Pub Date : 2024-02-29DOI: 10.1186/s13068-024-02482-9
Pedro Montenegro-Silva, Tom Ellis, Fernando Dourado, Miguel Gama, Lucília Domingues
Background
Bacterial cellulose (BC) is a biocompatible material with unique mechanical properties, thus holding a significant industrial potential. Despite many acetic acid bacteria (AAB) being BC overproducers, cost-effective production remains a challenge. The role of pyrroloquinoline quinone (PQQ)-dependent membrane dehydrogenases (mDH) is crucial in the metabolism of AAB since it links substrate incomplete oxidation in the periplasm to energy generation. Specifically, glucose oxidation to gluconic acid substantially lowers environmental pH and hinders BC production. Conversely, ethanol supplementation is known to enhance BC yields in Komagataeibacter spp. by promoting efficient glucose utilization.
Results
K. sucrofermentans ATCC 700178 was engineered, knocking out the four PQQ-mDHs, to assess their impact on BC production. The strain KS003, lacking PQQ-dependent glucose dehydrogenase (PQQ-GDH), did not produce gluconic acid and exhibited a 5.77-fold increase in BC production with glucose as the sole carbon source, and a 2.26-fold increase under optimal ethanol supplementation conditions. In contrast, the strain KS004, deficient in the PQQ-dependent alcohol dehydrogenase (PQQ-ADH), showed no significant change in BC yield in the single carbon source experiment but showed a restrained benefit from ethanol supplementation.
Conclusions
The results underscore the critical influence of PQQ-GDH and PQQ-ADH and clarify the effect of ethanol supplementation on BC production in K. sucrofermentans ATCC 700178. This study provides a foundation for further metabolic pathway optimization, emphasizing the importance of diauxic ethanol metabolism for high BC production.
背景:细菌纤维素(BC)是一种生物相容性材料,具有独特的机械性能,因此具有巨大的工业潜力。尽管许多醋酸菌(AAB)都能生产过量的纤维素,但要生产出具有成本效益的纤维素仍是一项挑战。依赖吡咯喹啉醌(PQQ)的膜脱氢酶(mDH)在醋酸细菌的新陈代谢中起着至关重要的作用,因为它将底物在周质中的不完全氧化与能量生成联系在一起。具体来说,葡萄糖氧化成葡萄糖酸会大大降低环境 pH 值,阻碍 BC 的产生。相反,已知乙醇补充可通过促进葡萄糖的有效利用来提高 Komagataeibacter 属的 BC 产量:结果:对 K. sucrofermentans ATCC 700178 进行了改造,敲除了四个 PQQ-mDHs,以评估它们对 BC 生产的影响。缺乏 PQQ 依赖性葡萄糖脱氢酶(PQQ-GDH)的菌株 KS003 不产生葡萄糖酸,在以葡萄糖为唯一碳源的条件下,其 BC 产量增加了 5.77 倍,在最佳乙醇补充条件下增加了 2.26 倍。相比之下,缺乏 PQQ 依赖性乙醇脱氢酶(PQQ-ADH)的菌株 KS004 在单一碳源实验中 BC 产量没有显著变化,但从乙醇补充中获益有限:结果强调了 PQQ-GDH 和 PQQ-ADH 的关键影响,并阐明了乙醇补充对蔗糖球菌 ATCC 700178 BC 产量的影响。这项研究为进一步优化代谢途径奠定了基础,强调了双乙醇代谢对高产萃取物的重要性。
{"title":"Enhanced bacterial cellulose production in Komagataeibacter sucrofermentans: impact of different PQQ-dependent dehydrogenase knockouts and ethanol supplementation","authors":"Pedro Montenegro-Silva, Tom Ellis, Fernando Dourado, Miguel Gama, Lucília Domingues","doi":"10.1186/s13068-024-02482-9","DOIUrl":"10.1186/s13068-024-02482-9","url":null,"abstract":"<div><h3>Background</h3><p>Bacterial cellulose (BC) is a biocompatible material with unique mechanical properties, thus holding a significant industrial potential. Despite many acetic acid bacteria (AAB) being BC overproducers, cost-effective production remains a challenge. The role of pyrroloquinoline quinone (PQQ)-dependent membrane dehydrogenases (mDH) is crucial in the metabolism of AAB since it links substrate incomplete oxidation in the periplasm to energy generation. Specifically, glucose oxidation to gluconic acid substantially lowers environmental pH and hinders BC production. Conversely, ethanol supplementation is known to enhance BC yields in <i>Komagataeibacter spp.</i> by promoting efficient glucose utilization.</p><h3>Results</h3><p><i>K. sucrofermentans</i> ATCC 700178 was engineered, knocking out the four PQQ-mDHs, to assess their impact on BC production. The strain KS003, lacking PQQ-dependent glucose dehydrogenase (PQQ-GDH), did not produce gluconic acid and exhibited a 5.77-fold increase in BC production with glucose as the sole carbon source, and a 2.26-fold increase under optimal ethanol supplementation conditions. In contrast, the strain KS004, deficient in the PQQ-dependent alcohol dehydrogenase (PQQ-ADH), showed no significant change in BC yield in the single carbon source experiment but showed a restrained benefit from ethanol supplementation.</p><h3>Conclusions</h3><p>The results underscore the critical influence of PQQ-GDH and PQQ-ADH and clarify the effect of ethanol supplementation on BC production in <i>K. sucrofermentans</i> ATCC 700178. This study provides a foundation for further metabolic pathway optimization, emphasizing the importance of diauxic ethanol metabolism for high BC production.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":null,"pages":null},"PeriodicalIF":6.3,"publicationDate":"2024-02-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-024-02482-9","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139998552","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-02-27DOI: 10.1186/s13068-024-02477-6
Xuebin Feng, Siyi Hong, Hongbo Zhao, Thu V. Vuong, Emma R. Master
Background
Chitin, the main form of aminated polysaccharide in nature, is a biocompatible, polycationic, and antimicrobial biopolymer used extensively in industrial processes. Despite the abundance of chitin, applications thereof are hampered by difficulties in feedstock harvesting and limited structural versatility. To address these problems, we proposed a two-step cascade employing carbohydrate oxidoreductases and amine transaminases for plant polysaccharide aminations via one-pot reactions. Using a galactose oxidase from Fusarium graminearum for oxidation, this study compared the performance of CvATA (from Chromobacterium violaceum) and SpATA (from Silicibacter pomeroyi) on a range of oxidized carbohydrates with various structures and sizes. Using a rational enzyme engineering approach, four point mutations were introduced on the SpATA surface, and their effects on enzyme activity were evaluated.
