Higher titer hyaluronic acid production in recombinant Lactococcus lactis

Abstract

Hyaluronic acid (HA) is a natural biopolymer and has long been attracting the attention of biotechnology industry due to its various biological functions. HA production with natural producer Streptococcus equi subsp. zooepidemicus has not been preferred because it has many drawbacks due to its pathogenicity. Therefore, in the present study, Streptococcal hyaluronan synthase gene (hasA) was introduced and expressed in Lactococcus lactis, through the auto inducible NICE system and the effect of nisin amount on the production of HA was examined. Newly constructed plasmid was transformed into L. lactis CES15, produced 6.09 g/l HA in static flask culture after three hours of induction period with initial 7.5 ng/ml nisin concentration within total six hours of incubation. The highest HA titer value ever was reported for recombinant HA-producing L. lactis by examining the effect of initial nisin concentration. We have shown that initial nisin concentration, which used to initiate the auto-inducing mechanism of NICE system and consequently hyaluronan synthase expression, has a direct and significant effect on the produced HA amount. Recently constructed recombinant L. lactis CES15 strain provide significant advantages for industrial HA production than those in literature in terms of production time, energy demand, carbon usage, and safety status.     

Repeated fed-batch lactic acid production in a packed bed-stirred fermentor system using a pH feedback feeding method

Lactic acid production by repeated fed-batch fermentation using free and immobilized cells of Lactobacillus lactis-11 in a packed bed-stirred fermentor (PBSF) system filled with different support materials including ceramic beads, macro-activated carbon cylinders and glass fiber balls was investigated. The results showed that the optimal support materials were the ceramic beads with diameters of 1–2 mm. Compared with the free cell fermentation system, lactic acid production and volumetric productivity in the PBSF system increased by 16.6 and 12.5%, respectively. Though the concentration of free cells decreased sharply, lactic acid production remained stable in five consecutive fed-batch runs using the PBSF system. pH gradients, immobilized cell concentration and mass diffusion in the packed bed were all affected by the recirculation rate of the culture broth. Maximum lactic acid production, productivity and yield occurred at a recirculation rate of 50 mL min⁻¹.     

HtrA Is Essential for Efficient Secretion of Recombinant Proteins by Lactococcus lactis

HtrA is a unique protease on the extracellular surface of Lactococcus lactis. It is known to take part in the proteolysis of many secreted recombinant proteins, and the mutation of htrA can lead to the complete stabilization of recombinant proteins. In this work, we have shown that htrA mutation also leads to significant reduction of the efficiency of recombinant-protein secretion. We also show that the level of HtrA can be lowered by the suppression of the acid tolerance response (ATR) in L. lactis. Instead of using an L. lactis htrA mutant, the reduction of the HtrA level in wild-type recombinant cultures of L. lactis by ATR suppression may serve as a better strategy for the production of secreted recombinant proteins.     

Influence of pH on growth and bacteriocin production byLactococcus lactis subsp.lactis 14ONWC during batch fermentation

The influence of pH on growth, and lactic acid and bacteriocin production byLactococcus lactis subsp.lactis 140 NWC was studied during batch fermentation in a lactose-based complex medium. Growth and lactic acid production were modelled using a simple logistic equation while substrate consumption was found to be a function growth and lactic acid production rate. The optimal pH for growth and lactic acid production was between 6.0 and 6.5. Bacteriocin production showed primary metabolite kinetics. pH had a dramatic effect on the production of the bacteriocin, lactococcin 140. A maximum activity of 15.4 × 10⁶ AU (arbitrary units) 1^–1 was obtained after 7 h at pH 5.5. Maximum bacteriocin activity was achieved before the end of growth and was followed by a decrease in activity, which was due to adsorption to the cells of the producing organism, possibly followed by degradation by specific proteases. Both bacteriocin production and degradation rates were higher at pH 5.0 and 5.5, resulting in sharper activity peaks than at pH 6.0 or 6.5. On the basis of the experimental results a qualitative model for bacteriocin production is proposed.     

