Metabolic Engineering

Budding yeast Saccharomyces cerevisiae is a workhorse chassis for producing added food and agricultural compounds. However, building multi-enzymatic pathways for these chemicals often requires iterative genomic integration, underscoring the need for efficient, rapid genome-editing tools that can reliably target transcriptionally active chromosomal regions. In this study, to accelerate strain construction, we established a genome-editing toolkit to rapidly engineer eight loci, highly expressed hot-spots, but nonessential genomic sites suitable for stable pathway assembly. Our approach integrates three key design features: (i) selectable markers to enable rapid screening of edited cells, (ii) extended homology arms that leverage the yeast homology-directed repair machinery for robust genomic integration, and (iii) co-delivery of Cas9 and guide RNAs to promote efficient double-stranded DNA breaks at specific integration sites. The sequence independence of FASTOP relies on the release of integration cassettes from integrative vectors, mediated by restriction digestion at two flanking multiple-cutting sites in the integration module to minimize the risk of introducing sequence errors during PCR amplification of the integration cassettes. Following the introduction of a fluorescent reporter cassette, we observed high integration efficiencies across the target sites. We then integrated the biosynthetic pathway of plant-derived flavonoid naringenin into the hot-spots of the yeast genome using the FASTOP toolkit. Our results demonstrated that upon expressing the five essential genes in simple shake flask culture, naringenin production reached 505.7 mg/L, representing a significant (69-fold) increase over previously reported titers for comparable minimal heterologous pathways in S. cerevisiae. Together, the FATSOP toolkit provides a user-friendly platform for reliably modifying hot-spot loci to rapidly construct multi-enzymatic metabolic pathways in S. cerevisiae, while achieving high production levels for high-value food-relevant metabolites.

Malic acid is a C4 dicarboxylic acid traditionally produced from petroleum and widely used in the food industry. As a sustainable alternative, it can also be produced as a value-added platform chemical from biomass. Previously, the Escherichia coli strain XZ658 was engineered to produce L-malate via the carbon-fixation reductive branch of the TCA cycle. In this study, we further improved this system by relieving allosteric regulation of citrate synthase, addressing redox imbalance, and enhancing malate export. These modifications approximately doubled the L-malate titer in the final strain MO128 compared to XZ658 under simple batch fermentation conditions. The process achieved a high mass yield of 1.2 g malate g-{superscript 1} glucose, highlighting the carbon-fixation capacity of the reductive TCA pathway for fermentative malate production.

The fast-growing cyanobacterium Synechococcus sp. PCC 11901 is emerging as a promising chassis for photosynthetic biomanufacturing. Here we report recombinant protein production in PCC 11901 via signal peptide-mediated secretion, enabling direct recovery of target proteins from the culture medium without cell disruption. Seven signal peptides spanning both Sec and Tat pathways are screened using eYFP as a reporter, with secretion quantified daily over seven days by fluorescence measurements. FutA, belonging to the Tat pathway from Synechocystis sp. PCC 6803, achieves 92.2% extracellular export by day 7, substantially outperforming all Sec candidates, including the best Sec signal peptide thermitase from Cyanobacterium aponinum PCC 10605 (55.7%). Signal peptide-bearing strains exhibit growth reductions of up to 26% relative to the wild-type, with FutA most affected, indicating a general metabolic cost correlated with secretion efficiency. The best-performing signal peptides from both pathways, FutA and thermitase, are validated with secretion of lichenase. Notably, the rank order of signal peptide performance is reversed for lichenase: thermitase demonstrates 2.6-fold higher extracellular activity than FutA, indicating that optimal signal peptide selection is cargo-dependent. These results establish PCC 11901 as a secretion-competent chassis and provide a rational framework for matching signal peptide pathways to target protein properties.

Cyanobacterial natural products are a rich source of bioactive compounds, yet their heterologous production remains challenging. This study investigates the feasibility of expressing the lyngbyatoxin A (LTXA) biosynthetic gene cluster in a fungal host. The lyngbyatoxin biosynthetic genes (ltxA, ltxB, ltxC) were individually cloned and expressed in Aspergillus oryzae NSAR1 under the control of an inducible promoter. Metabolite production was assessed using LC- MS, and transcriptional analysis was performed by RT-PCR. Codon-optimized constructs and precursor feeding experiments were employed to evaluate pathway functionality. No production of LTXA or pathway intermediates was detected upon co-expression of ltxA-C despite confirmed transcription of ltxB and ltxC. RT-PCR analysis revealed truncation of the ltxA transcript, suggesting incompatibility with fungal transcriptional or splicing machinery. In contrast, expression of a codon-optimized ltxC enabled biotransformation of indolactam V to LTXA in A. oryzae, confirming functional expression of the prenyltransferase. These results highlight transcriptional limitations as a key barrier to heterologous expression of cyanobacterial NRPS pathways in fungal hosts, while demonstrating that downstream tailoring enzymes can remain functional. This work provides insights for future engineering of fungal platforms for cyanobacterial natural product biosynthesis.

