Production Of D-amino Acids Research Articles

Efficient Fermentative Production of d-Alanine and Other d-Amino Acids by Metabolically Engineered Corynebacterium glutamicum.

d-Amino acids (d-AAs) have wide applications in industries such as pharmaceutical, food, and cosmetics due to their unique properties. Currently, the production of d-AAs has relied on chemical synthesis or enzyme catalysts, and it is challenging to produce d-AAs via direct fermentation from glucose. We observed that Corynebacterium glutamicum exhibits a remarkable tolerance to high concentrations of d-Ala, a crucial characteristic for establishing a successful fermentation process. By optimizing meso-diaminopilmelate dehydrogenases in different C. glutamicum strains and successively deleting l-Ala biosynthetic pathways, we developed an efficient d-Ala fermentation system. The d-Ala titer was enhanced through systems metabolic engineering, which involved strengthening glucose assimilation and pyruvate supply, reducing the formation of organic acid byproducts, and attenuating the TCA cycle. During fermentation in a 5-L bioreactor, a significant accumulation of l-Ala was observed in the broth, which was subsequently diminished by introducing an l-amino acid deaminase. Ultimately, the engineered strain DA-11 produced 85 g/L d-Ala with a yield of 0.30 g/g glucose, accompanied by an optical purity exceeding 99%. The fermentation platform has the potential to be extended for the synthesis of other d-AAs, as demonstrated by the production of d-Val and d-Glu.

Structural and functional analysis of hyper-thermostable ancestral L-amino acid oxidase that can convert Trp derivatives to D-forms by chemoenzymatic reaction

Production of D-amino acids (D-AAs) on a large-scale enables to provide precursors of peptide therapeutics. In this study, we designed a novel L-amino acid oxidase, HTAncLAAO2, by ancestral sequence reconstruction, exhibiting high thermostability and long-term stability. The crystal structure of HTAncLAAO2 was determined at 2.2 Å by X-ray crystallography, revealing that the enzyme has an octameric form like a “ninja-star” feature. Enzymatic property analysis demonstrated that HTAncLAAO2 exhibits three-order larger kcat/Km values towards four L-AAs (L-Phe, L-Leu, L-Met, and L-Ile) than that of L-Trp. Through screening the variants, we obtained the HTAncLAAO2(W220A) variant, which shows a > 6-fold increase in kcat value toward L-Trp compared to the original enzyme. This variant applies to synthesizing enantio-pure D-Trp derivatives from L- or rac-forms at a preparative scale. Given its excellent properties, HTAncLAAO2 would be a starting point for designing novel oxidases with high activity toward various amines and AAs.

Engineering acetylation platform for the total biosynthesis of D-amino acids

Optically pure D-amino acids are key chemicals with various applications. Although the production of specific D-amino acids has been achieved by chemical synthesis or with in vitro enzyme catalysts, it is challenging to convert a simple carbon source into D-amino acids with high efficiency. Here, we design an artificial metabolic pathway by engineering bacteria to heterologously express racemase and N-acetyltransferase to produce N-acetyl-D-amino acids from L-amino acids. This new platform allows the cytotoxicity of D-amino acids to be avoided. The universal potential of this acetylation protection strategy for effectively synthesizing optically pure D-amino acids is demonstrated by testing sixteen amino acid targets. Furthermore, we combine pathway optimization and metabolic engineering in Escherichia coli and achieve practically useful efficiency with four specific examples, including N-acetyl-D-valine, N-acetyl-D-serine, N-acetyl-D-phenylalanine and N-acetyl-D-phenylglycine, with titers reaching 5.65 g/L, 5.25 g/L, 8.025 g/L and 130 mg/L, respectively. This work opens up opportunities for synthesizing D-amino acids directly from simple carbon sources, avoiding costly and unsustainable conventional approaches.

Analysis of D-amino acid in Japanese post-fermented tea, Ishizuchi-kurocha.

