Abstract Arylmalonate decarboxylase (AMDase) stereoselectively converts disubstituted malonates to chiral carboxylic acids, but its substrate spectrum is very limited regarding the size of the smaller substituent. Inspired by the observation that ( S )-selective AMDase variants also convert larger substrates, we unlocked the synthesis of the ( R )-enantiomers of α-aryl and α-alkenyl n- butanoic and n- pentanoic acids, respectively, in exquisite enantiopurity.

Hydrophobic pocket engineering of arylmalonate decarboxylase (AMDase) unlocked the synthesis of the (R)-enantiomers of enantiomerically pure α-aryl and α-alkenyl n-butanoic acids and one n-pentanoic acid.

Enzyme engineering has produced numerous methods to optimize enzymes for biotechnological processes; however, less is known about how natural evolution creates new functionalities. We investigate the evolutionary emergence of enantioselectivity in plant borneol dehydrogenases, which feature hydrophobic active-sites and are enantioselective towards dibornane-type monoterpenols. Ancestral sequence reconstruction provided a trajectory from the oldest unselective ancestor N30 (E = 12) toward a more recent selective ancestor N32, involving 19 mutations: 18 mutations are peripheral, one (I111L) occurs in the active-site. The mutation L111I in the hydrophobic pocket increased the selectivity of N30, while the back-mutation I111L decreased the selectivity of N32. Additional peripheral mutations (V136L/G169A/V183I) were required for high selectivity. Crystal structures suggested that protein dynamics rather than structural changes shape these catalytic properties; this was confirmed by Machine Learning/Molecular Mechanics simulations of ligand binding. Funnel-metadynamics simulations revealed a correlation between the active-site’s solvent-accessible surface area and selectivity. This potential evolutionary pathway shapes enantioselectivity, and guides future enzyme engineering campaigns. Enzyme engineering has enabled optimization of enzymes for biotechnological processes, however, less is known about how natural evolution creates new functionalities. Here, the authors investigate the evolutionary emergence of enantioselectivity in plant borneol dehydrogenases using ancestral sequence reconstruction, structural analysis, site-directed mutagenesis and computational simulations.

The regio- and stereoselective hydroxylation of unactivated C(sp3)-H bonds is an important reaction in organic synthesis. While bacterial alkane monooxygenase AlkB catalyzes the terminal hydroxylation of aliphatic esters with excellent regioselectivity, the molecular principles of substrate recognition and selectivity of this integral membrane enzyme are still poorly understood. In this study, we investigated the substrate scope and engineered the medium-chain alkane monooxygenase from Marinobacter sp. (M_AlkB) for the terminal hydroxylation of linear and branched esters of fatty acids and alcohols. For the first time, we demonstrated the stereoselectivity of AlkB toward prochiral substrates containing terminal gem-dimethyl groups, leading to the corresponding chiral β-methyl primary alcohols in good optical purity (51%-79% ee). The hydroxylation products can be further derivatized to chiral diols and lactones. Substitution of the highly conserved active site residue F169 to leucine increased the activity toward short and medium-chain esters up to two-fold. While the wildtype enzyme displayed very low conversion of long-chain substrates, activity toward n-dodecyl acetate could be increased 11-fold by reducing the size of the tryptophan residue 60 situated in the putative substrate tunnel. Substitution of the peripheral I238 with valine increased activity regardless of the chain length of the substrate. Our results provide insight into the strong substrate dependency of the reported mutations and lay the groundwork for the establishment of a whole-cell process for the regio- and stereoselective hydroxylation of linear and branched esters, leading to valuable bifunctionalized products. The deeper understanding gained from mutating key residues and the substrate acceptance of AlkB will guide future protein engineering campaigns.