Results
Herein, a quantitative colorimetric assay was developed to enable simple and accurate time-course measurement of the yield of transamination reactions. With higher operational stability, SpATA produced higher product yields in 36 h reactions despite its lower initial activity. Successful amination of oxidized galactomannan by SpATA was confirmed using a deuterium labeling method; higher aminated carbohydrate yields achieved with SpATA compared to CvATA were verified using HPLC and XPS. By balancing the oxidase and transaminase loadings, improved operating conditions were identified where the side product formation was largely suppressed without negatively impacting the product yield. SpATA mutants with multiple alanine substitutions besides E407A showed improved product yield. The E407A mutation reduced SpATA activity substantially, supporting its predicted role in maintaining the dimeric enzyme structure.
Conclusions
Using oxidase–amine transaminase cascades, the study demonstrated a fully enzymatic route to polysaccharide amination. Although the activity of SpATA may be further improved via enzyme engineering, the low operational stability of characterized amine transaminases, as a result of low retention of PMP cofactors, was identified as a key factor limiting the yield of the designed cascade. To increase the process feasibility, future efforts to engineer improved SpATA variants should focus on improving the cofactor affinity, and thus the operational stability of the enzyme.
{"title":"Biocatalytic cascade to polysaccharide amination","authors":"Xuebin Feng, Siyi Hong, Hongbo Zhao, Thu V. Vuong, Emma R. Master","doi":"10.1186/s13068-024-02477-6","DOIUrl":"10.1186/s13068-024-02477-6","url":null,"abstract":"<div><h3>Background</h3><p>Chitin, the main form of aminated polysaccharide in nature, is a biocompatible, polycationic, and antimicrobial biopolymer used extensively in industrial processes. Despite the abundance of chitin, applications thereof are hampered by difficulties in feedstock harvesting and limited structural versatility. To address these problems, we proposed a two-step cascade employing carbohydrate oxidoreductases and amine transaminases for plant polysaccharide aminations via one-pot reactions. Using a galactose oxidase from <i>Fusarium graminearum</i> for oxidation, this study compared the performance of CvATA (from <i>Chromobacterium violaceum</i>) and SpATA (from <i>Silicibacter pomeroyi</i>) on a range of oxidized carbohydrates with various structures and sizes. Using a rational enzyme engineering approach, four point mutations were introduced on the SpATA surface, and their effects on enzyme activity were evaluated.</p><h3>Results</h3><p>Herein, a quantitative colorimetric assay was developed to enable simple and accurate time-course measurement of the yield of transamination reactions. With higher operational stability, SpATA produced higher product yields in 36 h reactions despite its lower initial activity. Successful amination of oxidized galactomannan by SpATA was confirmed using a deuterium labeling method; higher aminated carbohydrate yields achieved with SpATA compared to CvATA were verified using HPLC and XPS. By balancing the oxidase and transaminase loadings, improved operating conditions were identified where the side product formation was largely suppressed without negatively impacting the product yield. SpATA mutants with multiple alanine substitutions besides E407A showed improved product yield. The E407A mutation reduced SpATA activity substantially, supporting its predicted role in maintaining the dimeric enzyme structure.</p><h3>Conclusions</h3><p>Using oxidase–amine transaminase cascades, the study demonstrated a fully enzymatic route to polysaccharide amination. Although the activity of SpATA may be further improved via enzyme engineering, the low operational stability of characterized amine transaminases, as a result of low retention of PMP cofactors, was identified as a key factor limiting the yield of the designed cascade. To increase the process feasibility, future efforts to engineer improved SpATA variants should focus on improving the cofactor affinity, and thus the operational stability of the enzyme.</p><h3>Graphical Abstract</h3><div><figure><div><div><picture><source><img></source></picture></div></div></figure></div></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":null,"pages":null},"PeriodicalIF":6.3,"publicationDate":"2024-02-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-024-02477-6","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139974982","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The present work models the fermentation process parameters of the newly isolated, Meyerozyma caribbica CP02 for enhanced xylitol production and its fermentability study on rice straw hydrolysate. The study examined the impact of each of the process variables by one variable at a time optimization followed by statistical validation. Temperature of 32 °C, pH of 3.5, agitation of 200 rpm, 1.5% (v/v) inoculum, 80 gL−1 initial xylose was optimized. Subsequently, a sequential two-stage agitation approach was adopted for fermentation. At these optimized conditions, xylitol yield of 0.77 gg−1 and 0.64 gg−1 was achieved using media containing commercial and rice straw derived xylose, respectively. For scale up, in 3L batch bioreactor, the highest xylitol yield (0.63 gg−1) was attained at 72 h with rice straw hydrolysate media containing initial xylose (59.48 ± 0.82 gL−1) along with inhibitors (1.55 ± 0.10 gL−1 aliphatic acids, 0.0.048 ± 0.11 gL−1 furans, 0.64 ± 0.23 gL−1 total phenols). The results imply that even under circumstances characterized by an acidic pH and elevated initial xylose level, M. caribbica CP02, as an isolate, displays robustness and shows favorable fermentability of rice straw hydrolysate. Therefore, isolate CP02 has potential to be used in bio-refineries for high yield xylitol production with minimal hydrolysate processing requirements.
