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Article(id=1259888463424381482, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1259888457367806489, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20250922, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1765382400000, receivedDateStr=2025-12-11, revisedDate=null, revisedDateStr=null, acceptedDate=1768406400000, acceptedDateStr=2026-01-15, onlineDate=1778310417276, onlineDateStr=2026-05-09, pubDate=1777824000000, pubDateStr=2026-05-04, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1778310417276, onlineIssueDateStr=2026-05-09, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1778310417276, creator=13701087609, updateTime=1778310417276, updator=13701087609, issue=Issue{id=1259888457367806489, tenantId=1146029695717560320, journalId=1192105938417971205, year='2026', volume='66', issue='5', pageStart='2031', pageEnd='2556', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=0, articleOrder=1, issueType=-1, specialIssue=null, createTime=1778310415832, creator=13701087609, updateTime=1778320153326, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1259929299465921482, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1259888457367806489, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1259929299465921483, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1259888457367806489, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=2148, endPage=2158, ext={EN=ArticleExt(id=1259888464896582200, articleId=1259888463424381482, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=Reconstruction of methanol utilization pathway enhances co-utilization of methanol and CO2 in Komagataella phaffii, columnId=1192149543992045670, journalTitle=Acta Microbiologica Sinica, columnName=Research Article, runingTitle=null, highlight=null, articleAbstract=

Objective The engineering of the reductive glycine pathway (rGlyP) in Komagataella phaffii (syn. Pichia pastoris) represents a promising strategy for the co-utilization of methanol and CO2. However, the efficiency of this pathway is constrained by the insufficient supply of intracellular reduced nicotinamide adenine dinucleotide (NADH), as the native alcohol oxidase (AOX) pathway generates hydrogen peroxide rather than NADH, leading to energy loss and oxidative stress. To overcome this bottleneck, this study reconstructed the methanol oxidation pathway and employed a subcellular compartmentalization strategy to optimize the carbon flux and energy metabolism. Methods Five different sources of NAD+-dependent methanol dehydrogenase (MDH) were screened in an aox1/aox2-deficient strain by using the growth curve and methanol utilization rate as indicators to determine the optimal MDH, and the methanol induction concentration was optimized. Subsequently, a compartmentalization strategy was employed by fusing the peroxisomal targeting signal 1 (PTS1) to MDHN1T, which targeted the enzyme to the peroxisome to spatially couple methanol oxidation with formaldehyde detoxification. Results The MDHN1T derived from Cupriavidus necator had the best catalytic performance, and the optimum methanol induction concentration was optimized to be 0.6%. Under co-utilization conditions, the engineered strain achieved a methanol consumption rate of 28.98 mg/d, with the total intracellular NADtotal pool, NADH/NAD+ ratio, and biomass being 1.3, 1.2, and 2.2 folds, respectively, of those in the parental strain. Conclusion This study successfully alleviates the redox cofactor imbalance in the rGlyP and enhances co-utilization of methanol and CO2 in K. phaffii, providing a robust chassis and a theoretical basis for the development of microbial cell factories utilizing one-carbon resources.

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E-mail: BAI Zhonghu, ;
YANG Yankun,
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目的 工程化构建还原性甘氨酸途径(reductive glycine pathway, rGlyP)是实现甲醇与CO2协同利用的有效策略，但该途径的高效运转受限于胞内还原型烟酰胺腺嘌呤二核苷酸(reduced nicotinamide adenine dinucleotide, NADH)供给不足。毕赤酵母(Komagataella phaffii)内源醇氧化酶(alcohol oxidase, AOX)途径氧化甲醇产生过氧化氢而非NADH，导致能量浪费并引发氧化胁迫。为解决这一瓶颈，本研究通过重构甲醇氧化途径并结合亚细胞区室化策略，优化碳通量与能量代谢。 方法 以生长曲线和甲醇利用速率为指标，在敲除内源aox1和aox2的底盘菌株中对5种不同来源的NAD+依赖型甲醇脱氢酶(methanol dehydrogenase, MDH)进行筛选，以确定最佳MDH，并对其甲醇诱导浓度进行优化。进一步采用区室化策略，利用1型过氧化物酶体靶向信号(peroxisomal targeting signal 1, PTS1)信号肽将MDHN1T靶向定位至过氧化物酶体，实现了甲醇氧化与甲醛解毒的空间偶联。 结果 筛选确定源自杀虫贪铜菌(Cupriavidus necator)的MDHN1T具有最佳催化性能，并优化出其最适甲醇诱导浓度为0.6%。重组菌株在甲醇/CO2共利用条件下，甲醇消耗速率提升至28.98 mg/d，胞内NAD+和NADH总量提升至原来的1.3倍，NADH/NAD+提升至原来的1.2倍，生物量达到出发菌株的2.2倍。 结论 本研究成功缓解了rGlyP的还原力匮乏问题，促进了毕赤酵母对甲醇和CO2的共利用，为构建高效利用一碳资源的微生物细胞工厂提供了优质的底盘细胞及理论依据。

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作者贡献声明

王家孟：实验操作、研究构思设计、数据收集和处理，论文撰写与修改；张贝宁：实验操作和数据收集；李远：协助实验操作；白仲虎：研究构思、实验指导、论文润色；杨艳坤：基金支持、项目管理、提出构想、实验思路设计、论文修改。

