Exploiting the natural poly(3-hydroxyalkanoates) production capacity of Antarctic Pseudomonas strains: from unique phenotypes to novel biopolymers

Journal of Industrial Microbiology & Biotechnology - Extreme environments are a unique source of microorganisms encoding metabolic capacities that remain largely unexplored. In this work, we...     

Comparison of mcl-Poly(3-hydroxyalkanoates) synthesis by different Pseudomonas putida strains from crude glycerol: citrate accumulates at high titer under PHA-producing conditions

Background

Eukaryotic ubiquitin and SUMO are frequently used as tags to enhance the fusion protein expression in microbial host. They increase the solubility and stability, and protect the peptides from proteolytic degradation due to their stable and highly conserved structures. Few of prokaryotic ubiquitin-like proteins was used as fusion tags except ThiS, which enhances the fusion expression, however, reduces the solubility and stability of the expressed peptides in E. coli. Hence, we investigated if MoaD, a conserved small sulfur carrier in prokaryotes with the similar structure of ubiquitin, could also be used as fusion tag in heterologous expression in E. coli.

Results

Fusion of MoaD to either end of EGFP enhanced the expression yield of EGFP with a similar efficacy of ThiS. However, the major parts of the fusion proteins were expressed in the aggregated form, which was associated with the retarded folding of EGFP, similar to ThiS fusions. Fusion of MoaD to insulin chain A or B did not boost their expression as efficiently as ThiS tag did, probably due to a less efficient aggregation of products. Interestingly, fusion of MoaD to the murine ribonuclease inhibitor enhanced protein expression by completely protecting the protein from intracellular degradation in contrast to ThiS fusion, which enhanced degradation of this unstable protein when expressed in E. coli.

Conclusions

Prokaryotic ubiquitin-like protein MoaD can act as a fusion tag to promote the fusion expression with varying mechanisms, which enriches the arsenal of fusion tags in the category of insoluble expression.     

Industrial biotechnology of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> and related species

Since their discovery many decades ago, Pseudomonas putida and related subspecies have been intensively studied with regard to their potential application in industrial biotechnology. Today, these Gram-negative soil bacteria, traditionally known as well-performing xenobiotic degraders, are becoming efficient cell factories for various products of industrial relevance including a full range of unnatural chemicals. This development is strongly driven by systems biotechnology, integrating systems metabolic engineering approaches with novel concepts from bioprocess engineering, including novel reactor designs and renewable feedstocks.     

Biochemistry,genetics and biotechnology of glycerol utilization in Pseudomonas species

The use of renewable waste feedstocks is an environment-friendly choice contributing to the reduction of waste treatment costs and increasing the economic value of industrial by-products. Glycerol (1,2,3-propanetriol), a simple polyol compound widely distributed in biological systems, constitutes a prime example of a relatively cheap and readily available substrate to be used in bioprocesses. Extensively exploited as an ingredient in the food and pharmaceutical industries, glycerol is also the main by-product of biodiesel production, which has resulted in a progressive drop in substrate price over the years. Consequently, glycerol has become an attractive substrate in biotechnology, and several chemical commodities currently produced from petroleum have been shown to be obtained from this polyol using whole-cell biocatalysts with both wild-type and engineered bacterial strains. Pseudomonas species, endowed with a versatile and rich metabolism, have been adopted for the conversion of glycerol into value-added products (ranging from simple molecules to structurally complex biopolymers, e.g. polyhydroxyalkanoates), and a number of metabolic engineering strategies have been deployed to increase the number of applications of glycerol as a cost-effective substrate. The unique genetic and metabolic features of glycerol-grown Pseudomonas are presented in this review, along with relevant examples of bioprocesses based on this substrate – and the synthetic biology and metabolic engineering strategies implemented in bacteria of this genus aimed at glycerol valorization.     

Limited life cycle and cost assessment for the bioconversion of lignin-derived aromatics into adipic acid

Lignin is an abundant and heterogeneous waste byproduct of the cellulosic industry, which has the potential of being transformed into valuable biochemicals via microbial fermentation. In this study, we applied a fast-pyrolysis process using softwood lignin resulting in a two-phase bio-oil containing monomeric and oligomeric aromatics without syringol. We demonstrated that an additional hydrodeoxygenation step within the process leads to an enhanced thermochemical conversion of guaiacol into catechol and phenol. After steam bath distillation, Pseudomonas putida KT2440-BN6 achieved a percent yield of cis, cis-muconic acid of up to 95 mol% from catechol derived from the aqueous phase. We next established a downstream process for purifying cis, cis-muconic acid (39.9 g/L) produced in a 42.5 L fermenter using glucose and benzoate as carbon substrates. On the basis of the obtained values for each unit operation of the empirical processes, we next performed a limited life cycle and cost analysis of an integrated biotechnological and chemical process for producing adipic acid and then compared it with the conventional petrochemical route. The simulated scenarios estimate that by attaining a mixture of catechol, phenol, cresol, and guaiacol (1:0.34:0.18:0, mol ratio), a titer of 62.5 (g/L) cis, cis-muconic acid in the bioreactor, and a controlled cooling of pyrolysis gases to concentrate monomeric aromatics in the aqueous phase, the bio-based route results in a reduction of CO₂-eq emission by 58% and energy demand by 23% with a contribution margin for the aqueous phase of up to 88.05 euro/ton. We conclude that the bio-based production of adipic acid from softwood lignins brings environmental benefits over the petrochemical procedure and is cost-effective at an industrial scale. Further research is essential to achieve the proposed cis, cis-muconic acid yield from true lignin-derived aromatics using whole-cell biocatalysts.     

