State Key Laboratory of Microbial Resources

Nature has provided a fantastic array of enzymes that are responsible for essential biochemical functions but not usually suitable for technological applications. Not content with the natural repertoire, protein engineering holds promise to extend the applications of improved enzymes with tailored properties. However, engineering of robust proteins remains a difficult task since the positive mutation library may not cooperate to reach the target function in most cases owing to the ubiquity of epistatic effects. The main demand lies in identifying an efficient path of accumulated mutations. Herein, we devised a computational strategy (greedy accumulated strategy for protein engineering, GRAPE) to improve the robustness of a PETase from Ideonella sakaiensis. A systematic clustering analysis combined with greedy accumulation of beneficial mutations in a computationally derived library enabled the redesign of a variant, DuraPETase, which exhibits an apparent melting temperature that is drastically elevated by 31 °C and a strikingly enhanced degradation toward semicrystalline poly(ethylene terephthalate) (PET) films (30%) at mild temperatures (over 300-fold). Complete biodegradation of 2 g/L microplastics to water-soluble products under mild conditions is also achieved, opening up opportunities to steer the biological degradation of uncollectable PET waste and further conversion of the resulting monomers to high-value molecules. The crystal structure revealed the individual mutation match with the design model. Concurrently, synergistic effects are captured, while epistatic interactions are alleviated during the accumulation process. We anticipate that our design strategy will provide a broadly applicable strategy for global optimization of enzyme performance.

Besides genome editing, CRISPR-Cas12a has recently been used for DNA detection applications with attomolar sensitivity but, to our knowledge, it has not been used for the detection of small molecules. Bacterial allosteric transcription factors (aTFs) have evolved to sense and respond sensitively to a variety of small molecules to benefit bacterial survival. By combining the single-stranded DNA cleavage ability of CRISPR-Cas12a and the competitive binding activities of aTFs for small molecules and double-stranded DNA, here we develop a simple, supersensitive, fast and high-throughput platform for the detection of small molecules, designated CaT-SMelor (CRISPR-Cas12a- and aTF-mediated small molecule detector). CaT-SMelor is successfully evaluated by detecting nanomolar levels of various small molecules, including uric acid and p-hydroxybenzoic acid among their structurally similar analogues. We also demonstrate that our CaT-SMelor directly measured the uric acid concentration in clinical human blood samples, indicating a great potential of CaT-SMelor in the detection of small molecules.

Abstract Acetyl-CoA is a fundamental metabolite for all life on Earth, and is also a key starting point for the biosynthesis of a variety of industrial chemicals and natural products. Here we design and construct a Synthetic Acetyl-CoA (SACA) pathway by repurposing glycolaldehyde synthase and acetyl-phosphate synthase. First, we design and engineer glycolaldehyde synthase to improve catalytic activity more than 70-fold, to condense two molecules of formaldehyde into one glycolaldehyde. Second, we repurpose a phosphoketolase to convert glycolaldehyde into acetyl-phosphate. We demonstrated the feasibility of the SACA pathway in vitro, achieving a carbon yield ~50%, and confirmed the SACA pathway by 13 C-labeled metabolites. Finally, the SACA pathway was verified by cell growth using glycolaldehyde, formaldehyde and methanol as supplemental carbon source. The SACA pathway is proved to be the shortest, ATP-independent, carbon-conserving and oxygen-insensitive pathway for acetyl-CoA biosynthesis, opening possibilities for producing acetyl-CoA-derived chemicals from one-carbon resources in the future.

Covering: up to 2018 α-Ketoglutarate (αKG, also known as 2-oxoglutarate)-dependent mononuclear non-haem iron (αKG-NHFe) enzymes catalyze a wide range of biochemical reactions, including hydroxylation, ring fragmentation, C-C bond cleavage, epimerization, desaturation, endoperoxidation and heterocycle formation. These enzymes utilize iron(ii) as the metallo-cofactor and αKG as the co-substrate. Herein, we summarize several novel αKG-NHFe enzymes involved in natural product biosyntheses discovered in recent years, including halogenation reactions, amino acid modifications and tailoring reactions in the biosynthesis of terpenes, lipids, fatty acids and phosphonates. We also conducted a survey of the currently available structures of αKG-NHFe enzymes, in which αKG binds to the metallo-centre bidentately through either a proximal- or distal-type binding mode. Future structure-function and structure-reactivity relationship investigations will provide crucial information regarding how activities in this large class of enzymes have been fine-tuned in nature.

