Directed Evolution of Sequence-Regulating Polyhydroxyalkanoate Synthase to Synthesize a Medium-Chain-Length-Short-Chain-Length (MCL-SCL) Block Copolymer.

Sequence-regulating polyhydroxyalkanoate synthase PhaCAR is a chimeric enzyme comprising PhaCs from Aeromonas caviae and Ralstonia eutropha (Cupriavidus necator). It spontaneously synthesizes a short-chain-length (SCL, ≤C5) block copolymer poly(2-hydroxybutyrate)-b-poly(3-hydroxybutyrate) [P(2HB)-b-P(3HB)] from a mixture of monomer substrates. In this study, directed evolution of PhaCAR was performed to increase its activity toward a medium-chain-length (MCL, C6-12) monomer, 3-hydroxyhexanoyl (3HHx)-coenzyme A (CoA). Random mutagenesis and selection based on P(3HB-co-3HHx) production in Escherichia coli found that beneficial mutations N149D and F314L increase the 3HHx fraction. The site-directed saturation mutagenesis at position 314, which is adjacent to the catalytic center C315, demonstrated that F314H synthesizes the P(3HHx) homopolymer. The F314H mutant exhibited increased activity toward 3HHx-CoA compared with the parent enzyme, whereas the activity toward 3HB-CoA decreased. The predicted tertiary structure of PhaCAR by AlphaFold2 provided insight into the mechanism of the beneficial mutations. In addition, this finding enabled the synthesis of a new PHA block copolymer, P(3HHx)-b-P(2HB). Solvent fractionation indicated the presence of a covalent linkage between the polymer segments. This novel MCL-SCL block copolymer considerably expands the range of the molecular design of PHA block copolymers.

Versatile aliphatic polyester biosynthesis system for producing random and block copolymers composed of 2-, 3-, 4-, 5-, and 6-hydroxyalkanoates using the sequence-regulating polyhydroxyalkanoate synthase PhaCAR.

BACKGROUND: Polyhydroxyalkanoates (PHAs) are microbial polyesters synthesized by PHA synthases. Naturally occurring PHA copolymers possess a random monomer sequence. The development of PhaCAR, a unique sequence-regulating PHA synthase, has enabled the spontaneous biosynthesis of PHA block copolymers. PhaCAR synthesizes both a block copolymer poly(2-hydroxybutyrate)-b-poly(3-hydroxybutyrate) [P(2HB)-b-P(3HB)], and a random copolymer, poly(3HB-co-3-hydroxyhexanoate), indicating that the combination of monomers determines the monomer sequence. Therefore, in this study, we explored the substrate scope of PhaCAR and the monomer sequences of the resulting copolymers to identify the determinants of the monomer sequence. PhaCAR is a class I PHA synthase that is thought to incorporate long-main-chain hydroxyalkanoates (LMC HAs, > C3 in the main [backbone] chain). Thus, the LMC monomers, 4-hydroxy-2-methylbutyrate (4H2MB), 5-hydroxyvalerate (5HV), and 6-hydroxyhexanoate (6HHx), as well as 2HB, 3HB, and 3-hydroxypropionate (3HP) were tested. RESULTS: Recombinant Escherichia coli harboring PhaCAR, CoA transferase and CoA ligase genes was used for PHA production. The medium contained the monomer precursors, 2HB, 3HB, 3HP, 4H2MB, 5HV, and 6HHx, either individually or in combination. As a result, homopolymers were obtained only for 3HB and 3HP. Moreover, 3HB and 3HP were randomly copolymerized by PhaCAR. 3HB-based binary copolymers P(3HB-co-LMC HA)s containing up to 2.9 mol% 4H2MB, 4.8 mol% 5HV, or 1.8 mol% 6HHx were produced. Differential scanning calorimetry analysis of the copolymers indicated that P(3HB-co-LMC HA)s had a random sequence. In contrast, combining 3HP and 2HB induced the synthesis of P(3HP)-b-P(2HB). Similarly, P(2HB) segment-containing block copolymers P(3HB-co-LMC HA)-b-P(2HB)s were synthesized. Binary copolymers of LMC HAs and 2HB were not obtained, indicating that the 3HB or 3HP unit is essential to the polymer synthesis. CONCLUSION: PhaCAR possesses a wide substrate scope towards 2-, 3-, 4-, 5-, and 6-hydroxyalkanoates. 3HB or 3HP units are essential for polymer synthesis using PhaCAR. The presence of a 2HB monomer is key to synthesizing block copolymers, such as P(3HP)-b-P(2HB) and P(3HB-co-LMC HA)-b-P(2HB)s. The copolymers that did not contain 2HB units had a random sequence. This study's results provide insights into the mechanism of sequence regulation by PhaCAR and pave the way for designing PHA block copolymers.