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2.江南大学，粮食发酵与食品生物制造国家工程研究中心，江苏 无锡
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Metabolic Engineering, 2024, 84: 1-12., articleTitle=Engineering yeasts to co-utilize methanol or formate coupled with CO2 fixation, refAbstract=null)], funds=[Fund(id=1259928496558694829, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, awardId=32370054, language=EN, fundingSource=The National Natural Science Foundation of China(32370054), fundOrder=null, country=null), Fund(id=1259928497045234099, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, awardId=32370054, language=CN, fundingSource=国家自然科学基金(32370054), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1259928391260693157, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, xref=1., ext=[AuthorCompanyExt(id=1259928391587848876, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, companyId=1259928391260693157, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1.Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China), AuthorCompanyExt(id=1259928391688512173, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, companyId=1259928391260693157, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1.江南大学 生物工程学院，工业生物技术教育部重点实验室，江苏 无锡)]), AuthorCompany(id=1259928394620330685, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, xref=2., ext=[AuthorCompanyExt(id=1259928394628719294, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, companyId=1259928394620330685, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing, Jiangnan University, Wuxi, Jiangsu, China), AuthorCompanyExt(id=1259928394637107904, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, companyId=1259928394620330685, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.江南大学，粮食发酵与食品生物制造国家工程研究中心，江苏 无锡)]), AuthorCompany(id=1259928395530494676, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, xref=3., ext=[AuthorCompanyExt(id=1259928395673101015, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, companyId=1259928395530494676, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=3.Jiangsu Provincial Engineering Research Center for Bioactive Product Processing, Jiangnan University, Wuxi, Jiangsu, China), AuthorCompanyExt(id=1259928395878621915, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, companyId=1259928395530494676, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=3.江南大学，江苏省生物活性制品加工工程技术研究中心，江苏 无锡)]), AuthorCompany(id=1259928396767814379, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, xref=4., ext=[AuthorCompanyExt(id=1259928396780397294, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, companyId=1259928396767814379, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=4.Zhengzhou University of Technology, Zhengzhou, Henan, China), AuthorCompanyExt(id=1259928396784591599, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, companyId=1259928396767814379, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=4.郑州工程技术学院，河南 郑州)])], figs=[ArticleFig(id=1259928465214660833, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, language=EN, label=Figure 1, caption=Growth curves of different strains at varying methanol concentrations. A, C: 1.2% methanol concentration; B, D: 1.6% methanol concentration., figureFileSmall=AzwRlPxNWXZgDApJwrCbzg==, figureFileBig=erkbzwAjRmr53d5QORu+8Q==, tableContent=null), ArticleFig(id=1259928466837856494, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, language=CN, label=图1, caption=不同菌株在不同甲醇浓度下的生长曲线, figureFileSmall=AzwRlPxNWXZgDApJwrCbzg==, figureFileBig=erkbzwAjRmr53d5QORu+8Q==, tableContent=null), ArticleFig(id=1259928469702566142, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, language=EN, label=Figure 2, caption=Growth curves of 2Δ-GS115 and 2Δ-MDHN1T under different methanol concentration gradients. A: 0.3%; B: 0.4%; C: 0.5%; D: 0.6%; E: 0.7%., figureFileSmall=xyHslw2IkV3qPxRWvaVIwA==, figureFileBig=DAnoNjFWyYoUudX1yZ7C7A==, tableContent=null), ArticleFig(id=1259928470952468743, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, language=CN, label=图2, caption=2Δ-GS115和2Δ-MDHN1T在不同甲醇浓度梯度的生长曲线, figureFileSmall=xyHslw2IkV3qPxRWvaVIwA==, figureFileBig=DAnoNjFWyYoUudX1yZ7C7A==, tableContent=null), ArticleFig(id=1259928472676327702, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, language=EN, label=Figure 3, caption=Growth curves and methanol consumption of 2Δ-GS115 strain expressing MDH at 0.6% methanol concentration. A: Growth curves; B: Methanol consumption., figureFileSmall=eHs4yKAdqJOosL//yOBHxA==, figureFileBig=XWYUZRY4eIKWPgssZwjP9g==, tableContent=null), ArticleFig(id=1259928474026893606, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, language=CN, label=图3, caption=0.6%甲醇浓度下表达MDH的2Δ-GS115菌株的生长曲线和甲醇消耗, figureFileSmall=eHs4yKAdqJOosL//yOBHxA==, figureFileBig=XWYUZRY4eIKWPgssZwjP9g==, tableContent=null), ArticleFig(id=1259928475633312053, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, language=EN, label=Figure 4, caption=Diagram of the pathway of NAD+/NADH metabolism in A05. Red indicates overexpressed enzymes, including: formate-THF ligase (MIS1-1); Blue indicates knocked-out enzymes, including: glyoxylate aminotransferase 1 (Agt1), serine hydroxymethyl transferase 1/2 (Shm1/2), and threonine aldolase 1 (Gly1)., figureFileSmall=tF/eGvgmXqHikAXYtvqd8w==, figureFileBig=pA3qzS1po2Q22kndGYNqAg==, tableContent=null), ArticleFig(id=1259928476543476030, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, language=CN, label=图4, caption=A05菌株NAD+/NADH代谢途径示意图, figureFileSmall=tF/eGvgmXqHikAXYtvqd8w==, figureFileBig=pA3qzS1po2Q22kndGYNqAg==, tableContent=null), ArticleFig(id=1259928481018798413, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, language=EN, label=Figure 5, caption=Effect of introducing MDHN1T at a 0.6% methanol concentration on the A05 strain. A: Growth curves; B: Methanol consumption; C: Total NAD; D: NADH/NAD+. **: P<0.01; *: P<0.05., figureFileSmall=7jtebsosCV4BQDGry+AOzw==, figureFileBig=3W3j1mhDcz/+HtNXAkJLAA==, tableContent=null), ArticleFig(id=1259928483162087763, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, language=CN, label=图5, caption=0.6%甲醇浓度下引入MDHN1T对A05菌株的影响, figureFileSmall=7jtebsosCV4BQDGry+AOzw==, figureFileBig=3W3j1mhDcz/+HtNXAkJLAA==, tableContent=null), ArticleFig(id=1259928484466516320, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, language=EN, label=Table 1, caption=