DNA sequence analysis of a 7.5 kb XhoI DNA fragment from the region flanking the nikkomycin biosynthesis gene cluster in Streptomyces ansochromogenes revealed one 3.3 kb open reading frame (ORF), designated sanG. The deduced product of sanG (1061 amino acids), which is similar to PimR of Streptomyces natalensis, contains an OmpR-like DNA binding domain in its N-terminal portion and A- and B-type nucleotide binding motifs in the middle of the protein. Disruption of sanG abolished nikkomycin biosynthesis, reduced sporulation and led to brown pigment accumulation. All aspects of this complex phenotype were complemented by a single copy sanG which was integrated into the chromosome. The introduction of multiple copies of sanG resulted in increased nikkomycin production. S1 mapping results indicated that sanG is transcribed from at least three promoters (P1, P2 and P3), P1 being strongly upregulated when production of nikkomycins starts. Two putative transcription units for nikkomycin biosynthesis, starting from sanN and sanO, are dependent on the expression of sanG, whereas a putative transcription unit starting from sanF was not regulated by sanG. These results suggested that sanG encodes a transcriptional activator important for nikkomycin biosynthesis that, unusually, also has pleiotropic effects on secondary metabolism and development.

Intercellular signaling is indispensable for single cells to form complex biological structures, such as biofilms, tissues and organs. The genetic tools available for engineering intercellular signaling, however, are quite limited. Here we exploit the chemical diversity of biological small molecules to de novo design a genetic toolbox for high-performance, multi-channel cell-cell communications and biological computations. By biosynthetic pathway design for signal molecules, rational engineering of sensing promoters and directed evolution of sensing transcription factors, we obtain six cell-cell signaling channels in bacteria with orthogonality far exceeding the conventional quorum sensing systems and successfully transfer some of them into yeast and human cells. For demonstration, they are applied in cell consortia to generate bacterial colony-patterns using up to four signaling channels simultaneously and to implement distributed bio-computation containing seven different strains as basic units. This intercellular signaling toolbox paves the way for engineering complex multicellularity including artificial ecosystems and smart tissues.

Verticillium wilt of cotton is a vascular disease mainly caused by the soil-born filamentous fungus Verticillium dahliae. To study the mechanisms associated with defense responses in wilt-resistant sea-island cotton (Gossypium barbadense) upon V. dahliae infection, a comparative proteomic analysis between infected and mock-inoculated roots of G. barbadense var. Hai 7124 (a cultivar showing resistance against V. dahliae) was performed by 2-DE combined with local EST database-assisted PMF and MS/MS analysis. A total of 51 upregulated and 17 downregulated proteins were identified, and these proteins are mainly involved in defense and stress responses, primary and secondary metabolisms, lipid transport, and cytoskeleton organization. Three novel clues regarding wilt resistance of G. barbadense are gained from this study. First, ethylene signaling was significantly activated in the cotton roots attacked by V. dahliae as shown by the elevated expression of ethylene biosynthesis and signaling components. Second, the Bet v 1 family proteins may play an important role in the defense reaction against Verticillium wilt. Third, wilt resistance may implicate the redirection of carbohydrate flux from glycolysis to pentose phosphate pathway (PPP). To our knowledge, this study is the first root proteomic analysis on cotton wilt resistance and provides important insights for establishing strategies to control this disease.

With nearly three decades of development, Pichia pastoris ( P . pastoris ) has become a powerful eukaryotic protein expression system for the expression of thousands of proteins both on a laboratory and industrial scale. In addition, it has also been extensively used as a cell factory for the production of a variety of chemicals. This review summarizes the bottlenecks and solutions in achieving high‐level secretory protein expression with P. pastoris and then outlines its applications on chemical production with an emphasis on its role as whole‐cell biocatalyst. Furthermore, current challenges and future directions of this important system are also discussed.

