Genomic Modifications of Lactic Acid Bacteria and Their Applications in Dairy Fermentation.

Lactic Acid Bacteria (LAB) have a long history of safe use in milk fermentation and are generally recognized as health-promoting microorganisms when present in fermented foods. LAB are also important components of the human intestinal microbiota and are widely used as probiotics. Considering their safe and health-beneficial properties, LAB are considered appropriate vehicles that can be genetically modified for food, industrial and pharmaceutical applications. Here, this review describes (1) the potential opportunities for application of genetically modified LAB strains in dairy fermentation and (2) the various genomic modification tools for LAB strains, such as random mutagenesis, adaptive laboratory evolution, conjugation, homologous recombination, recombineering, and CRISPR (clustered regularly interspaced short palindromic repeat)- Cas (CRISPR-associated protein) based genome engineering. Lastly, this review also discusses the potential future developments of these genomic modification technologies and their applications in dairy fermentations.

Effects of 2'-fucosyllactose on the viability of starter cultures and Bifidobacterium strains of human origin in yogurt during refrigerated storage.

2'-Fucosyllactose (2'-FL) is postulated to provide health benefits and promote the growth of probiotics. This work was undertaken to study the effects of 2'-FL on the viability of starter cultures and Bifidobacterium strains of human origin in yogurt during refrigerated storage. Yogurts were produced containing 2'-FL (0 or 2 g/L) and Bifidobacterium strains of human origin (Bifidobacterium longum subsp. longum BB536 or Bifidobacterium longum subsp. infantis ATCC 15697) at a concentration of at least 109 CFU/mL. All yogurts were stored at 4°C for 5 weeks. Results showed that 2'-FL was stable in yogurts for at least 5 weeks of cold storage, and the addition of 2'-FL did not significantly alter yogurt fermentation parameters, associated metabolites, and the viability of mixed yogurt starter cultures and Bifidobacterium strains (p > 0.05). The addition of bifidobacteria had a negative impact (p < 0.05) on the survival rate of starter cultures, Streptococcus thermophilus and Lactobacillus delbureckii subsp. bulgaricus. Meanwhile, it is difficult to maintain a high survival rate of bifidobacteria in final yogurt products, and the addition of 2'-FL could not enhance the viability of bifidobacteria. B. longum BB536 survived at a level higher than 106 CFU/g for 28 days, while B. infantis ATCC15697 maintained this level for only 7 days. In summary, this study has shown the impact of 2'-FL and bifidobacterial species on yogurt properties, and results suggest that it is promising to use 2'-FL in yogurt products as a prebiotic. PRACTICAL APPLICATION: Yogurt is known for its beneficial effects on human health and nutrition. This study reported the production of symbiotic yogurt containing bifidobacteria and 2'-fucosyllactose (2'-FL) as a functional food for specified health uses. The viability of yogurt starter cultures and probiotic bifidobacterial strains was analyzed in this study. Moreover, this research demonstrated that 2'-FL could be added to yogurt without affecting the characteristics of yogurt significantly.

A CRISPR/Cas12a-Based System for Sensitive Detection of Antimicrobial-Resistant Genes in Carbapenem-Resistant Enterobacterales.

Antimicrobial-resistant (AMR) bacteria pose a significant global health threat, and bacteria that produce New Delhi metallo-ß-lactamase (NDM) are particularly concerning due to their resistance to most ß-lactam antibiotics, including carbapenems. The emergence and spread of NDM-producing genes in food-producing animals highlight the need for a fast and accurate method for detecting AMR bacteria. We therefore propose a PCR-coupled CRISPR/Cas12a-based fluorescence assay that can detect NDM-producing genes (blaNDM) in bacteria. Thanks to its designed gRNA, this CRISPR/Cas12a system was able to simultaneously cleave PCR amplicons and ssDNA-FQ reporters, generating fluorescence signals. Our method was found to be highly specific when tested against other foodborne pathogens that do not carry blaNDM and also demonstrated an excellent capability to distinguish single-nucleotide polymorphism. In the case of blaNDM-1 carrying E. coli, the assay performed exceptionally well, with a detection limit of 2.7 × 100 CFU/mL: 100 times better than conventional PCR with gel electrophoresis. Moreover, the developed assay detected AMR bacteria in food samples and exhibited enhanced performance compared to previously published real-time PCR assays. Thus, this novel PCR-coupled CRISPR/Cas12a-based fluorescence assay has considerable potential to improve current approaches to AMR gene detection and thereby contribute to mitigating the global threat of AMR.