The D-amino acid content of Ishizuchi-kurocha, a post-fermented tea produced in Ehime, Japan, was measured. Ishizuchi-kurocha mainly contains D-glutamic acid and D-alanine, but it also contains a small amount of D-aspartic acid. Two types of lactic acid bacteria, Lactiplantibacillus plantarum and Levilactobacillus brevis, are the main species involved in lactic acid fermentation during the tea fermentation process. Therefore, the D-amino acid-producing abilities of strains of these two species isolated from Ishizuchi-kurocha were examined. Specifically, the production of D-aspartic acid, D-alanine, and D-glutamic acid by L. brevis and L. plantarum strains was observed. The amount of D-aspartic acid produced by L. plantarum was low. D-glutamine was detected in culture supernatant but not in bacterial cells. D-arginine was detected in bacterial cells of the L. plantarum strains but not in the culture supernatant. Both the L. brevis and L. plantarum strains possessed at least three kinds of putative racemase genes: alanine racemase, glutamate racemase, and aspartate racemase. However, their expression and enzyme activity remain unknown. L. plantarum and L. brevis could play an important role in the production of D-amino acids in Ishizuchi-kurocha. In fact, Ishizuchi-kurocha is expected to possess the effective physiological activities of D-amino acids.

Metabolome analysis to investigate the effect of controlled fermentation on taste-related metabolites in terasi.

Terasi is a fermented shrimp paste unique to Indonesia and is used in dishes to add umami and saltiness. In a previous study, the controlled fermentation of terasi was optimized using starters containing three bacterial isolates: Staphylococcus saprophyticus, Bacillus subtilis, and Lactobacillus murinus. However, the influence of controlled fermentation using these starters on the metabolites in terasi has not yet been studied. Therefore, this study aimed to investigate the effect of controlled fermentation on taste-related metabolites in terasi using a metabolomics approach. Non-targeted analysis indicated that amino acids contributed to variations during fermentation. Subsequently, targeted analysis of amino acids revealed that terasi subjected to controlled fermentation using a starter with a 2:1:2 ratio of S. saprophyticus, B. subtilis, and L. murinus, respectively, resulted in a product containing D-amino acids, such as D-Asp, D-Gln, and D-Leu that was unique when compared to other terasi products prepared using controlled fermentation. Genetic analysis of isolates from the terasi produced using controlled fermentation was also carried out, and this is the first study to suggest that Staphylococcus spp. has the potential to produce D-amino acids. In conclusion, the ratio of bacterial species in starter cultures used in controlled fermentation influenced the amino acid profile of the product and starters with a higher ratio of Staphylococcus spp. may result in the production of D-amino acids.

One-pot biosynthesis of aromatic D-amino acids and neuroactive monoamines via enantioselective decarboxylation under in situ product removal using ion exchange resin

Living organisms have evolved a myriad number of enzymes, which inspires reaction engineers to reroute native biocatalytic utility into scalable synthetic purposes. One of such examples is aromatic L-amino acid decarboxylase (AADC), displaying several enzymatic properties desirable for synthetic applications, including a high turnover rate for C-C bond cleavage and strict enantioselectivity. However, exploitation of the catalytic potential has been limited to the preparation of monoamines and has not yet been proven to afford the synthesis of chiral compounds. Here we report AADC-catalyzed kinetic resolution of diverse aromatic amino acids to demonstrate one-pot production of enantiopure D-amino acids as well as neuroactive monoamines. To this end, we used AADC from Bacillus atrophaeus (AADC-BA) displaying broad substrate specificity and excellent enantioselectivity. Kinetic resolution using AADC-BA afforded > 99% ee of D-amino acid leftover at ≈ 50% conversion of racemic substrates such as phenylalanine, tryptophan, tyrosine, homophenylalanine, and substituted phenylalanine analogues. Product inhibition of AADC-BA led us to develop in situ product removal (ISPR) using cation exchange resin, demonstrating that the ISPR strategy facilitated the kinetic resolution owing to selective removal of the inhibitory monoamine. We also demonstrated that low solubility of racemic substrate could be overcome by fed-batch operation.

Identification of a novel d‐amino acid aminotransferase involved in d‐glutamate biosynthetic pathways in the hyperthermophile Thermotoga maritima