Plasmids used in this study

, figureFileSmall=null, figureFileBig=null, tableContent=
PlasmidsCharacteristicsSource
pBB1 MDH2 Ⅳ 9CamRThis study
pBB1 MDH3 Ⅳ 9CamRThis study
pBB1 MDHBS Ⅳ 9CamRThis study
pA0R MDHLXT Ⅱ 7AmpRThis study
pA0R MDHN1T Ⅱ 7AmpRThis study
pA0R ACT1 Ⅱ 7AmpRThis study
pGAPZB MDHN1TZeocin+This study
pGAPZB MDHN1T(p)Zeocin+This study
pAI05-sg PNS Ⅳ 9Zeocin+Lab stock
pAI05-sg PNS Ⅱ 7Zeocin+Lab stock
pAI05-sg aox1Zeocin+This study
pAI05-sg aox2Zeocin+This study
), ArticleFig(id=1259928486064546152, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, language=CN, label=表1, caption=

本研究所用的质粒

, figureFileSmall=null, figureFileBig=null, tableContent=
PlasmidsCharacteristicsSource
pBB1 MDH2 Ⅳ 9CamRThis study
pBB1 MDH3 Ⅳ 9CamRThis study
pBB1 MDHBS Ⅳ 9CamRThis study
pA0R MDHLXT Ⅱ 7AmpRThis study
pA0R MDHN1T Ⅱ 7AmpRThis study
pA0R ACT1 Ⅱ 7AmpRThis study
pGAPZB MDHN1TZeocin+This study
pGAPZB MDHN1T(p)Zeocin+This study
pAI05-sg PNS Ⅳ 9Zeocin+Lab stock
pAI05-sg PNS Ⅱ 7Zeocin+Lab stock
pAI05-sg aox1Zeocin+This study
pAI05-sg aox2Zeocin+This study
), ArticleFig(id=1259928487742267756, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, language=EN, label=Table 2, caption=

Strains used in this study

, figureFileSmall=null, figureFileBig=null, tableContent=
StrainsCharacteristicsSource
K. phaffii GS115PNS Ⅰ 6::Ppa. Rad52Lab stock
1Δ-GS115K. phaffii GS115, aox1ΔThis study
2Δ-GS115K. phaffii GS115, aox1Δ aox2ΔThis study
2Δ-MDH2K. phaffii GS115, aox1Δ aox2Δ, P AOX1 -MDH2, P AOX1 -ACT1This study
2Δ-MDH3K. phaffii GS115, aox1Δ aox2Δ, P AOX1 -MDH3, P AOX1 -ACT1This study
2Δ-MDHBSK. phaffii GS115, aox1Δ aox2Δ, P AOX1 -MDHBS, P AOX1 -ACT1This study
2Δ-MDHLXTK. phaffii GS115, aox1Δ aox2Δ, P AOX1 -MDHLXTThis study
2Δ-MDHN1TK. phaffii GS115, aox1Δ aox2Δ, P AOX1 -MDHN1TThis study
A05K. phaffii GS115-Shm1Δ Shm2Δ Gly1Δ AgtΔ, P GAP -MIS1-1Lab stock
A05-MDHN1T(p)A05, P AOX1 -MDHN1T (PTS1)This study
), ArticleFig(id=1259928489893945720, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, language=CN, label=表2, caption=

本研究所用的菌株

, figureFileSmall=null, figureFileBig=null, tableContent=
StrainsCharacteristicsSource
K. phaffii GS115PNS Ⅰ 6::Ppa. Rad52Lab stock
1Δ-GS115K. phaffii GS115, aox1ΔThis study
2Δ-GS115K. phaffii GS115, aox1Δ aox2ΔThis study
2Δ-MDH2K. phaffii GS115, aox1Δ aox2Δ, P AOX1 -MDH2, P AOX1 -ACT1This study
2Δ-MDH3K. phaffii GS115, aox1Δ aox2Δ, P AOX1 -MDH3, P AOX1 -ACT1This study
2Δ-MDHBSK. phaffii GS115, aox1Δ aox2Δ, P AOX1 -MDHBS, P AOX1 -ACT1This study
2Δ-MDHLXTK. phaffii GS115, aox1Δ aox2Δ, P AOX1 -MDHLXTThis study
2Δ-MDHN1TK. phaffii GS115, aox1Δ aox2Δ, P AOX1 -MDHN1TThis study
A05K. phaffii GS115-Shm1Δ Shm2Δ Gly1Δ AgtΔ, P GAP -MIS1-1Lab stock
A05-MDHN1T(p)A05, P AOX1 -MDHN1T (PTS1)This study
), ArticleFig(id=1259928491982709132, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, language=EN, label=Table 3, caption=

