DNA flexible chain modified MOFs as a versatile platform for chemoenzymatic cascade reactions in glucose catalysis.

Glucose oxidase (GOD) is widely used in the pharmaceutical industry, fermentation products and glucose biosensors for its essential role in catalyzing the conversion of glucose to gluconic acid and hydrogen peroxide (H2O2). As H2O2 is the by-product and will have a toxic effect on glucose oxidase, so introducing another enzyme that could consume H2O2 to form an enzymatic cascade reaction is a practical solution. However, this decision will lead to extra expenses and complex condition optimization such as the specific mass ratio, temperature and pH to improve the activity, stability and recyclability. Herein, we describe a mild and versatile strategy by anchoring GOD on carboxyl-activated MOF (Cu-TCPP(Fe)) through DNA-directed immobilization (DDI) technology. Robust MOF nanosheets were utilized as not only the carrier for the immobilization of GOD, but also a peroxidase-like catalyst for the decomposition of H2O2 to reduce its harmful impacts. In this work, the immobilized GOD retained 55.78% of its initial activity after being used for 7 times. More than 60% of the immobilized enzyme's catalytic activity was still maintained after 96 h of being stored at 50 â„ƒ. This study provides a new idea for preparing immobilized enzymes with enhanced stability, fast diffusion and high activity, which can be used in fields such as biocatalysis and biotechnology.

Modulated Arabinose Uptake and cAMP Signaling Synergistically Improve Glucose and Arabinose Consumption in Recombinant Yeast.

During the production of ethanol from lignocellulose-derived sugars, recombinant yeasts tend to utilize xylose and arabinose after glucose exhaustion. So far, many glucose-insensitive pentose transporters have been reported to counteract this phenomenon, but few studies have described intracellular factors. In this study, the combination of adaptive evolution, comparative genomics, and genetic complementation revealed that the hexokinase-deficient (Hxk0) arabinose-fermenting Saccharomyces cerevisiae requires the arabinose transporter variant Gal2-N376T and the mutations of guanine nucleotide exchange factor Cdc25 to overcome glucose restriction during arabinose assimilation. The results showed that the Hxk0 recombinant yeasts could lower the metabolic/physiological threshold of cell proliferation by downregulating the intracellular cAMP levels, resulting in smaller cells and increased arabinose assimilation under glucose restriction. In the medium containing 80 g/L glucose and 20 g/L arabinose, the evolved strain restoring the hexokinase activity completed fermentation at 22 h, compared to 24 h for the parental strain. Overall, the experimental results provide new insights into glucose repression of biorefinery yeasts.

Trace elements' deficiency in energy production through methanogenesis process: Focus on the characteristics of organic solid wastes.

Excessive or insufficient supplementation of trace elements (TEs) limits the progression of anaerobic digestion. The main reason for this is the lack of sufficient understanding of digestion substrate characteristics, which significantly affects the demand for TEs. In this review, the relationship between TEs requirements and substrate characteristics is discussed. We mainly focus on three aspects. 1) The basis for TE optimization and existing problems: The optimization of TEs often based on the total solids (TS) or volatile solids (VS) of substrates, does not fully consider substrate characteristics. 2) TE deficiency mechanisms for different types of substrates: nitrogen-rich, sulfur-rich, TE-poor, and easily hydrolyzed substrates are the four main types of substrates. The mechanisms underlying TEs deficiency in the different substrates are investigated. 3) Regulation of TE bioavailability: characteristics of substrates affect digestion parameters, which disturb the bioavailability TE. Therefore, methods for regulating bioavailability of TEs are discussed.

Reversible Adsorption and Detachment of Saccharomyces cerevisiae on Thermoresponsive Poly(N-isopropylacrylamide)-Grafted Fibers for Continuous Immobilized Fermentation.