Improving the reconstituted translation system is a key requirement for bottom-up synthetic biology. Here, we developed a two-step in vitro evolutionary method that can be used for improving translational proteins. In this method, two distinct conditions were sequentially applied while maintaining genotype-phenotype linkage in water-in-oil droplets. Using this method, we performed in vitro evolution of four translation factors, IleRS, PheRS, EF-G, and EF-Tu, and identified mutations that modestly enhanced translation activity in in vitro expression assays. One of the EF-G mutations (P610S) increased activity per protein approximately 2-fold for the recombinant protein purified from E. coli. This selection method is useful for improving translational proteins for bottom-up synthetic biology.

Human N-glycoproteins constitute a market worth hundreds of billions of dollars. However, their production in yeast is often limited by misfolding and subsequent degradation, largely due to differences in N-glycan-dependent protein quality control (QC) systems between humans and yeast. Notably, yeast lacks the UGGT-mediated reglucosylation-refolding cycle that rescues misfolded glycoproteins, and its degradation pathway involves fewer rate-limiting steps. To address this, we engineer the glycoprotein QC system in Kluyveromyces marxianus, a promising host for protein production, by introducing key human components and modifying native pathways. Expression of human UGGT1 or UGGT2 enhances the soluble and secretory production of glycoproteins in an activity-dependent manner. This effect is further improved by co-expression of the UGGT cochaperone SEP15 and by reducing native glucosidase II trimming activity. In addition, introduction of human EDEM2, a rate-limiting enzyme in glycoprotein degradation, delays ER-associated degradation and increases secretion. Integration of these engineering strategies substantially enhances the production of several high-value human-derived glycoprotein therapeutics, including etanercept, dulaglutide, and abatacept, with up to a [~]12-fold increase. These findings demonstrate that engineering a human-like glycoprotein QC network in yeast is an effective strategy to improve glycoprotein folding and secretion.

Hydrogenophilus thermoluteolus TH-1 is a thermophilic hydrogen-oxidizing bacterium capable of producing poly(3-hydroxybutyrate) (PHB) from CO2. To redirect carbon flux for producing other useful biomaterials, we disrupted the acetoacetyl-CoA reductase genes (phaB1 and phaB2), which are central to the primary PHB synthesis pathway. Unexpectedly, the resulting {Delta}phaB1B2 mutant still accumulated PHB under autotrophic conditions, reaching approximately 25-35 % of the wild-type level. Furthermore, PHB accumulation in the mutant was significantly restored when fatty acids (butyrate and oleate) were used as carbon sources, whereas acetate and malate resulted in reduced accumulation. These results suggest the existence of a PhaB-independent PHB synthesis pathway. We propose that intermediates from the {beta}-oxidation of fatty acids are converted to (R)-3-hydroxybutyryl-CoA, bypassing the disrupted PhaB enzymes. Additionally, the basal PHB production from non-fatty acid sources implies the involvement of a reverse {beta}-oxidation pathway. This study highlights the metabolic versatility of strain TH-1 for future metabolic engineering.

Human milk fat (HMF) contains triacylglycerol (TAG) as its primary component, providing over 50% of the calories for infant nutrition, along with structural and bioactive lipids that are important for immune and nervous system development. Palmitic acid, comprising 20-25% of the fatty acid complement of HMF, is predominantly esterified to the sn-2 position on the glycerol backbone. This regiospecific positioning facilitates absorption as 2-palmitoyl-monoacylglycerol after hydrolysis of the fatty acids at sn-1 and sn-2 by gut lipases. Other features of HMF include enrichment in structured medium- and long-chain triglycerides (MLCTs), and variation in the ratio of oleic acid to linoleic acid with maternal diet and geography. We have engineered Auxenochlorella, an oleaginous green alga, for biosynthesis of an MLCT- and sn-2 palmitate-enriched HMF substitute for infant formula, matching the regioisomeric composition and proportions of the most abundant fatty acids in HMF.

