Multi-copy integration is a core strategy for redirecting metabolic flux toward target compounds. However, its application has been hampered by the absence of methods for systematically identifying native multi-copy genomic loci. To overcome this, we developed a computational procedure for genome-wide identification of such loci. Theoretically, this method is potentially applicable to any genome-sequenced species as it only requires the genomic assembly of the target species as input. Applying the procedure to Kluyveromyces marxianus, we identified four groups of loci (KmCS1-4). Combining these loci-KmCS1-4 and the traditional 26S rDNA-with 14 markers with graded selection strengths, we established a versatile multi-copy integration toolkit comprising 70 plasmids. Each plasmid exhibits a unique integration pattern, collectively forming an integration profile. This profile serves as a manual, enabling users to select appropriate tools tailored to the expression requirements of rate-limiting enzymes in their pathways. Applying representative plasmids exhibiting low-, medium-, and high-copy integration patterns to lycopene biosynthesis modules resulted in lycopene titers of 3.5, 6.8 and 40.5 mg/L, corresponding to 2, 6 and 9 genomic copies, respectively, demonstrating a positive correlation between lycopene titers, genomic copy numbers and integration patterns, which highlights the versatility of the toolkit and its supporting manual. Our study not only provides a broadly applicable methodology for genome-wide identification of multi-copy loci, but also an efficient integration platform for K. marxianus.
CYP11A1-mediated catalysis is the rate-limiting step in the de novo biosynthesis of progesterone in yeast, and its relatively low catalytic efficiency largely results from insufficient electron transfer efficiency. In this study, we addressed this bottleneck by systematically reconstructing the electron transfer system (ETS) of CYP11A1. We found that, beyond its native Class I-type ETS (Adx/AdR), CYP11A1 can also function with diverse Class II-type ETSs (CPRs) across species. Screening identified the combination of CYP11A1 from Sus scrofa (SsCYP11A1) and CPR from Absidia orchidis (AoCPR) as the most effective pair. Furthermore, fusion of the two proteins via a flexible GGGS linker significantly enhanced catalytic efficiency. Subsequently, single-site mutational scanning of SsCYP11A1 using the protein language model ESM-2 identified the F75S variant, which increased progesterone titer by 2.43-fold compared to the wild type. Further analysis integrating residue flexibility prediction and site-directed mutagenesis revealed that the F75S mutation improves overall catalytic efficiency by strengthening interfacial interactions between SsCYP11A1 and AoCPR and stabilizing a catalytically favorable conformation. In addition, deletion of the vacuolar iron transporter CCC1 increased the availability of [2Fe-2S] clusters in the yeast cytosol, thereby enhancing Adx functionality and enabling the coordinated operation of Class I-type and Class II-type ETSs. Ultimately, in a 5 L bioreactor fermentation system, the engineered yeast chassis achieved a progesterone titer of 1594.15 ± 71.36 mg/L, representing the highest reported level of de novo progesterone production in yeast to date.
L-tryptophan is a high-value aromatic amino acid widely used in the food, feed, and pharmaceutical industries. However, large-scale microbial production is constrained by insufficient precursor supply and limited strain tolerance to high product concentrations. In this study, modular metabolic engineering was first employed to enhance the availability of key precursors, including shikimate, serine, and glutamine, yielding strain TRPJ-13 with a 34.6% increase in L-tryptophan titer. To enhance strain tolerance, an indigo-based high-throughput reporter system was constructed and coupled with genome-scale overexpression library screening, leading to the identification of soxS as a tolerance-conferring target. Mechanistic analysis demonstrated that soxS upregulated lpxC to enhance lipopolysaccharide biosynthesis, thereby reinforcing membrane integrity and improving L-tryptophan tolerance. Combinatorial engineering of soxS and lpxC generated strain TRPJ-23, which increased L-tryptophan tolerance by 74.8% and L-tryptophan titer by 10.3%. Furthermore, YicL was identified as a novel transmembrane protein involved in L-tryptophan transport that effectively promoted L-tryptophan efflux, further increasing the titer by 9.0%. After fermentation optimization, strain TRPJ-28 produced 74.3 g/L L-tryptophan in a 5-L bioreactor, with a yield of 0.26 g/g and a productivity of 1.24 g/L/h. In a 1000-L pilot-scale bioreactor, TRPJ-28 reached a titer, yield, and productivity of 70.4 g/L, 0.25 g/g, and 1.17 g/L/h, respectively. This study provides new engineering insights for developing industrially promising L-tryptophan-producing strains.