A microfluidic platform for in situ investigation of biofilm formation and its treatment under controlled conditions.

BACKGROUND: Studying bacterial adhesion and early biofilm development is crucial for understanding the physiology of sessile bacteria and forms the basis for the development of novel antimicrobial biomaterials. Microfluidics technologies can be applied in such studies since they permit dynamic real-time analysis and a more precise control of relevant parameters compared to traditional static and flow chamber assays. In this work, we aimed to establish a microfluidic platform that permits real-time observation of bacterial adhesion and biofilm formation under precisely controlled homogeneous laminar flow conditions. RESULTS: Using Escherichia coli as the model bacterial strain, a microfluidic platform was developed to overcome several limitations of conventional microfluidics such as the lack of spatial control over bacterial colonization and allow label-free observation of bacterial proliferation at single-cell resolution. This platform was applied to demonstrate the influence of culture media on bacterial colonization and the consequent eradication of sessile bacteria by antibiotic. As expected, the nutrient-poor medium (modified M9 minimal medium) was found to promote bacterial adhesion and to enable a higher adhesion rate compared to the nutrient-rich medium (tryptic soy broth rich medium ). However, in rich medium the adhered cells colonized the glass surface faster than those in poor medium under otherwise identical conditions. For the first time, this effect was demonstrated to be caused by a higher retention of newly generated bacteria in the rich medium, rather than faster growth especially during the initial adhesion phase. These results also indicate that higher adhesion rate does not necessarily lead to faster biofilm formation. Antibiotic treatment of sessile bacteria with colistin was further monitored by fluorescence microscopy at single-cell resolution, allowing in situ analysis of killing efficacy of antimicrobials. CONCLUSION: The platform established here represents a powerful and versatile tool for studying environmental effects such as medium composition on bacterial adhesion and biofilm formation. Our microfluidic setup shows great potential for the in vitro assessment of new antimicrobials and antifouling agents under flow conditions.

Development of Biopolyesters (PHA) as Part of the Swiss Priority Program in Biotechnology.

The Swiss Priority Program in Biotechnology of the Swiss National Science Foundation that lasted between 1992 and 2001 had a boosting effect on many biotech disciplines and on the developments of polyhydroxyalkanoates (PHAs) in Switzerland in particular. The funding organization led by Prof. Oreste Ghisalba enabled a better understanding of the PHA biosynthesis and the development, as well as the implementation of novel bioprocesses (e.g. two-phase fermentations, multiple nutrient limited growth conditions, multi-stage chemostats, and product formation in different host organisms). However, production of PHA in Switzerland appeared to be impossible for cost reasons due to the strong competition from cheaper, petrol-based plastics. The recent reports on environmental issues with non-degradable plastics has triggered a general change in the perception of biodegradable plastics, giving them an added value and thus justifying a higher price. Ongoing research focuses on the sustainable production of PHAs using carbon waste streams, synthesis gas or even CO2.