The hyperthermophilic bacterium Thermotoga maritima has an atypical peptidoglycan that contains d-lysine alongside the usual d-alanine and d-glutamate. We previously identified a lysine racemase involved in d-lysine biosynthesis, and this enzyme also possesses alanine racemase activity. However, T. maritima has neither alanine racemase nor glutamate racemase enzymes; hence, the precise biosynthetic pathways of d-alanine and d-glutamate remain unclear in T. maritima. In the present study, we identified and characterized a novel d-amino acid aminotransferase (TM0831) in T. maritima. TM0831 exhibited aminotransferase activity towards 23 d-amino acids, but did not display activity towards l-amino acids. It displayed high specific activities towards d-homoserine and d-glutamine as amino donors. The most preferred acceptor was 2-oxoglutarate, followed by glyoxylate. Additionally, TM0831 displayed racemase activity towards four amino acids including aspartate and glutamate. Catalytic efficiency (kcat /Km ) for aminotransferase activity was higher than for racemase activity, and pH profiles were distinct between these two activities. To evaluate the functions of TM0831, we constructed a TTHA1643 (encoding glutamate racemase)-deficient Thermus thermophilus strain (∆TTHA1643) and integrated the TM0831 gene into the genome of ∆TTHA1643. The growth of this TM0831-integrated strain was promoted compared with ∆TTHA1643 and was restored to almost the same level as that of the wild-type strain. These results suggest that TM0831 is involved in d-glutamate production. TM0831 is a novel d-amino acid aminotransferase with racemase activity that is involved in the production of d-amino acids in T. maritima.

Altered Cofactor Preference of Thermostable StDAPDH by a Single Mutation at K159.

D-amino acid production from 2-keto acid by reductive amination is an attractive pathway because of its high yield and environmental safety. StDAPDH, a meso-diaminopimelate dehydrogenase (meso-DAPDH) from Symbiobacterium thermophilum, was the first meso-DAPDH to show amination of 2-keto acids. Furthermore, StDAPDH shows excellent thermostability compared to other meso-DAPDHs. However, the cofactor of StDAPDH is NADP(H), which is less common than NAD(H) in industrial applications. Therefore, cofactor engineering for StDAPDH is needed. In this study, the highly conserved cofactor binding sites around the adenosine moiety of NADPH were targeted to determine cofactor specificity. Lysine residues within a loop were found to be critical for the cofactor specificity of StDAPDH. Replacement of lysine with arginine resulted in the activity of pyruvic acid with NADH as the cofactor. The affinity of K159R to pyruvic acid was equal with NADH or NADPH as the cofactor, regardless of the mutation. Molecular dynamics simulations revealed that the large steric hindrance of arginine and the interaction of the salt bridge between NADH and arginine may have restricted the free movement of NADH, which prompted the formation of a stable active conformation of mutant K159R. These results provide further understanding of the catalytic mechanism of StDAPDH and guidance for the cofactor engineering of StDAPDH.

Cystathionine β-lyase is involved in d-amino acid metabolism.

Non-canonical d-amino acids play important roles in bacteria including control of peptidoglycan metabolism and biofilm disassembly. Bacteria appear to produce non-canonical d-amino acids to adapt to various environmental changes, and understanding the biosynthetic pathways is important. We identified novel amino acid racemases possessing the ability to produce non-canonical d-amino acids in Escherichia coli and Bacillus subtilis in our previous study, whereas the biosynthetic pathways of these d-amino acids still remain unclear. In the present study, we demonstrated that two cystathionine β-lyases (MetC and MalY) from E. coli produce non-canonical d-amino acids including non-proteinogenic amino acids. Furthermore, MetC displayed d- and l-serine (Ser) dehydratase activity. We characterised amino acid racemase, Ser dehydratase and cysteine lyase activities, and all were higher for MetC. Interestingly, all three activities were at a comparable level for MetC, although optimal conditions for each reaction were distinct. These results indicate that MetC and MalY are multifunctional enzymes involved in l-methionine metabolism and the production of d-amino acids, as well as d- and l-Ser metabolism. To our knowledge, this is the first evidence that cystathionine β-lyase is a multifunctional enzyme with three different activities.

New Insights Into the Mechanisms and Biological Roles of D-Amino Acids in Complex Eco-Systems.

In the environment bacteria share their habitat with a great diversity of organisms, from microbes to humans, animals and plants. In these complex communities, the production of extracellular effectors is a common strategy to control the biodiversity by interfering with the growth and/or viability of nearby microbes. One of such effectors relies on the production and release of extracellular D-amino acids which regulate diverse cellular processes such as cell wall biogenesis, biofilm integrity, and spore germination. Non-canonical D-amino acids are mainly produced by broad spectrum racemases (Bsr). Bsr’s promiscuity allows it to generate high concentrations of D-amino acids in environments with variable compositions of L-amino acids. However, it was not clear until recent whether these molecules exhibit divergent functions. Here we review the distinctive biological roles of D-amino acids, their mechanisms of action and their modulatory properties of the biodiversity of complex eco-systems.

Bacterial Competition Assay Based on Extracellular D-amino Acid Production.