Cell death during late-stage culture remains a major limitation in mammalian manufacturing processes for antibody therapeutics, constraining yield and process robustness. Here, we identified the predominant apoptotic signalling axis associated with culture decline in Chinese hamster ovary (CHO) cells and engineering against it. Recombinant CHO lines were engineered to overexpress BCL-2 (intrinsic pathway and benchmark control), CFLAR (death receptor pathway regulator), or TPT1 (a multifunctional stress-response protein). Apoptosis profiling across fed-batch cultures indicated that viability loss is predominantly associated with intrinsic pathway activation, characterised by increased cleavage of caspase-9, caspase-7 and caspase-3, with minimal activation of caspase-8. In batch and fed-batch studies, CFLAR and TPT1 improved late-stage viability and extended culture lifespan relative to the control, with TPT1 providing the most consistent benefit and outperforming BCL-2 in overall process performance. Under apoptosis challenges, including chemical induction, pro-apoptotic BAK overexpression and caspase 3 activation, TPT1-expressing cells maintained higher viability, decreased apoptosis and attenuated caspase 3 activation. Finally, TPT1 overexpression was transferred to industrially relevant CHO DG44 production platforms expressing monoclonal and bispecific antibodies and improved culture longevity and titres in both formats, without altering cell-specific productivity and N-glycan profile. TPT1 also presented similar behaviour to a BAK/BAX double knockout on viability and exceeded it on growth and titre, and combining the two gave no further gain in unfed batch culture. TPT1 overexpression therefore offers a single-cassette route to longer culture and high volumetric output inCHO-based bioprocesses.
Lignin has potential as a sustainable feedstock to replace fossil fuels in chemical manufacturing. The coupling of chemical fractionation and biocatalysis has emerged as a promising technology to realize this potential. In this process, chemocatalytic fractionation of biomass or lignin yields heterogeneous mixtures of lignin-derived aromatic compounds (LDACs), which are subsequently funneled to target chemicals by microbial cell factories. The recent expansion of genetic toolkits for non-model bacteria offers burgeoning possibilities for engineering bespoke biocatalysts using natural LDAC degraders such as Rhodococcus aromaticivorans RHA1. Herein, we describe the development of an RHA1 biocatalyst to convert a softwood kraft lignin stream containing vanillin, vanillate and acetovanillone to muconic acid by leveraging Serine integrase-Assisted Genome Engineering (SAGE). We increased vanillin metabolism by co-expressing ligV, encoding a vanillin dehydrogenase, and RHA1's endogenous vanACB, encoding a vanillate O-demethylase, partially overcoming the vanillate bottleneck observed when expressing ligV alone. To funnel aromatics to muconic acid, we tested two aromatic acid decarboxylases, finding that AroY with EcdBD efficiently decarboxylated protocatechuate to catechol. We then integrated the Hpe pathway of Rhodococcus rhodochrous GD02 to enable acetovanillone conversion. Finally, deletion of catB enabled muconic acid accumulation. Our biocatalyst, strain RHAAL14, transformed the LDACs derived from the oxidation of softwood kraft lignin to muconic acid with a 97% molar yield and a titer of 1.4 g/L. The iterative, integrated metabolic engineering strategies described in this work advance the development of rhodococcal strains for microbial cell factories.
Methylotrophic yeasts are outstanding platforms for the production of recombinant proteins through precision fermentation. Growing demand for protein-based food ingredients requires economically viable secretion efficiency and space-time yields, as well as correct post-translational modifications to ensure proper food functionality. Here, we review recent advances and future potential in engineering methylotrophic yeasts for food protein production, with particular focus on Komagataella phaffii. We first summarise the production of three categories of protein food ingredients: recombinant dairy proteins, proteins for plant-based and cultivated meat applications, and sweet-tasting proteins. Addressing the dual goals of improving both yield and quality, we then review synthetic biology and metabolic engineering strategies in methylotrophic yeasts, with emphasis on genome engineering and gene expression tools, protein secretory pathway engineering, and glycoengineering. Further advances in synthetic biology, integrated with industrial process development, will be critical to unlocking future precision fermentation systems based on methylotrophic yeasts.
Polyketides are a diverse class of natural products with broad pharmacological activities and substantial industrial relevance; however, their scalable biosynthesis is often constrained by inefficient heterologous expression platforms. In this study, a systematic evaluation of multiple microbial hosts identified Aspergillus niger HL-1 as an efficient fungal chassis for polyketide production. Subcellular compartmentalization analysis revealed that cytosolic biosynthesis significantly outperformed peroxisomal localization, leading to a 5.88-fold increase in triacetic acid lactone (TAL) production. To further enhance production, intracellular acetyl-CoA and malonyl-CoA supplies were engineered and integrated with compartmentalized metabolic rewiring to redirect carbon flux toward polyketide biosynthesis. In addition, increasing the copy number of polyketide synthase genes further improved titers. As a result, monacolin J and TAL reached 1.68 g/L and 7.50 g/L in shake-flask cultures, respectively, and were further elevated to 2.61 g/L and 25.08 g/L in a 1.5-L bioreactor. Finally, a versatile type III polyketide synthase (PKS) expression platform was established, enabling the functional expression of ten heterologous PKSs. By further disrupting the endogenous katA gene to decelerate p-coumaric acid consumption, the competitive catabolic pathway was successfully blocked, yielding a naringenin shake-flask titer of 384.91 mg/L from p-coumaric acid. This work establishes a robust and scalable fungal platform for the efficient production of both complex polyketides and flavonoids, providing a promising strategy for biotechnological applications.