Genetic diversity is a result of evolution, enabling multiple ways for one particular physiological activity. Here, we introduce this strategy into bioengineering. We design two hydroxytyrosol biosynthetic pathways using tyrosine as substrate. We show that the synthetic capacity is significantly improved when two pathways work simultaneously comparing to each individual pathway. Next, we engineer flavin-dependent monooxygenase HpaBC for tyrosol hydroxylase, tyramine hydroxylase, and promiscuous hydroxylase active on both tyrosol and tyramine using directed divergent evolution strategy. Then, the mutant HpaBCs are employed to catalyze two missing steps in the hydroxytyrosol biosynthetic pathways designed above. Our results demonstrate that the promiscuous tyrosol/tyramine hydroxylase can minimize the cell metabolic burden induced by protein overexpression and allow the biosynthetic carbon flow to be divided between two pathways. Thus, the efficiency of the hydroxytyrosol biosynthesis is significantly improved by rearranging the metabolic flux among multiple pathways.

Hydroxytyrosol is an antioxidant free radical scavenger that is biosynthesized from tyrosine. In metabolic engineering efforts, the use of the mouse tyrosine hydroxylase limits its production. Here, we design an efficient whole-cell catalyst of hydroxytyrosol in Escherichia coli by de-bottlenecking two rate-limiting enzymatic steps. First, we replace the mouse tyrosine hydroxylase by an engineered two-component flavin-dependent monooxygenase HpaBC of E. coli through structure-guided modeling and directed evolution. Next, we elucidate the structure of the Corynebacterium glutamicum VanR regulatory protein complexed with its inducer vanillic acid. By switching its induction specificity from vanillic acid to hydroxytyrosol, VanR is engineered into a hydroxytyrosol biosensor. Then, with this biosensor, we use in vivo-directed evolution to optimize the activity of tyramine oxidase (TYO), the second rate-limiting enzyme in hydroxytyrosol biosynthesis. The final strain reaches a 95% conversion rate of tyrosine. This study demonstrates the effectiveness of sequentially de-bottlenecking rate-limiting steps for whole-cell catalyst development.

Members of the gram-negative, strictly aerobic genus Comamonas occur in various environments. Here we report the complete genome of Comamonas testosteroni strain CNB-2. Strain CNB-2 has a circular chromosome that is 5,373,643 bp long and has a G+C content of 61.4%. A total of 4,803 open reading frames (ORFs) were identified; 3,514 of these ORFs are functionally assigned to energy production, cell growth, signal transduction, or transportation, while 866 ORFs encode hypothetical proteins and 423 ORFs encode purely hypothetical proteins. The CNB-2 genome has many genes for transportation (22%) and signal transduction (6%), which allows the cells to respond and adapt to changing environments. Strain CNB-2 does not assimilate carbohydrates due to the lack of genes encoding proteins involved in glycolysis and pentose phosphate pathways, and it contains many genes encoding proteins involved in degradation of aromatic compounds. We identified 66 Tct and nine TRAP-T systems and a complete tricarboxylic acid cycle, which may allow CNB-2 to take up and metabolize a range of carboxylic acids. This nutritional bias for carboxylic acids and aromatic compounds enables strain CNB-2 to occupy unique niches in environments. Four different sets of terminal oxidases for the respiratory system were identified, and they putatively functioned at different oxygen concentrations. This study conclusively revealed at the genomic level that the genetic versatility of C. testosteroni is vital for competition with other bacteria in its special niches.

The production of chemicals from renewable biomass resources is usually limited by factors including high-cost processes and low efficiency of biosynthetic pathways. Fatty acids (FAs) are an ideal alternative biomass. Their advantages include high-efficiently producing acetyl-CoA and reducing power, coupling chemical production with CO2 fixation, and the fact that they are readily obtained from inexpensive feedstocks. The important platform chemical 3-hydroxypropionate (3HP) can be produced from FAs as the feedstock with a theoretical yield of 2.49 g/g, much higher than the theoretical yield from other feedstocks. In this study, we first systematically analyzed the limiting factors in FA-utilization pathways in Escherichia coli. Then, we optimized FA utilization in Escherichia coli by using a combination of metabolic engineering and optimization of fermentation conditions. The 3HP biosynthesis module was introduced into a FA-utilizing strain, and the flux balance was finely optimized to maximize 3HP production. The resulting strain was able to produce 3HP from FAs with a yield of 1.56 g/g, and was able to produce 3HP to a concentration of 52 g/L from FAs in a 5-L fermentation process. The strain also could produce 3HP from various type of FAs feedstock including gutter oil. This is the first report of a technique for the efficient production of the platform chemical 3HP from FAs.