Primers for PCR

, figureFileSmall=null, figureFileBig=null, tableContent=
Primer namesPrimer sequences (5′→3′)
MDH2-FCATATGAAGAACACCCTGTCTGCA
MDH2-RTTAATGGTGATGATGATGATGCATCG
MDH3-FATGACCAACACTCAATCTGCTTTC
MDH3-RTTAATGATGATGATGATGATGCATAGCG
MDHBs-FCATATGAAGGCCGCAGTGGT
MDHBs-RTTAATGGTGATGGTGATGATGATCTTCTT
MDHLXT-FCATATGAGCGATGTTCTGAAACAGTT
MDHLXT-RTTAATGGTGATGATGATGATGACTCAGG
MDHN1T-FCATATGACCCATCTGAATATCGCAAAT
MDHN1T-RTTAATGGTGATGATGATGATGCATGG
ACT1-FCATATGGGTAAACTGTTCGAAGAAAAGAC
ACT1-RTTAATGATGGTGGTGATGGTGCA
), ArticleFig(id=1259928493266166165, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888463424381482, language=CN, label=表3, caption=

PCR所用引物

, figureFileSmall=null, figureFileBig=null, tableContent=
Primer namesPrimer sequences (5′→3′)
MDH2-FCATATGAAGAACACCCTGTCTGCA
MDH2-RTTAATGGTGATGATGATGATGCATCG
MDH3-FATGACCAACACTCAATCTGCTTTC
MDH3-RTTAATGATGATGATGATGATGCATAGCG
MDHBs-FCATATGAAGGCCGCAGTGGT
MDHBs-RTTAATGGTGATGGTGATGATGATCTTCTT
MDHLXT-FCATATGAGCGATGTTCTGAAACAGTT
MDHLXT-RTTAATGGTGATGATGATGATGACTCAGG
MDHN1T-FCATATGACCCATCTGAATATCGCAAAT
MDHN1T-RTTAATGGTGATGATGATGATGCATGG
ACT1-FCATATGGGTAAACTGTTCGAAGAAAAGAC
ACT1-RTTAATGATGGTGGTGATGGTGCA
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重构甲醇利用途径促进毕赤酵母甲醇和CO2 的共利用
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王家孟 1, 2, 3 , 张贝宁 1, 2, 3 , 李远 1, 2, 3 , 白仲虎 1, 2, 3, 4 , 杨艳坤 1, 2, 3
微生物学报 | 研究报告 2026,66(5): 2148-2158
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微生物学报 | 研究报告 2026, 66(5): 2148-2158
重构甲醇利用途径促进毕赤酵母甲醇和CO2 的共利用
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王家孟1, 2, 3, 张贝宁1, 2, 3, 李远1, 2, 3, 白仲虎1, 2, 3, 4 , 杨艳坤1, 2, 3
作者信息
  • 1.江南大学 生物工程学院，工业生物技术教育部重点实验室，江苏 无锡
  • 2.江南大学，粮食发酵与食品生物制造国家工程研究中心，江苏 无锡
  • 3.江南大学，江苏省生物活性制品加工工程技术研究中心，江苏 无锡
  • 4.郑州工程技术学院，河南 郑州
Reconstruction of methanol utilization pathway enhances co-utilization of methanol and CO2 in Komagataella phaffii
Jiameng WANG1, 2, 3, Beining ZHANG1, 2, 3, Yuan LI1, 2, 3, Zhonghu BAI1, 2, 3, 4 , Yankun YANG1, 2, 3
Affiliations
  • 1.Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China
  • 2.National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing, Jiangnan University, Wuxi, Jiangsu, China
  • 3.Jiangsu Provincial Engineering Research Center for Bioactive Product Processing, Jiangnan University, Wuxi, Jiangsu, China
  • 4.Zhengzhou University of Technology, Zhengzhou, Henan, China
出版时间: 2026-05-04 doi: 10.13343/j.cnki.wsxb.20250922
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摘要
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目的 工程化构建还原性甘氨酸途径(reductive glycine pathway, rGlyP)是实现甲醇与CO2协同利用的有效策略，但该途径的高效运转受限于胞内还原型烟酰胺腺嘌呤二核苷酸(reduced nicotinamide adenine dinucleotide, NADH)供给不足。毕赤酵母(Komagataella phaffii)内源醇氧化酶(alcohol oxidase, AOX)途径氧化甲醇产生过氧化氢而非NADH，导致能量浪费并引发氧化胁迫。为解决这一瓶颈，本研究通过重构甲醇氧化途径并结合亚细胞区室化策略，优化碳通量与能量代谢。 方法 以生长曲线和甲醇利用速率为指标，在敲除内源aox1和aox2的底盘菌株中对5种不同来源的NAD+依赖型甲醇脱氢酶(methanol dehydrogenase, MDH)进行筛选，以确定最佳MDH，并对其甲醇诱导浓度进行优化。进一步采用区室化策略，利用1型过氧化物酶体靶向信号(peroxisomal targeting signal 1, PTS1)信号肽将MDHN1T靶向定位至过氧化物酶体，实现了甲醇氧化与甲醛解毒的空间偶联。 结果 筛选确定源自杀虫贪铜菌(Cupriavidus necator)的MDHN1T具有最佳催化性能，并优化出其最适甲醇诱导浓度为0.6%。重组菌株在甲醇/CO2共利用条件下，甲醇消耗速率提升至28.98 mg/d，胞内NAD+和NADH总量提升至原来的1.3倍，NADH/NAD+提升至原来的1.2倍，生物量达到出发菌株的2.2倍。 结论 本研究成功缓解了rGlyP的还原力匮乏问题，促进了毕赤酵母对甲醇和CO2的共利用，为构建高效利用一碳资源的微生物细胞工厂提供了优质的底盘细胞及理论依据。