This study develops and optimizes a high-cell-density cultivation (HCDC) of E. coli BL21 (DE3) to improve enzyme production utilizing DASGIP multibioreactor systems. By shifting from traditional shake flask methods to HCDC, we observed a 43-fold increase in biomass production, with a comparable mass-specific activity of the cell-free extract. The HCDC demonstrated a 30-fold increase in the active, soluble expression of arylmalonate decarboxylase (AMDase) from Bordetella bronchiseptica (BbAMDase) compared to conventional methodology. The protocol was designed to be readily adapted to various expression approaches and to provide a robust foundation for broader use in recombinant protein production. The HCDC was achieved with a linear glucose feed to promote robust cell growth. We optimized the glycerol feeding strategy during isopropyl β-D-1-thiogalactopyranoside (IPTG) induction to maximize AMDase production. Throughout the induction phase, we monitored both soluble expression yield and enzymatic activity, aiming to establish a process that is not only profitable but also efficient and stable.

Protein stabilization is a "Holy Grail" of biocatalysis, and stability design is an area of intense research interest. While it is increasingly feasible to effectively increase enzyme thermostability, optimization without compromising activity or selectivity remains a significant challenge. Here, we use full-atom protein sequence design with sidechain conditioning (FAMPNN) to engineer thermostable variants of the borneol dehydrogenase from Salvia rosmarinus (SrBDH1), an enzyme from a family where unselective enzymes dominate, and selectivity is determined by dynamical considerations. By combining FAMPNN design with residue conservation analysis and avoiding active site residues, we were able to computationally design SrBDH1 variants with up to 10 °C enhanced thermostability and strongly increased half-life time at elevated temperature, while retaining selectivity towards (+)-borneol. This design framework, integrating de novo and physics-based protein design tools, demonstrates that stability can be enhanced without disrupting functionally relevant dynamics, providing a route to engineer robust and selective biocatalysts.

Harnessing nature’s ingenuity with microorganisms for industrial production is an attractive solution to today’s climate concerns. Nature’s innate diversity allows the production of many value-added chemicals and can be expanded on through genetic engineering. Although the use of microbial cell factories (MCFs) has been extremely successful at lab scale, the numbers of successful bioprocesses remain limited. High cell densities and long cultivation times lead to reductions in productivity over the course of the cultivation through the effects of genetic and expression instability of the strain. This instability leads to population diversification. In this review, we explore the roots of genetic instability in microorganisms, focusing on prokaryotic bioprocesses, and how organisms cope with this instability. We spotlight single-cell detection methods capable of monitoring populations within the bioprocess both in- and on-line. We also examine different approaches to minimizing population diversification, both through strain development and bioprocess engineering. With this review, we highlight the fact that population-averaged metrics overlook the single-cell stresses driving genetic and functional instability, leading to an overestimation of microbial bioprocess robustness. High-throughput single-cell monitoring in industry-like conditions remains essential to identify and select truly stable microbial cell factories and bioprocesses.

This review summarizes recent progress in cell@MOF, cell@COF, and cell@HOF composites from a synthetic biology and materials science perspective. It outlines key synthetic strategies for the synthesis of porous abiotic exoskeletons, focusing on framework-based materials. Additionally, it discusses the cell surface chemistry and current methods for assessing cell viability. Major applications, including cell therapy, biocatalysis, biosensing, and CO2 mitigation, are examined alongside approaches for composite preparation and characterization. This review concludes with prospects and challenges for using framework materials to engineer synthetic cells and enhance cellular functions.

Photobiocatalysis with photoautotrophic whole cells has demonstrated strong potential for producing chiral molecules and platform chemicals using sustainable inputs such as light, water and CO2 under mild reaction conditions. Coupling enzymatic transformations directly to natural photosynthesis enables higher atom efficiency compared with heterotrophic systems. However, large-scale application remains challenging, particularly due to light attenuation in photobioreactors. In this review, we summarize recent advances in whole-cell photobiotransformations with emphasis on process conditions. We also discuss strategies for intensifying photobiocatalysis through improved reactor design and new immobilization materials, along with developments in fast-growing photoautotrophic strains. Sustainability analyses indicate that organic electron donors represent only one factor influencing environmental performance, and simply replacing them with photosynthetic water splitting does not inherently yield a carbon-negative process. Nonetheless, our calculations show that when high substrate loadings are combined with wastewater use and optimized downstream processing, photosynthesis-driven biotechnology can offer substantial reductions in CO2 emissions.

