X c = 27.80 %, which might have been due to the higher fractions of unsaturated building blocks found in the PHA produced by B. cepacia IPT400 than by C. necator IPT027. 99An even longer building block was detected only recently by Impallomeni et al. 100 These authors cultivated P. aeruginosa ATCC 27853 on long odd-numbered fatty acids with 17 to 21 carbon atoms (heptadecanoic acid, nonadecanoic acid, and heneicosanoic acid) under nitrogen-limited conditions.The resulting polyesters contained odd-numbered 3HAs from 3HV to the previously unprecedented monomer 3-hydroxyheptadecanoate (3HHpD).The materials were reported as sticky, soft, rubber-like materials having T g values between −45 and -39 °C, T m values between 48 and 52 °C, melting enthalpies of about 11 J g -1 , and low molar masses between 77 and 188 kg mol -1 .This study also showed, for the first time, that presence of magnesia ions in the cultivation medium is needed to allow the metabolization of such long-chain fatty acids towards PHA. 100 The longest PHA building block detected to date in natural PHA, 3-hydroxyoctadecanoate (3HOD), was described by Ray and Kalia, who obtained PHA copolyesters consisting of 3HD, 3-hydroxyhexadecanoate, and 3HOD after co-feeding Bacillus thuringiensis EGU45 with glucose, CGP, and propionic acid. 101The same building block was also detected by Guo and colleagues in PHA samples produced by a genetically engineered Pseudomonas mendocina cultivated on glucose as sole carbon source. 102Fig. 4 illustrates the lcl-PHA building blocks discussed in the present review.

3HV-containing heteropolyesters
The most frequently described member of the PHA family is PHB, the homopolyester of 3HB.This material was discovered about 90 years ago by Maurice Lemoigne (reviewed by 103 ), and, until 1974, it was considered the only type of natural PHA existing in microbes.Due to its high brittleness, high melting point (about 180 °C), and high crystallinity (typically 60-70 %), the application of PHB homopolyester, especially for packaging purposes, was strongly limited. 104In 1974, Wallen and Rohwedder isolated microbes from sewage water; the authors remarked that properties of the PHA produced by this microbial community differed considerably from those of PHB homopolyester; significantly lower T m below 100 °C and solubility in cold ethanol surprised these authors.Based on GC-MS analyses, it was confirmed that the polymer contained, beside 3HB, also 3HV units.To a minor extent, even 3HHx was discovered as the first unambiguously identified mcl-PHA building block; moreover, presence of 3HHp was evidenced, but not confirmed. 105The number of PHA monomers grew further in 1983, when Findley and White reported the composition of PHA extracted from estuarine sediments; these biopolyesters consisted of at least 11 different monomeric building blocks, among them, 3HB, 3HV, 3HHx, 3HHp, and 3HO were unambiguously identified by GC-MS; evidence was also given for iso-branched building blocks with five and seven carbon atoms, moreover, 3 peaks in the chromatogram were not assigned to a defined structure of a monomer.Parallel investigation described in this study revealed the complex composition of a PHA extracted from Bacillus megaterium ssp.globiguii, an organism containing Class IV synthase, when cultivated on a nitrogen-free minimal medium; surprisingly, this polymer turned out to contain, beside 3HB (95 %), also 3HHp (3 %), traces of 3HV and an additional, yet unidentified building block. 106This was in contrast to other B. megaterium ssp.isolated later, such as B. megaterium uyuni isolated from a Bolivian salt lake, which accumulated PHB homopolyester when grown on glucose-based, nitrogen-free minimal medium. 45Soon after, it was shown that the best-described scl-PHA production strains, like C. necator, require supplementation of precursor compounds F i g .4 -Monomers of mcl-PHA in PHA polyesters discussed in the present review structurally related to 3HV, if the production of poly(3HB-co-3HV) copolyesters instead of brittle PHB homopolyester is aspired; this was demonstrated for the first time by co-feeding butyric acid and the 3HV precursor pentanoic (= valeric) acid to A. eutrophus (today: C. necator); using pentanoic (=valeric) acid as sole carbon source, the molar 3HV fraction in PHA reached up to 90 %; based on the use of 13 C-labeled substrates, the authors elucidated the pathway for poly(3HB-co-3HV) copolyester biosynthesis by this strain, namely the conversion towards valeryl-CoA, the subsequent β-oxidation to 3-hydroxyvaleryl-CoA, and direct incorporation of the latter into PHA. 107Later, propionic acid was studied as another 3HV-precursor compound for C. necator; this compound is intracellularly converted to propionly-CoA, which couples with acetyl-CoA, thus forming the oxoacyl-thioester 3-keto-valeryl-CoA, which, after reduction to 3-hydroxyvalery-CoA, is incorporated as 3HV into growing PHA chains by class I PHA synthase (reviewed by Braunegg et al. 50).It was demonstrated by Lefebvre et al. that increased 3HV fractions in PHA can be achieved when restricting the dissolved oxygen concentration during cultivations supplied with glucose and sodium propanoate; this strategy prevents the oxidative decarboxylation of 3-hydroxyvalery-CoA towards 3-hydroxybutyryl-CoA, which generates 3HB at the expense of 3HV.However, this restricted oxygen supply results in reduced overall PHA productivity. 