Publications by authors named "James C Liao"

190 Publications

Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish.

J Vis Exp 2021 02 10(168). Epub 2021 Feb 10.

Department of Biology, University of Florida, The Whitney Laboratory for Marine Bioscience;

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates reafferent cues that animals must disentangle from relevant sensory cues of the surrounding environment. For example, when a fish swims, flow generated from body undulations is detected by the mechanoreceptive neuromasts, comprising hair cells, that compose the lateral line system. The hair cells then transmit fluid motion information from the sensor to the brain via the sensory afferent neurons. Concurrently, corollary discharge of the motor command is relayed to hair cells to prevent sensory overload. Accounting for the inhibitory effect of predictive motor signals during locomotion is, therefore, critical when evaluating the sensitivity of the lateral line system. We have developed an in vivo electrophysiological approach to simultaneously monitor posterior lateral line afferent neuron and ventral motor root activity in zebrafish larvae (4-7 days post fertilization) that can last for several hours. Extracellular recordings of afferent neurons are achieved using the loose patch clamp technique, which can detect activity from single or multiple neurons. Ventral root recordings are performed through the skin with glass electrodes to detect motor neuron activity. Our experimental protocol provides the potential to monitor endogenous or evoked changes in sensory input across motor behaviors in an intact, behaving vertebrate.
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http://dx.doi.org/10.3791/62233DOI Listing
February 2021

A numerical study of fish adaption behaviors in complex environments with a deep reinforcement learning and immersed boundary-lattice Boltzmann method.

Sci Rep 2021 Jan 18;11(1):1691. Epub 2021 Jan 18.

School of Engineering and Information Technology, University of New South Wales, Canberra, ACT, 2600, Australia.

Fish adaption behaviors in complex environments are of great importance in improving the performance of underwater vehicles. This work presents a numerical study of the adaption behaviors of self-propelled fish in complex environments by developing a numerical framework of deep learning and immersed boundary-lattice Boltzmann method (IB-LBM). In this framework, the fish swimming in a viscous incompressible flow is simulated with an IB-LBM which is validated by conducting two benchmark problems including a uniform flow over a stationary cylinder and a self-propelled anguilliform swimming in a quiescent flow. Furthermore, a deep recurrent Q-network (DRQN) is incorporated with the IB-LBM to train the fish model to adapt its motion to optimally achieve a specific task, such as prey capture, rheotaxis and Kármán gaiting. Compared to existing learning models for fish, this work incorporates the fish position, velocity and acceleration into the state space in the DRQN; and it considers the amplitude and frequency action spaces as well as the historical effects. This framework makes use of the high computational efficiency of the IB-LBM which is of crucial importance for the effective coupling with learning algorithms. Applications of the proposed numerical framework in point-to-point swimming in quiescent flow and position holding both in a uniform stream and a Kármán vortex street demonstrate the strategies used to adapt to different situations.
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http://dx.doi.org/10.1038/s41598-021-81124-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7814145PMC
January 2021

Role of cyanobacterial phosphoketolase in energy regulation and glucose secretion under dark anaerobic and osmotic stress conditions.

Metab Eng 2020 Dec 14. Epub 2020 Dec 14.

Institute of Biological Chemistry, Academia Sinica, Taiwan. Electronic address:

Primary metabolism in cyanobacteria is built on the Calvin-Benson-Bassham (CBB) cycle, oxidative pentose phosphate (OPP) pathway, Embden-Meyerhof-Parnas (EMP) pathway, and the tricarboxylic acid (TCA) cycle. Phosphoketolase (Xpk), commonly found in cyanobacteria, is an enzyme that is linked to all these pathways. However, little is known about its physiological role. Here, we show that most of the cyanobacterial Xpk surveyed are inhibited by ATP, and both copies of Xpk in nitrogen-fixing Cyanothece ATCC51142 are further activated by ADP, suggesting their role in energy regulation. Moreover, Xpk in Synechococcus elongatus PCC7942 and Cyanothece ATCC51142 show that their expressions are dusk-peaked, suggesting their roles in dark conditions. Finally, we find that Xpk in S. elongatus PCC7942 is responsible for survival using ATP produced from the glycogen-to-acetate pathway under dark, anaerobic condition. Interestingly, under this condition, xpk deletion causes glucose secretion in response to osmotic shock such as NaHCO, KHCO and NaCl as part of incomplete glycogen degradation. These findings unveiled the role of this widespread enzyme and open the possibility for enhanced glucose secretion from cyanobacteria.
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http://dx.doi.org/10.1016/j.ymben.2020.12.004DOI Listing
December 2020

Analysis of genomic distributions of SARS-CoV-2 reveals a dominant strain type with strong allelic associations.

Proc Natl Acad Sci U S A 2020 12 12;117(48):30679-30686. Epub 2020 Nov 12.

Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID 19, continues to evolve since its first emergence in December 2019. Using the complete sequences of 1,932 SARS-CoV-2 genomes, various clustering analyses consistently identified six types of the strains. Independent of the dendrogram construction, 13 signature variations in the form of single nucleotide variations (SNVs) in protein coding regions and one SNV in the 5' untranslated region (UTR) were identified and provided a direct interpretation for the six types (types I to VI). The six types of the strains and their underlying signature SNVs were validated in two subsequent analyses of 6,228 and 38,248 SARS-CoV-2 genomes which became available later. To date, type VI, characterized by the four signature SNVs C241T (5'UTR), C3037T (nsp3 F924F), C14408T (nsp12 P4715L), and A23403G (Spike D614G), with strong allelic associations, has become the dominant type. Since C241T is in the 5' UTR with uncertain significance and the characteristics can be captured by the other three strongly associated SNVs, we focus on the other three. The increasing frequency of the type VI haplotype 3037T-14408T-23403G in the majority of the submitted samples in various countries suggests a possible fitness gain conferred by the type VI signature SNVs. The fact that strains missing one or two of these signature SNVs fail to persist implies possible interactions among these SNVs. Later SNVs such as G28881A, G28882A, and G28883C have emerged with strong allelic associations, forming new subtypes. This study suggests that SNVs may become an important consideration in SARS-CoV-2 classification and surveillance.
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http://dx.doi.org/10.1073/pnas.2007840117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7720151PMC
December 2020

Converting Escherichia coli to a Synthetic Methylotroph Growing Solely on Methanol.

Cell 2020 08 10;182(4):933-946.e14. Epub 2020 Aug 10.

Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan. Electronic address:

Methanol, being electron rich and derivable from methane or CO, is a potentially renewable one-carbon (C1) feedstock for microorganisms. Although the ribulose monophosphate (RuMP) cycle used by methylotrophs to assimilate methanol differs from the typical sugar metabolism by only three enzymes, turning a non-methylotrophic organism to a synthetic methylotroph that grows to a high cell density has been challenging. Here we reprogrammed E. coli using metabolic robustness criteria followed by laboratory evolution to establish a strain that can efficiently utilize methanol as the sole carbon source. This synthetic methylotroph alleviated a so far uncharacterized hurdle, DNA-protein crosslinking (DPC), by insertion sequence (IS)-mediated copy number variations (CNVs) and balanced the metabolic flux by mutations. Being capable of growing at a rate comparable with natural methylotrophs in a wide range of methanol concentrations, this synthetic methylotrophic strain illustrates genome editing and evolution for microbial tropism changes and expands the scope of biological C1 conversion.
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http://dx.doi.org/10.1016/j.cell.2020.07.010DOI Listing
August 2020

Oxygen consumption of drift-feeding rainbow trout: the energetic tradeoff between locomotion and feeding in flow.

J Exp Biol 2020 06 26;223(Pt 12). Epub 2020 Jun 26.

The Whitney Laboratory for Marine Bioscience, Department of Biology, University of Florida, 9505 Oceanshore Blvd, St Augustine, FL 32080, USA

To forage in fast, turbulent flow environments where prey is abundant, fishes must deal with the high associated costs of locomotion. Prevailing theory suggests that many species exploit hydrodynamic refuges to minimize the cost of locomotion while foraging. Here, we challenge this theory based on direct oxygen consumption measurements of drift-feeding trout () foraging in the freestream and from behind a flow refuge at velocities up to 100 cm s We demonstrate that refuging is not energetically beneficial when foraging in fast flows because of a high attack cost and low prey capture success associated with leaving a station-holding refuge to intercept prey. By integrating optimum foraging theory with empirical data from respirometry and video tracking, we developed a mathematical model to predict when drift-feeding fishes should exploit or avoid refuges based on prey density, size and flow velocity. Our optimum foraging and refuging model provides new mechanistic insights into locomotor costs, habitat use and prey choice of fish foraging in current-swept habitats.
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http://dx.doi.org/10.1242/jeb.220962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7328162PMC
June 2020

An Algorithmic Approach to Natural Behavior.

Curr Biol 2020 Jun;30(11):R663-R675

Department of Zoology, University of Oxford, 11a Mansfield Road, Oxford OX1 3SZ, UK.

Uncovering the mechanisms and implications of natural behavior is a goal that unites many fields of biology. Yet, the diversity, flexibility, and multi-scale nature of these behaviors often make understanding elusive. Here, we review studies of animal pursuit and evasion - two special classes of behavior where theory-driven experiments and new modeling techniques are beginning to uncover the general control principles underlying natural behavior. A key finding of these studies is that intricate sequences of pursuit and evasion behavior can often be constructed through simple, repeatable rules that link sensory input to motor output: we refer to these rules as behavioral algorithms. Identifying and mathematically characterizing these algorithms has led to important insights, including the discovery of guidance rules that attacking predators use to intercept mobile prey, and coordinated neural and biomechanical mechanisms that animals use to avoid impending collisions. Here, we argue that algorithms provide a good starting point for studies of natural behavior more generally. Rather than beginning at the neural or ecological levels of organization, we advocate starting in the middle, where the algorithms that link sensory input to behavioral output can provide a solid foundation from which to explore both the implementation and the ecological outcomes of behavior. We review insights that have been gained through such an algorithmic approach to pursuit and evasion behaviors. From these, we synthesize theoretical principles and lay out key modeling tools needed to apply an algorithmic approach to the study of other complex natural behaviors.
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http://dx.doi.org/10.1016/j.cub.2020.04.018DOI Listing
June 2020

Efferent modulation of spontaneous lateral line activity during and after zebrafish motor commands.

J Neurophysiol 2019 12 23;122(6):2438-2448. Epub 2019 Oct 23.

The Whitney Laboratory for Marine Bioscience, Department of Biology, University of Florida, Saint Augustine, Florida.

