Protein acetylation-mediated cross regulation of acetic acid and ethanol synthesis in the gas-fermenting Clostridium ljungdahlii

Abstract

The autotrophic acetogen Clostridium ljungdahlii has emerged as a major candidate in the biological conversion of one-carbon gases (CO2/CO) to bulk chemicals and fuels. Nevertheless, the regulatory pathways and downstream metabolic changes responsible for product formation and distribution in this bacterium remain minimally explored. Protein lysine acetylation (PLA), a prevalent posttranslational modification, controls numerous crucial cellular functions. Herein, we revealed a novel cross-regulatory mechanism that uses both the PLA system and transcription factors to regulate the carbon flow distribution for product formation in C. ljungdahlii. The dominant acetylation/deacetylation system (At2/Dat1) in C. ljungdahlii was found to regulate the ratio of two major products, acetic acid and ethanol. Subsequent genetic and biochemical analyses revealed that the activities of Pta and AdhE1, two crucial enzymes responsible for acetic acid and ethanol synthesis, respectively, were greatly affected by their levels of PLA. We found that the acetylation statuses of Pta and AdhE1 underwent significant dynamic changes during the fermentation process, leading to differential synthesis of acetic acid and ethanol. Furthermore, the crucial redox-sensing protein Rex was shown to be regulated by PLA, which subsequently altered its transcriptional regulation on genes responsible for acetic acid and ethanol formation and distribution. Based on our understanding of this cross-regulatory module, we optimized the ethanol synthetic pathway by modifying the acetylation status (deacetylation-mimicked mutations of crucial lysine residues) of the related key enzyme, achieving significantly increased titer and yield of ethanol, an important chemical and fuel, by C. ljungdahlii in gas fermentation. The autotrophic acetogen Clostridium ljungdahlii has emerged as a major candidate in the biological conversion of one-carbon gases (CO2/CO) to bulk chemicals and fuels. Nevertheless, the regulatory pathways and downstream metabolic changes responsible for product formation and distribution in this bacterium remain minimally explored. Protein lysine acetylation (PLA), a prevalent posttranslational modification, controls numerous crucial cellular functions. Herein, we revealed a novel cross-regulatory mechanism that uses both the PLA system and transcription factors to regulate the carbon flow distribution for product formation in C. ljungdahlii. The dominant acetylation/deacetylation system (At2/Dat1) in C. ljungdahlii was found to regulate the ratio of two major products, acetic acid and ethanol. Subsequent genetic and biochemical analyses revealed that the activities of Pta and AdhE1, two crucial enzymes responsible for acetic acid and ethanol synthesis, respectively, were greatly affected by their levels of PLA. We found that the acetylation statuses of Pta and AdhE1 underwent significant dynamic changes during the fermentation process, leading to differential synthesis of acetic acid and ethanol. Furthermore, the crucial redox-sensing protein Rex was shown to be regulated by PLA, which subsequently altered its transcriptional regulation on genes responsible for acetic acid and ethanol formation and distribution. Based on our understanding of this cross-regulatory module, we optimized the ethanol synthetic pathway by modifying the acetylation status (deacetylation-mimicked mutations of crucial lysine residues) of the related key enzyme, achieving significantly increased titer and yield of ethanol, an important chemical and fuel, by C. ljungdahlii in gas fermentation. Autotrophic acetogens can use the reductive acetyl-CoA pathway for the production of acetic acid and ethanol as well as some other minor metabolites such as butanol, lactate, butyrate, hexanoate, and 2,3-butanediol using CO2 and CO as the carbon sources (1Yang Y. Nie X. Jiang Y. Yang C. Gu Y. Jiang W. Metabolic regulation in solventogenic clostridia: Regulators, mechanisms and engineering.Biotechnol. Adv. 2018; 36: 905-914Google Scholar). Some acetogens are human gut bacteria that are closely associated with human intestinal health (2Kasubuchi M. Hasegawa S. Hiramatsu T. Ichimura A. Kimura I. Dietary gut microbial metabolites, short-chain fatty acids, and host metabolic regulation.Nutrients. 