Engineering of the DNA replication and repair machinery to develop binary mutators for rapid genome evolution of Corynebacterium glutamicum.

Corynebacterium glutamicum is an important industrial workhorse for production of amino acids and chemicals. Although recently developed genome editing technologies have advanced the rational genetic engineering of C. glutamicum, continuous genome evolution based on genetic mutators is still unavailable. To address this issue, the DNA replication and repair machinery of C. glutamicum was targeted in this study. DnaQ, the homolog of ϵ subunit of DNA polymerase III responsible for proofreading in Escherichia coli, was proven irrelevant to DNA replication fidelity in C. glutamicum. However, the histidinol phosphatase (PHP) domain of DnaE1, the α subunit of DNA polymerase III, was characterized as the key proofreading element and certain variants with PHP mutations allowed elevated spontaneous mutagenesis. Repression of the NucS-mediated post-replicative mismatch repair pathway or overexpression of newly screened NucS variants also impaired the DNA replication fidelity. Simultaneous interference with the DNA replication and repair machinery generated a binary genetic mutator capable of increasing the mutation rate by up to 2352-fold. The mutators facilitated rapid evolutionary engineering of C. glutamicum to acquire stress tolerance and protein overproduction phenotypes. This study provides efficient tools for evolutionary engineering of C. glutamicum and could inspire the development of mutagenesis strategy for other microbial hosts.

Novel 15N Metabolic Labeling-Based Large-Scale Absolute Quantitative Proteomics Method for Corynebacterium glutamicum.

With fast growth, synthetic biology powers us with the capability to produce high commercial value products in an efficient resource/energy-consuming manner. Comprehensive knowledge of the protein regulatory network of a bacterial host chassis, e.g., the actual amount of the given proteins, is the key to building cell factories for certain target hyperproduction. Many talent methods have been introduced for absolute quantitative proteomics. However, for most cases, a set of reference peptides with isotopic labeling (e.g., SIL, AQUA, QconCAT) or a set of reference proteins (e.g., commercial UPS2 kit) needs to be prepared. The higher cost hinders these methods for large sample research. In this work, we proposed a novel metabolic labeling-based absolute quantification approach (termed nMAQ). The reference Corynebacterium glutamicum strain is metabolically labeled with 15N, and a set of endogenous anchor proteins of the reference proteome is quantified by chemically synthesized light (14N) peptides. The prequantified reference proteome was then utilized as an internal standard (IS) and spiked into the target (14N) samples. SWATH-MS analysis is performed to obtain the absolute expression levels of the proteins from the target cells. The cost for nMAQ is estimated to be less than 10 dollars per sample. We have benchmarked the quantitative performance of the novel method. We believe this method will help with the deep understanding of the intrinsic regulatory mechanism of C. glutamicum during bioengineering and will promote the process of building cell factories for synthetic biology.

120-GBaud 16-QAM silicon photonics IQ modulator for data center interconnection.

We demonstrated all-silicon IQ modulators (IQMs) operating at 120-GBaud 16-QAM with suitable bandwidth, and output power. We required optical signal-to-noise-ratio (rOSNR) that have promising potential to be used in 800-Gbps small-form-factor pluggable transceivers for data center interconnection. First, we tested an IQM chip using discrete drivers and achieved a per-polarization TX output power of -18.74 dBm and an rOSNR of 23.51 dB over a 100-km standard SMF. Notably, a low BER of 1.4e-3 was obtained using our SiP IQM chip without employing nonlinear compensation, optical equalization, or an ultra-wide-bandwidth, high-ENOB OMA. Furthermore, we investigated the performance of a 3D packaged transmitter by emulating its frequency response using an IQM chip, discrete drivers, and a programmable optical filter. With a laser power of 17 dBm, we achieved a per-polarization output power of -15.64 dBm and an rOSNR of 23.35 dB.

N-Doped Nonalternant Nanoribbons with Excellent Nonlinear Optical Performance.