108Using mixed microbial cultures supplied with volatile fatty acids, Montano-Herrera and colleagues noticed that the fraction of 3HV in PHA strongly depended on the instantaneous availability of reducing equivalents NAD(P) H. 109 In the meanwhile, poly(3HB-co-3HV) production is described for a range of other different strains, such as Burkholderia parafungorum (previously known as Pseudomonas hydrogenovora), 110 Hydrogenophaga pseudoflava, 110 etc.In addition to propionic acid and valeric acid, inexpensive 3HV precursors are currently under investigation in order to save material costs for the bioprocess; in this context, levulinic acid, a material easily accessible from conversion of lignocellulosic biomass, was successfully used in its pure form, 111,112 or as constituent of acid hydrolyzed spruce wood 113 for poly(3HB-co-3HV) production.More recently, waste lipids were ozonolytically hydrolyzed towards a cocktail containing a certain amount of compounds with an odd number of carbon atoms, which in turn acted as 3HV precursor for C. necator. 114Kumar and colleagues reported an intriguing study on the use of microbial hydrolyzed agro-food waste for poly(3HB-co-3HV) production by different Gram-positive production strains.Among the investigated strains, Bacillus thuringensis showed expedient potential for PHA accumulation, with composition of the biopolyesters differing depending on the applied agro-food waste; while application of hydrolyzed pea shells generated PHB homopolyester, poly(3HB-co-3HV) production was achieved using hydrolyzed apple pomace, onion peels or potato peals. 115A different direction is possible when using representatives of the haloarchaea, a group of ancient organisms occupying highly saline habitats.As demonstrated in the case of Hfx.mediterranei and Halogeometricum borinquense, such organisms produce poly(3HB-co-3HV) without the need for 3HV-related precursor compounds; feeding these strains with simple pure sugars, 116 hydrolyzed bagasse, 117 or glycerol 74 is sufficient to make them produce copolyesters.Only recently, copolyester production from inexpensive unrelated substrates was detected also in eubacteria, when Bacillus licheniformis PL26 was shown to accumulate poly(3HB-co-3HV) from crude glycerol from the biodiesel production. 118

Nodax-copolyesters
Although the incorporation of 3HV into the crystalline PHB matrix can reduce X c and T m of resulting poly(3HB-co-3HV) copolyesters, this effect is not sufficient for many applications due to the so-called "isodimorphism", describing the phenomenon of 3HV units being well integrated into the 3HB crystal lattice and vice versa, which prevents expedient disruption of crystallinity.Higher lattice disruption is obtained when integrating mcl-PHA building blocks, such as 3HHx or longer analogues.Resulting poly(3HB-co-mcl-3HA) with a rather low fraction of mcl-HA copolyesters are termed "Nodax"-PHA.These polymers are accessible either via bacterial fermentation of the wild type bacteria Aeromonas hydrophila or Aeromonas caviae, hence, organisms containing class III PHA synthases, on long chain fatty acids, or by genetically engineered C. necator harboring both the natural class I PHA synthase and Pseudomonas fluorescens GK-13 class II PHA synthase on sugars and fatty acids.Poly(3HB-co-3HHx) is the most simple type of "Nodax"-PHA.Based on the decreased crystallinity, Nodax copolymers are characterized by high toughness and ductility, and show convenient thermal properties similar to those of PE.Incorporation of mcl-3HA monomers efficiently lowers the X c and T m similar as observed for α-olefins (1-alkenes) in linear LDPE, resulting in a broad window of processibility, hence, a large difference between T m and decomposition temperature T d , which facilitates a range of processing techniques.While the chain length of the mcl-3HA component has only insignificant effect on reduction of T m and X c , it strongly dictates the polymers flexibility.Nodax copolyes-ters are reported to be completely biodegradable in both aerobic and anaerobic environments, are compatible with other (bio)polymers such as PLA, and exert expedient oxygen and flavor barrier properties, which makes them auspicious for packaging application in the food sector (reviewed by Noda et al. 119 ).