Ene‐reductases (EREDs) are commonly employed for the asymmetric synthesis of important industrial chemicals. In this research, we characterise two EREDs (HSI and HSII) belonging to the ‘thermophilic‐like’ Old Yellow Enzyme (OYE) family from the beta‐proteobacterium Herbaspirillum seropedicae. Both HSI and HSII show promiscuity towards the coenzymes NADH and NADPH. Despite high sequence identities of 46% and 76% with NADH/NADPH‐dependent xenobiotic reductase A (XenA) from Pseudomonas putida, HSI prefers NADPH, whereas HSII shows equal preference for both coenzymes. To study coenzyme specificity, ancestral sequence reconstruction was performed. Two ancestral enzymes (N29 and N38) displayed a strong preference for NADH over NADPH, with N29 exhibiting the highest specific activity with NADH (12 U mg−1). We conclude that oligomerisation and a conserved ‘arginine finger’ contribute to NADPH specificity, and demonstrate the practical utility of HSI and HSII by using them as coenzyme regeneration catalysts for 5‐hydroxymethylfurfural oxidation, resulting in yields of 34.5% and 25.0%.

Phenolic acid decarboxylases (PADs) convert bio-based hydroxycinnamic acids into valuable hydroxystyrene monomers under mild reaction conditions. These compounds are in high demand in polymer production, cosmetics, and flavoring. Especially 4-vinyl syringol, the decarboxylation product from sinapic acid, generates polymers with similar thermal stability and higher glass transition temperatures than vinyl guaiacol, the decarboxylation product from ferulic acid. However, natural PAD enzymes typically show slow turnover with sinapic acid. In addition, establishing a viable industrial process requires enzymes operating under elevated temperatures. To tackle these issues, we assessed five thermostable ancestral PADs towards their activity and stability for the conversion of ferulic acid and sinapic acid at different temperatures. A combinatorial active site library was prepared for the most thermostable ancestor. We expanded the substrate scope of a selected PAD ancestor to include sinapic acid through directed mutagenesis. A trade-off between ferulic-/caffeic acid and sinapic acid was observed and investigated via molecular dynamics simulations. The most stable ancestor was identified with a half-life of 3.65 days, analyzed at 50°C. We found the Ile29Ser-Leu80Ser-Ile93Ala triple mutation (SSA) to effectively expand the substrate scope with an 11-fold increase in catalytic efficiency for sinapic acid, and a half-life of 1.12 days at 50°C, being approximately 1.6-fold higher than the frequently used PAD from Bacillus subtilis.

Bacterial aryl malonate decarboxylase is a cofactor-free enzyme that generates a wide spectrum of α-chiral carboxylic acids in outstanding optical purity, including several non-steroidal anti-inflammatory drugs and chiral building blocks. The well-characterized AMDase from Bordetella bronchiseptica (Bb AMDase) and related enzymes of the same family have three main limitations: (i) low stability, both operational and thermal, and (ii) limited substrate spectrum regarding the size of the smaller substituent on the α-C-atom and (iii) low stereoselectivity towards α-alkenyl-α-alkyl malonic acids. To address these limitations, we expanded the structural diversity of the AMDase family by ancestral sequence reconstruction (ASR). The phylogenetic analysis of the decarboxylase revealed conserved structural motifs and key amino acids in the hydrophobic active-site cavity, a catalytic motif crucial for activity and selectivity of the enzyme. The analysis highlighted the natural distribution of amino acid exchanges that had been previously identified in enzyme engineering campaigns. AMDase ancestors showed higher stability, activity, and, in one case, also stereoselectivity than Bb AMDase. While the up to 10 °C higher unfolding temperature of AMDase ancestors is a frequent result in ASR, the improvement of the half-life time of 294-fold of ancestor N131 was surprising. Ancestor N31 formed 2-methyl-but-3-enoic acid from its corresponding malonic acid in an optical purity of 99.7% eeR . The extant Bb AMDase produces this compound in much lower optical purity (96.8% eeR ), which corresponds to a 1.4 kcal·mol-[1][1] difference of the transition state free energy of the two reaction paths leading to the different enantiomers. Furthermore, the stereoselectivity of the ancestors was completely inverted by switch of the catalytic cysteine residues G74C/C188G. ### Competing Interest Statement The authors have declared no competing interest. FWF Austrian Science Fund, https://ror.org/013tf3c58, 10.55776/P34280, 10.55776/6954-N Ministry for Science and Culture for Lower Saxony, 12.5-76251-17-9/20 [1]: #ref-1

