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.

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.

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.

Efficient biosynthesis of cinnamyl alcohol by engineered Escherichia coli overexpressing carboxylic acid reductase in a biphasic system.

BACKGROUND: Cinnamyl alcohol is not only a kind of flavoring agent and fragrance, but also a versatile chemical applied in the production of various compounds. At present, the preparation of cinnamyl alcohol depends on plant extraction and chemical synthesis, which have several drawbacks, including limited scalability, productivity and environmental impact. It is therefore necessary to develop an efficient, green and sustainable biosynthesis method. RESULTS: Herein, we constructed a recombinant Escherichia coli BLCS coexpressing carboxylic acid reductase from Nocardia iowensis and phosphopantetheine transferase from Bacillus subtilis. The strain could convert cinnamic acid into cinnamyl alcohol without overexpressing alcohol dehydrogenase or aldo-keto reductase. Severe product inhibition was found to be the key limiting factor for cinnamyl alcohol biosynthesis. Thus, a biphasic system was proposed to overcome the inhibition of cinnamyl alcohol via in situ product removal. With the use of a dibutyl phthalate/water biphasic system, not only was product inhibition removed, but also the simultaneous separation and concentration of cinnamyl alcohol was achieved. Up to 17.4 mM cinnamic acid in the aqueous phase was totally reduced to cinnamyl alcohol with a yield of 88.2%, and the synthesized cinnamyl alcohol was concentrated to 37.4 mM in the organic phase. This process also demonstrated robust performance when it was integrated with the production of cinnamic acid from L-phenylalanine. CONCLUSION: We developed an efficient one-pot two-step biosynthesis system for cinnamyl alcohol production, which opens up possibilities for the practical biosynthesis of natural cinnamyl alcohol at an industrial scale.

A versatile Pseudomonas putida KT2440 with new ability: selective oxidation of 5-hydroxymethylfurfural to 5-hydroxymethyl-2-furancarboxylic acid.

Currently, biotransformation of 5-hydroxymethylfurfural (HMF) into a series of high-value bio-based platform chemicals is massively studied. In this study, selective biooxidation of HMF to 5-hydroxymethyl-2-furancarboxylic acid (HMFCA) by Pseudomonas putida KT2440 with superior titer, yield, and productivity was reported. The biocatalytic performances of P. putida KT2440 were optimized separately. Under optimal conditions, 100% yield of HMFCA was obtained when HMF concentration was less than 150 mM, while the maximum concentration of 155 mM was achieved from 160 mM HMF in 12 h. P. putida KT2440 was highly tolerate to HMF, up to 190 mM. Besides, it was capable of selective oxidation of other furan aldehydes to the corresponding carboxylic acids with good yield of 100%. This study further demonstrates the potential of P. putida KT2440 as a biocatalyst for biomass conversion, as this strain has been proved the capacity to convert and utilize many kinds of biomass-derived sugars and ligin-derived aromatic compounds.

Enhanced biosynthesis of chiral phenyllactic acid from L-phenylalanine through a new whole-cell biocatalyst.

Phenyllactic acid (PLA) is a high-value compound, which was usually produced by lactic acid bacteria (LAB) as biocatalysts and glucose or phenylpyruvic acid (PPA) as starting materials for PLA synthesis in previous studies. However, the PLA produced using LAB is a racemic mixture. Besides, both glucose and PPA were unsatisfactory substrates, as the former could not produce high concentrations of PLA while the latter is not a renewable and green substrate. To overcome these drawbacks, in this study, a new biotransformation process was developed for chiral PLA production from L-phenylalanine via the intermediate PPA using recombinant Escherichia coli co-expressing L-amino acid deaminase, NAD-dependent L-lactate dehydrogenase or NAD-dependent D-lactate dehydrogenase, and formate dehydrogenase. After optimization, the recombinant E. coli produced L- and D-PLA at concentrations of 59.9 and 60.3 mM in 6 h, respectively. Hence, this process provides an effective and promising alternative method for chiral PLA production.