{"title":"Bioprocess optimization for enhanced xylitol synthesis by new isolate Meyerozyma caribbica CP02 using rice straw","authors":"Saumya Singh, Shailendra Kumar Arya, Meena Krishania","doi":"10.1186/s13068-024-02475-8","DOIUrl":"10.1186/s13068-024-02475-8","url":null,"abstract":"<div><p>The present work models the fermentation process parameters of the newly isolated, <i>Meyerozyma caribbica</i> CP02 for enhanced xylitol production and its fermentability study on rice straw hydrolysate. The study examined the impact of each of the process variables by one variable at a time optimization followed by statistical validation. Temperature of 32 °C, pH of 3.5, agitation of 200 rpm, 1.5% (v/v) inoculum, 80 gL<sup>−1</sup> initial xylose was optimized. Subsequently, a sequential two-stage agitation approach was adopted for fermentation. At these optimized conditions, xylitol yield of 0.77 gg<sup>−1</sup> and 0.64 gg<sup>−1</sup> was achieved using media containing commercial and rice straw derived xylose, respectively. For scale up, in 3L batch bioreactor, the highest xylitol yield (0.63 gg<sup>−1</sup>) was attained at 72 h with rice straw hydrolysate media containing initial xylose (59.48 ± 0.82 gL<sup>−1</sup>) along with inhibitors (1.55 ± 0.10 gL<sup>−1</sup> aliphatic acids, 0.0.048 ± 0.11 gL<sup>−1</sup> furans, 0.64 ± 0.23 gL<sup>−1</sup> total phenols). The results imply that even under circumstances characterized by an acidic pH and elevated initial xylose level, <i>M. caribbica</i> CP02, as an isolate, displays robustness and shows favorable fermentability of rice straw hydrolysate. Therefore, isolate CP02 has potential to be used in bio-refineries for high yield xylitol production with minimal hydrolysate processing requirements.</p><h3>Graphical Abstract</h3><div><figure><div><div><picture><source><img></source></picture></div></div></figure></div></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":null,"pages":null},"PeriodicalIF":6.3,"publicationDate":"2024-02-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-024-02475-8","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139944783","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-02-24DOI: 10.1186/s13068-024-02471-y
Yu-Lei Jia, Qing-Ming Zhang, Fei Du, Wen-Qian Yang, Zi-Xu Zhang, Ying-Shuang Xu, Wang Ma, Xiao-Man Sun, He Huang
Background
Eicosapentaenoic acid (EPA) is widely used in the functional food and nutraceutical industries due to its important benefits to human health. Oleaginous microorganisms are considered a promising alternative resource for the production of EPA lipids. However, the storage of EPA in triglyceride (TG) becomes a key factor limiting its level.
Results
This study aimed to incorporate more EPA into TG storage through metabolic engineering. Firstly, key enzymes for TG synthesis, the diacylglycerol acyltransferase (DGAT) and glycerol-3-phosphate acyltransferase (GPAT) genes from Schizochytrium sp. HX-308 were expressed in Yarrowia lipolytica to enhance lipid and EPA accumulation. In addition, engineering the enzyme activity of DGATs through protein engineering was found to be effective in enhancing lipid synthesis by replacing the conserved motifs “HFS” in ScDGAT2A and “FFG” in ScDGAT2B with the motif “YFP”. Notably, combined with lipidomic analysis, the expression of ScDGAT2C and GPAT2 enhanced the storage of EPA in TG. Finally, the accumulation of lipid and EPA was further promoted by identifying and continuing to introduce the ScACC, ScACS, ScPDC, and ScG6PD genes from Schizochytrium sp., and the lipid and EPA titer of the final engineered strain reached 2.25 ± 0.03 g/L and 266.44 ± 5.74 mg/L, respectively, which increased by 174.39% (0.82 ± 0.02 g/L) and 282.27% (69.70 ± 0.80 mg/L) compared to the initial strain, respectively.
Conclusion
This study shows that the expression of lipid synthesis genes from Schizochytrium sp. in Y. lipolytica effectively improves the synthesis of lipids and EPA, which provided a promising target for EPA-enriched microbial oil production.
{"title":"Identification of lipid synthesis genes in Schizochytrium sp. and their application in improving eicosapentaenoic acid synthesis in Yarrowia lipolytica","authors":"Yu-Lei Jia, Qing-Ming Zhang, Fei Du, Wen-Qian Yang, Zi-Xu Zhang, Ying-Shuang Xu, Wang Ma, Xiao-Man Sun, He Huang","doi":"10.1186/s13068-024-02471-y","DOIUrl":"10.1186/s13068-024-02471-y","url":null,"abstract":"<div><h3>Background</h3><p>Eicosapentaenoic acid (EPA) is widely used in the functional food and nutraceutical industries due to its important benefits to human health. Oleaginous microorganisms are considered a promising alternative resource for the production of EPA lipids. However, the storage of EPA in triglyceride (TG) becomes a key factor limiting its level.</p><h3>Results</h3><p>This study aimed to incorporate more EPA into TG storage through metabolic engineering. Firstly, key enzymes for TG synthesis, the diacylglycerol acyltransferase (<i>DGAT</i>) and glycerol-3-phosphate acyltransferase (<i>GPAT</i>) genes from <i>Schizochytrium</i> sp. HX-308 were expressed in <i>Yarrowia lipolytica</i> to enhance lipid and EPA accumulation. In addition, engineering the enzyme activity of <i>DGAT</i>s through protein engineering was found to be effective in enhancing lipid synthesis by replacing the conserved motifs “HFS” in <i>ScDGAT2A</i> and “FFG” in <i>ScDGAT2B</i> with the motif “YFP”. Notably, combined with lipidomic analysis, the expression of <i>ScDGAT2C</i> and <i>GPAT2</i> enhanced the storage of EPA in TG. Finally, the accumulation of lipid and EPA was further promoted by identifying and continuing to introduce the <i>ScACC</i>, <i>ScACS</i>, <i>ScPDC</i>, and <i>ScG6PD</i> genes from <i>Schizochytrium</i> sp., and the lipid and EPA titer of the final engineered strain reached 2.25 ± 0.03 g/L and 266.44 ± 5.74 mg/L, respectively, which increased by 174.39% (0.82 ± 0.02 g/L) and 282.27% (69.70 ± 0.80 mg/L) compared to the initial strain, respectively.</p><h3>Conclusion</h3><p>This study shows that the expression of lipid synthesis genes from <i>Schizochytrium</i> sp. in <i>Y. lipolytica</i> effectively improves the synthesis of lipids and EPA, which provided a promising target for EPA-enriched microbial oil production.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":null,"pages":null},"PeriodicalIF":6.3,"publicationDate":"2024-02-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-024-02471-y","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139944784","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-02-24DOI: 10.1186/s13068-024-02478-5
Jinsha Huang, Xiaoman Xie, Wanlin Zheng, Li Xu, Jinyong Yan, Ying Wu, Min Yang, Yunjun Yan
Background
Biodiesel, an emerging sustainable and renewable clean energy, has garnered considerable attention as an alternative to fossil fuels. Although lipases are promising catalysts for biodiesel production, their efficiency in industrial-scale application still requires improvement.
