Bacillus methanolicus: an emerging chassis for low-carbon biomanufacturing.

Bacillus methanolicus is a thermophilic methylotrophic bacterium that grows quickly on methanol in sea water-based media. It has been engineered for chemical bioproduction from methanol, but its efficiency needs improvement for industrialization. Synthetic biology approaches such as metabolic modeling and genome editing can reprogram B. methanolicus for low-carbon biomanufacturing.

Sustainable wheat straw pretreatment process by self-produced and cyclical crude lactic acid.

The purpose of this study was to investigate an environmentally friendly and recyclable pretreatment approach that would enhance the enzymatic digestibility of wheat straw. Wheat straw was pretreated using self-produced crude lactic acid obtained from enzymatic hydrolysate fermentation by Bacillus coagulans. Experimentally, crude lactic acid at low concentration could achieve a pretreatment effect comparable to that of commercial lactic acid. After pretreatment at 180 °C for 60 min with 2.0 % crude lactic acid, hemicellulose could be effectively separated and high recovery of cellulose was ensured, achieving cellulose recovery rate of 95.5 % and hemicellulose removal rate of 92.7 %. Excellent enzymatic hydrolysis was accomplished with a glucose yield of 99.7 %. Moreover, the crude lactic acid demonstrated acceptable pretreatment and enzymatic hydrolysis performance even after three repeated cycles. This not only effectively utilizes the pretreatment solution, but also offers insights into biomass pretreatment using other fermentable acids.

Biological Valorization of Lignin-Derived Aromatics in Hydrolysate to Protocatechuic Acid by Engineered Pseudomonas putida KT2440.

Alongside fermentable sugars, weak acids, and furan derivatives, lignocellulosic hydrolysates contain non-negligible amounts of lignin-derived aromatic compounds. The biological funnel of lignin offers a new strategy for the "natural" production of protocatechuic acid (PCA). Herein, Pseudomonas putida KT2440 was engineered to produce PCA from lignin-derived monomers in hydrolysates by knocking out protocatechuate 3,4-dioxygenase and overexpressing vanillate-O-demethylase endogenously, while acetic acid was used for cell growth. The sugar catabolism was further blocked to prevent the loss of fermentable sugar. Using the engineered strain, a total of 253.88 mg/L of PCA was obtained with a yield of 70.85% from corncob hydrolysate 1. The highest titer of 433.72 mg/L of PCA was achieved using corncob hydrolysate 2 without any additional nutrients. This study highlights the potential ability of engineered strains to address the challenges of PCA production from lignocellulosic hydrolysate, providing novel insights into the utilization of hydrolysates.

Promoting microbial fermentation in lignocellulosic hydrolysates by removal of inhibitors using MTES and PEI-modified chitosan-chitin nanofiber hybrid aerogel.

To further enhance the removal efficiency for furanic and phenolic compounds in lignocellulosic hydrolysates, a new detoxification strategy was proposed, which retained fermentable sugars and promoted the growth and metabolism of subsequent bacteria. The best adsorbent (P/M-CCA) was prepared by hybrid chitosan-chitin nanofiber, graft modification with polyethylenimine, and silanization with methyl triethoxylsilane in order. Taken corn cob hydrolysate as object, the removal rates of HMF and furfural were 85.1 % and 99.0 %, respectively. The removal rates of six out of nine phenolic inhibitors were 100 %, and the other three were more than 65 %. Even better, the retention rates of glucose and xylose were both 100 %. In contrast to no growth in undetoxified hydrolysates, Bacillus coagulans grew normally in detoxified hydrolysates, and lactic acid reached 19.1 g/L after 12 h fermentation. P/M-CCA achieves both removal of multiple inhibitors and retain sugars, which would promote the valorization of highly toxic lignocellulosic hydrolysates.

Achieving efficient and rapid high-solids enzymatic hydrolysis for producing high titer ethanol with the assistance of di-rhamnolipids.

