piR-368 promotes odontoblastic differentiation of dental papilla cells via the Smad1/5 signaling pathway by targeting Smurf1.

PURPOSE: The important role of non-coding RNAs in odontoblastic differentiation of dental tissue-derived stem cells has been widely demonstrated; however, whether piRNA (a subclass of non-coding RNA) involved in the course of odontoblastic differentiation is not yet available. This study aimed to investigate the expression profile of piRNA during odontogenic differentiation of mDPCs and the potential molecular mechanism in vitro. MATERIALS AND METHODS: The primary mouse dental papilla cells (mDPCs) were isolated from the first molars of 1-day postnatal Kunming mice. Then, they were cultured in odontogenic medium for 9 days. The expression profile of piRNA was detected by Small RNA sequencing. RT-qPCR was used to verify the elevation of piR-368. The mRNA and protein levels of mineralization markers were examined by qRT-PCR and Western blot analysis. Alkaline phosphatase (ALP) activity and alizarin red S staining were conducted to assess the odontoblastic differentiation ability. RESULTS: We validated piR-368 was significantly upregulated and interference with piR-368 markedly inhibited the odontogenic differentiation of mDPCs. In addition, the relationship between Smad1/5 signaling pathway and piR-368-induced odontoblastic differentiation has been discovered. Finally, we demonstrated Smurf1 as a target gene of piR-368 using dual-luciferase assays. CONCLUSION: This study was the first to illustrate the participation of piRNA in odontoblastic differentiation. We proved that piR-368 promoted odontoblastic differentiation of mouse dental papilla cells via the Smad1/5 signaling pathway by targeting Smurf1.

Characterization of maltose-binding protein-fused heparinases with enhanced thermostability by application of rigid and flexible linkers.

Heparinases, including heparinases I-III (HepI, HepII, and HepIII, respectively), are important tools for producing low-molecular-weight heparin, an improved anticoagulant. The poor thermostability of heparinases significantly hinders their industrial and laboratory applications. To improve the thermostability of heparinases, we applied a rigid linker (EAAAK)5 (R) and a flexible linker (GGGGS)5 (F) to fuse maltose-binding protein (MBP) and HepI, HepII, and HepIII from Pedobacter heparinus, replacing the original linker from the plasmid pMAL-c2X. Compared with their parental fusion protein, MBP-fused HepIs, HepIIs, and HepIIIs with linkers (EAAAK)5 or (GGGGS)5 all displayed enhanced thermostability (half-lives at 30°C: 242%-464%). MBP-fused HepIs and HepIIs exhibited higher specific activity (127%-324%), whereas MBP-fused HepIIIs displayed activity similar to that of their parental fusion protein. Kinetics analysis revealed that MBP-fused HepIIs showed a significantly decreased affinity toward heparin with increased Km values (397%-480%) after the linker replacement, whereas the substrate affinity did not change significantly for MBP-fused HepIs and HepIIIs. Furthermore, it preliminarily appeared that the depolymerization mechanism of these fusion proteins may not change after linker replacement. These findings suggest the superior enzymatic properties of MBP-fused heparinases with suitable linker designs and their potential for the bioproduction of low-molecular-weight heparin.

CRISPRi-microfluidics screening enables genome-scale target identification for high-titer protein production and secretion.

Genome-scale target identification promises to guide microbial cell factory engineering for higher-titer production of biomolecules such as recombinant proteins (r-protein), but challenges remain due to the need not only for comprehensive genotypic perturbation but also in conjunction with high-throughput phenotypic screening strategies. Here, we developed a CRISPRi-microfluidics screening platform to systematically identify crucial gene targets that can be engineered to enhance r-protein secretion in Corynebacterium glutamicum. We created a CRISPR interference (CRISPRi) library containing 46,549 single-guide RNAs, where we aimed to unbiasedly target all genes for repression. Meanwhile, we developed a highly efficient droplet-based microfluidics system integrating the FlAsH-tetracysteine assay that enables screening of millions of strains to identify potential knockdowns conducive to nanobody VHH secretion. Among our highest-ranking candidates are a slew of previously unknown targets involved in transmembrane transport, amino-acid metabolism and redox regulation. Guided by these findings, we eventually constructed a hyperproducer for multiple proteins via combinatorial engineering of redox-response transcription factors. As the near-universal applicability of CRISPRi technology and the FlAsH-based screening platform, this procedure might be expanded to include a varied variety of microbial species and recombinant proteins.

