Strain engineering and bioprocessing strategies for biobased production of porphobilinogen in Escherichia coli

Strain engineering and bioprocessing strategies were applied for biobased production of porphobilinogen (PBG) using Escherichia coli as the cell factory. The non-native Shemin/C4 pathway was first implemented by heterologous expression of hemA from Rhodopseudomonas spheroids to supply carbon flux from the natural tricarboxylic acid (TCA) pathways for PBG biosynthesis via succinyl-CoA. Metabolic strategies were then applied for carbon flux direction from the TCA pathways to the C4 pathway. To promote PBG stability and accumulation, Clustered Regularly Interspersed Short Palindromic Repeats interference (CRISPRi) was applied to repress hemC expression and, therefore, reduce carbon flowthrough toward porphyrin biosynthesis with minimal impact to cell physiology. To further enhance PBG biosynthesis and accumulation under the hemC-repressed genetic background, we further heterologously expressed native E. coli hemB. Using these engineered E. coli strains for bioreactor cultivation based on ~ 30 g L−1 glycerol, we achieved high PBG titers up to 209 mg L−1, representing 1.73% of the theoretical PBG yield, with improved PBG stability and accumulation. Potential biochemical, genetic, and metabolic factors limiting PBG production were systematically identified for characterization. Graphical Abstract

The Design-Build-Test-Learn cycle for metabolic engineering of Streptomycetes

Streptomycetes are producers of a wide range of specialized metabolites of great medicinal and industrial importance, such as antibiotics, antifungals, or pesticides. Having been the drivers of the golden age of antibiotics in the 1950s and 1960s, technological advancements over the last two decades have revealed that very little of their biosynthetic potential has been exploited so far. Given the great need for new antibiotics due to the emerging antimicrobial resistance crisis, as well as the urgent need for sustainable biobased production of complex molecules, there is a great renewed interest in exploring and engineering the biosynthetic potential of streptomycetes. Here, we describe the Design-Build-Test-Learn (DBTL) cycle for metabolic engineering experiments in streptomycetes and how it can be used for the discovery and production of novel specialized metabolites.

Adaptive laboratory evolution restores solvent tolerance in plasmid-cured Pseudomonas putida S12; a molecular analysis

Pseudomonas putida S12 is inherently solvent-tolerant and constitutes a promising platform for biobased production of aromatic compounds and biopolymers. The megaplasmid pTTS12 of P. putida S12 carries several gene clusters involved in solvent tolerance and the removal of this megaplasmid caused a significant reduction in solvent tolerance. In this study, we succeeded in restoring solvent tolerance in the plasmid-cured P. putida S12 using adaptive laboratory evolution (ALE), underscoring the innate solvent-tolerance of this strain. Whole genome sequencing identified several single nucleotide polymorphisms (SNPs) and a mobile element insertion enabling ALE-derived strains to survive and sustain growth in the presence of a high toluene concentration (10% (vol/vol)). Mutations were identified in an RND efflux pump regulator arpR, resulting in constitutive upregulation of the multifunctional efflux pump ArpABC. SNPs were also found in the intergenic region and subunits of ATP synthase, RNA polymerase subunit β’, global two-component regulatory system (GacA/GacS) and a putative AraC-family transcriptional regulator Afr. Transcriptomic analysis further revealed a constitutive down-regulation of energy consuming activities in ALE-derived strains, such as flagellar assembly, F0F1 ATP synthase, and membrane transport proteins. In summary, constitutive expression of a solvent extrusion pump in combination with high metabolic flexibility enabled the restoration of solvent-tolerance trait in P. putida S12 lacking its megaplasmid. Importance: Sustainable production of high-value chemicals can be achieved by bacterial biocatalysis. However, bioproduction of biopolymers and aromatic compounds may exert stress on the microbial production host and limit the resulting yield. Having a solvent tolerance trait is highly advantageous for microbial hosts used in the biobased production of aromatics. The presence of a megaplasmid has been linked to the solvent tolerance trait of Pseudomonas putida, however, the extent of innate, intrinsic solvent tolerance in this bacterium remained unclear. Using adaptive laboratory evolution, we successfully adapted the plasmid-cured P. putida S12 strain to regain its solvent tolerance. Through these adapted strains, we begin to clarify the causes, origins, limitations, and trade-offs of the intrinsic solvent tolerance in P. putida. This work sheds a light on the possible genetic engineering targets to enhance solvent tolerance in Pseudomonas putida as well as other bacteria.