Results
In this study, a novel strategy for multi-site mutagenesis in the binding pocket was developed via FuncLib (for mutant enzyme design) and Rosetta Cartesian_ddg (for free energy calculation) to improve the reaction rate and yield of lipase-catalyzed biodiesel production. Thermomyces lanuginosus lipase (TLL) with high activity and thermostability was obtained using the Pichia pastoris expression system. The specific activities of the mutants M11 and M21 (each with 5 and 4 mutations) were 1.50- and 3.10-fold higher, respectively, than those of the wild-type (wt–TLL). Their corresponding melting temperature profiles increased by 10.53 and 6.01 °C, (T_{50}^{15}) (the temperature at which the activity is reduced to 50% after 15 min incubation) increased from 60.88 to 68.46 °C and 66.30 °C, and the optimum temperatures shifted from 45 to 50 °C. After incubation in 60% methanol for 1 h, the mutants M11 and M21 retained more than 60% activity, and 45% higher activity than that of wt–TLL. Molecular dynamics simulations indicated that the increase in thermostability could be explained by reduced atomic fluctuation, and the improved catalytic properties were attributed to a reduced binding free energy and newly formed hydrophobic interaction. Yields of biodiesel production catalyzed by mutants M11 and M21 for 48 h at an elevated temperature (50 °C) were 94.03% and 98.56%, respectively, markedly higher than that of the wt–TLL (88.56%) at its optimal temperature (45 °C) by transesterification of soybean oil.
Conclusions
An integrating strategy was first adopted to realize the co-evolution of catalytic efficiency and thermostability of lipase. Two promising mutants M11 and M21 with excellent properties exhibited great potential for practical applications for in biodiesel production.
{"title":"In silico design of multipoint mutants for enhanced performance of Thermomyces lanuginosus lipase for efficient biodiesel production","authors":"Jinsha Huang, Xiaoman Xie, Wanlin Zheng, Li Xu, Jinyong Yan, Ying Wu, Min Yang, Yunjun Yan","doi":"10.1186/s13068-024-02478-5","DOIUrl":"10.1186/s13068-024-02478-5","url":null,"abstract":"<div><h3>Background</h3><p>Biodiesel, an emerging sustainable and renewable clean energy, has garnered considerable attention as an alternative to fossil fuels. Although lipases are promising catalysts for biodiesel production, their efficiency in industrial-scale application still requires improvement.</p><h3>Results</h3><p>In this study, a novel strategy for multi-site mutagenesis in the binding pocket was developed via FuncLib (for mutant enzyme design) and Rosetta Cartesian_ddg (for free energy calculation) to improve the reaction rate and yield of lipase-catalyzed biodiesel production. <i>Thermomyces lanuginosus</i> lipase (TLL) with high activity and thermostability was obtained using the <i>Pichia pastoris</i> expression system. The specific activities of the mutants M11 and M21 (each with 5 and 4 mutations) were 1.50- and 3.10-fold higher, respectively, than those of the wild-type (wt–TLL). Their corresponding melting temperature profiles increased by 10.53 and 6.01 °C, <span>(T_{50}^{15})</span> (the temperature at which the activity is reduced to 50% after 15 min incubation) increased from 60.88 to 68.46 °C and 66.30 °C, and the optimum temperatures shifted from 45 to 50 °C. After incubation in 60% methanol for 1 h, the mutants M11 and M21 retained more than 60% activity, and 45% higher activity than that of wt–TLL. Molecular dynamics simulations indicated that the increase in thermostability could be explained by reduced atomic fluctuation, and the improved catalytic properties were attributed to a reduced binding free energy and newly formed hydrophobic interaction. Yields of biodiesel production catalyzed by mutants M11 and M21 for 48 h at an elevated temperature (50 °C) were 94.03% and 98.56%, respectively, markedly higher than that of the wt–TLL (88.56%) at its optimal temperature (45 °C) by transesterification of soybean oil.</p><h3>Conclusions</h3><p>An integrating strategy was first adopted to realize the co-evolution of catalytic efficiency and thermostability of lipase. Two promising mutants M11 and M21 with excellent properties exhibited great potential for practical applications for in biodiesel production.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":null,"pages":null},"PeriodicalIF":6.3,"publicationDate":"2024-02-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-024-02478-5","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139944785","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The recently discovered PcAA14A and B from white-rot basidiomycete Pycnoporus coccineus enriched our understanding of the oxidative degradation of xylan in fungi, however, the unusual mode of action of AA14 LPMOs has sparked controversy. The substrate specificity and functionality of AA14 LPMOs still remain enigmatic and need further investigation.