3-hydroxypropionate (3HP) containing scl-PHA hetero-and homopolyesters
In 1991, Nakamura and colleagues from Y. Doi´s research team discovered 3HP as a new PHA building block when supplying nitrogen-depriviated C. necator cultures with 3-hydroxypropionate (3HP), 1,5-pentanediol, or 1,7-heptanediol as sole carbon sources.Depending on the precursor, 3HP fractions in poly(3HB-co-3HP) copolyesters varied between 0 and 7 mol-%, with increasing 3HP fractions resulting in a decrease in T m from 178 °C (PHB homopolyester) to 150 °C for poly(3HB-co-7 %-3HP). 120Based on the fact that 3HP turned out as the precursor resulting in highest 3HP fractions, the authors carried out follow up studies by co-feeding Alcaligenes latus ATCC 29713 with sucrose and 3HP at different ratios in 48 h shaking flask cultivations.On sucrose only, PHB homopolyester was accumulated, whereas 50/50 mixtures of sucrose/precursor resulted in highest 3HP molar fractions of 26 % in poly(3HB-co-3HP).However, CDM dropped from 4 g L -1 (pure sucrose; 0.6 g g -1 PHA in CDM) to 2 g L -1 (50/50 mixtures; 0.29 g g -1 PHA in CDM); using 3HP as sole carbon source, bacterial growth was completely inhibited, which indicated the inability of A. latus to convert 3HP to acetyl-CoA, which is in contrast to the above report using C. necator, which is able to grow on 3HP as sole carbon source.With increasing 3HP fractions, T m dropped from 177 °C (PHB) to 85 °C (poly(3HBco-26 %-3HP)); at the same time, T g also decreased from 4 °C (PHB) to -8 °C (poly(3HB-co-26 %-3HP).Together with the trend of heat fusion enthalpy values (δH m ) also decreasing with increasing 3HP fraction, the authors assumed that 3HP lowers the crystallinity of copolyesters containing this monomer. 121his was confirmed in subsequent studies by Shimamura and colleagues, who observed decreasing crystallinity in A. latus poly(3HB-co-3HP) with up to 0.88 mol mol -1 3HP using X-ray diffraction characterization; this study also revealed improved enzyme-catalyzed biodegradability of poly(3HB-co-3HP) films containing increased 3HP fraction. 122ndreeßen and Steinbüchel explained this decreased crystallinity and increased biodegradability by the missing methyl group in the monomer´s backbone. 123Kang and colleagues discovered poly(3HBco-3HP) biosynthesis when supplementing the methylotrophic soil bacterium Methylobacterium sp.KCTC 0048 on methanol as carbon source plus the precursor 3HP. 124The first report on poly(3HB-co-3HP) by recombinant bacteria was provided by Valentin et al., who supplied rec.E. coli expressing PHA synthesis genes (from C. necator) and genes encoding for propionyl-CoA synthetase (from Salmonella enterica) with 3HP; in this study, outstandingly high molar 3HP fractions in poly(3HB-co-3HP) of up to 0.9 mol mol -1 were achieved when co-supplying mannitol and 3HP. 125In addition to E. coli, also C. necator was genetically engineered to make it produce poly(3HB-co-3HP) from structurally unrelated carbon sources.Fukui and colleagues introduced malonyl-CoA reductase and propionyl-CoA synthase from the CO 2 -fixation pathway of the green non-sulfur bacterium Chloroflexus aurantiacus.Via malonyl-CoA, 3HP-CoA was generated from acetyl-CoA in the recombinant strain.Poly(3HB-co-3HP) with 3HP fractions from 0.2 to 2.1 mol% were obtained using fructose, gluconate or alkanoates (octanoate, dodecanoate). 126A later study demonstrated the first biosynthesis of poly(3HP) homopolyester, a material of typical thermoplastic properties; anaerobic Clostridium butyricum genes encoding for glycerol dehydratase, Salmonella enterica genes encoding for propionaldehyde dehydrogenase, and C. necator PHA synthase genes were expressed in rec.E. coli.Up to 12 wt.%poly(3HP) were obtained in a two-step fedbatch fermentation process using glycerol as sole carbon source.Importantly, the second stage of the process (PHA accumulation) had to be carried out under anaerobic conditions due to the nature of the glycerol dehydratase, which resulted in rather low productivity. 127Considerably higher poly(3HP) yields were obtained by Wang et al. in optimized, entirely aerobic, 3-Liter bioreactor fed-batch fermentation setups; these authors used recombinant E. coli expressing the aerobic Klebsiella pneumonia genes encoding for glycerol dehydratase, propionaldehyde dehydrogenase from Salmonella typhimurium, and C. necator PHA synthase for glycerol-based cultivations.Up to 22 g L -1 CDM and 10.1 g L -1 P3HP, corresponding to 0.46 g PHA per g CDM, were achieved by this experiment. 128A new poly(3HP) biosynthesis pathway to biosynthesize poly(3HP) homopolyester by rec.E. coli was developed by Wang et al.Malonyl-CoA reductase from Chloroflexus aurantiacus, E. coli propionyl-CoA synthetase and acetyl-CoA carboxylase, and C. necator PHA synthase were expressed by this recombinant strain.When using glucose as sole, unrelated carbon source, CDM and poly(3HP) mass fraction in CDM amounted to 1.32 g L -1 and 0.01 g g -1 , respectively. 129

PHA heteropolyesters containing branched building blocks
Watanabe and colleagues described the production of PHA containing α-methylated, hence, branched building blocks in genetically engineered E. coli cells harboring the PHA synthase of A. caviae.When cultivated on tiglic acid [(2E)-2-methylbut-2-enoic acid)], this strain accumulated a new copolyester of 3HB and 3-hydroxy-2-methylbutyrate (3H2MB) with reduced T m , T g , melting enthalpy (δH m ), and T c if compared to PHB homopolyester.Especially the decreased T c indicated that poly(3HBco-3H2MB) showed higher affinity towards crystallization than previously described scl-PHA copolyesters. 130Further pathway engineering of this strain carried out by Furutate and colleagues, by weakening its β-oxidation metabolism and inserting a propionyl-CoA transferase gene, enabled the production of another new class of PHA copolyesters: cultivation in tiglic acid and hexanoic acid resulted in accumulation of poly(3H2MB-co-3HHx); these copolyesters, consisting of both branched scl-PHA and linear mcl-PHA building blocks, revealed low T g values and totally lacking T m , hence, these products show no crystallinity in contrast to poly(3HBco-3H2MB). 