Paula Gaspar

Engineering Lactococcus lactis for Production of Mannitol: High Yields from Food-Grade Strains Deficient in Lactate Dehydrogenase and the Mannitol Transport System

ABSTRACT Mannitol is a sugar polyol claimed to have health-promoting properties. A mannitol-producing strain of Lactococcus lactis was obtained by disruption of two genes of the phosphoenolpyruvate (PEP)-mannitol phosphotransferase system (PTSMtl). Genes mtlA and mtlF were independently deleted by double-crossover recombination in strain L. lactis FI9630 (a food-grade lactate dehydrogenase-deficient strain derived from MG1363), yielding two mutant (ΔldhΔmtlA and ΔldhΔmtlF) strains. The new strains, FI10091 and FI10089, respectively, do not possess any selection marker and are suitable for use in the food industry. The metabolism of glucose in nongrowing cell suspensions of the mutant strains was characterized by in vivo 13C-nuclear magnetic resonance. The intermediate metabolite, mannitol-1-phosphate, accumulated intracellularly to high levels (up to 76 mM). Mannitol was a major end product, one-third of glucose being converted to this hexitol. The double mutants, in contrast to the parent strain, were unable to utilize mannitol even after glucose depletion, showing that mannitol was taken up exclusively by PEP-PTSMtl. Disruption of this system completely blocked mannitol transport in L. lactis, as intended. In addition to mannitol, approximately equimolar amounts of ethanol, 2,3-butanediol, and lactate were produced. A mixed-acid fermentation (formate, ethanol, and acetate) was also observed during growth under controlled conditions of pH and temperature, but mannitol production was low. The reasons for the alteration in the pattern of end products under nongrowing and growing conditions are discussed, and strategies to improve mannitol production during growth are proposed.

From physiology to systems metabolic engineering for the production of biochemicals by lactic acid bacteria.

The lactic acid bacteria (LAB) are a functionally related group of low-GC Gram-positive bacteria known essentially for their roles in bioprocessing of foods and animal feeds. Due to extensive industrial use and enormous economical value, LAB have been intensively studied and a large body of comprehensive data on their metabolism and genetics was generated throughout the years. This knowledge has been instrumental in the implementation of successful applications in the food industry, such as the selection of robust starter cultures with desired phenotypic traits. The advent of genomics, functional genomics and high-throughput experimentation combined with powerful computational tools currently allows for a systems level understanding of these food industry workhorses. The technological developments in the last decade have provided the foundation for the use of LAB in applications beyond the classic food fermentations. Here we discuss recent metabolic engineering strategies to improve particular cellular traits of LAB and to design LAB cell factories for the bioproduction of added value chemicals.

High Yields of 2,3-Butanediol and Mannitol in Lactococcus lactis through Engineering of NAD+ Cofactor Recycling

ABSTRACT Manipulation of NADH-dependent steps, and particularly disruption of the las-located lactate dehydrogenase (ldh) gene in Lactococcus lactis, is common to engineering strategies envisaging the accumulation of reduced end products other than lactate. Reverse transcription-PCR experiments revealed that three out of the four genes assigned to lactate dehydrogenase in the genome of L. lactis, i.e., the ldh, ldhB, and ldhX genes, were expressed in the parental strain MG1363. Given that genetic redundancy is often a major cause of metabolic instability in engineered strains, we set out to develop a genetically stable lactococcal host tuned for the production of reduced compounds. Therefore, the ldhB and ldhX genes were sequentially deleted in L. lactis FI10089, a strain with a deletion of the ldh gene. The single, double, and triple mutants, FI10089, FI10089ΔldhB, and FI10089ΔldhBΔldhX, showed similar growth profiles and displayed mixed-acid fermentation, ethanol being the main reduced end product. Hence, the alcohol dehydrogenase-encoding gene, the adhE gene, was inactivated in FI10089, but the resulting strain reverted to homolactic fermentation due to induction of the ldhB gene. The three lactate dehydrogenase-deficient mutants were selected as a background for the production of mannitol and 2,3-butanediol. Pathways for the biosynthesis of these compounds were overexpressed under the control of a nisin promoter, and the constructs were analyzed with respect to growth parameters and product yields under anaerobiosis. Glucose was efficiently channeled to mannitol (maximal yield, 42%) or to 2,3-butanediol (maximal yield, 67%). The theoretical yield for 2,3-butanediol was achieved. We show that FI10089ΔldhB is a valuable basis for engineering strategies aiming at the production of reduced compounds.

Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae

ABSTRACT Phenylalanine and tyrosine ammonia-lyases form cinnamic acid and p-coumaric acid, which are precursors of a wide range of aromatic compounds of biotechnological interest. Lack of highly active and specific tyrosine ammonia-lyases has previously been a limitation in metabolic engineering approaches. We therefore identified 22 sequences in silico using synteny information and aiming for sequence divergence. We performed a comparative in vivo study, expressing the genes intracellularly in bacteria and yeast. When produced heterologously, some enzymes resulted in significantly higher production of p-coumaric acid in several different industrially important production organisms. Three novel enzymes were found to have activity exclusively for phenylalanine, including an enzyme from the low-GC Gram-positive bacterium Brevibacillus laterosporus, a bacterial-type enzyme from the amoeba Dictyostelium discoideum, and a phenylalanine ammonia-lyase from the moss Physcomitrella patens (producing 230 μM cinnamic acid per unit of optical density at 600 nm [OD600]) in the medium using Escherichia coli as the heterologous host). Novel tyrosine ammonia-lyases having higher reported substrate specificity than previously characterized enzymes were also identified. Enzymes from Herpetosiphon aurantiacus and Flavobacterium johnsoniae resulted in high production of p-coumaric acid in Escherichia coli (producing 440 μM p-coumaric acid OD600 unit−1 in the medium) and in Lactococcus lactis. The enzymes were also efficient in Saccharomyces cerevisiae, where p-coumaric acid accumulation was improved 5-fold over that in strains expressing previously characterized tyrosine ammonia-lyases.

Enhancement of trehalose production in dairy propionibacteria through manipulation of environmental conditions

We have shown that the ability to produce trehalose is widespread within the genus Propionibacterium. Eighteen strains isolated from dairy sources were screened for trehalose synthesis; the effect of environmental conditions on trehalose production was evaluated in Propionibacterium freudenreichii ssp. shermanii NIZO B365, a strain that accumulated high amounts of this disaccharide. Lactose was the best carbohydrate source for trehalose production, whereas lactate, the substrate that led to the highest specific growth rate, was a poor precursor. Trehalose was consumed after exhaustion of the carbon source in the medium, suggesting its role as a reserve compound. The production of trehalose was not affected by lowering the growth temperature from 30 to 20 degrees C. On the other hand, the maximum trehalose accumulation increased from about 200 to 400 mg of trehalose/g of cell protein upon decreasing the pH from 7.0 to 4.7, by increasing the concentration of NaCl to 2% (w/v), or during growth under aerobic conditions (50% air saturation, 24 microM O(2), pH 7.0). In the absence of NaCl, trehalose accumulated concomitantly with growth, but an increase in salinity triggered a high trehalose production already in the early exponential growth phase. The data provide evidence for a dual function of trehalose as a reserve compound and as a stress-response metabolite. Moreover, P. freudenreichii ssp. shermanii NIZO B365 was able to produce high levels of trehalose in skim milk, which is promising for the implementation of fermented dairy products.

The lactate dehydrogenases encoded by the ldh and ldhB genes in Lactococcus lactis exhibit distinct regulation and catalytic properties - comparative modeling to probe the molecular basis.

