An overview of siderophore biosynthesis among fluorescent Pseudomonads and new insights into their complex cellular organization.

Siderophores are iron-chelating molecules produced by bacteria to access iron, a key nutrient. These compounds have highly diverse chemical structures, with various chelating groups. They are released by bacteria into their environment to scavenge iron and bring it back into the cells. The biosynthesis of siderophores requires complex enzymatic processes and expression of the enzymes involved is very finely regulated by iron availability and diverse transcriptional regulators. Recent data have also highlighted the organization of the enzymes involved in siderophore biosynthesis into siderosomes, multi-enzymatic complexes involved in siderophore synthesis. An understanding of siderophore biosynthesis is of great importance, as these compounds have many potential biotechnological applications because of their metal-chelating properties and their key role in bacterial growth and virulence. This review focuses on the biosynthesis of siderophores produced by fluorescent Pseudomonads, bacteria capable of colonizing a large variety of ecological niches. They are characterized by the production of chromopeptide siderophores, called pyoverdines, which give the typical green colour characteristic of fluorescent pseudomonad cultures. Secondary siderophores are also produced by these strains and can have highly diverse structures (such as pyochelins, pseudomonine, yersiniabactin, corrugatin, achromobactin and quinolobactin).

Production and characterization of two medium-chain-length polydroxyalkanoates by engineered strains of Yarrowia lipolytica.

BACKGROUND: The oleaginous yeast Yarrowia lipolytica is an organism of choice for the tailored production of various compounds such as biofuels or biopolymers. When properly engineered, it is capable of producing medium-chain-length polyhydroxyalkanoate (mcl-PHA), a biobased and biodegradable polymer that can be used as bioplastics or biopolymers for environmental and biomedical applications. RESULTS: This study describes the bioproduction and the main properties of two different mcl-PHA polymers. We generated by metabolic engineering, strains of Y. lipolytica capable of accumulating more than 25% (g/g) of mcl-PHA polymers. Depending of the strain genetic background and the culture conditions, we produced (i) a mcl-PHA homopolymer of 3-hydroxydodecanoic acids, with a mass-average molar mass (Mw) of 316,000 g/mol, showing soft thermoplastic properties with potential applications in packaging and (ii) a mcl-PHA copolymer made of 3-hydroxyoctanoic (3HO), decanoic (3HD), dodecanoic (3HDD) and tetradecanoic (3TD) acids with a Mw of 128,000 g/mol, behaving like a thermoplastic elastomer with potential applications in biomedical material. CONCLUSION: The ability to engineer Y. lipolytica to produce tailored PHAs together with the range of possible applications regarding their biophysical and mechanical properties opens new perspectives in the field of PHA bioproduction.

Increasing medium chain fatty acids production in Yarrowia lipolytica by metabolic engineering.

BACKGROUND: Oleaginous yeast Yarrowia lipolytica is an organism of choice for the development of biofuel and oleochemicals. It has become a chassis for metabolic engineering in order to produce targeted lipids. Understanding the function of key-enzymes involved in lipid metabolism is essential to design better routes for enhanced lipid production and for strains producing lipids of interest. Because medium chain fatty acids (MCFA) are valuable compounds for biokerosene production, we previously generated strains capable of producing MCFA up to 12% of total lipid content (Rigouin et al. in ACS Synth Biol 6:1870-1879, 2017). In order to improve accumulation and content of C14 fatty acid (FA), the elongation, degradation and accumulation of these MCFA in Yarrowia lipolytica were studied. RESULTS: We brought evidence of the role of YALI0F0654 (YlELO1) protein in the elongation of exogenous or de novo synthesized C14 FA into C16 FA and C18 FA. YlELO1 deletion into a αFAS_I1220W expressing strain leads to the sole production of C14 FA. However, because this strain does not provide the FA essential for its growth, it requires being cultivated with essential fatty acids and C14 FA yield is limited. To promote MCFA accumulation in Y. lipolytica without compromising the growth, we overexpressed a plant diglyceride acyltransferase specific for MCFA and reached an accumulation of MCFA up to 45% of total lipid content. CONCLUSION: We characterized the role of YlELO1 in Y. lipolytica by proving its involvement in Medium chain fatty acids elongation. We showed that MCFA content can be increased in Yarrowia lipolytica by promoting their accumulation into a stable storage form (triacylglycerides) to limit their elongation and their degradation.

