Mixed Selectivity Coding of Content-Temporal Detail by Dorsomedial Posterior Parietal Neurons.

The dorsomedial posterior parietal cortex (dmPPC) is part of a higher-cognition network implicated in elaborate processes underpinning memory formation, recollection, episode reconstruction, and temporal information processing. Neural coding for complex episodic processing is however under-documented. Here, we recorded extracellular neural activities from three male rhesus macaques (Macaca mulatta) and revealed a set of neural codes of "neuroethogram" in the primate parietal cortex. Analyzing neural responses in macaque dmPPC to naturalistic videos, we discovered several groups of neurons that are sensitive to different categories of ethogram items, low-level sensory features, and saccadic eye movement. We also discovered that the processing of category and feature information by these neurons is sustained by the accumulation of temporal information over a long timescale of up to 30 s, corroborating its reported long temporal receptive windows. We performed an additional behavioral experiment with additional two male rhesus macaques and found that saccade-related activities could not account for the mixed neuronal responses elicited by the video stimuli. We further observed monkeys' scan paths and gaze consistency are modulated by video content. Taken altogether, these neural findings explain how dmPPC weaves fabrics of ongoing experiences together in real time. The high dimensionality of neural representations should motivate us to shift the focus of attention from pure selectivity neurons to mixed selectivity neurons, especially in increasingly complex naturalistic task designs.

Metabolic engineering strategies to enable microbial electrosynthesis utilization of CO2: recent progress and challenges.

Microbial electrosynthesis (MES) is a promising technology that mainly utilizes microbial cells to convert CO2 into value-added chemicals using electrons provided by the cathode. However, the low electron transfer rate is a solid bottleneck hindering the further application of MES. Thus, as an effective strategy, genetic tools play a key role in MES for enhancing the electron transfer rate and diversity of production. We describe a set of genetic strategies based on fundamental characteristics and current successes and discuss their functional mechanisms in driving microbial electrocatalytic reactions to fully comprehend the roles and uses of genetic tools in MES. This paper also analyzes the process of nanomaterial application in extracellular electron transfer (EET). It provides a technique that combines nanomaterials and genetic tools to increase MES efficiency, because nanoparticles have a role in the production of functional genes in EET although genetic tools can subvert MES, it still has issues with difficult transformation and low expression levels. Genetic tools remain one of the most promising future strategies for advancing the MES process despite these challenges.

A repackaged CRISPR platform increases homology-directed repair for yeast engineering.

Inefficient homology-directed repair (HDR) constrains CRISPR-Cas9 genome editing in organisms that preferentially employ nonhomologous end joining (NHEJ) to fix DNA double-strand breaks (DSBs). Current strategies used to alleviate NHEJ proficiency involve NHEJ disruption. To confer precision editing without NHEJ disruption, we identified the shortcomings of the conventional CRISPR platforms and developed a CRISPR platform-lowered indel nuclease system enabling accurate repair (LINEAR)-which enhanced HDR rates (to 67-100%) compared to those in previous reports using conventional platforms in four NHEJ-proficient yeasts. With NHEJ preserved, we demonstrate its ability to survey genomic landscapes, identifying loci whose spatiotemporal genomic architectures yield favorable expression dynamics for heterologous pathways. We present a case study that deploys LINEAR precision editing and NHEJ-mediated random integration to rapidly engineer and optimize a microbial factory to produce (S)-norcoclaurine. Taken together, this work demonstrates how to leverage an antagonizing pair of DNA DSB repair pathways to expand the current collection of microbial factories.

Disease-induced changes in bacterial and fungal communities from plant below- and aboveground compartments.

