Heavy metal removal by bioaccumulation using genetically engineered microorganisms

Wastewater effluents from mines and metal refineries are often contaminated with heavy metal ions, so they pose hazards to human and environmental health. Conventional technologies to remove heavy metal ions are well-established, but the most popular methods have drawbacks: chemical precipitation generates sludge waste, and activated carbon and ion exchange resins are made from unsustainable non-renewable resources. Using microbial biomass as the platform for heavy metal ion removal is an alternative method. Specifically, bioaccumulation is a natural biological phenomenon where microorganisms use proteins to uptake and sequester metal ions in the intracellular space to utilize in cellular processes (e.g., enzyme catalysis, signaling, stabilizing charges on biomolecules). Recombinant expression of these import-storage systems in genetically engineered microorganisms allows for enhanced uptake and sequestration of heavy metal ions. This has been studied for over two decades for bioremediative applications, but successful translation to industrial-scale processes is virtually non-existent. Meanwhile, demands for metal resources are increasing while discovery rates to supply primary grade ores are not. This review re-thinks how bioaccumulation can be used and proposes that it can be developed for bioextractive applications—the removal and recovery of heavy metal ions for downstream purification and refining, rather than disposal. This review consolidates previously tested import-storage systems into a biochemical framework and highlights efforts to overcome obstacles that limit industrial feasibility, thereby identifying gaps in knowledge and potential avenues of research in bioaccumulation

Harnessing extremophilic carboxylesterases for applications in polyester depolymerisation and plastic waste recycling

The steady growth in industrial production of synthetic plastics and their limited recycling have resulted in severe environmental pollution and contribute to global warming and oil depletion. Currently, there is an urgent need to develop efficient plastic recycling technologies to prevent further environmental pollution and recover chemical feedstocks for polymer re-synthesis and upcycling in a circular economy. Enzymatic depolymerization of synthetic polyesters by microbial carboxylesterases provides an attractive addition to existing mechanical and chemical recycling technologies due to enzyme specificity, low energy consumption, and mild reaction conditions. Carboxylesterases constitute a diverse group of serine-dependent hydrolases catalysing the cleavage and formation of ester bonds. However, the stability and hydrolytic activity of identified natural esterases towards synthetic polyesters are usually insufficient for applications in industrial polyester recycling. This necessitates further efforts on the discovery of robust enzymes, as well as protein engineering of natural enzymes for enhanced activity and stability. In this essay, we discuss the current knowledge of microbial carboxylesterases that degrade polyesters (polyesterases) with focus on polyethylene terephthalate (PET), which is one of the five major synthetic polymers. Then, we briefly review the recent progress in the discovery and protein engineering of microbial polyesterases, as well as developing enzyme cocktails and secreted protein expression for applications in the depolymerisation of polyester blends and mixed plastics. Future research aimed at the discovery of novel polyesterases from extreme environments and protein engineering for improved performance will aid developing efficient polyester recycling technologies for the circular plastics economy

Improving 1,3-butanediol production in E. coli using a protein engineering approach

Traditional chemical production processes have high yields but require harsh reaction conditions and use non-renewable feedstocks derived from petroleum [1, 2]. These processes have a negative impact on the environment, which motivates the development of more sustainable processes as replacements [2]. Advances in systems metabolic engineering over the past thirty years have given rise to bioprocesses where engineered microbes make chemicals from natural feedstocks under mild reaction conditions [1]. The promise of the field has also resulted in financial resources being made available to the development and commercialization of bioprocess. According to a recent report by Ontario Genomics [3], global investment in the field is projected to be at $38.7B in 2020, a 12-fold increase from what it was at in 2013. Recently, a novel aldolase-based pathway for producing 1,3-butanediol (BDO) in E. coli was reported by Nemr et. al [4, 5]. 1,3-BDO is a commercially viable product as it is used in formulations in cosmetics products, and as a precursor for pharmaceuticals [2]. This pathway involves the conversion of pyruvate to acetaldehyde via the EutE enzyme from E. coli, followed by the conversion of acetaldehyde to 3- hydroxybutanal via the enzyme BH1352 – a Deoxyribose-phosphate aldolase (DERA) – from Bacillus halodurans and subsequently by the conversion of 3-hydroxybutanal to 1,3-BDO via the enzyme PA1127 (an aldo-keto reductase) from Pseudomonas aeruginosa [5]. We examined the crystal structure of BH1352, which revealed key residues involved in catalytic activity in the substrate binding pocket. We show that two DERA mutants F160Y and F160Y/M173I improve the production of 1,3-BDO 5-fold and 6-fold respectively in bench-scale bioreactors [6]. References: 1. Bonk, B. M., Tarasova, Y., Hicks, M. A., Tidor, B., & Prather, K. L. (2018). “Rational design of thiolase substrate specificity for metabolic engineering applications”. Biotechnology and bioengineering, 115(9), 1–16. http://doi.org/10.1002/bit.26737 2. Burk, M. J. (2010). Sustainable production of industrial chemicals from sugars. International Sugar Journal, 112(1333), 30. 3. Ontario Genomics. (2017). Ontario Synthetic Biology Report 2016. Retrieved from: http://www.ontariogenomics.ca/syntheticbiology/Ontario_Synthetic_Biology_Report_2016.pdf 4. Nemr, K., Müller, J. E. N., Joo, J. C., Gawand, P., Choudhary, R., Mendonca, B., et al. (2018). Engineering a short, aldolase-based pathway for (R)-1,3-butanediol production in Escherichia coli. Metabolic Engineering, 48, 13–24. http://doi.org/10.1016/j.ymben.2018.04.013 5. Nemr, K. (2018). Metabolic Engineering of Lyase-Based Biosynthetic Pathways for Non-natural Chemical Production (Unpublished doctoral dissertation). University of Toronto, Toronto, ON, Canada. 6. Kim, T. (2019). Biochemical and Structural Studies of Microbial Enzymes for the Biosynthesis of 1,3-Butanediol (Unpublished doctoral dissertation). University of Toronto, Toronto, ON, Canad

