Formation mechanisms of ethyl acetate and organic acids in Kluyveromyces marxianus L1-1 in Chinese acid rice soup

This study aims to explore the formation mechanism of ethyl acetate and organic acids in acid rice soup(rice-acid soup)inoculated with Kluyveromyces marxianus L1-1 through the complementary analysis of transcriptome and proteome.The quantity of K.marxianus L1-1 varied significantly in the fermentation process of rice-acid soup and the first and third days were the two key turning points in the growth phase of K.marxianus L1-1.Importantly,the concentrations of ethyl acetate,ethanol,acetic acid,and L-lactic acid increased from day 1 to day 3.At least 4231 genes and 2937 proteins were identified and 610 differentially expressed proteins were annotated to 30 Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways based on the analysis results of transcriptome and proteome.The key genes and proteins including up-regulated alcohol dehydrogenase family,alcohol O-acetyltransferase,acetyl-CoA C-acetyltransferase,acyl-coenzyme A thioester hydrolase,and down-regulated aldehyde dehydrogenase family were involved in glycolysis/gluconeogenesis pathways,starch and sucrose metabolism pathways,amino sugar and nucleotide sugar metabolism pathways,tricarboxylic acid(TCA)cycle,and pyruvate metabolism pathways,thus promoting the formation of ethyl acetate,organic acids,alcohols,and other esters.Our results revealed the formation mechanisms of ethyl acetate and organic acids in rice-acid soup inoculated with K.marxianus L1-1.

Integrative proteomic-transcriptomic analysis revealed the lifestyles of Lactobacillus paracasei H4-11 and Kluyveromyces marxianus L1-1 under co-cultivation conditions

Compared with the rice-acid soup inoculated with single starter,the synergistically intensifi ed rice-acid soup inoculated with Lactobacillus paracasei H4-11(L.paracasei H4-11)and Kluyveromyces marxianus L1-1(K.marxianus L1-1)contained more fl avor compounds.Organic acids mainly included L-lactic acid and the main volatile fl avor component was ethyl acetate.Moreover,the signal intensity of astringency and bitterness and the total concentration of volatile sulfur compounds were reduced.The combined analysis results of RNA sequencing(RNA-seq)technology and 4D label-free quantitative(4D LFQ)proteomics explained the fl avor formation pathways in rice-acid soup inoculated with L.paracasei H4-11 and K.marxianus L1-1.In L.paracasei H4-11,L-lactate dehydrogenase,phosphoglucomutase,acetate kinase,alcohol dehydrogenase and acetyl-CoA were up-regulated and D-lactate dehydrogenase and N-Acetyltransferase were down-regulated.In K.marxianus L1-1,Acetyl-CoA,acetaldehyde dehydrogenase,acyl-coenzyme A,N-acetyltransferase,and L-lactate dehydrogenase were up-regulated and hexokinase,alcohol dehydrogenase,and alcohol O-acetyltransferase were down-regulated.The above up-regulation and down-regulation synergistically promoted the formation of characteristic fl avor compounds(mainly L-lactic acid and ethyl acetate).Enzyme-linked immunoassay(ELISA)and parallel reaction monitoring(PRM)quantitative analysis respectively verifi ed that 5 key metabolic enzymes and 27 proteins in L.paracasei H4-11 and K.marxianus L1-1 were associated with the characteristic fl avor of rice-acid soup,as confi rmed by the quantitative results of 4D LFQ.

Transfer of disulfide bond formation modules via yeast artificial chromosomes promotes the expression of heterologous proteins in Kluyveromyces marxianus

Kluyveromyces marxianus is a food-safe yeast with great potential for producing heterologous proteins.Improving the yield in K.marxianus remains a challenge and incorporating large-scale functional modules poses a technical obstacle in engineering.To address these issues,linear and circular yeast artificial chromosomes of K.marxianus(KmYACs)were constructed and loaded with disulfide bond formation modules from Pichia pastoris or K.marxianus.These modules contained up to seven genes with a maximum size of 15 kb.KmYACs carried telomeres either from K.marxianus or Tetrahymena.KmYACs were transferred successfully into K.marxianus and stably propagated without affecting the normal growth of the host,regardless of the type of telomeres and configurations of KmYACs.KmYACs increased the overall expression levels of disulfide bond formation genes and significantly enhanced the yield of various heterologous proteins.In high-density fermentation,the use of KmYACs resulted in a glucoamylase yield of 16.8 g/l,the highest reported level to date in K.marxianus.Transcriptomic and metabolomic analysis of cells containing KmYACs suggested increased flavin adenine dinucleotide biosynthesis,enhanced flux entering the tricarboxylic acid cycle,and a preferred demand for lysine and arginine as features of cells overexpressing heterologous proteins.Consistently,supplementing lysine or arginine further improved the yield.Therefore,KmYAC provides a powerful platform for manipulating large modules with enormous potential for industrial applications and fundamental research.Transferring the disulfide bond formation module via YACs proves to be an efficient strategy for improving the yield of heterologous proteins,and this strategy may be applied to optimize other microbial cell factories.