The plant microbes are an integral part of the host and play fundamental roles in plant growth and health. There is evidence indicating that plants have the ability to attract beneficial microorganisms through their roots in order to defend against pathogens. However, the mechanisms of plant microbial community assembly from below- to aboveground compartments under pathogen infection remain unclear. In this study, we investigated the bacterial and fungal communities in bulk soil, rhizosphere soil, root, stem, and leaf of both healthy and infected (Potato virus Y disease, PVY) plants. The results indicated that bacterial and fungal communities showed different recruitment strategies in plant organs. The number and abundance of shared bacterial ASVs between bulk and rhizosphere soils decreased with ascending migration from below- to aboveground compartments, while the number and abundance of fungal ASVs showed no obvious changes. Field type, plant compartments, and PVY infection all affected the diversity and structures of microbial community, with stronger effects observed in the bacterial community than the fungal community. Furthermore, PVY infection, rhizosphere soil pH, and water content (WC) contributed more to the assembly of the bacterial community than the fungal community. The analysis of microbial networks revealed that the bacterial communities were more sensitive to PVY infection than the fungal communities, as evidenced by the lower network stability of the bacterial community, which was characterized by a higher proportion of positive edges. PVY infection further increased the bacterial network stability and decreased the fungal network stability. These findings advance our understanding of how microbes respond to pathogen infections and provide a rationale and theoretical basis for biocontrol technology in promoting sustainable agriculture. KEY POINTS: • Different recruitment strategies between plant bacterial and fungal communities. • Bacterial community was more sensitive to PVY infection than fungal community. • pH and WC drove the microbial community assembly under PVY infection.

Mass Spectrometry-Based High-Throughput Quantification of Bioproducts in Liquid Culture.

To meet the ever-increasing need for high-throughput screening in metabolic engineering, information-rich, fast screening methods are needed. Mass spectrometry (MS) provides an efficient and general approach for metabolite screening and offers the capability of characterizing a broad range of analytes in a label-free manner, but often requires a range of sample clean-up and extraction steps. Liquid extraction surface analysis (LESA) coupled MS is an image-guided MS surface analysis approach that directly samples and introduces metabolites from a surface to MS. Here, we combined the advantages of LESA-MS and an acoustic liquid handler with stable isotope-labeled internal standards. This approach provides absolute quantitation of target chemicals from liquid culture-dried droplets and enables high-throughput quantitative screening for microbial metabolites. In this study, LESA-MS was successfully applied to quantify several different metabolites (itaconic acid, triacetic acid lactone, and palmitic acid) from different yeast strains in different mediums, demonstrating its versatility, accuracy, and efficiency across a range of microbial engineering applications.

Metabolic regulation of Shewanella oneidensis for microbial electrosynthesis: From extracellular to intracellular.

Shewanella oneidensis MR-1 (S. oneidensis MR-1) has been shown to benefit from microbial electrosynthesis (MES) due to its exceptional electron transfer efficiency. In this study, genes involved in both extracellular electron uptake (EEU) and intracellular CO2 conversion processes were examined and regulated to enhance MES performance. The key genes identified for MES in the EEU process were mtrB, mtrC, mtrD, mtrE, omcA and cctA. Overexpression of these genes resulted in 1.5-2.1 times higher formate productivity than that of the wild-type strains (0.63 mmol/(L·µg protein)), as 0.94-1.61 mmol/(L·µg protein). In the intracellular CO2 conversion process, overexpression of the nadE, nadD, nadR, nadV, pncC and petC genes increased formate productivity 1.3-fold-3.4-fold. Moreover, overexpression of the formate dehydrogenase genes fdhA1, fdhB1 and fdhX1 in modified strains led to a 2.3-fold-3.1-fold increase in formate productivity compared to wild-type strains. The co-overexpression of cctA, fdhA1 and nadV in the mutant strain resulted in 5.59 times (3.50 mmol/(L·µg protein)) higher formate productivity than that of the wild-type strains. These findings revealed that electrons of MES derived from the electrode were utilized in the energy module for synthesizing ATP and NADH, followed by the synthesis of formate in formate dehydrogenase by the combinatorial effects of ATP, NADH, electrons and CO2. The results provide new insights into the mechanism of MES in S. oneidensis MR-1 and pave the way for genetic improvements that could facilitate the further application of MES.

Directed Evolution: Methodologies and Applications.