131In addition, the production of PHA harboring 3HB, 3HV, 3H2MV and 3-hydroxy-2methylbutyrate (3H2MB) was reported by Dai and colleagues, who cultivated a mixed culture rich in glycogen-accumulating organisms with high phylogenetic similarity with Defluviicoccus vanus under anaerobic conditions in not nutrient-limited media; propionic acid and acetate were used as carbon sources, with higher propionic acid fractions favoring incorporation of the branched monomers.Depending on the fractions of the branched building blocks, the generated poly(3HB-co-3HV-co-3H2MB-co-3H2MV) copolyesters had a molecular mass of 390-560 kg mol -1 , T m of 70 -160 °C, and T g between -8 and 0 °C.Generally, the authors observed that incorporation of the branched monomers generated defects in the poly(3HB-co-3HV) lattice, thus resulting in decreased crystallinity of poly(3HB-co-3HV-co-3H2MB-co-3H2MV) copolyesters. 132opolyesters harboring 3-hydroxy-4-methylvalerate (3H4MV) as another branched building block are still scarcely described in literature; however, these materials are considered auspicious for applications due to the fact that they are not prone to increasing crystallization with time ("aging") as is the case with PHB or poly(3HB-co-3HV).Up to now, production of 3H4MV-containing PHA was reported for wild type and genetically modified organisms.The first report on 3H4MV-containing PHA was provided by Tanadchangsaeng et al., who equipped a genetically engineered, PHA-negative C. necator strain with A. caviae PHA synthesis genes.Feeding the recombinant organism with fructose and 4-methylvaleric acid resulted in accumulation of poly(3HB-co-3H4MV) with 3H4MV fractions in PHA up to 0.35 mol mol -1 .These polyesters were well fractionable into randomly distributed copolyesters with 3H4MV fractions up to 0.47 mol mol -1 ; crystallinity, T m and T g values strongly decreased with increasing 3H3HV fractions, whereas susceptibility to enzymatic degradation increased in parallel. 133Similar attempts were reported by Chia and colleagues, who equipped the same PHA-negative C. necator with Chromobacterium sp.USM2 PHA synthase genes.Feeding this recombinant strain with palm kernel oil and 4-methylvaleric acid resulted in accumulation of scl-mcl-PHA copolyesters containing 3HB, 3HV, 3HHx and 3H4MV.Exemplarily investigating films of one of the products, poly(3HB-co-1 mol% 3HV-co-3 mol% 3H4MVco-18 mol% 3HHx) revealed excellent rubber-like properties of this material in terms of higher thermal stability, higher elasticity, outstanding flexibility, and higher ductility if compared with poly(3HB) and even poly(3HB-co-3HHx). 134The wild type strain Chromobacterium sp.USM2, donator of the PHA synthase in the study of Chia et al., 134 was also directly used by Ling et al. to investigate production of 3H4MV-containing PHA.These authors reached a molar 3H4MV fraction of 22 % when using 4-methylvaleric acid as sole carbon source.In addition, these authors found that decreased oxygen concentration in the cultivation medium was beneficial for poly(3HB-co-3H4MV) accumulation without drastically affecting cell growth at the same time. 135Later, Saika and associates remarked that small amounts (0.5 mol%) of 3H4MV were produced by PHA-negative C. necator harboring Pseudomonas sp.61-3 PHA synthase when cultured on fructose as sole carbon source.Co-feeding with excess amounts of the branched amino acid leucine as 3H4MV structurally related precursor compound was found to increase the production of 3H3MV.Even higher fractions of this branched monomer were obtained after manipulating the leucine metabolism of the production strain by manipulating the leucine feedback inhibition; up to 3.1 mol% 3H3MV plus 1.2 mol% 3HV were obtained by this new engineered strain.Again, the copolyester displayed enhanced thermal properties in comparison to PHB or poly(3HB-co-3HV) due to decreased crystallinity. 136Lau and colleagues investigated the wild type organism Burkholderia sp.USM and its transformant harboring Aeromonas caviae PHA synthase gene for 3H4MV formation by cultivations on hexoses and 4-methylvaleric acid.Whereas the wild type produced only minor amounts (up to 1 mol%) of 3H4MV in poly(3HB-co-3H4MV) copolyesters, up to 40 mol% of 3H4MV were reached in the transformed organism.Moreover, the authors demonstrated that 3H4MV-containing PHA is intracellularly mobilized by the strain by culturing copolyester-rich cells in carbon-free medium. 137

PHA heteropolyesters containing other unusual building blocks
The first report on PHA harboring aromatic building blocks was provided in 1991 by Kim et al., who supplied P. oleovorans with mixtures of 5-phenylvaleric acid and the alkanoic acids n-nonanoic acid or n-octanoic acid, respectively.When using 5-phenylvaleric acid parallel with n-nonanoic acid, odd-numbered 3-hydroxyalkanoates (C5-C11) and 3H5PV building blocks were detected; in the case of using octanoic acid plus 5-phenylvaleric acid, even numbered 3-hydroxyalkanoates (C6-C10) were found parallel with 3H5PV.It was shown that the fraction of aromatic building blocks drastically increased with increasing concentrations of 5-phenylvaleric acid in the substrate feed.Surprisingly, the authors provided no comment on the fact of having detected building blocks longer than the provided substrates. 138oreover, the occurrence of PHA building blocks with diverse functional groups other than unsaturated or aromatic groups was described years ago for halogenated PHA or PHA harboring epoxy-, ester-, or cyano groups.In each of these exotic cases, P. putida acted as production strain.PHA with 9-cyano-3-hydroxynonanoate and 7-cyano-3-hydroxyheptanoate as monomers were obtained by co-feeding n-octanoic or n-nonanoic acid and 11-cyanoundecanoic acid as carbon sources, 139 whereas bromated PHA was produced using nonanoic or octanoic acid and 6-bromohexanoic acid, 8-bromo-octanoic acid or 11-bromoundecanoic acid as substrate for P. oleovorans. 