Identification and structural analysis of an L-asparaginase enzyme from guinea pig with putative tumor cell killing properties.

The initial observation that guinea pig serum kills lymphoma cells marks the serendipitous discovery of a new class of anti-cancer agents. The serum cell killing factor was shown to be an enzyme with L-asparaginase (ASNase) activity. As a direct result of this observation, several bacterial L-asparaginases were developed and are currently approved by the Food and Drug Administration for the treatment of the subset of hematological malignancies that are dependent on the extracellular pool of the amino acid asparagine. As drugs, these enzymes act to hydrolyze asparagine to aspartate, thereby starving the cancer cells of this amino acid. Prior to the work presented here, the precise identity of this guinea pig enzyme has not been reported in the peer-reviewed literature. We discovered that the guinea pig enzyme annotated as H0W0T5_CAVPO, which we refer to as gpASNase1, has the required low Km property consistent with that possessed by the cell-killing guinea pig serum enzyme. Elucidation of the ligand-free and aspartate complex gpASNase1 crystal structures allows a direct comparison with the bacterial enzymes and serves to explain the lack of L-glutaminase activity in the guinea pig enzyme. The structures were also used to generate a homology model for the human homolog hASNase1 and to help explain its vastly different kinetic properties compared with gpASNase1, despite a 70% sequence identity. Given that the bacterial enzymes frequently present immunogenic and other toxic side effects, this work suggests that gpASNase1 could be a promising alternative to these bacterial enzymes.

Engineering Siderophore Biosynthesis and Regulation Pathways to Increase Diversity and Availability.

Siderophores are small metal chelators synthesized by numerous organisms to access iron. These secondary metabolites are ubiquitously present on Earth, and because their production represents the main strategy to assimilate iron, they play an important role in both positive and negative interactions between organisms. In addition, siderophores are used in biotechnology for diverse applications in medicine, agriculture and the environment. The generation of non-natural siderophore analogs provides a new opportunity to create new-to-nature chelating biomolecules that can offer new properties to expand applications. This review summarizes the main strategies of combinatorial biosynthesis that have been used to generate siderophore analogs. We first provide a brief overview of siderophore biosynthesis, followed by a description of the strategies, namely, precursor-directed biosynthesis, the design of synthetic or heterologous pathways and enzyme engineering, used in siderophore biosynthetic pathways to create diversity. In addition, this review highlights the engineering strategies that have been used to improve the production of siderophores by cells to facilitate their downstream utilization.

Screening of enzymatic activities for the depolymerisation of the marine bacterial exopolysaccharide HE800.

The exopolysaccharide (EPS) HE800 is a marine-derived polysaccharide (from 8 × 10(5) to 1.5 × 10(6) g mol(-1)) produced by Vibrio diabolicus and displaying original structural features close to those of glycosaminoglycans. In order to confer new biological activities to the EPS HE800 or to improve them, structural modifications need to be performed. In particular, depolymerisation is required to generate low-molecular-weight derivatives. To circumvent the use of chemical methods that lack specificity and reproducibility, enzymes able to perform such reaction are sought. This study reports the screening for enzymes capable of depolymerising the EPS HE800. A large diversity of enzyme sources has been studied: commercially available glycoside hydrolases with broad substrate specificity, lyases, and proteases as well as growing microorganisms. Interestingly, we found that the genus Enterococcus and, more particularly, the strain Enterococcus faecalis were able to depolymerise the EPS HE800. Partial characterization of the enzymatic activity gives evidence for a random and incomplete depolymerisation pattern that yields low-molecular-weight products of 40,000 g mol(-1). Genomic analysis and activity assays allowed the identification of a relevant open reading frame (ORF) which encodes an endo-N-acetyl-galactosaminidase. This study establishes the foundation for the development of an enzymatic depolymerisation process.

