Concentration Recognition-Based Auto-Dynamic Regulation System (CRUISE) Enabling Efficient Production of Higher Alcohols.

Microbial factories lacking the ability of dynamically regulating the pathway enzymes overexpression, according to in situ metabolite concentrations, are suboptimal, especially when the metabolic intermediates are competed by growth and chemical production. The production of higher alcohols (HAs), which hijacks the amino acids (AAs) from protein biosynthesis, minimizes the intracellular concentration of AAs and thus inhibits the host growth. To balance the resource allocation and maintain stable AA flux, this work utilizes AA-responsive transcriptional attenuator ivbL and HA-responsive transcriptional activator BmoR to establish a concentration recognition-based auto-dynamic regulation system (CRUISE). This system ultimately maintains the intracellular homeostasis of AA and maximizes the production of HA. It is demonstrated that ivbL-driven enzymes overexpression can dynamically regulate the AA-to-HA conversion while BmoR-driven enzymes overexpression can accelerate the AA biosynthesis during the HA production in a feedback activation mode. The AA flux in biosynthesis and conversion pathways is balanced via the intracellular AA concentration, which is vice versa stabilized by the competition between AA biosynthesis and conversion. The CRUISE, further aided by scaffold-based self-assembly, enables 40.4 g L-1 of isobutanol production in a bioreactor. Taken together, CRUISE realizes robust HA production and sheds new light on the dynamic flux control during the process of chemical production.

Self-assembly systems to troubleshoot metabolic engineering challenges.

Enzyme self-assembly is a technology in which enzyme units can aggregate into ordered macromolecules, assisted by scaffolds. In metabolic engineering, self-assembly strategies have been explored for aggregating multiple enzymes in the same pathway to improve sequential catalytic efficiency, which in turn enables high-level production. The performance of the scaffolds is critical to the formation of an efficient and stable assembly system. This review comprehensively analyzes these scaffolds by exploring how they assemble, and it illustrates how to apply self-assembly strategies for different modules in metabolic engineering. Functional modifications to scaffolds will further promote efficient strategies for production.

[Interdisciplinary teaching-assisted education reform in "Principal Biology"].

General education in biological courses such as "Principal Biology" is an essential avenue for gaining an understanding of life science and developing an interest in the field. The reform of biological education teaching mode based on interdisciplinary approaches aims to foster cross-disciplinary talents, which is crucial for the rapid development of China's bioeconomy. Teaching method that simply superimposes different subjects is difficult to discover the value of interdisciplinary education. To address this, a novel teaching system and an innovative teaching mode were proposed for "Principal Biology" course by integrating science and engineering subjects, based on the cross-disciplinary feature in Beijing Institute of Technology. The system involves the design of cross-disciplinary course content and the integration of multiple disciplines and knowledge points based on students' majors, taking into account the characteristics of students' physical and mental development. To improve students' scientific literacy and interdisciplinary thinking ability, differentiated and major-driven teaching modes were applied by incorporating the "1+N" mixed and immersive cross-thinking training. The effectiveness of tailored cross-disciplinary teaching was evaluated using "in-teaching" and "post-teaching" data feedback models, which promote the optimization of teaching process and enhance the quality of education in cross-disciplinary biological science.

A consolidated review of commercial-scale high-value products from lignocellulosic biomass.

For decades, lignocellulosic biomass has been introduced to the public as the most important raw material for the environmentally and economically sustainable production of high-valued bioproducts by microorganisms. However, due to the strong recalcitrant structure, the lignocellulosic materials have major limitations to obtain fermentable sugars for transformation into value-added products, e.g., bioethanol, biobutanol, biohydrogen, etc. In this review, we analyzed the recent trends in bioenergy production from pretreated lignocellulose, with special attention to the new strategies for overcoming pretreatment barriers. In addition, persistent challenges in developing for low-cost advanced processing technologies are also pointed out, illustrating new approaches to addressing the global energy crisis and climate change caused by the use of fossil fuels. The insights given in this study will enable a better understanding of current processes and facilitate further development on lignocellulosic bioenergy production.

Metabolic Engineering and Regulation of Diol Biosynthesis from Renewable Biomass in Escherichia coli.