2015; 7: 2839-2849Google Scholar). The fermentation process of autotrophic acetogens typically proceeds in two phases, acidogenesis and alcohologenesis, during which acetic acid and ethanol accumulate as the major products (3Amador-Noguez D. Brasg I.A. Feng X.J. Roquet N. Rabinowitz J.D. Metabolome remodeling during the acidogenic-solventogenic transition in Clostridium acetobutylicum.Appl. Environ. Microbiol. 2011; 77: 7984-7997Google Scholar). Such a biphasic physiological process is an essential characteristic of these anaerobes. With the increasing interest in the biological usage of C1 gases, the detailed investigation of the biphasic metabolism of autotrophic acetogens has become a research focus. Our current knowledge includes an understanding of the metabolic pathways responsible for the formation of acids and alcohols as well as of the conversion of acids to alcohols in autotrophic acetogens (1Yang Y. Nie X. Jiang Y. Yang C. Gu Y. Jiang W. Metabolic regulation in solventogenic clostridia: Regulators, mechanisms and engineering.Biotechnol. Adv. 2018; 36: 905-914Google Scholar, 4Ragsdale S.W. Pierce E. Acetogenesis and the Wood-Ljungdahl pathway of CO2 fixation.Biochim. Biophys. Acta. 2008; 1784: 1873-1898Google Scholar, 5Koepke M. Held C. Hujer S. Liesegang H. Wiezer A. Wollherr A. Ehrenreich A. Liebl W. Gottschalk G. Duerre P. Clostridium ljungdahlii represents a microbial production platform based on syngas.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 13087-13092Google Scholar, 6Zhang L. Zhao R. Jia D. Jiang W. Gu Y. Engineering Clostridium ljungdahlii as the gas-fermenting cell factory for the production of biofuels and biochemicals.Curr. Opin. Chem. Biol. 2020; 59: 54-61Google Scholar, 7Furdui C. Ragsdale S.W. The role of pyruvate ferredoxin oxidoreductase in pyruvate synthesis during autotrophic growth by the Wood-Ljungdahl pathway.J. Biol. Chem. 2000; 275: 28494-28499Google Scholar). Furthermore, some key enzymes and the corresponding genes have been identified and characterized (5Koepke M. Held C. Hujer S. Liesegang H. Wiezer A. Wollherr A. Ehrenreich A. Liebl W. Gottschalk G. Duerre P. Clostridium ljungdahlii represents a microbial production platform based on syngas.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 13087-13092Google Scholar). In contrast, the regulatory aspects of the distinctive metabolism of these autotrophic acetogens still remain largely unknown. Some external environmental factors have been reported to affect the acid and alcohol synthesis of autotrophic acetogens. For example, low temperature and some metal ions (Ni2+, Co2+, etc.) could enable Clostridium carboxidivorans P7 to produce more ethanol (8Ramió-Pujol S. Ganigué R. Bañeras L. Colprim J. Incubation at 25 °C prevents acid crash and enhances alcohol production in Clostridium carboxidivorans P7.Bioresour. Technol. 2015; 192: 296-303Google Scholar, 9Shen S. Gu Y. Chai C. Jiang W. Zhuang Y. Wang Y. Enhanced alcohol titre and ratio in carbon monoxide-rich off-gas fermentation of Clostridium carboxidivorans through combination of trace metals optimization with variable-temperature cultivation.Bioresour. Technol. 2017; 239: 236-243Google Scholar); W6+ could help Sporomusa ovata to produce more ethanol (10Ammam F. Tremblay P.L. Lizak D.M. Zhang T. Effect of tungstate on acetate and ethanol production by the electrosynthetic bacterium Sporomusa ovata.Biotechnol. Biofuels. 2016; 9: 163Google Scholar); and the addition of some amino acids such as arginine in media could reduce the accumulation of acetic acid by boosting the ATP level in Clostridium autoethanogenum (11Valgepea K. Loi K.Q. Behrendorff J.B. Lemgruber R.S.P. Plan M. Hodson M.P. Köpke M. Nielsen L.K. Marcellin E. Arginine deiminase pathway provides ATP and boosts growth of the gas-fermenting acetogen Clostridium autoethanogenum.Metab. Eng. 2017; 41: 202-211Google Scholar). However, the molecular mechanisms underlying these findings are unclear. Obviously, to develop an efficient gas fermentation process, a detailed understanding of the regulatory mechanism of the metabolic processes in these autotrophic acetogens is indispensable. Protein