Xuping Shentu

Effects of NADH availability on the Klebsiella pneumoniae strain with 1,3‐propanediol operon over‐expression

It is generally known that genes dhaB and dhaT are responsible for 1,3‐propanediol (1,3‐PD) production in the presence of glycerol in Klebsiella pneumoniae and these genes are organized in one operon. In the present study, a genetic means of increasing the enzyme activities of 1,3‐PD formation pathway through the over‐expression of 1,3‐PD opeorn was performed in K. pneumoniae S6. The recombinant strain S6‐PD showed 27‐ and 15‐fold increase in enzymatic activities of DhaB and DhaT, respectively with respect to wild‐type strain while failed to improve the 1,3‐PD yield due to the inadequacy of cofactor NADH. Therefore, in order to increase NADH availability, a NADH regeneration system was constructed by heterologous expression of NAD+‐dependent formate dehydrogenase gene (fdh1) from Candida boidinii and introduced into S6‐PD to investigate its effects on the glycerol utilization and 1,3‐PD formation. The results demonstrated that the increase of NADH availability could efficiently improve glycerol metabolism and promote 1,3‐PD yield.

1,3-propanediol production from glucose by mixed-culture fermentation of Zygosacharomyces rouxii and Klebsiella pneumonia

1,3‐propanediol is an important chemical widely used in polymer production. In this study, two strains, Zygosacharomyces rouxii JL2011 and Klebsiella pneumoniae S6, were used as a mixed culture for 1,3‐propanediol production directly from glucose. Two important parameters including inoculation time of K. pneumoniae S6 at stage of mixed culture and initial cell ratio of Z. rouxii JL2011 to K. pneumoniae S6 in mixed fermentation were optimized in culture flasks. In those experiments, the best results were obtained with a yield of 6.8 g/L 1,3‐propanediol from glucose when K. pneumoniae S6 was inoculated after 48 h in the culture of Z. rouxii JL2011 by mixed culture of Z. rouxii JL2011 and K. pneumoniae S6 with initial cell ratio of 1:200. In a 7‐L bioreactor, the maximum 1,3‐propanediol production could reach up to 15.2 g/L. Thus, this study presents an effective process for 1,3‐propanediol microbial production from glucose by using mixed culture of Z. rouxii JL2011 and K. pneumoniae S6. This work does not only demonstrate a new way to produce 1,3‐propanediol from a low‐cost feedstock, but may also make a valuable contribution to the development of a cost‐effective fermentation based on renewable resources.

Engineering Streptomyces diastatochromogenes 1628 to increase the production of toyocamycin

Toyocamycin (TM) shows antifungal activity and can be used to treat plant diseases. TM is produced by Streptomyces diastatochromogenes 1628, but the production rate is always very low. Recently, the gene toyG was cloned; however, nothing was known about the influence of toyG on TM production. In this study, the rational combined co‐expression of toyG with frr encoding a ribosome recycling factor and vgb encoding Vitreoscilla hemoglobin was performed. Introduction of these genes in double‐ or triple‐combination into strain S. diastatochromogenes 1628 led to greatly increased TM production. Transcription analysis revealed that higher TM production in the constructed recombinant strains resulted from an increase in transcriptional levels of toyG gene. At the end, among all these different recombinant strains, the strain S. diastatochromogenes 1628‐VGF, harboring vgb, frr, and toyG placed under the control of PermE*, exhibited the largest increase in TM production. Notably, TM produced by S. diastatochromogenes 1628‐VGF reached the highest level of 319.2 mg/L at 72 h, while TM yield of the original strain 1628 was 147.1 mg/L at 84 h. The high‐yield, energy‐saving strain 1628‐VGF constructed in this study shall be beneficial for the industrial production of TM.

Optimization of electroporation conditions for toyocamycin producer Streptomyces diastatochromogenes 1628

Because of its structural similarity to nucleoside, toyocamycin exhibits potential of wide application and various biological activities. Streptomyces diastatochromogenes 1628, capable of producing toyocamycin, has exhibited a potential biocontrol effect in inhibiting the development of phytopathogens in the agriculture field. An efficient transformation system is a prerequisite for genetic and molecular study of S. diastatochromogenes 1628. In this study, we optimized experimental factors involved in the electroporation transformation process. Key features of this procedure, including collection of cells at the mid‐log phase stage and the treatment of cells with lysozyme and penicillin G prior to the electroporation and recovery medium and time, produced the greatest increase in the efficiency and consistency of results. The transformation efficiency also depends on field strength, cell concentration, and plasmid DNA quantity. Under the optimal conditions, a maximal efficiency of (3 ± 0.4) × 104 µg−1 DNA was obtained. The development of transformation method for S. diastatochromogenes 1628 will foster genetic manipulation of this important strain.

Production of 1,3-propanediol by Klebsiella pneumoniae using raw glycerol from Zygosacharomyces rouxii

The conversion of glycerol to 1,3-propanediol (1,3-PD) is an environmentally friendly biological process and has attracted great interests worldwide. Efficient utilization of raw glycerol could bring significant economic benefits for 1,3-PD production. In the present study, the glycerol broth from strain Zygosacharomyces rouxii JL2011 was first used as the sole carbon source for 1,3-PD production by Klebsiella pneumoniae . The result indicates that the glycerol broth affected K. pneumoniae growth and 1,3-PD yield dramatically. Therefore, the raw glycerol was further prepared by extraction and purification, and the effect of raw glycerol on 1,3-PD production was investigated. The results obtained showed that the purified glycerol could be directly utilized in shake-flask culture by K. pneumoniae with a result comparable with that attained from pure glycerol. In addition, the fed-batch culture using raw glycerol as the substrate was conducted giving 56.1 g×L -1 with a yield of 0.40 g×g -1 glycerol and productivity of 1.12 g×L -1 ×h -1 . Key words: 1,3-propanediol, Klebsiella pneumoniae, Zygosacharomyces rouxii , raw glycerol.