Accurate sensory processing during movement requires the animal to distinguish between external (exafferent) and self-generated (reafferent) stimuli to maintain sensitivity to biologically relevant cues. The lateral line system in fishes is a mechanosensory organ that experiences reafferent sensory feedback, via detection of fluid motion relative to the body generated during behaviors such as swimming. For the first time in larval zebrafish (), we employed simultaneous recordings of lateral line and motor activity to reveal the activity of afferent neurons arising from endogenous feedback from hindbrain efferent neurons during locomotion. Frequency of spontaneous spiking in posterior lateral line afferent neurons decreased during motor activity and was absent for more than half of swimming trials. Targeted photoablation of efferent neurons abolished the afferent inhibition that was correlated to swimming, indicating that inhibitory efferent neurons are necessary for modulating lateral line sensitivity during locomotion. We monitored calcium activity with fish and found synchronous activity between putative cholinergic efferent neurons and motor neurons. We examined correlates of motor activity to determine which may best predict the attenuation of afferent activity and therefore what components of the motor signal are translated through the corollary discharge. Swim duration was most strongly correlated to the change in afferent spike frequency. Attenuated spike frequency persisted past the end of the fictive swim bout, suggesting that corollary discharge also affects the glide phase of burst and glide locomotion. The duration of the glide in which spike frequency was attenuated increased with swim duration but decreased with motor frequency. Our results detail a neuromodulatory mechanism in larval zebrafish that adaptively filters self-generated flow stimuli during both the active and passive phases of locomotion. For the first time in vivo, we quantify the endogenous effect of efferent activity on afferent gain control in a vertebrate hair cell system during and after locomotion. We believe that this pervasive effect has been underestimated when afferent activity of octavolateralis systems is characterized in the current literature. We further identify a refractory period out of phase with efferent control and place this gain mechanism in the context of gliding behavior of freely moving animals.
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http://dx.doi.org/10.1152/jn.00594.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6966311PMC
December 2019

Corrigendum to "Synthetic methanol auxotrophy of Escherichia coli for methanol-dependent growth and production" [Metab. Eng. 49 (2018) 257-266].

Metab Eng 2019 Jul 16;54:292. Epub 2019 May 16.

Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, 128 Sec. 2, Academia Road, Taipei, 115, Taiwan. Electronic address:

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http://dx.doi.org/10.1016/j.ymben.2019.04.006DOI Listing
July 2019

The pursuit strategy of predatory bluefish ( Pomatomus saltatrix).

Proc Biol Sci 2019 02;286(1897):20182934

2 The Whitney Laboratory for Marine Bioscience, University of Florida , Gainesville, FL , USA.

A predator's ability to capture prey depends critically on how it coordinates its approach in response to a prey's motion. Flying insects, bats and raptors are capable of capturing prey with a strategy known as parallel navigation, which allows a predator to move directly towards the anticipated point of interception. It is unclear if predators using other modes of locomotion are employing this strategy when pursuing evasive prey. Using kinematic measurements and mathematical modelling, we tested whether bluefish ( Pomatomus saltatrix) pursue prey fish ( Fundulus heteroclitus) with parallel navigation. We found that the directional changes of bluefish were not consistent with this strategy, but rather were predicted by a strategy known as deviated pursuit. Although deviated pursuit requires few sensory cues and relatively modest motor coordination, a comparison of mathematical models suggested negligible differences in path length from parallel navigation, largely owing to the acceleration exhibited by bluefish near the end of a pursuit. Therefore, the strategy of bluefish is unlike flying predators, but offers comparable performance with potentially more robust control that may be well suited to the visual system and habitat of fishes. These findings offer a foundation for understanding the sensing and locomotor control of predatory fishes.
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http://dx.doi.org/10.1098/rspb.2018.2934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6408892PMC
February 2019

Metabolome analysis revealed the knockout of glyoxylate shunt as an effective strategy for improvement of 1-butanol production in transgenic Escherichia coli.

J Biosci Bioeng 2019 Mar 25;127(3):301-308. Epub 2018 Oct 25.

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address:

High 1-butanol titer has been achieved in a transgenic Escherichia coli strain JCL299FT with a heterologous 1-butanol pathway by deleting competing pathways, balancing of cofactor and resolving free CoA imbalance. However, further improvement of 1-butanol production is still possible in the highest producing strain JCL299FT as indicated by the accumulation of acetate, a major undesired by-product during bio-production by microorganisms that competes with 1-butanol production for the available acetyl-CoA and inhibits protein synthesis resulting in poor growth. In this study, liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based metabolome analysis was performed to identify new rate limiting steps in the 1-butanol production pathway of E. coli strain JCL299FT. The results of metabolome analysis showed increased amounts of glyoxylate in JCL299FT compared to the previous highest-producing strain JCL299F. Knocking out aceA successfully decreased the amount of glyoxylate and reduced acetate accumulation, resulting in the increased levels of TCA cycle and 1-butanol pathway metabolites. These observations indicated that there was a redirection of flux from acetate to TCA cycle and 1-butanol producing pathway, which led to better growth of the 1-butanol producing strain. Consequently, 1-butanol production titer was improved by 39% and the production yield was improved by 12% in M9 medium supplemented with yeast extract. This study is the first report of using the knockout of aceA, the first gene in the glyoxylate shunt that encodes isocitrate lyase, as an effective strategy to reduce acetate overflow in 1-butanol producing E. coli.
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http://dx.doi.org/10.1016/j.jbiosc.2018.08.013DOI Listing
March 2019

Metabolic repair through emergence of new pathways in Escherichia coli.