毕赤酵母  /  甲醇脱氢酶  /  还原性甘氨酸途径  /  区室化策略  /  CO2固定  /  一碳资源利用

Objective The engineering of the reductive glycine pathway (rGlyP) in Komagataella phaffii (syn. Pichia pastoris) represents a promising strategy for the co-utilization of methanol and CO2. However, the efficiency of this pathway is constrained by the insufficient supply of intracellular reduced nicotinamide adenine dinucleotide (NADH), as the native alcohol oxidase (AOX) pathway generates hydrogen peroxide rather than NADH, leading to energy loss and oxidative stress. To overcome this bottleneck, this study reconstructed the methanol oxidation pathway and employed a subcellular compartmentalization strategy to optimize the carbon flux and energy metabolism. Methods Five different sources of NAD+-dependent methanol dehydrogenase (MDH) were screened in an aox1/aox2-deficient strain by using the growth curve and methanol utilization rate as indicators to determine the optimal MDH, and the methanol induction concentration was optimized. Subsequently, a compartmentalization strategy was employed by fusing the peroxisomal targeting signal 1 (PTS1) to MDHN1T, which targeted the enzyme to the peroxisome to spatially couple methanol oxidation with formaldehyde detoxification. Results The MDHN1T derived from Cupriavidus necator had the best catalytic performance, and the optimum methanol induction concentration was optimized to be 0.6%. Under co-utilization conditions, the engineered strain achieved a methanol consumption rate of 28.98 mg/d, with the total intracellular NADtotal pool, NADH/NAD+ ratio, and biomass being 1.3, 1.2, and 2.2 folds, respectively, of those in the parental strain. Conclusion This study successfully alleviates the redox cofactor imbalance in the rGlyP and enhances co-utilization of methanol and CO2 in K. phaffii, providing a robust chassis and a theoretical basis for the development of microbial cell factories utilizing one-carbon resources.