Biofilm-mediated continuous fermentation with cells immobilized has gained much attention in recent years. In this study, thermoresponsive poly(N-isopropylacrylamide)-grafted cotton fibers (PNIPAM-CF) were prepared via an improved surface-initiated atom transfer radical polymerization. The modification process imparted switchable wettability to the surface while maintaining the thermal stability and biocompatibility of the CF. During the ethanol transformation, the rapid, reversible cell adsorption and detachment of Saccharomyces cerevisiae were performed through the modulation of wettability, displaying the enhancement of immobilized biomass and immobilization efficiency from 2.20 g/L and 59.43% to 2.81 g/L and 93.32%, respectively. Moreover, the biofilm adsorption matched well with the Freundlich model, indicating that multilayer adhesion was the main mode of biofilm formation. Based on the accumulation of the biofilm, the fabrication and utilization of PNIPAM-CF improved the efficiency of continuous immobilized fermentation, making the ethanol production reach 26.34 g/L in the sixth batch of fermentation. Meanwhile, wettability regulation further enhanced the reusability of the carrier. Therefore, the findings of this study revealed that the application of smart materials in cell immobilization systems had broad prospects for achieving sustainable and continuous catalysis.

Continuous Production of Human Epidermal Growth Factor Using Escherichia coli Biofilm.

Increasing demand for recombinant proteins necessitates efficient protein production processes. In this study, a continuous process for human epidermal growth factor (hEGF) secretion by Escherichia coli was developed by taking advantage of biofilm formation. Genes bcsB, fimH, and csgAcsgB that have proved to facilitate biofilm formation and some genes moaE, yceA, ychJ, and gshB potentially involved in biofilm formation were examined for their effects on hEGF secretion as well as biofilm formation. Finally, biofilm-based fermentation processes were established, which demonstrated the feasibility of continuous production of hEGF with improved efficiency. The best result was obtained from ychJ-disruption that showed a 28% increase in hEGF secretion over the BL21(DE3) wild strain, from 24 to 32 mg/L. Overexpression of bcsB also showed great potential in continuous immobilized fermentation. Overall, the biofilm engineering here represents an effective strategy to improve hEGF production and can be adapted to produce more recombinant proteins in future.

Mycolicibacterium cell factory for the production of steroid-based drug intermediates.

Steroid-based drugs have been developed as the second largest medical category in pharmaceutics. The well-established route of steroid industry includes two steps: the conversion of natural products with a steroid framework to steroid-based drug intermediates and the synthesis of varied steroid-based drugs from steroid-based drug intermediates. The biosynthesis of steroid-based drug intermediates from phytosterols by Mycolicibacterium cell factories bypasses the potential undersupply of diosgenin in the traditional steroid chemical industry. Moreover, the biosynthesis route shows advantages on multiple steroid-based drug intermediate products, more ecofriendly processes, and consecutive reactions carried out in one operation step and in one pot. Androsta-4-ene-3,17-dione (AD), androsta-1,4-diene-3,17-dione (ADD) and 9-hydroxyandrostra-4-ene-3,17-dione (9-OH-AD) are the representative steroid-based drug intermediates synthesized by mycolicibacteria. Other steroid metabolites of mycolicibacteria, like 4-androstene-17ß-ol-3-one (TS), 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC), 22-hydroxy-23,24-bisnorchol-1,4-diene-3-one (1,4-HBC), 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one (9-OH-HBC), 3aα-H-4α-(3'-propionic acid)-7aß-methylhexahydro-1,5-indanedione (HIP) and 3aα-H-4α-(3'-propionic acid)-5α-hydroxy-7aß-methylhexahydro-1-indanone-δ-lactone (HIL), also show values as steroid-based drug intermediates. To improve the bio-production efficiency of the steroid-based drug intermediates, mycolicibacterial strains and biotransformation processes have been continuously studied in the past decades. Many mycolicibacteria that accumulate steroid drug intermediates have been isolated, and subsequently optimized by conventional mutagenesis and genetic engineering. Especially, with the clarification of the mycolicibacterial steroid metabolic pathway and the developments on gene editing technologies, rational design is becoming an important measure for the construction and optimization of engineered mycolicibacteria strains that produce steroid-based drug intermediates. Hence, by reviewing researches in the past two decades, this article updates the overall process of steroid metabolism in mycolicibacteria and provides comprehensive schemes for the rational construction of mycolicibacterial strains that accumulate steroid-based drug intermediates. In addition, the special strategies for the bioconversion of highly hydrophobic steroid in aqueous media are discussed as well.

Recent Advances in Producing Sugar Alcohols and Functional Sugars by Engineering Yarrowia lipolytica.