Nat Chem Biol 2018 11 16;14(11):1005-1009. Epub 2018 Oct 16.

Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.

Escherichia coli can derive all essential metabolites and cofactors through a highly evolved metabolic system. Damage of pathways may affect cell growth and physiology, but the strategies by which damaged metabolic pathways can be circumvented remain intriguing. Here, we use a ΔpanD (encoding for aspartate 1-decarboxylase) strain of E. coli that is unable to produce the β-alanine required for CoA biosynthesis to demonstrate that metabolic systems can overcome pathway damage by extensively rerouting metabolic pathways and modifying existing enzymes for unnatural functions. Using directed cell evolution, rewiring and repurposing of uracil metabolism allowed formation of an alternative β-alanine biosynthetic pathway. After this pathway was deleted, a second was evolved that used a gain-of-function mutation on ornithine decarboxylase (SpeC) to alter reaction and substrate specificity toward an oxidative decarboxylation-deamination reaction. After deletion of both pathways, yet another independent pathway emerged using polyamine biosynthesis, demonstrating the vast capacity of metabolic repair.
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http://dx.doi.org/10.1038/s41589-018-0149-6DOI Listing
November 2018

A modified serine cycle in Escherichia coli coverts methanol and CO to two-carbon compounds.

Nat Commun 2018 09 28;9(1):3992. Epub 2018 Sep 28.

Academia Sinica, 128 Academia Road, Section 2, Taipei, 115, Taiwan, ROC.

Microbial utilization of renewable one-carbon compounds, such as methane, methanol, formic acid, and CO, has emerged as a potential approach to increase the range of carbon sources for bioproduction and address climate change issues. Here, we modify the natural serine cycle present in methylotrophs and build an adapted pathway for Escherichia coli, which allows microorganism to condense methanol (or formate) together with bicarbonate to produce various products. We introduce the modified cycle into E. coli and demonstrate its capability for one-carbon assimilation through growth complementation and isotope labeling experiments. We also demonstrate conversion of methanol to ethanol by utilizing the modified serine cycle in an engineered E. coli strain, achieving a reaction yet to be accomplished by a one-pot chemical process. This work provides a platform to utilize various renewable one-carbon compounds as carbon sources for biosynthesis through a modified serine cycle in E. coli.
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http://dx.doi.org/10.1038/s41467-018-06496-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6162302PMC
September 2018

Head width influences flow sensing by the lateral line canal system in fishes.

J Exp Biol 2018 10 29;221(Pt 21). Epub 2018 Oct 29.

The Whitney Laboratory for Marine Bioscience, Department of Biology, University of Florida, 9505 Ocean Shore Blvd, St Augustine, FL 32080, USA

The architecture of the cephalic lateral line canal system, with distinct lines for the supraorbital, infraorbital and mandibular canals, is highly conserved among fish species. Because these canals lie on a cranial platform, the sensory input they receive is expected to change based on how flow interacts with the head and how the canal pores are spatially distributed. In this study, we explored how head width, a trait that can vary greatly between species and across ontogeny, affects flow sensing. We inserted pressure sensors into physical fish head models of varying widths (narrow, intermediate and wide) and placed these models in steady and vortical flows. We measured sensory performance in terms of detecting flow parameters (flow speed, vortex shedding frequency and cylinder diameter), sensitivity (change in pressure gradient as a function of flow speed) and signal-to-noise ratio (SNR; strength of vortex shedding frequency with respect to background). Our results show that in all model heads the amount of hydrodynamic information was maximized at the anterior region regardless of what metric we used to evaluate the sensory performance. In addition, we discovered that all model heads had the highest SNR for vortices at the intermediate flow speeds but that each head width passively optimized the SNR for different sized vortices, which may have implications for refuge and prey seeking. Our results provide insight into the sensory ecology of fishes and have implications for the design of autonomous underwater vehicles.
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http://dx.doi.org/10.1242/jeb.180877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6240294PMC
October 2018

Synthetic methanol auxotrophy of Escherichia coli for methanol-dependent growth and production.

Metab Eng 2018 09 30;49:257-266. Epub 2018 Aug 30.

Institute of Biological Chemistry, Academia Sinica, 128 Sec. 2, Academia Road, Nankang, Taipei 115, Taiwan. Electronic address:

Methanol is a potentially attractive substrate for bioproduction of chemicals because of the abundance of natural gas and biogas-derived methane. To move towards utilizing methanol as a sole carbon source, here we engineer an Escherichia coli strain to couple methanol utilization with growth on five-carbon (C5) sugars. By deleting essential genes in the pentose phosphate pathway for pentose utilization and expressing heterologous enzymes from the ribulose-monophosphate (RuMP) pathway, we constructed a strain that cannot grow on xylose or ribose minimal media unless methanol is utilized, creating a phenotype termed "synthetic methanol auxotrophy". Our best strains were able to utilize methanol for growth at a rate of 0.17 ± 0.006 (h) with methanol and xylose co-assimilation at a molar ratio of approximately 1:1. Genome sequencing and reversion of mutations indicated that mutations on genes encoding for adenylate cyclase (cyaA) and the formaldehyde detoxification operon (frmRAB) were necessary for the growth phenotype. The methanol auxotrophic strain was further engineered to produce ethanol or 1-butanol to final titers of 4.6 g/L and 2.0 g/L, respectively. C tracing showed that 43% and 71% of ethanol and 1-butanol produced had labeled carbon derived from methanol, respectively.
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http://dx.doi.org/10.1016/j.ymben.2018.08.010DOI Listing
September 2018

Directed strain evolution restructures metabolism for 1-butanol production in minimal media.