High-solids enzymatic hydrolysis is the premise of obtaining high concentration ethanol by fermentation. In this study, corn stover was first pretreated with formic acid under mild conditions, and more than 70 % of xylan and lignin were removed within the first hour. 173.0 g/L glucose was achieved from total 30 % solid of the pretreated corn stover via fed-batch mode. Moreover, the glucose concentration rose to 194.5 g/L and the hydrolysis time was significantly reduced by 42.9 % with the addition of di-rhamnolipid. On this basis, 89.1 g/L ethanol was obtained by fermentation, and the presence of di-rhamnolipid had no negative effect on fermentation. The effective conversion of corn stover to high titer ethanol provides support for the conversion of stover to ethanol in industrial production.

ß-Galactosidase: a traditional enzyme given multiple roles through protein engineering.

ß-Galactosidases are crucial carbohydrate-active enzymes that naturally catalyze the hydrolysis of galactoside bonds in oligo- and disaccharides. These enzymes are commonly used to degrade lactose and produce low-lactose and lactose-free dairy products that are beneficial for lactose-intolerant people. ß-galactosidases exhibit transgalactosylation activity, and they have been employed in the synthesis of galactose-containing compounds such as galactooligosaccharides. However, most ß-galactosidases have intrinsic limitations, such as low transglycosylation efficiency, significant product inhibition effects, weak thermal stability, and a narrow substrate spectrum, which greatly hinder their applications. Enzyme engineering offers a solution for optimizing their catalytic performance. The study of the enzyme's structure paves the way toward explaining catalytic mechanisms and increasing the efficiency of enzyme engineering. In this review, the structure features of ß-galactosidases from different glycosyl hydrolase families and the catalytic mechanisms are summarized in detail to offer guidance for protein engineering. The properties and applications of ß-galactosidases are discussed. Additionally, the latest progress in ß-galactosidase engineering and the strategies employed are highlighted. Based on the combined analysis of structure information and catalytic mechanisms, the ultimate goal of this review is to furnish a thorough direction for ß-galactosidases engineering and promote their application in the food and dairy industries.

Addressing two major limitations in high-solids enzymatic hydrolysis by an ordered polyethylene glycol pre-incubated strategy: Rheological properties and lignin adsorption for enzyme.

High-solids enzymatic hydrolysis for biomass has currently received considerable interest. However, the solid effect during the process limits its economic feasibility. This work presented an ordered polyethylene glycol (PEG) pre-incubated strategy for enhancing the auxiliary effect of PEG in a high-solids enzymatic hydrolysis system. The substrate and enzyme were separately pre-incubated with PEG in this strategy. The ordered PEG pre-incubated strategies yielded a maximum glucose concentration of 166.6 g/L from 32 % (w/v) pretreated corncob with an enzymatic yield of 94.1 % by 72 h hydrolysis. Using this method, PEG not only lessened the lignin adsorption to cellulase but also altered particle rheological characteristics in the high-solids enzymatic hydrolysis system as a viscosity modifier. This study offered a new insight into the mechanism behind the PEG synergistic effect and would make it possible to achieve efficient high-solids loading hydrolysis in the commercial manufacture of cellulosic ethanol.

Antioxidants Improve the Proliferation and Efficacy of hUC-MSCs against H2O2-Induced Senescence.

