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Background: Polyhydroxyalkanoates (PHAs) can be degraded by many microorganisms using intra-or extracellular PHA depolymerases. PHA depolymerases are very diverse in sequence and substrate specificity, but share a common α/β-hydrolase fold and a catalytic triad, which is also found in other α/β-hydrolases.

Protein production in Escherichia coli is a fundamental activity for a large fraction of academic, pharmaceutical, and industrial research laboratories. Maximum production is usually sought, as this reduces costs and facilitates downstream purification steps. Frustratingly, many coding sequences are poorly expressed even when they are codon-optimized and expressed from vectors with powerful genetic elements. In this study, we show that poor expression can be caused by certain nucleotide sequences (e.g., cloning scars) at the junction between the vector and the coding sequence. Since these sequences lie between the Shine-Dalgarno sequence and the start codon, they are an integral part of the translation initiation region. To identify the most optimal sequences, we devised a simple and inexpensive PCR-based step that generates sequence variants at the vector-coding sequence junction. These sequence variants modulated expression by up to 1000-fold. FACS-seq analyses indicated that low GC content and relaxed mRNA stability (ΔG) in this region were important, but not the only, determinants for high expression.

Evolution can be harnessed to optimize synthetic biology designs. A prominent example is recombinant protein production-a dominating theme in biotechnology for more than three decades. Typically, a protein coding sequence (cds) is recombined with genetic elements, such as promoters, ribosome binding sites and terminators, which control expression in a cell factory. A major bottleneck during production is translational initiation. Previously we identified more effective translation initiation regions (TIRs) by creating sequence libraries and then selecting for a TIR that drives high-level expression-an example of synthetic evolution. However, manual screening limits the ability to assay expression levels of all putative sequences in the libraries. Here we have solved this bottleneck by designing a collection of translational coupling devices based on a RNA secondary structure. Exchange of different sequence elements in this device allows for different coupling efficiencies, therefore giving the devices a tunable nature. Sandwiching these devices between the cds and an antibiotic selection marker that functions over a broad dynamic range of antibiotic concentrations adds to the tunability and allows expression levels in large clone libraries to be probed using a simple cell survival assay on the respective antibiotic. The power of the approach is demonstrated by substantially increasing production of two commercially interesting proteins, a Nanobody and an Affibody. The method is a simple and inexpensive alternative to advanced screening techniques that can be carried out in any laboratory.

SummaryThe development of efficient recovery processes is essential to reduce the cost of polyhydroxyalkanoates (PHAs) production. In this work, a programmed self‐disruptive Pseudomonas putida BXHL strain, derived from the prototype medium‐chain‐length PHA producer bacterium P. putida KT2440, was constructed as a proof of concept for exploring the possibility to control and facilitate the release of PHA granules to the extracellular medium. The new autolytic cell disruption system is based on two simultaneous strategies: the coordinated action of two proteins from the pneumococcal bacteriophage EJ‐1, an endolysin (Ejl) and a holin (Ejh), and the mutation of the tolB gene, which exhibits alterations in outer membrane integrity that induce lysis hypersensitivity. The ejl and ejh coding genes were expressed under a XylS/Pm monocopy expression system inserted into the chromosome of the tolB mutant strain, in the presence of 3‐methylbenzoate as inducer molecule. Our results demonstrate that the intracellular presence of PHA granules confers resistance to cell envelope. Conditions to control the cell autolysis in P. putida BXHL in terms of optimal fermentation, PHA content and PHA recovery have been set up by exploring the sensitivity to detergents, chelating agents and wet biomass solubility in organic solvents such as ethyl acetate.

ABSTRACTThe obligate predatorBdellovibrio bacteriovorusHD100 shows a large set of proteases and other hydrolases as part of its hydrolytic arsenal needed for its predatory life cycle. We present genetic and biochemical evidence that open reading frame (ORF) Bd3709 ofB. bacteriovorusHD100 encodes a novel medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase (PhaZBd). The primary structure of PhaZBdsuggests that this enzyme belongs to the α/β-hydrolase fold family and has a typical serine hydrolase catalytic triad (serine-histidine-aspartic acid) in agreement with other PHA depolymerases and lipases. PhaZBdhas been extracellularly produced using different hypersecretor Tol-pal mutants ofEscherichia coliandPseudomonas putidaas recombinant hosts. The recombinant PhaZBdhas been characterized, and its biochemical properties have been compared to those of other PHA depolymerases. The enzyme behaves as a serine hydrolase that is inhibited by phenylmethylsulfonyl fluoride. It is also affected by the reducing agent dithiothreitol and nonionic detergents like Tween 80. PhaZBdis an endoexohydrolase that cleaves both large and small PHA molecules, producing mainly dimers but also monomers and trimers. The enzyme specifically degrades mcl-PHA and is inactive toward short-chain-length polyhydroxyalkanoates (scl-PHA) like polyhydroxybutyrate (PHB). These studies shed light on the potentiality of these predators as sources of new biocatalysts, such as an mcl-PHA depolymerase, for the production of enantiopure hydroxyalkanoic acids and oligomers as building blocks for the synthesis of biobased polymers.

This work examines the potential of the predatory bacterium Bdellovibrio bacteriovorus HD100, an obligate predator of other Gram-negative bacteria, as an external cell-lytic agent for recovering valuable intracellular bio-products produced by prey cultures. The bio-product targets to be recovered were polyhydroxyalkanoates (PHAs) produced naturally by Pseudomonas putida and Cupriavidus necator, or by recombinant Escherichia coli strains. B. bacteriovorus with a mutated PHA depolymerase gene to prevent the unwanted breakdown of the bio-product allowed the recovery of up to 80% of that accumulated by the prey bacteria, even at high biomass concentrations. This innovative downstream process highlights how B. bacteriovorus can be used as a novel, biological lytic agent for the inexpensive, industrial scale recovery of intracellular products from different Gram-negative prey cultures.

Bdellovibrio bacteriovorus HD100 is an obligate predator that invades and grows within the periplasm of Gram-negative bacteria, including mcl-polyhydroxyalkanoate (PHA) producers such as Pseudomonas putida. We investigated the impact of prey PHA content on the predator fitness and the potential advantages for preying on a PHA producer. Using a new procedure to control P. putida KT2442 cell size we demonstrated that the number of Bdellovibrio progeny depends on the prey biomass and not on the viable prey cell number or PHA content. The presence of mcl-PHA hydrolysed products in the culture supernatant after predation on P. putida KT42Z, a PHA producing strain lacking PhaZ depolymerase, confirmed the ability of Bdellovibrio to degrade the prey's PHA. Predator motility was higher when growing on PHA accumulating prey. External addition of PHA polymer (latex suspension) to Bdellovibrio preying on the PHA minus mutant P. putida KT42C1 restored predator movement, suggesting that PHA is a key prey component to sustain predator swimming speed. High velocities observed in Bdellovibrio preying on the PHA producing strain were correlated to high intracellular ATP levels of the predator. These effects brought Bdellovibrio fitness benefits as predation on PHA producers was more efficient than predation on non-producing bacteria.