Two novel N-doped nonalternant nanoribbons (NNNR-1 and NNNR-2) featuring multiple fused N-heterocycles and bulky solubilizing groups were prepared via bottom-up solution synthesis. NNNR-2 achieves a total molecular length of 33.8 Å, which represents the longest soluble N-doped nonalternant nanoribbon reported to date. The pentagon subunits and doping of N atoms in NNNR-1 and NNNR-2 have successfully regulated their electronic properties, achieving high electron affinity and good chemical stability enabled by the nonalternant conjugation and electronic effects. When applied a laser pulse of 532 nm, the 13-rings nanoribbon NNNR-2 shows outstanding nonlinear optical (NLO) responses, with the nonlinear extinction coefficient of 374 cm GW-1 , much higher than those of NNNR-1 (96 cm GW-1 ) and the well-known NLO material C60 (153 cm GW-1 ). Our findings indicate that the N-doping of nonalternant nanoribbons is an effective strategy to access another type of excellent material system for high-performance NLO applications, which can be extended to construct numerous heteroatom-doped nonalternant nanoribbons with fine-tunable electronic properties.

Crystal structure of 5-Aminolevulinate synthase HemA from Rhodopseudomonas palustris presents multiple conformations.

5-ALA is the precursor of all tetrapyrroles. 5-Aminolevulinate synthase (ALAS) catalyzes the production of 5-aminolevulinic acid (5-ALA) from glycine and succinyl-CoA. HemA from Rhodopseudomonas palustris (Rp-HemA) was reported to be a highly active ALAS. To understand the catalytic mechanism of Rp-HemA, the 2.05 Å resolution crystal structure of Rp-HemA was solved. Open, half close and close conformations were observed in the substrate-free structures. Structure comparison and sequence alignment suggest the newly observed half close conformation may also be conserved in ALAS family. The pre-existed close and half close conformations in Rp-HemA may play a key role for its high activity.

Metallated Graphynes as a New Class of Photofunctional 2D Organometallic Nanosheets.

Two-dimensional (2D) nanomaterials are attracting much attention due to their excellent electronic and optical properties. Here, we report the first experimental preparation of two free-standing mercurated graphyne nanosheets via the interface-assisted bottom-up method, which integrates both the advantages of metal center and graphyne. The continuous large-area nanosheets derived from the chemical growth show the layered molecular structural arrangement, controllable thickness and enhanced π-conjugation, which result in their stable and outstanding broadband nonlinear saturable absorption (SA) properties (at both 532 and 1064 nm). The passively Q-switched (PQS) performances of these two nanosheets as the saturable absorbers are comparable to or higher than those of the state-of-the-art 2D nanomaterials (such as graphene, black phosphorus, MoS2 , γ-graphyne, etc.). Our results illustrate that the two metallated graphynes could act not only as a new class of 2D carbon-rich materials, but also as inexpensive and easily available optoelectronic materials for device fabrication.

Disruption or reduced expression of the orotidine-5'-decarboxylase gene pyrG increases citric acid production: a new discovery during recyclable genome editing in Aspergillus niger.

BACKGROUND: Aspergillus niger is a filamentous fungus used for the majority of global citric acid production. Recent developments in genome editing now enable biotechnologists to engineer and optimize A. niger. Currently, however, genetic-leads for maximizing citric acid titers in industrial A. niger isolates is limited. RESULTS: In this study, we try to engineer two citric acid A. niger production isolates, WT-D and D353, to serve as platform strains for future high-throughput genome engineering. Consequently, we used genome editing to simultaneously disrupt genes encoding the orotidine-5'-decarboxylase (pyrG) and non-homologous end-joining component (kusA) to enable use of the pyrG selection/counter selection system, and to elevate homologous recombination rates, respectively. During routine screening of these pyrG mutant strains, we unexpectedly observed a 2.17-fold increase in citric acid production when compared to the progenitor controls, indicating that inhibition of uridine/pyrimidine synthesis may increase citric acid titers. In order to further test this hypothesis, the pyrG gene was placed under the control of a tetracycline titratable cassette, which confirmed that reduced expression of this gene elevated citric acid titers in both shake flask and bioreactor fermentation. Subsequently, we conducted intracellular metabolomics analysis, which demonstrated that pyrG disruption enhanced the glycolysis flux and significantly improved abundance of citrate and its precursors. CONCLUSIONS: In this study, we deliver two citric acid producing isolates which are amenable to high throughput genetic manipulation due to pyrG/kusA deletion. Strikingly, we demonstrate for the first time that A. niger pyrG is a promising genetic lead for generating citric acid hyper-producing strains. Our data support the hypothesis that uridine/pyrimidine biosynthetic pathway offer future avenues for strain engineering efforts.