Results
In this study, a novel AA14 LPMO was characterized from the ascomycete Talaromyces rugulosus. TrAA14A has a broad substrate specificity with strong oxidative activity on pure amorphous cellulose and xyloglucan. It could simultaneously oxidize cellulose, xylan and xyloglucan in natural hemi/cellulosic substrate such as fibrillated eucalyptus pulp, and released native and oxidized cello-oligosaccharides, xylo-oligosaccharides and xyloglucan oligosaccharides from this substrate, but its cellulolytic/hemicellulolytic activity became weaker as the contents of xylan increase in the alkaline-extracted hemi/cellulosic substrates. The dual cellulolytic/hemicellulolytic activity enables TrAA14A to possess a profound boosting effect on cellulose hydrolysis by cellulolytic enzymes. Structure modelling of TrAA14A revealed that it exhibits a relatively flat active-site surface similar to the active-site surfaces in AA9 LPMOs but quite distinct from PcAA14B, despite TrAA14A is strongly clustered together with AA14 LPMOs. Remarkable difference in electrostatic potentials of L2 and L3 surfaces was also observed among TrAA14A, PcAA14B and NcLPMO9F. We speculated that the unique feature in substrate-binding surface might contribute to the cellulolytic/hemicellulolytic activity of TrAA14A.
Conclusions
The extensive cellulolytic/hemicellulolytic activity on natural hemi/cellulosic substrate indicated that TrAA14A from ascomycete is distinctively different from previously characterized xylan-active AA9 or AA14 LPMOs. It may play as a bifunctional enzyme to decompose some specific network structures formed between cellulose and hemicellulose in the plant cell walls. Our findings shed new insights into the novel substrate specificities and biological functionalities of AA14 LPMOs, and will contribute to developing novel bifunctional LPMOs as the booster in commercial cellulase cocktails to efficiently break down the hemicellulose-cellulose matrix in lignocellulose.
{"title":"A novel AA14 LPMO from Talaromyces rugulosus with bifunctional cellulolytic/hemicellulolytic activity boosted cellulose hydrolysis","authors":"Kaixiang Chen, Xu Zhao, Peiyu Zhang, Liangkun Long, Shaojun Ding","doi":"10.1186/s13068-024-02474-9","DOIUrl":"10.1186/s13068-024-02474-9","url":null,"abstract":"<div><h3>Background</h3><p>The recently discovered <i>Pc</i>AA14A and B from white-rot basidiomycete <i>Pycnoporus coccineus</i> enriched our understanding of the oxidative degradation of xylan in fungi, however, the unusual mode of action of AA14 LPMOs has sparked controversy. The substrate specificity and functionality of AA14 LPMOs still remain enigmatic and need further investigation.</p><h3>Results</h3><p>In this study, a novel AA14 LPMO was characterized from the ascomycete <i>Talaromyces rugulosus</i>. <i>Tr</i>AA14A has a broad substrate specificity with strong oxidative activity on pure amorphous cellulose and xyloglucan. It could simultaneously oxidize cellulose, xylan and xyloglucan in natural hemi/cellulosic substrate such as fibrillated eucalyptus pulp, and released native and oxidized cello-oligosaccharides, xylo-oligosaccharides and xyloglucan oligosaccharides from this substrate, but its cellulolytic/hemicellulolytic activity became weaker as the contents of xylan increase in the alkaline-extracted hemi/cellulosic substrates. The dual cellulolytic/hemicellulolytic activity enables <i>Tr</i>AA14A to possess a profound boosting effect on cellulose hydrolysis by cellulolytic enzymes. Structure modelling of <i>Tr</i>AA14A revealed that it exhibits a relatively flat active-site surface similar to the active-site surfaces in AA9 LPMOs but quite distinct from <i>Pc</i>AA14B, despite <i>Tr</i>AA14A is strongly clustered together with AA14 LPMOs. Remarkable difference in electrostatic potentials of L2 and L3 surfaces was also observed among TrAA14A, <i>Pc</i>AA14B and <i>Nc</i>LPMO9F. We speculated that the unique feature in substrate-binding surface might contribute to the cellulolytic/hemicellulolytic activity of <i>Tr</i>AA14A.</p><h3>Conclusions</h3><p>The extensive cellulolytic/hemicellulolytic activity on natural hemi/cellulosic substrate indicated that <i>Tr</i>AA14A from ascomycete is distinctively different from previously characterized xylan-active AA9 or AA14 LPMOs. It may play as a bifunctional enzyme to decompose some specific network structures formed between cellulose and hemicellulose in the plant cell walls. Our findings shed new insights into the novel substrate specificities and biological functionalities of AA14 LPMOs, and will contribute to developing novel bifunctional LPMOs as the booster in commercial cellulase cocktails to efficiently break down the hemicellulose-cellulose matrix in lignocellulose.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":null,"pages":null},"PeriodicalIF":6.3,"publicationDate":"2024-02-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-024-02474-9","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139937230","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-02-22DOI: 10.1186/s13068-024-02480-x
Min Yao, Dan He, Wen Li, Xinghua Xiong, Xin He, Zhongsong Liu, Chunyun Guan, Lunwen Qian
Background
The primary objective of rapeseed breeding is to enhance oil content, which is predominantly influenced by environmental factors. However, the molecular mechanisms underlying the impact of these environmental factors on oil accumulation remain inadequately elucidated. In this study, we used transcriptome data from two higher (HOC) and two lower oil content (LOC) inbred lines at 35 days after pollination (DAP) to investigate genes exhibiting stable expression across three different environments. Meanwhile, a genome-wide association study (GWAS) was utilized to detect candidate genes exhibiting significant associations with seed oil content across three distinct environments.