Lactococcus lactis FI9078, a construct carrying a disruption of the ldh gene, converted approximately 90% of glucose into lactic acid, like the parental strain MG1363. This unexpected lactate dehydrogenase activity was purified, and ldhB was identified as the gene encoding this protein. The activation of ldhB was explained by the insertion of an IS905‐like element that created a hybrid promoter in the intergenic region upstream of ldhB. The biochemical and kinetic properties of this alternative lactate dehydrogenase (LDHB) were compared to those of the ldh‐encoded enzyme (LDH), purified from the parental strain. In contrast to LDH, the affinity of LDHB for NADH and the activation constant for fructose 1,6‐bisphosphate were strongly dependent on pH. The activation constant increased 700‐fold, whereas the Km for NADH increased more than 10‐fold, in the pH range 5.5–7.2. The two enzymes also exhibited different pH profiles for maximal activity. Moreover, inorganic phosphate acted as a strong activator of LDHB. The impact of replacing LDH by LDHB on the physiology of L. lactis was assessed by monitoring the evolution of the pools of glycolytic intermediates and cofactors during the metabolism of glucose by in vivo NMR. Structural analysis by comparative modeling of the two proteins showed that LDH has a slightly larger negative charge than LDHB and a greater concentration of positive charges at the interface between monomers. The calculated pH titration curves of the catalytic histidine residues explain why LDH maintains its activity at low pH as compared to LDHB, the histidines in LDH showing larger pH titration ranges.

Effect of Oxygen on Glucose Metabolism: Utilization of Lactate in Staphylococcus Aureus as Revealed by In Vivo NMR Studies.

The ability to successfully adapt to changing host conditions is crucial for full virulence of bacterial pathogens. Staphylococcus aureus has to cope with fluctuating oxygen concentrations during the course of infection. Hence, we studied the effect of oxygen on glucose metabolism in non-growing S. aureus COL-S cells by in vivo 13C-NMR. Glucose catabolism was probed at different oxygen concentrations in suspensions of cells grown aerobically (direct effects on metabolism) or anaerobically (transcriptional adjustment to oxygen deprivation). In aerobically-grown cells, the rate of glucose consumption diminished progressively with decreasing oxygen concentrations. Additionally, oxygen deprivation resulted in biphasic glucose consumption, with the second phase presenting a higher rate. The fructose-1,6-bisphosphate pool peaked while glucose was still abundant, but the transient maximum varied with the oxygen concentration. As oxygen became limiting mannitol/mannitol-1-phosphate were detected as products of glucose catabolism. Under anoxic conditions, accumulation of mannitol-1-phosphate ceased with the switch to higher glucose consumption rates, which implies the activation of a more efficient means by which NAD+ can be regenerated. The distribution of end-products deriving from glucose catabolism was dramatically affected by oxygen: acetate increased and lactate decreased with the oxygen concentration; ethanol was formed only anaerobically. Moreover, oxygen promoted the energetically favourable conversion of lactate into acetate, which was particularly noticeable under fully oxygenated conditions. Interestingly, under aerobiosis growing S. aureus cells also converted lactate to acetate, used simultaneously glucose and lactate as substrates for growth, and grew considerably well on lactate-medium. We propose that the efficient lactate catabolism may endow S. aureus with a metabolic advantage in its ecological niche.

The Lcn972 Bacteriocin-Encoding Plasmid pBL1 Impairs Cellobiose Metabolism in Lactococcus lactis