{beta}4-galactosylation is a critical quality attribute of therapeutic monoclonal antibodies (mAbs), enhancing complement-dependent cytotoxicity, antibody-dependent cytotoxicity, and antibody-dependent cellular phagocytosis. Despite its therapeutic importance, galactosylation remains the most variable glycosylation motif due to its sensitivity to cell culture conditions. Here, we describe a dual genetic engineering strategy applied to two mAb-producing CHO cell lines, DP12 and VRC01, to simultaneously overcome the cellular machinery and metabolic bottlenecks that limit {beta}4-galactosylation. The first engineering event knocks out COSMC, the chaperone required for core 1 {beta}-1,3-galactosyltransferase 1 activity, to redirect UDP-Gal consumption from O-linked {beta}3-galactosylation towards mAb Fc N-linked {beta}4-galactosylation. The second event overexpresses {beta}-1,4-galactosyltransferase 1 ({beta}4GalT1) to augment cellular galactosylation machinery. Each modification was characterised individually (COSMC- and GalT+) and in combination (C-/GT+) across both cell lines in batch and fed batch cultures. The combined C-/GT+ strategy consistently achieved greater than 90% mAb Fc {beta}4-galactosylation, irrespective of host cell line or culture mode. Metabolic characterisation confirmed that both engineering events alleviate their respective bottlenecks: COSMC knockout redirects UDP-Gal flux and {beta}4GalT1 overexpression increases N-galactosylation capacity. The C-/GT+ strategy also reduced production of Man5 glycans, which accelerate serum clearance and pose immunogenicity risks. Metabolic profiling suggests that the COSMC knockout attenuates UTP consumption and contributes to reduced Man5 production. C-/GT+ glycoengineering had no negative impact on mAb titre. Our results establish the C-/GT+ dual glycoengineering strategy as a robust approach for consistently achieving high mAb galactosylation across diverse cell culture conditions, with the additional benefit of reduced Man5 glycans. HighlightsO_LIDual COSMC KO and {beta}4GalT1 overexpression achieves >90% mAb Fc galactosylation. C_LIO_LICOSMC KO redirects UDP-Gal from O-glycans to mAb Fc without impacting cell growth. C_LIO_LIDual glycoengineering reduces production of undesired Man5 glycans. C_LI

Engineered microbial communities hold significant biotechnological potential because their collective metabolism can produce functions beyond those achievable by individual strains. However, multicellular synthetic gene circuits require orthogonal communication systems that enable precise, programmable signaling between cells. Quorum sensing (QS), where cells both produce and detect small diffusible signal molecules, offers a natural framework for such intercellular communication. However, the construction of complex multicellular circuits for applications such as biobased production is currently hampered by the limited number of orthogonal QS channels available in yeast. Here, we expand the QS toolkit in Saccharomyces cerevisiae by characterizing four LuxR-type biosensors based on EsaR, LasR, TraR and RpaR, alongside the previously established LuxR biosensor. We functionally expressed acyl-CoA-dependent HSL synthases in yeast, producing a diverse range of aliphatic and aromatic HSL signals. LuxR and RpaR, were compatible with in vivo ligand production and established as orthogonal QS signaling pair with synthases MesI and RpaI, respectively. Co-culture experiments demonstrated QS-dependent intercellular signaling, with 3.9-fold and 6.4-fold induction relative to monocultures. Together, these results establish a modular and extensible platform for orthogonal intercellular communication in yeast, enabling the construction of multicellular synthetic gene circuits.

Bacillus subtilis is an important chassis for biotechnology, but its use in multiplex genome engineering is limited by low natural transformation efficiency. Here, we compared inducible promoter systems for synthetic activation of the competence regulator ComK and evaluated their effects on the comG operon competence reporter and transformation efficiency. Xylose- and mannitol-inducible systems outperformed IPTG-based constructs and shifted 96-99% of cells into a reporter-positive competent state. However, reporter activation alone did not predict transformation potential. Optimization of culture density and induction timing increased transformant yield 45-fold relative to the initial protocol and 2800-fold relative to the conventional Spizizen method. Disruption of native competence regulatory genes did not improve performance and often reduced transformation output, highlighting the importance of endogenous regulatory circuitry. Using the optimized strain and protocol, we achieved co-transformation frequencies of 11-18% and constructed multiplex spore-display libraries containing fluorescent protein fusions integrated at multiple loci. Screening identified strong dual-display combinations and showed that cargo loading depends on anchor protein, integration locus, and genetic background. SscA fusions supported the highest display capacity and promoted synergistic co-display. Together, these results show improvements in natural transformation-based genome engineering in B. subtilis and provide insight into the construction of multifunctional engineered spores.

