Differential responses of leaf photosynthesis to insect and pathogen outbreaks: A global synthesis.

Outbreak of insects or pathogens (referred to as biotic disturbance), which is projected to continually increase in a warmer climate, may profoundly affect plant photosynthesis and production. However, the response of plant photosynthesis to biotic disturbance remains unclear, especially differences in response between insects and pathogens, which hinders the prediction of plant productivity in future climate. In this study, a meta-analysis approach was used to examine effects of insects and pathogens on photosynthetic rate per unit leaf area (Pn) and the associated characteristics from 115 studies. Our results showed that biotic disturbance significantly decreased Pn by 34.8% but increased Rd by 26.2%. Most of parameters associated with Pn were significantly reduced by biotic disturbance, including gs, Tr, photosynthetic pigments (e.g., a+b, a, and b), and chlorophyll fluorescence properties (Fv/Fm, qp). The disturbance type (insects vs pathogens) was the most important factor affecting the response of Pn, with a greater decrease in Pn by pathogens (-37.5%) than insects (-28.0%). The response ratio of Pn was positively correlated with that of gs and Tr for both insects and pathogens, while negatively with Ci and positively with Chl a+b, ΦPSII, and qp for only pathogens. In addition, the higher sensitivity of Pn to biotic disturbance in crop than non-crop plants poses a great challenge to agricultural system in the future. The weighted response ratio of Pn and relationships of Pn with other associated paramerters under insect and pathogen disturbance will facilitate vegetation models to integrate the effects of biotic disturbance on primary production, improving predicition of the ecosystem carbon cyling in combining with leaf area measurement.

Synthetic Life with Alternative Nucleic Acids as Genetic Materials.

DNA, the fundamental genetic polymer of all living organisms on Earth, can be chemically modified to embrace novel functions that do not exist in nature. The key chemical and structural parameters for genetic information storage, heredity, and evolution have been elucidated, and many xenobiotic nucleic acids (XNAs) with non-canonical structures are developed as alternative genetic materials in vitro. However, it is still particularly challenging to replace DNAs with XNAs in living cells. This review outlines some recent studies in which the storage and propagation of genetic information are achieved in vivo by expanding genetic systems with XNAs.

Production and Sensing of Butyrate in a Probiotic Escherichia coli Strain.

The short-chain fatty acid butyrate plays critical roles in human gut health, affecting immunomodulation, cell differentiation, and apoptosis, while also serving as the preferred carbon source for colon cells. In this work, we have engineered a model probiotic organism, Escherichia coli Nissle 1917 (EcN, serotype O6:K5:H1), to produce butyrate from genomic loci up to approximately 1 g/L (11 mM). Then, for real-time monitoring of butyrate production in cultures, we developed a high-throughput biosensor that responds to intracellular butyrate concentrations, with green fluorescent protein as the reporter. This work provides a foundation for studies of butyrate for therapeutic applications.

CRISPR-based curing and analysis of metabolic burden of cryptic plasmids in Escherichia coli Nissle 1917.

E. coli Nissle 1917 (EcN) has long been used as an over-the-counter probiotic and has shown potential to be used as a live biotherapeutic. It contains two stably replicating cryptic plasmids, pMUT1, and pMUT2, the function of which is unclear but the presence of which may increase the metabolic burden on the cell, particularly in the context of added recombinant plasmids. In this work, we present a clustered regularly interspaced short palindromic repeats-Cas9-based method of curing cryptic plasmids, producing strains cured of one or both plasmids. We then assayed heterologous protein production from three different recombinant plasmids in wild-type and cured EcN derivatives and found that production of reporter proteins was not significantly different across strains. In addition, we replaced pMUT2 with an engineered version containing an inserted antibiotic resistance reporter gene and demonstrated that the engineered plasmid was stable over 90 generations without selection. These findings have broad implications for the curing of cryptic plasmids and for stable heterologous expression of proteins in this host. Specifically, curing of cryptic plasmids may not be necessary for optimal heterologous expression in this host.

Fermentative Production of Phenolic Glucosides by Escherichia coli with an Engineered Glucosyltransferase from Rhodiola sachalinensis.

Three rosmarinic acid analogs produced by recombinant Escherichia coli, two xanthones from fungi and honokiol from plants, were explored as the substrates of E. coli harboring a glucosyltransferase mutant UGT73B6FS to generate phenolic glucosides. Six new and two known compounds were isolated from the fermentation broth of the recombinant strain of the feeding experiments, and the compounds were identified by spectroscopy. The biotransformation of rosmarinic acid analogs and xanthones into corresponding glucosides was presented for the first time. This study not only demonstrated the substrate flexibility of the glucosyltransferase mutant UGT73B6FS toward aromatic alcohols but also provided an effective and economical method to produce phenolic glucosides by fermentation circumventing the use of expensive precursor UDP-glucose.

Biosynthesis of phlorisovalerophenone and 4-hydroxy-6-isobutyl-2-pyrone in Escherichia coli from glucose.