Lysine acetylation (PLA) is to be a crucial metabolic regulatory mechanism in both and in which physiological and metabolic processes have been revealed S. W. Jiang W. W. Y. Zhang T. J. L. Y. H. Y. J. W. F. of cellular metabolism by protein lysine 2010; Scholar, F. acetylation to and 2016; Scholar, E. Lysine with other posttranslational 2008; Scholar). In a on Clostridium a autotrophic we revealed an regulation that uses both the transcriptional and acetylation/deacetylation system to carbon L. Y. Zhao R. Zhang C. Jiang W. Gu Y. regulation of during CO2 in gas-fermenting 2020; Scholar). in addition to PLA an important role in crucial cellular in autotrophic leading to the regulation on levels of the of PLA in metabolic the remain on and regulatory have been reported to be affected by PLA and L. Y. Zhao R. Zhang C. Jiang W. Gu Y. regulation of during CO2 in gas-fermenting 2020; Scholar, D. Y. P. lysine acetylation of an in Natl. Acad. Sci. U. S. A. 2016; Scholar, K. D. H. of the controls transcription a Scholar). to autotrophic and the PLA statuses of regulatory affect crucial cellular growth and product synthesis, remain explored. more such regulatory mechanisms our understanding of these important Herein, we the of the in PLA levels on the product synthesis in C. ljungdahlii. the genetic and biochemical a regulatory that uses both the lysine acetylation and transcriptional regulation to product synthesis in C. ljungdahlii was Based on this a metabolic the optimization of both the and acetylation levels of the pathway genes was to the product synthesis in C. achieving a yield and ratio of ethanol in gas fermentation. C. ljungdahlii two dominant products, acetic acid and ethanol, in gas fermentation. The crucial genes responsible for the synthesis of these two chemicals for for acetate for alcohol and and for We the PLA status of the enzymes by these genes during gas fermentation based on our L. Y. Zhao R. Zhang C. Jiang W. Gu Y. regulation of during CO2 in gas-fermenting 2020; Scholar). the and were at lysine lysine was found in L. Y. Zhao R. Zhang C. Jiang W. Gu Y. regulation of during CO2 in gas-fermenting 2020; a of PLA on the activities of the enzymes the of the protein acetylation level of C. ljungdahlii on product a C. ljungdahlii with of the which for the key in C. ljungdahlii L. Y. Zhao R. Zhang C. Jiang W. Gu Y. regulation of during CO2 in gas-fermenting 2020; was to product synthesis with that of the by of The that the and the synthesis of acetic acid and ethanol, growth The increased ethanol with the acetic acid was in the with the Furthermore, the titer of the acid and of the was that of the Such changes of the could be by the findings that PLA a role in the regulation of the carbon distribution in C. in product in gas fermentation. pathway genes and their PLA status are crucial for product formation in C. the with of of the genes and were and and to their with that of the in gas fermentation. The that the and the and the greatly ethanol production and In contrast, changes were for the and and that and are crucial to the growth and product synthesis of C. ljungdahlii in gas fermentation. Based on these we PLA the activities of the and AdhE1 The and AdhE1 were in by the significantly increased acetylation level was for these enzymes the in acetylation by The that the increased acetylation level to significant in the activities of Pta and AdhE1 and for its and alcohol and was for and and revealed the the increased acetylation levels and activities of these the of lysine acetylation on these activities based on the of the in Pta and AdhE1 are crucial for acetic acid and ethanol formation in C. ljungdahlii the regulation of these two enzymes affect the carbon distribution the synthetic pathways of acetic acid and ethanol, the ratio of these two However, we that the activities of Pta and AdhE1 by their increased acetylation levels the greatly increased ethanol production of C. ljungdahlii that PLA can the product synthesis other in C. ljungdahlii. of the our analyses of C. ljungdahlii that a of were at lysine L. Y. Zhao R. Zhang C. Jiang W. Gu Y. regulation of during CO2 in gas-fermenting 2020; that the regulatory activities of these be affected by their PLA Furthermore, we found that crucial genes and responsible for