Results
The study found a total of 405 stable differentially expressed genes (DEGs), including 25 involved in lipid/fatty acid metabolism and 14 classified as transcription factors. Among these genes, BnBZIP10-A09, BnMYB61-A06, BnAPA1-A08, BnPAS2-A10, BnLCAT3-C05 and BnKASIII-C09 were also found to exhibit significant associations with oil content across multiple different environments based on GWAS of 50 re-sequenced semi-winter rapeseed inbred lines and previously reported intervals. Otherwise, we revealed the presence of additive effects among BnBZIP10-A09, BnKASIII-C09, BnPAS2-A10 and BnAPA1-A08, resulting in a significant increase in seed oil content. Meanwhile, the majority of these stable DEGs are interconnected either directly or indirectly through co-expression network analysis, thereby giving rise to an elaborate molecular network implicated in the potential regulation of seed oil accumulation and stability.
Conclusions
The combination of transcription and GWAS revealed that natural variation in six environment-insensitive gene regions exhibited significant correlations with seed oil content phenotypes. These results provide important molecular marker information for us to further improve oil content accumulation and stability in rapeseed.
{"title":"Identification of environment-insensitive genes for oil content by combination of transcriptome and genome-wide association analysis in rapeseed","authors":"Min Yao, Dan He, Wen Li, Xinghua Xiong, Xin He, Zhongsong Liu, Chunyun Guan, Lunwen Qian","doi":"10.1186/s13068-024-02480-x","DOIUrl":"10.1186/s13068-024-02480-x","url":null,"abstract":"<div><h3>Background</h3><p>The primary objective of rapeseed breeding is to enhance oil content, which is predominantly influenced by environmental factors. However, the molecular mechanisms underlying the impact of these environmental factors on oil accumulation remain inadequately elucidated. In this study, we used transcriptome data from two higher (HOC) and two lower oil content (LOC) inbred lines at 35 days after pollination (DAP) to investigate genes exhibiting stable expression across three different environments. Meanwhile, a genome-wide association study (GWAS) was utilized to detect candidate genes exhibiting significant associations with seed oil content across three distinct environments.</p><h3>Results</h3><p>The study found a total of 405 stable differentially expressed genes (DEGs), including 25 involved in lipid/fatty acid metabolism and 14 classified as transcription factors. Among these genes, <i>BnBZIP10-</i>A09, <i>BnMYB61</i>-A06, <i>BnAPA1</i>-A08, <i>BnPAS2</i>-A10, <i>BnLCAT3</i>-C05 and <i>BnKASIII</i>-C09 were also found to exhibit significant associations with oil content across multiple different environments based on GWAS of 50 re-sequenced semi-winter rapeseed inbred lines and previously reported intervals. Otherwise, we revealed the presence of additive effects among <i>BnBZIP10-</i>A09, <i>BnKASIII</i>-C09, <i>BnPAS2</i>-A10 and <i>BnAPA1</i>-A08, resulting in a significant increase in seed oil content. Meanwhile, the majority of these stable DEGs are interconnected either directly or indirectly through co-expression network analysis, thereby giving rise to an elaborate molecular network implicated in the potential regulation of seed oil accumulation and stability.</p><h3>Conclusions</h3><p>The combination of transcription and GWAS revealed that natural variation in six environment-insensitive gene regions exhibited significant correlations with seed oil content phenotypes. These results provide important molecular marker information for us to further improve oil content accumulation and stability in rapeseed.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":null,"pages":null},"PeriodicalIF":6.3,"publicationDate":"2024-02-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-024-02480-x","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139916687","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Photosynthesis is a fundamental process that underlies the formation of crop yield, wherein light serves as the driving force and carbon dioxide (CO2) as the raw material. These two factors have a direct influence on the progress and efficiency of photosynthesis in crops. Rapeseed is one of the four major oilseed crops worldwide. Plateau rapeseed has now become a research hotspot. However, the lack of high-yielding rapeseed germplasm resources on the plateau and the highly efficient strategy for screening them severely affect the development of rapeseed industry in plateau.
Results
In the rapeseed experimental fields located on the plateau (Lhasa, Tibet), we measured abundant sunlight, characterized by an average daily photosynthetically active radiation (PAR) of 1413 μmol m−2 s−1. In addition, the atmospheric CO2 concentrations range from 300 to 400 ppm, which is only two-thirds of that in the plain (Chengdu, Sichuan). We found that under different measurement conditions of light intensity and CO2 concentration, different rapeseed genotypes showed significant differences in leaf photosynthetic efficiency during the seedling stage. Moreover, the rapeseed materials with high photosynthetic efficiency under low CO2 concentrations rather than high light intensity, exhibited significant advantages in biomass, yield, and oil content when cultivated on the plateau, indicating that the CO2 is the key environmental factor which limited rapeseed production in plateau. Based on photosynthetic efficiency screening under low CO2 concentrations, six rapeseed varieties SC3, SC10, SC25, SC27, SC29 and SC37, shown significantly higher yields in plateau environment compared to local control variety were obtained. In addition, the adaptability of rapeseed to plateau was found to be related to the activities of key Calvin cycle enzymes and the accumulation of photosynthetic products.
Conclusions
This study established a screening strategy for plateau high-yielding rapeseed materials, obtained six varieties which were suitable for plateau cultivation, explored the mechanism of rapeseed response to the plateau environment, and thus provides a feasible strategy for plateau-adapted rapeseed breeding.