As bulk chemicals, diols have wide applications in many fields, such as clothing, biofuels, food, surfactant and cosmetics. The traditional chemical synthesis of diols consumes numerous non-renewable energy resources and leads to environmental pollution. Green biosynthesis has emerged as an alternative method to produce diols. Escherichia coli as an ideal microbial factory has been engineered to biosynthesize diols from carbon sources. Here, we comprehensively summarized the biosynthetic pathways of diols from renewable biomass in E. coli and discussed the metabolic-engineering strategies that could enhance the production of diols, including the optimization of biosynthetic pathways, improvement of cofactor supplementation, and reprogramming of the metabolic network. We then investigated the dynamic regulation by multiple control modules to balance the growth and production, so as to direct carbon sources for diol production. Finally, we proposed the challenges in the diol-biosynthesis process and suggested some potential methods to improve the diol-producing ability of the host.

A high-throughput visual screening method for p-hydroxybenzoate hydroxylase to increase phenolic compounds biosynthesis.

BACKGROUND: Gallic acid (GA) and pyrogallol are phenolic hydroxyl compounds and have diverse biological activities. Microbial-based biosynthesis, as an ecofriendly method, has been used for GA and pyrogallol production. In GA and pyrogallol biosynthetic pathways, the low hydroxylation activity of p-hydroxybenzoate hydroxylase (PobA) towards 3,4-dihydroxybenzoic acid (3,4-DHBA) limited the high-level biosynthesis of GA and pyrogallol. RESULTS: This work reported a high activity PobA mutant (Y385F/T294A/V349A PobA) towards 3,4-DHBA. This mutant was screened out from a PobA random mutagenesis library through a novel naked eye visual screening method. In vitro enzyme assay showed this mutant has a kcat/Km of 0.059 µM-1 s-1 towards 3,4-DHBA, which was 4.92-fold higher than the reported mutant (Y385F/T294A PobA). Molecular docking simulation provided the possible catalytic mechanism explanation of the high activity mutant. Expression of this mutant in E. coli BW25113 (F') can generate 840 ± 23 mg/L GA from 1000 mg/L 3,4-DHBA. After that, this mutant was assembled into a de novo GA biosynthetic pathway. Subsequently, this pathway was introduced into a 3,4-DHBA-producing strain (E. coli BW25113 (F')ΔaroE) to achieve 301 ± 15 mg/L GA production from simple carbon sources. Similarly, assembling this mutant into a de novo pyrogallol biosynthetic pathway enabled 129 ± 15 mg/L pyrogallol production. CONCLUSIONS: This work established an efficient screening method and generated a high activity PobA mutant. Assembling this mutant into de novo GA and pyrogallol biosynthetic pathways achieved the production of these two compounds from glucose. Besides, this mutant has great potential for the production of GA or pyrogallol derivatives. The screening method could be used for other GA biosynthesis-related enzymes.

Engineering Transcription Factor BmoR Mutants for Constructing Multifunctional Alcohol Biosensors.

Native transcription factor-based biosensors (TFBs) have the potential for the in situ detection of value-added chemicals or byproducts. However, their industrial application is limited by their ligand promiscuity, low sensitivity, and narrow detection range. Alcohols exhibit similar structures, and no reported TFB can distinguish a specific alcohol from its analogues. Here, we engineered an alcohol-regulated transcription factor, BmoR, and obtained various mutants with remarkable properties. For example, the generated signal-molecule-specific BmoRs could distinguish the constitutional isomers n-butanol and isobutanol, with insensitivity up to an ethanol concentration of 800 mM (36.9 g/L). Linear detection of 0-60 mM of a specific higher alcohol could be achieved in the presence of up to 500 mM (23.0 g/L) ethanol as background noise. Furthermore, we obtained two mutants with raised outputs and over 107-fold higher sensitivity and one mutant with an increased upper detection limit (14.8 g/L n-butanol or isobutanol). Using BmoR as an example, this study systematically explored the ultimate detection limit of a TFB toward its small-molecule ligands, paving the way for in situ detection in biofuel and wine industries.

Metabolic engineering of Corynebacterium glutamicum for producing branched chain amino acids.