Single-cell microliter-droplet screening system (MISS Cell): An integrated platform for automated high-throughput microbial monoclonal cultivation and picking.

Solid plates have been used for microbial monoclonal isolation, cultivation, and colony picking since 1881. However, the process is labor- and resource-intensive for high-throughput requirements. Currently, several instruments have been integrated for automated and high-throughput picking, but complicated and expensive. To address these issues, we report a novel integrated platform, the single-cell microliter-droplet screening system (MISS Cell), for automated, high-throughput microbial monoclonal colony cultivation and picking. We verified the monoclonality of droplet cultures in the MISS Cell and characterized culture performance. Compared with solid plates, the MISS Cell generated a larger number of monoclonal colonies with higher initial growth rates using fewer resources. Finally, we established a workflow for automated high-throughput screening of Corynebacterium glutamicum using the MISS Cell and identified high glutamate-producing strains. The MISS Cell can serve as a universal platform to efficiently produce monoclonal colonies in high-throughput applications, overcoming the limitations of solid plates to promote rapid development in biotechnology.

Guide-target mismatch effects on dCas9-sgRNA binding activity in living bacterial cells.

As an effective programmable DNA targeting tool, CRISPR-Cas9 system has been adopted in varieties of biotechnological applications. However, the off-target effects, derived from the tolerance towards guide-target mismatches, are regarded as the major problems in engineering CRISPR systems. To understand this, we constructed two sgRNA libraries carrying saturated single- and double-nucleotide mismatches in living bacteria cells, and profiled the comprehensive landscape of in vivo binding affinity of dCas9 toward DNA target guided by each individual sgRNA with particular mismatches. We observed a synergistic effect in seed, where combinatorial double mutations caused more severe activity loss compared with the two corresponding single mutations. Moreover, we found that a particular mismatch type, dDrG (D = A, T, G), only showed moderate impairment on binding. To quantitatively understand the causal relationship between mismatch and binding behaviour of dCas9, we further established a biophysical model, and found that the thermodynamic properties of base-pairing coupled with strand invasion process, to a large extent, can account for the observed mismatch-activity landscape. Finally, we repurposed this model, together with a convolutional neural network constructed based on the same mechanism, as a predictive tool to guide the rational design of sgRNA in bacterial CRISPR interference.

Dynamics of transcription-translation coordination tune bacterial indole signaling.

Indole signaling is an important cross-species communication pathway in the mammalian gut. In bacteria, upon induction by tryptophan, the molecular sensor (tnaC) controls indole biosynthesis by precisely coordinating dynamics of the corresponding macromolecular machineries during its transcription and translation. Our understanding of this regulatory program is still limited owing to its rapid dynamic nature. To address this shortcoming, we adopted a massively parallel profiling method to quantify the responses of 1,450 synthetic tnaC variants in the presence of three concentrations of tryptophan in living bacterial cells. The resultant dataset enabled us to comprehensively probe the key intermediate states of macromolecular machineries during the transcription and translation of tnaC. We also used modeling to provide a systems-level understanding of how these critical states collectively shape the output of this regulatory program quantitatively. A similar methodology will likely apply to other poorly understood dynamics-dependent cis-regulatory elements.

Construction and characterization of novel bifunctional fusion proteins composed of alcohol dehydrogenase and NADH oxidase with efficient oxidized cofactor regeneration.

