Acetic acid acting as a signal molecule in quorum sensing system enhances production of 2,3- 1 butanediol in Saccharomyces cerevisiae

: 11 Objectives : 2,3-butanediol (2,3-BD) has been extensively used in chemical synthese. The traditional 2,3- 12 BD production method has low yield and high cost. This study aimed to explore the use of acetic acid as 13 a signal molecule to initiate a quorum sensing (QS) system in order to promote the production of 2,3-BD 14 in Saccharomyces cerevisiae W141. 15 Results : We found that the yield of 2,3-BD from S. cerevisiae W141 is proportional to the cell density. 16 S. cerevisiae W141 does not produce 2,3-BD when cell density was lower than the threshold 17 concentration ( OD 600 nm = 10 or cell density 4.4 × 10 8 CFU/mL). When 1.5 g/L acetic acid was added in 18 the fermentation process, the yield of 2,3-BD was the highest reaching 3.01 ± 0.04 g/L (84 h). 19 Subsequently, we found that S. cerevisiae W141 was co-cultured with Acetobacter pasteurianus Huniang 20 1.01 under the optimal conditions and the acetic acid production was increased by 76.7% and 30.6% 21 compared with the original strain and the strain with 1.5 g/L acetic acid, respectively. In addition, the 22 yield of 2,3-BD was respectively increased by 81.9% and 3.3%. The above results are attributable to the 23 increased activity of acetolactate synthase (ILV2) and 2,3-BD dehydrogenase (BDH1) and the increase 24 of the relative expression of ilv2 and bdh1 genes. 25 Conclusion : Our data showed that biosynthesis of 2,3-BD was regulated by acetic acid as a signaling molecule. Moreover the study provides a deeper understanding of the mechanisms underlying between 27 acetic acid and 2,3-BD production. co- the relative expression gene 4.52 times that ( p < 0.01), the relative expression gene after adding 2 g/L acid, which lower than that of control p It shows that there is QS between S. and the acetic acid of A. can used signal the expression of key enzyme genes in the 2,3- BD synthesis pathway, and subsequently improve the activity of related enzymes to achieve the purpose of increasing 2,3-BD.


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However, 2,3-BD was originally separated from nature. Its yield is low and cannot be used in industrial 50 production. Therefore, therefore the current research concentrates on seeking a new strain and using 51 metabolic engineering methods to increase the yield of 2,3-BD. In recent years, the production of 2,3-52 BD by microbial fermentation has become the focus of attention both at home and abroad (Celińska and 53 Grajek 2009). The advantage of the proposed method is that it cannot only efficiently use glucose and 54 other monosaccharides as carbon sources, but also use plant straws as raw materials to ferment to produce 55 2,3-BD. The method is cheap and safe ; Kim et al. 2016; Kim et al. 2015). Although 56 numerous microorganisms have been studied for the production of 2,3-BD, however Saccharomyces 57 cerevisiae has become the main platform microorganism for many related researches due to its 58 compliance with the principle of safe production (Turner et al. 2016). Although S. cerevisiae can 59 naturally produce 2,3-BD, yet S. cerevisiae uses glucose to produce 2,3-BD by fermentation, which is 60 usually accompanied by the formation of various metabolites and by-products (Fig. 1), such as ethanol 61 and glycerol, competing for carbon sources in metabolic pathways. Ultimately, the lower yields of 2,3-62 4 BD can be generated.

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In the early stage of the current project, molecular biology was used to modify the metabolic pathway of 64 S. cerevisiae, aiming to increase the yields of 2,3-BD. The results demonstrated that although the yield 65 of 2,3-BD was improved in a short time, but the yield of 2,3-BD presented a slight downward trend due 66 to defects such as poor genetic stability of the engineered strain. Consequently, in order to solve the 67 present problem, this research attempts to depend on ecological principles and make attempts to enhance  (Anane et al. 2017). Therefore, in the metabolic process, the production of 76 2,3-BD may be regulated by QS, and acetic acid as a signal molecule can initiate QS system in order to 77 increase the production of 2,3-BD. In other words, it is beneficial to the production of 2,3-BD under 78 acidic conditions. Therefore acetic acid exerts an indispensable role in the 2,3-BD production process.