Metab Eng 2018 09 11;49:153-163. Epub 2018 Aug 11.

Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan. Electronic address:

Engineering a microbial strain for production sometimes entails metabolic modifications that impair essential physiological processes for growth or production. Restoring these functions may require amending a variety of non-obvious physiological networks, and thus, rational design strategies may not be practical. Here we demonstrate that growth and production may be restored by evolution that repairs impaired metabolic function. Furthermore, we use genomics, metabolomics and proteomics to identify several underlying mutations and metabolic perturbations that allow metabolism to repair. Previously, high titers of butanol production were achieved by Escherichia coli using a growth-coupled, modified Clostridial CoA-dependent pathway after all native fermentative pathways were deleted. However, production was only observed in rich media. Native metabolic function of the host was unable to support growth and production in minimal media. We use directed cell evolution to repair this phenotype and observed improved growth, titers and butanol yields. We found a mutation in pcnB which resulted in decreased plasmid copy numbers and pathway enzymes to balance resource utilization. Increased protein abundance was measured for biosynthetic pathways, glycolytic enzymes have increased activity, and adenosyl energy charge was increased. We also found mutations in the ArcAB two-component system and integration host factor (IHF) that tune redox metabolism to alter byproduct formation. These results demonstrate that directed strain evolution can enable systematic adaptations to repair metabolic function and enhance microbial production. Furthermore, these results demonstrate the versatile repair capabilities of cell metabolism and highlight important aspects of cell physiology that are required for production in minimal media.
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http://dx.doi.org/10.1016/j.ymben.2018.08.004DOI Listing
September 2018

Behavior, Electrophysiology, and Robotics Experiments to Study Lateral Line Sensing in Fishes.

Integr Comp Biol 2018 11;58(5):874-883

The Whitney Laboratory for Marine Bioscience, Department of Biology, University of Florida, St. Augustine, FL 32080, USA.

The lateral line system is a sensory system unique to fishes and amphibians. It is composed of distributed mechanosensory hair cell organs on the head and body (neuromasts), which are sensitive to pressure gradients and water movements. Over the last decade, we have pursued an interdisciplinary approach by combining behavioral, electrophysiology, and robotics experiments to study this fascinating sensory system. In behavioral and electrophysiology experiments, we have studied the larval lateral line system in the model genetic organism, zebrafish (Danio rerio). We found that the lateral line system, even in 5-day-old larvae, is involved in an array of behaviors that are critical to survival, and the deflection of a single neuromast can elicit a swimming response. In robotics experiments, we used a range of physical models with distributed pressure sensors to better understand the hydrodynamic environments from the local perspective of a fish or robot. So far, our efforts have focused on extracting control-related information for a range of application scenarios including characterizing unsteady flows such as Kármán vortex streets for station holding. We also used robot models to test biological hypotheses on how morphology and movement of fishes affect lateral line sensing. Overall, with this review we aim to increase the visibility and accessibility of this multi-disciplinary research approach.
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http://dx.doi.org/10.1093/icb/icy066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6204992PMC
November 2018

Augmenting the Calvin-Benson-Bassham cycle by a synthetic malyl-CoA-glycerate carbon fixation pathway.

Nat Commun 2018 05 22;9(1):2008. Epub 2018 May 22.

Academia Sinica, 128 Academia Road, Section 2, 115, Taipei, Taiwan.

The Calvin-Benson-Bassham (CBB) cycle is presumably evolved for optimal synthesis of C3 sugars, but not for the production of C2 metabolite acetyl-CoA. The carbon loss in producing acetyl-CoA from decarboxylation of C3 sugar limits the maximum carbon yield of photosynthesis. Here we design a synthetic malyl-CoA-glycerate (MCG) pathway to augment the CBB cycle for efficient acetyl-CoA synthesis. This pathway converts a C3 metabolite to two acetyl-CoA by fixation of one additional CO equivalent, or assimilates glyoxylate, a photorespiration intermediate, to produce acetyl-CoA without net carbon loss. We first functionally demonstrate the design of the MCG pathway in vitro and in Escherichia coli. We then implement the pathway in a photosynthetic organism Synechococcus elongates PCC7942, and show that it increases the intracellular acetyl-CoA pool and enhances bicarbonate assimilation by roughly 2-fold. This work provides a strategy to improve carbon fixation efficiency in photosynthetic organisms.
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http://dx.doi.org/10.1038/s41467-018-04417-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5964204PMC
May 2018

Escherichia coli as a host for metabolic engineering.

Metab Eng 2018 11 22;50:16-46. Epub 2018 Apr 22.

Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan. Electronic address:

Over the past century, Escherichia coli has become one of the best studied organisms on earth. Features such as genetic tractability, favorable growth conditions, well characterized biochemistry and physiology, and availability of versatile genetic manipulation tools make E. coli an ideal platform host for development of industrially viable productions. In this review, we discuss the physiological attributes of E. coli that are most relevant for metabolic engineering, as well as emerging techniques that enable efficient phenotype construction. Further, we summarize the large number of native and non-native products that have been synthesized by E. coli, and address some of the future challenges in broadening substrate range and fighting phage infection.
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http://dx.doi.org/10.1016/j.ymben.2018.04.008DOI Listing
November 2018

Construction and evolution of an strain relying on nonoxidative glycolysis for sugar catabolism.

