Jian Zhuge

Expression of 1,3-propanediol oxidoreductase and its isoenzyme in Klebsiella pneumoniae for bioconversion of glycerol into 1,3-propanediol

In the Klebsiella pneumoniae reduction pathway for 1,3-propanediol (1,3-PD) synthesis, glycerol is first dehydrated to 3-hydroxypropionaldehyde (3-HPA) and then reduced to 1,3-PD with NADH consumption. Rapid conversion of 3-HPA to 1,3-PD is one of the ways to improve the yield of 1,3-PD from glycerol and to avoid 3-HPA accumulation, which depends on enzyme activity of the reaction and the amount of reducing equivalents available from the oxidative pathway of glycerol. In the present study, the yqhD gene, encoding 3-propanediol oxidoreductase isoenzyme from Escherichia coli and the dhaT gene, encoding 3-propanediol oxidoreductase from K. pneumoniae were expressed individually and co-expressed in K. pneumoniae using the double tac promoter expression plasmid pEtac-dhaT-tac-yqhD. The three resultant recombinant strains (K. pneumoniae/pEtac-yqhD, K. pneumoniae/pEtac-dhaT, and K. pneumoniae/pEtac-dhaT-tac-yqhD) were used for fermentation studies. Experimental results showed that the peak values for 3-HPA production in broth of the three recombinant strains were less than 25% of that of the parent strain. Expression of dhaT reduced formation of by-products (ethanol and lactic acid) and increased molar yield of 1,3-PD slightly, while expression of yqhD did not enhance molar yield of 1,3-PD, but increased ethanol concentration in broth as NADPH participation in transforming 3-HPA to 1,3-PD allowed more cellular NADH to be used to produce ethanol. Co-expression of both genes therefore decreased by-products and increased the molar yield of 1,3-PD by 11.8%, by catalyzing 3-HPA conversion to 1,3-propanediol using two cofactors (NADH and NADPH). These results have important implications for further studies involving use of YqhD and DhaT for bioconversion of glycerol into 1,3-PD.

Cloning and characterization of a NAD+‐dependent glycerol‐3‐phosphate dehydrogenase gene from Candida glycerinogenes, an industrial glycerol producer

The osmotolerant yeast Candida glycerinogenes produces glycerol as a major metabolite on an industrial scale, but the underlying molecular mechanisms are poorly understood. We cloned and characterized a 4900-bp genomic fragment containing the CgGPD gene encoding a glycerol-3-phosphate dehydrogenase homologous to GPD genes in other yeasts using degenerate primers in conjunction with inverse PCR. Sequence analysis revealed a 1167-bp open reading frame encoding a putative peptide of 388 deduced amino acids with a molecular mass of 42 695 Da. The CgGPD gene consisted of an N-terminal NAD(+)-binding domain and a central catalytic domain, whereas seven stress response elements were found in the upstream region. Functional analysis revealed that Saccharomyces cerevisiae gpd1Delta and gpd1Delta/gpd2Delta osmosensitive mutants transformed with CgGPD were restored to the wild-type phenotype when cultured in high osmolarity media, suggesting that it is a functional GPD protein. Transformants also accumulated glycerol intracellularly and GPD-specific activity increased significantly when stressed with NaCl, whereas the S. cerevisiae mutants transformed with the empty plasmid showed only slight increases. The full-length CgGPD gene sequence including upstream and downstream regions has been deposited in GenBank under accession no. EU186536.

Construction of a Novel Expression System in Klebsiella pneumoniae and its Application for 1,3-Propanediol Production

A novel expression system of Klebsiella pneumoniae was developed in order to improve 1,3-propanediol (1,3-PD) production using a K. pneumoniae–Escherichia coli shuttle vector pET28a consisting of the kanamycin-resistance gene promoter Pkan. The recombinant plasmid pETPkan-cat carrying the chloramphenicol acetyltransferase gene cat as selectable marker was constructed to test the availability of the promoter Pkan in K. pneumoniae. The results showed that the chloramphenicol acetyltransferase was apparently expressed in K. pneumoniae, and the recombinant strain had a high-level resistance to chloramphenicol, suggesting that the promoter Pkan was efficient in K. pneumoniae. Then, the expression system was applied to the expression of 1,3-PD oxidoreductase in K. pneumoniae. The enzyme was over-expressed, and the recombinant K. pneumoniae showed a nearly 3.0-fold decrease in peak level of the intermediary metabolite 3-hydroxypropionaldehyde and an increase of 16.5% in yield of 1,3-PD with respect to the wild-type strain. From these results, the first reported expression system has paved the way for improvement of 1,3-PD production and will be available and efficient for other heterologous gene expression in K. pneumoniae.