Engineering siderophore production in Pseudomonas to improve asbestos weathering.

Iron plays a key role in microbial metabolism and bacteria have developed multiple siderophore-driven mechanisms due to its poor bioavailability for organisms in the environment. Iron-bearing minerals generally serve as a nutrient source to sustain bacterial growth after bioweathering. Siderophores are high-affinity ferric iron chelators, of which the biosynthesis is tightly regulated by the presence of iron. Pyoverdine-producing Pseudomonas have shown their ability to extract iron and magnesium from asbestos waste as nutrients. However, such bioweathering is rapidly limited due to repression of the pyoverdine pathway and the low bacterial requirement for iron. We developed a metabolically engineered strain of Pseudomonas aeruginosa for which pyoverdine production was no longer repressed by iron as a proof of concept. We compared siderophore-promoted dissolution of flocking asbestos waste by this optimized strain to that by the wild-type strain. Interestingly, pyoverdine production by the optimized strain was seven times higher in the presence of asbestos waste and the dissolution of magnesium and iron from the chrysotile fibres contained in flocking asbestos waste was significantly enhanced. This innovative mineral weathering process contributes to remove toxic iron from the asbestos fibres and may contribute to the development of an eco-friendly method to manage asbestos waste.

Genome Editing in Y. lipolytica Using TALENs.

TALENs (Transcription Activator-Like EndoNuclease) are molecular scissors designed to recognize and introduce a double-strand break at a specific genome locus. They represent tools of interest in the frame of genome edition. Upon cleavage, two different pathways lead to DNA repair: Non-homologous End Joining (NHEJ) repair, leading to efficient introduction of short insertion/deletion mutations which can disrupt translational reading frame and Homology Recombination (HR)-directed repair that occurs when exogenous DNA is supplied. Here we introduce how to use TALENs in the oleaginous yeast Yarrowia lipolytica by presenting a step-by-step method allowing to knock out or to introduce in vivo a point mutation in a gene of Yarrowia lipolytica. This chapter describes the material required, the transformation procedure, and the screening process.

Engineering the Yeast Yarrowia lipolytica for Production of Polylactic Acid Homopolymer.

Polylactic acid is a plastic polymer widely used in different applications from printing filaments for 3D printer to mulching films in agriculture, packaging materials, etc. Here, we report the production of poly-D-lactic acid (PDLA) in an engineered yeast strain of Yarrowia lipolytica. Firstly, the pathway for lactic acid consumption in this yeast was identified and interrupted. Then, the heterologous pathway for PDLA production, which contains a propionyl-CoA transferase (PCT) converting lactic acid into lactyl-CoA, and an evolved polyhydroxyalkanoic acid (PHA) synthase polymerizing lactyl-CoA, was introduced into the engineered strain. Among the different PCT proteins that were expressed in Y. lipolytica, the Clostridium propionicum PCT exhibited the highest efficiency in conversion of D-lactic acid to D-lactyl-CoA. We further evaluated the lactyl-CoA and PDLA productions by expressing this PCT and a variant of Pseudomonas aeruginosa PHA synthase at different subcellular localizations. The best PDLA production was obtained by expressing the PCT in the cytosol and the variant of PHA synthase in peroxisome. PDLA homopolymer accumulation in the cell reached 26 mg/g-DCW, and the molecular weights of the polymer (Mw = 50.5 × 103 g/mol and Mn = 12.5 × 103 g/mol) were among the highest reported for an in vivo production.

Multiple Parameters Drive the Efficiency of CRISPR/Cas9-Induced Gene Modifications in Yarrowia lipolytica.