Human umbilical cord mesenchymal stem cells (hUC-MSCs) are broadly applied in clinical treatment due to convenient accessibility, low immunogenicity, and the absence of any ethical issues involved. However, the microenvironment of inflammatory tissues may cause oxidative stress and induce senescence in transplanted hUC-MSCs, which will further reduce the proliferation, migration ability, and the final therapeutic effects of hUC-MSCs. Beta-nicotinamide mononucleotide (NMN) and coenzyme Q10 (CoQ10) are famous antioxidants and longevity medicines that could reduce intracellular reactive oxygen species levels by different mechanisms. In this study, hUC-MSCs were treated in vitro with NMN and CoQ10 to determine if they could reduce oxidative stress caused by hydrogen peroxide (H2O2) and recover cell functions. The effects of NMN and CoQ10 on the cell proliferation, the mRNA levels of the inflammatory cytokine TNFα and the anti-inflammatory cytokine IL10, and the differentiation and cell migration ability of hUC-MSCs before and after H2O2 treatment were investigated. The findings revealed that NMN and CoQ10 reduced H2O2-induced senescence and increased hUC-MSCs' proliferation in the late phase as passage 12 and later. The TNFα mRNA level of hUC-MSCs induced by H2O2 was significantly decreased after antioxidant treatment. NMN and CoQ10 all reduced the adipogenic differentiation ability of hUC-MSCs. CoQ10 improved the chondrogenic differentiation ability of hUC-MSCs. Furthermore, NMN was found to significantly enhance the migration ability of hUC-MSCs. Transcriptomic analysis revealed that NMN and CoQ10 both increased DNA repair ability and cyclin expression and downregulated TNF and IL-17 inflammatory signaling pathways, thereby contributing to the proliferative promotion of senecent stem cells and resistance to oxidative stress. These findings suggest that antioxidants can improve the survival and efficacy of hUC-MSCs in stem cell therapy for inflammation-related diseases.

Rapid adaptive evolution of Bacillus coagulans to undetoxified corncob hydrolysates for lactic acid production and new insights into its high phenolic degradation.

Here, an adapted Bacillus coagulans (Weizmannia coagulans) strain CC17B-1 was developed for lignocellulosic lactic acid production through a short and rapid adaptive laboratory evolution technique. Without any detoxification, two actual corn cob hydrolysates from the factory were effectively fermented to lactic acid within 60 h. Strain CC17B-1 is capable of degrading all nine determined phenolic compounds in the hydrolysate, with the only exception being vanillic acid. Notably, its tolerances for ferulic acid and p-coumaric acid are the highest doses reported in anaerobic microbes. A proposed degradation pathway showed that strain CC17B-1 could convert phenolic aldehydes to phenolic alcohol and then further degrade them completely. This work provides new ideas for the microbe phenolic degradation pathway and paves the way for industrial lactic acid production from lignocellulosic biomass.

In-depth investigation of formic acid pretreatment for various biomasses: Chemical properties, structural features, and enzymatic hydrolysis.

Formic acid pretreatment is a promising approach for fractionating biomass, and it has the advantages of efficient recycling and removal of hemicellulose and lignin. Biomass is one of the most plentiful resources on earth, yet its chemical structure differs significantly between woody and herbaceous biomass. The influence of formic acid pretreatment on the fractionation of woody and herbaceous biomasses, as well as changes in physical-chemical properties, was investigated in this study. The results indicated that formic acid is universal in the biorefinery of different biomass, however, herbaceous biomass had greater xylan and lignin removal than woody biomass (especially softwood). Formic acid pretreatment not only considerably improved the enzymatic efficiency of herbaceous biomass, but also had a good effect on the enzymatic efficiency of poplar. This study also found that the correlation between residual xylan content and enzymatic efficiency after pretreatment was much higher than that of lignin content.

Efficient biosynthesis of pinosylvin from lignin-derived cinnamic acid by metabolic engineering of Escherichia coli.

BACKGROUND: The conversion of lignin-derived aromatic monomers into valuable chemicals has promising potential to improve the economic competitiveness of biomass biorefineries. Pinosylvin is an attractive pharmaceutical with multiple promising biological activities. RESULTS: Herein, Escherichia coli was engineered to convert the lignin-derived standard model monomer cinnamic acid into pinosylvin by introducing two novel enzymes from the wood plant: stilbene synthase from Pinus pinea (PpSTS) and 4-Coumarate-CoA ligase from Populus trichocarpa (Ptr4CL4). The expression of Ptr4CL4 drastically improved the production of pinosylvin (42.5 ± 1.1 mg/L), achieving values 15.7-fold higher than that of Ptr4CL5 (another 4-Coumarate-CoA ligase from Populus trichocarpa) in the absence of cerulenin. By adjusting the expression strategy, the optimized engineered strain produced pinosylvin at 153.7 ± 2.2 mg/L with an extremely high yield of 1.20 ± 0.02 mg/mg cinnamic acid in the presence of cerulenin, which is 83.9% ± 1.17 of the theoretical yield. This is the highest reported pinosylvin yield directly from cinnamic acid to date. CONCLUSION: Our work highlights the feasibility of microbial production of pinosylvin from cinnamic acid and paves the way for converting lignin-related aromatics to valuable chemicals.