Protoplast preparation and polyethylene glycol (PEG)-mediated transformation of Candida glycerinogenes

The regeneration of Candida glycerinogenes protoplasts is a major step following genetic manipulations such as fusion and DNA-mediated transformation. An investigation of protoplast formation and cytological examination was used to gain further insight into the loss of protoplast viability in osmotically stabilized support media. Protoplasts with the highest regeneration frequency (98.6% protoplasts/mL) were isolated, using lysozyme dissolved in 1M sorbitol osmoticum. The commercial enzyme preparations, osmotic stabilisers, and growth phase were effective in raising the protoplast yield. Sodium chloride was effective for protoplast preparation; however, sugars and sugar alcohols were better for protoplast regeneration. Sorbitol at a concentration of 1 M was used in regeneration agar for further studies. Regeneration of colonies from protoplasts was maximal (11 ~ 15%) when protoplasts were incorporated in cooled agar containing 0.5% glucose, supplemented with 1M sorbitol as osmotic stabilizer. C. glycerinogenes strain was highly sensitive to zeocin, so transformation of protoplasts and PEG-mediated was achieved with an improved transformation system, using plasmid pURGAP-gfp containing zeocin gene driven by a PCgGAP promoter from C. glycerinogenes to express gfp gene and be transformed into the 5.8S rDNA site of C. glycerinogenes in order to test the system for studying the yeast osmoregulation. We developed an efficient method for transformation of C. glycerinogenes, and parameters involved in transformation efficiency were optimized. Expressions of gfp at different levels were conducted under osmotic stress containing NaCl, KCl, sorbitol or glycerol for the recombinant strains. These improved procedures for protoplast isolation, regeneration and transformation proved to be useful applications in genetic studies for other Candida species and industrial yeast.

Role of CgHOG1 in Stress Responses and Glycerol Overproduction of Candida glycerinogenes

Candida glycerinogenes, the glycerol producer with excellent multi-stress tolerances, is considered to be a potential biotechnological host used in the production of glycerol and its derivatives under extreme fermentation conditions. In this study, to evaluate the multiple roles of mitogen-activated protein kinase CgHOG1, we constructed a gene disruption system in the diploid C. glycerinogenes to obtain CgHOG1 null mutant. Pseudohyphae generation of the CgHOG1 mutant under non-inducing condition indicated a repressor role in morphological transitions. Disruption of CgHOG1 resulted in increased sensitivities to osmotic, acetic acid, and oxidative stress but not involved in thermotolerance. In the CgHOG1 mutant, NaCl shock failed to stimulate the accumulation of intracellular glycerol and was fatal. In addition, the CgHOG1 mutant displayed a significant prolonged growth lag phase in YPD medium with no decrease in glycerol production, whereas the mutant cannot grow under hyperosmotic condition with no detectable glycerol in broth. These results suggested that CgHOG1 plays important roles in morphogenesis and multi-stress tolerance. The growth and glycerol overproduction under osmotic stress are heavily dependent on CgHOG1 kinase.

Cloning and characterization of a novel NAD+‐dependent glyceraldehyde‐3‐phosphate dehydrogenase gene from Candida glycerinogenes and use of its promoter

A 3950 bp genomic fragment from Candida glycerinogenes, WL2002‐5, containing the CgGAP gene encoding a glyceraldehyde‐3‐phosphate dehydrogenase homologous to GAP genes in other yeasts using degenerate primers, was cloned and characterized with inverse PCR. Sequence analysis revealed a 1164 bp open reading frame encoding a putative peptide of 387 deduced amino acids, with a molecular mass of 36 kDa. The CgGAP protein consisted of an N‐terminal NAD+‐binding domain and a central catalytic domain. Six stress‐response elements were found in the upstream region of the CgGAP gene. The influence of CgGAP on glycolysis was investigated. Functional analysis revealed that Saccharomyces cerevisiae transformed with CgGAP was restored to the wild‐type phenotype when cultured in high‐osmolarity medium, suggesting that it is a functional GAP protein. Promoter studies in S. cerevisiae using the green fluorescent protein (gfp) gene as a reporter showed that the GAP promoter (PCgGAP) is constitutively expressed in S. cerevisiae cells grown on glucose. Copyright

budC knockout in Klebsiella pneumoniae for bioconversion from glycerol to 1,3‐propanediol