product formation significant changes in transcription at and and of respectively, these findings that PLA affect the product synthesis of C. ljungdahlii the acetylation level of related we the in C. ljungdahlii L. Y. Zhao R. Zhang C. Jiang W. Gu Y. regulation of during CO2 in gas-fermenting 2020; Scholar). two which are to the Clostridium Rex and that are to product synthesis L. Nie X. J. Gu Y. Yang S. Jiang W. Yang C. Rex alcohol production and in Clostridium Scholar, of and and of on the transition acidogenesis to in Clostridium Environ. Microbiol. were these two and were for the in to can on the pathway and The that could to the of and the two more were to PLA the regulatory activities of and two were in by the the and for the of and as the were for the at protein in contrast, were for using the However, was for the of to the of and findings that the acetylation level could significantly affect the of to its we on and its in C. ljungdahlii. The was a to the transcriptional changes of the genes in the acetic acid and ethanol synthetic The the of and synthetic were at of and acid synthetic were significantly at this at and acid still levels to of the with the the regulation of on the genes in product synthetic pathways in C. ljungdahlii. The revealed that acetylation and We to the key acetylation lysine that the regulatory of mutations were at these lysine for is shown in and the to a to the of the the of acetylation at this lysine for the of the at this could the regulatory of in the protein and its two that and and at were the to their on product The that the of and both to the of the acetic acid titer and acetic ratio to that of the in of these were at a level the of the for the in of in C. ljungdahlii. Protein acetylation is a dynamic and posttranslational we have revealed that the in acetylation affected the activities of Pta and AdhE1 as well as the regulatory of and and their acetylation levels during the fermentation this we the dynamic changes of the acetylation levels of these in gas fermentation. that the AdhE1, and Rex were the C. ljungdahlii with the of and The were using and the at and growth were for the protein of AdhE1, and which were for is shown in the acetylation levels of Pta were low at the increased with the of in contrast, AdhE1 a acetylation level at the with the low acetylation levels at and is that the C. ljungdahlii ethanol acetic acid in gas fermentation C. Nie X. Zhang H. Y. H. Yang C. Jiang W. Gu Y. and of the protein for autotrophic growth of gas-fermenting Clostridium Scholar); the significantly increased AdhE1 acetylation level and the low AdhE1 in the fermentation to the production of ethanol at this the of fermentation. The acetylation levels of increased with the of fermentation a regulatory of the differential regulation of on and the acetylation level affect the carbon distribution the synthesis of acetic acid and in the the low acetylation level to the of and the of and to acidogenesis at this with the in its regulatory and the transcriptional on and and subsequently leading to more ethanol ethanol, is a dominant product in gas fermentation. the of ethanol as the we to a ethanol production and acid ratio by the of the of acetylation on the of AdhE1 key in ethanol synthetic mutations to the acetylation of AdhE1, was for in acetylation by and for the acetylation by The with our and a of acetylation lysine were found in AdhE1 and mutations were at these lysine AdhE1 for the of key acetylation lysine that the of shown in the genes were the C. ljungdahlii for and the that the and mutations to significant in ethanol production at mutations were at these the that the greatly ethanol changes were found for the other mutations these that the acetylation status of the and are crucial for the in AdhE1 is a crucial amino acid for the in AdhE1 be associated with its acetylation the crucial lysine acetylation on AdhE1, the metabolic was the ethanol production and acid ratio the as a a that a mutations at the and was and the for the responsible for acetic acid in C. ljungdahlii was the to was the of the that was in the of Rex the both and the increased ethanol and acetic acid to the the the and in gas fermentation leading to a acid ratio the fermentation of the and was the for a of this the and was to