Anaerobic Production of Poly(3-hydroxybutyrate) and Its Precursor 3-Hydroxybutyrate from Synthesis Gas by Autotrophic Clostridia.

Anaerobic production of the biopolymer poly(3-hydroxybutyrate) (PHB) and the monomer 3-hydroxybutyrate (3-HB) was achieved using recombinant clostridial acetogens supplied with syn(thesis) gas as the sole carbon and energy source. 3-HB production was successfully accomplished by a new synthetic pathway containing the genes thlA (encoding thiolase A), ctfA/B (encoding CoA-transferase A/B), and bdhA (encoding (R)-3-hydroxybutyrate dehydrogenase). The respective recombinant Clostridium coskatii [p83_tcb] strain produced autotrophically 0.98 ± 0.12 mM and heterotrophically 21.7 ± 0.27 mM 3-HB. As a proof of concept, production of PHB was achieved using recombinant C. coskatii and Clostridium ljungdahlii strains expressing a novel synthetic PHB pathway containing the genes thlA (encoding thiolase A), hbd (encoding 3-hydroxybutyryl-CoA dehydrogenase), crt (encoding crotonase), phaJ (encoding (R)-enoyl-CoA hydratase), and phaEC (encoding PHA synthase). The strain C. coskatii [p83_PHB_Scaceti] synthesized heterotrophically 3.4 ± 0.29% PHB per cell dry weight (CDW) and autotrophically 1.12 ± 0.12% PHB per CDW.

Microplastics - from Anthropogenic to Natural.

In the context of the ongoing discussion of accumulating plastic debris in the environment, this article summarizes recent reports on the topic of microplastic pollution. Moreover, it gives an overview of the results from the activities of the research group for Biotechnology and Sustainable Chemistry of the Institute of Life Technologies at the HES-SO ValaisWallis concerning the fabrication of biodegradable microstructured plastics and microplastics from renewable resources. Polyhydroxyalkanoate biopolyesters are proposed as one suitable alternative in specific applications to reduce the use of persistent plastics. Standard techniques are described that have been successfully applied to produce porous materials, fibers and particles in the micro- and nanometer range.

Continuous Processes and Flow Chemistry at the Universities of Applied Sciences in Switzerland.

This article provides an overview of activities in the fields of continuous processes, flow chemistry and microreactors at the Universities of Applied Sciences in Switzerland.

Characterization of a poly(butylene adipate-co-terephthalate)- hydrolyzing lipase from Pelosinus fermentans.

Certain α/ß hydrolases have the ability to hydrolyze synthetic polyesters. While their partial hydrolysis has a potential for surface functionalization, complete hydrolysis allows recycling of valuable building blocks. Although knowledge about biodegradation of these materials is important regarding their fate in the environment, it is currently limited to aerobic organisms. A lipase from the anaerobic groundwater organism Pelosinus fermentans DSM 17108(PfL1) was cloned and expressed in Escherichia coli BL21-Gold (DE3) and purified from the cell extract. Biochemical characterization with small substrates showed thermoalkalophilic properties (Topt=50 °C, pHopt=7.5) and higher activity towards para-nitrophenyl octanoate (12.7 U mg(-1)) compared to longer and shorter chain lengths (C14 0.7 U mg(-1) and C2 4.3 U mg(-1), respectively). Crystallization and determination of the 3-D structure displayed the presence of a lid structure and a zinc ion surrounded by an extra domain. These properties classify the enzyme into the I.5 lipase family. PfL1 is able to hydrolyze poly(1,4-butylene adipate-co-terephthalate) (PBAT) polymeric substrates. The hydrolysis of PBAT showed the release of small building blocks as detected by liquid chromatography mass spectrometry (LC-MS). Protein dynamics seem to be involved with lid opening for the hydrolysis of PBAT by PfL1.

Chemical Modification of Polyhydroxyalkanoates (PHAs) for the Preparation of Hybrid Biomaterials.

