Exploring the secretome of Corynebacterium glutamicum ATCC 13032.

The demand for alternative sources of food proteins is increasing due to the limitations and challenges associated with conventional food production. Advances in biotechnology have enabled the production of proteins using microorganisms, thus prompting the exploration of attractive microbial hosts capable of producing functional proteins in high titers. Corynebacterium glutamicum is widely used in industry for the production of amino acids and has many advantages as a host organism for recombinant protein production. However, its performance in this area is limited by low yields of target proteins and high levels of native protein secretion. Despite representing a challenge for heterologous protein production, the C. glutamicum secretome has not been fully characterized. In this study, state-of-the-art mass spectrometry-based proteomics was used to identify and analyze the proteins secreted by C. glutamicum. Both the wild-type strain and a strain that produced and secreted a recombinant ß-lactoglobulin protein were analyzed. A total of 427 proteins were identified in the culture supernatants, with 148 predicted to possess a secretion signal peptide. MS-based proteomics on the secretome enabled a comprehensive characterization and quantification (based on abundance) of the secreted proteins through label-free quantification (LFQ). The top 12 most abundant proteins accounted for almost 80% of the secretome. These are uncharacterized proteins of unknown function, resuscitation promoting factors, protein PS1, Porin B, ABC-type transporter protein and hypothetical membrane protein. The data can be leveraged for protein production by, e.g., utilizing the signal peptides of the most abundant proteins to improve secretion of heterologous proteins. In addition, secretory stress can potentially be alleviated by inactivating non-essential secreted proteins. Here we provide targets by identifying the most abundant, secreted proteins of which majority are of unknown function. The data from this study can thus provide valuable insight for researchers looking to improve protein secretion and optimize C. glutamicum as a host for secretory protein production.

Treatment with Supercritical CO2 Reduces Off-Flavour of White Alfalfa Protein Concentrate.

White alfalfa protein concentrate from alfalfa (Medicago sativa) is a promising substitute for milk and egg protein due to its functionality. However, it contains many unwanted flavours that limits the amount that can be added to a food without affecting its taste negatively. In this paper, we have demonstrated a simple method for the extraction of white alfalfa protein concentrate followed by a treatment with supercritical CO2. Two concentrates were produced at lab scale and pilot scale, with yields of 0.012 g (lab scale) and 0.08 g (pilot scale), of protein per g of total protein introduced into the process. The solubility of the protein produced at lab scale and pilot scale was approximately 30% and 15%, respectively. By treating the protein concentrate at 220 bar and 45 °C for 75 min with supercritical CO2, off-flavours were lowered. The treatment did not decrease the digestibility or alter the functionality of white alfalfa protein concentrate when it was used to substitute egg in chocolate muffins and egg white in meringues.

Valorization of Green Biomass: Alfalfa Pulp as a Substrate for Oyster Mushroom Cultivation.

In this study, the potential of alfalfa pulp as an alternative substrate to wheat straw for the cultivation of oyster mushroom (Pleurotus ostreatus) was investigated. The major components associated with different mushroom stages were evaluated, as well as changes in lignocellulolytic enzyme activities in substrates composed of alfalfa pulp, wheat straw or a combination of both. Based on the results, alfalfa pulp was demonstrated to be a better substrate than wheat straw for the production of oyster mushrooms, with a high biological efficiency of 166.3 ± 25.4%. Compared to the cultivation period on commercial straw (31 days), a shorter lifecycle for oyster mushroom was found on alfalfa pulp (24 days), which could help to reduce the risk of contamination during industrial production. Study of the spent substrate as well as the harvested mushrooms revealed that the biological efficiency was related to the higher protein content (17.42%) in the alfalfa pulp compared to wheat straw, as well as greater degradation of cellulose (57.58%) and hemicellulose (56.60%). This was, by and large, due to greater extracellular hydrolytic and oxidative enzyme activity from the mushroom growth in the alfalfa pulp. The quality and safety of the fruiting bodies produced on alfalfa pulp was evaluated, which showed that the protein content was 20.4%, of which 46.3% was essential amino acids, and levels of trace elements and heavy metals were below acceptable limits. Hence, oyster mushroom cultivation using alfalfa pulp provides an alternative method to produce a value-added product, while reducing the biomass wastes in the green protein bio-refinery, and may contribute to sustainable growth in the agricultural industry.