Komagataella phaffii  /  methanol dehydrogenase (MDH)  /  reductive glycine pathway (rGlyP)  /  compartmentalization strategy  /  CO2 fixation  /  one-carbon resource utilization
王家孟, 张贝宁, 李远, 白仲虎, 杨艳坤. 重构甲醇利用途径促进毕赤酵母甲醇和CO2 的共利用. 微生物学报, 2026 , 66 (5) : 2148 -2158 . DOI: 10.13343/j.cnki.wsxb.20250922
Jiameng WANG, Beining ZHANG, Yuan LI, Zhonghu BAI, Yankun YANG. Reconstruction of methanol utilization pathway enhances co-utilization of methanol and CO2 in Komagataella phaffii[J]. Acta Microbiologica Sinica, 2026 , 66 (5) : 2148 -2158 . DOI: 10.13343/j.cnki.wsxb.20250922
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甲醇和CO2是廉价且来源丰富的可再生一碳资源，在生物制造领域展现出替代化石燃料的巨大潜力。二氧化碳和甲醇的生物固定与利用对实现碳中和目标具有重要意义。构建高效的微生物细胞工厂，可在温和条件下将一碳资源转化为化学品、生物燃料、可降解材料等高价值产物[1]，这对实现全球碳中和目标具有重要的战略意义，已成为研究热点[2-4]。
人工设计的还原性甘氨酸途径(reductive glycine pathway, rGlyP)能够有效固碳，已在多种微生物中用于将CO2转化为生物量和化学品[5-7]。毕赤酵母(Komagataella phaffii)具有完备且较为高效的甲醇利用途径(methanol utilization pathway, MUTP)，通过工程化改造rGlyP，实现了对甲醇和CO2的共利用[8]。rGlyP的高效运转高度依赖充足的还原力供给，而毕赤酵母本身的甲醇代谢主要依赖于醇氧化酶(alcohol oxidase, AOX)系统，该系统氧化甲醇生成甲醛并伴随过氧化氢的积累，这一过程不仅不产生还原力，反而因抗氧化需求消耗额外的还原力[9]。因此，改造现有的甲醇利用途径，解决还原力供需失衡问题，并偶联rGlyP，是提高甲醇和CO2协同利用效率的关键科学问题。
甲醇脱氢酶(methanol dehydrogenase, MDH)存在于自然界的一类天然甲醇营养菌中，能催化甲醇生成甲醛。根据电子受体的不同，甲基营养菌中的MDH可分为3类：吡咯喹啉醌(pyrroloquinoline quinone, PQQ)依赖型MDH、氧依赖型醇氧化酶以及烟酰胺腺嘌呤双核苷酸NAD+依赖型MDH。其中，NAD+依赖型MDH氧化甲醇生成甲醛，消耗NAD+并生成还原力NADH[10-11]。MDH在氧化甲醇的同时能为rGlyP提供更多的还原力，是重构MUTP的理想分子。
K. phaffii A05是前期构建的含重组rGlyP的重组菌株，可协同利用甲醇和CO2，但其生长受限于还原力供给[8]。本研究将5种不同生物来源的NAD+依赖型MDH引入毕赤酵母，通过敲除内源aox1和aox2基因以消除背景干扰，筛选获得最优MDH。在此基础上，采用区室化策略，将最优MDH定位于过氧化物酶体，重构了甲醇利用途径。将其与rGlyP耦合，探究胞内NADH水平及甲醇利用效率的改变，以期为微生物发酵工业中一碳资源的综合利用提供依据和策略。
1 材料与方法
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1.1 材料
1.1.1 菌株及质粒
K. phaffii GS115购自ThermoFisher Scientific公司。K. phaffii A05为本课题组Li等[8]构建，其表型为甘氨酸缺陷型且强化了rGlyP，可在常压大气环境下协同固定甲醇和CO2。本研究使用的质粒模板有pBB1、pAI05、pA0R、pGAPZB等4种，均由本实验室构建并保存。
本研究所用的质粒如表1所示。本研究所用菌株如表2所示。
1.1.2 培养基
LLB (low-salt Luria-Bertani)培养基(g/L)：酵母提取物5.0，胰蛋白胨10.0，NaCl 5.0。
LB (Luria-Bertani)培养基(g/L)：酵母提取物5.0，胰蛋白胨10.0，NaCl 10.0。
YPD (yeast extract peptone dextrose)培养基(g/L)：酵母提取物10.0，胰蛋白胨20.0，葡萄糖20.0。
MDG (minimal dextrose glycine)培养基(g/L)：无氨基酵母氮源YNB 13.40，葡萄糖20.00，甘氨酸1.13。
若要配制固体培养基则需再加入20.0 g/L的琼脂。添加抗生素时氯霉素、氨苄青霉素工作浓度为100.0 μg/mL，博莱霉素工作浓度为25.0 μg/mL。
BMMH (buffered minimal methanol histidine)培养基(g/L)：磷酸钾缓冲液(pH 6.0) 0.100 mol/L，无氨基酵母氮源YNB 13.400，甲醇0.1%-2.0% (体积分数)，组氨酸0.004。
MMF (buffered minimal methanol formic acid)培养基(g/L)：无氨基酵母氮源YNB 13.40，甲醇0.6%，甲酸1.84。
1.2 基因的合成以及质粒的构建
外源甲醇脱氢酶基因经密码子优化后，均由江苏赛索飞生物科技有限公司合成。根据来源，分别命名为MDH2和MDH3 [甲醇芽孢杆菌(Bacillus methanolicus)[12-15]]、MDHLXT [解木糖赖氨酸芽孢杆菌(Lysinibacillus xylanilyticus)[16]]、MDHN1T [杀虫贪铜菌(Cupriavidus necator)[17-20]]以及MDHBS [嗜热嗜脂肪地芽孢杆菌(Geobacillus stearothermophilus)[21-23]]。
使用PCR扩增MDH基因片段以及GS115基因组Ⅱ 7同源臂、Ⅳ 9同源臂、P AOX 启动子、AOXtt终止子片段，以原始质粒为模板得到线性质粒骨架片段，再利用Gibson组装法连接质粒骨架和片段。