Rational engineering of natural polyhydroxyalkanoates producing microorganisms for improved synthesis and recovery

Microbial production of biopolymers derived from renewable substrates and waste streams reduces our heavy reliance on petrochemical plastics. One of the most important biodegradable polymers is the family of polyhydroxyalkanoates (PHAs), naturally occurring intracellular polyoxoesters produced for decades by bacterial fermentation of sugars and fatty acids at the industrial scale. Despite the advances, PHA production still suffers from heavy costs associated with carbon substrates and downstream processing to recover the intracellular product, thus restricting market positioning. In recent years, model-aided metabolic engineering and novel synthetic biology approaches have spurred our understanding of carbon flux partitioning through competing pathways and cellular resource allocation during PHA synthesis, enabling the rational design of superior biopolymer producers and programmable cellular lytic systems. This review describes these attempts to rationally engineering the cellular operation of several microbes to elevate PHA production on specific substrates and waste products. We also delve into genome reduction, morphology, and redox cofactor engineering to boost PHA biosynthesis. Besides, we critically evaluate engineered bacterial strains in various fermentation modes in terms of PHA productivity and the period required for product recovery.     

Opportunities and obstacles in microbial synthesis of metal nanoparticles

Metallic nanoparticles (MeNPs) are widely used in many areas such as biomedicine, packaging, cosmetics, colourants, agriculture, antimicrobial agents, cleaning products, as components of electronic devices and nutritional supplements. In addition, some MeNPs exhibit quantum properties, making them suitable materials in the photonics, electronic and energy industries. Through the lens of technology, microbes can be considered nanofactories capable of producing enzymes, metabolites and capping materials involved in the synthesis, assembly and stabilization of MeNPs. This bioprocess is considered more ecofriendly and less energy intensive than the current chemical synthesis routes. However, microbial synthesis of MeNPs as an alternative method to the chemical synthesis of nanomaterials still faces some challenges that need to be solved. Some of these challenges are described in this Editorial.     

Channelling carbon flux through the meta-cleavage route for improved poly(3-hydroxyalkanoate) production from benzoate and lignin-based aromatics in Pseudomonas putida H

Lignin-based aromatics are attractive raw materials to derive medium-chain length poly(3-hydroxyalkanoates) (mcl-PHAs), biodegradable polymers of commercial value. So far, this conversion has exclusively used the ortho-cleavage route of Pseudomonas putida KT2440, which results in the secretion of toxic intermediates and limited performance. Pseudomonas putida H exhibits the ortho- and the meta-cleavage pathways where the latter appears promising because it stoichiometrically yields higher levels of acetyl-CoA. Here, we created a double-mutant H-ΔcatAΔA2 that utilizes the meta route exclusively and synthesized 30% more PHA on benzoate than the parental strain but suffered from catechol accumulation. The single deletion of the catA2 gene in the H strain provoked a slight attenuation on the enzymatic capacity of the ortho route (25%) and activation of the meta route by nearly 8-fold, producing twice as much mcl-PHAs compared to the wild type. Inline, the mutant H-ΔcatA2 showed a 2-fold increase in the intracellular malonyl-CoA abundance – the main precursor for mcl-PHAs synthesis. As inferred from flux simulation and enzyme activity assays, the superior performance of H-ΔcatA2 benefited from reduced flux through the TCA cycle and malic enzyme and diminished by-product formation. In a benzoate-based fed-batch, P. putida H-ΔcatA2 achieved a PHA titre of 6.1 g l^–1 and a volumetric productivity of 1.8 g l^–1 day^–1. Using Kraft lignin hydrolysate as feedstock, the engineered strain formed 1.4 g l^{- 1} PHA. The balancing of carbon flux between the parallel catechol-degrading routes emerges as an important strategy to prevent intermediate accumulation and elevate mcl-PHA production in P. putida H and, as shown here, sets the next level to derive this sustainable biopolymer from lignin hydrolysates and aromatics.