Photosynthetic microorganisms are capable of oxygenic photosynthesis, delivering both oxygen and cofactors to drive enzymatic redox reactions. However, their dependence on visible light limits the tolerable cell densities to achieve high reaction rates. Immobilizing cells within a matrix often increases biocatalyst productivity while allowing facile retainment but also creates mass transfer limitations across the solid-liquid interface. Herein, we address these challenges and present the immobilization of recombinant cyanobacteria in 3D-printed hydrogels of varying geometries. In particular, whole cells of the cyanobacterium Synechocystis sp. PCC 6803, engineered to express the gene of the ene-reductase YqjM, were implemented in biocompatible hydrogels made out of nanofibrillated cellulose and alginate. The hydrogels were 3D-printed via extrusion into different geometries to alleviate light and mass transfer limitations and were applied for the reduction of prochiral 2-methylmaleimide to (R)-2-methylsuccinimide. The obtained reactors exhibit high mechanical stability (620 kPa), efficient flow and mass transfer characteristics, high specific surface area (up to 2129 mm(2) g(-1)), and retention times favorable to achieve high product formation. (R)-2-methylsuccinimide was obtained with a space-time yield of 0.28 g L-1 h(-1) and a high enantiomeric purity (>99%). The highly atom-efficient chemical process (88%) using only water to provide electrons for NADPH regeneration could be upscaled and can potentially be operated in extended periods to reduce wastewater associated with cell cultivation. Overall, 3D printing of photosynthetic microorganisms embedded in a hydrogel matrix holds significant promise for advancing the development of whole-cell solid-state photosynthetic cell factories. These are important steps toward improved reactor designs and higher efficiencies to improve crucial redox biotransformations.

The cofactor-free arylmalonate decarboxylase (AMDase) is a valuable biocatalyst for synthesizing α-aryl and α-alkenyl alkanoic acids with excellent stereoselectivity. We engineered a new hydrophobic pocket in (S)-selective AMDase mutants, creating AMDase ICPLLG with enhanced activity. For the investigation of the mechanism, we synthesized isotope-labeled, pseudochiral 2-methyl-2-vinyl malonate via an auxiliary-based asymmetric route using a chiral imidazolidinone to enable stereoselective bis-alkylation of malonates. Our results reveal striking substrate-dependent stereochemical behavior: AMDase ICPLLG decarboxylates prochiral aromatic malonates with retention of configuration at the α-carbon. The critical Cys residue adds a proton from the same face of the substrate as the leaving carboxylate. Interestingly, the same mutant decarboxylates the corresponding alkenyl malonate with inversion of configuration, i.e., with protonation from the opposite face. Kinetic isotope effect measurements and QM/MM metadynamics calculations suggest that alkenyl malonates adopt an alternative binding mode and undergo decarboxylation via a borderline concerted mechanism instead of a stepwise mechanism. This new pathway changes the stereochemical preference. We exploited this strategy to decarboxylate sterically hindered alkenyl malonates (substrates not converted by wild-type AMDase) with high stereoselectivity. The engineered hydrophobic pocket in (S)-selective AMDase mutants expands the substrate scope for synthesizing enantiomerically pure α-aryl and α-alkenyl butanoic acids. This work demonstrates a new approach (a mechanistic change) to engineer the substrate range and stereoselectivity of enzymes.