1.3 酵母工程菌株的构建
首先利用CRISPR-Cas9基因敲入/敲除系统敲除酵母中原有的aox1，获得1Δ-GS115。在此基础上再将aox2敲除，得到2Δ-GS115底盘细胞。
使用相应引物(表3)和质粒DNA模板进行PCR扩增，得到相应质粒上带有同源臂的MDH表达盒、ACT1表达盒片段，利用实验室现有的CRISPR-Cas9基因敲入/敲除系统敲入底盘菌株中。对于无需ACT1的2种MDH (MDHLXT、MDHN1T)，直接插入至中性位点Ⅱ 7；剩下3种MDH表达盒插入至Ⅳ 9位点，同时将ACT1表达盒插入菌株Ⅱ 7位点[24]。
质粒DNA模板：统一采用质粒DNA小提试剂盒(南京诺唯赞生物科技股份有限公司)进行提取，提取方法见试剂盒说明书。
PCR反应体系(50 μL)：PrimeSTAR® Max DNA Polymerase (TaKaRa公司) 25 µL，质粒DNA模板1 µL，上、下游引物(10 µmol/L)各2.5 µL，ddH2O 19 µL。PCR反应条件：98 ℃预变性3 min；98 ℃变性30 s，58 ℃退火30 s，72 ℃延伸2 min，共30个循环；72 ℃终延伸3 min。
过氧化物酶体区室化：将过氧化物酶体信号肽PTS1添加至MDH的C端。对于需要ACT1激活的MDH，在对MDH定位的同时也使用PTS1对ACT1进行定位。
1.4 菌株生长曲线的测定
先将菌株接种至5 mL YPD或MDG液体培养基中，培养1-2 d，4 ℃、5 000 r/min离心5 min收集菌体，用5 mL无菌水吹吸洗涤2遍。接种至BMMH培养基或MMF培养基，初始OD600为0.2、0.4或0.5，进行发酵培养。每隔1 d或2 d进行取样，使用酶标仪对菌株的OD600值进行测定，每个实验生物学重复3次。
1.5 菌株甲醇利用率和还原力的测定
使用高效液相色谱检测甲醇含量。对收集的菌液在4 ℃、12 000 r/min离心5 min，吸取上清液，用0.22 μm滤膜过滤处理。HPLC中色谱柱规格：Aminex HPX-87H色谱柱(9 µm，1 300 mm×7.8 mm，Bio-Rad公司)，流动相：5 mmol/L H2SO4，流速设置为0.6 mL/min，柱温设置为50 ℃，运行22 min。配制不同浓度梯度的甲醇溶液，根据峰面积与浓度的对应关系绘制标准曲线。
采用高通量破碎仪对细胞进行破碎，再使用NAD+/NADH检测试剂盒(WST-8法) (上海碧云天生物技术股份有限公司)对菌株NAD总量和NADH/NAD+进行测定，每个实验进行3次平行重复，采用t检验进行差异显著性分析。
2 结果与分析
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2.1 构建缺陷型酵母菌株并筛选最优MDH
毕赤酵母中存在AOX系统，该系统能将甲醇转化为甲醛，AOX系统包括AOX1和AOX2两种酶。研究表明，在同一菌株中不同来源的MDH表达效果并不相同[10]。为筛选出效果最佳的MDH，本研究采用CRSPR-Cas9技术敲除毕赤酵母中的aox1和aox2，构建双缺陷型底盘细胞2Δ-GS115，并将不同MDH分别转入2Δ-GS115，以便更好地观察MDH的作用。同时本研究构建了AOX1单缺陷型菌株，用于对比。
首先测定了甲醇浓度为1.2%和1.6%时的生长曲线。结果显示，只有2Δ-MDHN1T菌株的生长显著高于其余菌株，在1.2%甲醇培养基中，其OD600值最高达到1.3，而对照菌株的OD600值最高仅为0.6 (图1A)，MDHN1T显现出的优势最强，能够部分回补AOX缺失导致的生长缺陷。
缺陷型菌株在1.2%和1.6%甲醇浓度下的生物量显著低于GS115野生型菌株，这证实了AOX是毕赤酵母主要的甲醇氧化酶。通过与野生型、AOX1单缺陷和双缺陷型菌株对比发现，MDH能在毕赤酵母中催化甲醇脱氢氧化，但其活性弱于AOX (图1)。
如图1所示，2Δ-MDHN1T菌株在甲醇浓度为1.2%时的生长曲线普遍高于1.6%，推测可能存在一个最适甲醇浓度，能使2Δ-MDHN1T菌株的生长达到最优。
2.2 培养重组菌株的最适甲醇浓度
鉴于MDHN1T菌株在较低甲醇浓度下生长更优，进一步对甲醇诱导浓度进行了优化。选取0.3%、0.4%、0.5%、0.6%、0.7%这5个浓度进行梯度测定，最终确定了0.6%为最佳甲醇浓度。在0.6%甲醇浓度下，菌株OD600值最高达到2.3 (图2D)。
2.3 最适甲醇浓度下重组菌株的生长曲线和甲醇利用率
在优化的0.6%甲醇浓度下，评估了各重组菌株的生长及底物消耗情况。除2Δ-MDHN1T菌株外，其余菌株均无法有效利用甲醇生长。
第8天时，2Δ-MDHN1T菌株呈现最高的OD600值，其数值为2.5 (图3A)。与对照2Δ-GS115菌株相比，2Δ-MDHN1T菌株的甲醇消耗速率约为4.09 mg/d (图3B)，显著高于仅靠扩散挥发或微量代谢消耗的对照组。该结果进一步确立了MDHN1T作为毕赤酵母甲醇代谢重构的首选MDH。
2.4 重构MUTP偶联rGlyP促进甲醇和CO2 协同利用
四氢叶酸连接酶(formate-THF ligase, Mis1-1)具有3种功能，可催化甲酸与四氢叶酸(THF)结合生成甲酰-四氢叶酸(10-formyl THF)，还具有亚甲基四氢叶酸环化水解酶活性和亚甲基四氢叶酸脱氢酶活性。A05中过表达Mis1-1增强了rGlyP中的四氢叶酸再生系统的运行，导致细胞中还原力的不足。毕赤酵母A05中的醇氧化酶AOX系统发挥作用时产物之一为H2O2，H2O2的氧化同样需要还原力。同时，甲醇氧化为甲醛，甲醛是一种细胞毒性物质，对细胞有毒害作用。
为解决rGlyP面临的还原力瓶颈及甲醛毒性问题，将筛选出的最佳MDH即MDHN1T引入Li等[8]构建的毕赤酵母A05中。采用区室化策略，将MDHN1T定位于过氧化物酶体，从而重构甲醇利用途径，获得重组菌株A05-MDHN1T(p)。代谢设计原理如图4所示，MDHN1T在过氧化物酶体内将甲醇氧化为甲醛，同时将NAD+转化为NADH。这一设计具有双重优势：(1) 空间隔离，将有毒中间体甲醛限制在过氧化物酶体内，减轻对胞质的毒害；(2) 能量偶联，产生的NADH可直接供给rGlyP中甲酸还原反应及甘氨酸裂解系统(GCS)循环，并通过异化途径增强CO2固定效率。
使用MMF培养基对A05-MDHN1T(p)进行发酵，测定菌株的生长曲线。结果显示，经过10 d培养，A05菌株的OD600值达到了2.5，A05-MDHN1T(p)菌株的OD600值达到了5.5，提升了约1.2倍(图5A)。对于甲醇的消耗情况，A05菌株和A05-MDHN1T(p)菌株的甲醇利用率如图5B所示，引入MDHN1T后甲醇消耗速率明显加快，A05菌株的甲醇消耗速率为17.88 mg/d，而A05-MDHN1T(p)菌株的甲醇消耗速率为28.98 mg/d，是A05菌株的1.62倍。
