BACKGROUND: Polyketide synthases (PKSs) are classified into three types based on their enzyme structures. Among them, type III PKSs, catalyzing the iterative condensation of malonyl-coenzyme A (CoA) with a CoA-linked starter molecule, are important synthases of valuable natural products. However, low efficiency and byproducts formation often limit their applications in recombinant overproduction.
RESULTS: Herein, a rapid growth selection system is designed based on the accumulation and derepression of toxic acyl-CoA starter molecule intermediate products, which could be potentially applicable to most type III polyketides biosynthesis. This approach is validated by engineering both chalcone synthases (CHS) and host cell genome, to improve naringenin productions in Escherichia coli. From directed evolution of key enzyme CHS, beneficial mutant with ~ threefold improvement in capability of naringenin biosynthesis was selected and characterized. From directed genome evolution, effect of thioesterases on CHS catalysis is first discovered, expanding our understanding of byproduct formation mechanism in type III PKSs. Taken together, a whole-cell catalyst producing 1082 mg L-1 naringenin in flask with E value (evaluating product specificity) improved from 50.1% to 96.7% is obtained.
CONCLUSIONS: The growth selection system has greatly contributed to both enhanced activity and discovery of byproduct formation mechanism in CHS. This research provides new insights in the catalytic mechanisms of CHS and sheds light on engineering highly efficient heterologous bio-factories to produce naringenin, and potentially more high-value type III polyketides, with minimized byproducts formation.

UNASSIGNED: 2-Amino-1-methyl-6-phenylimidazole [4,5-b] pyridine (PhIP), a heterocyclic amine (HAA), is found in meat products heated at high temperatures. However, PhIP is a mutagenic and potential carcinogenic compound. Cassiae semen, a type of medicine and food homology plant, is abundant in China and has been less applied for inhibiting heterocyclic amines.
UNASSIGNED: To investigate the inhibitory effect of cassiae semen extract on PhIP formation within a model system and elucidate the inhibitory mechanism, an ultrasonic-assisted method with 70% ethanol was used to obtain cassiae semen extract, which was added to a model system (0.6 mmol of phenylalanine: creatinine, 1:1). PhIP was analyzed by LC-MS to determine inhibitory effect. The byproducts of the system and the mechanism of PhIP inhibition were verified by adding the extract to a model mixture of phenylacetaldehyde, phenylacetaldehyde and creatinine.
UNASSIGNED: The results indicated that PhIP production decreased as the concentration of cassiae semen extract increased, and the highest inhibition rate was 91.9%. Byproduct (E), with a mass-charge ratio of m/z 199.9, was detected in the phenylalanine and creatinine model system but was not detected in the other systems. The cassiae semen extract may have reacted with phenylalanine to produce byproduct (E), which prevented the degradation of phenylalanine by the Strecker reaction to produce phenylacetaldehyde.
UNASSIGNED: Cassiae semen extract consumed phenylalanine, which is the precursor for PhIP, thus inhibiting the formation of phenylacetaldehyde and ultimately inhibiting PhIP formation. The main objective of this study was to elucidate the mechanism by which cassiae semen inhibit PhIP formation and establish a theoretical and scientific foundation for practical control measures.

The effects of seaweed cellulose (SC) on high fat-sugar diet (HFSD)-induced glucolipid metabolism disorders in mice and potential mechanisms were investigated. SC was isolated from dealginated residues of giant kelp (Macrocystis pyrifera), with a crystallinity index of 85.51 % and an average particle size of 678.2 nm. Administering SC to C57BL/6 mice at 250 or 500 mg/kg BW/day via intragastric gavage for six weeks apparently inhibited the development of HFSD-induced obesity, dyslipidemia, insulin resistance, oxidative stress and liver damage. Notably, SC intervention partially restored the structure and composition of the gut microbiota altered by the HFSD, substantially lowering the Firmicutes to Bacteroidetes ratio, and greatly increasing the relative abundance of Lactobacillus, Bifidobacterium, Oscillospira, Bacteroides and Akkermansia, which contributed to improved short-chain fatty acid (SCFA) production. Supplementing with a higher dose of SC led to more significant increases in total SCFA (67.57 %), acetate (64.56 %), propionate (73.52 %) and butyrate (66.23 %) concentrations in the rectal contents of HFSD-fed mice. The results indicated that highly crystalline SC microparticles could modulate gut microbiota dysbiosis and ameliorate HFSD-induced obesity and related metabolic syndrome in mice. Furthermore, particle size might have crucial impact on the prebiotic effects of cellulose as insoluble dietary fiber.