To tune the efficiency of oxidized cofactor recycling between alcohol dehydrogenase (ADH) and NADH oxidase (NOX) for the production of aromatic chiral alcohols, we designed and constructed four novel bifunctional fusion proteins composed of thermostable ADH and NOX from Thermococcus kodakarensis KOD1. ADH was linked to the N- or C-terminus of NOX with a typical rigid linker (EAAAK)3 and a flexible linker (GGGGS)3 , respectively. Compared with the parental enzymes, the NOX moieties in the four fusion proteins exhibited higher specific activities (141%-282%), while the ADH moieties exhibited varying levels of specific activity (69%-167%). All fusion proteins showed decreased affinities toward the cofactors, with increased Km values toward NADH (159%-406%) and NAD+ (202%-372%). In the enantioselective oxidation of (RS)-1-phenylethanol coupled with cofactor regeneration, the four fusion proteins displayed different positive and negative effects on the recycling efficiency of the oxidized cofactor. The two fusion proteins composed of NOX at the N-terminus exhibited higher total turnover numbers than the corresponding mixtures of individual enzymes with equal activities, particularly at low cofactor concentrations. These findings suggest high cofactor recycling efficiencies of the fusion proteins with appropriate design and their potential application in the biosynthesis of chiral alcohols.

Imaging Sensor for the Detection of the Flow Battery Via Weak Value Amplification.

Flow battery electrodes are vital for performing redox reactions, and an in-depth understanding of reaction kinetics and spatial distribution differences in electrodes is very important for improving the efficiency of electrochemical reactions. In this study, a reflection-type phase-sensitive weak measurement imaging system was developed for the detection of flow batteries. The phase difference between two polarization components in total internal reflection caused by electrode redox processes was measured by weak value amplification. The resulting refractive index resolution of the imaging system was estimated to be 2.8-4.2 × 10-6 RIU. The real-time monitoring ability of the system was demonstrated by linear sweep voltammetry tests of vanadium redox batteries. Compared to traditional optical methods, the proposed weak measurement imaging sensor did not require coating, as it can be used in acid electrolytes of vanadium flow batteries. Meanwhile, the weak value amplification effect led to a higher resolution than the total internal reflection system shown in our previous work, thereby resulting in more accurate detection of electrochemical reactions. In sum, the proposed sensor looks very promising for the detection of electrochemical reactions in flow batteries, water splitting, electrochemical corrosion, and electrocatalysis.

Rewiring the native methanol assimilation metabolism by incorporating the heterologous ribulose monophosphate cycle into Methylorubrum extorquens.

Methanol is assimilated through the serine cycle to generate acetyl-CoA without carbon loss. However, a highly active serine cycle requires high consumption of reducing equivalents and ATP, thereby leading to the impaired efficiency of methanol conversion to reduced chemicals. In the present study, a genome-scale flux balance analysis (FBA) predicted that the introduction of the heterologous ribulose monophosphate (RuMP) cycle, a more energy-efficient pathway for methanol assimilation, could theoretically increase growth rate by 31.3% for the model alphaproteobacterial methylotroph Methylorubrum extorquens AM1. Based on this analysis, we constructed a novel synergistic assimilation pathway in vivo by incorporating the RuMP cycle into M. extroquens metabolism with the intrinsic serine cycle. We demonstrated that the operation of the synergistic pathway could increase cell growth rate by 16.5% and methanol consumption rate by 13.1%. This strategy rewired the central methylotrophic metabolism through adjusting core gene transcription, leading to a pool size increase of C2 to C5 central intermediates by 1.2- to 3.6-fold and an NADPH cofactor improvement by 1.3-fold. The titer of 3-hydroxypropionic acid (3-HP), a model product in the newly engineered chassis of M. extorquens AM1, was increased to 91.2 mg/L in shake-flask culture, representing a 3.1-fold increase compared with the control strain with only the serine cycle. The final titer of 3-HP was significantly improved to 0.857 g/L in the fed-batch bioreactor, which was more competitive compared with the other 3-HP producers using methane and CO2 as C1 sources. Collectively, our current study demonstrated that engineering the synergistic methanol assimilation pathway was a promising strategy to increase the carbon assimilation and the yields of reduced chemicals in diverse host strains for C1 microbial cell factories.