140Chlorinated PHA was produced using the same organism by Doi and Abe, who applied mixtures of octane and 1-chlorooctane as carbon source in bioreactor cultivations.According to 1 H-and 13 C-NMR analysis, a random copolyester containing 3HHx, 3HO, 6-chloro-3-hydroxyhexanoate and 8-chloro-3-hydroxyoctanoate was produced.This material turned out to be completely amorphous; no melting endotherms were observed in the respective thermograms.M n of this new PHA amounted to only 87 kDa. 141The same research group reported also on production of ω-fluorinated PHA building blocks when cultivating P. putida on mixtures of nonane and 1-fluorononane. 142Later, these scientists improved this process by substituting nonane with gluconic acid; thus, it was possible to produce new PHA copolyesters in a controlled manner with a molar fraction of fluorinated build-ing blocks between 0 and 40 %.The materials revealed melting temperature between 50 and 60 °C, and showed deceasing melting enthalpy with increasing fluorinated fraction. 143Using a mixture of 10-epoxyundecanoic acid and sodium octanoate as substrate enabled the formation of PHA with pendent epoxy groups; relative high molecular mass (M n about 140 kDa), and very low T g values (-30 to -60 °C) were determined for these novel polyesters.These materials can further undergo chemical modification, e.g., towards carboxy groups, which provides PHA of outstanding hydrophilicity, making it even water-soluble. 144It has to be emphasized that those exotic PHAs were produced at low productivity, and only tiny amounts of these polymers were de facto generated; based on the high costs for the required functionalized co-substrates, no significant activities were reported over the last two decades on such unusual biopolymers.By chemical means, PHA bearing epoxide groups were produced by post-synthetic conversion of terminal alkene groups of unsaturated mcl-PHA in order to facilitate crosslinking of these polymers, 132 or the terminal groups were oxidized to diol 145 or carboxyl 146 groups to increase the hydrophilicity of the products.Scholz and colleagues utilized methyl, ethyl, and tert-butyl esters of octanoic, nonanoic and decanoic acid as feedstocks for P. putida.All applied substrates resulted in accumulation of PHA copolyesters with up to eight different monomers.Especially methyl esters gave copolyesters with a high degree of monomers with pendent methyl ester groups, while ethyl esters resulted in generation of copolyesters with unsubstituted monomers, monomers with pendant methyl-and ethyl ester groups.In contrast, the tert-butyl ester group was found in the generated PHA samples.Remarkably, the authors elucidated that the fraction of ester groups found in the polymers strongly depended on the oxygen supply; while restricted oxygen availability resulted in conversion of the substrate´s carboxylate group rather than in formation of a new carboxylate group, hence to formation of 3-hydroxyalkanoates, excess supply of oxygen resulted in an attack of the substrate´s alkane end, thus preserving the carboxylate group, therefore found in the polyesters. 147

PHA quarterpolyesters
As a rather new class of PHA, so-called "PHA quarterpolymers" were recently reported by Zhila and Shishatskaya. 148This group of heteropolyesters contains four different types of monomers, namely chiral scl-PHA building blocks with different side chains (3HB and 3HV), an achiral scl-monomer (4HB), and a mcl-PHA building block (3HHx).Using C. eutrophus B10646 as production strain in twostage batch cultivation setups, the physicochemical, mechanical, and biological properties of poly(3HBco-3HV) copolyesters (10.4 -75.0 mol% 4HB; in the publication: "bi-polymers"), poly(3HB-co-3HVco-4HB) terpolyesters (28.7 -55.6 mol% 4HB), and poly(3HB-co-3HV-co-4HB-co-3HHx) quarterpolyesters (9.3 -13.3 mol% 4HB) were studied.Glucose acted as main carbon source for all produced polyesters, valeric acid, GBL, and hexanoic acid were used as precursors for 3HV, 4HB, and 3HHx production, respectively.By comparison with PHB homopolyester prepared in the same experimental series, it was shown that increasing fractions of 4HB improved the processability of the polyesters by lowering crystallization temperature, melting point, and crystallinity, and by increasing the flexibility of the material, expressed as elongation at break.These beneficial effects were enhanced by the presence of 3HV in ter-and quarterpolymers, and by 3HHx in quarter-polymers.Remarkably, all heteropolyesters had significantly lower molecular mass (M w between 540 and 830 kDa) than the homopolyester (M w almost 1 MDa), but almost identical thermal stability.Films produced from the different PHA samples showed similar microstructure and porosity; none of them revealed any toxic effect on fibroblasts, thus underlining the biocompatibility of these materials.As shown previously, co-and terpolyesters containing, beside 3HB, one or two of the monomers 3HV, 4HB and 3HHx, can also be produced by this strain under mixotrophic cultivation conditions by replacing glucose with CO 2 in a recycled-gas closed-circuit cultivation system. 148

mcl-PHA homopolyesters
In vivo synthesis of mcl-PHA homopolyesters to date is described exclusively with the use of genetically engineered microorganisms. 150In most cases, mcl-PHA are synthesized starting with β-oxidation of fatty acids (vide supra), which produces a cocktail of 3-hydroxyalkyl-CoAs, encompassing 3-hydroxyhexanoyl-CoA and other monomers with longer side chains, with the maximum length of monomers depending on the chain length of the metabolized fatty acid. 88β-oxidation-weakened mutants of P. putida were generated by Liu and colleagues, who knocked out the genes encoding for the key enzymes catalyzing fatty acid degradation and for the 3-hydroxyacyl-CoA-acyl carrier protein.