Continuous <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid production by a novelthermotolerant <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> QU 41

We isolated and characterized a d-lactic acid-producing lactic acid bacterium (d-LAB), identified as Lactobacillus delbrueckii subsp. lactis QU 41. When compared to Lactobacillus coryniformis subsp. torquens JCM 1166 ^T and L. delbrueckii subsp. lactis JCM 1248 ^T, which are also known as d-LAB, the QU 41 strain exhibited a high thermotolerance and produced d-lactic acid at temperatures of 50 °C and higher. In order to optimize the culture conditions of the QU 41 strain, we examined the effects of pH control, temperature, neutralizing reagent, and initial glucose concentration on d-lactic acid production in batch cultures. It was found that the optimal production of 20.1 g/l d-lactic acid was acquired with high optical purity (>99.9% of d-lactic acid) in a pH 6.0-controlled batch culture, by adding ammonium hydroxide as a neutralizing reagent, at 43 °C in MRS medium containing 20 g/l glucose. As a result of product inhibition and low cell density, continuous cultures were investigated using a microfiltration membrane module to recycle flow-through cells in order to improve d-lactic acid productivity. At a dilution rate of 0.87 h⁻¹, the high cell density continuous culture exhibited the highest d-lactic acid productivity of 18.0 g/l/h with a high yield (ca. 1.0 g/g consumed glucose) and a low residual glucose (<0.1 g/l) in comparison with systems published to date.     

Culture method to enhance the productivity of hepatitis B surface antigen (pre S2 + S Ag) with recombinant Saccharomyces cerevisiae

Summary The optimum culture condition for the production of recombinant hepatitis B surface antigen (pre S2 + S Ag) was investigated. The productivity of hepatitis B surface antigen in recombinant Saccharomyces cerevisiae, which contains GAP (glyceraldehyde - 3 - phosphate dehydrogenase) promoter, was increased by 25% when the pH was changed from 5 to 8 during the stationary growth phase and by 46% when phenyl methyl sulfonyl fluoride was added at 0.344mM in the stationary phase.     

Increased erythritol production in fed-batch cultures of Torula sp. by controlling glucose concentration

The effect of glucose concentration on erythritol production by Torula sp. was investigated. The maximum volumetric productivity of erythritol was obtained at an initial glucose concentration of 300 g l⁻¹ in batch culture. The volumetric productivity was maximal at a controlled glucose concentration of 225 g l⁻¹, reducing the lag time of the erythritol production. A fed-batch culture was established with an initial glucose concentration of 300 g l⁻¹ and with a controlled glucose concentration of 225 g l⁻¹ in medium containing phytic acid as a phosphate source. In this fed-batch culture, a final erythritol production of 192 g l⁻¹ was obtained from 400 g l⁻¹ glucose in 88 h. This corresponded to a volumetric productivity of 2.26 g l⁻¹ h⁻¹ and a 48% yield. Journal of Industrial Microbiology & Biotechnology (2001) 26, 248–252. Received 26 September 2000/ Accepted in revised form 16 January 2001     

Improvement of nisin production in pH feed-back controlled,fed-batch culture by <Emphasis Type="Italic">Lactococcus lactis</Emphasis>subsp<Emphasis Type="Italic">. lactis</Emphasis>

Nisin production by Lactococcus lactis subsp. lactisin fed-batch culture was doubled by using a pH feed-back controlled method. Sucrose concentration was controlled at 10 g l^–1 giving 5010 IU nisin ml^–1 compared to 2660 IU nisin ml^–1 in batch culture.     

Production of Superoxide Dismutase from Streptococcus lactis Using a Bioreactor with a Microfiltration Module

The performance of a bioreactor with a microfiltration module for the production of an intracellular enzyme, superoxide dismutase (SOD), by Streptococcus lactis is described. The fermentation system involving the bioreactor enables the continuous removal of metabolites inhibitory for cell growth and the complete recycling of the cells to the bioreactor. In a fed-batch (FB) culture with filtration, in which the main metabolite, lactic acid, in the culture broth was maintained at a low concentration, S. lactis was cultivated to the high concentration of 15.5 g-dry cells/1. The SOD content of the cells remained at almost a constant level throughout the cultivation and the productivity of SOD as well as cells per unit time was 4.3-fold as high as that in the case of a conventional batch culture without filtration. Repeating the FB culture with filtration enhanced the productivities of SOD and cells further, as compared with those in the case of the FB culture with filtration.     