Scale-Up of Alfalfa (Medicago sativa) Protein Recovery Using Screw Presses.

As a consequence of the increased demand for proteins for both feed and food, alternative protein sources from green plants such as alfalfa (Medicago sativa) have come into focus, together with methods to recover these proteins. In this study, we have investigated the use of screw presses for protein recovery from alfalfa at laboratory and pilot scale. We found that using a pilot scale screw press, with a working pressure of 6 bar, 16% of the total protein was recovered in one pressing, and that after rehydrating and repressing the alfalfa up to ten times, 48% of the total protein could be recovered. The green alfalfa protein concentrate was analyzed for total protein, amino acid profile, protein digestibility, color, ash, fiber and fat content. It was found that repetitive pressings lowered the digestibility of the protein pool and reduced the total protein concentration due to dilution. To achieve the best quality protein at the highest concentrations, it is recommended to press the alfalfa no more than twice, which results in an alfalfa protein concentrate with more than 32% soluble protein and greater than 82% digestibility.

Harnessing lactic acid bacteria in synthetic microbial consortia.

Lactic acid bacteria (LAB) are important members in synthetic microbial consortia due to their 'generally recognized as safe' status and diverse metabolic activities. Defined communities with LAB show great potential in elucidating metabolic interactions that drive their assembly and demonstrating power to address sustainability challenges in food, environment, and health.

Food grade microbial synthesis of the butter aroma compound butanedione using engineered and non-engineered Lactococcus lactis.

The design-build-test-learn (DBTL) cycle has been implemented in metabolic engineering processes for optimizing the production of valuable compounds, including food ingredients. However, the use of recombinant microorganisms for producing food ingredients is associated with different challenges, e.g., in the EU, a content of more than 0.9% of such ingredients requires to be labeled. Therefore, we propose to expand the DBTL cycle and use the "learn" module to guide the development of non-engineered strains for clean label production. Here, we demonstrate how this approach can be used to generate engineered and natural cell factories able to produce the valuable food flavor compound - butanedione (diacetyl). Through comprehensive rerouting of the metabolism of Lactococcus lactis MG1363 and re-installment of the capacity to metabolize lactose and dairy protein, we managed to achieve a high titer of diacetyl (6.7 g/L) in pure dairy waste. Based on learnings from the engineering efforts, we successfully achieved the production of diacetyl without using recombinant DNA technology. We accomplish the latter by process optimization and by relying on high-throughput screening using a microfluidic system. Our results demonstrate the great potential that lies in combining metabolic engineering and natural approaches for achieving efficient production of food ingredients.

Deciphering the Regulation of the Mannitol Operon Paves the Way for Efficient Production of Mannitol in Lactococcus lactis.

Lactococcus lactis has great potential for high-yield production of mannitol, which has not yet been fully realized. In this study, we characterize how the mannitol genes in L. lactis are organized and regulated and use this information to establish efficient mannitol production. Although the organization of the mannitol genes in L. lactis was similar to that in other Gram-positive bacteria, mtlF and mtlD, encoding the enzyme IIA component (EIIAmtl) of the mannitol phosphotransferase system (PTS) and the mannitol-1-phosphate dehydrogenase, respectively, were separated by a transcriptional terminator, and the mannitol genes were found to be organized in two transcriptional units: an operon comprising mtlA, encoding the enzyme IIBC component (EIIBCmtl) of the mannitol PTS, mtlR, encoding a transcriptional activator, and mtlF, as well as a separately expressed mtlD gene. The promoters driving expression of the two transcriptional units were somewhat similar, and both contained predicted catabolite responsive element (cre) genes. The presence of carbon catabolite repression was demonstrated and was shown to be relieved in stationary-phase cells. The transcriptional activator MtlR (mtlR), in some Gram-positive bacteria, is repressed by phosphorylation by EIIAmtl, and when we knocked out mtlF, we indeed observed enhanced expression from the two promoters, which indicated that this mechanism was in place. Finally, by overexpressing the mtlD gene and using stationary-phase cells as biocatalysts, we attained 10.1 g/liter mannitol with a 55% yield, which, to the best of our knowledge, is the highest titer ever reported for L. lactis. Summing up, the results of our study should be useful for improving the mannitol-producing capacity of this important industrial organism. IMPORTANCE Lactococcus lactis is the most studied species of the lactic acid bacteria, and it is widely used in various food fermentations. To date, there have been several attempts to persuade L. lactis to produce mannitol, a sugar alcohol with important therapeutic and food applications. Until now, to achieve mannitol production in L. lactis with significant titer and yield, it has been necessary to introduce and express foreign genes, which precludes the use of such strains in foods, due to their recombinant status. In this study, we systematically characterize how the mannitol genes in L. lactis are regulated and demonstrate how this impacts mannitol production capability. We harnessed this information and managed to establish efficient mannitol production without introducing foreign genes.