Directed evolution aims to expedite the natural evolution process of biological molecules and systems in a test tube through iterative rounds of gene diversifications and library screening/selection. It has become one of the most powerful and widespread tools for engineering improved or novel functions in proteins, metabolic pathways, and even whole genomes. This review describes the commonly used gene diversification strategies, screening/selection methods, and recently developed continuous evolution strategies for directed evolution. Moreover, we highlight some representative applications of directed evolution in engineering nucleic acids, proteins, pathways, genetic circuits, viruses, and whole cells. Finally, we discuss the challenges and future perspectives in directed evolution.

Copper-inducible expression system for metabolic engineering of Escherichia coli.

AIMS: The inducible expression system plays an important role in engineering Escherichia coli for chemical production. However, it still heavily relies on expensive chemical inducers, like IPTG. There is a pressing need to develop alternative expression systems with more affordable inducers. MATERIALS AND RESULTS: We herein report a copper-inducible expression system in E. coli based on the two-component Cus system and T7 RNA polymerase (RNAP). By integrating the gene encoding T7 RNAP at the CusC locus, we managed to program eGFP expression under the T7 promoter in response to different concentrations of Cu2+ (0-20 µM). Subsequently, we demonstrated that the copper-inducible expression system was suitable for the metabolic engineering of E. coli toward protocatechuic acid overproduction, and the resulting strain with combined manipulation of the central metabolism via CRISPRi produced 4.12 g L-1 PCA under the optimal copper concentration and induction time. CONCLUSIONS: We have established a copper-inducible T7 RNAP expression system in E. coli. The copper-inducible expression system could rationally control metabolic pathways in a temporal and dose-dependent manner. The gradient expression system based on copper inducer could be widely used in E. coli cell factories, and the design principle reported here would also be applicable in other prokaryotes.

CUT&RUN identifies centromeric DNA regions of Rhodotorula toruloides IFO0880.

Rhodotorula toruloides has been increasingly explored as a host for bioproduction of lipids, fatty acid derivatives and terpenoids. Various genetic tools have been developed, but neither a centromere nor an autonomously replicating sequence (ARS), both necessary elements for stable episomal plasmid maintenance, has yet been reported. In this study, cleavage under targets and release using nuclease (CUT&RUN), a method used for genome-wide mapping of DNA-protein interactions, was used to identify R. toruloides IFO0880 genomic regions associated with the centromeric histone H3 protein Cse4, a marker of centromeric DNA. Fifteen putative centromeres ranging from 8 to 19 kb in length were identified and analyzed, and four were tested for, but did not show, ARS activity. These centromeric sequences contained below average GC content, corresponded to transcriptional cold spots, were primarily nonrepetitive and shared some vestigial transposon-related sequences but otherwise did not show significant sequence conservation. Future efforts to identify an ARS in this yeast can utilize these centromeric DNA sequences to improve the stability of episomal plasmids derived from putative ARS elements.

Metabolic engineering of oleaginous yeast Rhodotorula toruloides for overproduction of triacetic acid lactone.

The plant-sourced polyketide triacetic acid lactone (TAL) has been recognized as a promising platform chemical for the biorefinery industry. However, its practical application was rather limited due to low natural abundance and inefficient cell factories for biosynthesis. Here, we report the metabolic engineering of oleaginous yeast Rhodotorula toruloides for TAL overproduction. We first introduced a 2-pyrone synthase gene from Gerbera hybrida (GhPS) into R. toruloides and investigated the effects of different carbon sources on TAL production. We then systematically employed a variety of metabolic engineering strategies to increase the flux of acetyl-CoA by enhancing its biosynthetic pathways and disrupting its competing pathways. We found that overexpression of ATP-citrate lyase (ACL1) improved TAL production by 45% compared to the GhPS overexpressing strain, and additional overexpression of acetyl-CoA carboxylase (ACC1) further increased TAL production by 29%. Finally, we characterized the resulting strain I12-ACL1-ACC1 using fed-batch bioreactor fermentation in glucose or oilcane juice medium with acetate supplementation and achieved a titer of 28 or 23 g/L TAL, respectively. This study demonstrates that R. toruloides is a promising host for the production of TAL and other acetyl-CoA-derived polyketides from low-cost carbon sources.