When supplied with decanoic acid as a carbon source, the obtained β-oxidation-weakened mutant strain successfully accumulated poly(3-hydroxydecanoate) homopolyester, while poly(3HDco-84 %-3HDD) was generated when feeding with dodecanoic acid; this is in contrast to the wildtype strain, which produces copolyesters composed of three (3HHx, 3HO, 3HD) or four (3HHx, 3HO, 3HD, 3HDD) different monomers, respectively. 151pplying this β-oxidation weakening approach to Pseudomonas entomophila L48, similar results were reported.Using the new strain P. entomophila LAC23, an almost pure (3HDD molar fraction >99 %) poly(3HDD) homopolyester was obtained from dodecanoic acid, with polymer fractions in biomass exceeding 0.9 g g -1 . 152Later, Wang et al. used this organism for cultivations on defined C7-C14 fatty acids (heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, and tetradecanoic acid) as sole carbon sources in order to obtain the homopolyesters of corresponding 3HAs.Although productivities were rather modest due the high toxicity of the substrates, the authors succeeded in producing the desired homopolyesters in almost pure form; only traces of monomers shortened by two carbon atoms due to β-oxidation were detected, which allowed them to study the properties of different mcl-PHA homopolyesters for the very first time.As a remark-able finding, it was shown that T g and thermal stability of mcl-PHA homopolyesters of odd-numbered monomers (C7, C9, C11, C13) by trend increase with increasing number of carbon atoms in the monomer, while the opposite trend was observed for homopolyesters with even number of carbon in monomers.Independent of the monomers being odd or even, T m increased with increasing side chains of monomers.The authors emphasized that, after hydrolysis of these homopolyesters, pure chiral bioactive monomers can be obtained, which are of high pharmaceutical value. 153crostructure of PHA: Random distribution vs. Blocky structure In addition to the monomeric composition, the microstructure of PHA is another decisive parameter for the material properties of the biopolymer.Generally, blocky structured PHA heteropolyesters (b-PHA), PHA heteropolyesters with monomers randomly distributed, and polymer blends can occur in living cells (see Fig. 5).
Chains of b-PHA consist of at least two distinctive, covalently linked polymer sections (blocks).Properties of each block are encompassed by this structure, which opens the door to new polymer properties, not accessible by simple blending of polymers with the structure of the individual bocks.In a nutshell, production of b-PHA offers the possi-bility of biopolyesters with tunable, pre-defined microstructure.Moreover, b-PHA are reported to be less prone to polymer aging. 154Examples of b-PHA copolyester structures are diblocks (e.g., [3HB] x -[3HV] y ), triblocks (e.g., [3HB] x −[3HV] y −[3HB] z or [3HB] x −[3HV] y −[4HB] z ), or repeating multiblocks (e.g., [3HB-3HV] n ). 155Apart from microbial biosynthesis, traditional chemical synthesis techniques can be applied to generate artificial b-PHA, as shown when linking blocks of atactic PHB (a-PHB), a material obtained by ring opening polymerization of β-butyrolactone, to blocks of poly(6-hydroxyhexanoate). 156In addition, polyesterurethanes (PEU) consisting of "hard" (PHB) and "soft" (PHO) PHA segments, covalently linked by diisocyanate, or PHB blocks linked to poly(ethylene glycol) blocks were generated.Via racemic polymerization, amphiphilic triblock copolymers, such as poly(a-PHBb-ethylene glycol-b-a-PHB) are accessible, which overcome the insufficient hydrophilicity of a-PHB. 157More recently, novel diblock copolymers of different types of PHA (PHB, PHBV, PHO) and a-PHB were generated, which hold high promise as blend compatibilizers for cardiovascular engineering. 158n addition to chemical synthesis of PHA-based block polymers, b-PHA is also directly accessible by adapting the biotechnological production strategy, although PHA heteropolyesters are typically synthe- sized randomly with arbitrary interspaces of the individual monomers along the PHA chain, which makes control or predefinition of polyester properties difficult.In this context, Pederson and co-workers described the impact of adjusting the supply of C. necator H16 with fructose and the 3HV precursor valeric acid.It was shown that parallel availability of fructose and valeric acid, realized by permanent supply of fructose and periodic valerate pulse feeding after its exhaust, generated poly(3HB-co-3HV) copolyesters of random distribution, whereas during periods of fructose-only availability, PHB homopolyester blocks were generated.Now, the authors developed a feedback-controlled bioreactor cultivation system, which triggered the valerate supply as response to DO in fermentation broth and to CO 2 concentration in the exhaust air stream from the bioreactor, and succeeded in generating poly(PHB-b-PHBHV) diblock copolyesters of defined length of the individual blocks. 155 recent study provided by Ferre-Guell and Winterburn investigated cultivation strategies for the haloarcheon Hfx.mediterranei to produce randomly distributed and blocky structured poly(3HBco-3HV) copolyesters. 