Binding ability of methylene blue with heparin dependent on its sulfate level rather than its sulfation location or basic saccharide structure.

Methylene blue (MB) is one of the most common cationic dyes to detect heparin. As the sulfate residue presented in heparin was the main contributor to bind with MB, the UV performance of the MB with selectively desulfated heparin derivatives was investigated. It was found that the sulfate residue in different heparin analogues did not show the equal ability to attract MB binding. The stoichiometry of sulfate with MB among the heparin and derivatives was verified as a non-constant number. For the two selectively desulfated heparin derivatives: sulfate elimination at 6-O (6-OdeS) and N-acetylated heparin (N-deS-Acetyl), the MB to sulfate ratios were significantly higher than for heparin. For the not fully diminished sulfate at 2-O heparin derivative (2-OdeS), the MB-SO3- ratio of 2-OdeS was between 6-OdeS, N-deS-Acetlyl and heparin. Although in a distinct sulfation position, the MB-SO3- ratio of 6-OdeS and N-deS-Acetyl was almost equal, which agreed with the comparable total desulfation degree between 6-OdeS and N-deS-Acetyl. In addition, compared to heparin groups, the non-desulfated gs-HP showed no significantly different MB-SO3- ratio with heparin. The above results demonstrated that compared with the sulfate location and glycan composition of heparin, the content of sulfate was the most essential factor for the MB binding.

A versatile toolbox for CRISPR-based genome engineering in Pichia pastoris.

Pichia pastoris has gained much attention as a popular microbial cell factory for the production of recombinant proteins and high-value chemicals from laboratory to industrial scale. However, the lack of convenient and efficient genome engineering tools has impeded further applications of Pichia pastoris towards metabolic engineering and synthetic biology. Here, we report a CRISPR-based toolbox for gene editing and transcriptional regulation in P. pastoris. Based on the previous attempts in P. pastoris, we constructed a CRISPR/Cas9 system for gene editing using the RNA Pol-III-driven expression of sgRNA. The system was used to rapidly recycle the selectable marker with an eliminable episomal plasmid and achieved up to 100% knockout efficiency. Via dCas9 fused with transcriptional repressor (Mix1/RD1152) or activator (VPR), a flexible toolbox for regulation of gene expression was developed. The reporter gene eGFP driven by yeast pGAP or pCYC1 promoter showed strong inhibition (above 70%) and up to ~ 3.5-fold activation. To implement the combinatorial genetic engineering strategy, the CRISPR system contained a single Cas9-VPR protein, and engineered gRNA was introduced in P. pastoris for simultaneous gene activation, repression, and editing (CRISPR-ARE). We demonstrated that CRISPR-ARE was highly efficient for eGFP activation, mCherry repression, and ADE2 disruption, individually or in a combinatorial manner with a stable expression of multiplex sgRNAs. The simple and multifunctional toolkit demonstrated in this study will accelerate the application of P. pastoris in metabolic engineering and synthetic biology. KEY POINTS: • An eliminable CRISPR/Cas9 system yielded a highly efficient knockout of genes. • Simplified CRISPR/dCas9-based tools enabled transcriptional regulation of targeted genes. • CRISPR-ARE system achieved simultaneous gene activation, repression, and editing in P. pastoris.

Microbial microdroplet culture system (MMC): An integrated platform for automated, high-throughput microbial cultivation and adaptive evolution.