BACKGROUND: Branched chain amino acids (BCAAs) are widely applied in the food, pharmaceutical, and animal feed industries. Traditional chemical synthetic and enzymatic BCAAs production in vitro has been hampered by expensive raw materials, harsh reaction conditions, and environmental pollution. Microbial metabolic engineering has attracted considerable attention as an alternative method for BCAAs biosynthesis because it is environmentally friendly and delivers high yield. MAIN TEXT: Corynebacterium glutamicum (C. glutamicum) possesses clear genetic background and mature gene manipulation toolbox, and has been utilized as industrial host for producing BCAAs. Acetohydroxy acid synthase (AHAS) is a crucial enzyme in the BCAAs biosynthetic pathway of C. glutamicum, but feedback inhibition is a disadvantage. We therefore reviewed AHAS modifications that relieve feedback inhibition and then investigated the importance of AHAS modifications in regulating production ratios of three BCAAs. We have comprehensively summarized and discussed metabolic engineering strategies to promote BCAAs synthesis in C. glutamicum and offer solutions to the barriers associated with BCAAs biosynthesis. We also considered the future applications of strains that could produce abundant amounts of BCAAs. CONCLUSIONS: Branched chain amino acids have been synthesized by engineering the metabolism of C. glutamicum. Future investigations should focus on the feedback inhibition and/or transcription attenuation mechanisms of crucial enzymes. Enzymes with substrate specificity should be developed and applied to the production of individual BCAAs. The strategies used to construct strains producing BCAAs provide guidance for the biosynthesis of other high value-added compounds.

In Vitro Biosynthesis of Isobutyraldehyde Through the Establishment of a One-Step Self-Assembly-Based Immobilization Strategy.

The in vitro biosynthesis of high-value compounds has become popular and attractive. The convenient and simple strategy of enzyme immobilization has been significant for continuous and efficient in vitro biosynthesis. On the basis of that, this work established a one-step self-assembly-based immobilization strategy to efficiently biosynthesize isobutyraldehyde in vitro. Isobutyraldehyde is a crucial precursor for the synthesis of foods and spices. The established CipA scaffold-based strategy can express and immobilize enzymes at the same time, and purification requires only one centrifugation step. Structural simulations indicated that this scaffold-dependent self-assembly did not influence the structure or catalytic mechanisms of the isobutyraldehyde production-related enzymes leucine dehydrogenase (LeuDH) and ketoisovalerate decarboxylase (Kivd). Immobilized LeuDH and Kivd displayed a higher conversion capacity and thermal stability than the free enzymes. Batch conversion experiments demonstrated that the recovered immobilized LeuDH and Kivd have similar conversion capacities to the enzymes used in the first round of reaction. The continuous production of isobutyraldehyde was achieved by filling the immobilized enzymes into the column of a constructed device. This study not only expands the application range of self-assembly systems but also provides guidance for the in vitro production of value-added compounds.

Metabolic engineering of E. coli for producing phloroglucinol from acetate.

Phloroglucinol is a three-hydroxyl phenolic compound and has diverse physiological and pharmacological activities such as antivirus and anti-inflammatory activities. Chemical synthesis of phloroglucinol suffered from many drawbacks such as high cost and environmental pollution. To avoid the above issues, microbial phloroglucinol biosynthesis was successfully accomplished in this study, while the abundant and low-cost acetate was used as the main carbon source. Firstly, the toxicity of phloroglucinol was tested, and E. coli BL21(DE3) could tolerate 5 g/L phloroglucinol. The ability of phloroglucinol synthase (PhlD) for catalyzing malonyl-CoA to phloroglucinol was confirmed, and E. coli BL21(DE3) expressing PhlD and acetyl-CoA carboxylase (ACCase) could produce 1107 ± 12 mg/L phloroglucinol from glucose. Then, E. coli BL21(DE3) was engineered to utilize acetate to produce 228 ± 15 mg/L phloroglucinol. Then, the endogenous citrate synthase (GltA) which could catalyze oxaloacetate and acetyl-CoA generated from acetate to citrate was knocked down by CRISPRi system in order to enhance the carbon flux for phloroglucinol production, and the titer was improved to 284 ± 8 mg/L. This work demonstrated that acetate could be used as low-cost substrate to achieve the biosynthesis of phloroglucinol and provided an example of effective utilization of acetate.

Light trapping structures and plasmons synergistically enhance the photovoltaic performance of full-spectrum solar cells.