Plant cell culture is a growing platform for sustainable biomanufacturing of plant-derived compounds, including the chemotherapeutic paclitaxel, which is produced industrially using Taxus chinensis plant cell culture. While strategies for improving yields of natural products in plant cell culture through elicitation of secondary metabolism have been widely studied, genetic approaches for manipulating metabolism have remained underexplored. Here, we constitutively overexpress four different genes in the paclitaxel biosynthetic pathway in T. chinensis plant cell culture and elucidate effects on pathway regulation and taxane biosynthesis. Overexpression of each of these four genes - taxadiene synthase (TASY), 10-deacetylbaccatin III-10-O-transferase (DBAT), baccatin-aminophenylpropanoyl-13-O-transferase (BAPT), and 3'-N-debenzoyltaxol N-benzoyltransferase (DBTNBT) - resulted in a 2-8-fold increase in paclitaxel accumulation, with overexpression of DBAT resulting in the largest increase. We also identified DBAT as a key rate-controlling step in biosynthesis of not only paclitaxel, but other taxane-derived compounds, as its overexpression resulted in significantly increased accumulation of taxane impurities. Expression profiling of all four transgenic cell lines revealed concerted activation of taxane metabolism, with upregulation of nearly all pathway genes and transcription factors. DBAT was the notable exception to this concerted activation and was only upregulated in the cell line specifically targeting that gene, indicating that DBAT is likely regulated differently and much more tightly than all other genes in the pathway. While this work specifically developed tools for engineering Taxus plant cell cultures, more broadly, these results illustrate the importance of genetic engineering of plant cell systems to achieve optimum yields. Through the application of modern synthetic biology and metabolic engineering tools to plant cell culture, we can enable rapid cell line optimization for production of specialized metabolites.
Against the backdrop of green biomanufacturing, engineering methanol-utilizing Komagataella phaffii (K. phaffii) represents an effective strategy to expand the one carbon (C1) product profile and speed up the industrialization of C1-based bioeconomy. To address the technical challenges of low efficiency and cumbersome experimental procedures for multiplex gene editing and precise large-fragment integration during the reconstruction of complex metabolic pathways in K. phaffii, this study established a CRISPR toolkit — Efficient Multi-Gene Editing System 3.0 (EMGES 3.0) — which enabled one-step large-fragment integration coupled with multiplex gene knockout. EMGES 3.0 was constructed through the synergistic optimization of a repair-engineered chassis and an episomal CRISPR vector. For chassis engineering, five DNA repair modules: Δlig4 (DNA Ligase IV, non-homologous end joining end ligation), ppMRE11(The endogenous MRE11 gene from Pichia pastoris) overexpression (The Meiotic Recombination 11, DNA double-strand break end resection), Δrad9 (Radiation-Sensitive 9, DNA damage checkpoint regulation), Δmph1 (Mutator Phenotype Helicase 1, improvement of homologous recombinant strand extension), and PapRecT-PaSSB co-expression (stabilization of recombination intermediates) were integrated to generate the highly recombinogenic strain Y09. For vector engineering, cenARS was replaced by panARS and the endogenous promoter PGAP was employed to drive the double hammerhead ribozyme-single guide RNA-hepatitis delta virus ribozyme (double HH-sgRNA-HDV: dHgH)-mediated sgRNA expression, yielding the optimized vector Nov_pGAP_panARS_pLAT1_Cas9. These two features on K. phaffii together enhanced the EMGES 3.0 to a higher standard of transformation rate and editing efficiency. According to our results, EMGES 3.0 achieved dual-functional gene knockout efficiencies between 76.6% and 100%. For insertion of medium-long fragments (>4.5 kb), the efficiency achieved 93.3%. In addition, the one-step integration of ultra-long fragments (>16 kb) achieved 14.8%, which was reported for the first time. Furthermore, the efficiency of simultaneous long-fragment integration at three neutral loci reached 38.4% (>15 kb). We applied the system for one-step production of free fatty acids (FFAs, yield: 5.82 ∼ 7.30 mg/L/OD600) and resveratrol (yield: 1.14 ∼ 1.28 mg/L) using methanol as the sole carbon source. EMGES 3.0 provides a robust technical foundation for complex compounds biosynthesis and high-yield industrial strains, while also advancing K. phaffii as an industrial synthetic biology chassis for efficient C1 utilization.