Purified lactases versus whole-cell lactases-the winner takes it all.

Lactose-free dairy products are in great demand worldwide due to the high prevalence of lactose intolerance. To make lactose-free dairy products, commercially available ß-galactosidase enzymes, also termed lactases, are used to break down lactose to its constituent monosaccharides, glucose and galactose. In this mini-review, the characteristics of lactase enzymes, their origin, and ways of use are discussed in light of their potential for hydrolyzing lactose. We also discuss whole-cell lactase catalysts, which appear to have great potential in terms of cost reduction and convenience, and which are more natural alternatives to purified enzymes. Lactic acid bacteria (LAB) already used in food fermentations seem to be optimal candidates for whole-cell lactases. However, they have not been industrially exploited yet due to technical hurdles. For whole-cell lactases to be efficient, the lactase enzymes inside the cells must be made available for lactose hydrolysis, and thus, cells need to be permeabilized or disrupted prior to use. Here we review state-of-the-art approaches for disrupting or permeabilizing microorganisms. Lastly, based on recent scientific achievements, we propose a novel, resource-efficient, and low-cost scenario for achieving lactose hydrolysis at a dairy plant using a LAB whole-cell lactase.Key points• Lactases (ß-galactosidase) are essential for producing lactose-free dairy products• Novel permeabilization techniques facilitate the use of LAB lactases• Whole-cell lactase catalysts have great potential for reducing costs and resources Graphical abstract.

Energy Starvation Induces a Cell Cycle Arrest in Escherichia coli by Triggering Degradation of the DnaA Initiator Protein.

During steady-state Escherichia coli growth, the amount and activity of the initiator protein, DnaA, controls chromosome replication tightly so that initiation only takes place once per origin in each cell cycle, regardless of growth conditions. However, little is known about the mechanisms involved during transitions from one environmental condition to another or during starvation stress. ATP depletion is one of the consequences of long-term carbon starvation. Here we show that DnaA is degraded in ATP-depleted cells. A chromosome replication initiation block is apparent in such cells as no new rounds of DNA replication are initiated while replication events that have already started proceed to completion.

Droplet-Based Microfluidic High Throughput Screening of Corynebacterium glutamicum for Efficient Heterologous Protein Production and Secretion.

With emerging interests in heterologous production of proteins such as antibodies, growth factors, nanobodies, high-quality protein food ingredients, etc. the demand for efficient production hosts increases. Corynebacterium glutamicum is an attractive industrial host with great secretion capacity to produce therapeutics. It lacks extracellular protease and endotoxin activities and easily achieves high cell density. Therefore, this study focuses on improving protein production and secretion in C. glutamicum with the use of droplet-based microfluidic (DBM) high throughput screening. A library of C. glutamicum secreting ß-glucosidase was generated using chemical mutagenesis coupled with DBM screening of 200,000 mutants in just 20 min. Among 100 recovered mutants, 16 mutants exhibited enhanced enzyme secretion capacity, 13 of which had unique mutation profiles. Whole-genome analysis showed that approximately 50-150 SNVs had occurred on the chromosome per mutant. Functional enrichment analysis of genes with non-synonymous mutations showed overrepresentation of genes involved in protein synthesis and secretion relevant biological processes, such as DNA and ribosome RNA synthesis, protein secretion and energy turnover. Two mutants JCMT1 and JCMT8 exhibited the highest secretion with a six and a fivefold increase in the ß-glucosidase activity in the supernatant, respectively, relative to the reference strain JC0190. After plasmid curing, a new plasmid with the gene encoding α-amylase was cloned into these two mutants. The new strains SB024 and SB025 also exhibited a five and a sixfold increase in α-amylase activity in the supernatant, respectively, relative to the reference strain SB023. The results demonstrate how DBM screening can serve as a powerful development tool to improve cell factories for the production and secretion of heterologous proteins.