Self-Buffering system for Cost-Effective production of lactic acid from glucose and xylose using Acid-Tolerant Issatchenkia orientalis.

This study presents a cost-effective strategy for producing organic acids from glucose and xylose using the acid-tolerant yeast, Issatchenkia orientalis. I. orientalis was engineered to produce lactic acid from xylose, and the resulting strain, SD108XL, successfully converted sorghum hydrolysates into lactic acid. In order to enable low-pH fermentation, a self-buffering strategy, where the lactic acid generated by the SD108XL strain during fermentation served as a buffer, was developed. As a result, the SD108 strain produced 67 g/L of lactic acid from 73 g/L of glucose and 40 g/L of xylose, simulating a sugar composition of sorghum biomass hydrolysates. Moreover, techno-economic analysis underscored the efficiency of the self-buffering strategy in streamlining the downstream process, thereby reducing production costs. These results demonstrate the potential of I. orientalis as a platform strain for the cost-effective production of organic acids from cellulosic hydrolysates.

Precision fermentation for improving the quality, flavor, safety, and sustainability of foods.

Precision fermentation involves the rewiring of metabolic pathways in generally recognized as safe microorganisms, fermentation scale-up, and downstream processing to produce food ingredients from abundant and inexpensive substrates. Using precise genome editing of food-fermenting microorganisms, precision fermentation can also produce fermented foods with more desirable properties. These genetic tools allow for the manipulation of flavors and nutritional content in fermented foods, the economic production of functional food ingredients, and the sustainable production of otherwise-costly macronutrients. By introducing the metabolic designs, genetic modifications, and resulting products of engineered microorganisms developed through academic and industrial research, this review aims to provide insights into the potentials and challenges of precision fermentation for the economic, safe, and sustainable production of foods.

Compositional and temporal division of labor modulates mixed sugar fermentation by an engineered yeast consortium.

Synthetic microbial communities have emerged as an attractive route for chemical bioprocessing. They are argued to be superior to single strains through microbial division of labor (DOL), but the exact mechanism by which DOL confers advantages remains unclear. Here, we utilize a synthetic Saccharomyces cerevisiae consortium along with mathematical modeling to achieve tunable mixed sugar fermentation to overcome the limitations of single-strain fermentation. The consortium involves two strains with each specializing in glucose or xylose utilization for ethanol production. By controlling initial community composition, DOL allows fine tuning of fermentation dynamics and product generation. By altering inoculation delay, DOL provides additional programmability to parallelly regulate fermentation characteristics and product yield. Mathematical models capture observed experimental findings and further offer guidance for subsequent fermentation optimization. This study demonstrates the functional potential of DOL in bioprocessing and provides insight into the rational design of engineered ecosystems for various applications.

Toward rapid and efficient utilization of nonconventional substrates by nonconventional yeast strains.

Economic and sustainable production of biofuels and chemicals necessitates utilizing abundant and inexpensive lignocellulosic biomass. Yet, Saccharomyces cerevisiae, a workhorse strain for industrial biotechnology based on starch and sugarcane-derived sugars, is not suitable for lignocellulosic bioconversion due to a lack of pentose metabolic pathways and severe inhibition by toxic inhibitors in cellulosic hydrolysates. This review underscores the potential of nonconventional yeast strains, specifically Yarrowia lipolytica and Rhodotorula toruloides, for converting underutilized carbon sources, such as xylose and acetate, into high-value products. Multi-omics studies with nonconventional yeast have elucidated the structure and regulation of metabolic pathways for efficient and rapid utilization of xylose and acetate. The review delves into the advantages of using xylose and acetate for producing biofuels and chemicals. Collectively, value-added biotransformation of nonconventional substrates by nonconventional yeast strains is a promising strategy to improve both economics and sustainability of bioproduction.