Enhancing 5-aminolevulinic acid tolerance and production by engineering the antioxidant defense system of Escherichia coli.

5-Aminolevulinic acid (ALA) is a value-added compound with potential applications in the fields of agriculture and medicine. Although massive efforts have recently been devoted to building microbial producers of ALA through metabolic engineering, few studies focused on the cellular response and tolerance to ALA. In this study, we demonstrated that ALA caused severe cell damage and morphology change of Escherichia coli via generating reactive oxygen species (ROS), which were further determined to be mainly hydrogen peroxide and superoxide anion radical. ALA treatment activated the native antioxidant defense system by upregulating catalase (CAT) and superoxide dismutase (SOD) expression to combat ROS. Further overexpressing CAT (encoded by katG and katE) and SOD (encoded by sodA, sodB, and sodC) not only improved ALA tolerance but also its production level. Notably, coexpression of katE and sodB in an ALA synthase expressing strain enhanced the biomass and final ALA titer by 81% and 117% (11.5 g/L) in a 5 L bioreactor, respectively. This study demonstrates the importance of tolerance engineering in strain development. Reinforcing the antioxidant defense system holds promise to improve the bioproduction of chemicals that cause oxidative stress.

Expanding targeting scope, editing window, and base transition capability of base editing in Corynebacterium glutamicum.

CRISPR/Cas9-guided cytidine deaminase enables C:G to T:A base editing in bacterial genome without introduction of lethal double-stranded DNA break, supplement of foreign DNA template, or dependence on inefficient homologous recombination. However, limited by genome-targeting scope, editing window, and base transition capability, the application of base editing in metabolic engineering has not been explored. Herein, four Cas9 variants accepting different protospacer adjacent motif (PAM) sequences were used to increase the genome-targeting scope of bacterial base editing. After a comprehensive evaluation, we demonstrated that PAM requirement of bacterial base editing can be relaxed from NGG to NG using the Cas9 variants, providing 3.9-fold more target loci for gene inactivation in Corynebacterium glutamicum. Truncated or extended guide RNAs were employed to expand the canonical 5-bp editing window to 7-bp. Bacterial adenine base editing was also achieved with Cas9 fused to adenosine deaminase. With these updates, base editing can serve as an enabling tool for fast metabolic engineering. To demonstrate its potential, base editing was used to deregulate feedback inhibition of aspartokinase via amino acid substitution for lysine overproduction. Finally, a user-friendly online tool named gBIG was provided for designing guide RNAs for base editing-mediated inactivation of given genes in any given sequenced genome (www.ibiodesign.net/gBIG).

Systems metabolic engineering for citric acid production by Aspergillus niger in the post-genomic era.

Citric acid is the world's largest consumed organic acid and is widely used in beverage, food and pharmaceutical industries. Aspergillus niger is the main industrial workhorse for citric acid production. Since the release of the genome sequence, extensive multi-omic data are being rapidly obtained, which greatly boost our understanding of the citric acid accumulation mechanism in A. niger to a molecular and system level. Most recently, the rapid development of CRISPR/Cas9 system facilitates highly efficient genome-scale genetic perturbation in A. niger. In this review, we summarize the impact of systems biology on the citric acid molecular regulatory mechanisms, the advances in metabolic engineering strategies for enhancing citric acid production and discuss the development and application of CRISPR/Cas9 systems for genome editing in A. niger. We believe that future systems metabolic engineering efforts will redesign and engineer A. niger as a highly optimized cell factory for industrial citric acid production.

GREACE-assisted adaptive laboratory evolution in endpoint fermentation broth enhances lysine production by Escherichia coli.