In vivo and in vitro FMN prenylation and (de)carboxylase activation under aerobic conditions

Prenylated FMN (prFMN) is a newly discovered redox cofactor required for activity of the large family of reversible UbiD (de)carboxylases involved in biotransformation of aromatic, heteroaromatic, and unsaturated aliphatic acids (White et al., 2015). Despite the growing demand for decarboxylases in the pulp/paper industry and in forest biorefineries, the vast majority of UbiD-like decarboxylases remain uncharacterized. Functional characterization of the novel UbiD decarboxylases is hindered by the lack of prFMN generating system. prFMN cofactor is synthesized by the UbiX family of FMN prenyltransferases, which use reduced FMN as substrate under anaerobic conditions and dimethylallyl-monophosphate (DMAP) as the prenyl group donor. Here, we report the in vivo and in vitro biosynthesis of prFMN and UbiD activation under aerobic conditions. For in vivo biosynthesis, we used newly discovered UbiX proteins from Salmonella typhimurium and Klebsiella pneumonia, which activated ferulic acid UbiD decarboxylase Fdc1 from Aspergillus niger under aerobic conditions (0.5-1.5 U/mg). For in vitro biosynthesis of prFMN and UbiD activation, we established a one-pot enzyme cascade system that uses prenol, polyphosphate, formate, and riboflavin as starting substrates and (re)generates DMAP, ATP, FMN and NADH. The system contains 6 different enzymes: prenol kinase, polyphosphate kinase, formate dehydrogenase, FMN reductase, riboflavin kinase and FMN prenyltransferase. Under aerobic conditions, this system showed up to 80% conversion of FMN to prFMN and generated active Fdc1 decarboxylase (0.2-1 U/mg). Thus, both systems represent robust approaches for in vivo and in vitro prFMN biosynthesis and UbiD activation under aerobic conditions. The developed FMN prenylation systems will facilitate the exploration and biochemical characterization of UbiD-like decarboxylases and their applications in biocatalysis. Please click Additional Files below to see the full abstract

A novel C-terminal protein degron identified in bacterial aldehyde decarbonylases using directed enzyme evolution

Metabolic engineers have successfully synthesized alkanes, the bulk component of gasoline, using microbial cell factories as a sustainable alternative to petroleum-based fuels. Aldehyde decarbonylases (AD), enzymes which transform acyl aldehydes into alkanes, have been identified as the bottleneck in these alkane producing pathways. Previous studies demonstrated degradation of AD in E. coli cells via unknown molecular mechanism. Here, we present the discovery of a degradation tag (degron) in AD from Prochlorococcus marinus. AD variants were generated by random mutation using error-prone PCR, transferred into E. coli, and grown in chemostat culture with 2g/L hexanal to select for positive mutations. A short C-terminal sequence of AD from P. marinus was proven to be an intact degron by fusing to fluorescent proteins. Statistical analysis of C-terminal sequences of 371 non-redundant ADs from bacteria revealed a conserved sequence in this region, which was proven to be an effective degron. We also showed that ATP-dependent proteases clpAP and lon are responsible for the degradation of AD degron tagged protein. Furthermore, our results indicate that the AD degron caused 91.4% of green fluorescent protein (GFP) degradation when fused to its C-terminus, whereas its elimination in AD enhanced alkane production in vivo. Thus, our work demonstrated the presence of a protein degron tag in bacterial ADs, thereby facilitating further improvements in AD-based alkane production pathways. Please click Additional Files below to see the full abstract

Ni(II)-binding affinity of CcNikZ-II and its homologs:the role of the HH-prong and variable loop revealed by structural and mutational studies