Metabolic engineering of Saccharomyces cerevisiae for second-generation ethanol production from xylo-oligosaccharides and acetate.

Simultaneous intracellular depolymerization of xylo-oligosaccharides (XOS) and acetate fermentation by engineered Saccharomyces cerevisiae offers significant potential for more cost-effective second-generation (2G) ethanol production. In the present work, the previously engineered S. cerevisiae strain, SR8A6S3, expressing enzymes for xylose assimilation along with an optimized route for acetate reduction, was used as the host for expressing two ß-xylosidases, GH43-2 and GH43-7, and a xylodextrin transporter, CDT-2, from Neurospora crassa, yielding the engineered SR8A6S3-CDT-2-GH34-2/7 strain. Both ß-xylosidases and the transporter were introduced by replacing two endogenous genes, GRE3 and SOR1, that encode aldose reductase and sorbitol (xylitol) dehydrogenase, respectively, and catalyse steps in xylitol production. The engineered strain, SR8A6S3-CDT-2-GH34-2/7 (sor1Δ gre3Δ), produced ethanol through simultaneous XOS, xylose, and acetate co-utilization. The mutant strain produced 60% more ethanol and 12% less xylitol than the control strain when a hemicellulosic hydrolysate was used as a mono- and oligosaccharide source. Similarly, the ethanol yield was 84% higher for the engineered strain using hydrolysed xylan, compared with the parental strain. Xylan, a common polysaccharide in lignocellulosic residues, enables recombinant strains to outcompete contaminants in fermentation tanks, as XOS transport and breakdown occur intracellularly. Furthermore, acetic acid is a ubiquitous toxic component in lignocellulosic hydrolysates, deriving from hemicellulose and lignin breakdown. Therefore, the consumption of XOS, xylose, and acetate expands the capabilities of S. cerevisiae for utilization of all of the carbohydrate in lignocellulose, potentially increasing the efficiency of 2G biofuel production.

Draft genome sequence of Yarrowia lipolytica NRRL Y-64008, an oleaginous yeast capable of growing on lignocellulosic hydrolysates.

Yarrowia lipolytica is an oleaginous yeast that produces high titers of fatty acid-derived biofuels and biochemicals. It can grow on hydrophobic carbon sources and lignocellulosic hydrolysates. The genome sequence of Y. lipolytica NRRL Y-64008 is reported to aid in its development as a biotechnological chassis for producing biofuels and bioproducts.

Near-complete genome sequence of Lipomyces tetrasporous NRRL Y-64009, an oleaginous yeast capable of growing on lignocellulosic hydrolysates.

Lipomyces tetrasporous is an oleaginous yeast that can utilize a variety of plant-based sugars. It accumulates lipids during growth on lignocellulosic biomass hydrolysates. We present the annotated genome sequence of L. tetrasporous NRRL Y-64009 to aid in its development as a platform organism for producing lipids and lipid-based bioproducts.

Construction of a Compact Array of Microplasma Jet Devices and Its Application for Random Mutagenesis of Rhodosporidium toruloides.

A small and efficient DNA mutation-inducing machine was constructed with an array of microplasma jet devices (7 × 1) that can be operated at atmospheric pressure for microbial mutagenesis. Using this machine, we report disruption of a plasmid DNA and generation of mutants of an oleaginous yeast Rhodosporidium toruloides. Specifically, a compact-sized microplasma channel (25 × 20 × 2 mm3) capable of generating an electron density of greater than 1013 cm-3 was constructed to produce reactive species (N2*, N2+, O, OH, and Hα) under helium atmospheric conditions to induce DNA mutagenesis. The length of microplasma channels in the device played a critical role in augmenting both the volume of plasma and the concentration of reactive species. First, we confirmed that microplasma treatment can linearize a plasmid by creating nicks in vitro. Second, we treated R. toruloides cells with a jet device containing 7 microchannels for 5 min; 94.8% of the treated cells were killed, and 0.44% of surviving cells showed different colony colors as compared to their parental colony. Microplasma-based DNA mutation is energy-efficient and can be a safe alternative for inducing mutations compared to conventional methods using toxic mutagens. This compact and scalable device is amenable for industrial strain improvement involving large-scale mutagenesis.