The sugar alcohols and functional sugars have wide applications in food, pharmaceutical, and chemical industries. However, the smaller quantities of natural occurring sugar alcohols and functional sugars restricted their applications. The enzymatic and whole-cell catalyst production is emerging as the predominant alternatives. The properties of Yarrowia lipolytica make it a promising sugar alcohol and functional sugar producer. However, there are still some issues to be resolved. As there exist reviews about the chemical structures, physicochemical properties, biological functions, applications, and biosynthesis of sugar alcohols and/or functional sugars in Y. lipolytica, this mini review will not only update the recent advances in enzymatic and microbial production of sugar alcohols (erythritol, D-threitol, and xylitol) and functional sugars (isomaltulose, trehalose, fructo-oligosaccharides, and galacto-oligosaccharides) by using recombinant Y. lipolytica but also focus on the studies of gene discovery, pathway engineering, expanding substrate scope, bioprocess engineering, and novel breeding methods to resolve the aforementioned issues.

Optimization of n-butanol synthesis in Lactobacillus brevis via the functional expression of thl, hbd, crt and ter.

N-butanol is an important chemical and can be naturally synthesized by Clostridium species; however, the poor n-butanol tolerance of Clostridium impedes the further improvement in titer. In this study, Lactobacillus brevis, which possesses a higher butanol tolerance, was selected as host for heterologous butanol production. The Clostridium acetobutylicum genes thl, hbd, and crt which encode thiolase, ß-hydroxybutyryl-CoA dehydrogenase, and crotonase, and the Treponema denticola gene ter, which encodes trans-enoyl-CoA reductase were cloned into a single plasmid to express the butanol synthesis pathway in L. brevis. A titer of 40 mg/L n-butanol was initially achieved with plasmid pLY15-opt, in which all pathway genes are codon-optimized. A titer of 450 mg/L of n-butanol was then synthesized when ter was further overexpressed in this pathway. The role of metabolic flux was reinforced with pLY15, in which only the ter gene was codon-optimized, which greatly increased the n-butanol titer to 817 mg/L. Our strategy significantly improved n-butanol synthesis in L. brevis and the final titer is the highest achieved amongst butanol-tolerant lactic acid bacteria.

[Transport Characteristics of Air Pollution in Tianjin Based on Weather Background].

Regional transport is an important factor when considering the prevention and control of air pollution. The aim of this study was to provide support for the joint prevention and control of air pollution in the Beijing-Tianjin-Hebei region. With a focus on an analysis of the relationship between regional transport and meteorological conditions based on the weather background, an atmospheric chemical model was developed to quantitatively estimate the impact of regional transport on Tianjin from October 2016 to September 2017. The results showed that the contribution percentage of regional transport in cities in plains in the Beijing-Tianjin-Hebei region was significantly higher than in cities in mountains. The local contribution of PM2.5 in the Tianjin area was 62.9% and the contribution of regional transport was 37.1%. This was mainly affected by transmissions of Chanzhou, Langfang, central and southern Hebei, Beijing, Tanshan, and Shandong. Regional transport was the most significant from April to June, the weakest from July to August, and the highest contributor to local emissions. Regional transport was closely related to weather situation, wind field, precipitation, and other meteorological conditions. Post-high pressure and pre-frontal low pressure were the two types of pollution weather with the highest proportion in regional transport, and the impact of air pollution transport under the southwest wind, westerly wind and south wind was the most apparent. Wind speed of 2-3 m·s-1 was beneficial to the regional transport of PM2.5, and precipitation above 5 mm will effectively reduce the regional transport of air pollutants. For different pollution types and heavy pollution stages, the contribution of regional transport was the most apparent in light pollution weather, being 20.5% higher than the average. The heavy pollution weather was controlled by static stable air mass, and because of the migration of high PM2.5 concentrations, pollution air mass in the surrounding area had a significant impact on the accumulation of pollution and transport in the region. The contribution ratio of PM2.5 transport in the heavy pollution period was more than the average and was approximately 10% and 15% higher. In the process of heavy pollution, the proportion of transport contribution in the initial accumulation stage and peak stage were higher than in other periods, and 14.5% and 19.5% higher than in the outbreak stage. The contribution of local emissions in the outbreak stage was more significant, being 9.9% higher than average.

Metabolic Engineering and Adaptive Evolution of Clostridium beijerinckii To Increase Solvent Production from Corn Stover Hydrolysate.