Extracytoplasmic Ni(II)-binding proteins (NiBPs) are molecular shuttles involved in cellular nickel uptake. Here, we determined the crystal structure of apo CcNikZ-II at 2.38 Å, which revealed a Ni(II)-binding site comprised of the double His (HH-)prong (His511, His512) and a short variable (v-)loop nearby (Thr59-Thr64, TEDKYT). Mutagenesis of the site identified Glu60 and His511 as critical for high affinity Ni(II)-binding. Phylogenetic analysis showed 15 protein clusters with two groups containing the HH-prong. Metal-binding assays with 11 purified NiBPs containing this feature yielded higher Ni(II)-binding affinities. Replacement of the wild type v-loop with those from other NiBPs improved the affinity by up to an order of magnitude. This work provides molecular insights into the determinants for Ni(II) affinity and paves way for NiBP engineering.</p

Complete biosynthesis of adipic acid in Saccharomyces cerevisiae

Adipic acid is an important industrial chemical that is produced in significant quantities every year. However, conventional processes for its production are not sustainable due to a heavy dependence on petroleum derived feedstocks and emission of greenhouse gases. Biotechnological production of adipic acid in a yeast host is a sustainable alternative that can overcome these issues. While many heterologous pathways have been proposed to achieve this, significant progress has been made only using the muconic acid pathway which has been implemented by many research groups in both E. coli and S. cerevisiae. However, the in vivo conversion of muconic acid to adipic acid has not been reported. In this work, we describe the isolation of a novel enzyme: 2-enoate reductase that is capable of reducing the pi bond of an alpha unsaturated carboxylic acid such as muconic acid. We have characterized the substrate profile of these novel enzymes and have identified an oxygen tolerant enoate reductase that has significant potential for adipic acid production. This enzyme was tested for muconic acid activity in S. cerevisiae and was then expressed in a muconic acid producing yeast strain to construct a yeast host that is capable of complete biosynthesis of adipic acid using glucose as the only feedstock. To our knowledge, this is the first reported yeast strain that is capable of adipic acid biosynthesis using glucose as the only feedstock. We anticipate that adipic acid production can be improved further through metabolic engineering. Please click Additional Files below to see the full abstract

Enzymatic biotransformation of adipic acid to 6-aminocaproic acid and 1,6- hexamethylenediamine using engineered carboxylic acid reductases and aminotransferases

Biocatalytic reduction of carboxylic acids is gaining importance for the production of polymer precursors and different chemicals. Carboxylic acid reductases (CARs) reduce carboxylic acids to aldehydes using ATP and NADPH as cofactors under mild conditions. Recently, we demonstrated that several bacterial CARs can reduce a broad range of bifunctional carboxylic acids containing amino group or second carboxylic group including adipic acid, which is a precursor for nylon-6-6 (Khusnutdinova et al., 2017). In this project, we demonstrate application of CARs and aminotransferases for further bioconversion of adipic acid to 6-aminocaproic acid and hexamethylenediamine, two other important precursors for nylon synthesis. Based on the crystal structure of the adenylating domain of the CAR enzyme MCH22995 from Mycobacterium chelonae, we generated a structural model of the CAR enzyme MAB4714 from M. abscessus, which is active toward adipic acid. Aiming at improving MAB4714 activity toward 6-aminocaproic acid, we used structure-based protein engineering and generated 16 MAB4714 mutant proteins. Screening of 16 purified MAB4714 variants against 6-aminocaproic acid,identified one protein, which was 10 times more active than the wild-type protein. We also identified several bacterial aminotransferases producing 6-aminocaproic acid from adipic acid in combination with CARs. Further optimization of reaction conditions and application of cofactor regeneration systems resulted in efficient biotransformation of adipic acid to 6-aminocaproic acid (88% conversion) and further to 1,6-hexamethylenediamine (78% conversion). Please click Additional Files below to see the full abstract

Experimental validation of in silico model-predicted isocitrate dehydrogenase and phosphomannose isomerase from Dehalococcoides mccartyi

Gene sequences annotated as proteins of unknown or non-specific function and hypothetical proteins account for a large fraction of most genomes. In the strictly anaerobic and organohalide respiring Dehalococcoides mccartyi, this lack of annotation plagues almost half the genome. Using a combination of bioinformatics analyses and genome-wide metabolic modelling, new or more specific annotations were proposed for about 80 of these poorly annotated genes in previous investigations of D. mccartyi metabolism. Herein, we report the experimental validation of the proposed reannotations for two such genes (KB1_0495 and KB1_0553) from D. mccartyi strains in the KB-1 community. KB1_0495 or DmIDH was originally annotated as an NAD[superscript +]-dependent isocitrate dehydrogenase, but biochemical assays revealed its activity primarily with NADP[superscript +] as a cofactor. KB1_0553, also denoted as DmPMI, was originally annotated as a hypothetical protein/sugar isomerase domain protein. We previously proposed that it was a bifunctional phosphoglucose isomerase/phosphomannose isomerase, but only phosphomannose isomerase activity was identified and confirmed experimentally. Further bioinformatics analyses of these two protein sequences suggest their affiliation to potentially novel enzyme families within their respective larger enzyme super families.University of TorontoNatural Sciences and Engineering Research Council of CanadaGenome Canada (Firm) (Ontario Genomics Institute 2009-OGI-ABC-1405)United States. Dept. of Defense. Strategic Environmental Research and Development Progra