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According to the result of single factor, the four variables of inoculation ratio, temperature, rotation speed 119 6 and inoculation time are further optimized. This study uses four-factor L9(3 4 ) orthogonal design, each 120 factor has three different levels (Table 1).

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The strains were individually precultured as described above. The strains S. cerevisiae W141 and A.

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The threshold concentration is defined as the culture condition that does not produce 2,3-BD when the 175 cell density of S. cerevisiae W141 is lower than the threshold. It can be observed from Table 2 that  8 different inoculation amounts exert no obvious effect on the cell density, and the carbon source (glucose) 177 in the medium is the main factor which can affect the bacterial count. When the carbon source is not 178 changed (control group), the cell density (OD600 nm value) in the fermentation broth is the highest, the 179 second is when the carbon source is 1/2, and the cell density of 1/5 is the lowest. In the meanwhile, the 180 cup and dish method was employed to detect the relationship between the yield of 2,3-BD and cell density 181 under 9 different culture conditions (Table 2). Under 1/5 carbon source culture conditions, the yield of 182 2,3-BD cannot be detected regardless of the inoculum amount of 5%, 2%, or 0.5%, and the OD600 nm 183 values were respectively 9.683 ± 0.732, 9.613 ± 0.611 and 9.014 ± 0.571, all of which are less than 10. 184 Therefore, it can be proved that the yield of 2,3-BD within the certain range is directly proportional to 185 the cell density of the fermentation broth. The production process of 2,3-BD is dependent on cell density, 186 and the threshold concentration to be reached is OD600 nm is 10 (cell density is 4.4 × 10 8 CFU/mL). In 187 addition, it can also be determined that the threshold density culture condition is 1/5 carbon source 188 medium and the inoculation amount is 5%. Therefore, this condition can be used to determine whether 189 co-cultivation with other microorganisms can promote S. cerevisiae W141 in order to produce more 2,3-

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In the present study, we chose to add 1 g/L, 1.5 g/L and 2 g/L acetic acid to determine the changes in 203 biomass and 2,3-BD yield in the fermentation process of S. cerevisiae W141 (Fig. 2). The results 204 demonstrated that the OD600 nm value ended to first rise and then maintain stable, at the same time, with 205 S. cerevisiae W141 as the control group, OD600 nm value reached the maximum of 15.29 ± 0.31. After 206 9 adding 1g/L and 1.5 g/L acetic acid, the highest yields of 2 3-BD reached respectively 2.93 ± 0.02 g/L 207 (84 h) and 3.01 ± 0.04 g/L (84 h), which were compared with the control group (1.71 ± 0.02 g/L). In 208 addition, it was increased respectively by 71.4% and 76.0% (p < 0.01). At the same time, the OD600 nm 209 value has no obvious effect after adding 1 g/L acetic acid, while the OD600 nm value dropped to 14.46 ± 210 0.93 (cell density is 6 × 10 8 CFU/mL) after adding 1.5 g/L. After adding 2 g/L acetic acid, the 2,3-BD 211 yield decreased to 1.45 ± 0.02 g/L (84 h), which was 17.9% lower than the control group (p < 0.01).

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Meanwhile, the OD600 nm value dropped to 13.62 ± 0.48. (cell density is 5.7 × 10 8 CFU/mL). Consequently, 213 the relationship between acetic acid and 2,3-BD during the fermentation of S. cerevisiae W141 has a 214 certain coupling, that is, when acetic acid reaches a concentration of 1.5 g/L, it can effectively promote 215 the production of 2,3-BD, and excessive addition of acetic acid will lead to a decrease in the production 216 and cell density of 2,3-BD and inhibit its growth.

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The effect of acetic acid on microorganisms under co-culture conditions

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According to the results of the previous single-factor experiment, the fermentation conditions for co-219 cultivation include the inoculation ratio is 4:2, the fermentation temperature is 30℃, the rotation speed 220 is 140 r/min, and the inoculation time is 12 h. Besides, the output of 2,3-BD reached its maximum at that 221 time. We further carry out orthogonal optimization test for four factors, and each factor has three different 222 levels of orthogonal experimental design (Table 1). In accordance with the production of 2,3-BD 223 determined by each experimental design and the influence of these factors on the production of acetic 224 acid, according to the R value (Table 3)

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Based on the order of K value (Table 3)

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Under optimal culture conditions, samples were taken from the culture medium in order to analyze 232 changes in glucose concentration, biomass, ethanol, acetic acid, and 2,3-BD content. As shown in Fig.   233 3a, during the fermentation process, glucose was used as a carbon source and was quickly utilized by S.