Conventional microbial cell cultivation techniques are typically labor intensive, low throughput, and poorlyparallelized, rendering them inefficient. The development of automated, modular microbial cell micro-cultivation systems, particularly those employing droplet microfluidics, have gained attention for their high-throughput, highly paralellized and efficient cultivation capabilities. Here, we report the development of a microbial microdroplet culture system (MMC), which is an integrated platform for automated, high-throughput cultivation and adaptive evolution of microorganisms. We demonstrated that the MMC yielded both accurate and reproducible results for the manipulation and detection of droplets. The superior performance of MMC for microbial cell cultivation was validated by comparing the growth curves of six microbial strains grown in MMC, conventional shake flasks or well plates. The highest incipient growth rate for all six microbial strains was achieved by using MMC. We also conducted an 18-day process of adaptive evolution of methanol-essential Escherichia coli strain in MMC and obtained two strains exhibiting higher growth rates compared with the parent strain. Our study demonstrates the power of MMC to provide an efficient and reliable approach for automated, high-throughput microbial cultivation and adaptive evolution.

Establishment of CRISPR interference in Methylorubrum extorquens and application of rapidly mining a new phytoene desaturase involved in carotenoid biosynthesis.

The methylotrophic bacterium Methylorubrum extorquens AM1 holds a great potential of a microbial cell factory in producing high value chemicals with methanol as the sole carbon and energy source. However, many gene functions remain unknown, hampering further rewiring of metabolic networks. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been demonstrated to be a robust tool for gene knockdown in diverse organisms. In this study, we developed an efficient CRISPRi system through optimizing the promoter strength of Streptococcus pyogenes-derived deactivated cas9 (dcas9). When the dcas9 and sgRNA were respectively controlled by medium PR/tetO and strong PmxaF-g promoters, dynamic repression efficacy of cell growth through disturbing a central metabolism gene glyA was achieved from 41.9 to 96.6% dependent on the sgRNA targeting sites. Furthermore, the optimized CRISPRi system was shown to effectively decrease the abundance of exogenous fluorescent protein gene mCherry over 50% and to reduce the expression of phytoene desaturase gene crtI by 97.7%. We then used CRISPRi technology combined with 26 sgRNAs pool to rapidly discover a new phytoene desaturase gene META1_3670 from 2470 recombinant mutants. The gene function was further verified through gene deletion and complementation as well as phylogenetic tree analysis. In addition, we applied CRISPRi to repress the transcriptional level of squalene-hopene cyclase gene shc involved in hopanoid biosynthesis by 64.9%, which resulted in enhancing 1.9-fold higher of carotenoid production without defective cell growth. Thus, the CRISPRi system developed here provides a useful tool in mining functional gene of M. extorquens as well as in biotechnology for producing high-valued chemicals from methanol. KEY POINTS: Developing an efficient CRISPRi to knockdown gene expression in C1-utilizing bacteria CRISPRi combined with sgRNAs pool to rapidly discover a new phytoene desaturase gene Improvement of carotenoid production by repressing a competitive pathway.

Highly Efficient Capture of Marine Microbial Strains in Seawater Using Bare Fe3O4 Magnetic Beads.

We develop a method to capture marine bacterial strains at high efficiency to replace the conventional two-step collecting method. Lab-made, Fe3O4 magnetic beads were used to firstly verify the feasibility of capture in artificial seawater, using Bacillus velezensis. Almost 100% of the bacteria could be captured and separated within 10 min. Then, the salinity of capture medium was proved to have the most marked effect on the capture process. After that, the broad application and high efficiency of capture were verified using four different bacterial strains from the Pacific Ocean. Subsequently, through adjusting the salinity, the capture efficiency for Pseudoalteromonas sp. and Halomonas meridiana was increased from 20 to ~ 80% in a seawater system, which was used to simulate the in-situ capture conditions. Finally, mixed strains in seawater were successfully captured, and their genomic DNAs were isolated and analyzed. Bare Fe3O4 magnetic beads were initially applied to capture marine microorganisms and this method is convenient and highly efficient and thus has great potential to replace the conventional two-step method.

Improved sgRNA design in bacteria via genome-wide activity profiling.