The production of acetone-butanol-ethanol by solventogenic Clostridium using lignocellulosic biomass can be a potential alternative to petroleum-based butanol. However, previous studies on nondetoxified lignocellulose hydrolysate could not provide better results when compared to those in synthetic medium. In this study, we engineered the pentose pathway of Clostridium beijerinckii NCIMB 8052, which was then subjected to adaptive laboratory evolution in the gradient mixture of synthetic medium and pretreated corn stover enzymatic hydrolysate (CSH) prepared according to the National Renewable Energy Laboratory (NREL) standard. The final resultant strain CIBTS1274A produced 20.7 g/L of total solvents in NREL CSH diluted to 6% initial total sugars, supplemented with ammonium acetate. This performance was comparable with that of corn-based butanol. In addition, this strain was successfully used in the scale-up operation using nondetoxified corn stover and corncob hydrolysate at Lignicell Refining Biotechnologies Ltd., which once was the only commercial biobutanol industry in the world.

Consolidated bioprocessing for butanol production of cellulolytic Clostridia: development and optimization.

Butanol is an important bulk chemical, as well as a promising renewable gasoline substitute, that is commonly produced by solventogenic Clostridia. The main cost of cellulosic butanol fermentation is caused by cellulases that are required to saccharify lignocellulose, since solventogenic Clostridia cannot efficiently secrete cellulases. However, cellulolytic Clostridia can natively degrade lignocellulose and produce ethanol, acetate, butyrate and even butanol. Therefore, cellulolytic Clostridia offer an alternative to develop consolidated bioprocessing (CBP), which combines cellulase production, lignocellulose hydrolysis and co-fermentation of hexose/pentose into butanol in one step. This review focuses on CBP advances for butanol production of cellulolytic Clostridia and various synthetic biotechnologies that drive these advances. Moreover, the efforts to optimize the CBP-enabling cellulolytic Clostridia chassis are also discussed. These include the development of genetic tools, pentose metabolic engineering and the improvement of butanol tolerance. Designer cellulolytic Clostridia or consortium provide a promising approach and resource to accelerate future CBP for butanol production.

Enhanced solvent production by metabolic engineering of a twin-clostridial consortium.

The efficient fermentative production of solvents (acetone, n-butanol, and ethanol) from a lignocellulosic feedstock using a single process microorganism has yet to be demonstrated. Herein, we developed a consolidated bioprocessing (CBP) based on a twin-clostridial consortium composed of Clostridium cellulovorans and Clostridium beijerinckii capable of producing cellulosic butanol from alkali-extracted, deshelled corn cobs (AECC). To accomplish this a genetic system was developed for C. cellulovorans and used to knock out the genes encoding acetate kinase (Clocel_1892) and lactate dehydrogenase (Clocel_1533), and to overexpress the gene encoding butyrate kinase (Clocel_3674), thereby pulling carbon flux towards butyrate production. In parallel, to enhance ethanol production, the expression of a putative hydrogenase gene (Clocel_2243) was down-regulated using CRISPR interference (CRISPRi). Simultaneously, genes involved in organic acids reassimilation (ctfAB, cbei_3833/3834) and pentose utilization (xylR, cbei_2385 and xylT, cbei_0109) were engineered in C. beijerinckii to enhance solvent production. The engineered twin-clostridia consortium was shown to decompose 83.2g/L of AECC and produce 22.1g/L of solvents (4.25g/L acetone, 11.5g/L butanol and 6.37g/L ethanol). This titer of acetone-butanol-ethanol (ABE) approximates to that achieved from a starchy feedstock. The developed twin-clostridial consortium serves as a promising platform for ABE fermentation from lignocellulose by CBP.

CRISPR-based genome editing and expression control systems in Clostridium acetobutylicum and Clostridium beijerinckii.

Solventogenic clostridia are important industrial microorganisms that produce various chemicals and fuels. Effective genetic tools would facilitate physiological studies aimed both at improving our understanding of metabolism and optimizing solvent productivity through metabolic engineering. Here we have developed an all-in-one, CRISPR-based genome editing plasmid, pNICKclos, that can be used to achieve successive rounds of gene editing in Clostridium acetobutylicum ATCC 824 and Clostridium beijerinckii NCIMB 8052 with efficiencies varying from 6.7% to 100% and 18.8% to 100%, respectively. The plasmid specifies the requisite target-specific guide RNA, the gene encoding the Streptococcus pyogenes Cas9 nickase and the genome editing template encompassing the gene-specific homology arms. It can be used to create single target mutants within three days, with a further two days required for the curing of the pNICKclos plasmid ready for a second round of mutagenesis. A S. pyogenes dCas9-mediated gene regulation control system, pdCASclos, was also developed and used in a CRISPRi strategy to successfully repress the expression of spo0A in C. acetobutylicum and C. beijerinckii. The combined application of the established high efficiency CRISPR-Cas9 based genome editing and regulation control systems will greatly accelerate future progress in the understanding and manipulation of metabolism in solventogenic clostridia.