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Comparison of the 2,3-BD yield changes under the optimal conditions of co-cultivation, yeast 249 monoculture, and addition of 1.5 g/L acetic acid, can be seen from the results of Fig. 4a. Under the 250 optimal culture conditions, the maximum yield of 2,3-BD tested was 3.11 ± 0.02 g/L (72 h), which was 251 81.9% higher than the original strain (p < 0.01). Simultaneously, compared with adding 1.5 g/L acetic 252 acid, the yield increased by 3.3% (p < 0.05). Additionally, it could also be found that under the optimal 253 culture conditions, the acetic acid produced was higher than that of S. cerevisiae W141 monoculture and 254 1.5 g/L acetic acid (Fig. 4b). Therefore, it can be proved that S. cerevisiae W141 will use the acetic acid 255 produced by A. pasteurianus HN 1.01 to promote the production of 2,3-BD. The decrease of acetic acid 256 content will lead to the production of 2,3-BD decline, also further explaining that acetic acid can be used 257 as a signal molecule under co-culture conditions to affect the content of 2,3-BD produced by S. cerevisiae

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This study used qRT-PCR technology to detect the expression of ilv2 and bdh1 gene. The CT value of 278 each sample was analyzed by employing the relative quantitative 2 −ΔΔCT method, and the relative 279 expression of the ilv2 and bdh1 genes was calculated (Fig. 6). According to the results, the relative 280 expression levels of ilv2 gene were higher than those in the control group after adding 1 g/L and 1.5 g/L 281 acetic acid, which were 3.37 times and 5.56 times the gene expression levels respectively, and the relative 282 expression level of ilv2 gene under optimal co-culture condition was 4.52 times that of control group.

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The relative expression of ilv2 gene was 4.52 times that of the control group (p < 0.01). After adding 2 284 g/L acetic acid, the relative expression of the ilv2 gene was down-regulated, which was 20.5% lower than 285 that of the control group (p < 0.01). At the same time, the relative expression of bdh1 gene in S. cerevisiae 286 W141 after the addition of 1 g/L and 1.5 g/L acetic acid was also higher than that of the control group, 287 which was respectively 1.35 times and 1.12 times that of its gene expression. Under the optimal co-288 cultured conditions, the relative expression of bdh1 gene was 4.52 times that of the control group (p < 289 0.01), while the relative expression of bdh1 gene was down-regulated after adding 2 g/L acetic acid, 290 which was 85.2% lower than that of the control group (p < 0.01). It shows that there is QS between S.

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Some studies have found that the production process of 2,3-BD is affected by a lot of factors, such as 314 ventilation, pH and cell density (Tong et al. 2015;Kalia 2013). Based on our obtained data, the yield of 315 2,3-BD is affected by the cell density of S. cerevisiae. When the cell density of the fermentation broth is 316 lower than the threshold concentration (OD600 nm = 10 or cell density of 4.4 × 10 8 cells/mL), the 317 production of 2,3-BD cannot be detected. Under the same conditions, when the cell density is higher than 318 the threshold concentration, the production of 2,3-BD is found. Therefore, high cell density facilitates 319 the production of high levels of 2,3-BD. However, it has been found that the signal molecules produced 320 by some strains have a certain relationship with cell density. As a result, it shows that the production of the production of 2,3-BD by bacteria, they found that when a certain concentration of acetic acid was 335 added to E. aerogenes, the pH of the medium was adjusted to 6.5 and the yield of 2,3-BD was increased,

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indicating that the addition of acetic acid exerted a certain stimulating effect on 2,3-BD.

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Through conducting further co-cultivation experiments, we found that 2,3-BD production will be 338 affected by the growth and metabolism of A. pasteurianus HN1.01 to produce acetic acid. Under the 339 optimal co-cultivation conditions, the yield of 2,3-BD was higher than the original strain and the 1.