2,3‐Butanediol (2,3‐BD) is a major by‐product of 1,3‐propanediol (1,3‐PDO) fermentation by Klebsiella pneumoniae ZG25. It not only consumes large amounts of its carbon source and nicotinamide adenine dinucleotide to diminish synthesis of 1,3‐PDO, but also serves as an obstacle to high‐purity 1,3‐PDO in downstream processes. To decrease the formation of 2,3‐BD and make an intrinsic improvement in 1,3‐PDO production, the budC gene in K. pneumoniae, coding 2,3‐BD dehydrogenase, which is a key gene of the 2,3‐BD pathway, was successfully knocked out using the Red recombination system described in this paper. The results of the mutant fed‐batch fermentation showed that the 1,3‐PDO concentration, productivity per cell dry weight, and conversion rate increased to 880 mmol L−1, 22.0 mmol L−1 h−1, and 0.700 mol mol−1, respectively, increasing by 10%, 15%, and 11% compared with the parent strain. Meanwhile, 2,3‐BD was still found in fermentation broth with the 2,3‐BD metabolic pathway blocked, which implies that K. pneumoniae possesses a pathway of the 2,3‐BD cycle as a replenishment pathway.

Advances in the bioconversion mechanism of lovastatin to wuxistatin by Amycolatopsis sp. CGMCC 1149

Wuxistatin, a novel statin and more potent than lovastatin, was converted from lovastatin by Amycolatopsis sp. (CGMCC 1149). Product I, an intermediate product, was found in the fermentation broth, and the structure analysis showed that product I had an additional hydroxyl group at the methyl group attached to C3 compared to lovastatin, which indicates that product I is one isomer of wuxistatin. Isotope tracing experiment proved that hydroxyl group of wuxistatin was provided by product I and the reaction from product I to wuxistatin was an intramolecular transfer. Hydroxylation reaction established in a cell-free system could be inhibited by CO and enhanced by ATP, Fe2+, and ascorbic acid, which were consistent with the presumption that the hydroxylase was an induced cytochrome P450. Study on proteomics of Amycolatopsis sp. CGMCC 1149 suggested that three identified proteins, including integral membrane protein, Fe-S oxidoreductase, and GTP-binding protein YchF, were induced by lovastatin and required during hydroxylation reaction. In conclusion, bioconversion mechanism of wuxistatin by Amycolatopsis sp. CGMCC 1149 was proposed: lovastatin is firstly hydroxylated to product I by a hydroxylase, namely cytochrome P450, and then product I is rearranged to wuxistatin by isomerases.

Isolation and sequence analysis of the gene URA3 encoding the orotidine-5'-phosphate decarboxylase from Candida glycerinogenes WL2002-5, an industrial glycerol producer

The URA3 gene of Candida glycerinogenes WL2002‐5, an industrial glycerol producer encoding orotidine‐5′‐phosphate decarboxylase enzyme, was isolated by complementation cloning in Saccharomyces cerevisiae. DNA sequence analysis revealed the presence of an open reading frame (ORF) of 786 bp, encoding a 262 amino acid protein, which shares 71.65% amino acid sequence similarity to the S. cerevisiae URA3 protein. Furthermore, the cloned ORF fully complemented the ura3 mutation of S. cerevisiae, confirming that it encodes for the C. glycerinogenes Ura3 (CgUra3) protein. The GenBank Accession No. for CgUra3 is AY623794. Copyright

Facilitated Expression and Function Identification of Key Genes Converting Cyanocobalamin to Adenosylcobalamin

Klebsiella pneumoniae degrades glycerol generating 1,3-propanediol(1,3-PD) and 3-hydroxypropionic acid(3-HP) by a pathway dependent on coenzyme B12.Previous studies showed that glycerol could cause the irreversible cleavage of Co— C of coenzyme B12,inactivating glycerol dehydratase(GDHt).For further study on the course of converting inactive vitamin B12(CNCbI) to active coenzyme B12(AdoCbI),ATP:cob(I) alamin adenosyltransferase(ACA) gene btuR and dehydrogenase gene yciK from K.pneumoniae were cloned and a dual-promoter expression plasmid pUC18-tac-btuR-tac-yciK,including gene btuR and yciK,were successfully constructed.Finally,a strain of recombinant bacterium was obtained by transferring the plasmid into Escherichia coli JM109.The results of the improved analytical methods showed that(i) overexpressed proteins had cob(I) alamin adenosyltransferase activity;(ii) the recombinant E.coli JM109 could convert inactive cobalamin,such as vitamin B12,to active coenzyme B12very well.