Identification of the enantiomeric nature of 2-keto-3-deoxy-galactonate in the catabolic pathway of 3,6-anhydro-L-galactose.

A novel metabolic pathway of 3,6-anhydro-L-galactose (L-AHG), the main sugar component in red macroalgae, was first discovered in the marine bacterium Vibrio sp. EJY3. L-AHG is converted to 2-keto-3-deoxy-galactonate (KDGal) in two metabolic steps. Here, we identified the enantiomeric nature of KDGal in the L-AHG catabolic pathway via stereospecific enzymatic reactions accompanying the biosynthesis of enantiopure L-KDGal and D-KDGal. Enantiopure L-KDGal and D-KDGal were synthesized by enzymatic reactions derived from the fungal galacturonate and bacterial oxidative galactose pathways, respectively. KDGal, which is involved in the L-AHG pathway, was also prepared. The results obtained from the reactions with an L-KDGal aldolase, specifically acting on L-KDGal, showed that KDGal in the L-AHG pathway exists in an L-enantiomeric form. Notably, we demonstrated the utilization of L-KDGal by Escherichia coli for the first time. E. coli cannot utilize L-KDGal as the sole carbon source. However, when a mixture of L-KDGal and D-galacturonate was used, E. coli utilized both. Our study suggests a stereoselective method to determine the absolute configuration of a compound. In addition, our results can be used to explore the novel L-KDGal catabolic pathway in E. coli and to construct an engineered microbial platform that assimilates L-AHG or L-KDGal as substrates. KEY POINTS: • Stereospecific enzyme reactions were used to identify enantiomeric nature of KDGal • KDGal in the L-AHG catabolic pathway exists in an L-enantiomeric form • E. coli can utilize L-KDGal as a carbon source when supplied with D-galacturonate.

Microbiome Engineering Using Probiotic Yeast: Saccharomyces boulardii and the Secreted Human Lysozyme Lead to Changes in the Gut Microbiome and Metabolome of Mice.

The probiotic yeast Saccharomyces boulardii has great potential for use as a chassis for microbiome engineering because of its high resistance to environmental stress, well-developed genetic tools, and the ability to secrete recombinant proteins in the intestine. As oral feeding of lysozyme has been reported to change the gut microbiome and fecal metabolites, we engineered S. boulardii to secrete human lysozyme, and investigated the changes in the microbiome and fecal metabolites in response to the administration of the engineered probiotic yeast into mice. Administration of S. boulardii changed the structure of the gut microbiome by promoting the growth of clostridia and increasing the diversity of strains. The human lysozyme secreted by S. boulardii in the intestine resulted in a unique gut microbiome structure through selective growth. In addition, the administration of probiotic yeast S. boulardii affected host energy metabolism and decreased blood urea and fructose levels, suggesting a mechanism of health benefits in mice. IMPORTANCE Our study identified changes in the microbiome by administering wild-type S. boulardii in mice to healthy mice based on long-read sequencing and demonstrated that a recombinant protein secreted by engineered S. boulardii in the intestine could change the microbiome. Our results provide valuable information for the development of therapeutics using engineered S. boulardii that changes the gut microbiome and host physiology.

Engineering xylose fermentation in an industrial yeast: continuous cultivation as a tool for selecting improved strains.

Production of second-generation ethanol from lignocellulosic residues should be fueling the energy matrix in the near future. Lignocellulosic biomass has received considerable attention as an alternative renewable resource toward reducing the demand for fossil energy sources, contributing to a future sustainable bio-based economy. Fermentation of lignocellulosic hydrolysates poses many scientific and technological challenges as the drawback of Saccharomyces cerevisiae's inability in fermenting pentose sugars (derived from hemicellulose). To overcome the inability of S. cerevisiae to ferment xylose and increase yeast robustness in the presence of inhibitory compound-containing media, the industrial S. cerevisiae strain SA-1 was engineered using CRISPR-Cas9 with the oxidoreductive xylose pathway from Scheffersomyces stipitis (encoded by XYL1, XYL2, and XYL3). The engineered strain was then cultivated in a xylose-limited chemostat under increasing dilution rates (for 64 days) to improve its xylose consumption kinetics under aerobic conditions. The evolved strain (DPY06) and its parental strain (SA-1 XR/XDH) were evaluated under microaerobic in a hemicellulosic hydrolysate-based medium. DPY06 exhibited 35% higher volumetric ethanol productivity compared to its parental strain.

