Xiaoyuan Wang

Previously we have characterized a threonine dehydratase mutant TDF383V (encoded by ilvA1) and an acetohydroxy acid synthase mutant AHASP176S, D426E, L575W (encoded by ilvBN1) in Corynebacterium glutamicum IWJ001, one of the best l-isoleucine producing strains. Here, we further characterized an aspartate kinase mutant AKA279T (encoded by lysC1) and a homoserine dehydrogenase mutant HDG378S (encoded by hom1) in IWJ001, and analyzed the consequences of all these mutant enzymes on amino acids production in the wild type background. In vitro enzyme tests confirmed that AKA279T is completely resistant to feed-back inhibition by l-threonine and l-lysine, and that HDG378S is partially resistant to l-threonine with the half maximal inhibitory concentration between 12 and 14 mM. In C. glutamicum ATCC13869, expressing lysC1 alone led to exclusive l-lysine accumulation, co-expressing hom1 and thrB1 with lysC1 shifted partial carbon flux from l-lysine (decreased by 50.1 %) to l-threonine (4.85 g/L) with minor l-isoleucine and no l-homoserine accumulation, further co-expressing ilvA1 completely depleted l-threonine and strongly shifted carbon flux from l-lysine (decreased by 83.0 %) to l-isoleucine (3.53 g/L). The results demonstrated the strongly feed-back resistant TDF383V might be the main driving force for l-isoleucine over-synthesis in this case, and the partially feed-back resistant HDG378S might prevent the accumulation of toxic intermediates. Information exploited from such mutation-bred production strain would be useful for metabolic engineering.

Flagella assembly was investigated in the Escherichia coli W3110 wild‐type strain and ΔwaaF, ΔwaaC, and ΔwaaG mutant strains that only synthesize lipopolysaccharide with different lengths, using transmission electron microscopy and whole genome transcriptome profiling. Under the electron microscope, the flagella were observed on the cell surface of the W3110 strain but not the ΔwaaC, ΔwaaF, or ΔwaaG strains. Transcriptional profiling showed that 1382 genes in ΔwaaC, 526 genes in ΔwaaF, and 965genes in ΔwaaG were significantly regulated compared to the control W3110 strain. These genes were further analyzed by gene ontology and KEGG pathway. Although there were significant transcriptional differences among the ΔwaaC, ΔwaaF, and ΔwaaG strains, genes related to flagella assembly and bacterial chemotaxis (the linkage between the flagella and the environment) were significantly down‐regulated in all three strains. The data demonstrated that flagella assembly in E. coli depends on the length of lipopolysaccharide.

Corynebacterium crenatum SYPA 5 is the industrial strain for l-arginine production. Poly-β-hydroxybutyrate (PHB) is a kind of biopolymer stored as bacterial reserve materials for carbon and energy. The introduction of the PHB synthesis pathway into several strains can regulate the global metabolic pathway. In addition, both the pathways of PHB and l-arginine biosynthesis in the cells are NADPH-dependent. NAD kinase could upregulate the NADPH concentration in the bacteria. Thus, it is interesting to investigate how both PHB and NAD kinase affect the l-arginine biosynthesis in C. crenatum SYPA 5. C. crenatum P1 containing PHB synthesis pathway was constructed and cultivated in batch fermentation for 96 h. The enzyme activities of the key enzymes were enhanced comparing to the control strain C. crenatum SYPA 5. More PHB was found in C. crenatum P1, up to 12.7 % of the dry cell weight. Higher growth level and enhanced glucose consumptions were also observed in C. crenatum P1. With respect to the yield of l-arginine, it was 38.54 ± 0.81 g/L, increasing by 20.6 %, comparing to the control under the influence of PHB accumulation. For more NADPH supply, C. crenatum P2 was constructed with overexpression of NAD kinase based on C. crenatum P1. The NADPH concentration was increased in C. crenatum P2 comparing to the control. PHB content reached 15.7 % and 41.11 ± 1.21 g/L l-arginine was obtained in C. crenatum P2, increased by 28.6 %. The transcription levels of key l-arginine synthesis genes, argB, argC, argD and argJ in recombinant C. crenatum increased 1.9–3.0 times compared with the parent strain. Accumulation of PHB by introducing PHB synthesis pathway, together with up-regulation of coenzyme level by overexpressing NAD kinase, enables the recombinant C. crenatum to serve as high-efficiency cell factories in the long-time l-arginine fermentation. Furthermore, batch cultivation of the engineered C. crenatum revealed that it could accumulate both extracellular l-arginine and intracellular PHB simultaneously. All of these have a potential biotechnological application as a strategy for high-yield l-arginine.