BACKGROUND: Late-stage fermentation broth contains high concentrations of target chemicals. Additionally, it contains various cellular metabolites which have leaked from lysed cells, which would exert multifactorial stress to industrial hyperproducers and perturb both cellular metabolism and product formation. Although adaptive laboratory evolution (ALE) has been wildly used to improve stress tolerance of microbial cell factories, single-factor stress condition (i.e. target product or sodium chloride at a high concentration) is currently provided. In order to enhance bacterial stress tolerance to actual industrial production conditions, ALE in late-stage fermentation broth is desired. Genome replication engineering assisted continuous evolution (GREACE) employs mutants of the proofreading element of DNA polymerase complex (DnaQ) to facilitate mutagenesis. Application of GREACE coupled-with selection under stress conditions is expected to accelerate the ALE process. RESULTS: In this study, GREACE was first modified by expressing a DnaQ mutant KR5-2 using an arabinose inducible promoter on a temperature-sensitive plasmid, which resulted in timed mutagenesis introduction. Using this method, tolerance of a lysine hyperproducer E. coli MU-1 was improved by enriching mutants in a lysine endpoint fermentation broth. Afterwards, the KR5-2 expressing plasmid was cured to stabilize acquired genotypes. By subsequent fermentation evaluation, a mutant RS3 with significantly improved lysine production capacity was selected. The final titer, yield and total amount of lysine produced by RS3 in a 5-L batch fermentation reached 155.0 ± 1.4 g/L, 0.59 ± 0.02 g lysine/g glucose, and 605.6 ± 23.5 g, with improvements of 14.8%, 9.3%, and 16.7%, respectively. Further metabolomics and genomics analyses, coupled with molecular biology studies revealed that mutations SpeBA302V, AtpBS165N and SecYM145V mainly contributed both to improved cell integrity under stress conditions and enhanced metabolic flux into lysine synthesis. CONCLUSIONS: Our present study indicates that improving a lysine hyperproducer by GREACE-assisted ALE in its stressful living environment is efficient and effective. Accordingly, this is a promising method for improving other valuable chemical hyperproducers.

Simultaneously improving the activity and thermostability of a new proline 4-hydroxylase by loop grafting and site-directed mutagenesis.

trans-Proline 4-hydroxylases (trans-P4Hs) hydroxylate free L-proline to trans-4-hydroxy-L-proline (trans-4-Hyp) is a valuable chiral synthon for important pharmaceuticals such as carbapenem antibiotics. However, merely few microbial trans-P4Hs have been identified, and trans-4-Hyp fermentations using engineered Escherichia coli strains expressing trans-P4Hs are usually performed at temperatures below 37 °C, which is likely due to poor stability and low activities. In the present study, a new trans-P4H from uncultured bacterium esnapd13 (UbP4H) with potential in the fermentative production of trans-4-Hyp at 37 °C was reported. In order to enhance the activity and thermostability of UbP4H, the replacement of its putative "lid" loop in combination with site-directed mutagenesis was performed. Consequently, four loop hybrids were designed by substituting a loop of UbP4H (A162-K178) with the corresponding sequences of four other known trans-P4Hs, respectively. Among them, UbP4H-Da exhibited a doubled activity when compared to the wild type (81.6 ± 1.9 vs. 40.4 ± 4.6 U/mg) but with reduced thermostability (t1/2, 11 vs. 47 min). Meanwhile, 10 single variants were designed through sequence alignments and folding free energy calculations. Three best point substitutions were respectively combined with UbP4H-Da, resulting in UbP4H-Da-R90G, UbP4H-Da-E112P, and UbP4H-Da-A260P. UbP4H-Da-E112P exhibited a 1.8-fold higher activity (85.2 ± 0.6 vs. 46.6 ± 4.0 U/mg), a 7.6-fold increase in t1/2 (359 vs. 47 min), and a 3 °C rise in Tm (46 vs. 43 °C) when compared to UbP4H. The fed-batch fermentations of trans-4-Hyp at 37 °C using trans-4-Hyp producing chassis cells expressing UbP4H or its variants were evaluated, and a 3.3-fold increase in trans-4-Hyp titer was obtained for UbP4H-Da-E112P (12.9 ± 0.1 vs. 3.9 ± 0.0 g/L for UbP4H). These results demonstrate the potential application of UbP4H-Da-E112P in the industrial production of trans-4-Hyp.

Growth-coupled evolution of phosphoketolase to improve L-glutamate production by Corynebacterium glutamicum.

