Microbial Cell Factories

3,4-Dihydroxybenzoate (protocatechuate, PCA) is a phenolic compound naturally found in edible vegetables and medicinal herbs. PCA is of interest in the chemical industry as a building block for novel polymers and has wide potential for pharmaceutical applications due to its antioxidant, anti-inflammatory, and antiviral properties. In the present study, we designed and constructed a novel Corynebacterium glutamicum strain to enable the efficient utilization of O_SCPLOWDC_SCPLOW-xylose for microbial production of PCA. The engineered strain showed a maximum PCA titer of 62.1 {+/-} 12.1 mM (9.6 {+/-} 1.9 g L-1) from O_SCPLOWDC_SCPLOW-xylose as the primary carbon and energy source. The corresponding yield was [Formula], which corresponds to 38 % of the maximum theoretical yield and is 14-fold higher compared to the parental producer strain on O_SCPLOWDC_SCPLOW-glucose. By establishing a one-pot bioreactor cultivation process followed by subsequent process optimization, the same maximum titer and a total amount of 16.5 {+/-} 1.1 g was reached. Downstream processing of PCA from this fermentation broth was realized via electrochemically induced crystallization by taking advantage of the pH-dependent properties of PCA. Since PCA turned out to be electrochemically unstable in combination with several anode materials, a threechamber electrolysis setup was established to crystallize PCA and to avoid direct anode contact. This resulted in a maximum final purity of 95.4 %. In summary, the established PCA production process represents a highly sustainable approach, which will serve as a blueprint for the bio-based production of other hydroxybenzoic acids from alternative sugar feedstocks.

Eubacterium limosum is a Clostridial acetogen that efficiently utilizes a wide range of single-carbon substrates and contributes to metabolism of health-associated compounds in the human gut microbiota. These traits have led to interest in developing it as a platform for sustainable CO2-based biofuel production to combat carbon emissions, and for exploring the importance of the microbiota in human health. However, synthetic biology and metabolic engineering in E. limosum have been hindered by the inability to rapidly make precise genomic modifications. Here, we screened a diverse library of recombinase proteins to develop a highly efficient oligonucleotide-based recombineering system based on the viral recombinase RecT. Following optimization, the system is capable of catalyzing ssDNA recombination at an efficiency of up to 2%. Addition of a Cas9 counterselection system allows recombination to reach an efficiency of up to 100%, enabling creation of genomic point mutations in a scarless and markerless manner. We deployed this system to create a clean knockout of the extracellular polymeric substance (EPS) gene cluster, generating a strain incapable of biofilm formation. This approach is rapid and simple, not requiring laborious homology arm cloning, and can readily be retargeted to almost any genomic locus. This work overcomes a major bottleneck Eubacterium limosum genetic engineering by enabling precise genomic modifications, and provides both a roadmap and associated recombinase plasmid library for developing similar systems in other Clostridia of interest. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/588731v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1366103org.highwire.dtl.DTLVardef@11b1f0borg.highwire.dtl.DTLVardef@1931600org.highwire.dtl.DTLVardef@1896e4c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Combinatorial DNA design and assembly is an efficient and pragmatic way to obtain high-performing metabolic pathway designs quickly. However, implementation may require organism-specific technical barriers to be overcome. Firstly, suitable expression control parts such as promoters and ribosome-binding sites (RBSs) which provide a suitable range of expression levels need to be identified or developed. Secondly, these need to be assembled into pathway-encoding combinatorial libraries of sufficient size, quality and diversity. For organisms with transformation frequencies too low to allow direct transformation of library assembly reactions, such as many Clostridium spp., assembly and amplification is typically carried out using Escherichia coli. However, if constructs are deleterious (or burdensome) to E. coli, which is often the case when using Clostridium genetic parts, poor libraries may be obtained. Here we develop a new approach called integration-coupled activation of promoterless sequences (ICAPS) to overcome this barrier and therefore enable combinatorial assembly in Clostridium. Libraries were designed and assembled as promoterless synthetic operons, preventing expression during DNA assembly, and expression was only activated later, when constructs were integrated into the host genome downstream of a promoter. Variation of expression levels was achieved using a range of context-resistant RBS sequences. This approach was used to produce a Clostridium acetobutylicum library with combinatorial expression variants of an introduced hexanol pathway. This proof of concept provides a generally-applicable approach to implement combinatorial metabolic pathway-encoding libraries in Clostridium spp., circumventing the excessive strength of Clostridium expression control parts in E. coli, and is applicable to other organisms.

