In the context of global carbon emission reduction and carbon neutrality strategies, microbial carbon-fixing enzymes have emerged as a research frontier in biological carbon fixation technologies due to their unique advantages, including high efficiency, low energy consumption, and the ability to produce value-added chemicals. This review systematically summarizes the classification, structural characteristics, catalytic mechanisms, and regulatory networks of both natural and engineered carbon-fixing enzymes. It focuses on the roles and kinetic regulation of key enzymes such as ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), carbonic anhydrase (CA), formolase (FLS), and carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) in CO₂ fixation. From a structural biology perspective, the review discusses how static structures and dynamic conformational changes determine catalytic efficiency. Furthermore, this paper reviews the recent advances in protein engineering strategies including rational design, directed evolution, high-throughput screening, and artificial intelligence (AI)-assisted prediction for carbon-fixing enzymes, along with the enabling role of specialized databases such as Greenase in enzyme mining and pathway reconstruction. Representative case studies, including RuBisCO, CA, FLS, and multi-enzyme cascade systems, are adopted to illustrate engineering pathways from single-enzyme optimization to integrated systems. Finally, future directions are discussed, including dynamic catalytic mechanism simulation, in vivo fitness engineering, de novo enzyme design, and electro-/photo-enzyme coupling systems. This review provides a theoretical reference for the development of efficient, stable, and industrially adaptable novel biological carbon fixation technologies.

The efficient conversion of CO₂ into fuels and chemicals represents a key route toward sustainable biomanufacturing, and microbial cell factories offer significant potential for this purpose. Despite substantial advances in carbon fixation pathway design and carbon flux rewiring, the performance of microbial CO₂-fixing systems remains largely constrained by the efficiency of energy supply and utilization. At the core of this limitation is a systemic mismatch between energy metabolism and carbon fixation, involving discrepancies in energy carrier types [ATP, NAD(P)H, and low-potential electrons], supply ratios, and redox potential, which ultimately restrict carbon flux and energy utilization efficiency. From the perspective of energy-carbon flux coupling, this review proposes an energy conversion chain framework that conceptualizes carbon fixation as a cascade network comprising energy input, electron transfer, and carbon assimilation. On the basis of this framework, a quantitative evaluation system is established, encompassing energy input efficiency (EE), redox/electron utilization efficiency (RE), and energy-to-carbon conversion efficiency (ECE). By integrating experimental measurements with metabolic modeling, we systematically analyze the distribution of energy carriers and electron fluxes and identify key bottlenecks, including limitations in energy input flux, electron transfer losses, cofactor mismatches, and reaction network efficiency. At the engineering level, we further summarize strategies for improving energy-carbon compatibility, including chassis selection under energy constraints, construction of exogenous energy-supplying modules, and rewiring of endogenous energy networks. Building upon these advances, a multi-scale energy-carbon co-design framework is proposed to achieve precise matching between energy supply and carbon fixation demand. Overall, this review redefines energy metabolism as a central design dimension in carbon fixation engineering and provides both a theoretical foundation and engineering strategies for the development of efficient and robust CO₂ bioconversion systems.

Microbial carbon fixation represents a crucial negative emission technology for achieving the carbon peaking and carbon neutrality goals and serves as a primary source of raw materials for the next generation of biomanufacturing. However, naturally occurring carbon fixation pathways generally suffer from limitations such as slow carboxylation rates, harsh reaction conditions, and difficulties in heterologous reconstruction. In recent years, scientists have designed and constructed several synthetic carbon fixation pathways, establishing a foundation for developing efficient biological carbon fixation systems. This article systematically reviews the naturally discovered carbon fixation pathways and summarizes the key advances in the modification of natural pathways and the design of synthetic carbon fixation pathways empowered by synthetic biology. Furthermore, it summarizes recent progress in energy supply strategies for microbial carbon fixation. Finally, we discuss the bottlenecks and optimization strategies, providing scientific insights and perspectives for the development of efficient carbon fixation systems for practical applications.