High-titre production of aromatic amines in metabolically engineered Escherichia coli.

AIMS: Aromatic amines with diverse physical characteristics are often employed as antioxidants and precursors to pharmaceutical products. As the traditional chemical methods pose serious environmental pollution, there is an arising interest in biomanufacturing aromatic amines from renewable feedstocks. MATERIALS AND RESULTS: We report the establishment of a bacterial platform for synthesizing three types of aromatic amines, namely, tyramine, dopamine and phenylethylamine. First, we expressed aromatic amino acid decarboxylase from Enterococcus faecium (pheDC) in an Escherichia coli strain with increasing shikimate (SHK) pathway flux towards L-tyrosine. We found that glycerol served as a better carbon source than glucose, resulting in 940 ± 46 mg/L tyramine from 4% glycerol. Next, the genes of lactate dehydrogenase (ldhA), pyruvate formate lyase (pflB), phosphate acetyltransferase (pta) and alcohol dehydrogenase (adhE) were deleted to mitigate the fermentation by-product formation. The tyramine level was further increased to 1.965 ± 0.205 g/L in the shake flask, which was improved by 2.1 times compared with that of the parental strain. By using a similar strategy, we also managed to produce 703 ± 21 mg/L dopamine and 555 ± 50 mg/L phenethylamine. CONCLUSIONS: We demonstrated that the knockout of ldhA-pflB-pta-adhE is an effective strategy for improving aromatic amine productions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study achieved the highest aromatic amine titres in E. coli under shake flask reported to date.

Development of shuttle vectors for rapid prototyping of engineered Synechococcus sp. PCC7002.

Cyanobacteria are of particular interest for chemical production as they can assimilate CO2 and use solar energy to power chemical synthesis. However, unlike the model microorganism of Escherichia coli, the availability of genetic toolboxes for rapid proof-of-concept studies in cyanobacteria is generally lacking. In this study, we first characterized a set of promoters to efficiently drive gene expressions in the marine cyanobacterium Synechococcus sp. PCC7002. We identified that the endogenous cpcBA promoter represented one of the strongest promoters in PCC7002. Next, a set of shuttle vectors was constructed based on the endogenous pAQ1 plasmid to facilitate the rapid pathway assembly. Moreover, we used the shuttle vectors to modularly optimize the amorpha-4,11-diene synthesis in PCC7002. By modularly optimizing the metabolic pathway, we managed to redistribute the central metabolism toward the amorpha-4,11-diene production in PCC7002 with enhanced product titer. Taken together, the plasmid toolbox developed in this study will greatly accelerate the generation of genetically engineered PCC7002. KEY POINTS: • Promoter characterization revealed that the endogenous cpcBA promoter represented one of the strongest promoters in PCC7002 • A set of shuttle vectors with different antibiotic selection markers was constructed based on endogenous pAQ1 plasmid • By modularly optimizing the metabolic pathway, amorpha-4,11-diene production in PCC7002 was improved.

Revisiting the unique structure of autonomously replicating sequences in Yarrowia lipolytica and its role in pathway engineering.

Production of industrially relevant compounds in microbial cell factories can employ either genomes or plasmids as an expression platform. Selection of plasmids as pathway carriers is advantageous for rapid demonstration but poses a challenge of stability. Yarrowia lipolytica has attracted great attention in the past decade for the biosynthesis of chemicals related to fatty acids at titers attractive to industry, and many genetic tools have been developed to explore its oleaginous potential. Our recent studies on the autonomously replicating sequences (ARSs) of nonconventional yeasts revealed that the ARSs from Y. lipolytica showcase a unique structure that includes a previously unannotated sequence (spacer) linking the origin of replication (ORI) and the centromeric (CEN) element and plays a critical role in modulating plasmid behavior. Maintaining a native 645-bp spacer yielded a 2.2-fold increase in gene expression and 1.7-fold higher plasmid stability compared to a more universally employed minimized ARS. Testing the modularity of the ARS sub-elements indicated that plasmid stability exhibits a pronounced cargo dependency. Instability caused both plasmid loss and intramolecular rearrangements. Altogether, our work clarifies the appropriate application of various ARSs for the scientific community and sheds light on a previously unexplored DNA element as a potential target for engineering Y. lipolytica.