BackgroundAmidst the escalating carbon dioxide levels resulting from fossil fuel consumption, there is a pressing need for sustainable, bio-based alternatives to underpin future global economies. Single carbon feedstocks, derived from CO2, represent promising substrates for biotechnological applications. Especially methanol is gaining prominence for bio-production of commodity chemicals. ResultsIn this study, we show the potential of Komagataella phaffii as a production platform for itaconic acid using methanol as the carbon source. Successful integration of heterologous genes from Aspergillus terreus (cadA, mttA and mfsA) alongside fine-tuning of the mfsA gene expression, led to promising initial itaconic acid titers of 28 g{middle dot}L-1 after five days of fed-batch cultivation. Through the combined efforts of process optimization and strain engineering strategies we further boosted the itaconic acid production reaching titers of 55 g{middle dot}L-1 after less than five days of methanol feed, whilst increasing the product yield on methanol from 0.06 g{middle dot}g- 1 to 0.24 g{middle dot}g-1. ConclusionOur results highlight the potential of K. phaffii as a methanol-based platform organism for sustainable biochemical production.

BackgroundOxygen tolerant complex metal-dependent formate dehydrogenases hold potential for biotechnological applications. Principle FindingsIn this work, we report the functional expression of the complex, molybdenum-dependent soluble formate dehydrogenase encoded by the fdsGBACD operon from Cupriavidus necator (CnFDH) in Escherichia coli. Expression of the operon from plasmids or from a copy integrated in the chromosome enabled growth of an energy-auxotrophic selection strain on formate as sole energy source under aerobic conditions. Growth could be accelerated in turbidostat, leading to a drop of the generation time of 1 hour. While no mutation was found in the operon of evolved isolates, genome sequencing revealed non-synonymous point mutations in the gene focA coding for a bidirectional formate transporter carried in all isolates sequenced. Reverting the mutations led to a drop in the growth rate demonstrating the focA mutation as principle target of continuous culture adaptation. SignificanceA member of the oxygen-tolerant subclass of complex FDH showed stable formate oxidation activity when expressed in the heterologous host E. coli, a model organism of biotechnology. The integration of the operon in the chromosome offers the possibility of structure/function studies and activity enhancements through in vivo mutagenesis, which can also be applied to CO2 reduction in appropriate selection hosts.

The efficient production of food and biochemicals using microorganisms that utilize single-carbon feedstocks presents a promising approach for advancing a circular bioeconomy. Komagataella phaffii (formerly Pichia pastoris) is a methylotrophic yeast already widely used in industry, making it an attractive host for such applications. Recently, K. phaffii was converted into an autotrophic strain capable of assimilating CO2 into both biomass and secreted organic acids, using energy derived from dissimilation of methanol to CO2. In these strains, methanol oxidation is catalysed by an alcohol oxidase (Aox2), which transfers electrons to oxygen without conserving reducing equivalents. To address this limitation, in this study we explored redirecting methanol dissimilation through the native alcohol dehydrogenase (Adh2), coupling methanol oxidation with NADH generation to improve carbon efficiency. By deleting AOX2 and overexpressing ADH2, we generated Adh2-based autotrophic strains that exhibited growth rates comparable to the parental strain (0.007 h-{superscript 1}), while reducing specific CO2 production by 53% and increasing biomass yield (YX/MeOH) by 59%. We further applied this strategy to convert previously developed autotrophic strains producing itaconic acid and lactic acid into Adh2-dependent strains. Optimizing ADH2 expression through multicopy integration resulted in strains with approximately two-fold higher molar carbon efficiency (Y(X+P)/CO2) while achieving elevated product titers--2.2-fold for itaconic acid and 3.8-fold for lactic acid--relative to the parental strains. Our findings demonstrate that alcohol dehydrogenase-mediated methanol dissimilation can significantly improve yield and productivity of autotrophic K. phaffii strains, with broad implications for sustainable bioproduction from one-carbon substrates.