BACKGROUND: Type III polyketide synthases (PKSs) contribute to the synthesis of many economically important natural products, which are typically produced by direct extraction from plants or synthesized chemically. For example, humulone and lupulone (Fig. 1a) in hops (Humulus lupulus) account for the characteristic bitter taste of beer and display multiple pharmacological effects. 4-Hydroxy-6-methyl-2-pyrone is a precursor of parasorboside contributing to insect and disease resistance of plant Gerbera hybrida, and was recently demonstrated to be a potential platform chemical. Fig. 1 Examples of phloroglucinols (a) and 2-pyrones (b) synthesized by type III PKS. PIBP phlorisobutyrophenone; PIVP phlorisovalerophenone; TAL 4-hydroxy-6-methyl-2-pyrone (triacetic acid lactone); HIPP 4-hydroxy-6-isopropyl-2-pyrone; HIBP 4-hydroxy-6-isobutyl-2-pyrone RESULTS: In this study, we achieved simultaneous biosynthesis of phlorisovalerophenone, a key intermediate of humulone biosynthesis and 4-hydroxy-6-isobutyl-2-pyrone in Escherichia coli from glucose. First, we constructed a biosynthetic pathway of isovaleryl-CoA via hydroxy-3-methylglutaryl CoA followed by dehydration, decarboxylation and reduction in E. coli. Subsequently, the type III PKSs valerophenone synthase or chalcone synthase from plants were introduced into the above E. coli strain, to produce phlorisovalerophenone and 4-hydroxy-6-isobutyl-2-pyrone at the highest titers of 6.4 or 66.5 mg/L, respectively. CONCLUSIONS: The report of biosynthesis of phlorisovalerophenone and 4-hydroxy-6-isobutyl-2-pyrone in E. coli adds a new example to the list of valuable compounds synthesized in E. coli from renewable carbon resources by type III PKSs.

De novo biosynthesis of Gastrodin in Escherichia coli.

Gastrodin, a phenolic glycoside, is the key ingredient of Gastrodia elata, a notable herbal plant that has been used to treat various conditions in oriental countries for centuries. Gastrodin is extensively used clinically for its sedative, hypnotic, anticonvulsive and neuroprotective properties in China. Gastrodin is usually produced by plant extraction or chemical synthesis, which has many disadvantages. Herein, we report unprecedented microbial synthesis of gastrodin via an artificial pathway. A Nocardia carboxylic acid reductase, endogenous alcohol dehydrogenases and a Rhodiola glycosyltransferase UGT73B6 transformed 4-hydroxybenzoic acid, an intermediate of ubiquinone biosynthesis, into gastrodin in Escherichia coli. Pathway genes were overexpressed to enhance metabolic flux toward precursor 4-hydroxybenzyl alcohol. Furthermore, the catalytic properties of the UGT73B6 toward phenolic alcohols were improved through directed evolution. The finally engineered strain produced 545mgl(-1) gastrodin in 48h. This work creates a new route to produce gastrodin, instead of plant extractions and chemical synthesis.

Production of salidroside in metabolically engineered Escherichia coli.

Salidroside (1) is the most important bioactive component of Rhodiola (also called as "Tibetan Ginseng"), which is a valuable medicinal herb exhibiting several adaptogenic properties. Due to the inefficiency of plant extraction and chemical synthesis, the supply of salidroside (1) is currently limited. Herein, we achieved unprecedented biosynthesis of salidroside (1) from glucose in a microorganism. First, the pyruvate decarboxylase ARO10 and endogenous alcohol dehydrogenases were recruited to convert 4-hydroxyphenylpyruvate (2), an intermediate of L-tyrosine pathway, to tyrosol (3) in Escherichia coli. Subsequently, tyrosol production was improved by overexpressing the pathway genes, and by eliminating competing pathways and feedback inhibition. Finally, by introducing Rhodiola-derived glycosyltransferase UGT73B6 into the above-mentioned recombinant strain, salidroside (1) was produced with a titer of 56.9 mg/L. Interestingly, the Rhodiola-derived glycosyltransferase, UGT73B6, also catalyzed the attachment of glucose to the phenol position of tyrosol (3) to form icariside D2 (4), which was not reported in any previous literatures.

Engineered short branched-chain acyl-CoA synthesis in E. coli and acylation of chloramphenicol to branched-chain derivatives.

Short branched-chain acyl-CoAs are important building blocks for a wide variety of pharmaceutically valuable natural products. Escherichia coli has been used as a heterologous host for the production of a variety of natural compounds for many years. In the current study, we engineered synthesis of isobutyryl-CoA and isovaleryl-CoA from glucose in E. coli by integration of the branched-chain α-keto acid dehydrogenase complex from Streptomyces avermitilis. In the presence of the chloramphenicol acetyltransferase (cat) gene, chloramphenicol was converted to both chloramphenicol-3-isobutyrate and chloramphenicol-3-isovalerate by the recombinant E. coli strains, which suggested successful synthesis of isobutyryl-CoA and isovaleryl-CoA. Furthermore, we improved the α-keto acid precursor supply by overexpressing the alsS gene from Bacillus subtilis and the ilvC and ilvD genes from E. coli and thus enhanced the synthesis of short branched-chain acyl-CoAs. By feeding 25 mg/L chloramphenicol, 2.96 ± 0.06 mg/L chloramphenicol-3-isobutyrate and 3.94 ± 0.06 mg/L chloramphenicol-3-isovalerate were generated by the engineered E. coli strain, which indicated efficient biosynthesis of short branched-chain acyl-CoAs. HPLC analysis showed that the most efficient E. coli strain produced 80.77 ± 3.83 nmol/g wet weight isovaleryl-CoA. To our knowledge, this is the first report of production of short branched-chain acyl-CoAs in E. coli and opens a way to biosynthesize various valuable natural compounds based on these special building blocks from renewable carbon sources.