Pseudomonas putida KT2442 is an important bacterium for producing various types of polyhydroxyalkanoate polymers. Phospholipids and lipid A in membranes of P. putida play important roles in stress responses, but detailed structural information of these lipids is not known. In this study, phospholipids and lipid A were isolated from P. putida KT2442, and their structures were analyzed using thin layer chromatography, high performance liquid chromatography and electrospray ionization/mass spectrometry. Major phospholipids in P. putida KT2442 were phosphatidylethanolamine (79.9%), phosphatidylglycero1 (12.7%) and cardiolipin (7.4%), with C_16:1 and/or C_18:1 acyl chains. Four lipid A species were found in P. putida KT2442: two are hexa-acylated and the other two are penta-acylated. Compared with lipid A of P. aeruginosa, P. putida lipid A has less hydroxylation on the secondary acyl chains and less modification. Therefore, P. putida lipid A could be used as a base structure to investigate lipid A modification of P. aeruginosa for understanding its pathogenesis.

Lipopolysaccharides, major molecules in the outer membrane of Gram-negative bacteria, play important roles on membrane integrity of the cell. However, how the core oligosaccharide of lipopolysaccharide affect the membrane behavior is not well understood. In this study, the relationship between the core oligosaccharide of lipopolysaccharide and the membrane behavior was investigated using a series of Escherichia coli mutants defective in genes to affect the biosynthesis of core oligosaccharide of lipopolysaccharide. Cell surface hydrophobicity, outer membrane permeability, biofilm formation and auto-aggregation of these mutant cells were compared. Compared to the wild type W3110, cell surface hydrophobicities of mutant ΔwaaC, ΔwaaF, ΔwaaG, ΔwaaO, ΔwaaP, ΔwaaY and ΔwaaB were enhanced, outer membrane permeabilities of ΔwaaC, ΔwaaF, ΔwaaG and ΔwaaP were significantly increased, abilities of biofilm formation by ΔwaaC, ΔwaaF, ΔwaaG, ΔwaaO, ΔwaaR, ΔwaaP, ΔwaaQ and ΔwaaY decreased, and auto-aggregation abilities of ΔwaaC, ΔwaaF, ΔwaaG, ΔwaaO, ΔwaaR, ΔwaaU, ΔwaaP and ΔwaaY were strongly enhanced. These results give new insight into the influence of core oligosaccharide of lipopolysaccharide on bacterial cell membrane behavior.

Ribosome elongation factor G encoded by fusA promotes the translocation step of protein synthesis in bacteria; ribosome recycling factor encoded by frr, together with the elongation factor G, dissociates ribosomes from messenger RNA after the termination of translation. Both factors play important roles during protein synthesis in bacteria. In this study, we found that overexpression of fusA and/or frr led to the increase of l-isoleucine production in Corynebacterium glutamicum IWJ001, an l-isoleucine production strain generated by random mutagenesis. Reverse transcription polymerase chain reaction analysis showed that transcriptional levels of genes lysC, hom, thrB, ilvA, ilvBN, and ilvE encoding the key enzymes in the biosynthetic pathway of l-isoleucine increased in C. glutamicum IWJ001 when fusA and/or frr were overexpressed. Co-overexpression of fusA and frr, together with genes ilvA, ilvB, ilvN, and ppnk in C. glutamicum IWJ001, led to 76.5 % increase of l-isoleucine production in flask cultivation and produced 28.5 g/L l-isoleucine in 72-h fed-batch fermentation. The results demonstrate that overexpressing ribosome elongation factor G and ribosome recycling factor is an efficient approach to enhance l-isoleucine production in C. glutamicum.