Unraveling the mechanism of furfural tolerance in engineered Pseudomonas putida by genomics.

As a dehydration product of pentoses in hemicellulose sugar streams derived from lignocellulosic biomass, furfural is a prevalent inhibitor in the efficient microbial conversion process. To solve this obstacle, exploiting a biorefinery strain with remarkable furfural tolerance capability is essential. Pseudomonas putida KT2440 (P. putida) has served as a valuable bacterial chassis for biomass biorefinery. Here, a high-concentration furfural-tolerant P. putida strain was developed via adaptive laboratory evolution (ALE). The ALE resulted in a previously engineered P. putida strain with substantially increased furfural tolerance as compared to wild-type. Whole-genome sequencing of the adapted strains and reverse engineering validation of key targets revealed for the first time that several genes and their mutations, especially for PP_RS19785 and PP_RS18130 [encoding ATP-binding cassette (ABC) transporters] as well as PP_RS20740 (encoding a hypothetical protein), play pivotal roles in the furfural tolerance and conversion of this bacterium. Finally, strains overexpressing these three striking mutations grew well in highly toxic lignocellulosic hydrolysate, with cell biomass around 9-, 3.6-, and two-fold improvement over the control strain, respectively. To our knowledge, this study first unravels the furan aldehydes tolerance mechanism of industrial workhorse P. putida, which provides a new foundation for engineering strains to enhance furfural tolerance and further facilitate the valorization of lignocellulosic biomass.

Valorization of cheese whey to lactobionic acid by a novel strain Pseudomonas fragi and identification of enzyme involved in lactose oxidation.

BACKGROUND: Efficient upgrading of inferior agro-industrial resources and production of bio-based chemicals through a simple and environmentally friendly biotechnological approach is interesting Lactobionic acid is a versatile aldonic acid obtained from the oxidation of lactose. Several microorganisms have been used to produce lactobionic acid from lactose and whey. However, the lactobionic acid production titer and productivity should be further improved to compete with other methods. RESULTS: In this study, a new strain, Pseudomonas fragi NL20W, was screened as an outstanding biocatalyst for efficient utilization of waste whey to produce lactobionic acid. After systematic optimization of biocatalytic reactions, the lactobionic acid productivity from lactose increased from 3.01 g/L/h to 6.38 g/L/h in the flask. In batch fermentation using a 3 L bioreactor, the lactobionic acid productivity from whey powder containing 300 g/L lactose reached 3.09 g/L/h with the yield of 100%. Based on whole genome sequencing, a novel glucose dehydrogenase (GDH1) was determined as a lactose-oxidizing enzyme. Heterologous expression the enzyme GDH1 into P. putida KT2440 increased the lactobionic acid yield by 486.1%. CONCLUSION: This study made significant progress both in improving lactobionic acid titer and productivity, and the lactobionic acid productivity from waste whey is superior to the ever reports. This study also revealed a new kind of aldose-oxidizing enzyme for lactose oxidation using P. fragi NL20W for the first time, which laid the foundation for further enhance lactobionic acid production by metabolic engineering.

Extending galactose-oxidation pathway of Pseudomonas putida for utilization of galactose-rich red macroalgae as sustainable feedstock.