Yarrowia lipolytica is an oleaginous yeast of growing industrial interest for biotechnological applications. In the last few years, genome edition has become an easier and more accessible prospect with the world wild spread development of CRISPR/Cas9 technology. In this study, we focused our attention on the production of the two key elements of the CRISPR-Cas9 ribonucleic acid protein complex in this non-conventional yeast. The efficiency of NHEJ-induced knockout was measured by time-course monitoring using multiple parameters flow cytometry, as well as phenotypic and genotypic observations, and linked to nuclease production levels showing that its strong overexpression is unnecessary. Thus, the limiting factor for the generation of a functional ribonucleic acid protein complex clearly resides in guide expression, which was probed by testing different linker lengths between the transfer RNA promoter and the sgRNA. The results highlight a clear deleterious effect of mismatching bases at the 5' end of the target sequence. For the first time in yeast, an investigation of its maturation from the primary transcript was undertaken by sequencing multiple sgRNAs extracted from the host. These data provide insights into of the yeast small RNA processing, from synthesis to maturation, and suggests a pathway for their degradation in Y. lipolytica. Subsequently, a whole-genome sequencing of a modified strain detected no abnormal modification due to off-target effects, confirming CRISPR/Cas9 as a safe strategy for editing Y. lipolytica genome. Finally, the optimized system was used to promote in vivo directed mutagenesis via homology-directed repair with a ssDNA oligonucleotide.

Discovery of human-like L-asparaginases with potential clinical use by directed evolution.

L-asparaginase is a chemotherapy drug used to treat acute lymphoblastic leukemia (ALL). The main prerequisite for clinical efficacy of L-asparaginases is micromolar KM for asparagine to allow for complete depletion of this amino acid in the blood. Since currently approved L-asparaginases are of bacterial origin, immunogenicity is a challenge, which would be mitigated by a human enzyme. However, all human L-asparaginases have millimolar KM for asparagine. We recently identified the low KM guinea pig L-asparaginase (gpASNase1). Because gpASNase1 and human L-asparaginase 1 (hASNase1) share ~70% amino-acid identity, we decided to humanize gpASNase1 by generating chimeras with hASNase1 through DNA shuffling. To identify low KM chimeras we developed a suitable bacterial selection system (E. coli strain BW5Δ). Transforming BW5Δ with the shuffling libraries allowed for the identification of several low KM clones. To further humanize these clones, the C-terminal domain of gpASNase1 was replaced with that of hASNase1. Two of the identified clones, 63N-hC and 65N-hC, share respectively 85.7% and 87.1% identity with the hASNase1 but have a KM similar to gpASNase1. These clones possess 100-140 fold enhanced catalytic efficiency compared to hASNase1. Notably, we also show that these highly human-like L-asparaginases maintain their in vitro ALL killing potential.

Production of Medium Chain Fatty Acids by Yarrowia lipolytica: Combining Molecular Design and TALEN to Engineer the Fatty Acid Synthase.

Yarrowia lipolytica is a promising organism for the production of lipids of biotechnological interest and particularly for biofuel. In this study, we engineered the key enzyme involved in lipid biosynthesis, the giant multifunctional fatty acid synthase (FAS), to shorten chain length of the synthesized fatty acids. Taking as starting point that the ketoacyl synthase (KS) domain of Yarrowia lipolytica FAS is directly involved in chain length specificity, we used molecular modeling to investigate molecular recognition of palmitic acid (C16 fatty acid) by the KS. This enabled to point out the key role of an isoleucine residue, I1220, from the fatty acid binding site, which could be targeted by mutagenesis. To address this challenge, TALEN (transcription activator-like effector nucleases)-based genome editing technology was applied for the first time to Yarrowia lipolytica and proved to be very efficient for inducing targeted genome modifications. Among the generated FAS mutants, those having a bulky aromatic amino acid residue in place of the native isoleucine at position 1220 led to a significant increase of myristic acid (C14) production compared to parental wild-type KS. Particularly, the best performing mutant, I1220W, accumulates C14 at a level of 11.6% total fatty acids. Overall, this work illustrates how a combination of molecular modeling and genome-editing technology can offer novel opportunities to rationally engineer complex systems for synthetic biology.