A High-Efficiency Artificial Synthetic Pathway for 5-Aminovalerate Production From Biobased L-Lysine in Escherichia coli

Bioproduction of 5-aminovalerate (5AVA) from renewable feedstock can support a sustainable biorefinery process to produce bioplastics, such as nylon 5 and nylon 56. In order to achieve the biobased production of 5AVA, a 2-keto-6-aminocaproate-mediated synthetic pathway was established. Combination of L-Lysine α-oxidase from Scomber japonicus, α-ketoacid decarboxylase from Lactococcus lactis and aldehyde dehydrogenase from Escherichia coli could achieve the biosynthesis of 5AVA from biobased L-Lysine in E. coli. The H2O2 produced by L-Lysine α-oxidase was decomposed by the expression of catalase KatE. Finally, 52.24 g/L of 5AVA were obtained through fed-batch biotransformation. Moreover, homology modeling, molecular docking and molecular dynamic simulation analyses were used to identify mutation sites and propose a possible trait-improvement strategy: the expanded catalytic channel of mutant and more hydrogen bonds formed might be beneficial for the substrates stretch. In summary, we have developed a promising artificial pathway for efficient 5AVA synthesis.

Role of Microbial Hydrolysis in Anaerobic Digestion

There is a growing need of substrate flexibility for biobased production of energy and value-added products that allows the application of variable biodegradable residues within a circular economy. It can be used to balance fluctuating energy provision of other renewable sources. Hydrolysis presents one of the biggest limitations during anaerobic digestion. Methods to improve it will result in broader process applicability and improved integration into regional material cycles. Recently, one focus of anaerobic digestion research has been directed to systems with a separate hydrolysis–acidogenesis stage as it might be promised to improve process performance. Conditions can be adjusted to each class of microorganisms individually without harming methanogenic microorganisms. Extensive research of separate biomass pretreatment via biological, chemical, physical or mixed methods has been conducted. Nevertheless, several methods lack economic efficiency, have a high environmental impact or focus on specific substrates. Pretreatment via a separate hydrolysis stage as cell-driven biotransformation in a suspension might be an alternative that enables high yields, flexible feeding and production, and a better process control. In this review, we summarize existing technologies for microbial hydrolytic biotransformation in a separate reactor stage and the impacts of substrate, operational parameters, combined methods and process design as well as remaining challenges.

Integrating Value Considerations in the Decision Making for the Design of Biorefineries

Biobased production has been promoted as a sustainable alternative to fossil resources. However, controversies over its impact on sustainability highlight societal concerns, value tensions and uncertainties that have not been taken into account during its development. In this work, the consideration of stakeholders’ values in a biorefinery design project is investigated. Value sensitive design (VSD) is a promising approach to the design of technologies with consideration of stakeholders’ values, however, it is not directly applicable for complex systems like biorefineries. Therefore, some elements of VSD, such as the identification of relevant values and their connection to a technology’s features, are brought into biorefinery design practice. Midstream modulation (MM), an approach to promoting the consideration of societal aspects during research and development activities, is applied to promote reflection and value considerations during the design decision making. As result, it is shown that MM interventions during the design process led to new design alternatives in support of stakeholders' values, and allowed to recognize and respond to emerging value tensions within the scope of the project. In this way, the present work shows a novel approach for the technical investigation of VSD, especially for biorefineries. Also, based on this work it is argued that not only reflection, but also flexibility and openness are important for the application of VSD in the context of biorefinery design.

Turning the screw: engineering extreme pH resistance in Escherichia coli through combinatorial synthetic operons

Adoption of microorganisms as platforms for sustainable biobased production requires host cells to be able to withstand harsh industrial conditions, which are usually far from the ones where these organisms are naturally adapted to thrive. However, novel survival mechanisms unearthed by the study of microbiomes from extreme habitats may be exploited to enhance microbial robustness under the strict conditions needed for different applications. In this work, synthetic biology approaches were used to engineer enhanced acidic tolerance in Escherichia coli under extreme conditions through the characterization of a library of twenty-seven unique operons composed of combinatorial assemblies of three novel genes from an extreme environment and three synthetic ribosome binding sites. The results here presented illustrate the efficacy of combining different metagenomic genes for tolerance in truly synthetic genetic operons, as expression of these gene clusters increased hundred-fold the survival percentage of cells exposed to an acidic shock in minimal media at pH 1.9 under aerobic conditions.