Marine red macroalgae has attracted researchers' consideration as a non-lignocellulosic feedstock for microbial growth to produce biofuels and biochemical products. Gelidium amansii is representative galactose-rich red macroalgae biomass but studies on its galactose utilization are currently scarce. Herein, we engineered Pseudomonas putida KT2440 as a functional chassis for assimilation of galactose in addition to glucose in G. amansii hydrolysate. P. putida KT2440 was confirmed owning high ability to oxidize galactose to galactonate by glucose dehydrogenase. Thereafter galactose-oxidation pathway was extended by introducing galactonate transport and metabolism modules from Pseudomonas rhodesiae NL2019. The recombinant strains NL910 and NL911 were able to grow on galactose with high cell densities and growth rates, and simultaneously upgrade all red macroalgae streams, which is essential to develop a sustainable and cost-effective bioprocess for valorization of red macroalgae.

[Production of mucic acid from pectin-derived D-galacturonic acid: a review].

Mucic acid is a hexaric acid that can be biosynthesized by oxidation of D-galacturonic acid, which is the main constituent of pectin. The structure and properties of mucic acid are similar to that of glucaric acid, and can be widely applied in the preparation of important platform compounds, polymers and macromolecular materials. Pectin is a cheap and abundant renewable biomass resource, thus developing a process enabling production of mucic acid from pectin would be of important economic value and environmental significance. This review summarized the structure and hydrolysis of pectin, the catabolism and regulation of D-galacturonic acid in microorganisms, and the strategy for mucic acid production based on engineering of corresponding pathways. The future application of mucic acid are prospected, and future directions for the preparation of mucic acid by biological method are also proposed.

Removal of inhibitory furan aldehydes in lignocellulosic hydrolysates via chitosan-chitin nanofiber hybrid hydrogel beads.

To obtain fermentable sugars from lignocellulose, various inhibitors, especially furan aldehydes, are usually generated during the pretreatment process. These inhibitors are harmful to subsequent microbial growth and fermentation. In this study, a novel detoxification strategy was proposed to remove 5-hydroxymethylfurfural (HMF) and furfural while retaining glucose and xylose using self-prepared chitosan-chitin nanofiber hybrid hydrogel beads (C-CNBs). After C-CNBs treatment, the removal rates of HMF and furfural from sugarcane bagasse hydrolysates reached 63.1% and 68.4%, while the loss rates of glucose and xylose were only 6.3% and 8.2%, respectively. Two typical industrial strains grew well in monosaccharide-rich detoxified hydrolysates, with a specific growth rate at least 4.1 times that of undetoxified hydrolysates. Furthermore, adsorption mechanism analysis revealed that the Schiff base reaction and mesopore filling were involved in furan aldehyde adsorption. In total, C-CNBs provide an efficient and practical approach for the removal of furan aldehydes from lignocellulosic hydrolysates.

Efficient lactic acid production from dilute acid-pretreated lignocellulosic biomass by a synthetic consortium of engineered Pseudomonas putida and Bacillus coagulans.

BACKGROUND: Lignocellulosic biomass is an attractive and sustainable alternative to petroleum-based feedstock for the production of a range of biochemicals, and pretreatment is generally regarded as indispensable for its biorefinery. However, various inhibitors that severely hinder the growth and fermentation of microorganisms are inevitably produced during the pretreatment of lignocellulose. Presently, there are few reports on a single microorganism that can detoxify or tolerate toxic mixtures of pretreated lignocellulose hydrolysate while effectively transforming sugar components into valuable compounds. Alternatively, microbial coculture provides a simpler and more efficacious way to realize this goal by distributing metabolic functions among different specialized strains. RESULTS: In this study, a novel synthetic microbial consortium, which is composed of a responsible for detoxification bacterium engineered Pseudomonas putida KT2440 and a lactic acid production specialist Bacillus coagulans NL01, was developed to directly produce lactic acid from highly toxic lignocellulosic hydrolysate. The engineered P. putida with deletion of the sugar metabolism pathway was unable to consume the major fermentable sugars of lignocellulosic hydrolysate but exhibited great tolerance to 10 g/L sodium acetate, 5 g/L levulinic acid, 10 mM furfural and HMF as well as 2 g/L monophenol compound. In addition, the engineered strain rapidly removed diverse inhibitors of real hydrolysate. The degradation rate of organic acids (acetate, levulinic acid) and the conversion rate of furan aldehyde were both 100%, and the removal rate of most monoaromatic compounds remained at approximately 90%. With detoxification using engineered P. putida for 24 h, the 30% (v/v) hydrolysate was fermented to 35.8 g/L lactic acid by B. coagulans with a lactic acid yield of 0.8 g/g total sugars. Compared with that of the single culture of B. coagulans without lactic acid production, the fermentation performance of microbial coculture was significantly improved. CONCLUSIONS: The microbial coculture system constructed in this study demonstrated the strong potential of the process for the biosynthesis of valuable products from lignocellulosic hydrolysates containing high concentrations of complex inhibitors by specifically recruiting consortia of robust microorganisms with desirable characteristics and also provided a feasible and attractive method for the bioconversion of lignocellulosic biomass to other value-added biochemicals.