The valorization of one-carbon (C1) gases such as CO, CO₂,_{and syngas (a mixture with H2) represents a promising route for responding to global climate change and establishing a sustainable circular economy. Acetogens, a group of obligate anaerobic microorganisms capable of efficiently assimilating C1 gases via the Wood-Ljungdahl pathway (WLP), offer a natural conversion route with high carbon atom economy for biological carbon fixation. This paper provides a systematic review of the latest advancements in C1 gases conversion by acetogens, spanning from fundamental research to industrial applications. We first elucidate the metabolic coupling between acetogens and C1 gases, delving into the biochemical basis for utilizing C1 sources. In terms of fundamental research, we highlight recent breakthroughs in energy conservation mechanisms and metabolic flux regulation. Then, we summarize how the development of advanced genetic manipulation systems, exemplified by CRISPR-Cas tools, has enhanced the titers of natural products such as acetate and ethanol and accelerated the transformation of acetogens into chassis cells for the production of high-value chemicals such as butanol and 3-hydroxybutyrate. Finally, this review analyzes the engineering challenges, including gas-liquid mass transfer limitations and energy supply bottlenecks, associated with the scale-up of gas fermentation while offering a perspective on the broad application prospects of acetogens in achieving carbon peaking and carbon neutrality goals and advancing next-generation green biomanufacturing.}

Methane, the second most abundant greenhouse gas after carbon dioxide, represents a critical target in climate change mitigation efforts. Owing to their unique ability to utilize methane, methanotrophs have received substantial attention in greenhouse gas mitigation and low-carbon biomanufacturing, making the understanding of their metabolic pathways and ecological functions a vibrant research area worldwide. In recent years, the rapid development of high-throughput omics technologies and the integration of multi-omics analysis have accelerated a paradigm shift in methanotroph research, enabling the deep evolution from the identification of single gene functions to the elucidation of systemic metabolic processes. This review systematically summarizes the progress in the application of genomics, transcriptomics, proteomics, and metabolomics in methanotroph research, highlighting the decisive roles of omics technologies in elucidating carbon assimilation flux distribution in aerobic methanotrophs, uncovering novel metabolic pathways of anaerobic methane oxidation, and deciphering cross-domain interactions and electron transfer mechanisms within microbial consortia. To address current bottlenecks in the industrial application of methanotrophs, including poorly understood environmental adaptation mechanisms, imbalanced metabolic flux in chassis cells, and limited gas-liquid mass transfer efficiency, this review proposes a shift from descriptive omics toward function-driven and precision-intervention research. By integrating single-cell multi-omics and artificial intelligence-enhanced modeling, future studies may construct efficient artificial methanotrophic cell factories and synthetic microbial communities, thereby providing innovative biological solutions for achieving the global carbon neutrality goal.

Against the backdrop of global climate change and energy security, developing efficient carbon dioxide utilization technologies has become a strategic core for achieving the “dual carbon” goals. Photosynthetic biomanufacturing, which uses photoautotrophic organisms as chassis to directly convert solar energy and carbon dioxide into biofuels and bio-based chemicals, enables simultaneous carbon fixation, emission reduction, and green synthesis. It stands as a pivotal technology for advancing carbon neutrality. Cyanobacteriota, with their efficient photosynthetic capacity, well-defined genetic background, and mature operational systems, have emerged as highly promising photosynthetic cell chassis. To advance their large-scale application, there is an urgent need to develop highly efficient cyanobacteria-driven photosynthetic carbon fixation cell factories. This review systematically summarizes recent research progress from two dimensions: enhancing photosynthetic carbon fixation efficiency and achieving precise carbon flux direction toward target products. In terms of photosynthetic carbon fixation, this review mainly focuses on strategies such as endogenous pathway optimization, external material-enabled enhancement, and chassis exploration and reshaping for constructing efficient and stable photosynthetic carbon fixation systems. Regarding carbon flux direction and product synthesis, this review analyzes the implementation pathways for precise carbon resource orientation and efficient synthesis of complex products, centered on single-strain metabolic engineering and multi-strain modular collaboration. Finally, this review outlines future development directions in this field. The review aims to provide theoretical references and technical pathways for the systematic construction of cyanobacteria-driven photosynthetic carbon fixation cell factories, thereby contributing to the achievement of carbon neutrality.

Against the backdrop of global climate change and resource scarcity, biological carbon fixation technologies driven by clean energy have emerged and attracted widespread attention. Hydrogen (H₂), as a renewable and clean energy carrier with abundant sources and easy production, can serve as both the energy and the electron donor for microbial carbon fixation. This review summarizes the metabolic foundations of hydrogen-driven carbon-fixing chassis cells represented by hydrogen-oxidizing bacteria (HOB), and highlights recent advances in several representative strains. Furthermore, engineering strategies for HOB are discussed, including the development of enabling technologies, the reconstruction and reinforcement of metabolic pathways, and microbe-material coupling approaches. Additionally, this paper analyzes the current challenges facing HOB and proposes the future perspectives. Despite existing technical bottlenecks, with the advancement of metabolic engineering and systems biology, HOB are expected to play an important role in carbon recycling and sustainable biomanufacturing.

