A mutant fitness compendium in Bifidobacteria reveals molecular determinants of colonization and host-microbe interactions.
bioRxiv : the preprint server for biology
Bifidobacteria commonly represent a dominant constituent of human gut microbiomes during infancy, influencing nutrition, immune development, and resistance to infection. Despite interest as a probiotic therapy, predicting the nutritional requirements and health-promoting effects of Bifidobacteria is challenging due to major knowledge gaps. To overcome these deficiencies, we used large-scale genetics to create a compendium of mutant fitness in Bifidobacterium breve (Bb). We generated a high density, randomly barcoded transposon insertion pool in Bb, and used this pool to determine Bb fitness requirements during colonization of germ-free mice and chickens with multiple diets and in response to hundreds of in vitro perturbations. To enable mechanistic investigation, we constructed an ordered collection of insertion strains covering 1462 genes. We leveraged these tools to improve models of metabolic pathways, reveal unexpected host- and diet-specific requirements for colonization, and connect the production of immunomodulatory molecules to growth benefits. These resources will greatly reduce the barrier to future investigations of this important beneficial microbe.
View details for DOI 10.1101/2023.08.29.555234
View details for PubMedID 37693407
View details for PubMedCentralID PMC10491234
Engineering an efficient whole-cell catalyst for D-allulose production from glycerol.
D-Allulose has many health-benefiting properties and therefore sustainably applied in food, pharmaceutical, and nutrition industries. The aldol reaction based route is a very promising alternative to Izumoring strategy in D-allulose production. Remarkable studies reported in the past cannot get rid of by-product formation and costly purified enzyme usage. In the present study, we explored the glycerol assimilation by modularly assembling the D-allulose synthetic cascade in Escherichia coli envelop. We achieved an efficient whole-cell catalyst that produces only D-allulose from cheap glycerol feedstock, eliminating the involvement of purified enzymes. Detailed process optimization improved the D-allulose titer by 1500.00%. Finally, the production was validated in 3-L scale using a 5-L fermenter, and 5.67 g/L D-allulose was produced with a molar yield of 31.43%. This article is protected by copyright. All rights reserved.
View details for DOI 10.1002/biot.202200600
View details for PubMedID 37079661
Regulation of Ethanol Assimilation for Efficient Accumulation of Squalene in Saccharomyces cerevisiae.
Journal of agricultural and food chemistry
Squalene is a triterpene that can be obtained from fish and plant oils. It is important in cosmetics and vaccines and is a precursor for many high-value terpenes and steroids. In order to increase squalene accumulation, the mevalonate pathway was systematically enhanced. Accumulation of squalene tended to increase when ethanol was added as a carbon source during fermentation, but a high concentration of ethanol affected both the strain growth and accumulation of products. By overexpressing the key trehalose synthesis gene TPS1 and the heat shock protein gene HSP104, the content of trehalose by Saccharomyces cerevisiae (S. cerevisiae) was enhanced, and stress caused by ethanol was relieved. The OD600 value of the modified S. cerevisiae strain was increased by 80.2%, its ethanol tolerance was increased to 30 g/L, and it retained excellent activity with 50 g/L ethanol. After optimizing the fermentation conditions, the squalene titer in a 5 L bioreactor reached 27.3 g/L and the squalene content was 650 mg/g dry cell weight, the highest squalene production parameters reported to date for a microorganism.
View details for DOI 10.1021/acs.jafc.3c00515
View details for PubMedID 37052370
Produce D-allulose from non-food biomass by integrating corn stalk hydrolysis with whole-cell catalysis.
Frontiers in bioengineering and biotechnology
2023; 11: 1156953
D-allulose is a high-value rare sugar with many health benefits. D-allulose market demand increased dramatically after approved as generally recognized as safe (GRAS). The current studies are predominantly focusing on producing D-allulose from either D-glucose or D-fructose, which may compete foods against human. The corn stalk (CS) is one of the main agricultural waste biomass in the worldwide. Bioconversion is one of the promising approach to CS valorization, which is of significance for both food safety and reducing carbon emission. In this study, we tried to explore a non-food based route by integrating CS hydrolysis with D-allulose production. Firstly we developed an efficient Escherichia coli whole-cell catalyst to produce D-allulose from D-glucose. Next we hydrolyzed CS and achieved D-allulose production from the CS hydrolysate. Finally we immobilized the whole-cell catalyst by designing a microfluidic device. Process optimization improved D-allulose titer by 8.61 times, reaching 8.78 g/L from CS hydrolysate. With this method, 1 kg CS was finally converted to 48.87 g D-allulose. This study validated the feasibility of valorizing corn stalk by converting it to D-allulose.
View details for DOI 10.3389/fbioe.2023.1156953
View details for PubMedID 36911188
View details for PubMedCentralID PMC9998921
Engineering heterologous enzyme secretion in Yarrowia lipolytica
MICROBIAL CELL FACTORIES
2022; 21 (1): 134
Eukaryotic cells are often preferred for the production of complex enzymes and biopharmaceuticals due to their ability to form post-translational modifications and inherent quality control system within the endoplasmic reticulum (ER). A non-conventional yeast species, Yarrowia lipolytica, has attracted attention due to its high protein secretion capacity and advanced secretory pathway. Common means of improving protein secretion in Y. lipolytica include codon optimization, increased gene copy number, inducible expression, and secretory tag engineering. In this study, we develop effective strategies to enhance protein secretion using the model heterologous enzyme T4 lysozyme.By engineering the commonly used native lip2prepro secretion signal, we have successfully improved secreted T4 lysozyme titer by 17-fold. Similar improvements were measured for other heterologous proteins, including hrGFP and [Formula: see text]-amylase. In addition to secretion tag engineering, we engineered the secretory pathway by expanding the ER and co-expressing heterologous enzymes in the secretion tag processing pathway, resulting in combined 50-fold improvement in T4 lysozyme secretion.Overall, our combined strategies not only proved effective in improving the protein production in Yarrowia lipolytica, but also hint the possible existence of a different mechanism of secretion regulation in ER and Golgi body in this non-conventional yeast.
View details for DOI 10.1186/s12934-022-01863-9
View details for Web of Science ID 000820223100001
View details for PubMedID 35786380
View details for PubMedCentralID PMC9252082
- Rational engineering of low temperature activity in thermoalkalophilic Geobacillus thermocatenulatus lipase BIOCHEMICAL ENGINEERING JOURNAL 2021; 174