produce of ethanol and of acetic the of ethanol and of acetic acid a product titer acetic and acid ratio were by the that the in activities of the enzymes responsible for product synthesis in C. ljungdahlii could be by the lysine the titer of In this we reported a regulatory which is by both the redox-sensing protein Rex and the PLA system for the regulation of product synthesis in the autotrophic acetogen C. ljungdahlii. The of PLA for the activities of crucial enzymes in product synthetic pathways and the regulatory of Rex was findings our understanding of the metabolic regulation of product synthesis in autotrophic acetogens. the a more efficient synthesis of the our still at the molecular level autotrophic regulate the carbon distribution in product and have been found to in this (8Ramió-Pujol S. Ganigué R. Bañeras L. Colprim J. Incubation at 25 °C prevents acid crash and enhances alcohol production in Clostridium carboxidivorans P7.Bioresour. Technol. 2015; 192: 296-303Google Scholar, X. M. R. S. of for of acid on production in fermentation of Clostridium Scholar, S. Y. Zhang Y. Y. the of the of Clostridium to an of increasing Microbiol. Scholar). In as C. some have been reported to regulate product synthesis and affect the yield of products, and L. Nie X. J. Gu Y. Yang S. Jiang W. Yang C. Rex alcohol production and in Clostridium Scholar, of and and of on the transition acidogenesis to in Clostridium Environ. Microbiol. Scholar, and fermentation of and in Clostridium Scholar, Y. Clostridium and its 2015; Scholar, C. Gu Y. Y. Zhang W. Yang C. Yang S. Jiang W. of protein in Clostridium Scholar, Y. Zhang L. H. Yang C. Yang S. Gu Y. Jiang W. provides regulation in 2017; Scholar, affect in Clostridium Scholar). the of these can be found in autotrophic C. ljungdahlii However, the product and gas-fermenting Clostridium the mechanisms of these in C. ljungdahlii be which The of posttranslational in crucial physiological and metabolic processes of autotrophic has been Herein, the findings the of PLA on the activities of Pta and AdhE1 as well as the Rex in C. ljungdahlii revealed the physiological of PLA in these autotrophic acetogens. PLA has on we found that an important role in the regulation of product synthetic pathways in C. ljungdahlii. AdhE1, and Rex low acetylation status in the this increased with the of fermentation that the major in C. ljungdahlii more in the The low acetylation levels of these key enzymes in the be of the low of acetyl-CoA at the of gas an transcriptional factors have been reported to be affected by PLA and altered regulatory the that metabolism D. Y. P. lysine acetylation of an in Natl. Acad. Sci. U. S. A. 2016; the of the system K. D. H. of the controls transcription a and the in C. ljungdahlii L. Y. Zhao R. Zhang C. Jiang W. Gu Y. regulation of during CO2 in gas-fermenting 2020; Scholar). Rex is as a of the in bacteria L. Nie X. J. Gu Y. Yang S. Jiang W. Yang C. Rex alcohol production and in Clostridium Scholar, D. novel of in J. Scholar, K. D. for by a Rex 2010; Scholar, M. H. The redox-sensing protein a transcriptional of in Clostridium acetobutylicum.Appl. Microbiol. Scholar, R. the associated with of the pathway in 2016; Scholar, of in Clostridium and its on the to Scholar). The of the and revealed that could a that the Rex to in which that are important for were identified K. D. for by a Rex 2010; Scholar). The that the of a key lysine the C. ljungdahlii Rex was to the responsible for can be that the acetylation level of the the regulatory of detailed understanding of the regulatory mechanism of lysine acetylation on the synthesis of acetic acid and ethanol provides a for the cellular of C. ljungdahlii in gas fermentation. In the metabolic of the optimization of the transcription of genes still represents the However, the optimization of posttranslational is an that be lysine acetylation is crucial for the activities of some key enzymes and related to product formation in C. the of their acetylation levels be in genetic to the synthetic of In this an cross-regulatory mechanism that controls the synthesis of acids and alcohols in autotrophic acetogens a physiological role and of PLA in In gut are to be the major human intestinal bacteria J. Jia H. X. H. Feng S. M. E. Nielsen T. C. Zhang W. F. of genes in the human gut and of short-chain fatty acids and alcohols that a to human intestinal health R. by gut for Microbiol. 