A full-spectrum solar cell exhibits potential as an effective strategy to enhance the absorption of incident solar light. To ensure the absorption capability of solar cells, trapping structures or plasmons have emerged as two main ways of utilizing the full spectrum of solar energy. First, recent progress in the full-spectrum solar cells based on NCs was reviewed from the aspects of trapping structures and plasmon design. Moreover, the effects of light trapping and surface plasmon resonance on light absorption and photoelectronic conversion were emphasized and discussed. Finally, the application prospect of their combination in the field of full-spectrum solar cells was examined. It was pointed out that the deep exploration of the physical mechanism of photoelectric conversion, controllable preparation of the interface and stability of composite structures will become the main directions of future research.

Engineering transcription factor BmoR for screening butanol overproducers.

The wild-type transcription factors are sensitive to their corresponding signal molecules. Using wild-type transcription factors as biosensors to screen industrial overproducers are generally impractical because of their narrow detection ranges. This study took transcription factor BmoR as an example and aimed to expand the detection range of BmoR for screening alcohols overproducers. Firstly, a BmoR mutation library was established, and the mutations distributed randomly in all predicted functional domains of BmoR. Structure of BmoR-isobutanol complex were modelled, and isobutanol binding sites were confirmed by site-directed mutagenesis. Subsequently, the effects of the mutations on the detection range or output were confirmed in the BmoR mutants. Four combinatorial mutants containing one increased-detection-range mutation and one enhanced-output mutation were constructed. Compared with wild-type BmoR, F276A/E627N BmoR and D333N/E627N BmoR have wider detection ranges (0-100 mM) and relatively high outputs to the isobutanol added quantitatively or produced intracellularly, demonstrating they have potential for screening isobutanol overproduction strains. This work presented an example of engineering the wild-type transcription factors with physiological significance for industrial utilization.

A novel protein purification strategy mediated by the combination of CipA and Ssp DnaB intein.

Protein purification is an indispensable step in diverse fields of biological research or production process. Conventional purification methods including the affinity purification or the usage of self-aggregating tags suffered from many drawbacks such as the complicated steps, high cost and low efficiency. Moreover, the fusion tag usually had negative effects on the activity of the target protein. To address the above issues, here we propose a novel protein purification method which needs simple operation steps, and this method is mediated by the combination of CipA protein and a mini-intein (Synechocystis sp. PCC6803 DnaB, Ssp DnaB), depending on the assembly function of CipA and the self-cleavage function of Ssp DnaB. To realize the purification, CipA-DnaB-eGFP protein was expressed and assembled into protein crystalline inclusions (PCIs) in E. coli. Then, only cell lysis, cleavage and centrifugation steps were required to purify eGFP. Purified eGFP was in the supernatant with a purity of over 90%. The cleavage efficiency and the yield of eGFP reached 51.96% and 13.99 ±â€¯0.88 mg/L fermentation broth, respectively. Furthermore, to broaden the application of this approach, three other proteins which were maltose binding protein (MBP), ketoisovalerate decarboxylase (Kivd) and alcohol dehydrogenase (AdhP) were purified with high cleavage efficiency. The purified Kivd and AdhP remained high specific activities. This work demonstrated an effective and convenient protein purification method.

Establishment of BmoR-based biosensor to screen isobutanol overproducer.

BACKGROUND: Isobutanol, a C4 branched-chain higher alcohol, is regarded as an attractive next-generation transport fuel. Metabolic engineering for efficient isobutanol production has been achieved in many studies. BmoR, an alcohol-regulated transcription factor, mediates a σ54-dependent promoter Pbmo of alkane monooxygenase in n-alkane metabolism of Thauera butanivorans and displays high sensitivity to C4-C6 linear alcohols and C3-C5 branched-chain alcohols. In this study, to achieve the high-level production of isobutanol, we established a screening system which relied on the combination of BmoR-based biosensor and isobutanol biosynthetic pathway and then employed it to screen isobutanol overproduction strains from an ARTP mutagenesis library. RESULTS: Firstly, we constructed and verified a GFP-based BmoR-Pbmo device responding to the isobutanol produced by the host. Then, this screening system was employed to select three mutants which exhibited higher GFP/OD600 values than that of wild type. Significantly, GFP/OD600 of mutant 10 was 190.7 ± 4.8, a 1.4-fold higher value than that of wild type. Correspondingly, the isobutanol titer of that strain was 1597.6 ± 129.6 mg/L, 2.0-fold higher than the wild type. With the overexpression of upstream pathway genes, the isobutanol production from mutant 10 reached 14.0 ± 1.0 g/L after medium optimization in shake flask. The isobutanol titer reached 56.5 ± 1.8 g/L in a fed-batch production experiment. CONCLUSIONS: This work screened out isobutanol overproduction strains from a mutagenesis library by using a screening system which depended on the combination of BmoR-based biosensor and isobutanol biosynthetic pathway. Optimizing fermentation condition and reinforcing upstream pathway could realize the increase of isobutanol production from the overproducer. Lastly, fed-batch fermentation of the mutant enhanced the isobutanol production to 56.5 ± 1.8 g/L.