Terpenes constitute the largest and most structurally diverse class of plant secondary metabolites, with critical roles in plant-environment interactions and broad industrial applications. Although nuclear genome engineering of terpene pathways has been extensively explored, chloroplast genome engineering remains largely undeveloped, with all reported studies restricted to the model plant Nicotiana. Here we report successful chloroplast genome engineering for diterpene production in the crop plant potato (Solanum tuberosum). First we identified the trnT/trnL plastomic locus as optimal for minimizing integration-associated growth penalties. Insertion of a bifunctional diterpene synthase gene into this plastomic site yielded transplastomic plants with successful diterpene production, but with reduced growth. The co-expression of a geranylgeranyl diphosphate synthase gene to enhance precursor supply restored normal growth while elevating diterpene accumulation. Transplastomic plants were otherwise agronomically comparable to wild-type. This work expands chloroplast engineering as a viable strategy for terpene pathway engineering in crop improvement and high-value terpene production.

Wickerhamomyces ciferrii is a non-model diploid yeast that naturally produces tetraacetyl phytosphingosine (TAPS), a sphingoid base used in cosmetic and dermatological applications. However, its strong preference for non-homologous end joining (NHEJ) over homologous recombination (HR) limits conventional genome editing, while disruption of LIG4, a core NHEJ gene, compromises cellular fitness. Here, we repurposed native NHEJ activity to develop a homology-independent multicopy genome integration platform for W. ciferrii. The platform combines three optimized donor-design features: telomeric end-shielding with two tandem copies of an 11 bp repeat to improve linear donor persistence, a defective URA5 auxotrophic marker to enrich multicopy integrants, and 5'-phosphorylated donor termini to enhance transformant recovery and integration output. These features were consolidated into the platform vector pTdmVU5. As a metabolic engineering demonstration, multicopy integration of LCB1 and LCB2, encoding the two subunits of serine palmitoyltransferase, increased TAPS titer by 2.7-fold. This work converts the native NHEJ bias of W. ciferrii from a barrier to precise genome editing into a practical tool for pathway amplification and establishes a framework for engineering NHEJ-dominant non-model yeasts.

Odd-chain carboxylates such as valerate and heptanoate are ecologically relevant metabolites and promising platform chemicals, yet the factors leading to their formation during secondary lactate fermentations remain poorly understood. Here, a continuous anaerobic bioreactor was operated for 297 days under mildly acidic conditions to evaluate how lactate:propionate molar ratios shape product spectrum in lactate fermentations. Valerate was the predominant odd-chain product under all conditions, reaching concentrations up to 110 mM, while heptanoate accumulated only at low levels (<10 mM). At low lactate concentrations (10-20 g/L), product selectivity strongly depended on the lactate:propionate ratio. When lactate:propionate ratios were around 1.2 mol/mol, odd-chain products were favored, whereas higher ratios (up to 4.8 mol/mol) shifted metabolism toward caproate and butyrate formation. However, this trend was not maintained at higher lactate concentrations (30-40 g/L; lactate not fully consumed), where odd-chain selectivities remained high even at lactate:propionate ratios of 4.8 mol/mol. Pathway analysis indicated that under high-lactate conditions up to 30% of lactate was redirected toward propionate and acetate formation, likely via the acrylate pathway. Microbial community analysis revealed a stable dominance of Caproiciproducens spp., that could be correlated to valerate production. Overall, this work provides mechanistic insights into the ecology of lactate fermentations and offers a framework for steering product selectivity in engineered anaerobic systems. HighlightsValerate was the dominant product, reaching up to 110 mM. Lactate:propionate ratios drive product selectivities. High lactate concentrations activated in situ propionate formation pathways. Caproiciproducens dominance was associated with sustained valerate production.