Efficient regeneration of NAD+ remains a significant challenge for oxidative biotransformations. In order to identify enzymes with higher activity and stability, a panel of NADH oxidases (Nox) was investigated in the regeneration of nicotinamide cofactors for the oxidation of hydroxymethyl furfural (HMF) to 5-hydroxymethyl-2-furancarboxylic acid (HMFCA). We present novel Nox that exhibit remarkable catalytic activities, elevated thermal and pH stabilities, and higher intrinsic flavin loadings, thus eliminating the need for external flavin addition. The kinetic analysis of the NADH oxidases indicates that AdNox, GdNox, CmNox, and LvNox exhibit Vmax values of 86 U/mg, 50 U/mg, 4.3 U/mg, and 23 U/mg, respectively. When these NADH oxidases were applied in a HMF oxidation reaction, LvNox demonstrated the highest HMFCA yield of 97% in the presence of 0.1mM NAD and 10mM HMF. In contrast to previously reported NADH oxidases from the same family, these NADH oxidases naturally accept NADPH as a substrate. Rapid kinetics experiments identified the oxidative reaction as the rate-limiting step of the reaction. NADH oxidases achieved high atom economy, a high reaction mass efficiency and a low process mass intensity. The findings contribute significantly to the field of biocatalysis and offer potential avenues for more environmentally friendly cofactor regeneration in chemical synthesis.

Oxygenases catalyze C-H oxyfunctionalization under mild reaction conditions and often display outstanding selectivity. However, their utilization is hampered by the difficulty of transporting oxygen across the gas-liquid interface, which is particularly problematic for continuous reactor systems and can only be alleviated by high pressure or the use of complex oxygen-permeable materials. Herein, oxygen is directly released into the medium by the phototrophic cyanobacterium Synechocystis sp. PCC 6803 expressing the genes of a Baeyer-Villiger Monooxygenase from Burkholderia xenovorans to drive the oxidation of cyclohexanone for the production of the polymer precursor, ε-caprolactone. The rates at which photosynthetic oxygen can solely drive the oxidation were determined by performing the reaction in a continuous coil reactor with a very limited external oxygen supply. In heterotrophic nonoxygen-producing Escherichia coli expressing the same gene, a 10-fold lower specific activity was observed when the oxidation was performed in the coil reactor compared with batch mode underlining the impact of oxygen-limitation on the volumetric productivity. In contrast, cyanobacterial whole cells showed activities of 16.7 and 13.5 U gDCW -1 in nonoxygen-limited batch and oxygen-limited continuous flow, respectively. Net oxygen production of the whole-cell biocatalyst during the reaction led to a steady-state oxygen concentration allowing volumetric productivities as high as 3 mmol L-1 h-1 highlighting the advantages of photoautotrophic production systems for oxyfunctionalization under oxygen-limiting conditions. Moreover, the space-time yield of the reaction was improved 7-fold (2.8 vs 0.4 g L-1 h-1) by utilizing the continuous coil reactor compared to the batch mode. The combination of flow catalysis and photosynthetic oxygen production can overcome current limitations in photo(bio)oxidation and achieve significant improvements in terms of volumetric productivity enabling more sustainable chemical synthesis. This approach using whole-cells of cyanobacteria achieves a notably lower ratio of waste to product (E-factor) and higher atom economy compared with oxidation mediated by Escherichia coli .