A qPCR Method to Assay Endonuclease Activity of Cas9-sgRNA Ribonucleoprotein Complexes.

The CRISPR-Cas system has emerged as the most efficient genome editing technique for a wide range of cells. Delivery of the Cas9-sgRNA ribonucleoprotein complex (Cas9 RNP) has gained popularity. The objective of this study was to develop a quantitative polymerase chain reaction (qPCR)-based assay to quantify the double-strand break reaction mediated by Cas9 RNP. To accomplish this, the dextransucrase gene (dsr) from Leuconostoc citreum was selected as the target DNA. The Cas9 protein was produced using recombinant Escherichia coli BL21, and two sgRNAs were synthesized through in vitro transcription to facilitate binding with the dsr gene. Under optimized in vitro conditions, the 2.6 kb dsr DNA was specifically cleaved into 1.1 and 1.5 kb fragments by both Cas9-sgRNA365 and Cas9-sgRNA433. By monitoring changes in dsr concentration using qPCR, the endonuclease activities of the two Cas9 RNPs were measured, and their efficiencies were compared. Specifically, the specific activities of dsr365RNP and dsr433RNP were 28.74 and 34.48 (unit/µg RNP), respectively. The versatility of this method was also verified using different target genes, uracil phosphoribosyl transferase (upp) gene, of Bifidobacterium bifidum and specific sgRNAs. The assay method was also utilized to determine the impact of high electrical field on Cas9 RNP activity during an efficient electroporation process. Overall, the results demonstrated that the qPCR-based method is an effective tool for measuring the endonuclease activity of Cas9 RNP.

Production of Ethyl-agarobioside, a Novel Skin Moisturizer, by Mimicking the Alcoholysis from the Japanese Sake-Brewing Process.

Agarobiose (AB; d-galactose-ß-1,4-AHG), produced by one-step acid hydrolysis of agarose of red seaweed, is considered a promising cosmetic ingredient due to its skin-moisturizing activity. In this study, the use of AB as a cosmetic ingredient was found to be hampered due to its instability at high temperature and alkaline pH. Therefore, to increase the chemical stability of AB, we devised a novel process to synthesize ethyl-agarobioside (ethyl-AB) from the acid-catalyzed alcoholysis of agarose. This process mimics the generation of ethyl α-glucoside and glyceryl α-glucoside by alcoholysis in the presence of ethanol and glycerol during the traditional Japanese sake-brewing process. Ethyl-AB also showed in vitro skin-moisturizing activity similar to that of AB, but showed higher thermal and pH stability than AB. This is the first report of ethyl-AB, a novel compound produced from red seaweed, as a functional cosmetic ingredient with high chemical stability.

L-Fucose is involved in human-gut microbiome interactions.

L-Fucose is one of the key metabolites in human-gut microbiome interactions. It is continuously synthesized by humans in the form of fucosylated glycans and fucosyl-oligosaccharides and delivered into the gut throughout their lifetime. Gut microorganisms metabolize L-fucose and produce short-chain fatty acids, which are absorbed by epithelial cells and used as energy sources or signaling molecules. Recent studies have revealed that the carbon flux in L-fucose metabolism by gut microorganisms is distinct from that in other sugar metabolisms because of cofactor imbalance and low efficiencies in energy synthesis of L-fucose metabolism. The large amounts of short-chain fatty acids produced during microbial L-fucose metabolism are used by epithelial cells to recover most of the energy used up during L-fucose synthesis. In this review, we present a detailed overview of microbial L-fucose metabolism and a potential solution for disease treatment and prevention using genetically engineered probiotics that modulate fucose metabolism. Our review contributes to the understanding of human-gut microbiome interactions through L-fucose metabolism. KEY POINTS: • Fucose-metabolizing microorganisms produce large amounts of short-chain fatty acids • Fucose metabolism differs from other sugar metabolisms by cofactor imbalance • Modulating fucose metabolism is the key to control host-gut microbiome interactions.