Efficient in situ separation and production of L-lactic acid by Bacillus coagulans using weak basic anion-exchange resin.

To get rid of the dependence on lactic acid neutralizer, a simple and economical approach for efficient in situ separation and production of L-lactic acid was established by Bacillus coagulans using weak basic anion-exchange resin. During ten tested resins, the 335 weak basic anion-exchange resins demonstrated the highest adsorption capacity and selectivity for lactic acid recovery. The adsorption study of the 335 resins for lactic acid confirmed that it is an efficient adsorbent under fermentation condition. Langmuir models gave a good fit to the equilibrium data at 50 °C and the maximum adsorption capacity for lactic acid by 335 resins was about 402 mg/g. Adsorption kinetic experiments showed that pseudo-second-order kinetics model gave a good fit to the adsorption rate. When it was used for in situ fermentation, the yield of L-lactic acid by B. coagulans CC17 was close to traditional fermentation and still maintained at about 82% even after reuse by ten times. These results indicated that in situ separation and production of L-lactic acid using the 335 resins were efficient and feasible. This process could greatly reduce the dosage of neutralizing agent and potentially be used in industry.

Characterization of an L-arabinose isomerase from Bacillus coagulans NL01 and its application for D-tagatose production.

BACKGROUND: L-arabinose isomerase (AI) is a crucial catalyst for the biotransformation of D-galactose to D-tagatose. In previous reports, AIs from thermophilic bacterial strains had been wildly researched, but the browning reaction and by-products formed at high temperatures restricted their applications. By contrast, AIs from mesophilic Bacillus strains have some different features including lower optimal temperatures and lower requirements of metallic cofactors. These characters will be beneficial to the development of a more energy-efficient and safer production process. However, the relevant data about the kinetics and reaction properties of Bacillus AIs in D-tagatose production are still insufficient. Thus, in order to support further applications of these AIs, a comprehensive characterization of a Bacillus AI is needed. RESULTS: The coding gene (1422 bp) of Bacillus coagulans NL01 AI (BCAI) was cloned and overexpressed in the Escherichia coli BL21 (DE3) strain. The enzymatic property test showed that the optimal temperature and pH of BCAI were 60 °C and 7.5 respectively. The raw purified BCAI originally showed high activity in absence of outsourcing metallic ions and its thermostability did not change in a low concentration (0.5 mM) of Mn(2+) at temperatures from 70 °C to 90 °C. Besides these, the catalytic efficiencies (k cat/K m) for L-arabinose and D-galactose were 8.7 mM(-1) min(-1) and 1.0 mM(-1) min(-1) respectively. Under optimal conditions, the recombinant E. coli cell containing BCAI could convert 150 g L(-1) and 250 g L(-1) D-galactose to D-tagatose with attractive conversion rates of 32 % (32 h) and 27 % (48 h). CONCLUSIONS: In this study, a novel AI from B. coagulans NL01was cloned, purified and characterized. Compared with other reported AIs, this AI could retain high proportions of activity at a broader range of temperatures and was less dependent on metallic cofactors such as Mn(2+). Its substrate specificity was understood deeply by carrying out molecular modelling and docking studies. When the recombinant E. coli expressing the AI was used as a biocatalyst, D-tagatose could be produced efficiently in a simple one-pot biotransformation system.

Genomic analysis of a xylose operon and characterization of novel xylose isomerase and xylulokinase from Bacillus coagulans NL01.

OBJECTIVE: To investigate the xylose operon and properties of xylose isomerase and xylulokinase in Bacillus coagulans that can effectively ferment xylose to lactic acid. RESULTS: The xylose operon is widely present in B. coagulans. It is composed of four putative ORFs. Novel xylA and xylB from B. coagulans NL01 were cloned and expressed in Escherichia coli. Sequence of xylose isomerase was more conserved than that of xylulokinase. Both the enzymes exhibited maximum activities at pH 7-8 but with a high temperature maximum of 80-85 °C, divalent metal ion was prerequisite for their activation. Xylose isomerase and xylulokinase were most effectively activated by Ni(2+) and Co(2+), respectively. CONCLUSIONS: Genomic analysis of xylose operon has contributed to understanding xylose metabolism in B. coagulans and the novel xylose isomerase and xylulokinase might provide new alternatives for metabolic engineering of other strains to improve their fermentation performance on xylose.