BackgroundThe compound 1,2-propanediol is an important industrial bulk chemical that has proven particularly recalcitrant to bio-production. Solvent-producing Clostridium species represent promising candidates for engineering 1,2-propaediol production. Co-production of 1,2-popanediol and butanol has the potential to improve the economics of the acetone-butanol-ethanol (ABE) fermentation. ResultsIn this study, the methylglyoxal synthase gene (mgsA) from Clostridium beijerinckii NCIMB 8052 was homologously expressed in this organism. Additionally, a separate strain of Clostridium beijerinckii NCIMB 8052 was engineered by cloning and expressing mgsA and methylglyoxal/glyoxal reductase (mgR) from Clostridium pasteurianum ATCC 6013 as a fused protein linked by polyglycine linker in the former. Both strains of C. beijerinckii NCIMB 8052 failed to produce 1,2-propaneol. Instead, traces of acetol--the precursor of 1,2-propanediol--were detected in cultures of both strains. When the recombinant strains were exposed to acetol, both strains exhibited [~]100% acetol-to-1,2-propanediol conversion efficiency. Conversely, methylglyoxal supplementation led to the production of traces of acetol but not lactaldehyde or 1,2-propanediol. When wildtype C. beijerinckii NCIMB 8052, C. pasteurianum ATCC 6013 and Clostridium tyrobutyricum ATCC 25755 were challenged with methylglyoxal, C. beijerinckii produced [~]0.1 g/L (S)-(+)-1,2-Propanediol, while C. tyrobutyricum produced traces of lactate. C. pasteurianum produced neither 1,2-propanediol nor lactate. The wild types of all three species above exhibited [~]100% acetol-to-1,2-propanediol conversion efficiency. The recombinant strain of C. beijerinckii expressing fused MgsA and MgR from C. pasteurianum ATCC 6013 showed enhanced growth and solvent production, producing as high as 88% more butanol on both glucose and lactose than the control strain and the recombinant strain of the same organism expressing the native MgsA. ConclusionsRecombinant and native strains of C. beijerinckii, C. pasteurianum and C. tyrobutyricum studied in this work exhibit extremely poor capacity to catalyze the conversion of the intermediates of the methylglyoxal bypass to 1,2-propanediol. This is indicative of lack of appropriate enzymes to catalyze the reactions from methylglyoxal to acetol or lactaldehyde. Inability to detect methylglyoxal in the recombinant strains harboring mgsA (both homologous and heterologous)-- whereas the strain expressing both mgsA and mgR from C. pasteurianum, under the same promoter (Padc) produced higher concentrations of butanol--suggests that C. beijerinckii might possess a regulatory mechanism that limits the activity of methylglyoxal-producing MgsA. The protein product of mgR from C. pasteurianum represents a promising metabolic engineering candidate towards increasing butanol production.