Fermentation of C1 gases is an emerging technology where waste gases are bio converted into value-added products. This study navigates the gas fermentation potential of Gordonia rubripertincta to produce carotenoids. The crucial carbon monoxide dehydrogenase (CODH) enzyme, necessary for gas uptake by the microbe, was found to be present in G. rubripertincta through blastp on NCBI website. The organism was then used for gas fermentation experiments in a continuous stirred tank reactor (CSTR) in different modes of reactor operation resulting in the production of about 500 mg pigment/g WCW (wet cell weight). Two important reactor parameters, molybdenum content and pH, were optimized for enhanced carotenoid production. Overall, G. rubripertincta was observed to be an efficient candidate organism for C1 gas fermentation. KEY HIGHLIGHTSO_LIGordonia rubripertincta synthesises aerobic carbon monoxide dehydrogenase enzyme. C_LIO_LIIt is a potential gas fermenting microbe that gives carotenoids as product. C_LIO_LIThe gas uptake efficiency of the microbe is more in fed-batch discontinued mode. C_LIO_LIIn FB-D, the resultant carotenoids are 500+9 mg/g wet cell weight (WCW). C_LIO_LIMo/pH of 20 mg/7.0 resulted in highest carotenoids, i.e., 134+41 mg/g WCW. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/722808v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@8b1185org.highwire.dtl.DTLVardef@2b6f90org.highwire.dtl.DTLVardef@1a9697dorg.highwire.dtl.DTLVardef@14c9dc8_HPS_FORMAT_FIGEXP M_FIG C_FIG

{beta}-galactosidases (BGs) are essential enzymes widely used in the food industry, particularly in the production of lactose-free products. Among them, the BG from Aspergillus oryzae is of industrial relevance due to its activity at acidic pH and moderate thermal tolerance. However, enhancing its catalytic performance remains a key challenge. Traditional enzyme engineering methods are time-consuming and resource-intensive, limiting their scalability. Recent advances in Artificial Intelligence (AI), particularly those based on Natural Language Processing, offer a promising alternative by enabling efficient exploration of protein sequence space and prediction of beneficial mutations. In this study, we introduce an ensemble-based, zero-shot Protein Language Model pipeline that reconciles predictions from six independent models (ESM2 and the five ESM1v variants) combined with a diversity-aware candidate selection strategy. Applied to the BG from A. oryzae, this approach identified beneficial mutations leading to novel enzyme variants with up to a four-fold increase in catalytic efficiency on oNPGal, a two-fold increase on lactose, and, independently, a T338I variant with markedly enhanced thermostability ({approx}80% residual activity after 24 h at 60 {degrees}C), all without requiring supervised fine-tuning on experimental fitness data. Our results demonstrate that consensus across an ensemble of PLMs can efficiently enrich beneficial substitutions in industrially relevant enzymes and substantially reduce the number of wet-lab candidates that need to be screened. Table of Contents graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/726739v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@18084f7org.highwire.dtl.DTLVardef@99a102org.highwire.dtl.DTLVardef@19a64forg.highwire.dtl.DTLVardef@1f59cff_HPS_FORMAT_FIGEXP M_FIG C_FIG

Primary human hepatocytes (PHHs) are the gold standard for toxicology and drug metabolism studies in industry. However, their limited availability, substantial batch-to-batch variability, and high cost restrict their use. Here, we report a novel culture condition that reprograms PHHs into a proliferative state. These proliferating cells, termed precursors of chemically expanded hepatocytes (pre-cHep), expand over 106-fold within 30 days while retaining liver repopulation capacity comparable to PHHs. pre-cHep can further differentiate into chemically expanded hepatocytes (cHep) as three-dimensional (3D) spheroids within 7 days in vitro, exhibiting global gene expression profiles, albumin production, and cytochrome P450 (CYP) activities similar to 3D-cultured PHH spheroids (3D PHH). Efficient genetic manipulation of pre-cHep using CRISPR/Cas9 is also achievable. Together, pre-cHep and cHep represent a promising alternative to high-quality PHHs, providing a more affordable, reproducible, and scalable source of human hepatocytes for toxicology, drug metabolism studies, disease modelling, towards precision drug development.

