Advancements in Medium-Chain fatty acids production through chain Elongation: Key mechanisms and innovative solutions for overcoming Rate-Limiting steps.

The depletion of fossil fuels has prompted an urgent search for alternative chemicals from renewable sources. Current technology in medium chain fatty acids (MCFAs) production though chain elongation (CE) is becoming increasingly sustainable, hence the motivation for this review, which provides the detailed description, insights and analysis of the metabolic pathways, substrates type, inoculum and fermentation process. The main rate-limiting steps of microbial MCFAs production were comprehensively revealed and the corresponding innovative solutions were also critically evaluated. Innovative strategies such as substrate pretreatment, electrochemical regulation, product separation, fermentation parameter optimization, and electroactive additives have shown significant advantages in overcoming the rate-limiting steps. Furthermore, novel regulatory strategies such as quorum sensing and electronic bifurcation are expected to further increase the MCFAs yield. Finally, the techno-economic analysis was carried out, and the future research focuses were also put forward.

Unraveling the rate-limiting step in microorganisms' mediation of denitrification and phosphorus absorption/transport processes in a highly regulated river-lake system.

River-lake ecosystems are indispensable hubs for water transfers and flow regulation engineering, which have frequent and complex artificial hydrological regulation processes, and the water quality is often unstable. Microorganisms usually affect these systems by driving the nutrient cycling process. Thus, understanding the key biochemical rate-limiting steps under highly regulated conditions was critical for the water quality stability of river-lake ecosystems. This study investigated how the key microorganisms and genes involving nitrogen and phosphorus cycling contributed to the stability of water by combining 16S rRNA and metagenomic sequencing using the Dongping river-lake system as the case study. The results showed that nitrogen and phosphorus concentrations were significantly lower in lake zones than in river inflow and outflow zones (p < 0.05). Pseudomonas, Acinetobacter, and Microbacterium were the key microorganisms associated with nitrate and phosphate removal. These microorganisms contributed to key genes that promote denitrification (nirB/narG/narH/nasA) and phosphorus absorption and transport (pstA/pstB/pstC/pstS). Partial least squares path modeling (PLS-PM) revealed that environmental factors (especially flow velocity and COD concentration) have a significant negative effect on the key microbial abundance (p < 0.001). Our study provides theoretical support for the effective management and protection of water transfer and the regulation function of the river-lake system.

PRO-IP-seq Tracks Molecular Modifications of Engaged Pol II Complexes at Nucleotide Resolution.

RNA Polymerase II (Pol II) is a multi-subunit complex that undergoes covalent modifications as transcription proceeds through genes and enhancers. Rate-limiting steps of transcription control Pol II recruitment, site and degree of initiation, pausing duration, productive elongation, nascent transcript processing, transcription termination, and Pol II recycling. Here, we developed Precision Run-On coupled to Immuno-Precipitation sequencing (PRO-IP-seq) and tracked phosphorylation of Pol II C-terminal domain (CTD) at nucleotide-resolution. We uncovered precise positional control of Pol II CTD phosphorylation as transcription proceeds from the initiating nucleotide, through early and late promoter-proximal pause, and into productive elongation. Pol II CTD was predominantly unphosphorylated in the early pause-region, whereas serine-2- and serine-5-phosphorylations occurred preferentially in the later pause-region. Serine-7-phosphorylation dominated after the pause-release in a region where Pol II accelerates to its full elongational speed. Interestingly, tracking transcription upon heat-induced reprogramming demonstrated that Pol II with phosphorylated CTD remains paused on heat-repressed genes.

Metabolomics-Driven Identification of the Rate-Limiting Steps in 1-Propanol Production.

The concerted effort for bioproduction of higher alcohols and other commodity chemicals has yielded a consortium of metabolic engineering techniques to identify targets to enhance performance of engineered microbial strains. Here, we demonstrate the use of metabolomics as a tool to systematically identify targets for improved production phenotypes in Escherichia coli. Gas chromatography/mass spectrometry (GC/MS) and ion-pair LC-MS/MS were performed to investigate metabolic perturbations in various 1-propanol producing strains. Two initial strains were compared that differ in the expression of the citramalate and threonine pathways, which hold a synergistic relationship to maximize production yields. While this results in increased productivity, no change in titer was observed when the threonine pathway was overexpressed beyond native levels. Metabolomics revealed accumulation of upstream byproducts, norvaline and 2-aminobutyrate, both of which are derived from 2-ketobutyrate (2KB). Eliminating the competing pathway by gene knockouts or improving flux through overexpression of glycolysis gene effectively increased the intracellular 2KB pool. However, the increase in 2KB intracellular concentration yielded decreased production titers, indicating toxicity caused by 2KB and an insufficient turnover rate of 2KB to 1-propanol. Optimization of alcohol dehydrogenase YqhD activity using an ribosome binding site (RBS) library improved 1-propanol titer (g/L) and yield (g/g of glucose) by 38 and 29% in 72 h compared to the base strain, respectively. This study demonstrates the use of metabolomics as a powerful tool to aid systematic strain improvement for metabolically engineered organisms.