Building microbial factories for the production of aromatic amino acid pathway derivatives: From commodity chemicals to plant-sourced natural products.

The aromatic amino acid biosynthesis pathway, together with its downstream branches, represents one of the most commercially valuable biosynthetic pathways, producing a diverse range of complex molecules with many useful bioactive properties. Aromatic compounds are crucial components for major commercial segments, from polymers to foods, nutraceuticals, and pharmaceuticals, and the demand for such products has been projected to continue to increase at national and global levels. Compared to direct plant extraction and chemical synthesis, microbial production holds promise not only for much shorter cultivation periods and robustly higher yields, but also for enabling further derivatization to improve compound efficacy by tailoring new enzymatic steps. This review summarizes the biosynthetic pathways for a large repertoire of commercially valuable products that are derived from the aromatic amino acid biosynthesis pathway, and it highlights both generic strategies and specific solutions to overcome certain unique problems to enhance the productivities of microbial hosts.

A genetic toolbox for metabolic engineering of Issatchenkia orientalis.

The nonconventional yeast Issatchenkia orientalis can grow under highly acidic conditions and has been explored for production of various organic acids. However, its broader application is hampered by the lack of efficient genetic tools to enable sophisticated metabolic manipulations. We recently constructed an episomal plasmid based on the autonomously replicating sequence (ARS) from Saccharomyces cerevisiae (ScARS) in I. orientalis and developed a CRISPR/Cas9 system for multiplexed gene deletions. Here we report three additional genetic tools including: (1) identification of a 0.8 kb centromere-like (CEN-L) sequence from the I. orientalis genome by using bioinformatics and functional screening; (2) discovery and characterization of a set of constitutive promoters and terminators under different culture conditions by using RNA-Seq analysis and a fluorescent reporter; and (3) development of a rapid and efficient in vivo DNA assembly method in I. orientalis, which exhibited ~100% fidelity when assembling a 7 kb-plasmid from seven DNA fragments ranging from 0.7 kb to 1.7 kb. As proof of concept, we used these genetic tools to rapidly construct a functional xylose utilization pathway in I. orientalis.

Development of a CRISPR/Cas9 system for high efficiency multiplexed gene deletion in Rhodosporidium toruloides.

The oleaginous yeast Rhodosporidium toruloides is considered a promising candidate for production of chemicals and biofuels thanks to its ability to grow on lignocellulosic biomass, and its high production of lipids and carotenoids. However, efforts to engineer this organism are hindered by a lack of suitable genetic tools. Here we report the development of a CRISPR/Cas9 system for genome editing in R. toruloides based on a fusion 5S rRNA-tRNA promoter for guide RNA (gRNA) expression, capable of greater than 95% gene knockout for various genetic targets. Additionally, multiplexed double-gene knockout mutants were obtained using this method with an efficiency of 78%. This tool can be used to accelerate future metabolic engineering work in this yeast.

Construction of a series of episomal plasmids and their application in the development of an efficient CRISPR/Cas9 system in Pichia pastoris.

The methylotrophic yeast Pichia pastoris is widely used in recombinant expression of eukaryotic proteins owing to the ability of post-translational modification, tightly regulated promoters, and high cell density fermentation. However, episomal plasmids for heterologous gene expression and the CRISPR/Cas9 system for genome editing have not been well developed in P. pastoris. In the present study, a panel of episomal plasmids containing various autonomously replicating sequences (ARSs) were constructed and their performance in transformation efficiency, copy numbers, and propagation stability were systematically compared. Among the five ARSs with different origins, panARS isolated from Kluyveromyces lactis was determined to have the best performance and used to develop an efficient CRISPR/Cas9 based genome editing system. Compared with a previously reported system using the endogenous and most commonly used ARS (PARS1), the CRISPR/Cas9 genome editing efficiency was increased for more than tenfold. Owing to the higher plasmid stability with panARS, efficient CRISPR/Cas9-mediated genome editing with a type III promoter (i.e. SER promoter) to drive the expression of the single guide RNA (sgRNA) was achieved for the first time. The constructed episomal plasmids and developed CRISPR/Cas9 system will be important synthetic biology tools for both fundamental studies and industrial applications of P. pastoris.