Efficient Production of Nisin A from Low-Value Dairy Side Streams Using a Nonengineered Dairy Lactococcus lactis Strain with Low Lactate Dehydrogenase Activity.

Nisin is commonly used as a biopreservative in foods. For industrial production, nisin-producing Lactococcus lactis strains are usually grown to high cell densities to achieve the highest possible nisin titer. However, accumulation of lactic acid eventually halts production, even in pH-controlled fermentations. Here, we describe a nisin-producing L. lactis strain Ge001, which was obtained after transferring the nisin gene cluster from L. lactis ATCC 11454, by conjugation, into the natural mutant L. lactis RD1M5, with low lactate dehydrogenase activity. The ability of Ge001 to produce nisin was tested using dairy waste as the fermentation substrate. To accommodate redox cofactor regeneration, respiration conditions were used, and to alleviate oxidative stress and to reduce adsorption of nisin onto the producing cells, we found it to be beneficial to add 1 mM Mn2+ and 100 mM Ca2+, respectively. A high titer of 12 084 IU/mL nisin could be reached, which is comparable to the highest titers reported using expensive, rich media. Summing up, we here present a 100% natural, robust, and sustainable approach for producing food-grade nisin and acetoin from readily available dairy waste.

Synthesis of high-titer alka(e)nes in Yarrowia lipolytica is enabled by a discovered mechanism.

Alka(e)nes are ideal fuel components for aviation, long-distance transport, and shipping. They are typically derived from fossil fuels and accounting for 24% of difficult-to-eliminate greenhouse gas emissions. The synthesis of alka(e)nes in Yarrowia lipolytica from CO2-neutral feedstocks represents an attractive alternative. Here we report that the high-titer synthesis of alka(e)nes in Yarrowia lipolytica harboring a fatty acid photodecarboxylase (CvFAP) is enabled by a discovered pathway. We find that acyl-CoAs, rather than free fatty acids (FFAs), are the preferred substrate for CvFAP. This finding allows us to debottleneck the pathway and optimize fermentation conditions so that we are able to redirect 89% of acyl-CoAs from the synthesis of neutral lipids to alka(e)nes and reach titers of 1.47 g/L from glucose. Two other CO2-derived substrates, wheat straw and acetate, are also demonstrated to be effective in producing alka(e)nes. Overall, our technology could advance net-zero emissions by providing CO2-neutral and energy-dense liquid biofuels.

Disruption of the Oxidative Pentose Phosphate Pathway Stimulates High-Yield Production Using Resting Corynebacterium glutamicum in the Absence of External Electron Acceptors.

Identifying and overcoming the limitations preventing efficient high-yield production of chemicals remain important tasks in metabolic engineering. In an attempt to rewire Corynebacterium glutamicum to produce ethanol, we attained a low yield (63% of the theoretical) when using resting cells on glucose, and large amounts of succinate and acetate were formed. To prevent the by-product formation, we knocked out the malate dehydrogenase and replaced the native E3 subunit of the pyruvate dehydrogenase complex (PDHc) with that from Escherichia coli, which is active only under aerobic conditions. However, this tampering resulted in a 10-times-reduced glycolytic flux as well as a greatly increased NADH/NAD+ ratio. When we replaced glucose with fructose, we found that the glycolytic flux was greatly enhanced, which led us to speculate whether the source of reducing power could be the pentose phosphate pathway (PPP) that is bypassed when fructose is metabolized. Indeed, after shutting down the PPP by deleting the zwf gene, encoding glucose-6-phosphate dehydrogenase, the ethanol yield on glucose increased significantly, to 92% of the theoretical. Based on that, we managed to rechannel the metabolism of C. glutamicum into d-lactate with high yield, 98%, which is the highest that has been reported. It is further demonstrated that the PPP-inactivated platform strain can offer high-yield production of valuable chemicals using lactose contained in dairy waste as feedstock, which paves a promising way for potentially turning dairy waste into a valuable product.IMPORTANCE The widely used industrial workhorse C. glutamicum possesses a complex anaerobic metabolism under nongrowing conditions, and we demonstrate here that the PPP in resting C. glutamicum is a source of reducing power that can interfere with otherwise redox-balanced metabolic pathways and reduce yields of desired products. By harnessing this physiological insight, we employed the PPP-inactivated platform strains to produce ethanol, d-lactate, and alanine using the dairy waste whey permeate as the feedstock. The production yield was high, and our results show that inactivation of the PPP flux in resting cells is a promising strategy when the aim is to use nongrowing C. glutamicum cells for producing valuable compounds. Overall, we describe the benefits of disrupting the oxidative PPP in nongrowing C. glutamicum and provide a feasible approach toward waste valorization.