Influence of the phosphorus and nitrogen source on nisin production in Lactococcus lactis subsp. lactis batch fermentations using a complex medium

The influence of different phosphorus and nitrogen sources on Lactococcus lactis subsp. lactis NIZO 22186 growth and nisin production was studied in batch fermentations using a complex medium. KH₂PO₄ was found to be the best phosphorus source for nisin production. Increasing initial phosphate concentrations from 0 to 5% KH₂PO₄ exerted a double effect, creating favourable pH conditions and particularly stimulating the nisin production levels, which were highest at 5% KH₂PO₄. Up to now, no such high initial phosphate concentrations have been reported for the production of other antibiotics or bacteriocins. Nisin, a lanthionine-containing peptide antibiotic with bacteriocin properties, clearly behaved as a primary metabolite, since its formation was linked with active growth and was not suppressed by phosphate concentrations up to 5%. A complex medium supplemented with cotton seed meal as nitrogen source also gave very high nisin yields. Correspondence to: L. De Vuyst     

Production of lactic acid from date juice extract with free cells of single and mixed cultures of Lactobacillus casei and Lactococcus lactis

The production of lactic acid from date juice by single and mixed cultures of Lactobacillus casei and Lactococcus lactis was investigated. In the present conditions, the highest concentration of lactic acid (60.3 g l⁻¹) was obtained in the mixed culture system while in single culture fermentations of Lactobacillus casei or Lactococcus lactis, the maximum concentration of lactic acid was 53 and 46 g l⁻¹, respectively. In the case of single Lactobacillus casei or Lactococcus lactis, the total percentage of glucose and fructose utilized were 82.2; 94.4% and 93.8; 60.3%, respectively, whereas in the case of mixed culture, the total percentage of glucose and fructose were 96 and 100%, respectively. These results showed that the mixed culture system gave better results than single cultures regarding lactic acid concentration, and sugar consumption.     

Generalized model of the effect of pH on lactate fermentation and citrate bioconversion in Lactococcus lactis ssp. Lactis biovar. diacetylactis

An aroma-imparting mesophilic lactic starter (Lactococcus lactis ssp. lactis biovar. diacetylactis) was studied in batch culture in medium with 50 g·l^–1 lactose and 2 g·l^–1 citrate. The effect of pH on the physiology of growth and the production of flavour compounds was investigated with a mathematical model. The specific rates of growth and of lactose fermentation obeyed a law of non-competitive inhibition by lactic acid produced, inhibition increasing as the pH of the medium decreased. The pH thus acted indirectly by increasing the proportion of non-dissociated lactic acid, identified as the inhibiting form of lactic acid. The generalized model, taking into account the effect of pH, was tested using fermentations at pH controlled at different values (4.5–6.5), as well as with a fermentation conducted at non-regulated pH. These simulations supported the working hypotheses. The effect of pH on the fermentation of citric acid resulted in an increase in the maximal specific rate of citrate utilization, in the bioconversion yield, and in the constant of diacetyl and acetoin reduction at acid pH. The production of flavour compounds is a complex phenomenon resulting from the interaction of pH, citric acid concentration, and the physiological state of the cells. These results are discussed with respect to the use of this strain in the preparation of manufactured dairy products.     

Application of medium optimization tools for improving recombinant human interferon gamma production from Kluyveromyces lactis

The present study is focused upon improving biomass of Kluyveromyces lactis cells expressing recombinant human interferon gamma (hIFN-γ), with the aim of augmenting hIFN-γ concentration using statistical and artificial intelligence approach. Optimization of medium components viz., lactose, yeast extract, and trace elements were performed with Box–Behnken design (BBD) and artificial neural network linked genetic algorithm (ANN-GA) for maximizing biomass of recombinant K. lactis (objective function). The studies resulted over 1.5-fold improvement in the biomass concentration in a medium composed of 80?g/L lactose, 10.353?g/L yeast extract, and 15?mL/L trace elements as compared with initial biomass value. In the same study hIFN-γ concentration reached 881?µg/L which was 2.28-fold higher as compared with initial hIFN-γ concentration obtained in unoptimized medium. Further the batch fermentation study displayed mixed growth associated kinetics with the maximum hIFN-γ production rate of 1.1?mg/L. BBD and ANN-GA, both optimization techniques predicted a higher lactose concentration was clearly beneficial for augmenting K. lactis biomass which in turn increased hIFN-γ concentration.     