Multiple-site genetic modifications in Escherichia coli using lambda-Red recombination and I-SceI cleavage.

OBJECTIVES: Genetic modifications to bacterial chromosomes are important for research; recently we reported a two-plasmid system for single locus modification in Escherichia coli and an improved method for simultaneous multiple-loci modification is needed. RESULTS: An intermediate bacterial strain was generated with different resistance marker genes flanked by I-SceI recognition sites at multiple target loci. Then a donor plasmid carrying several alleles with desired modifications was transformed into the intermediate strain together with a bifunctional helper plasmid encoding λ-Red recombinase and I-SceI endonuclease. I-SceI would induce double-strand breaks (DSBs) in the chromosome and λ-Red would induce recombination between chromosome DSBs and allele fragments from the donor plasmid, resulting in genomic modifications. CONCLUSIONS: This method has been used to successfully perform three different loci modifications simultaneously.

Current status and prospects of industrial bio-production of n-butanol in China.

n-Butanol is an important bulk chemical. Commercial fermentative production of n-butanol has been applied more than 100 years ago but is currently more costly than production from propylene and syngas. Renewed interest in biobutanol as a biofuel has spurred technological advances to the fermentation process. This article reviewed the recent status including the commercialization, pilot production and R&D activities of n-butanol fermentation in China. Long-term bio-production of n-butanol as a next generation biofuel and biochemical from biomass waste and steel mill off-gas needs technology breakthroughs and more environmental concerns from policymakers.

I-SceI-mediated scarless gene modification via allelic exchange in Clostridium.

Although gene disruption in Clostridium spp. with the TargeTron technology is much more effective than single-crossover integration, it cannot achieve gene modification via allelic exchange. Here, we developed a targeted, nonpolar, scarless gene modification system based on the I-SceI endonuclease. First, a replicative plasmid containing homology arms on either side of the target sequence and I-SceI recognition sites was integrated into the Clostridium chromosome, resulting in single-crossover integrants containing a mutant allele. Second, the cells were transformed with plasmids containing the synthetic gene (sceC) encoding the I-SceI enzyme, resulting in double-stranded breaks at the I-SceI recognition sites, which stimulated homologous recombination and yielded double-crossover mutants. Application of the method was demonstrated by deleting two genes (adc and glcG) from C. acetobutylicum ATCC 824 and one gene (adc) from C. beijerinckii NCIMB 8052, and by introducing point mutations into xylR of C. beijerinckii NCIMB 8052. The double-crossover mutants displayed similar fermentation phenotypes to those constructed with the TargeTron technology.

High-efficiency scarless genetic modification in Escherichia coli by using lambda red recombination and I-SceI cleavage.

Genetic modifications of bacterial chromosomes are important for both fundamental and applied research. In this study, we developed an efficient, easy-to-use system for genetic modification of the Escherichia coli chromosome, a two-plasmid method involving lambda Red (λ-Red) recombination and I-SceI cleavage. An intermediate strain is generated by integration of a resistance marker gene(s) and I-SceI recognition sites in or near the target gene locus, using λ-Red PCR targeting. The intermediate strain is transformed with a donor plasmid carrying the target gene fragment with the desired modification flanked by I-SceI recognition sites, together with a bifunctional helper plasmid for λ-Red recombination and I-SceI endonuclease. I-SceI cleavage of the chromosome and the donor plasmid allows λ-Red recombination between chromosomal breaks and linear double-stranded DNA from the donor plasmid. Genetic modifications are introduced into the chromosome, and the placement of the I-SceI sites determines the nature of the recombination and the modification. This method was successfully used for cadA knockout, gdhA knock-in, seamless deletion of pepD, site-directed mutagenesis of the essential metK gene, and replacement of metK with the Rickettsia S-adenosylmethionine transporter gene. This effective method can be used with both essential and nonessential gene modifications and will benefit basic and applied genetic research.