Candida yeasts represent a versatile yet underexploited platform for industrial biotechnology. These yeasts utilize a remarkably broad range of carbon sources, particularly for hydrophobic carbon sources, coupled with robust growth and diverse biosynthetic capacities, making them promising hosts for sustainable production of chemicals, fuels, and proteins. Despite these advantages, industrial deployment of Candida species has been hindered by concerns regarding opportunistic pathogenicity and the historical lack of efficient genetic manipulation tools, leading to a substantial gap between metabolic potential and practical utilization. Recent advances in functional genomics, genome editing, and systems metabolic engineering are rapidly overcoming these barriers, enabling more precise and efficient strain development. In this review, we systematically summarize recent progress in the metabolic engineering of Candida species as microbial cell factories, with particular emphasis on expanding genetic toolkits, utilizting renewable and non-conventional carbon sources, and biosynthesizing high-value compounds. In addition, we propose a biosafety-oriented classification framework to support their safe industrial deployment. Finally, we discuss current challenges and emerging opportunities, emphasizing that the synergy of synthetic biology and artificial intelligence-driven design holds the key to unlocking the biotechnological potential of Candida yeasts.
Branched-chain fatty acids (BCFAs), naturally synthesized by Gram-positive bacteria, are promising feedstocks for the production of advanced biofuels. However, efficient BCFA biosynthesis in Gram-negative bacteria such as Escherichia coli remains challenging because of insufficient supply of branched-chain acyl-CoA precursors, poor compatibility of the endogenous fatty acid synthesis pathway with branched-chain substrates, and limited cellular robustness toward non-native fatty acids. In this study, we first engineered an orthogonal isovaleryl-CoA biosynthetic pathway in E. coli and demonstrated its functionality in supporting BCFA production. Importantly, we identified a strong synergistic interaction between the isovaleryl-CoA pathway and the branched-chain α-keto acid dehydrogenase pathway, and thereby established a dual-route strategy for precursor supply. To further enhance BCFA production, we rewired central carbon metabolism by eliminating competing pathways and introducing a non-oxidative glycolysis pathway to increase acetyl-CoA availability while minimizing byproduct formation. We additionally optimized the fatty acid biosynthetic module through expression of a highly active 'TesA (R65C) variant and the transcriptional regulator FadR, and enhanced cellular robustness via introduction of N138H mutation into PcnB and overexpression of the stress resistance associated genes rfaY and yafL. The final engineered strain produced 2.96 g/L BCFAs (approximately 10-fold higher than the previously reported titers), representing 55% of total fatty acids, with a yield of 0.08 g/g glucose. Overall, this work established a dual-precursor supply strategy combined with systems metabolic engineering for BCFA production, providing a foundation for the development of sustainable bioprocesses for advanced branched-chain biofuels.
Tricyclic sesquiterpenes are bioactive natural products with broad applications in pharmaceuticals, fragrances, and sustainable aviation biofuels. However, the repertoire of characterized tricyclic sesquiterpene synthases (tSTSs) remains limited. To systematically expand this enzyme class, we developed a multi-step bioinformatics workflow to identify genes encoding for cyclic sesquiterpene synthase integrating BLAST-based homology filtering, conserved motif validation, AlphaFold2-based structural modeling, and molecular docking. This workflow progressively refined an initial set of 1063 tSTS candidates to six putative enzymes that satisfied a C1-C10 distance criterion. Heterologous expression of all six candidates in Corynebacterium glutamicum JP-2 overexpressing a methylerythritol 4-phosphate pathway gene module confirmed biosynthetic activity, yielding five known cyclic sesquiterpenes and tricyclic avermitilol produced by a tSTS (AtTPS) identified from Actinokineospora terrae by NMR spectroscopy and high-resolution electrospray ionization mass spectrometry. To improve industrial feasibility, the chemical-inducible promoter was replaced with constitutive leaderless synthetic σB promoters, and fusion of avermitilol synthase with farnesyl pyrophosphate synthase via a GGGGS linker enhanced the local concentration of intermediates between enzymes. In addition, a plasmid-free, antibiotic selection-free strain was constructed via the CRISPR-associated transposons, enabling dual chromosomal integrations of the avermitilol synthase expression cassette and achieving 81.52 mg/L avermitilol production. The chromosomally integrated strain maintained stable production over serial passages, whereas the plasmid-based strain exhibited greater than 90% loss of productivity. Fed-batch fermentation of AVM-int02cp in a 2-L bioreactor achieved a final avermitilol titer of 100.99 mg/L. This work demonstrates a sequence- and 3D structure-assisted gene discovery-to-production workflow for a tricyclic sesquiterpene through the identification of an uncharacterized synthase gene in a microbial host.