同时对发酵至第6天的A05菌株和A05-MDHN1T(p)菌株进行收集，并测定其NAD总量和NADH/NAD+。2种菌株的NADtotal分别为1.64 μmol/L和2.21 μmol/L，A05-MDHN1T(p)菌株的NAD总含量是出发菌株的1.3倍(图5C)。对于NADH/NAD+比值，A05-MDHN1T(p)菌株(2.08)是A05菌株(1.75)的1.2倍(图5D)。这说明MDH能够显著改善胞内还原力供给。
上述数据表明，通过过氧化物酶体区室化表达MDHN1T，成功重构了甲醇利用途径，显著改善了胞内还原力供给，偶联rGlyP，大幅提升了毕赤酵母对甲醇和CO2的协同利用效率。
3 讨论与结论
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一碳化合物的综合利用是发酵工程领域的重要研究方向。本研究将具有高催化效率的甲醇脱氢酶(MDH)与区室化代谢工程策略相结合，成功强化了毕赤酵母菌株A05的甲醇代谢能力。改造后的A05菌株NAD总量提升了30% (图5C)，NADH/NAD+提升了20% (图5D)，OD600值提升了120% (图5A)，甲醇利用率提升了62% (图5B)，重组菌株繁殖速率加快、发酵效率提升，这标志着在开发能够高效共利用甲醇与CO2的毕赤酵母细胞工厂方面取得了关键进展，增强了后续重组菌株作为底盘细胞利用甲醇和CO2生产单细胞蛋白或高附加值化合物的优势和潜能。
本研究通过区室化策略对甲醇利用途径进行空间重构，能够有效解耦甲醇氧化的能量生成过程与有毒中间体甲醛的代谢过程，从而系统性提升细胞性能[12]。具体而言，将外源优化的MDH酶与内源途径进行区室化整合，解决了2个主要问题：甲醇氧化的能量需求与甲醛同化的还原力需求之间的冲突，以及甲醛毒性对细胞活力的抑制[25]。
MDH的区室化设计可能优化了胞内的氧化还原平衡。在天然甲醇代谢中，甲醛既作为同化前体，又是异化途径底物，碳流分配存在固有损耗[25-26]。同时，区室化可能将甲醛限制在特定细胞器(过氧化物酶体)内，减少了其扩散至胞质造成的氧化应激和酶抑制。当胞内NADH水平提高后，依赖还原力的CO2固定途径(重构的rGlyP)得以更有效地运行，进而提升生物量。然而，胞内甲醛含量的提升可能为异化途径提供更多底物，实现了甲醇衍生还原力与CO2固定过程的耦合。同时，毕赤酵母本身存在的甲醛同化途径可能也因更多的甲醇利用而得到加强，重组工程菌株生物量得到进一步提升。
细胞生长(OD600)与甲醇利用率的大幅提升也是上述代谢再平衡的直接体现。减少的碳损失和毒性压力为细胞积累了更多可用于生长的前体和能量。这解释了本工程菌株相较于仅优化单一途径的菌株，在整体碳转化效率上表现出协同增强效应。
此外，表达水平的差异也是导致不同外源MDH表现差异的关键因素之一。本研究采取了一些措施尽量减少表达水平带来的差异：不同来源的MDH在相同的表达载体基础上构建，采用相同的重组菌株构建方法、相同的诱导型启动子、相同的诱导表达条件，且对MDH基因进行密码子优化以适应毕赤酵母偏好。在此基础上重组菌株表现出的显著差异主要由MDH的活性引起。对于筛选出的较优突变体，未来可以通过蛋白质工程改造、分子进化等技术，获得在毕赤酵母中更稳定、更易折叠的MDH突变体，以及共表达分子伴侣帮助蛋白正确折叠等措施使MDH在毕赤酵母中得到更好的应用。
Wang等[27]在Butyribacterium methylotrophicum中重构rGlyP，成功协同利用甲醇和CO2，本研究在真核宿主毕赤酵母中取得了类似的协同效应，但途径不同。这证明了能量/还原力供给强化是实现C1化合物共利用的一个普适性关键。与在Saccharomyces cerevisiae中通过区室化构建一碳营养型微生物的开创性工作[12]相比，本研究直接在天然甲基营养菌毕赤酵母中进行优化，起点更高，获得的性能提升幅度也更为显著。最近的一项前沿工作通过在毕赤酵母中组装由甲醇/甲酸氧化模块和还原甘氨酸途径(rGlyP)组成的人工MFORG途径，成功实现了甲醇与CO2的共固定[28]，进一步证明利用毕赤酵母固定CO2的可行性。本研究在结合rGlyP的基础上对毕赤酵母固有甲醇利用途径进行改造和区室化，使其更适应于rGlyP。
基于本研究成果，后续研究可尝试进行生物反应器水平的扩大培养。还原力与生长的提升是CO2同化增强的间接证据，后续可以精确量化重组工程菌株对CO2的利用。未来研究可以集中于利用此底盘生产特定高附加值产物，验证其工业应用潜力；进一步通过系统生物学方法(如转录组分析等)解析改造后全局代谢网络的动态变化，为理性设计提供更深入的见解。
基金
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  • 国家自然科学基金(32370054)
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2026年第66卷第5期
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doi: 10.13343/j.cnki.wsxb.20250922
  • 接收时间：2025-12-11
  • 首发时间：2026-05-09
  • 出版时间：2026-05-04
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  • 收稿日期：2025-12-11
  • 录用日期：2026-01-15
基金
The National Natural Science Foundation of China(32370054)
国家自然科学基金(32370054)
作者信息
    1.江南大学 生物工程学院，工业生物技术教育部重点实验室，江苏 无锡
    2.江南大学，粮食发酵与食品生物制造国家工程研究中心，江苏 无锡
    3.江南大学，江苏省生物活性制品加工工程技术研究中心，江苏 无锡
    4.郑州工程技术学院，河南 郑州
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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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