Direct RBS Engineering of the biosynthetic gene cluster for efficient productivity of violaceins in E. coli.

BACKGROUND: Violaceins have attracted much attention as potential targets used in medicines, food additives, insecticides, cosmetics and textiles, but low productivity was the key factor to limit their large-scale applications. This work put forward a direct RBS engineering strategy to engineer the violacein biosynthetic gene cluster cloned from Chromobacterium violaceum ATCC 12,472 to efficiently improve the fermentation titers. RESULTS: Through four-rounds of engineering of the native RBSs within the violaceins biosynthetic operon vioABCDE, this work apparently broke through the rate-limiting steps of intermediates conversion, resulting in 2.41-fold improvement of violaceins production compared to the titers of the starting strain Escherichia coli BL21(DE3) (Vio12472). Furthermore, by optimizing the batch-fermentation parameters including temperature, concentration of IPTG inducer and fermentation time, the maximum yield of violaceins from (BCDE)m (tnaA-) reached 3269.7 µM at 2 mM tryptophan in the medium. Interestingly, rather than previous reported low temperature (20 ℃), we for the first time found the RBS engineered Escherichia coli strain (BCDE)m worked better at higher temperature (30 ℃ and 37 ℃), leading to a higher-level production of violaceins. CONCLUSIONS: To our knowledge, this is the first time that a direct RBS engineering strategy is used for the biosynthesis of natural products, having the potential for a greater improvement of the product yields within tryptophan hyperproducers and simultaneously avoiding the costly low temperature cultivation for large-scale industrial production of violaciens. This direct RBS engineering strategy could also be easily and helpfully used in engineering the native RBSs of other larger and value-added natural product biosynthetic gene clusters by widely used site-specific mutagenesis methods represented by inverse PCR or CRISPR-Cas9 techniques to increase their fermentation titers in the future.

Engineering Haloferax mediterranei as an Efficient Platform for High Level Production of Lycopene.

Lycopene attracts increasing interests in the pharmaceutical, food, and cosmetic industries due to its anti-oxidative and anti-cancer properties. Compared with other lycopene production methods, such as chemical synthesis or direct extraction from plants, the biosynthesis approach using microbes is more economical and sustainable. In this work, we engineered Haloferax mediterranei, a halophilic archaeon, as a new lycopene producer. H. mediterranei has the de novo synthetic pathway for lycopene but cannot accumulate this compound. To address this issue, we reinforced the lycopene synthesis pathway, blocked its flux to other carotenoids and disrupted its competitive pathways. The reaction from geranylgeranyl-PP to phytoene catalyzed by phytoene synthase (CrtB) was identified as the rate-limiting step in H. mediterranei. Insertion of a strong promoter PphaR immediately upstream of the crtB gene, or overexpression of the heterologous CrtB and phytoene desaturase (CrtI) led to a higher yield of lycopene. In addition, blocking bacterioruberin biosynthesis increased the purity and yield of lycopene. Knock-out of the key genes, responsible for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) biosynthesis, diverted more carbon flux into lycopene synthesis, and thus further enhanced lycopene production. The metabolic engineered H. mediterranei strain produced lycopene at 119.25 ± 0.55 mg per gram of dry cell weight in shake flask fermentation. The obtained yield was superior compared to the lycopene production observed in most of the engineered Escherichia coli or yeast even when they were cultivated in pilot scale bioreactors. Collectively, this work offers insights into the mechanism involved in carotenoid biosynthesis in haloarchaea and demonstrates the potential of using haloarchaea for the production of lycopene or other carotenoids.

Added value of autoregulation and multi-step kinetics of transcription initiation.

Bacterial gene expression regulation occurs mostly during transcription, which has two main rate-limiting steps: the close complex formation, when the RNA polymerase binds to an active promoter, and the subsequent open complex formation, after which it follows elongation. Tuning these steps' kinetics by the action of e.g. transcription factors, allows for a wide diversity of dynamics. For example, adding autoregulation generates single-gene circuits able to perform more complex tasks. Using stochastic models of transcription kinetics with empirically validated parameter values, we investigate how autoregulation and the multi-step transcription initiation kinetics of single-gene autoregulated circuits can be combined to fine-tune steady state mean and cell-to-cell variability in protein expression levels, as well as response times. Next, we investigate how they can be jointly tuned to control complex behaviours, namely, time counting, switching dynamics and memory storage. Overall, our finding suggests that, in bacteria, jointly regulating a single-gene circuit's topology and the transcription initiation multi-step dynamics allows enhancing complex task performance.