Kinetic characterization of recombinant Bacillus coagulans FDP-activated l-lactate dehydrogenase expressed in Escherichia coli and its substrate specificity.

Bacillus coagulans is a homofermentative, acid-tolerant and thermophilic sporogenic lactic acid bacterium, which is capable of producing high yields of optically pure lactic acid. The l-(+)-lactate dehydrogenase (l-LDH) from B. coagulans is considered as an ideal biocatalyst for industrial production. In this study, the gene ldhL encoding a thermostable l-LDH was amplified from B. coagulans NL01 genomic DNA and successfully expressed in Escherichia coli BL21 (DE3). The recombinant enzyme was partially purified and its enzymatic properties were characterized. Sequence analysis demonstrated that the l-LDH was a fructose 1,6-diphosphate-activated NAD-dependent lactate dehydrogenase (l-nLDH). Its molecular weight was approximately 34-36kDa. The Km and Vmax values of the purified l-nLDH for pyruvate were 1.91±0.28mM and 2613.57±6.43µmol(minmg)(-1), respectively. The biochemical properties of l-nLDH showed that the specific activity were up to 2323.29U/mg with optimum temperature of 55°C and pH of 6.5 in the pyruvate reduction and 351.01U/mg with temperature of 55°C and pH of 11.5 in the lactate oxidation. The enzyme also showed some activity in the absence of FDP, with a pH optimum of 4.0. Compared to other lactic acid bacterial l-nLDHs, the enzyme was found to be relatively stable at 50°C. Ca(2+), Ba(2+), Mg(2+) and Mn(2+) ions had activated effects on the enzyme activity, and the enzyme was greatly inhibited by Ni(2+) ion. Besides these, l-nLDH showed the higher specificity towards pyruvate esters, such as methyl pyruvate and ethyl pyruvate.

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.

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.

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.

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.

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.

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.

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.

Genome sequence of Pseudomonas stutzeri SDM-LAC, a typical strain for studying the molecular mechanism of lactate utilization.

Pseudomonas stutzeri SDM-LAC is an efficient lactate utilizer with various applications in biocatalysis. Here we present a 4.2-Mb assembly of its genome. The annotated four adjacent genes form a lactate utilization operon, which could provide further insights into the molecular mechanism of lactate utilization.

Lactate utilization is regulated by the FadR-type regulator LldR in Pseudomonas aeruginosa.

NAD-independent L-lactate dehydrogenase (l-iLDH) and NAD-independent D-lactate dehydrogenase (D-iLDH) activities are induced coordinately by either enantiomer of lactate in Pseudomonas strains. Inspection of the genomic sequences of different Pseudomonas strains revealed that the lldPDE operon comprises 3 genes, lldP (encoding a lactate permease), lldD (encoding an L-iLDH), and lldE (encoding a D-iLDH). Cotranscription of lldP, lldD, and lldE in Pseudomonas aeruginosa strain XMG starts with the base, C, that is located 138 bp upstream of the lldP ATG start codon. The lldPDE operon is located adjacent to lldR (encoding an FadR-type regulator, LldR). The gel mobility shift assays revealed that the purified His-tagged LldR binds to the upstream region of lldP. An XMG mutant strain that constitutively expresses D-iLDH and L-iLDH was found to contain a mutation in lldR that leads to an Ile23-to-serine substitution in the LldR protein. The mutated protein, LldR(M), lost its DNA-binding activity. A motif with a hyphenated dyad symmetry (TGGTCTTACCA) was identified as essential for the binding of LldR to the upstream region of lldP by using site-directed mutagenesis. L-Lactate and D-lactate interfered with the DNA-binding activity of LldR. Thus, L-iLDH and D-iLDH were expressed when the operon was induced in the presence of L-lactate or D-lactate.