Phenotypic design choices for enhanced two-stage microbial production processes

Microbial metabolism can be harnessed to produce a broad range of industrially important chemicals. Many microorganisms naturally produce some important compounds but do so with low efficiency. To target a more diverse range of chemicals, pathways for non-natural products can be designed and implemented. However, in order to improve these microbes toward the target of industrial production, their metabolism must be engineered by controlling metabolic flux through key pathways. The merits of microbial production processes are often measured using three key variables: titer, rate and yield (TRY). Each of these variables has an impact on the economic viability of any microbial production process. Previous research into improving these TRY metrics have examined the efficacy of decoupling microbial growth from chemical production to achieve enhanced production rates. However, there has been limited research into the choice of microbial phenotype for the growth and production stages of two-stage production processes. Moreover, the substrate uptake rates of microbes drop significantly upon reducing the growth rate, adding to the need for intelligent phenotype selection while designing strains for two-stage processes. In this work, we present a two-stage optimization framework that scans the phenotypic space of microbial metabolism to identify the correct choice of phenotypes during growth and production stages, along with the optimal time to switch between these stages to achieve required TRY values. Through this framework and using Escherichia coli as a model organism, we compare the performance of two-stage fermentation processes where dynamic pathway regulation is involved with one-stage fermentation processes that have static intervention strategies implemented for a range of naturally produced chemicals. Our results indicate that while one stage processes are better at achieving optimal yields, two-stage processes outperform them in achieving optimal production rates even after incorporating the effects of reduced substrate uptake rates during the production stage. We anticipate that this optimization framework would be invaluable in designing microbial strains and fermentation processes for industrial chemical productio

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

Characterization of Proton Production and Consumption Associated with Microbial Metabolism

BACKGROUND: Production or consumption of protons in growth medium during microbial metabolism plays an important role in determining the pH of the environment. Such pH changes resulting from microbial metabolism may influence the geochemical speciation of many elements in subsurface environments. Protons produced or consumed during microbial growth were measured by determining the amount of acid or base added in a 5 L batch bioreactor equipped with pH control for different species including Escherichia coli, Geobacter sulfurreducens, and Geobacter metallireducens. RESULTS: An in silico model was used to predict the proton secretion or consumption rates and the results were compared with the data. The data was found to confirm predictions of proton consumption during aerobic growth of E. coli with acetate as the carbon source. However, in contrast to proton consumption observed during aerobic growth of E. coli with acetate, proton secretion was observed during growth of Geobacter species with acetate as the donor and Fe(III) as the extracellular electron acceptor. CONCLUSIONS: In this study, we have also shown that the final pH of the medium can be either acidic or basic depending on the choice of the electron acceptor for the same electron donor. In all cases, the in silico model could predict qualitatively the proton production/consumption rates obtained from the experimental data. Therefore, measurements of pH equivalents generated or consumed during growth can help characterize the microbial physiology further and can be valuable for optimizing practical applications such as microbial fuel cells, where growth associated pH changes can limit current generation rates

In silico characterization of microbial electrosynthesis for metabolic engineering of biochemicals

<p>Abstract</p> <p>Background</p> <p>A critical concern in metabolic engineering is the need to balance the demand and supply of redox intermediates such as NADH. Bioelectrochemical techniques offer a novel and promising method to alleviate redox imbalances during the synthesis of biochemicals and biofuels. Broadly, these techniques reduce intracellular NAD⁺to NADH and therefore manipulate the cell's redox balance. The cellular response to such redox changes and the additional reducing power available to the cell can be harnessed to produce desired metabolites. In the context of microbial fermentation, these bioelectrochemical techniques can be used to improve product yields and/or productivity.</p> <p>Results</p> <p>We have developed a method to characterize the role of bioelectrosynthesis in chemical production using the genome-scale metabolic model of <it>E. coli</it>. The results in this paper elucidate the role of bioelectrosynthesis and its impact on biomass growth, cellular ATP yields and biochemical production. The results also suggest that strain design strategies can change for fermentation processes that employ microbial electrosynthesis and suggest that dynamic operating strategies lead to maximizing productivity.</p> <p>Conclusions</p> <p>The results in this paper provide a systematic understanding of the benefits and limitations of bioelectrochemical techniques for biochemical production and highlight how electrical enhancement can impact cellular metabolism and biochemical production.</p

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

Economics of membrane occupancy and respiro-fermentation

The authors propose that prokaryotic metabolism is fundamentally constrained by the cytoplasmic membrane surface area available for protein expression, and show that this constraint can explain previously puzzling physiological phenomena, including respiro-fermentation

उत्तर तमिलनाडु के कूटल्लूर, पषयार और कावेरिपट्टणम से तारली की भारी पकड

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The oil sardine fishery along northern Tamil Nadu coast with a note on unusually heavy landings at Cuddalore,Pazhayar and Kaveripattinam

The Indian Oil sardine, Sardinella longiceps is one of the important exploited fishery resources along the Tamil Nadu. The bulk of oil sardine were landed along the coast by the bag-net, Eda valai while lesser quantities were caught by the gill nets, Kavala valai and Thattakavala valai. Unusually heavy landings of oil sardine by the Eda valai units have been recorded at Cuddalore and Pazhayar fisheries harbours and Kaveripattinam during certain months of the period, 1989-'90. Abundance of juvenile oil sardines of the size group 110 - 114 mm was noticed during May - June, '90 at Pazhayar and in July at Kaveripattinam. Larger size groups of 175 - 179 mm predominated in September, '89 whereas in the same period of the succeeding year, 165 – 169 mm length groups supported the fishery. The unprecedented heavy landings of the oil sardine in most of the centers did not benefit the fishermen monetarily to any significant extent. Due to lack of demand for fresh fish, the bulk of catches was sun dried. Oil sardine catches are obtained along the east coast of India in areas close to harbours, backwaters and river mouths and this discontinuous distribution of fish appears to indicate its affinity, particularly juvenile phase, to areas where there is admixture of fresh and brackish water

Recent exploitation trend of oil sardine along Tamil nadu - Pondicherry coast

The Indian oil sardine SardineUa lorgiceps.though a non-conventional resource of the east coast, supports now a regular fishery of high magnitude especially along Andhra Pradesh and TanJl Nadu - Pondicheny coasts. The estimated annual average landing from east coast during the period 1993-97 was 60.638 tonnes against 46.000 t obtained along west coast thereby showing the potential of this new resource, especially along the southern maritime states of the east coast. It has been observed that the oil sardine catch during 1993-97 increased to more than three fold in Andhra Pradesh from that of the previous five year period 1988-92 while along Tamil Nadu - Pondicheny coast the catch almost doubled and recorded 80% of the total oil sardine production of east coast

तमिलनाडु-पोण्डिचेरी तट की तारली मात्स्यिकी-हाल की विदोहन प्रवणता और कुछ जैविक सूचनाऐं

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