Abstract: The uptake of glucose by Corynebacterium glutamicum is mainly through the high affinity (Km=0.35 mM) mannose phosphotransferase system (PTS) for glucose. Select mutants that can tolerate 2-deoxyglucose (2DG) and lack PEP dependent glucose transport activity, which can grow on culture media with glucose as a single carbon and energy source. Due to the use of a low affinity (Ks=11 mM) non PTS uptake system, the glucose uptake rate is significantly reduced. Wild type cells grow in media with different osmotic pressures, and their glucose consumption rate and growth rate decrease. When measured under standard conditions, the cells of these cultures showed similar PTS activity. However, when cells were resuspended in buffer solutions with different osmotic pressures, PTS activity showed a correlation with osmotic pressure. This inhibitory effect can explain the observed decrease in sugar absorption and growth rate in high osmotic pressure fermentation medium. However, non PTS glucose transport systems are not affected by the osmotic pressure of the culture medium. Under industrial production conditions, a similar inhibition of sugar transport capacity was observed as the osmotic pressure of the culture medium increased with the accumulation of glutamate. This phenomenon thus limits the fermentation process, as a decrease in specific rate leads to an increase in the proportion of sugar consumed for cell maintenance, accompanied by a decrease in product yield.

Keywords: glucose transport; Osmotic pressure; Glutamic acid fermentation; Corynebacterium glutamicum;

Research content:

1. The effect of osmotic pressure on the activity of PTS glucose uptake system

The PTS activity remains constant within an osmotic pressure of 800 mOsmol/Kg, and then decreases linearly with increasing osmotic pressure. At an osmotic pressure of 1900mOsmol/Kg, the PTS activity was only 15% of its highest level.

2. The effect of osmotic pressure on glutamic acid fermentation

The accumulation of glutamate and secondary metabolites can cause a significant increase in the osmotic pressure of the culture medium. In order to maintain a constant pH value, the addition of alkali (ammonia in this case) further exacerbates the increase in osmotic pressure. This phenomenon was observed in the temperature sensitive strain 2262 of Corynebacterium glutamicum, which can achieve high product concentrations (glutamic acid>85g/L) and osmotic pressures exceeding 2000mOsmol · kg-1. The consumption rate of sugar remains unchanged in the first few hours, and then gradually increases

Gradually decreasing. To test whether the decrease in activity is related to the increase in osmotic pressure caused by the accumulation of glutamate, the contribution of PTS activity to total sugar uptake was measured, and the contribution measured in the wild bacterium Corynebacterium glutamicum ATCC17965 was consistent, at 4.5 mmol · g-1 · h-1. In glutamate producing strains, the total sugar uptake is faster, indicating higher osmotic enzyme activity. The glucose uptake rate measured under non coercive conditions indicates that the contribution of osmotic enzymes in Corynebacterium glutamicum 2262 is as high as 25% of the total glucose uptake. According to the in vitro measurement method of the PTS transport system, the inhibitory effect of osmotic stress on PTS activity was determined. Under glucose supplementation conditions (where the contribution of osmotic enzymes can be ignored) (low residual glucose, i.e. less than 2g · l-1), it can be observed that the decrease in glucose consumption is very close to the predicted decrease in PTS activity (Figure 3a). When using a glucose supplementation strategy that maintains a high concentration (50 g · l-1), the improvement in glucose consumption may be due to the high saturation of osmotic enzyme activity and glucose present in the reactor to the system (Figure 3b). Under these conditions, high glucose consumption rates lead to the early appearance of additional metabolites, such as lactate.

3. The impact on the yield of glutamic acid

One consequence of decreased metabolic activity once the osmotic pressure in the culture medium increases is a significant decrease in glutamate production at the end of fermentation. The decrease in glucose consumption rate will lead to a decrease in glutamate yield, which is due to an increase in the proportion of glucose converted into CO2. The CO2 production remains roughly constant throughout the entire production stage, indicating that obtaining biochemical energy from sugar metabolism to meet the constant demand for cell maintenance is proportional to the biomass inside the fermentation tank,

Therefore, the decrease in sugar consumption rate requires an increase in the proportion of sugar used. When using the maintenance coefficient of 0.6 mmol glucose/g cell/h for Corynebacterium glutamicum, the proportion of glucose required for cell maintenance increased from 15% of the total sugar consumption in the initial stage of glutamate production to 40% at the highest osmotic pressure in the later stage of fermentation (Figure 4). The observed decrease in glutamate yield during the later stages of fermentation is consistent with changes in the carbon metabolic flow required to maintain demand. If the metabolic oxygen demand decreases during the period of reduced metabolic activity, controlling the residual sugar concentration based on oxygen changes may restore some of the yield loss.

4. Discussion

This article explains that the inhibition of glutamic acid fermentation in the later stage is directly related to the accumulation of products and the control effect of osmotic environment on sugar absorption capacity. This phenomenon is not due to changes in gene expression levels, but rather to alterations in the biochemical activity of transport systems under osmotic stress conditions. In addition, this effect is reversible because once osmotic stress is eliminated, washed cells can still restore their ability to transport sugars. Similarly, when resuspended in a hypertonic buffer, the glucose transport capacity of normally growing cells is significantly reduced. As osmotic pressure increases, this physiological effect leads to a decrease in glucose consumption capacity and a slower metabolic rate. Due to the constant energy requirements for maintaining biomass and the need to increase additional energy in many cases to counteract such pressures, this will also lead to a decrease in product yield. More and more sugar consumption will be used for energy production, so it will be lost in the form of carbon dioxide at the cost of reducing products. For glutamic acid fermentation, as the osmotic pressure of the culture medium increases, both yield and productivity significantly decrease. The biochemical mechanism underlying the decrease in PTS protein transport capacity remains to be determined, but it may be related to structural changes in the protein/membrane complex. Strains that produce glutamic acid more efficiently or quickly will reach the osmotic threshold faster, which may result in no increase in yield after 24 hours of fermentation. Therefore, screening strains with high pressure resistance is beneficial for fermentation, ensuring that industrial strains are robust enough to maintain high metabolic activity while achieving high product yields. Another path to overcome this metabolic inhibition is to develop a fermentation system that can integrate downstream extraction processes to remove products during fermentation accumulation.