Efficient conversion of aromatic and phenylpropanoid alcohols to acids by the cascade biocatalysis of alcohol and aldehyde dehydrogenases.

Benzyl and phenylpropanoid acids are widely used in organic synthesis of fine chemicals, such as pharmaceuticals and condiments. However, biocatalysis of these acids has received less attention than chemical synthesis. One of the main challenges for biological production is the limited availability of alcohol dehydrogenases and aldehyde dehydrogenases. Environmental microorganisms are potential sources of these enzymes. In this study, 129 alcohol dehydrogenases and 42 aldehyde dehydrogenases from Corynebacterium glutamicum, Pseudomonas aeruginosa, and Bacillus subtilis were identified and explored with various benzyl and phenylpropanoid alcohol and aldehyde substrates, among which four alcohol dehydrogenases and four aldehyde dehydrogenases with broad substrate specificity and high catalytic activity were obtained. Moreover, a cascade whole-cell catalytic system including ADH-90, ALDH-40, and the NAD(P)H oxidase LreNox was established, which showed high efficiency in converting cinnamyl alcohol and p-methylbenzyl alcohol into the respective carboxylic acids. Remarkably, this biocatalytic system can be easily scaled up to gram-level production, facilitating preparation purposes.

Experimentally-calibrated estimation of CO2 removal potentials of enhanced weathering.

Enhanced weathering (EW) of (ultra)mafic rocks is widely considered as a promising option for carbon dioxide removal (CDR). However, accurately measuring its CDR potential remains unavailable due to sluggish weathering process. Previous models have estimated annual CDR potentials ranging from 1 to 95 Gt by 2100, with the maximum significantly exceeding the anthropogenic CO2 emissions in 2021 (approximately 41 Gt). This raises concerns that a misconception may arise, suggesting active mitigations of CO2 emissions might not be necessary. Herein, we address this issue by partitioning the CDR potential of EW into two components, flow-through and non-flow-through processes, and develop an experimentally-calibrated model to reduce discrepancies between previous theoretical and experimental weathering rates. Our model estimates the upper bound of CDR potentials to be 0.22 (±0.16) Gt annually and 17 (±13) Gt cumulatively by 2100, thereby emphasizing the significance and urgency to advance ultra-enhanced weathering strategies.

Metabolic Engineering for Efficient Production of Z,Z-Farnesol in E. coli.

Z,Z-farnesol (Z,Z-FOH), the all-cis isomer of farnesol, holds enormous potential for application in cosmetics, daily chemicals, and pharmaceuticals. In this study, we aimed to metabolically engineer Escherichia coli to produce Z,Z-FOH. First, we tested five Z,Z-farnesyl diphosphate (Z,Z-FPP) synthases that catalyze neryl diphosphate to form Z,Z-FPP in E. coli. Furthermore, we screened thirteen phosphatases that could facilitate the dephosphorylation of Z,Z-FPP to produce Z,Z-FOH. Finally, through site-directed mutagenesis of cis-prenyltransferase, the optimal mutant strain was able to produce 572.13 mg/L Z,Z-FOH by batch fermentation in a shake flask. This achievement represents the highest reported titer of Z,Z-FOH in microbes to date. Notably, this is the first report on the de novo biosynthesis of Z,Z-FOH in E. coli. This work represents a promising step toward developing synthetic E. coli cell factories for the de novo biosynthesis of Z,Z-FOH and other cis-configuration terpenoids.

A reusable, impurity-tolerant and noble metal-free catalyst for hydrocracking of waste polyolefins.

One-step conversion of low-purity polyolefins to value-added products without pretreatments represents a great opportunity for chemical recycling of waste plastics. However, additives, contaminants, and heteroatom-linking polymers tend to be incompatible with catalysts that break down polyolefins. Here, we disclose a reusable, noble metal-free and impurity-tolerant bifunctional catalyst, MoSx-Hbeta, for hydroconversion of polyolefins into branched liquid alkanes under mild conditions. The catalyst works for a wide scope of polyolefins, including different kinds of high-molecular weight polyolefins, polyolefins mixed with various heteroatom-linking polymers, contaminated polyolefins, and postconsumer polyolefins with/without cleaning under 250°C and 20 to 30 bar H2 in 6 to 12 hours. A 96% yield of small alkanes was successfully achieved even at a temperature as low as 180°C. These results demonstrate the great potentials of hydroconversion in practical use of waste plastics as a largely untapped carbon feedstock.

Diversification of phenolic glucosides by two UDP-glucosyltransferases featuring complementary regioselectivity.