Currently, levan is attracting attention due to its promising applications in the food and biomedical fields. Levansucrase synthesizes levan by polymerizing the fructosyl unit in sucrose. However, a large amount of the byproduct glucose is produced during this process. In this paper, an engineered oleaginous yeast (Yarrowia lipolytica) strain was constructed using a surface display plasmid containing the LevS gene of Gluconobacter sp. MP2116. The levansucrase activity of the engineered yeast strain reached 327.8 U/g of cell dry weight. The maximal levan concentration (58.9 g/l) was achieved within 156 h in the 5-liter fermentation. Over 81.2 % of the sucrose was enzymolyzed by the levansucrase, and the byproduct glucose was converted to 21.8 g/l biomass with an intracellular oil content of 25.5 % (w/w). The obtained oil was comprised of 91.3 % long-chain fatty acids (C16-C18). This study provides new insight for levan production and comprehensive utilization of the byproduct in levan biosynthesis.

The ubiquitous occurrence of microplastics in water and wastewater is a growing concern. In this study, the chemical transformation and organic release of virgin and UV-aged thermoplastic polyurethane (TPU) polymers during chlorination were investigated. As compared to virgin TPU polymer, the UV-aged TPU polymer exhibited high chlorine reactivity with noticeable destruction on its surface functional groups after chlorination, which could be ascribed to the UV-induced activation of hard segment of TPU backbone and increased contact area. The concentrations of leached organics increased by 1.6-fold with obviously high abundances of low-molecular-weight components. Additives, monomers, compounds relating to TPU chain extension, and their chlorination byproducts contributed to the increased organic release. Meanwhile, the formation of chloroform, haloacetic acids, trichloroacetaldehyde, and dichloroacetonitrile increased by 3.8-, 1.7-, 4.9-, and 2.4-fold, respectively. Two additives and six chlorination byproducts in leachate from chlorinated UV-aged TPU were predicted as highly toxic, e.g., butyl octyl phthalate, palmitic acid, 2,6-di-tert-butyl-1,4-benzoquinone, and chlorinated aniline. Evaluated by human hepatocarcinoma cells, the 50% lethal concentration factor of organics released from chlorinated UV-aged TPU was approximately 10% of that from its virgin counterpart, indicating a substantially increased level of cytotoxicity. This study highlights that the release of additives and chlorination byproducts from the chemical transformation of UV-aged microplastics during chlorination may be of potentially toxic concern.

2\'-Fucosyllactose (2\'-FL), the most typical human milk oligosaccharide, is used as an additive in premium infant formula. Herein, we constructed two highly effective 2\'-FL synthesis producers via a de novo GDP-fucose pathway from engineered Escherichia coli MG1655. First, lacZ and wcaJ, two competitive pathway genes, were disrupted to block the invalid consumption of lactose and GDP-fucose, respectively. Next, the lacY gene was strengthened by switching its native promoter to PJ23119. To enhance the supply of endogenous GDP-fucose, the promoters of gene clusters manC-manB and gmd-fcl were strengthened individually or in combination. Subsequently, chromosomal integration of a constitutive PJ23119 promoter-based BKHT expression cassette (PJ23119-BKHT) was performed in the arsB and recA loci. The most productive plasmid-based and plasmid-free strains produced 76.9 and 50.1 g/L 2\'-FL by fed-batch cultivation, respectively. Neither of them generated difucosyl lactose nor 3-fucosyllactose as byproducts.