{"title":"Low CO2 concentration, a key environmental factor for developing plateau adapted rapeseed","authors":"Sha Liu, Lin Tang, Jingyan Fu, Caixia Zhao, Ying Zhang, Meng Yin, Maolin Wang, Rui Wang, Yun Zhao","doi":"10.1186/s13068-024-02481-w","DOIUrl":"10.1186/s13068-024-02481-w","url":null,"abstract":"<div><h3>Background</h3><p>Photosynthesis is a fundamental process that underlies the formation of crop yield, wherein light serves as the driving force and carbon dioxide (CO<sub>2</sub>) as the raw material. These two factors have a direct influence on the progress and efficiency of photosynthesis in crops. Rapeseed is one of the four major oilseed crops worldwide. Plateau rapeseed has now become a research hotspot. However, the lack of high-yielding rapeseed germplasm resources on the plateau and the highly efficient strategy for screening them severely affect the development of rapeseed industry in plateau.</p><h3>Results</h3><p>In the rapeseed experimental fields located on the plateau (Lhasa, Tibet), we measured abundant sunlight, characterized by an average daily photosynthetically active radiation (PAR) of 1413 μmol m<sup>−2</sup> s<sup>−1</sup>. In addition, the atmospheric CO<sub>2</sub> concentrations range from 300 to 400 ppm, which is only two-thirds of that in the plain (Chengdu, Sichuan). We found that under different measurement conditions of light intensity and CO<sub>2</sub> concentration, different rapeseed genotypes showed significant differences in leaf photosynthetic efficiency during the seedling stage. Moreover, the rapeseed materials with high photosynthetic efficiency under low CO<sub>2</sub> concentrations rather than high light intensity, exhibited significant advantages in biomass, yield, and oil content when cultivated on the plateau, indicating that the CO<sub>2</sub> is the key environmental factor which limited rapeseed production in plateau. Based on photosynthetic efficiency screening under low CO<sub>2</sub> concentrations, six rapeseed varieties SC3, SC10, SC25, SC27, SC29 and SC37, shown significantly higher yields in plateau environment compared to local control variety were obtained. In addition, the adaptability of rapeseed to plateau was found to be related to the activities of key Calvin cycle enzymes and the accumulation of photosynthetic products.</p><h3>Conclusions</h3><p>This study established a screening strategy for plateau high-yielding rapeseed materials, obtained six varieties which were suitable for plateau cultivation, explored the mechanism of rapeseed response to the plateau environment, and thus provides a feasible strategy for plateau-adapted rapeseed breeding.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":null,"pages":null},"PeriodicalIF":6.3,"publicationDate":"2024-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-024-02481-w","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139915601","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The demand for melatonin is increasing due to its health-promoting bioactivities such as antioxidant and sleep benefits. Although melatonin is present in various organisms, its low content and high extraction cost make it unsustainable. Biosynthesis is a promising alternative method for melatonin production. However, the ectopic production of melatonin in microorganisms is very difficult due to the low or insoluble expression of melatonin synthesis genes. Hence, we aim to explore the biosynthesis of melatonin using Escherichia coli as a cell factory and ways to simultaneously coordinated express genes from different melatonin synthesis pathways.
Results
In this study, the mXcP4H gene from Xanthomonas campestris, as well as the HsAADC, HsAANAT and HIOMT genes from human melatonin synthesis pathway were optimized and introduced into E. coli via a multi-monocistronic vector. The obtained strain BL7992 successfully synthesized 1.13 mg/L melatonin by utilizing L-tryptophan (l-Trp) as a substrate in a shake flask. It was determined that the rate-limiting enzyme for melatonin synthesis is the arylalkylamine N-acetyltransferase, which is encoded by the HsAANAT gene. Targeted metabolomics analysis of l-Trp revealed that the majority of l-Trp flowed to the indole pathway in BL7992, and knockout of the tnaA gene may be beneficial for increasing melatonin production.
Conclusions
A metabolic engineering approach was adopted and melatonin was successfully synthesized from low-cost l-Trp in E. coli. This study provides a rapid and economical strategy for the synthesis of melatonin.
{"title":"Biosynthesis of melatonin from l-tryptophan by an engineered microbial cell factory","authors":"Lijuan Wang, Yongdong Deng, Jianjie Gao, Bo Wang, Hongjuan Han, Zhenjun Li, Wenhui Zhang, Yu Wang, Xiaoyan Fu, Rihe Peng, Quanhong Yao, Yongsheng Tian, Jing Xu","doi":"10.1186/s13068-024-02476-7","DOIUrl":"10.1186/s13068-024-02476-7","url":null,"abstract":"<div><h3>Background</h3><p>The demand for melatonin is increasing due to its health-promoting bioactivities such as antioxidant and sleep benefits. Although melatonin is present in various organisms, its low content and high extraction cost make it unsustainable. Biosynthesis is a promising alternative method for melatonin production. However, the ectopic production of melatonin in microorganisms is very difficult due to the low or insoluble expression of melatonin synthesis genes. Hence, we aim to explore the biosynthesis of melatonin using <i>Escherichia coli</i> as a cell factory and ways to simultaneously coordinated express genes from different melatonin synthesis pathways.</p><h3>Results</h3><p>In this study, the <i>mXcP4H</i> gene from <i>Xanthomonas campestris</i>, as well as the <i>HsAADC</i>, <i>HsAANAT</i> and <i>HIOMT</i> genes from human melatonin synthesis pathway were optimized and introduced into <i>E. coli</i> via a multi-monocistronic vector. The obtained strain BL7992 successfully synthesized 1.13 mg/L melatonin by utilizing L-tryptophan (<span>l</span>-Trp) as a substrate in a shake flask. It was determined that the rate-limiting enzyme for melatonin synthesis is the arylalkylamine N-acetyltransferase, which is encoded by the <i>HsAANAT</i> gene. Targeted metabolomics analysis of <span>l</span>-Trp revealed that the majority of <span>l</span>-Trp flowed to the indole pathway in BL7992, and knockout of the <i>tnaA</i> gene may be beneficial for increasing melatonin production.</p><h3>Conclusions</h3><p>A metabolic engineering approach was adopted and melatonin was successfully synthesized from low-cost <span>l</span>-Trp in <i>E. coli</i>. This study provides a rapid and economical strategy for the synthesis of melatonin.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":null,"pages":null},"PeriodicalIF":6.3,"publicationDate":"2024-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-024-02476-7","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139898749","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-02-15DOI: 10.1186/s13068-024-02470-z
Shuting Zhao, Dongtao Deng, Tianzheng Wan, Jie Feng, Lei Deng, Qianyi Tian, Jiayu Wang, Umm E. Aiman, Balym Mukhaddi, Xiaofeng Hu, Shaolin Chen, Ling Qiu, Lili Huang, Yahong Wei
Background
Bioconversion of plant biomass into biofuels and bio-products produces large amounts of lignin. The aromatic biopolymers need to be degraded before being converted into value-added bio-products. Microbes can be environment-friendly and efficiently degrade lignin. Compared to fungi, bacteria have some advantages in lignin degradation, including broad tolerance to pH, temperature, and oxygen and the toolkit for genetic manipulation.