The tetracenomycins are aromatic anticancer polyketides that inhibit peptide translation via binding to the large ribosomal subunit. Here, we expressed the elloramycin biosynthetic gene cluster in the heterologous host Streptomyces coelicolor M1146 to facilitate the downstream production of tetracenomycin analogs. We developed a BioBricks(R) genetic toolbox of genetic parts for substrate precursor engineering in S. coelicolor M1146::cos16F4iE. We cloned a series of integrating vectors based on the VWB, TG1, and SV1 integrase systems to interrogate gene expression in the chromosome. We genetically engineered three separate genetic constructs to modulate tetracenomycin biosynthesis: 1) the vhb hemoglobin from obligate aerobe Vitreoscilla stercoraria to improve oxygen utilization; (2) the accA2BE acetyl-CoA carboxylase to enhance condensation of malonyl-CoA; (3) lastly, the sco6196 acyltransferase, which is a "metabolic regulatory switch" responsible for mobilizing triacylglycerols to {beta}-oxidation machinery for acetyl-CoA. In addition, we engineered the tcmO 8-O-methyltransferase and newly identified tcmD 12-O-methyltransferase from Amycolatopsis sp. A23 to generate tetracenomycins C and X. We also co-expressed the tcmO methyltransferase with oxygenase urdE to generate the analog 6-hydroxy-tetracenomycin C. Altogether, this system is compatible with the BioBricks(R) [RFC 10] cloning standard for the co-expression of multiple gene sets for metabolic engineering of Streptomyces coelicolor M1146::cos16F4iE.

Low efficiency of DNA integration into the chromosome limits multiplexed bacterial genetic engineering. We address this by establishing multiplexed Serine recombinase-Assisted Genome Engineering (mSAGE), enabling simultaneous, site-specific insertion of three DNAs into the chromosome of Pseudomonas putida KT2440. We construct a combinatorial library of isophthalate catabolism and transport genes through a single, pooled transformation of three gene libraries, rapidly identifying the best combination of genes for biodegradation of this plastic comonomer.

Peptide drugs have seen rapid advancement in biopharmaceutical development, with over 80 candidates approved globally. Despite their therapeutic potential, the clinical translation of peptide drugs is hampered by challenges in production yields and stability. Engineered bacterial therapeutics is a unique approach being explored to overcome these issues by using bacteria to produce and deliver therapeutic compounds at the body site of use. A key advantage of this technology is the possibility to control drug delivery within the body in real time using genetic switches. However, the performance of such genetic switches suffers when used to control drugs that require post-translational modifications or are toxic to the host. In this study, these challenges were experienced when attempting to establish a thermal switch for the production of a ribosomally synthesized and post-translationally modified peptide antibiotic, darobactin, in probiotic E. coli. These challenges were overcome by developing a thermo-amplifier circuit that combined the thermal-switch with a T7 RNA Polymerase and its promoter that overcame limitations imposed by the host transcriptional machinery due to its orthogonality to it. This circuit enabled production of pathogen-inhibitory levels of darobactin at 40{degrees}C while maintaining leakiness below the detection limit at 37{degrees}C. More impressively, the thermo-amplifier circuit sustained production beyond the thermal induction duration. Thus, raised temperature for 2 h was sufficient for the bacteria to produce pathogen-inhibitory levels of darobactin even in the physiologically relevant simulated conditions of the intestines that include bile salts and low nutrient levels. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/579303v2_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1cdb311org.highwire.dtl.DTLVardef@1a7523aorg.highwire.dtl.DTLVardef@77b68org.highwire.dtl.DTLVardef@136f52b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Production of secretory protein in Gram-negative bacteria simplifies downstream processing in recombinant protein production, accelerates protein engineering, and advances synthetic biology. Signal peptides and secretory carrier proteins are commonly used to effect the secretion of heterologous recombinant protein in Gram-negative bacteria. The Escherichia coli osmotically-inducible protein Y (OsmY) is a carrier protein that secretes a target protein extracellularly, and we have successfully applied it in the Bacterial Extracellular Protein Secretion System (BENNY) to accelerate the directed evolution workflow. In this study, we applied directed evolution on OsmY to enhance its total secretory protein production. After just one round of directed evolution followed by combining the mutations found, OsmY(M3) (L6P, V43A, S154R, V191E) was identified as the best carrier protein. OsmY(M3) produced 3.1 {+/-} 0.3 fold and 2.9 {+/-} 0.8 fold more secretory Tfu0937 {beta}-glucosidase than its wildtype counterpart in E. coli strains BL21(DE3) and C41(DE3), respectively. OsmY(M3) also produced more secretory Tfu0937 at different cultivation temperatures (37 {degrees}C, 30 {degrees}C and 25 {degrees}C). Subcellular fractionation of the expressed protein confirmed the essential role of OsmY in protein secretion. Up to 80.8 {+/-} 12.2% of total soluble protein was secreted after 15 h of cultivation. When fused to a red fluorescent protein or a lipase from Bacillus subtillis, OsmY(M3) also produced more secretory protein compared to the wildtype. This is the first report of applying directed evolution on a carrier protein to enhance total secretory protein production. The methodology can be further extended to evolve other signal peptides or carrier proteins for secretory protein production in E. coli and other bacteria. In this study, OsmY(M3) improved the production of three proteins, originating from diverse organisms and with diverse properties, in secreted form, clearly demonstrating its wide-ranging applications.