One-carbon (C1) compounds, including CO₂, methanol, and formate, are regarded as promising feedstocks for sustainable biomanufacturing. Diverse natural carbon fixation pathways, such as the Calvin-Benson-Bassham (CBB) cycle, the reductive tricarboxylic acid (rTCA) cycle, and the Wood-Ljungdahl pathway (WLP), have been extensively investigated. However, their inherent limitations in energy efficiency and carbon fixation rate have constrained the development of efficient C1 biomanufacturing systems, thereby motivating the rapid development of artificial C1 assimilation pathways. In recent years, a variety of artificial C1 assimilation pathways have been designed for the utilization of CO₂, methanol, and formate, and their functions have been validated in both in vitro and in vivo systems, providing new approaches for the construction of efficient C1-based biomanufacturing platforms. This review systematically summarizes recent advances in artificial C1 assimilation pathways using CO₂, methanol, and formate as substrates, with a particular focus on pathway design, the engineering and optimization of key enzymatic components, and the construction and application of artificial pathways in microbial chassis. In addition, this paper discusses the future perspectives on artificial carbon fixation, providing guidance and insights for the development of efficient and sustainable C1 biomanufacturing systems.

With the proposal of carbon neutrality goals, microbial conversion of CO₂ into high-value chemicals, fuels, and biomaterials has emerged as an important strategy for clean energy and green manufacturing. Although single-strain carbon fixation systems have ideal controllability, they still face limitations, including heavy metabolic burden, limited carbon fixation flux, insufficient energy and reducing power supply, low yield, and challenges in industrial-scale application. In contrast, synthetic microbial consortia enable rational metabolic division of labor among functionally distinct microorganisms, thereby alleviating metabolic burden, enhancing carbon fixation efficiency, and expanding product diversity. These advantages further improve product value and promote the industrial application of CO₂ fixation. This review summarizes recent advances in the application of synthetic microbial consortia for CO₂ conversion, including photosynthesis-driven systems, hydrogen-driven systems, and potentially safer chemoautotrophic systems classified according to carbon fixation modules. In addition, this paper discusses the design principles for constructing carbon cycling systems based on synthetic microbial consortia, together with current challenges and future perspectives, providing theoretical and practical guidance for the development of efficient and stable artificial carbon cycling systems.

Under the strategic goal of carbon neutrality, enhancing the carbon atom utilization efficiency of microbial cell factories has emerged as a core scientific challenge and imperative technical demand for advanced green biomanufacturing. Conventional fermentation processes possess mature industrial applicability, yet they are constrained by inevitable carbon losses via oxidative decarboxylation in central carbon metabolism. Such inherent limitation hinders fundamental improvement in substrate carbon conversion efficiency and restricts the green and high-quality advancement of the biomanufacturing industry. Multi-carbon source co-fermentation enables the rational integration of carbohydrate substrates with C1 feedstocks (formic acid, methanol, and syngas) and C2 feedstocks (acetic acid), constructing a mixotrophic metabolic system featured with carbon skeleton complementation, energy supply synergy, and intracellular redox balance. This strategy offers an innovative technological paradigm to break the theoretical carbon yield bottleneck of conventional bioprocesses. This paper systematically reviews the advances in multi-carbon source co-fermentation driven by synthetic biology. We emphatically elaborate on metabolic pathway reconstruction and regulatory mechanisms of typical co-fermentation systems consisting of organic substrates, C1/C2 compounds, and industrial waste gas. The state-of-the-art applications in synthetic fuels, bio-based materials, and high-value natural product biosynthesis are summarized. Furthermore, this paper discusses the industrial potential of this technology in integrating carbon capture and utilization and high-value biomanufacturing, aiming to provide a theoretical basis and strategic references for the innovation of efficient biomanufacturing towards carbon neutrality.