The introduction of the key non-oxidative glycolytic (NOG) pathway enzyme, phosphoketolases (PKTs), into heterologous hosts can improve the yield of a variety of acetyl CoA-derived products of interest. However, the low specific activity of existing PKTs compared with that of 6-phosphofructokinase (PFK), the key EMP pathway enzyme, largely limits their potential applications. To improve PKT activity, previous attempts have focused on increasing intracellular PKT concentration via the use of strong promoters. Herein, we report the establishment of a growth-coupled evolution strategy for the enrichment and selection of PKT mutants with improved specific activity in Corynebacterium glutamicum hosts with defective PFK. Five mutants from 9 Bifidobacterium adolescentis-source PKT (BA-PKT) mutant libraries were obtained. Site-directed mutagenesis analysis revealed 11 mutant sites which contributed to improved BA-PKT specific activity. Further structural analysis revealed that the mutant sites were located far away from the enzyme active site, which makes them almost unpredictable using a rational design approach. Mutant site recombination led to the construction of a novel mutant, PKTT2A/I6T/H260Y, with Vmax 29.77 ± 1.58 U/mg and Kcat/Km 0.32 ± 0.01 s-1/mM, which corresponds to 73.27 ± 3.25% and 80.16 ± 3.38% improvements, respectively, compared with the wildtype (Vmax; 17.17 ± 0.59 U/mg, Kcat/Km; 0.17 ± 0.01 s-1/mM). Expression of PKTT2A/I6T/H260 in C. glutamicum Z188 resulted in 16.67 ± 2.24% and 18.19 ± 0.53% improvement in L-glutamate titer and yield, respectively, compared with the wildtype BA-PKT. Our findings provide an efficient approach for improving the activity of PKTs. Furthermore, the novel mutants could serve as useful tools in improving the yield of L-glutamate and other acetyl CoA-associated products.

Enhancing thermostability and removing hemin inhibition of Rhodopseudomonas palustris 5-aminolevulinic acid synthase by computer-aided rational design.

OBJECTIVE: To enhance the thermostability and deregulate the hemin inhibition of 5-aminolevulinic acid (ALA) synthase from Rhodopseudomonas palustris (RP-ALAS) by a computer-aided rational design strategy. RESULTS: Eighteen RP-ALAS single variants were rationally designed and screened by measuring their residual activities upon heating. Among them, H29R and H15K exhibited a 2.3 °C and 6.0 °C higher melting temperature than wild-type, respectively. A 6.7-fold and 10.3-fold increase in specific activity after 1 h incubation at 37 °C was obtained for H29R (2.0 U/mg) and H15K (3.1 U/mg) compared to wild-type (0.3 U/mg). Additionally, higher residual activities in the presence of hemin were obtained for H29R and H15K (e.g., 64% and 76% at 10 µM hemin vs. 27% for wild-type). The ALA titer was increased by 6% and 22% in fermentation using Corynebacterium glutamicum ATCC 13032 expressing H29R and H15K, respectively. CONCLUSION: H29R and H15K showed high thermostability, reduced hemin inhibition and slightly high activity, indicating that these two variants are good candidates for bioproduction of ALA.

Metabolic engineering of Corynebacterium glutamicum by synthetic small regulatory RNAs.

Corynebacterium glutamicum is an important platform strain that is wildly used in industrial production of amino acids and various other biochemicals. However, due to good genomic stability, C. glutamicum is more difficult to engineer than genetically tractable hosts. Herein, a synthetic small regulatory RNA (sRNA)-based gene knockdown strategy was developed for C. glutamicum. The RNA chaperone Hfq from Escherichia coli and a rationally designed sRNA consisting of the E. coli MicC scaffold and a target binding site were proven to be indispensable for repressing green fluorescent protein expression in C. glutamicum. The synthetic sRNA system was applied to improve glutamate production through knockdown of pyk, ldhA, and odhA, resulting almost a threefold increase in glutamate titer and yield. Gene transcription and enzyme activity were down-regulated by up to 80%. The synthetic sRNA system developed holds promise to accelerate C. glutamicum metabolic engineering for producing valuable chemicals and fuels.