β-Arbutin is a plant-derived glycoside and widely used in cosmetic and pharmaceutical industries because of its safe and effective skin-lightening property as well as anti-oxidant, anti-microbial, and anti-inflammatory activities. In recent years, microbial fermentation has become a highly promising method for the production of β-arbutin. However, this method suffers from low titer and low yield, which has become the bottleneck for its widely industrial application. In this study, we used β-arbutin to demonstrate methods for improving yields for industrial-scale production in Escherichia coli. First, the supply of precursors phosphoenolpyruvate and uridine diphosphate glucose was improved, leading to a 4.6-fold increase in β-arbutin production in shaking flasks. The engineered strain produced 36.12 g/L β-arbutin with a yield of 0.11 g/g glucose in a 3-L bioreactor. Next, based on the substrate and product\'s structural similarity, an endogenous O-acetyltransferase was identified as responsible for 6-O-acetylarbutin formation for the first time. Eliminating the formation of byproducts, including 6-O-acetylarbutin, tyrosine, and acetate, resulted in an engineered strain producing 43.79 g/L β-arbutin with a yield of 0.22 g/g glucose in fed-batch fermentation. Thus, the yield increased twofold by eliminating byproducts formation. To the best of our knowledge, this is the highest titer and yield of β-arbutin ever reported, paving the way for the industrial production of β-arbutin. This study demonstrated a systematic strategy to alleviate undesirable byproduct accumulation and improve the titer and yield of target products. KEY POINTS: • A systematic strategy to improve titer and yield was showed • Genes responsible for 6-O-acetylarbutin formation were firstly identified • 43.79 g/L β-arbutin was produced in bioreactor, which is the highest titer so far.

With the steady increase in the consumption of ultra-processed foods, there is growing interest in sustainable diets that include more plant protein. However, little information is available regarding the structural and functional properties of cactus (Opuntia ficus-indica) seed protein (CSP), a by-product of the cactus seed food-processing chain. This study aimed to explore the composition and nutritional value of CSP and reveal the effects of ultrasound treatment on protein quality. Protein chemical structure analysis showed that an appropriate intensity of ultrasound treatment (450 W) could significantly increase protein solubility (96.46 ± 2.07%) and surface hydrophobicity (13.76 ± 0.85 μg), decrease the content of T-SH (50.25 ± 0.79 μmol/g) and free-SH (8.60 ± 0.30 μmol/g), and enhance emulsification characteristics. Circular dichroism analysis further confirmed that the ultrasonic treatment increased the α-helix and random coil content. Amino acid analysis also suggested that ultrasound treatment (450 W) increased the hydrophobic amino acid content. To evaluate the impact of changes in the chemical structure, its digestion behavior was studied. The results showed that ultrasound treatment increased the release rate of free amino acids. Furthermore, nutritional analysis showed that the digestive products of CSP by ultrasound treatment can significantly enhance the intestinal permeability, increase the expression of ZO-1, Occludin and Claudin-1, thus repairing LPS induced intestinal barrier disfunction. Hence, CSP is a functional protein with high value, and ultrasound treatment is recommended. These findings provide new insights into the comprehensive utilization of cactus fruits.

In this study, umami-rich seasoning powder was produced from the offcuts of Taiwanese tilapia (Oreochromis mossambicus) by cooking concentration and spray drying of granules while yielding an abundance of glutamic acid (0.23 mg/100 g), glycine (0.10 mg/100 g), aspartic acid (0.11 mg/100 g), lysine (0.10 mg/100 g), and 11 other aminic acids. It exhibited water content (3.81%), water activity (0.3), powder yields (68.83%), and a good water solubility index (99.89%), while the particle microstructure was a spherical powder. Additionally, it received the highest overall preference score (7.53) in the consumer-type sensory evaluation compared to commercially available seasonings. This study proves that offcuts may be part of the human diet after proper processing and can be widely used to flavor savory food. The producers involved could increase their economic returns while meeting the environmental challenges. The practical contribution could create incremental value for products to critical stakeholders at each point in the tilapia supply chain with an operational guide for transitioning from inefficient to innovative circular practices.

2\'-Fucosyllactose (2\'-FL) is a kind of fucosylated human milk oligosaccharide (HMO), representing the most abundant oligosaccharide in breast milk. We conducted systematic studies on three canonical α1,2-fucosyltransferases (WbgL, FucT2, and WcfB) to quantify the byproducts in a lacZ- and wcaJ-deleted Escherichia coli BL21(DE3) basic host strain. Further, we screened a highly active α1,2-fucosyltransferase from Helicobacter sp. 11S02629-2 (BKHT), which exhibits high in vivo 2\'-FL productivity without the formation of byproducts difucosyl lactose (DFL) and 3-FL. The maximum 2\'-FL titer and yield reached 11.13 g/L and 0.98 mol/mol of lactose, respectively, in shake-flask cultivation, both approaching the theoretical maximum value. In a 5 L fed-batch cultivation, the maximum 2\'-FL titer reached 94.7 g/L extracellularly with a yield of 0.98 mol of 2\'-FL/mol of lactose and productivity of 1.14 g L-1 h-1. Our reported 2\'-FL yield is the highest from lactose reported to date.