Solid-state photosynthetic cell factories (SSPCFs) are a new production concept that leverages the innate photosynthetic abilities of microbes to drive the production of valuable chemicals. It addresses practical challenges such as high energy and water demand and improper light distribution associated with suspension-based culturing; however, these systems often face significant challenges related to mass transfer. The approach focuses on overcoming these limitations by carefully engineering the microstructure of the immobilization matrix through freeze-induced assembly of nanochitin building blocks. The use of nanochitins with optimized size distribution enabled the formation of macropores with lamellar spatial organization, which significantly improves light transmittance and distribution, crucial for maximizing the efficiency of photosynthetic reactions. The biomimetic crosslinking strategy, leveraging specific interactions between polyphosphate anions and primary amine groups featured on chitin fibers, produced mechanically robust and wet-resilient cryogels that maintained their functionality under operational conditions. Various model biotransformation reactions leading to value-added chemicals are performed in chitin-based matrix. It demonstrates superior or comparable performance to existing state-of-the-art matrices and suspension-based systems. The findings suggest that chitin-based cryogel approach holds significant promise for advancing the development of solid-state photosynthetic cell factories, offering a scalable solution to improve the efficiency and productivity of light-driven biotransformation.

The reduction of carboxylic acids to alcohols is a key chemical transformation. We present a whole-cell biocatalyst employing carboxylic acid reductases and H2 for cofactor regeneration. H2 as a reductant ensures high atom and mass efficiency, enabling a more sustainable route to alcohols. Notably, we showcase that the reaction proceeds under O2-limited conditions, demonstrating potential for safer bioprocesses.

The α‐methylene‐γ‐butyrolactone tulipalin A, a defense compound found in tulips, can polymerize via addition at the vinyl group or via ring‐opening polymerization, making it a highly promising monomer for bio‐based polymers. Since the biosynthesis of tulipalin A in plants remains elusive, we propose an alternative pathway for its synthesis starting from the terpenoid intermediate isoprenyl acetate. While fungal unspecific peroxygenases showed a preference for the unwanted epoxidation of its exo‐olefin group, bacterial alkane monooxygenases were selective for terminal hydroxylation. By combining protein engineering based on de novo structure prediction of the membrane enzymes with cell engineering, the specific activity was increased 6‐fold to 1.83 U gcdw‐1. Oxidation of the formed allylic alcohol by a three‐enzyme cascade and subsequent lactonization yielded tulipalin A. Our results demonstrate the feasibility of producing the polymer precursor tulipalin A from terpenoid intermediates and provide a solid foundation for future metabolic engineering endeavors.

Light-driven biotransformations in recombinant cyanobacteria benefit from the atom-efficient regeneration of reaction equivalents like NADPH from water and light by oxygenic photosynthesis. The self-shading of photosynthetic cells throughout the reaction volume, along with the need for extended light paths, limits adequate light supply and significantly restricts the potential for upscaling. Here, we present a flat panel photobioreactor (1 cm optical path length) as a scalable system to provide efficient illumination at high cell densities. The genes of five ene-reductases from different classes were expressed in Synechocystis sp. PCC 6803. The strains were characterised in the light-driven reduction of a set of prochiral substrates. With specific activities up to 150 U gCDW-1 under standard conditions in small-scale reactions, the recombinant strains harbouring the ene-reductases TsOYE C25G I67T and OYE3 showed the highest specific activities observed so far in photobiotransformations and were selected for the up-scale in the flat panel photobioreactor in 120 mL-scale. The strain producing OYE3 exhibited a specific activity as high as 56.1 U gCDW-1. The corresponding volumetric productivity of 1 g L-1 h-1 compares favourably to other photosynthesis-driven processes. This setup facilitated the conversion of 50 mM over approximately 8 hours to an isolated yield of 87%. The atom economy of 88% compares favourably to the use of the sacrificial co-substrates glucose and formic acid with 49% and 78%, respectively. Determination of the complete E-Factor of 203 including water reveals that the volumetric yield and water required for cultivation are crucial for the sustainability. In summary, our results point out key factors for the sustainability of light-driven whole-cell biotransformations, and provide a solid basis for future optimisation and up-scale campaigns of photosynthesis-driven bioproduction.