Engineering Artificial Fusion Proteins for Enhanced Methanol Bioconversion.

Methanol is a low-cost and abundantly available feedstock derived from natural gas and syngas. Although bioconversion holds promise for producing desired chemicals from methanol under economically viable operating conditions, the efficiency is limited by unfavorable kinetics of methanol oxidation and assimilation. Herein, artificial fusion proteins were engineered to enhance methanol bioconversion. Nicotinamide adenine dinucleotide (NAD)-dependent methanol dehydrogenase (Mdh), 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloisomerase (Phi) from different sources were first screened for catalytic activity. Next, we designed six fusion proteins using the best enzyme candidates and flexible linkers. Fusing Mdh with Hps or Hps-Phi increased the Vmax of methanol oxidation up to 5.8-fold, and enhanced methanol conversion to fructose-6-phosphate up to 1.3-fold. Interestingly, fusion engineering changed the polymerization states of proteins and produced larger multimers, which may be responsible for the changed catalytic characteristics. This fusion engineering approach can be coupled with other metabolic engineering strategies for enhanced methanol bioconversion to valuable chemicals.

Engineering Corynebacterium glutamicum for methanol-dependent growth and glutamate production.

Methanol is a promising feedstock for bioproduction of fuels and chemicals, thus massive efforts have been devoted to engineering non-native methylotrophic platform microorganisms to utilize methanol. Herein, we rationally designed and experimentally engineered the industrial workhorse Corynebacterium glutamicum to serve as a methanol-dependent synthetic methylotroph. The cell growth of the methanol-dependent strain relies on co-utilization of methanol and xylose, and most notably methanol is an indispensable carbon source. Due to the methanol-dependent characteristic, adaptive laboratory evolution was successfully applied to improving methanol utilization. The evolved mutant showed a 20-fold increase in cell growth on methanol-xylose minimal medium and utilized methanol and xylose with a high mole ratio of 3.83:1. 13C-labeling experiments demonstrated that the carbon derived from methanol was assimilated into intracellular building blocks, high-energy carriers, cofactors, and biomass (up to 63% 13C-labeling). By inhibiting cell wall biosynthesis, methanol-dependent glutamate production was also achieved, demonstrating the potential application in bioconversion of methanol into useful chemicals. Genetic mutations detected in the evolved strains indicate the importance of intracellular NAD+/NADH ratio, substrate uptake, and methanol tolerance on methanol utilization. This study reports significant improvement in the area of developing fully synthetic methylotrophs.

MACBETH: Multiplex automated Corynebacterium glutamicum base editing method.

CRISPR/Cas9 or Cpf1-introduced double strand break dramatically decreases bacterial cell survival rate, which hampers multiplex genome editing in bacteria. In addition, the requirement of a foreign DNA template for each target locus is labor demanding and may encounter more GMO related regulatory hurdle in industrial applications. Herein, we developed a multiplex automated Corynebacterium glutamicum base editing method (MACBETH) using CRISPR/Cas9 and activation-induced cytidine deaminase (AID), without foreign DNA templates, achieving single-, double-, and triple-locus editing with efficiencies up to 100%, 87.2% and 23.3%, respectively. In addition, MACBETH was applied to generate a combinatorial gene inactivation library for improving glutamate production, and pyk&ldhA double inactivation strain was found to improve glutamate production by 3-fold. Finally, MACBETH was automated with an integrated robotic system, which would enable us to generate thousands of rationally engineered strains per month for metabolic engineering of C. glutamicum. As a proof of concept demonstration, the automation platform was used to construct an arrayed genome-scale gene inactivation library of 94 transcription factors with 100% success rate. Therefore, MACBETH would be a powerful tool for multiplex and automated bacterial genome editing in future studies and industrial applications.

A Novel Corynebacterium glutamicum l-Glutamate Exporter.