CRISPR/Cas9 is a promising tool in prokaryotic genome engineering, but its success is limited by the widely varying on-target activity of single guide RNAs (sgRNAs). Based on the association of CRISPR/Cas9-induced DNA cleavage with cellular lethality, we systematically profiled sgRNA activity by co-expressing a genome-scale library (∼70 000 sgRNAs) with Cas9 or its specificity-improved mutant in Escherichia coli. Based on this large-scale dataset, we constructed a comprehensive and high-density sgRNA activity map, which enables selecting highly active sgRNAs for any locus across the genome in this model organism. We also identified 'resistant' genomic loci with respect to CRISPR/Cas9 activity, notwithstanding the highly accessible DNA in bacterial cells. Moreover, we found that previous sgRNA activity prediction models that were trained on mammalian cell datasets were inadequate when coping with our results, highlighting the key limitations and biases of previous models. We hence developed an integrated algorithm to accurately predict highly effective sgRNAs, aiming to facilitate CRISPR/Cas9-based genome engineering, screenings and antimicrobials design in bacteria. We also isolated the important sgRNA features that contribute to DNA cleavage and characterized their key differences among wild type Cas9 and its mutant, shedding light on the biophysical mechanisms of the CRISPR/Cas9 system.

In vivo continuous evolution of metabolic pathways for chemical production.

Microorganisms have long been used as chemical plant to convert simple substrates into complex molecules. Various metabolic pathways have been optimised over the past few decades, but the progresses were limited due to our finite knowledge on metabolism. Evolution is a knowledge-free genetic randomisation approach, employed to improve the chemical production in microbial cell factories. However, evolution of large, complex pathway was a great challenge. The invention of continuous culturing systems and in vivo genetic diversification technologies have changed the way how laboratory evolution is conducted, render optimisation of large, complex pathway possible. In vivo genetic diversification, phenotypic selection, and continuous cultivation are the key elements in in vivo continuous evolution, where any human intervention in the process is prohibited. This approach is crucial in highly efficient evolution strategy of metabolic pathway evolution.

MiYA, an efficient machine-learning workflow in conjunction with the YeastFab assembly strategy for combinatorial optimization of heterologous metabolic pathways in Saccharomyces cerevisiae.

Facing boosting ability to construct combinatorial metabolic pathways, how to search the metabolic sweet spot has become the rate-limiting step. We here reported an efficient Machine-learning workflow in conjunction with YeastFab Assembly strategy (MiYA) for combinatorial optimizing the large biosynthetic genotypic space of heterologous metabolic pathways in Saccharomyces cerevisiae. Using ß-carotene biosynthetic pathway as example, we first demonstrated that MiYA has the power to search only a small fraction (2-5%) of combinatorial space to precisely tune the expression level of each gene with a machine-learning algorithm of an artificial neural network (ANN) ensemble to avoid over-fitting problem when dealing with a small number of training samples. We then applied MiYA to improve the biosynthesis of violacein. Feed with initial data from a colorimetric plate-based, pre-screened pool of 24 strains producing violacein, MiYA successfully predicted, and verified experimentally, the existence of a strain that showed a 2.42-fold titer improvement in violacein production among 3125 possible designs. Furthermore, MiYA was able to largely avoid the branch pathway of violacein biosynthesis that makes deoxyviolacein, and produces very pure violacein. Together, MiYA combines the advantages of standardized building blocks and machine learning to accelerate the Design-Build-Test-Learn (DBTL) cycle for combinatorial optimization of metabolic pathways, which could significantly accelerate the development of microbial cell factories.

Massilia violaceinigra sp. nov., a novel purple-pigmented bacterium isolated from glacier permafrost.