CipA-mediating enzyme self-assembly to enhance the biosynthesis of pyrogallol in Escherichia coli.

Pyrogallol is a valuable phenolic compound and displays various physiological and pharmaceutical functions. Chemical synthesis of pyrogallol suffered from many issues, including environmental pollution, high cost, and low yield. Here, to address the above drawbacks, an artificial pathway for de novo pyrogallol production was established and this pathway only needed two exogenous enzymes (Y385F/T294A PobA and 3,4-dihydroxybenzoic acid decarboxylase (PDC)). Y385F/T294A PobA is a mutant of PobA which is a hydroxylase from Pseudomonas aeruginosa, while PDC is a decarboxylase from Klebsiella pneumoniae subsp. pneumoniae. First, the conversion efficiency of PDC was tested and 1800 ± 100 mg/L pyrogallol was generated from 4 g/L gallic acid (GA). Subsequently, assembly of the whole pathway enabled 33 ± 6 mg/L pyrogallol production from simple carbon sources. After that, based on the assembling property of CipA (a hydrophobic protein) and to enhance the hydroxylation of 3,4-dihydroxybenzoic acid, CipA was employed to organize its fusion (Y385F/T294A PobA) into protein crystalline inclusions (PCIs). Remarkably, the formation of CipA-Y385F/T294A PobA PCIs increased the pyrogallol production to 60 ± 6 mg/L, a 1.8 ± 0.4-fold higher value as compared to the strain without enzyme self-assembly. Additionally, the titer of pyrogallol was enhanced to 80 ± 1 mg/L through yeast extract concentration optimization. This work not only realizes the biosynthesis of pyrogallol from renewable carbon sources but also demonstrates that using CipA-mediating enzyme self-assembly could reinforce the hydroxylation efficiency of Y385F/T294A PobA, resulting in the enhancement of pyrogallol production.

Sensor-regulator and RNAi based bifunctional dynamic control network for engineered microbial synthesis.

Writing artificial logic and dynamic function into complex cellular background to achieve desired phenotypes or improved outputs calls for the development of new genetic tools as well as their innovative use. In this study, we present a sensor-regulator and RNAi-based bifunctional dynamic control network that can provide simultaneous upregulation and downregulation of cellular metabolism for engineered biosynthesis. The promoter-regulator-mediated upregulation function and its transduced downregulation function through RNAi are systematically verified and characterized. We apply this dynamic control network to regulate the phosphoenolpyruvate metabolic node in Escherichia coli and achieve autonomous distribution of carbon flux between its native metabolism and the engineered muconic acid biosynthetic pathway. This allows muconic acid biosynthesis to reach 1.8 g L-1. This study also suggests the circumstances where dynamic control approaches are likely to take effects.

High-level production of ChSase ABC I by co-expressing molecular chaperones in Escherichia coli.

Chondroitinase ABC I (ChSase ABC I), as a polysaccharide lyase, can catalyze high molecular weight chondroitin sulfate (CS) to low molecular weight glycosaminoglycan which are easier to be absorbed and utilized by organisms. In this study, to enhance the production of ChSase ABC I and avoid the negative influence of tags on its catalytic efficiency, we employed molecular chaperones to co-express with ChSase ABC I. Firstly, different molecular chaperones and their combinations were screened and GroES exhibited the best positive effect. Consecutively, fermentation conditions were optimized to further improve the production. As a result, the production of ChSase ABC I was increased to 4640.44 ±â€¯896.26 IU/g wet weight, a 2.15-fold higher value when compared with that of control in the same fermentation conditions. After that, to testify the influence of GroES on characterization of ChSase ABC I, the optimal pH and temperature, and kinetic parameters were confirmed. The affinity to substrate of ChSase ABC I with GroES assist was increased 7 folds as compared to the native ChSase ABC I, and ChSase ABC I with GroES co-expression still has high catalytic activity. This work not only presents to date the first achievement of ChSase ABC I high-level production with molecular chaperone co-expression, but also serves as a potential basis for its industrial application.