Multilevel engineering of the upstream module of aromatic amino acid biosynthesis in Saccharomyces cerevisiae for high production of polymer and drug precursors.

A multilevel approach was implemented in Saccharomyces cerevisiae to optimize the precursor module of the aromatic amino acid biosynthesis pathway, which is a rich resource for synthesizing a great variety of chemicals ranging from polymer precursor, to nutraceuticals and pain-relief drugs. To facilitate the discovery of novel targets to enhance the pathway flux, we incorporated the computational tool YEASTRACT for predicting novel transcriptional repressors and OptForce strain-design for identifying non-intuitive pathway interventions. The multilevel approach consisted of (i) relieving the pathway from strong transcriptional repression, (ii) removing competing pathways to ensure high carbon capture, and (iii) rewiring precursor pathways to increase the carbon funneling to the desired target. The combination of these interventions led to the establishment of a S. cerevisiae strain with shikimic acid (SA) titer reaching as high as 2.5gL-1, 7-fold higher than the base strain. Further expansion of the platform led to the titer of 2.7gL-1 of muconic acid (MA) and its intermediate protocatechuic acid (PCA) together. Both the SA and MA production platforms demonstrated increases in titer and yield nearly 300% from the previously reported, highest-producing S. cerevisiae strains. Further examination elucidated the diverged impacts of disrupting the oxidative branch (ZWF1) of the pentose phosphate pathway on the titers of desired products belonging to different portions of the pathway. The investigation of other non-intuitive interventions like the deletion of the Pho13 enzyme also revealed the important role of the transaldolase in determining the fate of the carbon flux in the pathways of study. This integrative approach identified novel determinants at both transcriptional and metabolic levels that constrain the flux entering the aromatic amino acid pathway. In the future, this platform can be readily used for engineering the downstream modules toward the production of important plant-sourced aromatic secondary metabolites.

Regulation of bacteria population behaviors by AI-2 "consumer cells" and "supplier cells".

BACKGROUND: Autoinducer-2 (AI-2) is a universal signal molecule and enables an individual bacteria to communicate with each other and ultimately control behaviors of the population. Harnessing the character of AI-2, two kinds of AI-2 "controller cells" ("consumer cells" and "supplier cells") were designed to "reprogram" the behaviors of entire population. RESULTS: For the consumer cells, genes associated with the uptake and processing of AI-2, which includes LsrACDB, LsrFG, LsrK, were overexpressed in varying combinations. Four consumer cell strains were constructed: Escherichia coli MG1655 pLsrACDB (NK-C1), MG1655 pLsrACDBK (NK-C2), MG1655 pLsrACDBFG (NK-C3) and MG1655 pLsrACDBFGK (NK-C4). The key enzymes responsible for production of AI-2, LuxS and Mtn, were also overexpressed, yielding strains MG1655 pLuxS (NK-SU1), and MG1655 pLuxS-Mtn (NK-SU2). All the consumer cells could decrease the environmental AI-2 concentration. NK-C2 and NK-C4 were most effective in AI-2 uptake and inhibited biofilm formation. While suppliers can increase the environmental AI-2 concentration and NK-SU2 was most effective in supplying AI-2 and facilitated biofilm formation. Further, reporter strain, MG1655 pLGFP was constructed. The expression of green fluorescent protein (GFP) in reporter cells was initiated and guided by AI-2. Mixture of consumer cells and reporter cells suggest that consumer cells can decrease the AI-2 concentration. And the supplier cells were co-cultured with reporter cells, indicating that supplier cells can provide more AI-2 compared to the control. CONCLUSIONS: The consumer cells and supplier cells could be used to regulate environmental AI-2 concentration and the biofilm formation. They can also modulate the AI-2 concentration when they were co-cultured with reporter cells. It can be envisioned that this system will become useful tools in synthetic biology and researching new antimicrobials.