ABSTRACT pBL1 is a Lactococcus lactis theta-replicating 10.9-kbp plasmid that encodes the synthetic machinery of the bacteriocin Lcn972. In this work, the transcriptomes of exponentially growing L. lactis strains with and without pBL1 were compared. A discrete response was observed, with a total of 10 genes showing significantly changed expression. Upregulation of the lactococcal oligopeptide uptake (opp) system was observed, which was likely linked to a higher nitrogen demand required for Lcn972 biosynthesis. Strikingly, celB, coding for the membrane porter IIC of the cellobiose phosphoenolpyruvate-dependent phosphotransferase system (PTS), and the upstream gene llmg0186 were downregulated. Growth profiles for L. lactis strains MG1363, MG1363/pBL1, and MG1363 ΔcelB grown in chemically defined medium (CDM) containing cellobiose confirmed slower growth of MG1363/pBL1 and MG1363 ΔcelB, while no differences were observed with growth on glucose. The presence of pBL1 shifted the fermentation products toward a mixed acid profile and promoted substantial changes in intracellular pool sizes for glycolytic intermediates in cells growing on cellobiose as determined by high-pressure liquid chromatography (HPLC) and nuclear magnetic resonance (NMR). Overall, these data support the genetic evidence of a constriction in cellobiose uptake. Notably, several cell wall precursors accumulated, while other UDP-activated sugar pools were lower, which could reflect rerouting of precursors toward the production of structural or storage polysaccharides. Moreover, cells growing slowly on cellobiose and those lacking celB were more tolerant to Lcn972 than cellobiose-adapted cells. Thus, downregulation of celB could help to build up a response against the antimicrobial activity of Lcn972, enhancing self-immunity of the producer cells.

An extended dynamic model of Lactococcus lactis metabolism for mannitol and 2,3-butanediol production

Biomedical research and biotechnological production are greatly benefiting from the results provided by the development of dynamic models of microbial metabolism. Although several kinetic models of Lactococcus lactis (a Lactic Acid Bacterium (LAB) commonly used in the dairy industry) have been developed so far, most of them are simplified and focus only on specific metabolic pathways. Therefore, the application of mathematical models in the design of an engineering strategy for the production of industrially important products by L. lactis has been very limited. In this work, we extend the existing kinetic model of L. lactis central metabolism to include industrially relevant production pathways such as mannitol and 2,3-butanediol. In this way, we expect to study the dynamics of metabolite production and make predictive simulations in L. lactis. We used a system of ordinary differential equations (ODEs) with approximate Michaelis-Menten-like kinetics for each reaction, where the parameters were estimated from multivariate time-series metabolite concentrations obtained by our team through in vivo Nuclear Magnetic Resonance (NMR). The results show that the model captures observed transient dynamics when validated under a wide range of experimental conditions. Furthermore, we analyzed the model using global perturbations, which corroborate experimental evidence about metabolic responses upon enzymatic changes. These include that mannitol production is very sensitive to lactate dehydrogenase (LDH) in the wild type (W.T.) strain, and to mannitol phosphoenolpyruvate: a phosphotransferase system (PTS(Mtl)) in a LDH mutant strain. LDH reduction has also a positive control on 2,3-butanediol levels. Furthermore, it was found that overproduction of mannitol-1-phosphate dehydrogenase (MPD) in a LDH/PTS(Mtl) deficient strain can increase the mannitol levels. The results show that this model has prediction capability over new experimental conditions and offers promising possibilities to elucidate the effect of alterations in the main metabolism of L. lactis, with application in strain optimization.

BacHBerry: BACterial Hosts for production of Bioactive phenolics from bERRY fruits

BACterial Hosts for production of Bioactive phenolics from bERRY fruits (BacHBerry) was a 3-year project funded by the Seventh Framework Programme (FP7) of the European Union that ran between November 2013 and October 2016. The overall aim of the project was to establish a sustainable and economically-feasible strategy for the production of novel high-value phenolic compounds isolated from berry fruits using bacterial platforms. The project aimed at covering all stages of the discovery and pre-commercialization process, including berry collection, screening and characterization of their bioactive components, identification and functional characterization of the corresponding biosynthetic pathways, and construction of Gram-positive bacterial cell factories producing phenolic compounds. Further activities included optimization of polyphenol extraction methods from bacterial cultures, scale-up of production by fermentation up to pilot scale, as well as societal and economic analyses of the processes. This review article summarizes some of the key findings obtained throughout the duration of the project.