Achieving carbon peaking and carbon neutrality is a major strategic priority for China. Third-generation biomanufacturing, which uses one-carbon (C1) compounds such as methanol, carbon dioxide, and formic acid as feedstocks for bioconversion, has attracted increasing research interest. Among these C1 feedstocks, methanol is considered a promising substrate for biomanufacturing because of its ease of storage and transportation, high degree of reduction, and potential for large-scale production via CO₂ hydrogenation. This review systematically compares natural and synthetic methanol assimilation pathways and elucidates their advantages. It further summarizes methanol metabolism in natural methylotrophic microorganisms and pathway design strategies for constructing synthetic methylotrophs, while also discussing the major challenges associated with methanol utilization. On this basis, this paper reviews recent progress in the methanol-based biosynthesis of various high-value chemicals and discusses the bottlenecks and corresponding engineering strategies for methanol bioconversion. This review provides a theoretical foundation for methanol-driven green biomanufacturing.

Methanol is regarded as an ideal non-food feedstock for biomanufacturing due to its abundant supply and low cost. Methylorubrum extorquens AM1 has become a prominent model chassis strain in this field, owing to its natural ability to utilize methanol. This review summarizes recent advances in methanol-based biomanufacturing viaM. extorquens AM1 cell factories. First, the methanol metabolic pathways and compatible synthetic biology tools available for this strain are discussed. Subsequently, we highlight recent achievements in the metabolic engineering of this strain for the production of recombinant proteins, biodegradable materials, organic acids, and terpenoids. Building on these findings, we outline the metabolic engineering strategies that have been employed to enhance the biosynthetic capacity of engineered strains. Finally, we discuss the current limitations in constructing and applying M. extorquens AM1 cell factories and propose future research directions. This review will serve as a valuable reference for the development of M. extorquens cell factories and for the industrial production of methanol-derived products.

As efficient and sustainable proteins, single-cell proteins (SCPs) demonstrate significant potential in alleviating the constraints of traditional agricultural resources and reducing the carbon footprint of food manufacturing. Low-carbon C1/C2 feedstocks, such as carbon dioxide, methanol, and acetate, offer distinct advantages, including broad availability, low costs, and minimal carbon footprints. However, the assimilation of these feedstocks and the accumulation of proteins are still limited by key bottlenecks such as poor host cell adaptability, imbalanced metabolic flux distribution, and inefficient energy supply. In recent years, remarkable progress has been achieved in the discovery, evaluation, and rational engineering of host strains for the efficient bioconversion of C1/C2 feedstocks. The application of synthetic biology, metabolic engineering, and laboratory adaptive evolution has enabled the continuous improvements of the strain tolerance to C1/C2 feedstocks, assimilation efficiency, and intracellular protein accumulation capacity. On the basis of these advancements, various SCP production processes utilizing C1/C2 feedstocks are gradually advancing toward pilot-scale and industrial applications. This review systematically summarizes the strategies for constructing chassis cells that efficiently utilize C1/C2 feedstocks, key metabolic engineering technologies driving efficient SCP production, and the current technological and industrial status of SCP production from different C1/C2 feedstocks. Furthermore, it discusses challenges related to energy efficiency, nutritional quality, safety, and downstream scale-up, aiming to provide a theoretical foundation and technical reference for the sustainable development and industrial application of SCPs produced from C1/C2 feedstocks.

Against the dual constraints of shrinking carbon budgets and geopolitical rivalry for resource supplies, traditional manufacturing is confronted with various bottlenecks and needs to transform into a sustainable development mode imperatively. Microbial gas fermentation adopts feedstocks including industrial off-gas, gasification syngas, and natural gas to produce diversified products such as green biofuels, fine chemicals, and microbial proteins. With the advantages of high carbon utilization efficiency, mild reaction conditions, and environmental friendliness, it plays a vital role in carbon resource recycling and emission reduction of greenhouse gases. This paper systematically reviews the research and industrial progress in microbial gas fermentation. First, the performance and limitations of three generations of biomass feedstocks are analyzed and compared, and the technical advantages of microbial gas fermentation are illustrated. Secondly, the core metabolic pathways, growth preferences, and product synthesis characteristics of different gas-fermenting microorganisms are summarized. For commercial application, this study comprehensively sorts out the technical characteristics and limiting factors of industrial gas fermentation processes including feed gas pretreatment, gas-liquid mass transfer, multistage continuous fermentation, and in-situ product recovery, as well as their impacts on the fermentation process. Meanwhile, this work reviews global and domestic industrial cases, and elaborates on the characteristics of core microbial strains, typical technical routes, and product structures of various enterprises. In addition, in view of the technical shortcomings and further development needs of industrial gas fermentation, feasible strategies are proposed, mainly including strain performance improvement, fermentation equipment upgrading, intelligent process control, and improvement of economic benefits. This review aims to provide pivotal insights into the scaled-up and industrial application of microbial gas fermentation technology.