Polyhydroxyalkanoates (PHAs) are biopolyesters produced by bacteria as intracellular granules under metabolic stress conditions. Many carbon sources such as alkanes, alkenes, alcohols, sugars, fatty acids can be used as feedstock and thus a wide variety of polyesters and monomer units can be potentially synthetized. The work presented here describes the process to chemically modify such biopolymers in order to render them readily available for the preparation of bio-molecular conjugates as promising new classes of biocompatible biomaterials. Such hybrid biomaterials belong to the rapidly growing class of biocompatible polymers, which are of great interest for medical and therapeutic applications. In this work, the biosynthesis of a new PHA homopolymer and the chemical modification, an epoxidation reaction, are described.

PAT at the Universities of Applied Sciences.

An overview of activities in the field of Process Analytical Technologies (PAT) at the Universities of Applied Sciences in Switzerland is presented.

PAT at the Universities of Applied Sciences.

An overview of activities in the field of Process Analytical Technologies (PAT) at the Universities of Applied Sciences in Switzerland is presented.

Improved productivity of poly (4-hydroxybutyrate) (P4HB) in recombinant Escherichia coli using glycerol as the growth substrate with fed-batch culture.

BACKGROUND: The most successful polyhydroxyalkanoate (PHA) in medical applications is poly(4-hydroxybutyrate) (P4HB), which is due to its biodegradability, biocompatibility and mechanical properties. One of the major obstacles for wider applications of P4HB is the cost of production and purification. It is highly desired to obtain P4HB in large scale at a competitive cost. RESULTS: In this work, we studied the possibility to increase P4HB productivity by using high cell density culture. To do so, we investigated for the first time some of the most relevant factors influencing P4HB biosynthesis in recombinant Escherichia coli. We observed that P4HB biosynthesis correlated more with limitations of amino acids and less with nitrogen depletion, contrary to the synthesis of many other types of PHAs. Furthermore, it was found that using glycerol as the primary carbon source, addition of acetic acid at the beginning of a batch culture stimulated P4HB accumulation in E. coli. Fed-batch high cell density cultures were performed to reach high P4HB productivity using glycerol as the sole carbon source for cell growth and 4HB as the precursor for P4HB synthesis. A P4HB yield of 15 g L-1 was obtained using an exponential feeding mode, leading to a productivity of 0.207 g L-1 h-1, which is the highest productivity for P4HB reported so far. CONCLUSIONS: We demonstrated that the NZ-amines (amino acids source) in excess abolished P4HB accumulation, suggesting that limitation in certain amino acid pools promotes P4HB synthesis. Furthermore, the enhanced P4HB yield could be achieved by both the effective growth of E. coli JM109 (pKSSE5.3) on glycerol and the stimulated P4HB synthesis via exogenous addition of acetic acid. We have developed fermentation strategies for P4HB production by using glycerol, leading to a productivity of 0.207 g L-1 h-1 P4HB. This high P4HB productivity will decrease the total production cost, allowing further development of P4HB applications.

Poly(4-hydroxybutyrate) (P4HB) production in recombinant Escherichia coli: P4HB synthesis is uncoupled with cell growth.