Synthetic microbial cocultures, which combine the unique capabilities of multiple microbes into one process, have significant potential for sustainable production of fuels and chemicals. Most studies of defined cocultures have tested relatively low cell densities in lab-scale batch cultures, not the high cell density fed-batch or continuous processes with cell retention typically required to achieve industrially-relevant volumetric productivities. Here, we explore the impact of increased cell density on isopropanol production from the syntrophic coculture of genetically-modified Clostridium acetobutylicum [CACas9 {Delta}hbd (p95ace02_atoB), with deleted 4-C metabolism expressing an acetone-formation pathway on the plasmid] with WT Clostridium ljungdahlii using first a pseudo-perfusion approach followed by perfusion culture. CACas9 {Delta}hbd (p95ace02_atoB) produces acetone without any 4-C metabolites and C. ljungdahlii converts that acetone to isopropanol. To explore the mechanism by which these cultures enable supratheoretical isopropanol yields, we first identified NADH-driven hydrogen conversion in CACas9 {Delta}hbd (p95ace02_atoB) as the thermodynamically-limiting step for acetone and thus isopropanol production. We then demonstrated the ability of C. ljungdahlii to mitigate this issue by eliminating detectable hydrogen accumulation in the coculture. Pseudo-perfusion cocultures showed that high cell densities combined with a high population fraction of C. ljungdahlii enable dramatic increases in isopropanol yields beyond the thermodynamic limitation imposed in CACas9 {Delta}hbd (p95ace02_atoB) monocultures. Finally, we demonstrate carbon-negative fermentation of glucose to isopropanol as the sole alcohol product in a perfusion bioreactor.

AimsThis study aims to optimize and scale up the production of the chimeric antigen EIT through E. coli fed-batch fermentations. The approach seeks for simplicity and cost-effectiveness, considering EIT as a potential vaccine candidate against EHEC for cattle. Its ability to induce a humoral immune response has been recently verified in bovines in a proof-of-concept study. Methods and ResultsAn initial screening was conducted to select the optimal medium for EIT expression, using lactose for recombinant protein induction. M9 Minimal medium supplemented with yeast extract yielded the highest relative levels of EIT, as determined by Western blot analysis. E. coli cultures were subsequently grown in a stirred-tank bioreactor, and both biomass production and EIT expression were monitored. The process was reproducible across three independent fermentations, with all parameters being improved compared with shake-flask cultivations. Antigen recovery was achieved through thermal permeabilization, as the construction includes a periplasmic signal sequence. Overall, the process resulted in an average potential output of 350 doses per liter of fermented culture, while preserving the EIT antigenic capability, as confirmed by ELISA assays. ConclusionThe production of the recombinant EIT antigen was successfully scaled up using a stirred-tank bioreactor through a quite simple and cost-effective approach, achieving increased yields for supporting further studies and interventions. Impact StatementCattle are the major reservoir and source of dissemination of enterohemorrhagic E. coli (EHEC), a human pathogen responsible for outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) worldwide. A scalable and cost-effective preharvest vaccine for cattle could help with developing strategies aimed at reducing bacterial carriage and thus, the impact of this zoonosis.