Natural CO2 fixation cycles usually comprise multiple reactions, which may reduce the efficiency of the cycle. Here, we report the design and experimental demonstration of a minimized synthetic CO2 fixation cycle which contains only four reactions. The cycle comprises pyruvate carboxylase, oxaloacetate acetylhydrolase, acetate-CoA ligase, and pyruvate synthase and is named the POAP cycle. The POAP cycle can condense two molecules of CO2 into one molecule of oxalate in each step at the expense of two molecules of ATP and one reducing equivalent in the form of NAD(P)H. By identifying a ferredoxin from Hydrogenobacter thermophilus that can efficiently drive the rate-limiting reductive carboxylation step, the POAP cycle can be operated at 50 °C under anaerobic conditions, reaching a CO2 fixation rate of 8.0 nmol CO2 min–1 mg–1 CO2-fixing enzymes. The design and demonstration of the POAP cycle may provide a model to study CO2 fixation in the earliest organisms.

We have previously reported that the administration of Lactobacillus plantarum DR7 for 12 weeks reduced stress and anxiety in stressed adults as compared to the placebo group, in association with changes along the brain neurotransmitters pathways of serotonin and dopamine-norepinephrine. We now aim to evaluate the effects of DR7 on gut functions, gut microbiota compositional changes, and determine the correlations between microbiota changes and the pathways of brain neurotransmitters. The administration of DR7 prevented an increase of defecation frequency over 12 weeks as compared to the placebo (p = 0.044), modulating the increase of stress-induced bowel movement. Over 12 weeks, alpha diversity of gut microbiota was higher in DR7 than the placebo group across class (p = 0.005) and order (p = 0.018) levels, while beta diversity differed between groups at class and order levels (p < 0.001). Differences in specific bacterial groups were identified, showing consistency at different taxonomic levels that survived multiplicity correction, along the phyla of Bacteroides and Firmicutes and along the classes of Deltaproteobacteria and Actinobacteria. Bacteroidetes, Bacteroidia, and Bacteroidales which were reduced in abundance in the placebo group showed opposing correlation with gene expression of dopamine beta hydrolase (DBH, dopamine pathway; p < 0.001), while Bacteroidia and Bacteroidales showed correlation with tryptophan hydroxylase-II (TPH2, serotonin pathway; p = 0.001). A correlation was observed between DBH and Firmicutes (p = 0.002), Clostridia (p < 0.001), Clostridiales (p = 0.001), Blautia (p < 0.001), and Romboutsia (p < 0.001), which were increased in abundance in the placebo group. Blautia was also associated with TDO (p = 0.001), whereas Romboutsia had an opposing correlation with TPH2 (p < 0.001). Deltaproteobacteria and Desulfovibrionales which were decreased in abundance in the placebo group showed opposing correlation with DBH (p = 0.001), whereas Bilophila was associated with TPH2 (p = 0.001). Our present data showed that physiological changes induced by L. plantarum DR7 could be associated with changes in specific taxa of the gut microbiota along the serotonin and dopamine pathways.

The early identification of causative pathogens in clinical specimens that require no cultivation is essential for directing evidence-based antimicrobial treatments in resource limited settings. Here, we describe an integrated microfluidic device for the rapid identification of pathogens in complex physiological matrices such as blood. The device was designed and fabricated using SlipChip technologies, which integrated four channels processing independent samples and identifying up to twenty different pathogens. Briefly, diluted whole human blood samples were directly injected into the device for analysis. The pathogens were extracted from the blood by dielectrophoresis, retained in an array of grooves, and identified by multiplex array PCR in nanoliter volumes with end-point fluorescence detection. The universality of the dielectrophoretic separation of pathogens from physiological fluids was evaluated with a panel of clinical isolates covering predominant bacterial and fungal species. Using this system, we simultaneously identified Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli O157:H7 within 3 h. In addition to the prompt diagnosis of bloodstream infections, this method may also be utilized for differentiating microorganisms in contaminated water and environmental samples.