A Gram-stain-negative, motile and rod-shaped bacterium, designated strain B2T, which can synthesize purple pigments of violacein and dexyoviolacein, was isolated from Tianshan glacier in Xinjiang, China. Phylogenetic analysis based on 16S rRNA gene sequences indicated that it was grouped in the genus Massilia with Massilia glaciei B448-2T, Massilia eurypsychrophila B528-3T and Massilia psychrophila B1555-1T as its closest relatives (98.2, 97.9 and 97.0 % 16S rRNA gene sequence similarity, respectively). Genomic relatedness between strain B2T and its closest relatives was evaluated using average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity, with values of 77.93-85.08 %, 22.4-23.4 % and 71.54-72.99 %, respectively. Q-8 was the major ubiquinone. The major fatty acids (>5 %) of strain B2T were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C12 : 0 and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). The major polar lipids included phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The DNA G+C content of strain B2T was 63.51 mol%. Based on genomic relatedness, physiological, biochemical and chemotaxonomic data, strain B2T (=CGMCC 1.6993T=DSM 19531T=KCTC 32446T) is considered to represent a novel species within the genus Massilia, for which the name Massilia violaceinigra sp. nov. is proposed.

Genome-wide screening identifies promiscuous phosphatases impairing terpenoid biosynthesis in Escherichia coli.

Terpenoids are a large family of natural compounds that are important for both biotechnological applications and basic microorganism physiology. Inspired by the current literature, we hypothesized that recently deciphered phosphatase promiscuity may be an unexplored factor that negatively affects terpenoid biosynthesis by redirecting carbon flux away from the pathway via unrecognized catalytic activities on the phosphorylated intermediates. We used lycopene as a proof-of-concept to test this hypothesis. Based on an extensive bioinformatics analysis, we selected 56 phosphatase-encoding genes in Escherichia coli and constructed a knockdown library for these genes in a lycopene overproducer via CRISPR interference (CRISPRi). We screened this phosphatase knockdown library and observed enrichment (28 of 56) for genes that impair lycopene biosynthesis. Further scaled-up cultivation, combinatorial knockdown, and knockout assays in strains that overproduce lycopene or another terpenoid (ß-carotene) confirmed the proposed relationship between promiscuous phosphatases and impaired terpenoid biosynthesis. This study hence suggests the necessity of reconsidering the interactions of promiscuous phosphatases with ubiquitous phosphorylated components of metabolic networks with respect to engineering metabolism.

Biosensor-assisted transcriptional regulator engineering for Methylobacterium extorquens AM1 to improve mevalonate synthesis by increasing the acetyl-CoA supply.

Acetyl-CoA is not only an important intermediate metabolite for cells but also a significant precursor for production of industrially interesting metabolites. Methylobacterium extorquens AM1, a model strain of methylotrophic cell factories using methanol as carbon source, is of interest because it produces abundant coenzyme A compounds capable of directing to synthesis of different useful compounds from methanol. However, acetyl-CoA is not always efficiently accumulated in M. extorquens AM1, as it is located in the center of three cyclic central metabolic pathways. Here we successfully demonstrated a strategy for sensor-assisted transcriptional regulator engineering (SATRE) to control metabolic flux re-distribution to increase acetyl-CoA flux from methanol for mevalonate production in M. extorquens AM1 with introduction of mevalonate synthesis pathway. A mevalonate biosensor was constructed and we succeeded in isolating a mutated strain (Q49) with a 60% increase in mevalonate concentration (an acetyl-CoA-derived product) following sensor-based high-throughput screening of a QscR transcriptional regulator library. The mutated QscR-49 regulator (Q8*,T61S,N72Y,E160V) lost an N-terminal α-helix and underwent a change in the secondary structure of the RD-I domain at the C terminus, two regions that are related to its interaction with DNA. 13C labeling analysis revealed that acetyl-CoA flux was improved by 7% and transcriptional analysis revealed that QscR had global effects and that two key points, NADPH generation and fumC overexpression, might contribute to the carbon flux re-distribution. A fed-batch fermentation in a 5-L bioreactor for QscR-49 mutant yielded a mevalonate concentration of 2.67g/L, which was equivalent to an overall yield of 0.055mol acetyl-CoA/mol methanol, the highest yield among engineered strains of M. extorquens AM1. This work was the first attempt to regulate M. extorquens AM1 on transcriptional level and provided molecular insights into the mechanism of carbon flux regulation.