Glucose assimilation rate determines the partition of flux at pyruvate between lactic acid and ethanol in Saccharomyces cerevisiae.

Engineered Saccharomyces cerevisiae expressing a lactic acid dehydrogenase can metabolize pyruvate into lactic acid. However, three pyruvate decarboxylase (PDC) isozymes drive most carbon flux toward ethanol rather than lactic acid. Deletion of endogenous PDCs will eliminate ethanol production, but the resulting strain suffers from C2 auxotrophy and struggles to complete a fermentation. Engineered yeast assimilating xylose or cellobiose produce lactic acid rather than ethanol as a major product without the deletion of any PDC genes. We report here that sugar flux, but not sensing, contributes to the partition of flux at the pyruvate branch point in S. cerevisiae expressing the Rhizopus oryzae lactic acid dehydrogenase (LdhA). While the membrane glucose sensors Snf3 and Rgt2 did not play any direct role in the option of predominant product, the sugar assimilation rate was strongly correlated to the partition of flux at pyruvate: fast sugar assimilation favors ethanol production while slow sugar assimilation favors lactic acid. Applying this knowledge, we created an engineered yeast capable of simultaneously converting glucose and xylose into lactic acid, increasing lactic acid production to approximately 17 g L-1 from the 12 g L-1 observed during sequential consumption of sugars. This work elucidates the carbon source-dependent effects on product selection in engineered yeast.

Reshaping the 2-Pyrone Synthase Active Site for Chemoselective Biosynthesis of Polyketides.

Engineering enzymes with novel reactivity and applying them in metabolic pathways to produce valuable products are quite challenging due to the intrinsic complexity of metabolic networks and the need for high in vivo catalytic efficiency. Triacetic acid lactone (TAL), naturally generated by 2-pyrone synthase (2PS), is a platform molecule that can be produced via microbial fermentation and further converted into value-added products. However, these conversions require extra synthetic steps under harsh conditions. We herein report a biocatalytic system for direct generation of TAL derivatives under mild conditions with controlled chemoselectivity by rationally engineering the 2PS active site and then rewiring the biocatalytic pathway in the metabolic network of E. coli to produce high-value products, such as kavalactone precursors, with yields up to 17 mg/L culture. Computer modeling indicates sterics and hydrogen-bond interactions play key roles in tuning the selectivity, efficiency and yield.

Enhanced production of 3,4-dihydroxybutyrate from xylose by engineered yeast via xylonate re-assimilation under alkaline condition.

To realize lignocellulose-based bioeconomy, efficient conversion of xylose into valuable chemicals by microbes is necessary. Xylose oxidative pathways that oxidize xylose into xylonate can be more advantageous than conventional xylose assimilation pathways because of fewer reaction steps without loss of carbon and ATP. Moreover, commodity chemicals like 3,4-dihydroxybutyrate and 3-hydroxybutyrolactone can be produced from the intermediates of xylose oxidative pathway. However, successful implementations of xylose oxidative pathway in yeast have been hindered because of the secretion and accumulation of xylonate which is a key intermediate of the pathway, leading to low yield of target product. Here, high-yield production of 3,4-dihydroxybutyrate from xylose by engineered yeast was achieved through genetic and environmental perturbations. Specifically, 3,4-dihydroxybutyrate biosynthetic pathway was established in yeast through deletion of ADH6 and overexpression of yneI. Also, inspired by the mismatch of pH between host strain and key enzyme of XylD, alkaline fermentations (pH ≥ 7.0) were performed to minimize xylonate accumulation. Under the alkaline conditions, xylonate was re-assimilated by engineered yeast and combined product yields of 3,4-dihydroxybutyrate and 3-hydroxybutyrolactone resulted in 0.791 mol/mol-xylose, which is highest compared with previous study. These results shed light on the utility of the xylose oxidative pathway in yeast.