Transcriptomic analysis reveals hub genes and pathways in response to acetic acid stress in Kluyveromyces marxianus during high-temperature ethanol fermentation

The thermotolerant yeast Kluyveromyces marxianus is known for its potential in high-temperature ethanol fermentation,yet it suffers from excess acetic acid production at elevated temperatures,which hinders ethanol production.To better understand how the yeast responds to acetic acid stress during high-temperature ethanol fermentation,this study investigated its transcriptomic changes under this condition.RNA sequencing(RNA-seq)was used to identify differentially expressed genes(DEGs)and enriched gene ontology(GO)terms and pathways under acetic acid stress.The results showed that 611 genes were differentially expressed,and GO and pathway enrichment analysis revealed that acetic acid stress promoted protein catabolism but repressed protein synthesis during high-temperature fermentation.Protein-protein interaction(PPI)networks were also constructed based on the interactions between proteins coded by the DEGs.Hub genes and key modules in the PPI networks were identified,providing insight into the mechanisms of this yeast’s response to acetic acid stress.The findings suggest that the decrease in ethanol production is caused by the imbalance between protein catabolism and protein synthesis.Overall,this study provides valuable insights into the mechanisms of K.marxianus’s response to acetic acid stress and highlights the importance of maintaining a proper balance between protein catabolism and protein synthesis for high-temperature ethanol fermentation.

Novel Stereoselective Carbonyl Reductase from Kluyveromyces marxianus for Chiral Alcohols Synthesis

A novel nicotinamide adenine dinucleotide phosphate（NADPH）-dependent carbonyl reductase from Kluyverornyces marxianus（KmCR） was identified, which can convert various prochiral ketone esters and ketone substrates to their corresponding chiral alcohols. KmCR was over-expressed in E. coli BL21（DE3）, purified to homogeneity, and characterized. The purified enzyme exhibits the highest activity at 40℃ and pH=6.0. Based on the gel filtration and sodium dodecyl sulfate-polyacrylamide gel eiectrophoresis（SDS-PAGE） analysis, the monomeric protein was determined to have a molecular weight of approximate 39000. Vmax and Km of KmCR are 4.28 μmol.min^-1·mg^-1 and 0.41 mmol/L for ketone ester substrate ethyl 2-oxo-4-phenylbutyrate（OPBE）, 3.09μmol.min^-1·mg^-1 and 1.21 mmol/L for cofactor NADPH, respectively. Cofactor recycle was achieved by co-expression of KmCR and glucose dehydrogenase（GDH） in E. coli. Recombinant E. coli harboring KmCR and GDH showed moderate asymmetric reduction activity towards various α- and β-ketoesters, diaryl ketone substrates. In an aqueous/butyl acetate biphasic system, the whole-cell biocatalyst was used to prepare ethyl （R）-2-hydroxy-4- phenylbutanoate[（R）-HPBE] in an e.e. of 99.5% with a space-time yield of 433.6 g.L-1.d-1 and a yield of 80.3% at 270 g/L OPBE.

Genome and metabolic engineering in non-conventional yeasts:Current advances and applications

Microbial production of chemicals and proteins from biomass-derived andwaste sugar streams is a rapidly growing area of research and development.While the model yeast Saccharomyces cerevisiae is an excellent host for the conversion of glucose to ethanol,production of other chemicals from alternative substrates often requires extensive strain engineering.To avoid complex and intensive engineering of S.cerevisiae,other yeasts are often selected as hosts for bioprocessing based on their natural capacity to produce a desired product:for example,the efficient production and secretion of proteins,lipids,and primary metabolites that have value as commodity chemicals.Even when using yeasts with beneficial native phenotypes,metabolic engineering to increase yield,titer,and production rate is essential.The non-conventional yeasts Kluyveromyces lactis,K.marxianus,Scheffersomyces stipitis,Yarrowia lipolytica,Hansenula polymorpha and Pichia pastoris have been developed as eukaryotic hosts because of their desirable phenotypes,including thermotolerance,assimilation of diverse carbon sources,and high protein secretion.However,advanced metabolic engineering in these yeasts has been limited.This review outlines the challenges of using non-conventional yeasts for strain and pathway engineering,and discusses the developed solutions to these problems and the resulting applications in industrial biotechnology.