Alzheimer’s disease (AD) is a progressive disease and one of the most common forms of neurodegenerative disorders. Emerging evidence is supporting the use of various strategies that modulate gut microbiota to exert neurological and psychological changes. This includes the utilisation of probiotics as a natural and dietary intervention for brain health. Here, we showed the potential AD-reversal effects of Lactobacillus probiotics through feeding to our Drosophila melanogaster AD model. The administration of Lactobacillus strains was able to rescue the rough eye phenotype (REP) seen in AD-induced Drosophila , with a more prominent effect observed upon the administration of Lactobacillus plantarum DR7 (DR7). Furthermore, we analysed the gut microbiota of the AD-induced Drosophila and found elevated levels of Wolbachia . The administration of DR7 restored the gut microbiota diversity of AD-induced Drosophila with a significant reduction in Wolbachia ’s relative abundance, accompanied by an increase of Stenotrophomonas and Acetobacter . Through functional predictive analyses, Wolbachia was predicted to be positively correlated with neurodegenerative disorders, such as Parkinson’s, Huntington’s and Alzheimer’s diseases, while Stenotrophomonas was negatively correlated with these neurodegenerative disorders. Altogether, our data exhibited DR7’s ability to ameliorate the AD effects in our AD-induced Drosophila . Thus, we propose that Wolbachia be used as a potential biomarker for AD.

drops per h. We applied the pipeline to screen samples collected from different locations, including sediments from a hot spring in Tibet, soils from the Zoige wetland, contaminated soils from an abandoned oilfield, and a Chinese Daqu starter. In total, we obtained 47 lipase-producing bacterial strains belonging to seven genera, including Staphylococcus, Bacillus, Enterobacter, Serratia, Prolinoborus, Acinetobacter, and Leclercia. We believe that this FADS-based pipeline could be extended to screen various enzymes from the environment, and may find wide applications in breeding of industrial microorganisms.

Abstract Microbial biophotovoltaics (BPV) offers a biological solution for renewable energy production by using photosynthetic microorganisms as light absorbers. Although abiotic engineering approaches, e.g., electrode modification and device optimization, can enhance the electrochemical communication between living cells and electrodes, the power densities of BPV are still low due to the weak exoelectrogenic activity of photosynthetic microorganisms. Here, we develop a BPV based on a d -lactate mediated microbial consortium consisting of photosynthetic cyanobacteria and exoelectrogenic Shewanella . By directing solar energy from photons to d -lactate, then to electricity, this BPV generates a power density of over 150 mW·m −2 in a temporal separation setup. Furthermore, a spatial-temporal separation setup with medium replenishment enables stable operation for over 40 days with an average power density of 135 mW·m −2 . These results demonstrate the electron flow constrained microbial consortium can facilitate electron export from photosynthetic cells and achieve an efficient and durable power output.

Abstract Microbiota play important roles in the internal environment and health of humans, livestock and wild animals. Short-chain fatty acids (SCFAs) and lactate are primary metabolites that can impact the composition and function of human microbiota. According to the well-characterized key synthesis genes, many SCFA- and lactate-producing bacteria have been identified in the gut microbiota. However, unknown genes may also contribute to the formation of SCFAs and lactate. The identification of such genes will provide new engineering targets and new strategies for maintaining a stable structure of beneficial microbiota. In this study, we used Escherichia coli as a model to analyze possible genes related to SCFAs and lactate production besides the well-characterized ones. The functions of nineteen candidate genes were studied by targeted gene deletion and overexpression. Results indicated thioesterase genes such as yciA , tesA , tesB , and menI can contribute to acetate and/or butyrate formation. As for lactate, mgsA and lldD can function in addition to ldh gene. At the same time, the distribution of these functional genes in gut microbiota was investigated. Most bacteria contain the well-studied genes whereas some bacteria contain some of the described unusual ones. The results provide insights and genetic targets for the discovery of new SCFA- and lactate-producing bacteria in gut microbiota.

Plant plasma membrane (PM) plays important roles in immune response. Here, we utilized quantitative mass spectrometry to explore rice PM protein composition and dynamic changes during Magnaporthe oryzae infection. We report, thus far, the largest rice PM proteome dataset with 3,906 identified proteins, among which 484 proteins were differentially expressed after M. oryzae infection. One third of the identified proteins are predicted to have at least one transmembrane domain. Half of the identified proteins are predicted to have binding functions and over one third of the proteins have enzyme-related functions. In addition, Gene Ontology analyses revealed that abscisic acid (ABA) and cytokinin (CK) signaling were sequentially activated after M. oryzae infection in rice. We found that the activation of ABA signaling and the suppression of rice immune response occurred at the early infection stage, while the activation of CK signaling, the upregulation of sugar transporter genes expression, and the nutrient efflux of infected rice cells occurred at later infection stage. Thus, we further propose that M. oryzae activates ABA signaling to repress rice immune signaling for initial invasion and redirects nutrient efflux of infected cells for massive growth at the later infection stage.