Improved algal oil production from Botryococcus braunii by feeding nitrate and phosphate in an airlift bioreactor

To improve biomass and microalgal oil production of Botryococcus braunii, fed‐batch culture was investigated in an airlift photobioreactor. The optimal feeding time of the fed‐batch culture was after 15 days of cultivation, where 1.82 g/L of the microalgal biomass was obtained in the batch culture. Nitrate nutrient was the restrictive factor for the fed‐batch cultivation while phosphate nutrient with high concentration did not affect the microalgal growth. The optimal mole ratio of nitrate to phosphate was 34.7:1, where nitrate concentration reached the initial level and phosphate concentration was one quarter of its initial level. With one feeding, the biomass of B. braunii reached 2.56 g/L after 18 days. Two feedings in 2‐day interval enhanced the biomass production up to 2.87 g/L after 19 days of cultivation. The hydrocarbon content in dry biomass of B. braunii kept at high level of 64.3% w/w. Compared with the batch culture, biomass production and hydrocarbon productivity of B. braunii were greatly improved by the strategic fed‐batch cultivation.     

Process development of itaconic acid production by a natural wild type strain of <Emphasis Type="Italic">Aspergillus terreus</Emphasis> to reach industrially relevant final titers

Itaconic acid is a promising organic acid and is commercially produced by submerged fermentation of Aspergillus terreus. The cultivation process of the sensitive filamentous fungus has been studied intensively since 1932, with respect to fermentation media components, oxygen supply, shearing rate, pH value, or culture method. Whereas increased final titers were achieved over the years, the productivity has so far remained quite low. In this study, the impact of the pH on the itaconic acid production was investigated in detail. The pH during the growth and production phase had a significant influence on the final itaconic acid concentration and pellet diameter. The highest itaconic acid concentration of 160 g/L was achieved at a 1.5-L scale within 6.7 days by raising and controlling the pH value to pH 3.4 in the production phase. An ammonia solution and an increased phosphate concentration were used with an itaconic acid yield of 0.46 (w/w) and an overall productivity of 0.99 g/L/h in a fed-batch mode. A cultivation with a lower phosphate concentration resulted in an equal final concentration with an increased yield of 0.58 (w/w) after 11.8 days and an overall productivity of 0.57 g/L/h. This optimized process was successfully transferred from a 1.5-L scale to a 15-L scale. After 9.7 days, comparable pellet morphology and a final concentration of 150 g/L itaconic acid was reached. This paper provides a process strategy to yield a final titer of itaconic acid from a wild-type strain of A. terreus which is in the same range as the well-known citric acid production.     

Continuous culture with complete cell recycle to obtain high cell densities in product inhibited cultures; cultivation ofStreptococcus lactis for production of superoxide dismutase

Summary A cultivation system is described for cultivatingStreptococcus lactis in continuous culture with complete cell recycle. The aim was to obtain high cell densities for the production of su-peroxide dismutase (SOD) while avoiding the growth inhibiting effects of the lactic acid produced. This type of cultivation was performed both at constant and at increasing dilution rates. Comparisons made include those between cell mass productivity and SOD productivity in recycling cultivations and batch cultivations. In the recycling cultivation at increasing dilution rates a cell mass of 19 g/1 was obtained after 22 h of cultivation and the SOD productivity was 43.10³ U/1.h which is four times higher than for batch cultivations. The effect of recyclingS. lactis was also considered and no damage of the microorganisms was observed.     