Investigation of the Synergetic Effect of Xylose Metabolic Pathways on the Production of Glutaric Acid.

Efficient utilization of lignocellulose is pivotal for economically converting renewable feedstocks into value-added products. Xylose is the second most abundant sugar in lignocellulose, but it is quite challenging to ferment xylose as efficiently as glucose by microorganisms. Here, we investigated the metabolic potential of three xylose catabolic pathways (isomerase, Weimberg, and Dahms pathways) and illustrated the synergetic effect between the isomerase pathway and Weimberg pathway for the synthesis of chemicals derived from 2-ketoglutarate and acetyl-CoA. When using glutaric acid as the target product, employment of such synergetic pathways in combination resulted in an increased glutaric acid titer (602 mg/L) compared with using each pathway alone (104 or 209 mg/L), and this titer even outcompetes that obtained from the glucose catabolic pathway for glutaric acid synthesis (420 mg/L). This work validates a novel and powerful strategy for xylose metabolic utilization to overcome the inefficiency of using a single xylose metabolic pathway for the synthesis of TCA cycle derived chemicals.

Investigating the strategies for microbial production of trehalose from lignocellulosic sugars.

Trehalose, a multi-functional and value-added disaccharide, can be efficiently biosynthesized from glucose by using a synergetic carbon utilization mechanism (SynCar) which coupled phosphoenolpyruvate (PEP) generation from the second carbon source with PEP-dependent phosphotransferase system (PTS) to promote non-catabolic use of glucose. Considering glucose and xylose present in large amounts in lignocellulosic sugars, we explored new strategies for conversion of both sugars into trehalose. Herein, we first attempted trehalose production from xylose directly, based on which, synergetic utilization of glucose, and xylose prompted by SynCar was implemented in engineered Escherichia coli. As the results, the final titer of trehalose reached 5.55 g/L in shake flask experiments. The conversion ratio or utilization efficiency of glucose or xylose to trehalose was around fourfold higher than that of the original strain (YW-3). This work not only demonstrated the possibility of directly converting xylose (C5 sugar) into trehalose (C12 disaccharide), but also suggested a promising strategy for trehalose production from lignocellulosic sugars for the first time.

Establishing an Artificial Pathway for Efficient Biosynthesis of Hydroxytyrosol.

Hydroxytyrosol (HT) is a valuable natural phenolic compound with strong antioxidant activity and various physiological and pharmaceutical functions. In this study, we established an artificial pathway for HT biosynthesis. First, efficient enzymes were selected to construct a tyrosol biosynthetic pathway. Aro10 from Saccharomyces cerevisiae was shown to be a better ketoacid decarboxylase than Kivd from Lactococcus lactis for tyrosol production. While knockout of feaB significantly decreased accumulation of the byproduct 4-hydroxyphenylacetic acid, overexpression of alcohol dehydrogenase ADH6 further improved tyrosol production. The titers of tyrosol reached 1469 ± 56 mg/L from tyrosine and 620 ± 23 mg/L from simple carbon sources, respectively. The pathway was further extended for HT production by overexpressing Escherichia coli native hydroxylase HpaBC. To enhance transamination of tyrosine to 4-hydroxyphenylpyruvate, NH4Cl was removed from the culture media. To decrease oxidation of HT, ascorbic acid was added to the cell culture. To reduce the toxicity of HT, 1-dodecanol was selected as the extractant for in situ removal of HT. These efforts led to an additive increase in HT titer to 1243 ± 165 mg/L in the feeding experiment. Assembly of the full pathway resulted in 647 ± 35 mg/L of HT from simple carbon sources. This work provides a promising alternative for sustainable production of HT, which shows scale-up potential.