Rate-limiting steps in the Saccharomyces cerevisiae ergosterol pathway: towards improved ergosta-5,7-dien-3ß-ol accumulation by metabolic engineering.

Ergosterol is the predominant nature sterol constituent of plasma membrane in Saccharomyces cerevisiae. Herein, the biosynthetic pathway of ergosterol was proposed to be metabolically engineered for the efficient production of ergosta-5,7-dien-3ß-ol, which is the precursor of vitamin D4. By target disruption of erg5, involved in the end-steps of post-squalene formation, predominantly accumulated ergosta-5,7-dien-3ß-ol (4.12 mg/g dry cell weight). Moreover, the rate-limiting enzymes of ergosta-5,7-dien-3ß-ol biosynthesis were characterized. Overexpression of Hmg1p led to a significant accumulation of squalene, and induction of Erg1p/Erg11p expression raised the yield of both total sterols and ergosta-5,7-dien-3ß-ol with no obvious changes in growth behavior. Furthermore, the transcription factor allele upc2-1 was overexpressed to explore the effect of combined induction of rate-limiting enzymes. Compared with an obviously enhanced yield of ergosterol in the wild-type strain, decreases of both the ergosta-5,7-dienol levels and the total sterol yield were found in Δerg5-upc2-1, probably due to the unbalanced NADH/NAD+ ratio observed in the erg5 knockouts, suggesting the whole-cell redox homeostasis was also vital for end-product biosynthesis. The data obtained in this study can be used as reference values for the production of sterol-related intermediates involved in the post-squalene biosynthetic pathway in food-grade S. cerevisiae strains.

Dissecting the stochastic transcription initiation process in live Escherichia coli.

We investigate the hypothesis that, in Escherichia coli, while the concentration of RNA polymerases differs in different growth conditions, the fraction of RNA polymerases free for transcription remains approximately constant within a certain range of these conditions. After establishing this, we apply a standard model-fitting procedure to fully characterize the in vivo kinetics of the rate-limiting steps in transcription initiation of the Plac/ara-1 promoter from distributions of intervals between transcription events in cells with different RNA polymerase concentrations. We find that, under full induction, the closed complex lasts ∼788 s while subsequent steps last ∼193 s, on average. We then establish that the closed complex formation usually occurs multiple times prior to each successful initiation event. Furthermore, the promoter intermittently switches to an inactive state that, on average, lasts ∼87 s. This is shown to arise from the intermittent repression of the promoter by LacI. The methods employed here should be of use to resolve the rate-limiting steps governing the in vivo dynamics of initiation of prokaryotic promoters, similar to established steady-state assays to resolve the in vitro dynamics.

A probabilistic framework for the exploration of enzymatic capabilities based on feasible kinetics and control analysis.

BACKGROUND: Analysis of limiting steps within enzyme-catalyzed reactions is fundamental to understand their behavior and regulation. Methods capable of unravelling control properties and exploring kinetic capabilities of enzymatic reactions would be particularly useful for protein and metabolic engineering. While single-enzyme control analysis formalism has previously been applied to well-studied enzymatic mechanisms, broader application of this formalism is limited in practice by the limited amount of kinetic data and the difficulty of describing complex allosteric mechanisms. METHODS: To overcome these limitations, we present here a probabilistic framework enabling control analysis of previously unexplored mechanisms under uncertainty. By combining a thermodynamically consistent parameterization with an efficient Sequential Monte Carlo sampler embedded in a Bayesian setting, this framework yields insights into the capabilities of enzyme-catalyzed reactions with modest kinetic information, provided that the catalytic mechanism and a thermodynamic reference point are defined. RESULTS: The framework was used to unravel the impact of thermodynamic affinity, substrate saturation levels and effector concentrations on the flux control and response coefficients of a diverse set of enzymatic reactions. CONCLUSIONS: Our results highlight the importance of the metabolic context in the control analysis of isolated enzymes as well as the use of statistically sound methods for their interpretation. GENERAL SIGNIFICANCE: This framework significantly expands our current capabilities for unravelling the control properties of general reaction kinetics with limited amount of information. This framework will be useful for both theoreticians and experimentalists in the field.