Over a thousand fungal genomes have been sequenced, yet manually curated genome-scale metabolic models (GEMs) are available for only a limited number of species. Moreover, these models have often been developed independently, leading to inconsistencies in namespaces, compartment definitions, and pathway representations that hinder comparative analysis, the systematic reuse of prior curation efforts, and the integration of consolidated metabolic knowledge. Here, we present the Consolidated Fungal Core Metabolism Model (CFCMM), constructed by integrating thirteen published fungal models spanning Ascomycota, Mucoromycota, and both Crabtree-positive and Crabtree-negative yeasts. We harmonized metabolites and reactions into a non-redundant shared ModelSEED ontological space, standardized compartmentalization, and refined gene-protein-reaction (GPR) rules. Using pathway-level visualization and systematic gap detection, we further improved the integrated network through literature-guided curation to correct stoichiometry, stereospecificity, and pathway architecture. Orthologous protein family reconstruction and functional annotation workflows were used to validate and inform GPR associations, with particular emphasis on ambiguous enzyme superfamilies and membrane-associated components. Using the resulting CFCMM, we built high-quality central carbon core models for each fungus and performed flux balance analysis to quantify ATP-yield variation under aerobic and anaerobic conditions, explicitly evaluating scenarios driven by differences in electron transport chain (ETC) composition. Simulations reproduced the expected fermentative yield of approximately 2 mmol ATP per mmol glucose under anaerobic conditions and separated the thirteen fungi into two bioenergetic groups under aerobic respiration based on Complex I status, with predicted yields of approximately 30 versus 22 mmol ATP per mmol glucose. Forcing flux through the alternative oxidase bypass further reduced ATP yields to approximately 12 and 4 mmol ATP per mmol glucose in Complex I-containing and Complex I-lacking fungi, respectively. Collectively, this work provides a manually curated, ModelSEED-consistent, and extensible fungal core metabolic template, deployed in DOE KBase as a resource for automated reconstruction of central carbon core models from any sequenced fungal genome. In addition, the CFCMM provides modular components for developing GEMs with more accurate energy predictions and enables robust comparative analyses of fungal bioenergetics and core metabolic diversity.

Bacteroides thetaiotaomicron (Bt), a dominant bacterial species in the human gut, is a promising chassis for engineering in situ therapeutic delivery systems. Previously, we developed a secretion toolkit composed of endogenous lipoprotein signal peptides (SPs) and full-length secretory proteins from Bt. However, due to variations in length, structure, and amino-acid sequence, these SPs exhibited inconsistent secretion efficiencies across different cargo proteins. Because the activity of individual SP-cargo pairs is not readily predictable, screening and optimization is often required to achieve target secretion titers. To enhance the utility of our toolbox, we studied the impact of different SP sequence components on protein secretion, then applied this knowledge to develop a standardized toolkit to and enable predictable and tunable protein secretion across diverse SP-cargo pairs. To achieve this, we first identified the lipoprotein export sequence (LES) as the key determinant of efficient secretion of heterologous proteins by lipoprotein SPs. We next performed mutagenesis on the LES region of a representative lipoprotein SP to generate a pool of mutants featuring a standardized SP backbone with diversified LES regions. Screening and characterization of this mutant pool revealed a charge-dependent regulation of both secretion and surface display of heterologous cargo proteins. From these findings, we established a toolkit with improved tunability, enhanced predictability, and surface display capabilities that minimizes the need for iterative screening when developing protein secreting gene circuits for Bt and other Bacteroides species. By enhancing both the flexibility and control of therapeutic protein output, these results expand the potential of engineered living therapeutic applications, particularly those requiring tunable dosing or surface presentation of proteins.

Genetic code expansion introduces new-to-nature chemical moieties into ribosomally synthesized proteins. In practice, the scope of functional groups that can be accessed using this method is often limited by noncanonical amino acid (ncAA) availability. Producing ncAAs directly in cells can circumvent poor ncAA uptake or commercial unavailability, but limited enzymes suitable for this application exist. In vitro evolution campaigns have been remarkably successful in yielding synthetically useful "ncAA synthases." However, these enzymes are optimized for preparative-scale synthesis and their activities often do not translate well to cellular biosynthesis. Thus, expanding strategies to engineer enzymes specifically for ncAA production within cells will benefit further implementation of genetic code expansion. Here, we use phage-assisted noncontinuous and continuous evolution to evolve enzymes for improved synthesis of non-canonical tyrosine derivatives in E. coli. Using simple serial passaging, we uncovered mutations that doubled the production of an expensive ncAA, 3-methoxytyrosine, by tyrosine phenol lyase, and furthermore evolved variants that enable 3-iodotyrosine biosynthesis, a transformation the parent enzyme is unable to catalyze. Additionally, we evolved a recently reported tyrosine synthase for improved production of 3-halogenated tyrosines, identifying variants that exhibit high activity even at low substrate concentrations owing to a [~]8-fold reduction in KM. Our results demonstrate that phage assisted evolution can be used to rapidly improve the activity of enzymes for ncAA production in cells.