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Saccharomyces cerevisiae is a widely used strain for ethanol fermentation; meanwhile, efficient utilization of glucose could effectively promote ethanol production. The PFK1 gene is a key gene for intracellular glucose metabolism in S. cerevisiae. Our previous work suggested that although deletion of the PFK1 gene could confer higher oxidative tolerance to S. cerevisiae cells, the PFK1Δ strain was prone to contamination by other microorganisms. High interspecies microbial competition ability is vital for the growth and survival of microorganisms in co-cultures. The result of our previous studies hinted us a reasonable logic that the EMP (i.e., the Embden-Meyerhof-Parnas pathway, the glycolytic pathway) key gene PFK1 could be involved in regulating interspecies competitiveness of S. cerevisiae through the regulation of glucose utilization and ethanol production efficiency. The results suggest that under 2% and 5% glucose, the PFK1Δ strain showed slower growth than the S288c wild-type and TDH1Δ strains in the lag and exponential growth stages, but realized higher growth in the stationary stage. However, relative high supplement of glucose (10%) eliminated this phenomenon, suggesting the importance of glucose in the regulation of PFK1 in yeast cell growth. Furthermore, during the lag growth phase, the PFK1Δ strain displayed a decelerated glucose consumption rate (P < 0.05). The expression levels of the HXT2, HXT5, and HXT6 genes decreased by approximately 0.5-fold (P < 0.05) and the expression level of the ZWF1 exhibited a onefold increase in the PFK1Δ strain compared to that in the S. cerevisiae S288c wild-type strain (P < 0.05).These findings suggested that the PFK1 inhibited the uptake and utilization of intracellular glucose by yeast cells, resulting in a higher amount of residual glucose in the medium for the PFK1Δ strain to utilize for growth during the reverse overshoot stage in the stationary phase. The results presented here also indicated the potential of ethanol as a defensive weapon against S. cerevisiae. The lower ethanol yield in the early stage of the PFK1Δ strain (P < 0.001) and the decreased expression levels of the PDC5 and PDC6 (P < 0.05), which led to slower growth, resulted in the strain being less competitive than the wild-type strain when co-cultured with Escherichia coli. The lower interspecies competitiveness of the PFK1Δ strain further promoted the growth of co-cultured E. coli, which in turn activated the ethanol production efficiency of the PFK1Δ strain to antagonize it from E. coli at the stationary stage. The results presented clarified the regulation of the PFK1 gene on the growth and interspecies microbial competition behavior of S. cerevisiae and would help us to understand the microbial interactions between S. cerevisiae and other microorganisms. Key points: • PFK1Δ strain could realize reverse growth overshoot at the stationary stage • PFK1 deletion decreased ethanol yield and interspecific competitiveness • Proportion of E. coli in co-culture affected ethanol yield capacity of yeast cells [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae is a widely used strain for ethanol fermentation; meanwhile, efficient utilization of glucose could effectively promote ethanol production. The PFK1 gene is a key gene for intracellular glucose metabolism in S. cerevisiae. Our previous work suggested that although deletion of the PFK1 gene could confer higher oxidative tolerance to S. cerevisiae cells, the PFK1Δ strain was prone to contamination by other microorganisms. High interspecies microbial competition ability is vital for the growth and survival of microorganisms in co-cultures. The result of our previous studies hinted us a reasonable logic that the EMP (i.e., the Embden-Meyerhof-Parnas pathway, the glycolytic pathway) key gene PFK1 could be involved in regulating interspecies competitiveness of S. cerevisiae through the regulation of glucose utilization and ethanol production efficiency. The results suggest that under 2% and 5% glucose, the PFK1Δ strain showed slower growth than the S288c wild-type and TDH1Δ strains in the lag and exponential growth stages, but realized higher growth in the stationary stage. However, relative high supplement of glucose (10%) eliminated this phenomenon, suggesting the importance of glucose in the regulation of PFK1 in yeast cell growth. Furthermore, during the lag growth phase, the PFK1Δ strain displayed a decelerated glucose consumption rate (P < 0.05). The expression levels of the HXT2, HXT5, and HXT6 genes decreased by approximately 0.5-fold (P < 0.05) and the expression level of the ZWF1 exhibited a onefold increase in the PFK1Δ strain compared to that in the S. cerevisiae S288c wild-type strain (P < 0.05).These findings suggested that the PFK1 inhibited the uptake and utilization of intracellular glucose by yeast cells, resulting in a higher amount of residual glucose in the medium for the PFK1Δ strain to utilize for growth during the reverse overshoot stage in the stationary phase. The results presented here also indicated the potential of ethanol as a defensive weapon against S. cerevisiae. The lower ethanol yield in the early stage of the PFK1Δ strain (P < 0.001) and the decreased expression levels of the PDC5 and PDC6 (P < 0.05), which led to slower growth, resulted in the strain being less competitive than the wild-type strain when co-cultured with Escherichia coli. The lower interspecies competitiveness of the PFK1Δ strain further promoted the growth of co-cultured E. coli, which in turn activated the ethanol production efficiency of the PFK1Δ strain to antagonize it from E. coli at the stationary stage. The results presented clarified the regulation of the PFK1 gene on the growth and interspecies microbial competition behavior of S. cerevisiae and would help us to understand the microbial interactions between S. cerevisiae and other microorganisms. Key points: • PFK1Δ strain could realize reverse growth overshoot at the stationary stage • PFK1 deletion decreased ethanol yield and interspecific competitiveness • Proportion of E. coli in co-culture affected ethanol yield capacity of yeast cells [ABSTRACT FROM AUTHOR]