Proc Natl Acad Sci U S A 2018 04 19;115(14):3538-3546. Epub 2018 Mar 19.

Institute of Biological Chemistry, Academia Sinica, 115 Taipei, Taiwan

The Embden-Meyerhoff-Parnas (EMP) pathway, commonly known as glycolysis, represents the fundamental biochemical infrastructure for sugar catabolism in almost all organisms, as it provides key components for biosynthesis, energy metabolism, and global regulation. EMP-based metabolism synthesizes three-carbon (C3) metabolites before two-carbon (C2) metabolites and must emit one CO in the synthesis of the C2 building block, acetyl-CoA, a precursor for many industrially important products. Using rational design, genome editing, and evolution, here we replaced the native glycolytic pathways in with the previously designed nonoxidative glycolysis (NOG), which bypasses initial C3 formation and directly generates stoichiometric amounts of C2 metabolites. The resulting strain, which contains 11 gene overexpressions, 10 gene deletions by design, and more than 50 genomic mutations (including 3 global regulators) through evolution, grows aerobically in glucose minimal medium but can ferment anaerobically to products with nearly complete carbon conservation. We confirmed that the strain metabolizes glucose through NOG by C tracer experiments. This redesigned strain represents a different approach for carbon catabolism and may serve as a useful platform for bioproduction.
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http://dx.doi.org/10.1073/pnas.1802191115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5889684PMC
April 2018

Rearrangement of Coenzyme A-Acylated Carbon Chain Enables Synthesis of Isobutanol via a Novel Pathway in Ralstonia eutropha.

ACS Synth Biol 2018 03 15;7(3):794-800. Epub 2018 Feb 15.

Department of Chemical Engineering and Materials Science , University of California, Irvine , Irvine , California 90697 , United States.

Coenzyme A (CoA)-dependent pathways have been explored extensively for the biosynthesis of fuels and chemicals. While CoA-dependent mechanisms are widely used to elongate carbon chains in a linear fashion, branch-making chemistry has not been incorporated. In this study, we demonstrated the production of isobutanol, a branched-chain alcohol that can be used as a gasoline substitute, using a novel CoA-dependent pathway in recombinant Ralstonia eutropha H16. The designed pathway is constituted of three modules: chain elongation, rearrangement, and modification. We first integrated and optimized the chain elongation and modification modules, and we achieved the production of ∼200 mg/L n-butanol from fructose or ∼30 mg/L from formate by engineered R. eutropha. Subsequently, we incorporated the rearrangement module, which features a previously uncharacterized, native isobutyryl-CoA mutase in R. eutropha. The engineered strain produced ∼30 mg/L isobutanol from fructose. The carbon skeleton rearrangement chemistry demonstrated here may be used to expand the range of the chemicals accessible with CoA-dependent pathways.
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http://dx.doi.org/10.1021/acssynbio.7b00409DOI Listing
March 2018

Accelerating fishes increase propulsive efficiency by modulating vortex ring geometry.

Proc Natl Acad Sci U S A 2017 12 11;114(52):13828-13833. Epub 2017 Dec 11.

The Whitney Laboratory for Marine Bioscience, Department of Biology, University of Florida, St. Augustine, FL 32080;

Swimming animals need to generate propulsive force to overcome drag, regardless of whether they swim steadily or accelerate forward. While locomotion strategies for steady swimming are well characterized, far less is known about acceleration. Animals exhibit many different ways to swim steadily, but we show here that this behavioral diversity collapses into a single swimming pattern during acceleration regardless of the body size, morphology, and ecology of the animal. We draw on the fields of biomechanics, fluid dynamics, and robotics to demonstrate that there is a fundamental difference between steady swimming and forward acceleration. We provide empirical evidence that the tail of accelerating fishes can increase propulsive efficiency by enhancing thrust through the alteration of vortex ring geometry. Our study provides insight into how propulsion can be altered without increasing vortex ring size and represents a fundamental departure from our current understanding of the hydrodynamic mechanisms of acceleration. Our findings reveal a unifying hydrodynamic principle that is likely conserved in all aquatic, undulatory vertebrates.
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http://dx.doi.org/10.1073/pnas.1705968115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5748167PMC
December 2017

Fish Swimming in a Kármán Vortex Street: Kinematics, Sensory Biology and Energetics.

Mar Technol Soc J 2017 Sep-Oct;51(5):48-55

Department of Computer Science, Aberystwyth University.

Fishes often live in environments characterized by complex flows. To study the mechanisms of how fishes interact with unsteady flows, the periodic shedding of vortices behind cylinders has been employed to great effect. In particular, fishes that hold station in a vortex street (i.e., Kármán gaiting) show swimming kinematics that are distinct from their patterns of motion during freestream swimming in uniform flows, although both behaviors can be modeled as an undulatory body wave. Kármán gait kinematics are largely preserved across flow velocities. Larger fish have a shorter body wavelength and slower body wave speed than smaller fish, in contrast to freestream swimming where body wavelength and wave speed increases with size. The opportunity for Kármán gaiting only occurs under specific conditions of flow velocity and depends on the length of the fish; this is reflected in the highest probability of Kármán gaiting at intermediate flow velocities. Fish typically Kármán gait in a region of the cylinder wake where the velocity deficit is about 40% of the nominal flow. The lateral line plays a role in tuning the kinematics of the Kármán gait, since blocking it leads to aberrant kinematics. Vision allows fish to maintain a consistent position relative to the cylinder. In the dark, fish do not show the same preference to hold station behind a cylinder though Kármán gait kinematics are the same. When oxygen consumption level is measured, it reveals that Kármán gaiting represents about half of the cost of swimming in the freestream.
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http://dx.doi.org/10.4031/MTSJ.51.5.8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6324577PMC
January 2019

Orthogonal partial least squares/projections to latent structures regression-based metabolomics approach for identification of gene targets for improvement of 1-butanol production in Escherichia coli.