Besides metabolic pathways and regulatory networks, transport systems are also pivotal for cellular metabolism and hyperproduction of biochemicals using microbial cell factories. The identification and characterization of transporters are therefore of great significance for the understanding and engineering of transport reactions. Herein, a novel l-glutamate exporter, MscCG2, which exists extensively in Corynebacterium glutamicum strains but is distinct from the only known l-glutamate exporter, MscCG, was discovered in an industrial l-glutamate-producing C. glutamicum strain. MscCG2 was predicted to possess three transmembrane helices in the N-terminal region and located in the cytoplasmic membrane, which are typical structural characteristics of the mechanosensitive channel of small conductance. MscCG2 has a low amino acid sequence identity (23%) to MscCG and evolved separately from MscCG with four transmembrane helices. Despite the considerable differences between MscCG2 and MscCG in sequence and structure, gene deletion and complementation confirmed that MscCG2 also functioned as an l-glutamate exporter and an osmotic safety valve in C. glutamicum Besides, transcriptional analysis showed that MscCG2 and MscCG genes were transcribed in similar patterns and not induced by l-glutamate-producing conditions. It was also demonstrated that MscCG2-mediated l-glutamate excretion was activated by biotin limitation or penicillin treatment and that constitutive l-glutamate excretion was triggered by a gain-of-function mutation of MscCG2 (A151V). Discovery of MscCG2 will enrich the understanding of bacterial amino acid transport and provide additional targets for exporter engineering.IMPORTANCE The exchange of matter, energy, and information with surroundings is fundamental for cellular metabolism. Therefore, studying transport systems that are essential for these processes is of great significance. Besides, transport systems of bacterial cells are usually related to product excretion as well as product reuptake, making transporter engineering a useful strategy for strain improvement. The significance of our research is in identifying and characterizing a novel l-glutamate exporter from the industrial workhorse Corynebacterium glutamicum, which will enrich the understanding of l-glutamate excretion and provide a new target for studying bacterial amino acid transport and engineering transport reactions.

Mutations in Peptidoglycan Synthesis Gene ponA Improve Electrotransformation Efficiency of Corynebacterium glutamicum ATCC 13869.

Corynebacterium glutamicum is frequently engineered to serve as a versatile platform and model microorganism. However, due to its complex cell wall structure, transformation of C. glutamicum with exogenous DNA is inefficient. Although efforts have been devoted to improve the transformation efficiency by using cell wall-weakening agents, direct genetic engineering of cell wall synthesis for enhancing cell competency has not been explored thus far. Herein, we reported that engineering of peptidoglycan synthesis could significantly increase the transformation efficiency of C. glutamicum Comparative analysis of C. glutamicum wild-type strain ATCC 13869 and a mutant with high electrotransformation efficiency revealed nine mutations in eight cell wall synthesis-related genes. Among them, the Y489C mutation in bifunctional peptidoglycan glycosyltransferase/peptidoglycan dd-transpeptidase PonA dramatically increased the electrotransformation of strain ATCC 13869 by 19.25-fold in the absence of cell wall-weakening agents, with no inhibition on growth. The Y489C mutation had no effect on the membrane localization of PonA but affected the peptidoglycan structure. Deletion of the ponA gene led to more dramatic changes to the peptidoglycan structure but only increased the electrotransformation by 4.89-fold, suggesting that appropriate inhibition of cell wall synthesis benefited electrotransformation more. Finally, we demonstrated that the PonAY489C mutation did not cause constitutive or enhanced glutamate excretion, making its permanent existence in C. glutamicum ATCC 13869 acceptable. This study demonstrates that genetic engineering of genes involved in cell wall synthesis, especially peptidoglycan synthesis, is a promising strategy to improve the electrotransformation efficiency of C. glutamicumIMPORTANCE Metabolic engineering and synthetic biology are now the key enabling technologies for manipulating microorganisms to suit the practical outcomes desired by humankind. The introduction of exogenous DNA into cells is an indispensable step for this purpose. However, some microorganisms, including the important industrial workhorse Corynebacterium glutamicum, possess a complex cell wall structure to shield cells against exogenous DNA. Although genes responsible for cell wall synthesis in C. glutamicum are known, engineering of related genes to improve cell competency has not been explored yet. In this study, we demonstrate that mutations in cell wall synthesis genes can significantly improve the electrotransformation efficiency of C. glutamicum Notably, the Y489C mutation in bifunctional peptidoglycan glycosyltransferase/peptidoglycan dd-transpeptidase PonA increased electrotransformation efficiency by 19.25-fold by affecting peptidoglycan synthesis.