A comprehensive review on microbial production of 1,2-propanediol: micro-organisms, metabolic pathways, and metabolic engineering.

1,2-Propanediol is an important building block as a component used in the manufacture of unsaturated polyester resin, antifreeze, biofuel, nonionic detergent, etc. Commercial production of 1,2-propanediol through microbial biosynthesis is limited by low efficiency, and chemical production of 1,2-propanediol requires petrochemically derived routes involving wasteful power consumption and high pollution emissions. With the development of various strategies based on metabolic engineering, a series of obstacles are expected to be overcome. This review provides an extensive overview of the progress in the microbial production of 1,2-propanediol, particularly the different micro-organisms used for 1,2-propanediol biosynthesis and microbial production pathways. In addition, outstanding challenges associated with microbial biosynthesis and feasible metabolic engineering strategies, as well as perspectives on the future microbial production of 1,2-propanediol, are discussed.

[Biodegradation of furan aldehydes in lignocellulose hydrolysates].

Lignocellulose is the most abundant renewable organic carbon resource on earth. However, due to its complex structure, it must undergo a series of pretreatment processes before it can be efficiently utilized by microorganisms. The pretreatment process inevitably generates typical inhibitors such as furan aldehydes that seriously hinder the growth of microorganisms and the subsequent fermentation process. It is an important research field for bio-refining to recognize and clarify the furan aldehydes metabolic pathway of microorganisms and further develop microbial strains with strong tolerance and transformation ability towards these inhibitors. This article reviews the sources of furan aldehyde inhibitors, the inhibition mechanism of furan aldehydes on microorganisms, the furan aldehydes degradation pathways in microorganisms, and particularly focuses on the research progress of using biotechnological strategies to degrade furan aldehyde inhibitors. The main technical methods include traditional adaptive evolution engineering and metabolic engineering, and the emerging microbial co-cultivation systems as well as functional materials assisted microorganisms to remove furan aldehydes.

The key role of delignification in overcoming the inherent recalcitrance of Chinese fir for biorefining.

The enzymatic digestibility of softwood is hindered for its highly recalcitrant nature to enzymatic attack. In this study, the effects of dilute sulfuric acid pretreatment (DSAP), acidic sodium chlorite pretreatment (SCP), and their combined pretreatments (DSA-SCP and SC-DSAP) on Chinese fir sawdust were investigated, respectively. Results demonstrated that lignin was the most important obstacle, and digestibility increased linearly with lignin removal yield. Furthermore, the results revealed that the order of sequential pretreatment significantly affected the delignification, and hemicellulose should be removed first. Compared to SC-DSAP, DSA-SCP involving the hemicellulose-removal-first strategy exhibited higher delignification efficiency. DSA-SCP caused lignin removal of 92.3% and the enzymatic hydrolysis was high of 97.9%. Finally, a regression model with high reliability was established to quickly evaluate pretreatment process. In summary, this study highlighted the importance of delignification for saccharification of softwood and unveiled the effect of hemicellulose on delignification.