The monitoring of microbial cultivation in real time and controlling their cultivation aid in increasing the production yield of useful material in a jar fermenter. Common sensors such as dissolved oxygen (DO) and pH can easily provide general-purpose indexes but do not reveal the physiological states of microbes because of the complexity of measuring them in culture conditions. It is well known from microscopic observations that the microbial morphology changes in response to the intracellular state or extracellular environment. Recently, studies have focused on rapid and quantitative image analysis techniques using machine learning or deep learning for gleaning insights into the morphological, physiological or gene expression information in microbes. During image analysis, it is necessary to retrieve high-definition images to analyze the microbial morphology in detail. In this study, we have developed a microfluidic device with a high-speed camera for the microscopic observation of yeast, and have constructed a system capable of generating their morphological information in real-time and at high definition. This system was connected to a jar fermenter, which enabled the automatic sampling for monitoring the cultivation. We successfully acquired high-definition images of over 10,000 yeast cells in about 2.2 s during ethanol fermentation automatically for over 168 h. We recorded 33,600 captures containing over 1,680,000 cell images. By analyzing these images, the morphological changes of yeast cells through ethanol fermentation could be captured, suggesting the expansion of the application of this system in controlling microbial fermentation using the morphological information generated. Key points: • Enables real-time visualization of microbes in a jar fermenter using microscopy. • Microfluidic device for acquiring high-definition images. • Generates a large amount of image data by using a high-speed camera. [ABSTRACT FROM AUTHOR]

Cat8 is a C6 zinc cluster transcription activator in yeast. It is generally recognized that the transcription of CAT8 is inhibited and that Cat8 is inactive in the presence of high concentrations of glucose. However, our recent study found that constitutively overexpressed Cat8 played a regulatory role in Saccharomyces cerevisiae in the presence of 20 g/L glucose. To explore the regulatory network of Cat8 at high glucose concentrations, CAT8 was both overexpressed and deleted in this study. Cell growth and glucose consumption in different media were significantly accelerated by the deletion of CAT8, while the lag period was greatly shortened. RNA-seq and genetic modification showed that the deletion of CAT8 changed the type of energy metabolism in yeast cells. Many genes related to the mitochondrial respiratory chain were downregulated, resulting in a reduction in aerobic respiration and the tricarboxylic acid cycle. Meanwhile, both the energy supply of anaerobic ethanol fermentation and the Crabtree effect of S. cerevisiae were enhanced by the deletion of CAT8. CAT8 knockout cells show a higher sugar uptake rate, a higher cell growth rate, and higher tolerance to glucose than the wild-type strain YS58. This study expands the understanding of the regulatory network of Cat8 and provides guidance for modulating yeast cell growth. Key points: • The deletion of CAT8 promoted cell growth of S. cerevisiae. • Transcriptome analysis revealed the regulation network of Cat8 under 1% glucose condition. • CAT8 deletion increases the glucose tolerance of cells by enhancing the Crabtree effect. [ABSTRACT FROM AUTHOR]

Zymomonas mobilis may encounter various types of stress during ethanol fermentation, which reduces ethanol production efficiency. This situation may be mitigated by molecular chaperones, including the chaperonin GroESL, which confers enhanced protection against various stresses. In this study, we successfully developed a Z. mobilis strain R301 that harbors groESL genes and can be used for high-temperature ethanol production from sweet sorghum juice. Sequence analyses of GroES and GroEL from Z. mobilis TISTR548 demonstrated conserved residues at specific positions within GroES and conserved glycine-glycine-methionine (GGM) repeats at the C-terminus of GroEL. The Z. mobilis wild-type and R301 strains were then evaluated for their tolerance to stresses, including high temperatures, high sugar concentrations, and high ethanol concentrations up to 40°C, 300 g/L, and 13% (v/v), respectively. Z. mobilis R301 exhibited better growth performance than the wild-type strain under all stress conditions. This is the first report on ethanol production at 40°C by recombinant Z. mobilis using sweet sorghum juice; this strain produced an ethanol concentration of 41.66 g/L, with a productivity of 0.87 g/L/h and a theoretical ethanol yield of 88.9%. Overexpression of groESL resulted in increased ethanol production, with values approximately 11% higher than those of the wild type at 40°C. Additionally, at 37°C, Z. mobilis R301 gave a higher theoretical ethanol yield (92.6%) than that shown in previous research. This work illustrates the potential for future enhancement of industrial-scale ethanol production at high temperatures utilizing Z. mobilis R301 in the bioconversion of sweet sorghum juice, a promising energy crop. Key points: • The groESL-overexpressing Z. mobilis strain was successfully constructed. • The recombinant Z. mobilis exhibited higher stress tolerance than the wild-type strain. • Overexpression of groESL genes improved ethanol production efficiency at high temperatures. [ABSTRACT FROM AUTHOR]