159Cultures of these organisms were supplied with different VFAs of even (acetic, butyric, hexanoic, octatonic, and decanoic acid) and odd (propionic, valeric, heptanoic, nonanoic, undecanoic acid) number of carbon atoms.Growth and PHA production were observed only for those VFAs with less than seven carbon atoms.While acetic acid generated poly(3HB-co-3HV) with about 10 mol% 3HV (similar to setups on glucose), the 3HV fraction in polyesters produced from butyric acid was negligible.Using valeric acid as sole carbon source, copolyesters with outstandingly high (more than 90 mol%) 3HV content were obtained, whereas 3HV fraction in polyesters from propionic acid was lower due to a partial oxidative decarboxylation of propionyl-CoA towards the 3HB precursor acetyl-CoA, which is in accordance with the findings discussed previously.Different feeding strategies were carried out using butyric and valeric acid or defined mixtures thereof; it was found that sequential feeding of one VFA after the other generated blocky structured copolyesters, whereas co-feeding resulted in random distribution of the building blocks, as evidenced by 1 H and 13 C NMR measurements.Regarding the thermal properties of the copolyester samples, it was shown that increasing 3HV fraction in randomly distributed polymers resulted in a decrease in T g , thus in a higher mobility of PHA chains in the amorphous phase.Generally, increasing 3HV fractions in random poly(3HBco-3HV) samples favored the amorphous character of the polymers, as evidenced by lacking T c peaks in DSC thermograms.Moreover, 3HV-rich copolyes-ters exhibited lower T m , improved elasticity, and enhanced ductility. 159s shown by Ashby et al., sequential feeding of substrates to generate b-PHA can be overcome by using substrates that are not converted in parallel by the production strain, which results in a kind of "diauxic PHA biosynthesis".The authors used B. sacchari DSM 17165, a currently emerging PHA production strain (vide supra), on substrate mixtures of xylose and levulinic acid.112 As shown previously, xylose, as follow-up material of inexpensive lignocelluloses, was converted by this strain for PHB homopolyester production, 160 whereas levulinic acid, also accessible as platform chemical starting from renewable resources, performed successfully as 3HV precursor for other PHA-producing microbes.111,112 The study reported by Ashy et al. demonstrated that, when supplying both substrates in parallel, levulinic acid was converted preferentially to xylose by B. sacchari.By adapting the initial concentration of levulinic acid in the substrate mix and the fermentation length, the size of poly(3HV) blocks in poly(3HB-b-3HV) diblock copolyesters can be varied.Remarkably, these polyesters revealed outstandingly high molecular masses exceeding 1 MDa.112 As shown over the last couple of years, PHA can be produced at high productivity in continuously operated multistage bioreactor cascades; 161 these processes are well-investigated by diverse approaches of mathematical modelling.[162][163][164][165] Such bioreactor cascade systems are not only suggested for high-throughput PHA biosynthesis, but, although up to now investigated experimentally only for PHB production, also offer the possibility of feeding individual cascade stages with different carbon sources, thus generating designed PHA blocks.This approach requests complete consumption of a carbon source in a given stage of the cascade, and subsequent feeding of another carbon source in the next stage, etc. 161 This process engineering tool might be a decisive leap in direction of producing tailor-made PHA of pre-defined composition and properties.
When mcl-PHA is generated by Pseudomonades using β-oxidation of fatty acids, they typically display random distribution of the building blocks; this was recently demonstrated by Impallomeni and colleagues, who produced randomly distributed mcl-PHA copolyesters with building blocks up to 3HHpD. 100 Other researchers purposely aimed at production of randomly distributed copolyesters.In this context, mixotrophic cultivations in a 7-liter bubbled and stirred tank bioreactor was investigated by Ghysels and colleagues; CO 2 and valeric acid were metabolized by the well-described "Knallgasbacterium" C. necator DSM 545 towards poly(3HB-co-3HV) copolyesters of tailored composition and monomeric distribution.CO 2 was continuously sparged, whereas valeric acid was added as substrate pulses and by pH-stat feeding; thus, copolyesters containing more than 60 mol% 3HV were obtained.As confirmed by 1 H and 13 C NMR, the desired random monomer distribution can be obtained by keeping the valeric acid concentration below 1 g L -1 , as shown by the values for dyad sequence distribution and the degree of randomness both being close to 1. Based on validation by independent datasets, the authors developed a reproducible and highly predictive model for mixotrophic production of poly(3HB-co-3HV) of pre-defined composition. 166Also regarding poly(3HB-co-3HP) copolyesters reported previously herein, NMR measurements revealed that the parallel supply of C. necator and A. latus with sucrose and 3HP resulted in formation of copolyesters of random distribution. 121 PHA copolyester reported by Cespedes et al., produced using a genetically engineered Pseudomonas sp. from glucose, was found to consist of a single copolyester of two polymer blocks: the first block revealed the typical composition of a mcl-PHA from other described Pseudomonas sp., whereas the second block exhibited the properties of Nodax-type scl-mcl-copolyesters, containing 3HB and 3HHx with a high molar 3HHx fraction.These unexpected findings shed new light on the substrate specificity of Aeromonas sp.PHA synthase.167 Blocky structured PHA consisting of "soft" and "hard" segments were generated using genetically engineered E. coli.Sequential feeding with 1,4-butanediol (precursor for the "soft" segment poly(4HB)) and 1,3-propanediol (precursor of the "hard" segment poly(3HP)) resulted in biosynthesis of poly(poly(4HB)-b-poly(3HP)).The authors underlined the convenient