BACKGROUND: Glucoside natural products have been showing great medicinal values and potentials. However, the production of glucosides by plant extraction, chemical synthesis, and traditional biotransformation is insufficient to meet the fast-growing pharmaceutical demands. Microbial synthetic biology offers promising strategies for synthesis and diversification of plant glycosides. RESULTS: In this study, the two efficient UDP-glucosyltransferases (UGTs) (UGT85A1 and RrUGT3) of plant origin, that are capable of recognizing phenolic aglycons, are characterized in vitro. The two UGTs show complementary regioselectivity towards the alcoholic and phenolic hydroxyl groups on phenolic substrates. By combining a developed alkylphenol bio-oxidation system and these UGTs, twenty-four phenolic glucosides are enzymatically synthesized from readily accessible alkylphenol substrates. Based on the bio-oxidation and glycosylation systems, a number of microbial cell factories are constructed and applied to biotransformation, giving rise to a variety of plant and plant-like O-glucosides. Remarkably, several unnatural O-glucosides prepared by the two UGTs demonstrate better prolyl endopeptidase inhibitory and/or anti-inflammatory activities than those of the clinically used glucosidic drugs including gastrodin, salidroside and helicid. Furthermore, the two UGTs are also able to catalyze the formation of N- and S-glucosidic bonds to produce N- and S-glucosides. CONCLUSIONS: Two highly efficient UGTs, UGT85A1 and RrUGT3, with distinct regioselectivity were characterized in this study. A group of plant and plant-like glucosides were efficiently synthesized by cell-based biotransformation using a developed alkylphenol bio-oxidation system and these two UGTs. Many of the O-glucosides exhibited better PEP inhibitory or anti-inflammatory activities than plant-origin glucoside drugs, showing significant potentials for new glucosidic drug development.

Modular engineering of E. coli coculture for efficient production of resveratrol from glucose and arabinose mixture.

Resveratrol, a valuable plant-derived polyphenolic compound with various bioactivities, has been widely used in nutraceutical industries. Microbial production of resveratrol suffers from metabolic burden and low malonyl-CoA availability, which is a big challenge for synthetic biology. Herein, we took advantage of coculture engineering and divided the biosynthetic pathway of resveratrol into the upstream and downstream strains. By enhancing the supply of malonyl-CoA via CRISPRi system and fine-tuning the expression intensity of the synthetic pathway genes, we significantly improved the resveratrol productivity of the downstream strain. Furthermore, we developed a resveratrol addiction circuit that coupled the growth of the upstream strain and the resveratrol production of the downstream strain. The bidirectional interaction stabilized the coculture system and increased the production of resveratrol by 74%. Moreover, co-utilization of glucose and arabinose by the coculture system maintained the growth advantage of the downstream strain for production of resveratrol throughout the fermentation process. Under optimized conditions, the engineered E. coli coculture system produced 204.80 mg/L of resveratrol, 12.8-fold improvement over monoculture system. This study demonstrates the promising potential of coculture engineering for efficient production of natural products from biomass.

Metabolic Division in an Escherichia coli Coculture System for Efficient Production of Kaempferide.

Kaempferide, a plant-derived natural flavonoid, exhibits excellent pharmacological activities with nutraceutical and medicinal applications in human healthcare. Efficient microbial production of complex flavonoids suffers from metabolic crosstalk and burden, which is a big challenge for synthetic biology. Herein, we identified 4'-O-methyltransferases and divided the artificial biosynthetic pathway of kaempferide into upstream, midstream, and downstream modules. By combining heterologous genes from different sources and fine-tuning the expression, we optimized each module for the production of kaempferide. Furthermore, we designed and evaluated four division patterns of synthetic labor in coculture systems by plug-and-play modularity. The linear division of three modules in a three-strain coculture showed higher productivity of kaempferide than that in two-strain cocultures. The U-shaped division by co-distributing the upstream and downstream modules in one strain led to the best performance of the coculture system, which produced 116.0 ± 3.9 mg/L kaempferide, which was 510, 140, and 50% higher than that produced by the monoculture, two-strain coculture, and three-strain coculture with the linear division, respectively. This is the first report of efficient de novo production of kaempferide in a robust Escherichia coli coculture. The strategy of U-shaped pathway division in the coculture provides a promising way for improving the productivity of valuable and complex natural products.

[Microbial synthesis of plant polyphenols].