Bacteria live in polymicrobial communities under tough competition. To persist in a specific niche many species produce toxic extracellular effectors as a strategy to interfere with the growth of nearby microbes. One of such effectors are the non-canonical D-amino acids. Here we describe a method to test the effect of D-amino acid production in fitness/survival of bacterial subpopulations within a community. Co-cultivation methods usually involve the growth of the competing bacteria in the same container. Therefore, within such mixed cultures the effect on growth caused by extracellular metabolites cannot be distinguished from direct physical interactions between species (e.g., T6SS effectors). However, this problem can be easily solved by using a filtration unit that allows free diffusion of small metabolites, like L- and D-amino acids, while keeping the different subpopulations in independent compartments. With this method, we have demonstrated that D-arginine is a bactericide effector produced by Vibrio cholerae, which strongly influences survival of diverse microbial subpopulations. Moreover, D-arginine can be used as a cooperative instrument in mixed Vibrio communities to protect non-producing members from competing bacteria.

Characterization of d-succinylase from Cupriavidus sp. P4-10-C and its application in d-amino acid synthesis

d-Amino acids are important building blocks for various compounds, such as pharmaceuticals and agrochemicals. A more cost-effective enzymatic method for d-amino acid production is needed in the industry. We improved a one-pot enzymatic method for d-amino acid production by the dynamic kinetic resolution of N-succinyl amino acids using two enzymes: d-succinylase (DSA) from Cupriavidus sp. P4-10-C, which hydrolyzes N-succinyl-d-amino acids enantioselectively to their corresponding d-amino acid, and N-succinyl amino acid racemase (NSAR, EC.4.2.1.113) from Geobacillus stearothermophilus NCA1503. In this study, DSA and NSAR were purified and their properties were investigated. The optimum temperature of DSA was 50°C and it was stable up to 55°C. The optimum pH of DSA and NSAR was around 7.5. In d-phenylalanine production, the optical purity of product was improved to 91.6% ee from the examination about enzyme concentration. Moreover, 100mM N-succinyl-dl-tryptophan was converted to d-tryptophan at 81.8% yield with 94.7% ee. This enzymatic method could be useful for the industrial production of various d-amino acids.

Genome Sequence of Leuconostoc mesenteroides LK-151 Isolated from a Japanese Sake Cellar as a High Producer of d-Amino Acids.

ABSTRACTHere, we report the complete genome sequence of strain LK-151 of Leuconostoc mesenteroides, which was isolated from a Japanese sake cellar and has the potential to produce large amounts of d-amino acids, namely, d-Ala and d-Glu. The genome contains 4 genes related to d-amino acid production.

Structure-Based Engineering of an Artificially Generated NADP+-Dependent d-Amino Acid Dehydrogenase.

A stable NADP+-dependent d-amino acid dehydrogenase (DAADH) was recently created from Ureibacillus thermosphaericusmeso-diaminopimelate dehydrogenase through site-directed mutagenesis. To produce a novel DAADH mutant with different substrate specificity, the crystal structure of apo-DAADH was determined at a resolution of 1.78 Å, and the amino acid residues responsible for the substrate specificity were evaluated using additional site-directed mutagenesis. By introducing a single D94A mutation, the enzyme's substrate specificity was dramatically altered; the mutant utilized d-phenylalanine as the most preferable substrate for oxidative deamination and had a specific activity of 5.33 μmol/min/mg at 50°C, which was 54-fold higher than that of the parent DAADH. In addition, the specific activities of the mutant toward d-leucine, d-norleucine, d-methionine, d-isoleucine, and d-tryptophan were much higher (6 to 25 times) than those of the parent enzyme. For reductive amination, the D94A mutant exhibited extremely high specific activity with phenylpyruvate (16.1 μmol/min/mg at 50°C). The structures of the D94A-Y224F double mutant in complex with NADP+ and in complex with both NADPH and 2-keto-6-aminocapronic acid (lysine oxo-analogue) were then determined at resolutions of 1.59 Å and 1.74 Å, respectively. The phenylpyruvate-binding model suggests that the D94A mutation prevents the substrate phenyl group from sterically clashing with the side chain of Asp94. A structural comparison suggests that both the enlarged substrate-binding pocket and enhanced hydrophobicity of the pocket are mainly responsible for the high reactivity of the D94A mutant toward the hydrophobic d-amino acids with bulky side chains.IMPORTANCE In recent years, the potential uses for d-amino acids as source materials for the industrial production of medicines, seasonings, and agrochemicals have been growing. To date, several methods have been used for the production of d-amino acids, but all include tedious steps. The use of NAD(P)+-dependent d-amino acid dehydrogenase (DAADH) makes single-step production of d-amino acids from oxo-acid analogs and ammonia possible. We recently succeeded in creating a stable DAADH and demonstrated that it is applicable for one-step synthesis of d-amino acids, such as d-leucine and d-isoleucine. As the next step, the creation of an enzyme exhibiting different substrate specificity and higher catalytic efficiency is a key to the further development of d-amino acid production. In this study, we succeeded in creating a novel mutant exhibiting extremely high catalytic activity for phenylpyruvate amination. Structural insight into the mutant will be useful for further improvement of DAADHs.