BACKGROUND: Poly(4-hydroxybutyrate) (P4HB), belonging to the family of bacterial polyhydroxyalkanoates (PHAs), is a strong, flexible and absorbable material which has a large variety of medical applications like tissue engineering and drug delivery. For efficient production of P4HB recombinant Escherichia coli has been employed. It was previously found that the P4HB synthesis is co-related with the cell growth. In this study, we aimed to investigate the physiology of P4HB synthesis, and to reduce the total production cost by using cheap and widely available xylose as the growth substrate and sodium 4-hydroxybutyrate (Na-4HB) as the precursor for P4HB synthesis. RESULTS: Six different E. coli strains which are able to utilize xylose as carbon source were compared for their ability to accumulate P4HB. E. coli JM109 was found to be the best strain regarding the specific growth rate and the P4HB content. The effect of growth conditions such as temperature and physiological stage of Na-4HB addition on P4HB synthesis was also studied in E. coli JM109 recombinant in batch culture. Under the tested conditions, a cellular P4HB content in the range of 58 to 70% (w w(-1)) and P4HB concentrations in the range of 2.76 to 4.33 g L(-1) were obtained with a conversion yield (Y(P4HB/Na-4HB)) of 92% w w(-1) in single stage batch cultures. Interestingly, three phases were identified during P4HB production: the "growth phase", in which the cells grew exponentially, the "accumulation phase", in which the exponential cell growth stopped while P4HB was accumulated exponentially, and the "stagnation phase", in which the P4HB accumulation stopped and the total biomass remained constant. CONCLUSIONS: P4HB synthesis was found to be separated from the cell growth, i.e. P4HB synthesis mainly took place after the end of the exponential cell growth. High conversion rate and P4HB contents from xylose and precursor were achieved here by simple batch culture, which was only possible previously through fed-batch high cell density cultures with glucose.

New insights on the reorganization of gene transcription in Pseudomonas putida KT2440 at elevated pressure.

BACKGROUND: Elevated pressure, elevated oxygen tension (DOT) and elevated carbon dioxide tension (DCT) are readily encountered at the bottom of large industrial bioreactors and during bioprocesses where pressure is applied for enhancing the oxygen transfer. Yet information about their effect on bacteria and on the gene expression thereof is scarce. To shed light on the cellular functions affected by these specific environmental conditions, the transcriptome of Pseudomonas putida KT2440, a bacterium of great relevance for the production of medium-chain-length polyhydroxyalkanoates, was thoroughly investigated using DNA microarrays. RESULTS: Very well defined chemostat cultivations were carried out with P. putida to produce high quality RNA samples and ensure that differential gene expression was caused exclusively by changes of pressure, DOT and/or DCT. Cellular stress was detected at 7 bar and elevated DCT in the form of heat shock and oxidative stress-like responses, and indicators of cell envelope perturbations were identified as well.Globally, gene transcription was not considerably altered when DOT was increased from 40 ± 5 to 235 ± 20% at 7 bar and elevated DCT. Nevertheless, differential transcription was observed for a few genes linked to iron-sulfur cluster assembly, terminal oxidases, glutamate metabolism and arginine deiminase pathway, which shows their particular sensitivity to variations of DOT. CONCLUSIONS: This study provides a comprehensive overview on the changes occurring in the transcriptome of P. putida upon mild variations of pressure, DOT and DCT. Interestingly, whereas the changes of gene transcription were widespread, the cell physiology was hardly affected, which illustrates how efficient reorganization of the gene transcription is for dealing with environmental changes that may otherwise be harmful. Several particularly sensitive cellular functions were identified, which will certainly contribute to the understanding of the mechanisms involved in stress sensing/response and to finding ways of enhancing the stress tolerance of microorganisms.

Production of medium-chain-length polyhydroxyalkanoates by sequential feeding of xylose and octanoic acid in engineered Pseudomonas putida KT2440.