From Waste to Taste-Efficient Production of the Butter Aroma Compound Acetoin from Low-Value Dairy Side Streams Using a Natural (Nonengineered) Lactococcus lactis Dairy Isolate.

Lactococcus lactis subsp. lactis biovar diacetylactis is widely used in dairy fermentations as it can form the butter aroma compounds acetoin and diacetyl from citrate in milk. Here, we explore the possibility of producing acetoin from the more abundant lactose. Starting from a dairy isolate of L. lactis biovar diacetylactis, we obtained a series of mutants with low lactate dehydrogenase (ldh) activity. One isolate, RD1M5, only had a single insertion mutation in the ldh gene compared to its parental strain as revealed by whole genome resequencing. We tested the ability of RD1M5 to produce acetoin in milk. With aeration, all the lactose could be consumed, and the only product was acetoin. In a simulated cheese fermentation, a 50% increase in acetoin concentration could be achieved. RD1M5 turned out to be an excellent cell factory for acetoin and was able to convert lactose in dairy waste into acetoin with high titer (41 g/L) and high yield (above 90% of the theoretical yield). Summing up, RD1M5 was found to be highly robust and to grow excellently in milk or dairy waste. Being natural in origin opens up for applications within dairies as well as for safe production of food-grade acetoin from low-cost substrates.

No more cleaning up - Efficient lactic acid bacteria cell catalysts as a cost-efficient alternative to purified lactase enzymes.

ß-galactosidases, commonly referred to as lactases, are used for producing lactose-free dairy products. Lactases are usually purified from microbial sources, which is a costly process. Here, we explored the potential that lies in using whole cells of a food-grade dairy lactic acid bacterium, Streptococcus thermophilus, as a substitute for purified lactase. We found that S. thermophilus cells, when treated with the antimicrobial peptide nisin, were able to hydrolyze lactose efficiently. The rate of hydrolysis increased with temperature; however, above 50 °C, stability was compromised. Different S. thermophilus strains were tested, and the best candidate was able to hydrolyze 80% of the lactose in a 50 g/L solution in 4 h at 50 °C, using only 0.1 g/L cells (dry weight basis). We demonstrated that it was possible to grow the cell catalyst on dairy waste, and furthermore, that a cell-free supernatant of a culture of a nisin-producing Lactococcus lactis strain could be used instead of purified nisin, which reduced cost of use significantly. Finally, we tested the cell catalysts in milk, where lactose also was efficiently hydrolyzed. The method presented is natural and low-cost, and allows for production of clean-label and lactose-free dairy products without using commercial enzymes from recombinant microorganisms. KEY POINTS: • Nisin-permeabilized Streptococcus thermophilus cells can hydrolyze lactose efficiently. • A low-cost and more sustainable alternative to purified lactase enzymes. • Reduction of overall sugar content. • Clean-label production of lactose-free dairy products.

Harnessing Adaptive Evolution to Achieve Superior Mannitol Production by Lactococcus lactis Using Its Native Metabolism.