Optimization of d-amino acid production catalyzed by immobilized multi-enzyme system in polyelectrolyte complex gel capsules

Multi-enzyme systems containing d-hydantoinase and d-carboamylase encapsulated in alginate–chitosan polyelectrolyte complexes have been proved as interesting biocatalysts to produce d-p-hydroxyphenylglycine. These biocatalysts were produced in a one-step procedure (so called biocatalyst V-I) and in a two-step procedure (so called biocatalyst V-II). In this paper, the production of d-p-hydroxyphenylglycine by using these biocatalysts is optimized taking into account the special features of this system (low enzyme stability and both substrate poor solubility and low stability with temperature). The best operational conditions to produce d-p-hydroxyphenylglycine are identified. Moreover, the encapsulation allows us to carry out the reaction in heterogeneous conditions and to reuse the biocatalysts 3 and 5 times without losing their activity for biocatalyst V-II and V-I, respectively. Finally, the encapsulation dramatically protects the enzymatic activity during lyophilization improving biocatalysts storage.

Continuous colorimetric screening assay for detection of d-amino acid aminotransferase mutants displaying altered substrate specificity

d-Amino acid aminotransferase (DAAT) catalyzes the synthesis of numerous d-amino acids, making it an attractive biocatalyst for the production of enantiopure d-amino acids. To bolster its biocatalytic applicability, improved variants displaying increased activity toward non-native substrates are desired. Here, we report the development of a high-throughput, colorimetric, continuous coupled enzyme assay for the screening of DAAT mutant libraries that is based on the use of d-amino acid oxidase (DAAO). In this assay, the d-amino acid product of DAAT is oxidized by DAAO with concomitant release of hydrogen peroxide, which is detected colorimetrically by the addition of horseradish peroxidase and o-dianisidine. Using this assay, we measured apparent KM and kcat values for DAAT and identified mutants displaying altered substrate specificity via the screening of cell lysates in 96-well plates. The DAAO coupled assay is sensitive in that it allowed the detection of a DAAT mutant displaying an approximately 2000-fold decrease in kcat/KM relative to wild type. In addition, the DAAO assay enabled the identification of two DAAT mutants (V33Y and V33G) that are more efficient than wild type at transaminating the non-native acceptor phenylpyruvate. We expect that this assay will be useful for the engineering of additional mutants displaying increased activity toward non-native substrates.

Distribution of D-amino acids in vinegars and involvement of lactic acid bacteria in the production of D-amino acids

Levels of free D-amino acids were compared in 11 vinegars produced from different sources or through different manufacturing processes. To analyze the D- and L-amino acids, the enantiomers were initially converted into diastereomers using pre-column derivatization with o-phthaldialdehyde plus N-acethyl-L-cysteine or N-tert-butyloxycarbonyl-L-cysteine. This was followed by separation of the resultant fluorescent isoindol derivatives on an octadecylsilyl stationary phase using ultra-performance liquid chromatography. The analyses showed that the total D-amino acid level in lactic fermented tomato vinegar was very high. Furthermore, analysis of the amino acids in tomato juice samples collected after alcoholic, lactic and acetic fermentation during the production of lactic fermented tomato vinegar showed clearly that lactic fermentation is responsible for the D-amino acids production; marked increases in D-amino acids were seen during lactic fermentation, but not during alcoholic or acetic fermentation. This suggests lactic acid bacteria have a greater ability to produce D-amino acids than yeast or acetic acid bacteria.