BACKGROUND: Pseudomonas putida KT2440 is able to synthesize large amounts of medium-chain-length polyhydroxyalkanoates (mcl-PHAs). To reduce the substrate cost, which represents nearly 50% of the total PHA production cost, xylose, a hemicellulose derivate, was tested as the growth carbon source in an engineered P. putida KT2440 strain. RESULTS: The genes encoding xylose isomerase (XylA) and xylulokinase (XylB) from Escherichia coli W3110 were introduced into P. putida KT2440. The recombinant KT2440 exhibited a XylA activity of 1.47 U and a XylB activity of 0.97 U when grown on a defined medium supplemented with xylose. The cells reached a maximum specific growth rate of 0.24 h(-1) and a final cell dry weight (CDW) of 2.5 g L(-1) with a maximal yield of 0.5 g CDW g(-1) xylose. Since no mcl-PHA was accumulated from xylose, mcl-PHA production can be controlled by the addition of fatty acids leading to tailor-made PHA compositions. Sequential feeding strategy was applied using xylose as the growth substrate and octanoic acid as the precursor for mcl-PHA production. In this way, up to 20% w w(-1) of mcl-PHA was obtained. A yield of 0.37 g mcl-PHA per g octanoic acid was achieved under the employed conditions. CONCLUSIONS: Sequential feeding of relatively cheap carbohydrates and expensive fatty acids is a practical way to achieve more cost-effective mcl-PHA production. This study is the first reported attempt to produce mcl-PHA by using xylose as the growth substrate. Further process optimizations to achieve higher cell density and higher productivity of mcl-PHA should be investigated. These scientific exercises will undoubtedly contribute to the economic feasibility of mcl-PHA production from renewable feedstock.

Putting cells under pressure: a simple and efficient way to enhance the productivity of medium-chain-length polyhydroxyalkanoate in processes with Pseudomonas putida KT2440.

The success of bioprocess implementation relies on the ability to achieve high volumetric productivities and requires working with high-cell-density cultivations. Elevated atmospheric pressure might constitute a promising tool for enhancing the oxygen transfer rate (OTR), the major growth-limiting factor for such cultivations. However, elevated pressure and its effects on the cellular environment also represent a potential source of stress for bacteria and may have negative effects on product formation. In order to determine whether elevated pressure can be applied for enhancing productivity in the case of medium-chain-length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas putida KT2440, the impact of a pressure of 7 bar on the cell physiology was assessed. It was established that cell growth was not inhibited by this pressure if dissolved oxygen tension (DOT) and dissolved carbon dioxide tension (DCT) were kept below ∼30 and ∼90 mg L(-1), respectively. Remarkably, a little increase of mcl-PHA volumetric productivity was observed under elevated pressure. Furthermore, the effect of DCT, which can reach substantial levels during high-cell-density processes run under elevated pressure, was investigated on cell physiology. A negative effect on product formation could be dismissed since no significant reduction of mcl-PHA content occurred up to a DCT of ∼540 mg L(-1). However, specific growth rate exhibited a significant decrease, indicating that successful high-cell-density processes under elevated pressure would be restricted to chemostats with low dilution rates and fed-batches with a small growth rate imposed during the final part. This study revealed that elevated pressure is an adequate and efficient way to enhance OTR and mcl-PHA productivity. We estimate that the oxygen provided to the culture broth under elevated pressure would be sufficient to triple mcl-PHA productivity in our chemostat system from 3.4 (at 1 bar) to 11 g L(-1)h(-1) (at 3.2 bar).

Pressure to kill or pressure to boost: a review on the various effects and applications of hydrostatic pressure in bacterial biotechnology.

Much knowledge has been gained for the last 30 years about the effects of pressure on bacteria, and various pressure-based technologies have been designed. The development of modern molecular biology techniques (e.g., DNA microarrays) as well as the technological advances realized in the manufacturing of robust sampling and high-pressure devices has allowed these advances. Not only the direct effects on cell components (membranes, proteins, and nucleic acids) have been unraveled, but also the cellular response to pressure has been investigated by means of transcriptome and proteome analyses. Initially, research was performed by marine biologists who studied the microorganisms living in the deep sea at pressures of 1,000 bar. In parallel, food technologists developed pressure-based methods for inactivating microorganisms without altering the food properties as much as with temperature treatment. The preservation of specific product properties is also the rationale for pressure-based methods for the disinfection of biomaterials and for vaccine production. Therefore, attention was first focused on the "killing" potential of high pressure. On the other hand, there has been a growing interest in using elevated pressures (up to ~10 bar) for enhancing the productivity of bioprocesses. In this case, no killing effect was sought, but pressure was applied to "boost" the process by enhancing the oxygen transfer to the cell culture. This paper gives an overview on the effects of pressures in the range of 1 bar to 10 kbar on bacteria and presents the major and most recent achievements realized in the development of pressure-based biotechnological applications.