Results
Our previous study isolated a novel ligninolytic bacterial strain Erwinia billingiae QL-Z3. Under optimized conditions, its rate of lignin degradation was 25.24% at 1.5 g/L lignin as the sole carbon source. Whole genome sequencing revealed 4556 genes in the genome of QL-Z3. Among 4428 protein-coding genes are 139 CAZyme genes, including 54 glycoside hydrolase (GH) and 16 auxiliary activity (AA) genes. In addition, 74 genes encoding extracellular enzymes are potentially involved in lignin degradation. Real-time PCR quantification demonstrated that the expression of potential ligninolytic genes were significantly induced by lignin. 8 knock-out mutants and complementary strains were constructed. Disruption of the gene for ELAC_205 (laccase) as well as EDYP_48 (Dyp-type peroxidase), ESOD_1236 (superoxide dismutase), EDIO_858 (dioxygenase), EMON_3330 (monooxygenase), or EMCAT_3587 (manganese catalase) significantly reduced the lignin-degrading activity of QL-Z3 by 47–69%. Heterologously expressed and purified enzymes further confirmed their role in lignin degradation. Fourier transform infrared spectroscopy (FTIR) results indicated that the lignin structure was damaged, the benzene ring structure and groups of macromolecules were opened, and the chemical bond was broken under the action of six enzymes encoded by genes. The abundant enzymatic metabolic products by EDYP_48, ELAC_205 and ESOD_1236 were systematically analyzed via liquid chromatography–mass spectrometry (LC–MS) analysis, and then provide a speculative pathway for lignin biodegradation. Finally, The activities of ligninolytic enzymes from fermentation supernatant, namely, LiP, MnP and Lac were 367.50 U/L, 839.50 U/L, and 219.00 U/L by orthogonal optimization.
Conclusions
Our findings provide that QL-Z3 and its enzymes have the potential for industrial application and hold great promise for the bioconversion of lignin into bioproducts in lignin valorization.
{"title":"Lignin bioconversion based on genome mining for ligninolytic genes in Erwinia billingiae QL-Z3","authors":"Shuting Zhao, Dongtao Deng, Tianzheng Wan, Jie Feng, Lei Deng, Qianyi Tian, Jiayu Wang, Umm E. Aiman, Balym Mukhaddi, Xiaofeng Hu, Shaolin Chen, Ling Qiu, Lili Huang, Yahong Wei","doi":"10.1186/s13068-024-02470-z","DOIUrl":"10.1186/s13068-024-02470-z","url":null,"abstract":"<div><h3>Background</h3><p>Bioconversion of plant biomass into biofuels and bio-products produces large amounts of lignin. The aromatic biopolymers need to be degraded before being converted into value-added bio-products. Microbes can be environment-friendly and efficiently degrade lignin. Compared to fungi, bacteria have some advantages in lignin degradation, including broad tolerance to pH, temperature, and oxygen and the toolkit for genetic manipulation.</p><h3>Results</h3><p>Our previous study isolated a novel ligninolytic bacterial strain <i>Erwinia billingiae</i> QL-Z3. Under optimized conditions, its rate of lignin degradation was 25.24% at 1.5 g/L lignin as the sole carbon source. Whole genome sequencing revealed 4556 genes in the genome of QL-Z3. Among 4428 protein-coding genes are 139 CAZyme genes, including 54 glycoside hydrolase (GH) and 16 auxiliary activity (AA) genes. In addition, 74 genes encoding extracellular enzymes are potentially involved in lignin degradation. Real-time PCR quantification demonstrated that the expression of potential ligninolytic genes were significantly induced by lignin. 8 knock-out mutants and complementary strains were constructed. Disruption of the gene for <i>ELAC_205</i> (laccase) as well as <i>EDYP_48</i> (Dyp-type peroxidase), <i>ESOD_1236</i> (superoxide dismutase), <i>EDIO_858</i> (dioxygenase), <i>EMON_3330</i> (monooxygenase), or <i>EMCAT_3587</i> (manganese catalase) significantly reduced the lignin-degrading activity of QL-Z3 by 47–69%. Heterologously expressed and purified enzymes further confirmed their role in lignin degradation. Fourier transform infrared spectroscopy (FTIR) results indicated that the lignin structure was damaged, the benzene ring structure and groups of macromolecules were opened, and the chemical bond was broken under the action of six enzymes encoded by genes. The abundant enzymatic metabolic products by EDYP_48, ELAC_205 and ESOD_1236 were systematically analyzed via liquid chromatography–mass spectrometry (LC–MS) analysis, and then provide a speculative pathway for lignin biodegradation. Finally, The activities of ligninolytic enzymes from fermentation supernatant, namely, LiP, MnP and Lac were 367.50 U/L, 839.50 U/L, and 219.00 U/L by orthogonal optimization.</p><h3>Conclusions</h3><p>Our findings provide that QL-Z3 and its enzymes have the potential for industrial application and hold great promise for the bioconversion of lignin into bioproducts in lignin valorization.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":null,"pages":null},"PeriodicalIF":6.3,"publicationDate":"2024-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-024-02470-z","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139742946","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}