As an important branch of carbon capture, utilization, and storage (CCUS) technology, microbial carbon capture and utilization technology has become a vital bridge linking carbon neutrality and the sustainable bioeconomy. This paper systematically reviews the research progress in four technical pathways for converting CO₂ into starch, protein, microalgae biomass, and ethanol, and conducts carbon footprint and techno-economic analysis with ethanol as a typical case. The results indicate that the carbon reduction potential of microbial carbon capture and utilization technology depends on the supply mode of energy and value positioning of products. The in-depth integration of synthetic biology and electrocatalysis is reshaping the economic boundaries of carbon utilization. This study can provide references for technical pathway selection, research and development investment decision-making, and policy formulation.

L-threitol is a significant intermediate in pharmaceutical synthesis. Previously, we developed a one-pot, two-step multi-enzyme cascade for synthesizing L-threitol from formaldehyde. In this pathway, benzoylformate decarboxylase (BFD) and fructose-6-phosphate aldolase (FSA) catalyze the conversion of formaldehyde into L-erythrulose. This is followed by the reduction of L-erythrulose to L-threitol, driven by L-threitol dehydrogenase (TDH) and a methanol dehydrogenase (MDH)/isopropanol cofactor regeneration system. While this route boasts high atom economy and minimal by-products, the compatibility and optimal concentrations of the enzymes required optimization. [Objective] To systematically optimize the dosages and compatibility of enzymes in the multi-enzyme cascade to enhance both the reaction rate and conversion efficiency. [Methods] The activities of four key enzymes—BFD, FSA, TDH, and MDH—were assessed. Subsequently, factors including enzyme dosage, the cofactor regeneration system, reaction duration, and temperature were optimized step-by-step to improve the system compatibility. [Results] The optimal reaction conditions were determined as follows: enzyme dosages of BFD, FSA, TDH, and MDH being 10, 1, 1, and 8 mg/mL, respectively. Notably, the dosages of FSA and TDH were reduced by 87% and 67%, respectively, compared with pre-optimization levels. Other optimal parameters included a NAD⁺ concentration of 2 mmol/L, a reaction temperature of 30 ℃, and reaction duration of 8 h (representing a 60% decrease from that of the original system). Under these conditions, the maximum L-threitol concentration reached 166.76 mmol/L, with a yield of 89%. In a scale-up experiment, the L-threitol yield remained at 80%, representing a 43% increase compared with the pre-optimization level. [Conclusion] By systematically optimizing enzyme compatibility and reaction conditions, this study significantly reduces the enzyme dosages and reaction duration for converting formaldehyde to L-threitol. Simultaneously, it substantially improves the production efficiency and yield, establishing a robust foundation for the enzymatic synthesis of L-threitol from formaldehyde.

[Objective] 1,3-propanediol (PDO) is an important chemical monomer. Its biosynthetic routes mainly rely on carbon sources such as glycerol or glucose, which suffer from low carbon efficiency or reliance on food-based resources. Methanol, as a non-food renewable carbon source, offers advantages of a high reduction degree and low costs. However, the existing bioconversion of methanol to PDO is limited by low methanol utilization efficiency and cytotoxicity. [Methods] In this study, a novel route combining enzymatic and whole-cell catalysis for the conversion of methanol to PDO was designed. First, an in vitro multi-enzyme cascade system was used to convert methanol to glycerol, comprising alcohol oxidase (AOX), catalase (CAT), formaldehyde lyase (FLS), glycerol dehydrogenase (GldA), and formate dehydrogenase (FDH). After 4 h of reaction, the glycerol concentration reached 63.3 mmol/L, with a carbon conversion efficiency of 95.0% from methanol to glycerol. Second, the glycerol transporter GlpF, glycerol dehydratase DhaB123 and its activator GdrAB, and the NADPH-dependent aldehyde reductase YqhD were introduced into Corynebacterium glutamicum to construct a recombinant strain, enabling the whole-cell conversion of glycerol to PDO. Under optimized conditions, the carbon conversion efficiency from glycerol to PDO reached 96.0%. [Results] To convert methanol to PDO, we coupled the two processes, which achieved a final PDO titer of 30.4 mmol/L and the overall carbon conversion efficiency of 90.2% from methanol to PDO. [Conclusion] This study achieves efficient conversion of methanol to PDO and provides a new strategy for the green biomanufacturing of methanol-based high-value chemicals.