2008; Scholar). our a the of PLA in gut and were as the for and protein The were in an G. at and other with The C. ljungdahlii was at °C in a for and a for gas fermentation to H. Chai C. N. P. Yang S. Jiang W. Gu Y. efficient in Clostridium an autotrophic gas-fermenting Biol. 2016; Scholar). was the in this are in the in this are in The was as the H. Chai C. N. P. Yang S. Jiang W. Gu Y. efficient in Clostridium an autotrophic gas-fermenting Biol. 2016; the was with and the was through using the as the and the The two the of were C. ljungdahlii using the and to the through using the the the and the were using a the The other for the of and were the the was that the and were The was as the was with and the was through using C. ljungdahlii as the and the The was through using C. ljungdahlii as the and the the the and the were using the the The other were the and using The and for AdhE1, and respectively, were for the production of the corresponding For example, the was through using the C. ljungdahlii as the and the the and the which was by with and were using the the The other for and protein were in a that the of the genes were The protein that were the as reported C. Gu Y. Y. Zhang W. Yang C. Yang S. Jiang W. of protein in Clostridium Scholar). 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The of the were by using Protein In a for protein was by at for a of protein to The of protein was by to the the protein were through and to for to at The were in the as reported L. Y. Zhao R. Zhang C. Jiang W. Gu Y. regulation of during CO2 in gas-fermenting 2020; Scholar). The in this lysine and The were as J. R. P. and of two and acetate the with In a that and Pta protein was at °C for the was using a was with a and the was at temperature for The of Pta was by the changes in the at the of lysine acetylation on the the Pta protein was in by and a of the to Pta protein in the was for the Pta The were for the of lysine acetylation on the activities of the other The of was using a to the In the of was by synthesis, which was through with the of by pyruvate and The of of of of of of of and of pyruvate The was at 25 °C and for the of was by the in at by the of on a The were as reported with were the of AdhE1, we a and of the alcohol of AdhE1, we a and of The was at and the activities were by the changes in the at T. L. J. S. of the alcohol and in and Clostridium and 2015; Scholar). The were as reported with were The and of The was at and the activities were by the changes in the at Metabolic of Clostridium ljungdahlii to Environ. Microbiol. 2015; Scholar, J. K. and metabolic of ferredoxin oxidoreductase the and Scholar). In acetylation were as reported L. Y. Zhao R. Zhang C. Jiang W. Gu Y. regulation of during CO2 in gas-fermenting 2020; with In a system was by of the and and at °C for the was two for and acetylation lysine was In were as reported L. Y. Zhao R. Zhang C. Jiang W. Gu Y. regulation of during CO2 in gas-fermenting 2020; with In a system was by of the and and at °C for was using the at a flow of The was the was was with and was with were with a to of to in and to at for the were a The was in with a of at of of dynamic of and a of to for was to at and was was and the which the of the to be at was as The was with were using the C. ljungdahlii with was the modification, with and as dynamic The were as of of of of and the of The and gas fermentation of C. ljungdahlii were in and respectively, in which was The detailed were to H. Chai C. N. P. Yang S. Jiang W. Gu Y. efficient in Clostridium an autotrophic gas-fermenting Biol. 2016; Scholar). The growth of C. ljungdahlii was to the of the at using a The of acetic acid and ethanol were to H. Chai C. N. P. Yang S. Jiang W. Gu Y. efficient in Clostridium an autotrophic gas-fermenting Biol. 2016; using a gas with a and a The have been in with of and the findings of this research are in the and the In and in the are the corresponding The that have of interest with the of this was by the of the of the and Y. L. and Y. W. and Y. G. W. J. and Y. G. W. J. and Y. G. W. J. and Y. G. W. J. and Y. G. W. J. and Y. G. with and

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