Mannitol can be obtained as a by-product of certain heterolactic lactic acid bacteria, when grown on substrates containing fructose. Lactococcus lactis, a homolactic lactic acid bacterium, normally does not form mannitol but can be persuaded into doing so by expressing certain foreign enzyme activities. In this study, we find that L. lactis has an inherent capacity to form mannitol from glucose. By adaptively evolving L. lactis or derivatives blocked in NAD+ regenerating pathways, we manage to accelerate growth on mannitol. When cells of the adapted strains are resuspended in buffer containing glucose, 4-58% of the glucose metabolized is converted into mannitol, in contrast to nonadapted strains. The highest conversion was obtained for a strain lacking all major NAD+ regenerating pathways. Mannitol had an inhibitory effect on the conversion, which we speculated was due to the mannitol uptake system. After its inactivation, 60% of the glucose was converted into mannitol by cells suspended in glucose buffer. Using a two-stage setup, where biomass first was accumulated by aerated culturing, followed by a nonaerated phase (static conditions), it was possible to obtain 6.1 g/L mannitol, where 60% of the glucose had been converted into mannitol, which is the highest yield reported for L. lactis.

Complete Genome Sequence of Lactococcus lactis subsp. lactis bv. diacetylactis SD96.

The genome of Lactococcus lactis subsp. lactis bv. diacetylactis SD96, a strain used for cheese production, is presented. SD96 is refractory to phage attack, which is a desired property for starter bacteria. Its 10 plasmids provide industrially important traits, such as lactose and citrate metabolism, proteolytic activity, and phage resistance.

Harnessing biocompatible chemistry for developing improved and novel microbial cell factories.

White biotechnology relies on the sophisticated chemical machinery inside living cells for producing a broad range of useful compounds in a sustainable and environmentally friendly way. However, despite the impressive repertoire of compounds that can be generated using white biotechnology, this approach cannot currently fully replace traditional chemical production, often relying on petroleum as a raw material. One challenge is the limited number of chemical transformations taking place in living organisms. Biocompatible chemistry, that is non-enzymatic chemical reactions taking place under mild conditions compatible with living organisms, could provide a solution. Biocompatible chemistry is not a novel invention, and has since long been used by living organisms. Examples include Fenton chemistry, used by microorganisms for degrading plant materials, and manganese or ketoacids dependent chemistry used for detoxifying reactive oxygen species. However, harnessing biocompatible chemistry for expanding the chemical repertoire of living cells is a relatively novel approach within white biotechnology, and it could potentially be used for producing valuable compounds which living organisms otherwise are not able to generate. In this mini review, we discuss such applications of biocompatible chemistry, and clarify the potential that lies in using biocompatible chemistry in conjunction with metabolically engineered cell factories for cheap substrate utilization, improved cell physiology, efficient pathway construction and novel chemicals production.

Efficient production of α-acetolactate by whole cell catalytic transformation of fermentation-derived pyruvate.

BACKGROUND: Diacetyl provides the buttery aroma in products such as butter and margarine. It can be made via a harsh set of chemical reactions from sugarcane bagasse, however, in dairy products it is normally formed spontaneously from α-acetolactate, a compound generated by selected lactic acid bacteria in the starter culture used. Due to its bacteriostatic properties, it is difficult to achieve high levels of diacetyl by fermentation. Here we present a novel strategy for producing diacetyl based on whole-cell catalysis, which bypasses the toxic effects of diacetyl. RESULTS: By expressing a robust α-acetolactate synthase (ALS) in a metabolically optimized Lactococcus lactis strain we obtained a whole-cell biocatalyst that efficiently converted pyruvate into α-acetolactate. After process optimization, we achieved a titer for α-acetolactate of 172 ± 2 mM. Subsequently we used a two-stage production setup, where pyruvate was produced by an engineered L. lactis strain and subsequently used as the substrate for the biocatalyst. Using this approach, 122 ± 5 mM and 113 ± 3 mM α-acetolactate could be made from glucose or lactose in dairy waste, respectively. The whole-cell biocatalyst was robust and fully active in crude fermentation broth containing pyruvate. CONCLUSIONS: An efficient approach for converting sugar into α-acetolactate, via pyruvate, was developed and tested successfully. Due to the anaerobic conditions used for the biotransformation, little diacetyl was generated, and this allowed for efficient biotransformation of pyruvate into α-acetolactate, with the highest titers reported to date. The use of a two-step procedure for producing α-acetolactate, where non-toxic pyruvate first is formed, and subsequently converted into α-acetolactate, also simplified the process optimization. We conclude that whole cell catalysis is suitable for converting lactose in dairy waste into α-acetolactate, which favors resource utilization.