[Objective] To address the bottlenecks of heavy reliance on fossil resources and severe environmental pollution associated with chemical synthesis, circumvent the “competing with humans for food” dilemma of conventional sugar-based biorefineries, and contribute to the achievement of carbon neutrality. This study developed a low-carbon engineering strain that can directly capture one-carbon (C1) gases and synthesize high-value-added products via metabolic engineering, thereby realizing the sustainable and efficient biosynthesis of ethyl acetate (EA). [Methods] With the autotrophic microorganism Clostridium ljungdahlii, which possesses a natural carbon-fixing ability, as the expression chassis, the lipase B from Candida antarctica (CALB) exhibiting robust esterification activity was heterologously expressed, and thus a metabolic pathway for converting intracellular short-chain precursors (acetate and ethanol) into EA was constructed. To overcome the challenges of low cell density and misallocated precursor pools inherent in autotrophic carbon fixation, we systematically parsed and reshaped different trophic regimes for fermentation. The performance of the recombinant strain was evaluated across heterotrophic (fructose), autotrophic (CO/CO₂), and mixotrophic (fructose+CO/CO₂) regimes in terms of biomass accumulation, metabolic flux distribution, and target product synthesis. [Results] The foreign lipase CALB was successfully and functionally expressed inside the acetogenic chassis, effectively driving the precursor flux toward target ester synthesis. The engineered strain produced 16.49 mg/L of EA under heterotrophic conditions and 9.18 mg/L of EA under purely autotrophic conditions. Crucially, the dual-substrate mixotrophic fermentation regime not only bypassed the carbon catabolite repression (CCR) effect but also synergistically enhanced both cell growth and precursor supply. This approach boosted the ultimate EA titer to 36.25 mg/L, demonstrating superior catalytic efficiency and targeted esterification performance compared with single trophic modes. [Conclusion] The C. ljungdahlii strain engineeredthrough systematic metabolic modifications and fermentation mode remodeling can successfully capture and convert greenhouse gases/industrial off-gases into high-value-added ester products. This gas-fermenting cell factory represents a promising chassis for the production of high-value-added derivatives in the future, expanding the technical frontiers for low-carbon industrial blueprints driven by synthetic biology.

[Objective] To address the low yield and conversion rate in the biosynthesis of glycolic acid, we developed a whole-cell catalytic system with ethylene glycol as the substrate to obtain a strain with a high yield of glycolic acid and an efficient biosynthesis process. [Methods] With ethylene glycol as the substrate, wild-type strains were screened for their glycolic acid synthetic performance. The catalytic conditions of the optimal strain were systematically optimized to enhance the whole-cell catalytic efficiency in the production of glycolic acid. Ultraviolet mutagenesis combined with a glycolic acid biosensor-based high-throughput screening was employed to isolate high-yield strains, which were further validated in a 5-L fermenter. [Results] Pichia kudriavzevii GX-01 was successfully screened out, which could grow on glucose and produce glycolic acid from ethylene glycol via whole-cell catalysis. The whole-cell catalytic conditions were optimized by single-factor experiments as a catalytic temperature of 30 ℃, supplementation with 0.50 g MgCO₃ to adjust the system pH after 6 h of reaction, a loading volume of 50 mL in a 250-mL flask, an initial glucose concentration of 25 g/L, and an initial ethylene glycol concentration of 20 g/L. Under these conditions, the glycolic acid titer in shake flasks reached 19.30 g/L after 96 h of catalysis, and the strain showed favorable tolerance to glycolic acid. A positive mutant strain P. kudriavzevii GSUV-7 was obtained through UV mutagenesis and high-throughput screening, whose glycolic acid productivity was 2.90-fold higher than that of the original strain. In a 5-L fermenter, P. kudriavzevii GSUV-7 produced 117.60 g/L of glycolic acid after 106 h of catalysis, with a 100% conversion rate. [Conclusion] This study successfully obtains a mutant strain P. kudriavzevii GSUV-7 with a high yield of glycolic acid, providing strain resources and new insights for the research on biosynthesis of glycolic acid.