Plant polyphenols are phenylpropanoid derivatives including phenolic acids, stilbenes, curcumins and flavonoids. These compounds display a variety of biological and pharmacological activities such as antioxidation, vasorelaxation, anti-coagulation, anti-inflammation, anti-tumor and anti-virus, conferring a huge application potential in the sectors of drugs, foods, cosmetics, and chemicals. Microorganisms have become important hosts for heterologous synthesis of natural products due to the advantages of fast growth, easiness of culture and industrial operation. In recent years, the development of synthetic biology has boosted the microbial synthesis of plant natural products, achieving substantial progress. In this review, we summarize the synthesis of plant polyphenols in engineered Escherichia coli, Saccharomyces cerevisiae and other microorganisms equipped with the designed biosynthetic pathways of polyphenols. We also discuss the optimization strategies such as precursor engineering, dynamic regulation, and co-cultivation to improve the production of polyphenols and propose future prospects for polyphenol pathway engineering.

Plasticity engineering of plant monoterpene synthases and application for microbial production of monoterpenoids.

Plant monoterpenoids with structural diversities have extensive applications in food, cosmetics, pharmaceuticals, and biofuels. Due to the strong dependence on the geographical locations and seasonal annual growth of plants, agricultural production for monoterpenoids is less effective. Chemical synthesis is also uneconomic because of its high cost and pollution. Recently, emerging synthetic biology enables engineered microbes to possess great potential for the production of plant monoterpenoids. Both acyclic and cyclic monoterpenoids have been synthesized from fermentative sugars through heterologously reconstructing monoterpenoid biosynthetic pathways in microbes. Acting as catalytic templates, plant monoterpene synthases (MTPSs) take elaborate control of the monoterpenoids production. Most plant MTPSs have broad substrate or product properties, and show functional plasticity. Thus, the substrate selectivity, product outcomes, or enzymatic activities can be achieved by the active site mutations and domain swapping of plant MTPSs. This makes plasticity engineering a promising way to engineer MTPSs for efficient production of natural and non-natural monoterpenoids in microbial cell factories. Here, this review summarizes the key advances in plasticity engineering of plant MTPSs, including the fundamental aspects of functional plasticity, the utilization of natural and non-natural substrates, and the outcomes from product isomers to complexity-divergent monoterpenoids. Furthermore, the applications of plasticity engineering for improving monoterpenoids production in microbes are addressed.

Combining Metabolic and Monoterpene Synthase Engineering for de Novo Production of Monoterpene Alcohols in Escherichia coli.

The monoterpene alcohols acyclic nerol and bicyclic borneol are widely applied in the food, cosmetic, and pharmaceutical industries. The emerging synthetic biology enables microbial production to be a promising alternative for supplying monoterpene alcohols in an efficient and sustainable approach. In this study, we combined metabolic and plant monoterpene synthase engineering to improve the de novo production of nerol and borneol in prene-overproducing Escherichia coli. We engineered the growth-orthogonal neryl diphosphate (NPP) as the universal precursor of monoterpene alcohol biosynthesis and coexpressed nerol synthase (GmNES) from Glycine max to generate nerol or coexpressed the truncated bornyl diphosphate synthase (LdtBPPS) from Lippia dulcis for borneol production. Further, through site-directed mutation of LdtBPPS based on the structural simulation, we screened multiple variants that markedly elevated the production of acyclic nerol or bicyclic borneol, of which the LdtBPPSS488T mutant outperformed the wild-type LdtBPPS on borneol synthesis and the LdtBPPSF612A variant was superior to GmNES on nerol production. Subsequently, we overexpressed the endogenous Nudix hydrolase NudJ to facilitate the dephosphorylation of precursors and boosted the production of nerol and borneol from glucose. Finally, after the optimization of the fermentation process, the engineered strain ENO2 produced 966.55 mg/L nerol, and strain ENB57 generated 87.20 mg/L borneol in a shake flask, achieving the highest reported titers of nerol and borneol in microbes to date. This work shows a combinatorial engineering strategy for microbial production of natural terpene alcohols.

Scalable direct N-methylation of drug-like amines using 12CO2/13CO2 by simple inorganic base catalysis.

With the growing urgency of potential catastrophic climate changes due to anthropogenic CO2 emissions, numerous efforts have been devoted to development of synthetic protocols using CO2 as a building block in organic reactions, but the general applicability to complex drug-like substrates remains a challenge. We develop a general protocol for scalable direct N-methylation of a wide-scope drug-like amines using CO2 and polymethylhydrosiloxane-a nontoxic, aerobically-stable hydrosilane considered as an industrial waste-via simple inorganic base catalysis. A rare application of the Sabatier principle in organic chemistry led to the discovery of cheap, nontoxic K3PO4 as an efficient catalyst. Preparations of a wide-scope drug-like amines with carbon-isotope label were also successfully achieved, enabling direct use of CO2 in studies of drug absorption, distribution, metabolism and excretion.