Engineering the ethanologenic Gram-negative bacterium Z. mobilis for the secretion of hydrolytic enzymes is a key step towards establishing a biofuel cell factory that uses complex waste material as feedstock. Secretion strategies in Z. mobilis have exclusively relied on signal peptides, which limit protein transport to the periplasm. To achieve single-step secretion across the Z. mobilis double-layered membrane, we sought to identify a native Type I Secretion System (T1SS) tag for fusion to proteins of interest. While a T1SS operon had been identified in the Z. mobilis genome, its native cargo had remained unknown and the use of T1SS secretion tags had so far been unexplored. Here, bioinformatic analysis identified the Major Intrinsic Protein (MIP) as a putative T1SS cargo, and its role validated through fusion of C-terminal sequences of two lengths (61 and 141 amino acids) to a heterologous {beta}-galactosidase from Bacteroides thetaiotaomicron, expressed in Z. mobilis. The 141 amino acid tag, including two RTX domains, resulted in significantly higher secretion efficiency than the 61 amino acid tag lacking RTX repeats, consistent with the established role of RTX domains in preventing premature cytoplasmic folding, thus improving secretion. As extracellular secretion of hydrolytic enzymes has remained a major bottleneck in the development of Z. mobilis as a sustainable cell factory, the identification of a native T1SS secretion tag directly addresses this limitation, introducing a novel tool for enzyme delivery.

Acetogens are promising microbes for sustainable biomanufacturing but improving acetogen gas fermentation requires efficient conversion of CO and CO2 into fuels and chemicals. Carbon monoxide dehydrogenase (CODH) enzymes couple carbon fixation to energy conservation in acetogens and serve as potential regulatory modules for tuning autotrophic metabolism. Intriguingly, the model-acetogen Clostridium autoethanogenum lost its unique truncation in the bifunctional CODH (acsA), essential for autotrophy, during autotrophic adaptive laboratory evolution while obtaining superior phenotypes. Additionally, protein expression of the monofunctional CODH cooS1 is high and conditionally-regulated in C. autoethanogenum. Here, we genetically engineered CODHs in C. autoethanogenum by replacing the stop codon in acsA with leucine (strain Leu_SNP) or serine (Ser_SNP), and deleting cooS1 ({Delta}cooS1). Phenotyping in autotrophic batch and chemostat cultures revealed altered growth profiles and significant redistribution of carbon and redox flows in SNP strains, whereas {Delta}cooS1 showed moderate and condition-dependent effects. Surprisingly, structural modelling identified no conformational differences between wild-type and mutant AcsA proteins. While transcriptomics showed limited transcriptional changes in {Delta}cooS1, it suggested potential transcriptional adjustments linked to reduced robustness and altered product profile of Leu_SNP. Our results demonstrate the impact of CODHs on autotrophy and offer targets for rational engineering of acetogen cell factories.

Bioelectrochemical systems (BES) are promising for energy, sensing, environmental, and synthesis applications. Escherichia coli were previously bioengineered for application in BES by introduction of extracellular electron transfer (EET) pathways. Inspired by the metal-reducing (Mtr) pathway of Shewanella oneidensis MR-1, several of its cytochromes were heterologously expressed in E. coli, leading to increased EET rates and successful application in BES. Besides direct electron transfer, S. oneidensis MR-1 is known to secrete flavins that act as redox mediators and are crucial for high EET rates. Here we co-express the Mtr pathway and a flavin biosynthesis pathway in E. coli, to enhance EET in engineered strains. The secretion of both flavin mononucleotide and riboflavin was increased up to 3-fold in engineered strains. Chronoamperometry revealed an up to ~3.4-fold increase in current over the wild type when co-expressing cytochromes and flavin biosynthesis genes, and a ~2.3-fold increase when expressing flavin biosynthesis genes on their own. Thus, the introduction of flavin biosynthesis genes yields in a distinct, yet complementary EET mechanism, and holds promise for application in BES.