Acetic acid production from whey lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum

Summary Acetic acid was produced from anaerobic fermentation of lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum at 35° C and pHs between 7.0 and 7.6. Lactose was converted to lactic acid, and then to acetic acid in this mixed culture fermentation. The overall acetic acid yield from lactose was about 95% at pH 7.6 and 90% at pH 7.0. The fermentation rate was also higher at pH 7.6 than at pH 7.0. In batch fermentation of whey permeate containing about 5% lactose at pH 7.6, the concentration of acetic acid reached 20 g/l within 20 h. The production rate then became very slow due to end-product inhibition and high Na⁺ concentration. About 30 g/l acetate and 20 g/l lactate were obtained at a fermentation time of 80 h. However, when diluted whey permeate containing 2.5% lactose was used, all the whey lactose was converted to acetic acid within 30 h by this mixed culture.     

Modelling the influence of pH, temperature, glucose and lactic acid concentrations on the kinetics of lactic acid production by Lactococcus lactis ssp. lactis ATCC 19435 in whole-wheat flour

A kinetic model of the fermentative production of lactic acid from glucose by Lactococcus lactis ssp. lactis ATCC 19435 in whole-wheat flour has been developed. The model consists of terms for substrate and product inhibition as well as for the influence of pH and temperature. Experimental data from fermentation experiments under different physical conditions were used to fit and verify the model. Temperatures above 30 °C and pH levels below 6 enhanced the formation of by-products and d-lactic acid. By-products were formed in the presence of maltose only, whereas d-lactic acid was formed independently of the presence of maltose although the amount formed was greater when maltose was present. The lactic acid productivity was highest between 33 °C and 35 °C and at pH 6. In the concentration interval studied (up to 180 g l⁻¹ glucose and 89  g l⁻¹ lactic acid) simulations showed that both substances were inhibiting. Glucose inhibition was small compared with the inhibition due to lactic acid. Received: 28 October 1997 / Received revision: 3 February 1998 / Accepted: 6 February 1998     

Optimization of erythritol production by Candida magnoliae in fed-batch culture

A two-stage fed-batch process was designed to enhance erythritol productivity by the mutant strain of Candida magnoliae. The first stage (or growth stage) was performed in the fed-batch mode where the growth medium was fed when the pH of the culture broth dropped below 4.5. The second stage (or production stage) was started with addition of glucose powder into the culture broth when the cell mass reached about 75 g dry cell weight l⁻¹. When the initial glucose concentration was adjusted to 400 g l⁻¹ in the production stage, 2.8 g l⁻¹ h⁻¹ of overall erythritol productivity and 41% of erythritol conversion yield were achieved, which represented a fivefold increase in erythritol productivity compared with the simple batch fermentation process. A high glucose concentration in the production phase resulted in formation of organic acids including citrate and butyrate. An increase in dissolved oxygen level caused formation of gluconic acid instead of citric acid. Journal of Industrial Microbiology & Biotechnology (2000) 25, 100–103. Received 25 February 2000/ Accepted in revised form 08 June 2000     

Hyaluronic acid production is enhanced by the additional co-expression of UDP-glucose pyrophosphorylase in Lactococcus lactis

Hyaluronic acid (HA) production was metabolically engineered in Lactococcus lactis by introducing the HA synthetic machinery from the has operon of the pathogenic bacterium Streptococcus zooepidemicus. This study shows that the insertion of uridine diphosphate (UDP)-glucose pyrophosphorylase (hasC) gene in addition to the HA synthase (hasA) and UDP-glucose dehydrogenase (hasB) genes has a significant impact on increasing HA production. The recombinant L. lactis NZ9000 strain transformed with the plasmid pSJR2 (co-expressing hasA and hasB genes only) produced a maximum of 107 mg/l HA in static flask experiments with varying initial glucose concentrations, while the corresponding experiments with the transformant SJR3 (co-expressing hasA, hasB, and hasC genes) gave a maximum yield of 234 mg/l HA. The plasmid cloned with the insertion of the full has operon comprising of five different genes (hasA, hasB, hasC, hasD, and hasE) exhibited structural instability. The HA yield was further enhanced in batch bioreactor experiments with controlled pH and aeration, and a maximum of 1.8 g/l HA was produced by the SJR3 culture.