Active site model of (R)-selective ω-transaminase and its application to the production of d-amino acids

ω-Transaminase (ω-TA) is one of the important biocatalytic toolkits owing to its unique enzyme property which enables the transfer of an amino group between primary amines and carbonyl compounds. In addition to preparation of chiral amines, ω-TA reactions have been exploited for the asymmetric synthesis of L-amino acids using (S)-selective ω-TAs. However, despite the availability of (R)-selective ω-TAs, catalytic utility of the ω-TAs has not been explored for the production of D-amino acids. Here, we investigated the substrate specificity of (R)-selective ω-TAs from Aspergillus terreus and Aspergillus fumigatus and demonstrated the asymmetric synthesis of D-amino acids from α-keto acids. Substrate specificity toward D-amino acids and α-keto acids revealed that the two (R)-selective ω-TAs possess strict steric constraints in the small binding pocket that precludes the entry of a substituent larger than an ethyl group, which is reminiscent of (S)-selective ω-TAs. Molecular models of the active site bound to an external aldimine were constructed and used to explain the observed substrate specificity and stereoselectivity. α-Methylbenzylamine (α-MBA) showed the highest amino donor reactivity among five primary amines (benzylamine, α-MBA, α-ethylbenzylamine, 1-aminoindan, and isopropylamine), leading us to employ α-MBA as an amino donor for the amination of 5 reactive α-keto acids (pyruvate, 2-oxobutyrate, fluoropyruvate, hydroxypyruvate, and 2-oxopentanoate) among 17 ones tested. Unlike the previously characterized (S)-selective ω-TAs, the enzyme activity of the (R)-selective ω-TAs was not inhibited by acetophenone (i.e., a deamination product of α-MBA). Using racemic α-MBA as an amino donor, five D-amino acids (D-alanine, D-homoalanine, D-fluoroalanine, D-serine, and D-norvaline) were synthesized with excellent product enantiopurity (enantiomeric excess >99.7%).

Cloning, expression and characterization of d-aminoacylase from Achromobacter xylosoxidans subsp. denitrificans ATCC 15173

d-Aminoacylase catalyzes the conversion of N-acyl-d-amino acids to d-amino acids and fatty acids. The aim of this study was to identify the d-aminoacylase gene from Achromobacter xylosoxidans subsp. denitrificans ATCC 15173 and investigate the biochemical characterization of the enzyme. A previously uncharacterized d-aminoacylase gene (ADdan) from this organism was cloned and sequenced. The open reading frame (ORF) of ADdan was 1467bp in size encoding a 488-amino acid polypeptide. ADdan, with a high amino acid similarity to N-acyl-d-aspartate amidohydrolase from Alcaligenes A6, showed relatively low sequence similarities to other characterized d-aminoacylases. The recombinant ADdan protein was expressed in Escherichia coli BL21 (DE3) using pET-28a with a T7 promoter. The enzyme was purified in a single chromatographic step using nickel affinity gel column. The molecular mass of the expressed protein, calculated by SDS–PAGE, was about 52kDa. The purified ADdan showed optimal activity at pH 8.0 and 50°C, and was stable at pH 6.0–8.0 and up to 45°C. Its activity was inhibited by Cu2+, Fe2+, Ca2+, Mn2+, Ni2+, Zn2+ and Hg2+, whereas Mg2+ had no significant influence on this recombinant d-aminoacylase. This is the first report on the characterization of d-aminoacylase with activity towards both N-acyl derivatives of neutral d-amino acids and N-acyl-d-aspartate. The characteristics of ADdan could prove to be of interest in industrial production of d-amino acids.

Distinct pathways for modification of the bacterial cell wall by non-canonicalD-amino acids

Production of non-canonical D-amino acids (NCDAAs) in stationary phase promotes remodelling of peptidoglycan (PG), the polymer that comprises the bacterial cell wall. Impairment of NCDAAs production leads to excessive accumulation of PG and hypersensitivity to osmotic shock; however, the mechanistic bases for these phenotypes were not previously determined. Here, we show that incorporation of NCDAAs into PG is a critical means by which NCDAAs control PG abundance and strength. We identified and reconstituted in vitro two (of at least three) distinct processes that mediate NCDAA incorporation. Diverse bacterial phyla incorporate NCDAAs into their cell walls, either through periplasmic editing of the mature PG or via incorporation into PG precursor subunits in the cytosol. Production of NCDAAs in Vibrio cholerae requires the stress response sigma factor RpoS, suggesting that NCDAAs may aid bacteria in responding to varied environmental challenges. The widespread capacity of diverse bacteria, including non-producers, to incorporate NCDAAs suggests that these amino acids may serve as both autocrine- and paracrine-like regulators of chemical and physical properties of the cell wall in microbial communities.