properties of these blocky structured polyesters in comparison to randomly distributed analogues regarding lower melting points, and drastically better and higher Young's modulus, yield strengths, and tension strengths.Typical for b-PHA, the new materials displayed two T g and two T m peaks.168 The β-oxidation weakened P. entomophila mutant strain described by Wang et al. for mcl-PHA homopolyester production (vide supra), was cultivated on mixtures of octanoate and dodecanoic acid or tetradecanoic acid, respectively.Depending on the composition of the substrate mix and the cultivation time, fractions of poly(3HO-co-3HDD) and poly(3HO-co-3HTD) copolyesters of random distribution and adaptable 3HDD or 3HTD, respectively, content were obtained; only traces of other monomers (3HHx or 3HD) were detected due to the β-oxidation deficiency of the strain.In the same study, the authors demonstrated sequential feeding as a viable tool to generate blocky structured poly(3HO-b-3HDD) copolyesters by this organism by adding the second substrate (dodecanoic acid) only after depletion of the first (octanoate).153 A similar approach was used by Li et al., who cultured recombinant P. putida harboring Aeromonas caviae PHA synthesis genes (P. putida KTQQ20) on butyrate and heptanoate in a 5-L bioreactor.Again, sequential feeding (addition of heptanoate only after depletion of butyrate) resulted in the production of a new class of b-PHA, namely a copolyester containing alternating blocks of PHB homopolyester and random poly(3HV-co-3HHp); 3HV monomers were generated by the loss of one acetyl-CoA unit via β-oxidation.Feeding a mixture of butyrate and heptanoate resulted in formation of randomly distributed poly(3HB-co-3HV-co-3HHp) copolyesters.The authors underlined the expedient material quality of the b-PHA by comparing its tensile strength and Young´s modulus with data obtained from the randomly distributed copolyester and with a PHA blend of PHB and poly(3HV-co-3HHp) of similar overall monomeric composition.169 Later, two different β-oxidation weakened P. putida mutants were used for biosynthesis of mcl-PHA with tunable composition and microstructure.When cultivated on butyrate/hexanoate mixtures, poly(3HB-co-3HHx) copolyesters with random distribution were accumulated by P. putida KTOYO6ΔC, whereas using hexanoate/decanoate mixtures resulted in biosynthesis of randomly distributed poly(3HHx-co-3HD) copolyesters by P. putida KTQQ20.The monomeric compositions were easily tunable by varying the ration of fatty acids in the feed.In contrast, sequential feeding of P. putida KTQQ20 with hexanoate and dodecanoate resulted in production of a novel diblock copolymer poly(P(3HHx)-b-poly(3HD-co-3HDD)).170 The latest discovery in the field of b-PHA is the production of poly(2HB-b-3HB) by a recombinant E. coli harbouring an engineered enzyme designated as PhaC AR , which was a chimeric synthase of A. caviae and C. necator PHA synthases.Cultivation was performed by parallel availability of the two substrates sodium (R,S)-3HB and sodium (R,S)-2HB at varied concentrations. 1 H NMR ruled out the possibility of the polymer being a randomly distributed copolyester, while subsequent solvent (THF) fractionation studies confirmed that, de facto, a b-PHA was produced with the blocks of PHB and poly(2HB) being covalently linked.The authors assume rapid intracellular changes of 2HB-CoA and 3HB-CoA pools and fluctuating individual availability of these thioesters being responsible for formation of b-PHA instead of a random copolyester by this chimer synthase.171 A typical intracellular blend of PHA was obtained for the products produced by Kim and Lenz with P. putida on alkanoic acids and 5-phenylvaleric acid (vide supra).Based on fractionation studies and the comparison of T g and T m of the products with values for poly(3HN) and poly(3HO), respectively, it became clear that two different polymers were present in the cells in parallel, namely, a fraction rich in 3H5PV and a fraction poor in this aromatic compound. 138uch later, the production of PHA blends by Hfx.mediterranei was demonstrated by Don et al., who fractionated poly(3HB-co-3HV) produced by this organism from glucose and yeast extract with chloroform/acetone.The major fraction (about 93 %) contained 10.7 % 3HV and was of high molecular mass of 570 kDa, whereas the smaller fraction had a higher 3HV fraction (12.3 %) and a considerably low molecular mass of 78 kDa.172 These results were later confirmed by Koller et al., who extracted lyophilized Hfx.mediterranei biomass containing a poly(3HB-co-3HV-co-4HB) copolyester with acetone in a Soxleth apparatus; also in this case, a low molecular mass fraction (209 kDa, not even 1 % of the entire PHA on a mass basis) was extracted, while the major part of PHA (molecular mass of more than 1 MDa) remained in the biomass, and was acetone-extracted only under pressure and temperature above acetone boiling point.38

Conclusions
This review shows that PHA material properties strongly depend on the monomeric composition of these biopolyesters, and on their microstructure.Different microbiological, synthetic biological, process engineering, and biosynthetic approaches are available to manufacture tailor-made PHA according to individual application needs; thus, the properties of a considerable part of currently produced and applied plastics can be mimicked by biological alternatives.A huge variety of differently composed PHA with pre-defined material performance will be available in a not too distant future, enabling the already long awaited substitution of various petrol-based plastics contemporarily governing the polymer market.