Interactions of biodegradable poly([R]-3-hydroxy-10-undecenoate) with 1,2-dioleoyl-sn-glycero-3-phosphocholine lipid: a monolayer study.

Polyhydroxyalkanoates (PHAs) are biodegradable, biocompatible polyesters and very attractive candidates for biomedical applications as materials for tissue engineering. They have a hydrophobic character, but some are able to spread at the air-water interface to form monomolecularly thin films (Langmuir monolayers). This is a very convenient model to analyze PHA self-assembly in two dimensions and to study their molecular interactions with other amphiphilic compounds, which is very important considering compatibility between biomaterials and cell membranes. We used the Langmuir monolayer technique and Brewster angle microscopy to study the properties of poly([R]-3-hydroxy-10-undecenoate) (PHUE) films on the free water surface in various experimental conditions. Moreover, we investigated the interactions between the polymer and one of the main biomembrane components, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The addition of lipid to a polymer film does not change the monolayer phase behavior; however, the interactions between these two materials are repulsive and fall in two composition-dependent regimes. In summary, this is the first systematic study of the monolayer behavior of PHUE, thus forming a solid basis for a thorough understanding of material interactions, in particular in the context of biomaterials and implants.

A reduction in growth rate of Pseudomonas putida KT2442 counteracts productivity advances in medium-chain-length polyhydroxyalkanoate production from gluconate.

BACKGROUND: The substitution of plastics based on fossil raw material by biodegradable plastics produced from renewable resources is of crucial importance in a context of oil scarcity and overflowing plastic landfills. One of the most promising organisms for the manufacturing of medium-chain-length polyhydroxyalkanoates (mcl-PHA) is Pseudomonas putida KT2440 which can accumulate large amounts of polymer from cheap substrates such as glucose. Current research focuses on enhancing the strain production capacity and synthesizing polymers with novel material properties. Many of the corresponding protocols for strain engineering rely on the rifampicin-resistant variant, P. putida KT2442. However, it remains unclear whether these two strains can be treated as equivalent in terms of mcl-PHA production, as the underlying antibiotic resistance mechanism involves a modification in the RNA polymerase and thus has ample potential for interfering with global transcription. RESULTS: To assess PHA production in P. putida KT2440 and KT2442, we characterized the growth and PHA accumulation on three categories of substrate: PHA-related (octanoate), PHA-unrelated (gluconate) and poor PHA substrate (citrate). The strains showed clear differences of growth rate on gluconate and citrate (reduction for KT2442 > 3-fold and > 1.5-fold, respectively) but not on octanoate. In addition, P. putida KT2442 PHA-free biomass significantly decreased after nitrogen depletion on gluconate. In an attempt to narrow down the range of possible reasons for this different behavior, the uptake of gluconate and extracellular release of the oxidized product 2-ketogluconate were measured. The results suggested that the reason has to be an inefficient transport or metabolization of 2-ketogluconate while an alteration of gluconate uptake and conversion to 2-ketogluconate could be excluded. CONCLUSIONS: The study illustrates that the recruitment of a pleiotropic mutation, whose effects might reach deep into physiological regulation, effectively makes P. putida KT2440 and KT2442 two different strains in terms of mcl-PHA production. The differences include the onset of mcl-PHA production (nitrogen limitation) and the resulting strain performance (growth rate). It remains difficult to predict a priori where such major changes might occur, as illustrated by the comparable behavior on octanoate. Consequently, experimental data on mcl-PHA production acquired for P. putida KT2442 cannot always be extrapolated to KT2440 and vice versa, which potentially reduces the body of available knowledge for each of these two model strains for mcl-PHA production substantially.