With advancements in synthetic biology and metabolic engineering, microorganisms can now be engineered to perform increasingly complex functions, which may be limited by the resources available in individual cells. Division of labour in synthetic microbial communities offers a promising approach to enhance metabolic efficiency and resilience in bioproduction. By distributing complex metabolic pathways across multiple subpopulations, the resource competition and metabolic burden imposed on an individual cell is reduced, potentially enabling more efficient production of target compounds. Violacein is a high-value pigment with anti-tumour properties that exemplifies such a challenge due to its complex bioproduction pathway, imposing a significant metabolic burden on host cells. In this study we investigated the benefits of division of labour for violacein production by splitting the violacein bioproduction pathway between two subpopulations of Escherichia coli based synthetic communities. We tested several pathway splitting strategies and reported that splitting the pathway into two subpopulations expressing VioABE and VioDC at a final composition of 60:40 yields a 2.5 fold increase in violacein production as compared to a monoculture. We demonstrated that the coculture outperforms the monoculture when both subpopulations exhibit similar metabolic burden levels, resulting in comparable growth rates, and when both subpopulations are present in sufficiently high proportions.

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.

Extracellular electron transfer (EET) engineering in Escherichia coli holds great potential for bioremediation, energy and electrosynthesis applications fueled by readily available organic substrates. Due to its vast metabolic capabilities and availability of synthetic biology tools to adapt strains to specific applications, E. coli is of advantage over native exoelectrogens, but limited in electron transfer rates. We enhanced EET in engineered strains through systematic expression of electron transfer pathways differing in cytochrome composition, localization and origin. While a hybrid pathway harboring components of an E. coli nitrate reductase and the Mtr complex from the exoelectrogen Shewanella oneidensis MR-1 enhanced EET, the highest efficiency was achieved by implementing the complete Mtr pathway from S. oneidensis MR1 in E. coli. We show periplasmic electron shuttling through overexpression of a small tetraheme cytochrome to be central to the electroactivity of this strain, leading to enhanced degradation of the pollutant methyl orange and significantly increased electrical current to graphite electrodes.

Despite its industrial importance, microbial L-lysine production has largely been confined to classical producer strains, leaving the fast-growing, non-pathogenic marine microorganism V. natriegens largely untapped as an unconventional biosynthetic platform. In this work, we established an L-lysine-overproducing V. natriegens DSM759 strain through a step-wise, systematic rational engineering strategy targeting the native biosynthetic pathway. Guided by our prior systems-level analysis of the strains genetic and regulatory architecture, we identified key metabolic bottlenecks and implemented knowledge-driven interventions to relieve pathway constraints. Central to production was alleviation of feedback inhibition in the native key regulatory enzymes, aspartate kinase (AK, lysC) and dihydrodipicolinate synthase (DHDPS, dapA). Site-directed amino-acid substitutions, replicating established E. coli feedback-resistance mechanisms, were introduced into conserved regions of the V. natriegens DSM759 enzymes, producing L-lysine-insensitive variants with kinetic parameters comparable to that of corresponding wild type enzymes. Among the tested configurations, the strain co-expressing Vn.lysC2 and Vn.dapA1:E84T reached the highest L-lysine titer (9.0{+/-}0.6 mM) and yield (0.11{+/-}0.01 molLys molGlc-1), whereas overexpression of additional L-lysine pathway genes provided no further benefit. Leveraging the hosts metabolic versatility, L-lysine synthesis was also demonstrated from the chitin-derived amino-sugar N-acetylglucosamine (0.09{+/-}0.00 molLys molGlcNAc-1), highlighting the potential to valorize chitin-rich waste streams from the seafood industry. This work establishes a minimal, rational strategy for L-lysine biosynthesis in V. natriegens DSM759 and positions it as a promising platform for sustainable amino acid production.