J Biosci Bioeng 2017 Nov 29;124(5):498-505. Epub 2017 Jun 29.

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address:

Metabolomics is the comprehensive analysis of metabolites in biological systems that uses multivariate analyses such as principal component analysis (PCA) or partial least squares/projections to latent structures regression (PLSR) to understand the metabolome state and extract important information from biological systems. In this study, orthogonal PLSR (OPLSR) model-based metabolomics approach was applied to 1-butanol producing Escherichia coli to facilitate in strain improvement strategies. Here, metabolite data obtained by liquid chromatography/mass spectrometry (LC/MS) was used to construct an OPLSR model to correlate metabolite changes with 1-butanol production and rationally identify gene targets for strain improvement. Using this approach, acetyl-CoA was determined as the rate-limiting step of the pathway while free CoA was found to be insufficient for 1-butanol production. By resolving the problems addressed by the OPLSR model, higher 1-butanol productivity was achieved. In this study, the usefulness of OPLSR-based metabolomics approach for understanding the whole metabolome state and determining the most relevant metabolites was demonstrated. Moreover, it was able to provide valuable insights for selection of rational gene targets for strain improvement.
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http://dx.doi.org/10.1016/j.jbiosc.2017.05.015DOI Listing
November 2017

A non-toxic dose of cobalt chloride blocks hair cells of the zebrafish lateral line.

Hear Res 2017 07 12;350:17-21. Epub 2017 Apr 12.

Department of Biology, Whitney Laboratory for Marine Bioscience, University of Florida, St. Augustine, FL, USA. Electronic address:

Experiments on the flow-sensitive lateral line system of fishes have provided important insights into the function and sensory transduction of vertebrate hair cells. A common experimental approach has been to pharmacologically block lateral line hair cells and measure how behavior changes. Cobalt chloride (CoCl) blocks the lateral line by inhibiting calcium movement through the membrane channels of hair cells, but high concentrations can be toxic, making it unclear whether changes in behavior are due to a blocked lateral line or poor health. Here, we identify a non-toxic treatment of cobalt that completely blocks lateral line hair cells. We exposed 5-day post fertilization zebrafish larvae to CoCl concentrations ranging from 1 to 20 mM for 15 min and measured 1) the spiking rate of the afferent neurons contacting hair cells and 2) the larvae's health and long-term survival. Our results show that a 15-min exposure to 5 mM CoCl abolishes both spontaneous and evoked afferent firing. This treatment does not change swimming behavior, and results in >85% survival after 5 days. Weaker treatments of CoCl did not eliminate afferent activity, while stronger treatments caused close to 50% mortality. Our work provides a guideline for future zebrafish investigations where physiological confirmation of a blocked lateral line system is required.
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http://dx.doi.org/10.1016/j.heares.2017.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5495470PMC
July 2017

Metabolomics-driven approach to solving a CoA imbalance for improved 1-butanol production in Escherichia coli.

Metab Eng 2017 05 8;41:135-143. Epub 2017 Apr 8.

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address:

High titer 1-butanol production in Escherichia coli has previously been achieved by overexpression of a modified clostridial 1-butanol production pathway and subsequent deletion of native fermentation pathways. This strategy couples growth with production as 1-butanol pathway offers the only available terminal electron acceptors required for growth in anaerobic conditions. With further inclusion of other well-established metabolic engineering principles, a titer of 15g/L has been obtained. In achieving this titer, many currently existing strategies have been exhausted, and 1-butanol toxicity level has been surpassed. Therefore, continued engineering of the host strain for increased production requires implementation of alternative strategies that seek to identify non-obvious targets for improvement. In this study, a metabolomics-driven approach was used to reveal a CoA imbalance resulting from a pta deletion that caused undesirable accumulation of pyruvate, butanoate, and other CoA-derived compounds. Using metabolomics, the reduction of butanoyl-CoA to butanal catalyzed by alcohol dehydrogenase AdhE2 was determined as a rate-limiting step. Fine-tuning of this activity and subsequent release of free CoA restored the CoA balance that resulted in a titer of 18.3g/L upon improvement of total free CoA levels using cysteine supplementation. By enhancing AdhE2 activity, carbon flux was directed towards 1-butanol production and undesirable accumulation of pyruvate and butanoate was diminished. This study represents the initial report describing the improvement of 1-butanol production in E. coli by resolving CoA imbalance, which was based on metabolome analysis and rational metabolic engineering strategies.
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http://dx.doi.org/10.1016/j.ymben.2017.04.003DOI Listing
May 2017

Kinetically accessible yield (KAY) for redirection of metabolism to produce exo-metabolites.

Metab Eng 2017 05 5;41:144-151. Epub 2017 Apr 5.

Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, USA; UCLA-DOE Institute for Genomics and Proteomics, University of California, Los Angeles, USA; Academia Sinica, Taiwan. Electronic address:

The product formation yield (product formed per unit substrate consumed) is often the most important performance indicator in metabolic engineering. Until now, the actual yield cannot be predicted, but it can be bounded by its maximum theoretical value. The maximum theoretical yield is calculated by considering the stoichiometry of the pathways and cofactor regeneration involved. Here we found that in many cases, dynamic stability becomes an issue when excessive pathway flux is drawn to a product. This constraint reduces the yield and renders the maximal theoretical yield too loose to be predictive. We propose a more realistic quantity, defined as the kinetically accessible yield (KAY) to predict the maximum accessible yield for a given flux alteration. KAY is either determined by the point of instability, beyond which steady states become unstable and disappear, or a local maximum before becoming unstable. Thus, KAY is the maximum flux that can be redirected for a given metabolic engineering strategy without losing stability. Strictly speaking, calculation of KAY requires complete kinetic information. With limited or no kinetic information, an Ensemble Modeling strategy can be used to determine a range of likely values for KAY, including an average prediction. We first apply the KAY concept with a toy model to demonstrate the principle of kinetic limitations on yield. We then used a full-scale E. coli model (193 reactions, 153 metabolites) and this approach was successful in E. coli for predicting production of isobutanol: the calculated KAY values are consistent with experimental data for three genotypes previously published.
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http://dx.doi.org/10.1016/j.ymben.2017.03.011DOI Listing
May 2017

Metabolic systems modeling for cell factories improvement.

Curr Opin Biotechnol 2017 08 4;46:114-119. Epub 2017 Apr 4.

Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, Los Angeles, CA 90095, United States; Academia Sinica, Taipei 11529, Taiwan. Electronic address:

Techniques for modeling microbial bioproduction systems have evolved over many decades. Here, we survey recent literature and focus on modeling approaches for improving bioproduction. These techniques from systems biology are based on different methodologies, starting from stoichiometry only to various stoichiometry with kinetics approaches that address different issues in metabolic systems. Techniques to overcome unknown kinetic parameters using random sampling have emerged to address meaningful questions. Among those questions, pathway robustness seems to be an important issue for metabolic engineering. We also discuss the increasing significance of databases in biology and their potential impact for biotechnology.
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http://dx.doi.org/10.1016/j.copbio.2017.02.005DOI Listing
August 2017

Rational engineering of diol dehydratase enables 1,4-butanediol biosynthesis from xylose.

Metab Eng 2017 03 13;40:148-156. Epub 2017 Feb 13.

College of Engineering, The University of Georgia, Athens, GA 30602, USA. Electronic address:

Establishing novel synthetic routes for microbial production of chemicals often requires overcoming pathway bottlenecks by tailoring enzymes to enhance bio-catalysis or even achieve non-native catalysis. Diol dehydratases have been extensively studied for their interactions with C2 and C3 diols. However, attempts on utilizing these insights to enable catalysis on non-native substrates with more than two hydroxyl groups have been plagued with low efficiencies. Here, we rationally engineered the Klebsiella oxytoca diol dehydratase to enable and enhance catalytic activity toward a non-native C4 triol, 1,2,4-butanetriol. We analyzed dehydratase's interaction with 1,2-propanediol and glycerol, which led us to develop rationally conceived hypotheses. An in silico approach was then developed to identify and screen candidate mutants with desired activity. This led to an engineered diol dehydratase with nearly 5 fold higher catalytic activity toward 1,2,4-butanetriol than the wild type as determined by in vitro assays. Based on this result, we then expanded the 1,2,4-butanetriol pathway to establish a novel 1,4-butanediol production platform. We engineered Escherichia coli's xylose catabolism to enhance the biosynthesis of 1,2,4-butanetriol from 224mg/L to 1506mg/L. By introducing the complete pathway in the engineered strain we achieve de novo biosynthesis of 1,4-butanediol at 209mg/L from xylose. This work expands the repertoire of substrates catalyzed by diol dehydratases and serves as an elucidation to establish novel biosynthetic pathways involving dehydratase based biocatalysis.
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http://dx.doi.org/10.1016/j.ymben.2017.02.003DOI Listing
March 2017

Engineering a Thermostable Keto Acid Decarboxylase Using Directed Evolution and Computationally Directed Protein Design.

ACS Synth Biol 2017 04 11;6(4):610-618. Epub 2017 Jan 11.

UCLA-DOE Institute of Genomics and Proteomics , 420 Westwood Plaza, Los Angeles, California 90095, United States.

Keto acid decarboxylase (Kdc) is a key enzyme in producing keto acid derived higher alcohols, like isobutanol. The most active Kdc's are found in mesophiles; the only reported Kdc activity in thermophiles is 2 orders of magnitude less active. Therefore, the thermostability of mesophilic Kdc limits isobutanol production temperature. Here, we report development of a thermostable 2-ketoisovalerate decarboxylase (Kivd) with 10.5-fold increased residual activity after 1h preincubation at 60 °C. Starting with mesophilic Lactococcus lactis Kivd, a library was generated using random mutagenesis and approximately 8,000 independent variants were screened. The top single-mutation variants were recombined. To further improve thermostability, 16 designs built using Rosetta Comparative Modeling were screened and the most active was recombined to form our best variant, LLM4. Compared to wild-type Kivd, a 13 °C increase in melting temperature and over 4-fold increase in half-life at 60 °C were observed. LLM4 will be useful for keto acid derived alcohol production in lignocellulosic thermophiles.
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http://dx.doi.org/10.1021/acssynbio.6b00240DOI Listing
April 2017