Characterization of the phytochelatin synthase from the human parasitic nematode Ancylostoma ceylanicum.

Hookworm disease is a debilitating worm infection that affects hundreds of millions of people. Despite the existence of anthelmintic drugs, reports have testified of a decrease in efficacy of these drugs. Therefore, it is imperative to find new drugs and drug targets for hookworm disease treatment. In this study we identify the gene encoding the phytochelatin synthase in the human hookworm, Ancylostoma ceylanicum (AcePCS). Phytochelatin synthase catalyzes the production of metal chelating peptides, the phytochelatins, from glutathione (GSH). In plants, algae, and fungi phytochelatin production is important for metal tolerance and detoxification. Phytochelatin synthase proteins also function in the elimination of xenobiotics by processing GSH S-conjugates. We found that in vitro AcePCS could both synthesize phytochelatins and hydrolyze a GSH S-conjugate. Interestingly, the enzyme works through a thiol-dependent and, notably, metal-independent mechanism for both transpeptidase (phytochelatin synthesis) and peptidase (hydrolysis of GSH S-conjugates) activities. AcePCS mRNAs are expressed in vivo throughout the life cycle of A. ceylanicum. Mature adult male hookworms isolated from the small intestines of their hosts displayed significantly enhanced expression of AcePCS with transcript levels 5-fold greater than other developmental forms. Although the role of AcePCS in A. ceylanicum biology has yet to be fully investigated the results reported here provide encouraging evidence of the potential that this enzyme holds as a target for new chemotherapeutic intervention.

Towards an understanding of the function of the phytochelatin synthase of Schistosoma mansoni.

Phytochelatin synthase (PCS) is a protease-like enzyme that catalyzes the production of metal chelating peptides, the phytochelatins, from glutathione (GSH). In plants, algae, and fungi phytochelatin production is important for metal tolerance and detoxification. PCS proteins also function in xenobiotic metabolism by processing GSH S-conjugates. The aim of the present study is to elucidate the role of PCS in the parasitic worm Schistosoma mansoni. Recombinant S. mansoni PCS proteins expressed in bacteria could both synthesize phytochelatins and hydrolyze various GSH S-conjugates. We found that both the N-truncated protein and the N- and C-terminal truncated form of the enzyme (corresponding to only the catalytic domain) work through a thiol-dependant and, notably, metal-independent mechanism for both transpeptidase (phytochelatin synthesis) and peptidase (hydrolysis of GSH S-conjugates) activities. PCS transcript abundance was increased by metals and xenobiotics in cultured adult worms. In addition, these treatments were found to increase transcript abundance of other enzymes involved in GSH metabolism. Highest levels of PCS transcripts were identified in the esophageal gland of adult worms. Taken together, these results suggest that S. mansoni PCS participates in both metal homoeostasis and xenobiotic metabolism rather than metal detoxification as previously suggested and that the enzyme may be part of a global stress response in the worm. Because humans do not have PCS, this enzyme is of particular interest as a drug target for schistosomiasis.

The redox biology of schistosome parasites and applications for drug development.

Schistosomiasis caused by Schistosoma spp. is a serious public health concern, especially in sub-Saharan Africa. Praziquantel is the only drug currently administrated to treat this disease. However, praziquantel-resistant parasites have been identified in endemic areas and can be generated in the laboratory. Therefore, it is essential to find new therapeutics. Antioxidants are appealing drug targets. In order to survive in their hosts, schistosomes are challenged by reactive oxygen species from intrinsic and extrinsic sources. Schistosome antioxidant enzymes have been identified as essential proteins and novel drug targets and inhibition of the antioxidant response can lead to parasite death. Because the organization of the redox network in schistosomes is significantly different from that in humans, new drugs are being developed targeting schistosome antioxidants. In this paper the redox biology of schistosomes is discussed and their potential use as drug targets is reviewed. It is hoped that compounds targeting parasite antioxidant responses will become clinically relevant drugs in the near future.